研究者紹介システム
| 星 信彦 |  |
| ホシ ノブヒコ | |
| 大学院農学研究科 資源生命科学専攻 | |
| 教授 | |
| 獣医学畜産学関係 |
研究者情報
所属
【主配置】
大学院農学研究科 資源生命科学専攻【配置】
農学部 資源生命科学科
学位
授業科目
- 発生学, 医学部 医学科, 2022
- Global Topics in Animal Science, 農学部, 2022
- 高度教養セミナー農学部応用動物学, 農学部, 2022
- 卒業研究, 農学部, 2022
- 応用動物学実験, 農学部, 2022
- 外国書講読Ａ, 農学部, 2022
- 形態機能学Ⅰ－１, 農学部, 2022
- 資源生命科学入門Ⅰ－１, 農学部, 2022
- 実験動物の技術と応用, 農学部, 2022
- 実験動物の技術と応用１, 農学部, 2022
- 動物形態機能学Ⅰ, 農学部, 2022
- 初年次セミナー（応用動物学コース）, 農学部, 2022
- 形態機能学Ⅰ－２, 農学部, 2022
- 資源生命科学入門Ⅰ－２, 農学部, 2022
- 実験動物の技術と応用２, 農学部, 2022
- 実験動物マネジメント, 農学部, 2022
- 高度教養セミナー農学部応用動物学入門, 農学部, 2022
- 高度教養セミナー農学部応用動物学入門, 農学部, 2022
- 応用動物学各論, 農学部, 2022
- 外国書講読Ｂ, 農学部, 2022
- 形態機能学Ⅱ－１, 農学部, 2022
- 動物形態機能学Ⅱ, 農学部, 2022
- 形態機能学Ⅱ－２, 農学部, 2022
- 動物分子形態学, 大学院農学研究科, 2022
- 特定課題演習 I-1, 大学院農学研究科, 2022
- 課題開発演習, 大学院農学研究科, 2022
- 資源生命科学プレゼンテーション演習Ⅰ（オーラル・ペーパー）, 大学院農学研究科, 2022
- 特定課題演習 II-1, 大学院農学研究科, 2022
- 先端融合科学特論A（食料編）, 大学院農学研究科, 2022
- 動物生殖生理論, 大学院農学研究科, 2022
- 特定課題演習 I-2, 大学院農学研究科, 2022
- 資源生命科学プレゼンテーション演習Ⅱ（オーラル・ペーパー）, 大学院農学研究科, 2022
- 特定課題演習 II-2, 大学院農学研究科, 2022
ジャンル
コメントテーマ
研究活動
研究分野
論文
- Springer Science and Business Media LLC, 2023年05月06日, Cell and Tissue Research
研究論文（学術雑誌）
Neonicotinoid pesticides (NN) were recently reported to exhibit adverse effects in higher vertebrates. Moreover, NNs are routinely transferred from mother to offspring, raising concerns about their effects on future generations. The fetal and neonatal periods are the most critical to the formation of neural circuits in the brain through neurogenesis and differentiation, neuronal migration, axon guidance, and synaptogenesis. NN exposure throughout the fetal and neonatal periods was found to affect the neurobehavior of the offspring, but the stage-specific neurobehavioral effects are unclear. We exposed fetal and neonatal mice to a no-observed-adverse-effect level (NOAEL) of clothianidin (CLO) for 4 days during each of four developmental stages: neurite proliferation and differentiation (fetal days 9-12, CLO-1), neurite outgrowth (fetal days 15-18, CLO-2), synapse formation and astrocyte differentiation (days 1-4 after birth, CLO-3), and synapse remodeling (days 11-14 after birth, CLO-4). CLO's neurobehavioral effects were evaluated in juveniles and adults, revealing that CLO-1 and CLO-2 caused behavioral abnormalities in adult mice. CLO-3 significantly increased locomotor activity and decreased juvenile neurons in the hippocampal dentate gyrus in adulthood. Comprehensive gene analysis of CLO-3 revealed high expression of genes related to neurite outgrowth and axonal branching in the hippocampus in juveniles and adults. These results revealed developmental stage-specific effects of a NOAEL of CLO in the fetal and neonatal periods, suggesting that the susceptibility of the fetus and neonate to CLO varies by developmental stage.
2023年03月01日, The Journal of veterinary medical science, 85 (4), 486 - 496, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
The expression of sex determining region of the Y chromosome (Sry) in the fetal gonads is important for male development. In a mouse model of disorders of sex development (C57BL/6 (B6)-XYPOS), the gonadal phenotype and the timing of Sry expression differ due to differences among the B6 substrain as the genetic background. Since differences in Sry expression among B6 substrains have been speculated, the present study examined Sry expression in B6J, B6JJmsSlc, and B6NCrl mice. These substrains differed in the number of Sry-expressing cells in the gonads of embryonic mice at each developmental stage, with B6NCrl having more than the other strains. The substrains differed also in the number of Sry-expressing cells between the left and right gonads, with B6J and B6NCrl, but not B6JJmsSlc, showing left gonad-dominant Sry expression. Substrain differences existed also in the distribution of Sry-expressing cells in the medial and lateral directions of gonads. In addition, in the left gonad-dominant Sry-expressing substrains B6J and B6NCrl, the medial and central regions of the left gonad had more Sry-expressing cells than those of the right gonad. Substrains of B6 mice have not always been considered in sex differentiation studies. In the present study, however, we observed substrain differences in the number of Sry-expressing cells, left-right distribution, and medial/lateral distribution during the early stages of gonadal development in B6 mice. Therefore, future studies on sex differentiation in B6 mice should consider substrain differences.
2023年02月28日, The Journal of veterinary medical science, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Diamide insecticides activate ryanodine receptors expressed in lepidopteran skeletal muscle and promote Ca2+ release in the sarcoplasmic reticulum, causing abnormal contractions and paralysis, leading to death of the pest. Although they had been thought not to act on nontarget organisms, including mammals, adverse effects on vertebrates were recently reported, raising concerns about their safety in humans. We investigated the neurotoxicity of the acute no-observed-adverse-effect level of chlorantraniliprole (CAP), a diamide insecticide, in mice using clothianidin (CLO), a neonicotinoid insecticide, as a positive control. The CLO-administered group showed decreased locomotor activities, increased anxiety-like behaviors, and abnormal human-audible vocalizations, while the CAP-administered group showed anxiety-like behaviors but no change in locomotor activities. The CAP-administered group had greater numbers of c-fos‒immunoreactive cells in the hippocampal dentate gyrus, and similar to the results in a CLO-administered group in our previous study. Blood corticosterone levels increased in the CLO-administered group but did not change in the CAP-administered group. Additionally, CAP was found to decreased 3-Methoxytyramine and histamine in mice at the time to maximum concentration. These results suggest that CAP-administered mice are less vulnerable to stress than CLO-administered mice, and the first evidence that CAP exposure increases neuronal activity and induces anxiety-like behavior as well as neurotransmitter disturbances in mammals.
2023年02月28日, The Journal of veterinary medical science, 85 (4), 497 - 506, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Recently, the effects of exposure to clothianidin (CLO) on the thymus and gut microbiota have become clear, but no report has examined its next-generation impacts. Pregnant C57BL/6N mice were administered a no-observed-adverse-effect-level dose of CLO until weaning. We examined CLO's effects on the gut microbiota and immune organs of dams and their 3- and 10-week-old male offspring. CLO administration led to several alterations of the top 30 bacterial genera in the gut microbiota in dams and 3-week-old mice. Compared to controls, 10-week-old mice had more thymic Hassall's corpuscles, and both dams and 10-week-old mice had fewer macrophages. These results suggest that fetal and lactational CLO exposure may affect the immune system and gut microbiota of the next generation.
2023年02月17日, The Journal of veterinary medical science, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Our previous studies and others have revealed detailed characteristics of the mucosal nerve network in the small intestine, but much remains unknown about the corresponding network in the large intestine. We herein investigated regional differences in the expression of neurochemical markers, the nerve network structure, and the cells in contact with nerve fibers by histological analysis using both immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM). Immunohistochemistry revealed that immunopositive structures for protein gene product 9.5, vasoactive intestinal peptide (VIP), calretinin and vesicular acetylcholine transporter were more prevalent in the lamina propria of the ascending colon than the cecum and descending colon (DC). There was no significant difference in the frequency of most neurochemical markers between the cecum and DC, but the frequencies of VIP+ structures were higher in the cecum than in the DC. SBF-SEM analysis showed that the nerve network structure was more developed on the luminal side of the DC than the cecum. The cells that nerve fibers abundantly contacted were subepithelial and lamina propria fibroblast-like cells and macrophages. In addition, nerve fibers in the cecum were in more frequent contact with immune cells such as macrophages and plasma cells than nerve fibers in the DC. Thus, the present histological analysis suggested that the mucosal nerve network in the large intestine possessed both regional universality and various specificities, and revealed the intimate relationship between the nerve network and immune cells, especially in the cecum.
2022年12月13日, The Journal of veterinary medical science, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Abstract Neonicotinoid pesticides are a class of insecticides that reportedly have harmful effects on bees and dragonflies, causing a reduction in their numbers. Neonicotinoids act as neuroreceptor modulators, and some studies have reported their association with neurodevelopmental disorders. However, the precise effect of neonicotinoids on the central nervous system has not yet been identified. Herein, we conducted in vivo Ca2+ imaging using a two-photon microscope to detect the abnormal activity of neuronal circuits in the brain after neonicotinoid application. The oral administration of acetamiprid (ACE) (20 mg/kg body weight (BW) in mature mice with a quantity less than the no-observed-adverse-effect level (NOAEL) and a tenth or half of the median lethal dose (LD50) of nicotine (0.33 or 1.65 mg/kg BW, respectively), as a typical nicotinic acetylcholine receptor (nAChR) agonist, increased anxiety-like behavior associated with altered activities of the neuronal population in the somatosensory cortex. Furthermore, we detected ACE and its metabolites in the brain, 1 h after ACE administration. The results suggested that in vivo Ca2+ imaging using a two-photon microscope enabled the highly sensitive detection of neurotoxicant-mediated brain disturbance of nerves.
Springer Science and Business Media LLC, 2022年12月, Scientific Reports, 12 (1), 5114 - 5114, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Neonicotinoid pesticides (NNs) have been reported to have neurobehavioral effects on offspring after fetal and lactational exposure. In this study, clothianidin (CLO), an NN, was administered orally as a single dose (6.5mg/kg: 1/10 of the no-observed-adverse-effect level in the current Pesticide Evaluation Report) to 10-day post-partum ICR mice, and CLO and its metabolites were quantified using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) after collecting maternal breast milk and blood samples over time (1, 3, 6, 9, 12, and 24h after administration). CLO and its metabolite desmethyl-CLO (dm-CLO) were detected in the breast milk at 1h after the administration, and their concentrations were significantly higher than those in blood at all time points. The concentrations of CLO and dm-CLO in the breast milk were at their highest levels at 1 and 3h, respectively, and then decreased over time to become almost undetectable at 24h after the administration. These results show that CLO is metabolized in the mother's body and is rapidly transferred to and concentrated in the breast milk. Since CLO concentrations in breast milk are higher than those in the blood, there is concern about the effects of CLO during lactation.
2022年10月31日, Toxicology letters, 373, 33 - 40, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Although neonicotinoids are among the major classes of pesticides that affect mammalian nervous systems, little is known about sex differences in their effects. This study aimed to examine whether the neurobehavioral effects of a neonicotinoid, clothianidin (CLO), differed between sexes. Male and female C57BL/6 N mice were orally administered CLO (5 or 50 mg/kg) at or below the chronic no-observed-adverse-effect-level (NOAEL) and subjected to behavioral tests of emotional and learning functions. Changes in neuroactivity in several brain regions and the concentrations of CLO and its metabolites in blood and urine were measured. Acute CLO exposure caused sex-related behavioral effects; decreases in locomotor activities and elevation of anxiety-like behaviors were more apparent in males than in females. In addition, male-specific impairment of short- and long-term learning memory by CLO exposure was observed in both the novel recognition test and the Barnes maze test. Male-dominant increases in the number of c-fos positive cells were observed in the paraventricular thalamic nucleus in the thalamus and in the dentate gyrus in the hippocampus, which are related to the stress response and learning function, respectively. The concentrations of CLO and most metabolites in blood and urine were higher in males. These results support the notion that male mice are more vulnerable than females to the neurobehavioral effects of CLO and provide novel insights into the risk assessment of neonicotinoids in mammalian neuronal function.
2022年10月14日, Toxicology and applied pharmacology, 456, 116283 - 116283, 英語, 国際誌研究論文（学術雑誌）
Our previous studies using immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM) clarified that fibroblast-like cells (FBLCs) in the rat ileal mucosa are classifiable into several subtypes, but their characteristics throughout the large intestine remain unknown. In this study, we investigated the region-specific characteristics of FBLCs in the rat large intestine using histological analysis including SBF-SEM. Immunohistochemistry revealed that CD34+CD31- FBLCs were localized in the lamina propria beneath the crypt bases throughout the large intestine and were more abundant in the descending colon than in the other regions. In addition, platelet-derived growth factor receptor α (PDGFRα)+ FBLCs were ubiquitously present just below the epithelium throughout the large intestine, and those at the crypt base were slightly more abundant in the descending colon than in the other regions. SBF-SEM analysis revealed that there were two types of FBLCs around the crypt base in both the cecum and the descending colon: sub-epithelial FBLCs localizing just beneath the epithelium in the manner of PDGFRα+ FBLCs, and lamina propria FBLCs localizing farther away from the epithelium than sub-epithelial FBLCs in the manner of CD34+CD31- FBLCs. The lamina propria FBLCs were closely apposed to various immune cells in the lamina propria, and their endoplasmic reticulum in the descending colon exhibited various dilatation levels, unlike that in the cecum. These findings indicate that FBLCs, especially around the crypt base, differed in each region of the large intestine with respect to localization, abundance, and ultrastructure, which could lead to the region-specific microenvironment around the crypt base.
2022年09月, Cell and tissue research, 389 (3), 427 - 441, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
- Springer Science and Business Media LLC, 2022年04月11日, Cell and Tissue Research
[査読有り]
研究論文（学術雑誌）
Neonicotinoid pesticides (NNs) cause behavioral abnormalities in mammals, raising concerns about their effects on neural circuit activity. We herein examined the neurological effects of the NN clothianidin (CLO) by in vivo Ca2+ imaging using two-photon microscopy. Mice were fed the no-observed-adverse-effect-level (NOAEL) dose of CLO for 2 weeks and their neuronal activity in the primary somatosensory cortex (S1) was observed weekly for 2 weeks. CLO exposure caused a sustained influx of Ca2+ in neurons in the S1 2/3 layers, indicating hyperactivation of neurons. In addition, microarray gene expression analysis suggested the induction of neuroinflammation and changes in synaptic activity. These results demonstrate that exposure to the NOAEL dose of CLO can overactivate neurons and disrupt neuronal homeostasis.
Japanese Society of Veterinary Science, 2022年, Journal of Veterinary Medical Science, 84 (4), 585 - 592, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Pyrethroids are one of the most commonly used classes of synthetic pesticides in the world. Recent laboratory and epidemiological evidence suggested that pyrethroids have potential adverse effects in the mammalian brain; however, the underlying mechanisms of the neurotoxic effects of pyrethroids have not been fully elucidated. In the present study, we investigated the mechanisms of effects of a type II pyrethroid deltamethrin (DM) in a neuronal cell model focusing on the proteolytic function, including autophagy and the ubiquitin-proteasome system. We confirmed that a micromolar concentration of DM dose-dependently decreased the cell viability and induced apoptotic cell death. Our results showed that DM enhanced autophagy in association with an accumulation of autophagosomes and increase in the levels of autophagy markers LC3-II/LC3-I ratio and p62 which were much elevated in the presence of lysosomal inhibitors bafilomycin A1 and chloroquine. We also found that DM caused a dysfunction of mitochondria with a decrease of mitochondrial membrane potential and mitochondrial DNA copy number as well as colocalization with autophagosomes. Moreover, a decrease in the activities of three major proteasomal enzymes and an accumulation of ubiquitinated proteins were observed by the exposure to DM. Transcriptome analysis revealed that up-regulated genes supported the activation of autophagy with induction of cellular stress responses including oxidative stress and endoplasmic reticulum stress, while down-regulated genes related to the cell cycle and DNA replication. These findings provide novel insights into the neurotoxicity of DM which underlie the imbalance in proteolytic function caused by mitophagy activation and proteasome inhibition.
2021年11月01日, Toxicology and applied pharmacology, 430, 115723 - 115723, 英語, 国際誌研究論文（学術雑誌）
Fipronil is a phenylpyrazole insecticide that is widely used as a pesticide and a veterinary drug, although studies suggest that it could be toxic to mammals. The objectives of this study were to examine the pharmacokinetic profile of fipronil in mice, dogs, and cats, and to evaluate its effects on emotional and cognitive behaviors of dogs and cats using the data obtained from mice. The assessment of in vivo kinetics of fipronil was conducted in mice and dogs. We also performed behavioral tests (elevated plus-maze and Y-maze) and measured the levels of neurotransmitters in mice exposed to fipronil. In addition, the in vitro metabolism of fipronil were evaluated using liver microsomes of rats, mice, dogs, and cats. The results revealed that fipronil is distributed throughout the body (blood, brain, adipose tissue, and liver) of mice after dermal application. It was metabolized to fipronil sulfone primarily in the liver. The data on kinetics show that both fipronil and fipronil sulfone have a longer half-life in dogs and cats than in mice. The behavioral tests indicated that fipronil and fipronil sulfone could affect emotional and cognitive behaviors and alter the levels of neurotransmitters (dopamine in the striatum and serotonin in the hippocampus) in mice. Furthermore, we found that dogs and cats have a low ability to metabolize fipronil than mice and rats. However, further comprehensive studies are needed to determine whether fipronil affects the emotional and cognitive behaviors when administered to dogs and cats. To the best of our knowledge, this is the first study to examine the pharmacokinetic data and verify the effects of fipronil on emotional and cognitive behaviors of dogs and cats using the data obtained from mice.
2021年06月, Pesticide biochemistry and physiology, 175, 104847 - 104847, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Neonicotinoids, which act as agonists of the nicotinic acetylcholine receptors of insects, are widely used pesticides worldwide. Although epidemiological studies revealed that the detection amounts of neonicotinoids in urine are higher in the elderly population than other age-groups, there is no available information regarding the risks of neonicotinoids to older mammals. This study was aimed to investigate aging-related differences in the behavioral effects of the neonicotinoid pesticide clothianidin (CLO). We acutely administered a sub-NOAEL level (5 mg/kg) of CLO to adult (12-week-old) and aging (90-week-old) mice and conducted four behavioral tests focusing on the emotional behavior. In addition, we measured the concentrations of CLO and its metabolites in blood, brain and urine. There were age-related changes in most parameters in all behavioral tests, and CLO significantly decreased the locomotor activity in the open field test and elevated plus-maze test in the aging group, but not in the adult group. The concentrations of most CLO and its metabolites were significantly higher in the blood and brain and were slightly lower in the urine in the aging group compared to the adult group. These findings should contribute to our understanding of age-related differences in the adverse effects of neonicotinoids in mammals.
2021年05月15日, Toxicology letters, 342, 95 - 103, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Several types of macrophages have been reported in the intestinal mucosa, but their histological localization remains ambiguous. Here, we obtained detailed information about ultrastructural and phenotypical diversity of macrophage-like cells (MLCs) in the rat ileal mucosa using immunofluorescent analysis and serial block-face scanning electron microscopy (SBF-SEM). The results revealed that the cells immunopositive for CD68, the pan-macrophage marker, included CD163-CD4+, CD163+CD4+, and CD163-CD4- cells in the lamina propria (LP) of the intestinal villus and around the crypt. CD68+CD4+CD163- cells seemed to be preferentially localized in the intestinal villus, whereas CD68+CD163+CD4+ cells were frequently localized around the crypt. SBF-SEM analysis identified three types of MLCs in the ileal mucosa, which were tentatively named types I-III MLC based on aspects of the 3D-ultrastructure, such as the localization, quantity of lysosomes, endoplasmic reticulum, and exoplasm. Type I and II MLCs were localized in the villous LP, while type III MLCs were localized around the crypt, although type II MLCs were a minor population. All three MLC types extended their cellular processes into the epithelium, with type I MLCs showing the greatest abundance of extended processes. Type I MLCs in the upper portion of the intestinal villus showed a higher level of attachment to intraepithelial lymphocytes (IELs) compared to type III MLCs around the crypt. These findings suggest that macrophages of the rat ileal mucosa differed by region along the longitudinal axis of the villous tip-crypt from the perspective of ultrastructure, cellular composition, localization, and interactions with IELs.
2021年05月07日, Cell and tissue research, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Recently, developmental exposure to clothianidin (CLO) has been shown to cause reproductive toxicity in male mice, but the effects in female mice remain to be clarified. Pregnant C57BL/6N mice were given a no-observed-adverse-effect-level (NOAEL) dose of CLO until weaning. We then examined ovaries of 3- or 10-week-old female offspring. In the CLO-administered group, morphological changes, a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 (GPx4), and activation of genes in the steroid hormone biosynthesis pathway were observed in 3-week-old mice, and decreases of GPx4 immunoreactivity, 17OH-progesterone and corticosterone levels were observed in 10-week-old mice, along with high rates of infanticide and severe neglect, providing new evidence that developmental exposure to CLO affects juvenile and adult mice differently.
2021年04月24日, The Journal of veterinary medical science, 83 (4), 746 - 753, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Hepatic steatosis is known to precede a continuum of events that lead to hepatic metabolic dysfunction, inflammation and carcinogenesis. Recently, studies have linked xenobiotic exposures to hepatic steatogenesis and its associated metabolic disorders; however, the underlying mechanisms remain elusive. This study aimed to elucidate the mechanistic role of imidacloprid in the prevalence of high fat diet (HFD)-induced liver steatosis, using a C57BL/6J mice model. Mice (3 weeks old) were fed with HFD and treated with 0.6 mg/kg bw/day (one-tenth of the NOAEL) of imidacloprid through water or diet, for 24 weeks. In a controlled group, mice were fed with only HFD. At the end of the study, imidacloprid treatment significantly potentiated HFD-induced body weight gain in mice. Also, imidacloprid increased the liver weights of mice, with complimentary reductions in mesenteric and gonadal white adipose tissue weights. Histopathological analysis of liver revealed a drastic steatosis in imidacloprid treated mice. Following a real-time qPCR analysis, imidacloprid upregulated transcriptions of hepatic fatty acid biosynthesis-related transcription factors and genes. Imidacloprid also induced hepatic expression of the gene encoding pregnane X receptor; but had no significant effect on hepatic expressions of liver X receptor and aryl hydrocarbon receptor. The imidacloprid treatment further enhanced serum alanine aminotransferase levels but downregulated hepatic antioxidant mRNA expressions. Ultimately, this study suggested an imidacloprid-potentiation effects on prevalence of HFD-induced liver steatosis via transcriptional modulations of the hepatic FA biosynthesis pathway.
2021年04月03日, The Journal of veterinary medical science, 83 (3), 487 - 500, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Recently, it has been reported that neonicotinoid pesticides (NNs) are transferred from mother to child and are assumed to affect the next generation, but the behavioral effects of NN exposure at different developmental stages have not been investigated. We exposed mice to no-observed-adverse-effect level (NOAEL) doses of clothianidin (CLO) during the fetal and lactational period, and then evaluated the neurobehavioral effects in juvenile and adult mice. Significant increases in anxiety-like behavior and locomotor activity were observed in juveniles and adults, respectively, and neuronal activity and neurogenesis in the hippocampal dentate gyrus were affected in both stages. These results suggest that fetal and lactational exposure to CLO may inhibit neurogenesis and cause different behavioral abnormalities at different developmental stages.
2021年04月03日, The Journal of veterinary medical science, 83 (3), 542 - 548, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Fipronil (FPN) is a systemic insecticide that antagonizes the gamma-aminobutyric acid type A (GABAA) receptors in insects. Recently, adverse effects of FPN on mammals have been reported, but most of those were caused by high doses of FPN and additives in the products. We investigated the effects of low-dose pure FPN on the emotional behavior of mice. Nine-week-old male mice conducted behavioral tests 24 hr after FPN administration by gavage at doses of 0.05 or 5 mg/kg based on the no-observed-effect level (NOEL), showed a significant increase in locomotor activity and dose-dependent responses on the time they spent in the central zone in the open field test. Pure FPN below the NOEL dose may affect the emotional behavior of mice.
2021年03月05日, The Journal of veterinary medical science, 83 (2), 344 - 348, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Although low‑intensity ultrasound (LIUS) is a clinically established procedure, the early cellular effect of LIUS on a genetic level has not yet been studied. The current study investigated the early response genes elicited by LIUS in bone marrow stromal cells (BMSCs) using global‑scale microarrays and computational gene expression analysis tools. Mouse ST2 BMSCs were treated with LIUS [ISATA, 25 mW/cm2 for 20 min with a frequency of 1.11 MHz in a pulsed‑wave mode (0.2‑s burst sine waves repeated at 1 kHz)], then cultured for 0.5, 1 and 3 h at 37˚C. The time course of changes in gene expression was evaluated using GeneChip® high‑density oligonucleotide microarrays and Ingenuity® Pathway Analysis tools. The results were verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). A single exposure of LIUS did not affect cell morphology, cell growth or alkaline phosphatase activity. However, 61 upregulated and 103 downregulated genes were identified from 0.5 to 3 h after LIUS treatment. Two significant gene networks, labeled E and H, were identified from the upregulated genes, while a third network, labeled T, was identified from the downregulated genes. Gene network E or H containing the immediate‑early genes FBJ osteosarcoma oncogene and early growth response 1 or the heat shock proteins heat shock protein 1a/b was associated mainly with the biological functions of bone physiology and protein folding or apoptosis, respectively. Gene network T containing transcription factors fos‑like antigen 1 and serum response factor was also associated with the biological functions of the gene expression. RT‑qPCR indicated that the expression of several genes in the gene networks E and H were elevated in LIUS‑treated cells. LIUS was demonstrated to induce gene expression after short application in mouse ST2 BMSCs. The results of the present study provide a basis for the elucidation of the detailed molecular mechanisms underlying the cellular effects of LIUS.
2021年03月, Molecular medicine reports, 23 (3), 英語, 国際誌[査読有り]
研究論文（学術雑誌）
【背景・目的】農薬等の化学物質曝露は、異常タンパク質の蓄積やミトコンドリアの機能異常を原因とする神経変性疾患のリスク要因となる可能性が指摘されている。本研究では、細胞内のタンパク質分解を司るオートファジーおよびユビキチンプロテアソーム系に着目し、ピレスロイド系農薬デルタメトリン（DM）による神経毒性メカニズムの一端を解明することを目的とした。
【方法】Neuro2a細胞を1–100 µMのDMに曝露し、wst-8アッセイおよびフローサイトメーターを用いて生細胞数およびアポトーシス細胞数を測定した。ウェスタンブロットおよび免疫染色により各種オートファジーマーカー（LC3、p62）、ミトコンドリアマーカー（TOM20、UQCRC1）の発現量および細胞内局在を解析した。加えて、JC-10および定量的PCRを用いてミトコンドリアの機能および量的影響を評価し、ユビキチン化タンパク質量およびプロテアソーム活性の測定を行った。
【結果・考察】DM曝露により濃度依存的なLC3-IIおよびp62発現量の上昇がみられ、30 µM以上の濃度ではアポトーシス細胞数の増加が観察された。一方で、DM曝露によりUQCRC1発現およびmtDNA量が低下し、膜電位の脱分極を伴うミトコンドリア傷害が誘導された。加えて、DM曝露群の細胞質中においてドット上に凝集した各種マーカーが共局在することから、ミトコンドリアが取り込んだオートファゴソームが蓄積することが明らかとなった。DMによりユビキチン化タンパク質量は増加し、各種プロテアソーム活性は低下した。以上の結果から、DM曝露時においてはプロテアソーム活性の低下に伴い、傷害されたミトコンドリアを基質とするマイトファジーの活性が上昇していることが示され、両タンパク質分解系の不均衡状態がアポトーシス誘導に関与することが初めて明らかとなった。
日本毒性学会, 2021年, 日本毒性学会学術年会, 48, P - 2E, 日本語- (公社)日本獣医学会, 2020年10月, 日本獣医学会学術集会講演要旨集, 163回, 315 - 315, 日本語極微量ネオニコチノイド系農薬がマウスの雌性生殖器に及ぼす継世代影響
Eosinophils are abundantly present in intestinal mucosa. However, the morphological characteristics of their cellular population are still largely unknown. In this study, we examine their characteristics in the rat ileal mucosa using histological and ultrastructural methods. The results indicated that ileal eosinophils could be distinguished into two main groups based on their nuclear shapes and distribution: eosinophils with spheric or reniform nuclei mainly localized in the villous region and eosinophils with annular or bacilliform nuclei as the major population around crypts. Immunohistochemical analysis revealed that all eosinophils in the lamina propria (LP) were immunopositive for CD11b, whereas eosinophils in LP of the intestinal villus but not those in LP around the crypt, were immunopositive for CD11c. Three-dimensional ultrastructural analysis using serial block-face scanning electron microscopy showed that the eosinophils with spheric or reniform nuclei were abundant in the upper portions of the intestinal villus, whereas those with annular nuclei were abundant in the lower portions of the intestinal villus and around crypts. The eosinophils with spheric or reniform nuclei possessed broader cellular bodies with greater abundance of surface projections compared with those with annular nuclei. Eosinophils in the upper portions of intestinal villus frequently extended their cellular bodies into the intraepithelial space. The number of total and eosinophil-specific granules was positively correlated with the minor axis of the nuclear holes in the annular nuclei. These data suggest that ileal eosinophils exhibit not homogenous but rather diverse characteristics, possible due to the mixture of eosinophils at different maturation and/or activation stages.
2020年09月, Cell and tissue research, 381 (3), 439 - 450, 英語, 国際誌研究論文（学術雑誌）
The comprehensive targets of innervation in the intestinal mucosa are unknown, partly because of the diversity of cell types and the complexity of the neural circuits. Herein, we investigated the comprehensive targets of neural connectivity and analyzed the precise characteristics of their contact structures in the mucosa of rat ileum. We examined target cells of neural connections and the characteristics of their contact structures by serial block-face scanning electron microscopy at four portions of the rat ileal mucosa: the apical and basal portions in the villi, and the lateral and basal portions around/in the crypts. Nerve fibers were in contact with several types of fibroblast-like cells (FBLCs), macrophage-like cells, eosinophils, lymphocyte-like cells, and other types of cells. The nerve fibers almost always ran more inside of lamina propria than subepithelial FBLC, and thus contacts with epithelial cells were very scarce. The contact structures of the nerve fibers were usually contained synaptic vesicle-like structures, and we classified them into patterns based on the number of nerve fiber contacting the target cells at one site, the maximum diameter of the contact structures, and the relationship between nerve fibers and nerve bundles. The contact structures for each type of cells occasionally dug into the cellular bodies of the target cells. We revealed the comprehensive targets of neural connectivity based on the characteristics of contact structures, and identified FBLCs, immunocompetent cells, and eosinophils as the candidate targets for innervation in the rat ileal mucosa.
2020年07月31日, The Journal of veterinary medical science, 82 (7), 990 - 999, 英語, 国内誌研究論文（学術雑誌）
- (一社)日本毒性学会, 2020年06月, The Journal of Toxicological Sciences, 45 (Suppl.), S86 - S86, 日本語ニコチン性アセチルコリン受容体アゴニストによるシグナル毒性と2光子顕微鏡を用いた検出手法の開発
Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.
2020年04月09日, The Journal of veterinary medical science, 82 (4), 414 - 421, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Neonicotinoids (NNs), a widely used class of systemic pesticides, are regarded as exhibiting selective toxicity in insects. However, NNs are suspected of exerting adverse effects on mammals as well, including humans. To date, only adult male animal models have been subjected to general toxicity studies of NNs; fetuses have yet to be considered in this context. Here, we focused on the NN clothianidin (CLO) for the first quantitative LC-MS/MS analysis of maternal-to-fetal transfer and residual property of once-daily (single or multiple days), orally administered CLO and its metabolites in mice. The results revealed the presence of CLO and its five metabolites at approximately the same respective blood levels in both dams and fetuses. In the dams, CLO showed a peak value 1 h after administration, after which levels rapidly decreased at 3 and 6 h. In the fetuses of each group, levels of CLO were almost the same as those observed in the corresponding dams. The present results clearly demonstrated rapid passage of CLO through the placental barrier. However, metabolite-dependent differences observed in blood pharmacokinetics and residual levels. This is the first quantitative demonstration of the presence of CLO and its metabolites in fetal mouse blood.
2020年04月01日, Toxicology letters, 322, 32 - 38, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Neonicotinoid pesticides (NNs) act as agonists on nicotinic acetylcholine receptors (nAChRs) of insects, and there have been concerns about the effects of NNs on the health of mammals. Since nAChRs are expressed in immune cells, it is possible that NNs disturb the immune system. However, few reports have examined the immunotoxicity of clothianidin (CLO), a widely-used NN. Here, we report the effects of CLO on immune organs and type IV allergic reactions in ear auricles. We orally administered CLO at 0, 30 and 300 mg/kg/day (CLO-0, 30 and 300) to Sprague-Dawley rats for 28 days. The effects were evaluated by organ and body weights, histopathology, and immunohistochemistry (TCRαβ, CD4, CD8, CD11b, CD68, CD103). In addition, some cecal contents were subjected to preliminary gut microbiota analysis, because microbiota contribute to host homeostasis, including the immunity. Our results showed loose stool, suppression of body weight gain, significant changes in organ weights (thymus: decreased; liver: increased) and changes of the gut microbiota in the CLO-300 group. There were no obvious histopathological changes in immune organs. Granulomas of the ear auricles were found in one rat of each of the CLO-30 and 300 groups, but CLO had no apparent effect on the thickness or immunohistochemistry in the ear auricles. We present new evidence that CLO affects the thymus and intestine, and might enhance the local inflammatory response. These findings should contribute to the appropriate evaluation of the safety of NNs in the future.
2020年03月24日, The Journal of veterinary medical science, 82 (3), 360 - 372, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Dinotefuran (DIN) belongs to the neonicotinoids (NNs), a class of globally applied pesticides originally developed to exhibit selective toxicity in insects. However, several reports have suggested that NNs also exert neurotoxic effects in mammals. We previously demonstrated neurobehavioral effects of DIN on mice under non-stressful conditions. For further toxicity assessments in the present study, we investigated the effects of DIN on mice exposed to stressful conditions. After subacutely administering a no-observed-effect-level (NOEL) dose of DIN and/or chronic unpredictable mild stress (CUMS) to mice, we conducted three behavioral tests (i.e., open field test [OFT], tail suspension test [TST] and forced swimming test [FST]). In addition, serotonin (5-HT) and tryptophan hydroxylase 2 (TPH2) of the dorsal raphe nuclei (DRN) and median raphe nuclei (MRN) and tyrosine hydroxylase (TH) of the ventral tegmental area and substantia nigra (SN) were evaluated immunohistochemically. A NOEL dose of DIN or CUMS alone increased of the total distance in OFT, decreased or increased the immobility time in TST or FST, respectively, and increased the positive intensity of 5-HT and TPH2 in the DRN/MRN, and TH in the SN. These changes were suppressed under the conditions of combined exposure to DIN and CUMS, though the blood corticosterone level was increased depending on the blood DIN values and the presence of CUMS. The present study suggests the multifaceted toxicity of the neurotoxin DIN.
2020年03月24日, The Journal of veterinary medical science, 82 (3), 350 - 359, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
Two typical medium replacement methods are employed to culture cells, that is, traditional manual medium change, wherein complete medium changes are performed at short-term intervals; and perfusion, wherein continuous culture media delivery and discharge are performed. The former is associated with the issue of readily accumulation of metabolic waste products, which is resolved by the latter. However, the latter requires a specific chamber for cell culturing such as a microfluidic channel for flow stability. Generally, bio-medical researchers require a culturing process that utilizes commercial culture dishes to test their conventional manipulation know-how, experience, and protocols. Thus, we constructed a perfusion-culture system using commercial 35 mm culture dishes. To use commercial culture dishes, it is necessary to maintain the culturing conditions including a constant volume and uniform flow rate, and we invented a novel adapter, culture dish adapter (CD-Adapter), attached to a commercial culture dish. The CD-Adapter is fixed with a holding jig to keep the medium volume constant, so it can maintain a uniform flow in the culture dish. Additionally, we demonstrate the applicability of the system by applying it to a perfusion culture of 293T cells, revealing a 50% reduction in DDIT3, a cellular stress marker, compared with that in conventional manual medium change.
MDPI, 2020年, APPLIED SCIENCES-BASEL, 10 (1), 英語[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 65 - 65, 日本語クロチアニジンの亜急性摂取はラット腸内細菌叢を変化させる
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 66 - 66, 日本語クロチアニジンおよびその代謝産物の母子間移行における組織分布・蓄積性の定量的解明
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 67 - 67, 日本語低用量クロチアニジンによるマウスの物体認識・空間学習記憶障害とその性差
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 68 - 68, 日本語低用量クロチアニジンによる情動変容への加齢の影響評価
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 69 - 69, 日本語低用量クロチアニジンの胎子・授乳期曝露による神経行動学的影響
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2019年12月, 環境ホルモン学会研究発表会要旨集, 22回, 70 - 70, 日本語ネオニコチノイド系農薬クロチアニジンが次世代雌性生殖器に及ぼす影響
Neonicotinoids are one of most widely used pesticides targeting nicotinic acetylcholine receptors (nAChRs) of insects. Recent epidemiological evidence revealed increasing amounts of neonicotinoids detected in human samples, raising the critical question of whether neonicotinoids affect human health. We investigated the effects of a neonicotinoid pesticide clothianidin (CTD) on human neuroblastoma SH-SY5Y cells as in vitro models of human neuronal cells. Cellular and functional effects of micromolar doses of CTD were evaluated by changes in cell growth, intracellular signaling activities and gene expression profiles. We examined further the effects of CTD on neuronal differentiation by measuring neurite outgrowth. Exposure to CTD (1-100 μM) significantly increased the number of cells within 24 h of culture. The nAChRs antagonists, mecamylamine and SR16584, inhibited this effect, suggesting human α3β4 nAChRs could be targets of neonicotinoids. We observed a transient intracellular calcium influx and increased phosphorylation of extracellular signal-regulated kinase 1/2 shortly after exposure to CTD. Transcriptome analysis revealed that CTD down-regulated genes involved in neuronal function (e.g., formation of filopodia and calcium ion influx) and morphology (e.g., axon guidance signaling and cytoskeleton signaling); these changes were reflected by a finding of increased neurite length during neuronal differentiation. These findings provide novel insight into the potential risks of neonicotinoids to the human nervous system.
2019年11月15日, Toxicology and applied pharmacology, 383, 114777 - 114777, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2019年08月, 日本獣医学会学術集会講演要旨集, 162回, 504 - 504, 日本語GABA攪乱物質フィプロニルがマウスの情動行動に及ぼす影響
- (一社)日本毒性学会, 2019年06月, The Journal of Toxicological Sciences, 44 (Suppl.), S53 - S53, 日本語ネオニコチノイド系殺虫剤と農薬の現状と今後の展望〜毒性学・農学・行政の視点から〜 ネオニコチノイド系殺虫剤の曝露実態の解明と毒性評価
- (一社)日本毒性学会, 2019年06月, The Journal of Toxicological Sciences, 44 (Suppl.), S280 - S280, 日本語ヒト神経芽細胞腫におけるネオニコチノイド系農薬のシグナル毒性的影響評価と作用機序の解析
- (一社)日本毒性学会, 2019年06月, The Journal of Toxicological Sciences, 44 (Suppl.), S53 - S53, 日本語ネオニコチノイド系殺虫剤と農薬の現状と今後の展望〜毒性学・農学・行政の視点から〜 ネオニコチノイド系殺虫剤の曝露実態の解明と毒性評価
[査読有り]
C57BL/6J-XYPOS (B6J-XYPOS) mice, which have the Y chromosome derived from Mus musculus poschiavinus on a B6J genetic background, form ovotestes or ovaries. Previously, we replaced the genetic background of B6J-XYPOS mice with B6N and found that individuals with testes also appeared in addition to those with ovaries or ovotestes. To investigate the effect of the B6J genetic sequence on the testis differentiation, the genetic background of B6N-XYPOS mice was replaced with B6J again. The recovery of the B6J genetic background significantly decreased the incidence of testes; only ovaries developed. These results indicate that the testicular differentiation process tends to be perturbed especially in the B6J substrain. This shows the importance of substrain differences in mice usually treated as B6 collectively.
2019年04月27日, The Journal of veterinary medical science, 81 (4), 608 - 611, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
【背景・目的】1990年代以降に農薬登録されたネオニコチノイドは昆虫のニコチン性アセチルコリン受容体（nAChRs）に対するアゴニスト作用を示す新規農薬であるが、動物実験により哺乳類nAChRsを介して不測の影響を及ぼす例が報告されている。また、国内外における本農薬使用量の増加に伴い、ヒト尿からの検出率および検出量が急増している一方で、ヒトnAChRsに対する作用については十分に検証されていない。本研究では、ネオニコチノイド系農薬の1種クロチアニジン（CTD）がヒト神経芽細胞腫に及ぼすシグナル毒性的影響および作用機序を解明し、ヒト神経系に及ぼす未知のリスクを明らかにすることを目的とした。
【方法】ヒト神経芽細胞腫（SH-SY5Y）に1–100 µMのCTDを曝露し、細胞数の変化を計測するとともに、細胞内シグナル状態の変化から機能的影響を評価し、各種nAChRsアンタゴニストの作用を検討した。加えて、マイクロアレイを用いてCTD曝露による遺伝子発現プロファイルの変化を解析した。
【結果・考察】CTD曝露群においては、培養24時間後以降に濃度依存的な細胞数の増加がみられ、その作用は広域アンタゴニストである mecamylamineおよびα3β4特異的アンタゴニストであるSR16584により消失した。また、CTD曝露直後においては一過性の細胞内カルシウム濃度の上昇、ならびにERKリン酸化レベルの上昇がみられた。Gene Ontology解析およびパスウェイ解析の結果、検出された発現低下遺伝子群は「カルシウムイオン流入」や「糸状仮足形成」等の細胞機能に関与しており、「軸索誘導」や「細胞骨格」に関するパスウェイが変動していることが示唆された。以上の結果から、ヒト神経芽細胞腫においてネオニコチノイド系農薬が機能的影響を及ぼすリスクが初めて明らかとなり、そのメカニズムとしてヒトnAChRs α3β4を介した細胞内カルシウムシグナルのかく乱が関連することが示唆された。
日本毒性学会, 2019年, 日本毒性学会学術年会, 46, P - 88E, 日本語ネオニコチノイド（NNs）は、現在世界で最も使用されている殺虫剤の一つである。特に日本では、果物や野菜における残留基準値が諸外国と比べ高く設定され、かつ複数種が使用されるため、全体的にNNsの摂取量が多いと考えられる。
そこで本研究では、日本人におけるNNs曝露実態を明らかにする事を目的に、新生児、幼児を含む延べ数百人から尿を採取し、尿中に含まれる7種のNNs（アセタミプリド、イミダクロプリド、チアメトキサム、チアクロプリド、ニテンピラム、クロチアニジン、ジノテフラン）及び代謝物（N-デスメチルアセタミプリド）をLC/MS/MSで定量した。更に、得られた尿中データより、推定摂取量を算出し、一日摂取許容量（ADI）と比較した。
分析の結果、日本人の尿から何らかのNNsが検出され、特に、N-デスメチルアセタミプリド（90％）、クロチアニジン（50％）、ジノテフラン（50％）の検出頻度が高かった。一方、尿中濃度から推定した各NNs摂取量は、10～50 µg/dayであり、ADIに比べアセタミプリドで最大1％程度、他のNNsは1％未満であった。更に本研究では、生後48時間以内の新生児の尿も分析した。その尿中濃度は‹LOD～0.7 ng/mLと極めて低かったが、分析した57サンプルのうち14サンプルから検出された。
本研究の結果、多くの日本人は胎児を含めNNsの曝露を受けていることが明らかにされた。NNsの慢性低濃度曝露の健康影響については、胎児移行のメカニズムや神経発達を含む毒性に不明な点が多いが、最近の報告においてNNsは現在のNOAELの1/10程度の曝露でも実験動物に対し不安などの情動認知行動に影響を与える事が示されている。
NNsの毒性について、再度リスク評価を実施した上で、継続的なモニタリングを行う必要がある。
日本毒性学会, 2019年, 日本毒性学会学術年会, 46 (0), S7 - 2, 日本語[査読有り]
Cell culture medium replacement is necessary to replenish nutrients and remove waste products, and perfusion and batch media exchange methods are available. The former can establish an environment similar to that in vivo, and microfluidic devices are frequently used. However, these methods are hampered by incompatibility with commercially available circular culture dishes and the difficulty in controlling liquid flow. Here, we fabricated a culture dish adapter using polydimethylsiloxane that has a small recess structure for flow control compatible with commercially available culture dishes. We designed U-shaped and I-shaped recess structure adapters and we examined the effects of groove structure on medium flow using simulation. We found that the U-shaped and I-shaped structures allowed a uniform and uneven flow of medium, respectively. We then applied these adaptors to 293T cell culture and examined the effects of recess structures on cell proliferation. As expected, cell proliferation was similar in each area of a dish in the U-shaped structure adapter, whereas in the early flow area in the I-shaped structure adapter, it was significantly higher. In summary, we succeeded in controlling liquid flow in culture dishes with the fabricated adapter, as well as in applying the modulation of culture medium flow to control cell culture.
MDPI, 2019年, APPLIED SCIENCES-BASEL, 9 (2), 英語[査読有り]
研究論文（学術雑誌）
Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.
2019年, The Journal of Veterinary Medical Science, 81 (3), 454 - 465, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
The effect of bacterial colonies expanded into the intervillous spaces on the localization of several lymphocyte lineages was immunohistochemically investigated in two types of mucosa: ordinary mucosa of rat ileum, which consists of mucosa without any mucosal lymphatic tissue; and follicle-associated mucosa (FAM), which accompanies the parafollicular area under the muscularis mucosae in the rat ileal Peyer's patch. The results showed that bacterial colonies in the intervillous spaces induced increased populations of CD8+ cells in the epithelium of the intestinal villus in ordinary mucosa (IV) and intestinal villus in FAM (IV-FAM). Bacterial colonies in the intervillous spaces were also associated with increased numbers of IgA+ cells, which were mainly localized in the lamina propria of basal portions of IV and IV-FAM, and with expanded localization of IgA+ cells into the villous apex in both IV and IV-FAM. Moreover, IgA+ cells around the intestinal crypts adjacent to IV or IV-FAM were also increased in response to bacterial colonies. In the IV-FAM, but not IV, L-selectin+ cells, which were found to be immunopositive for TCRαβ or CD19, were drastically increased in the lamina propria from the crypt to middle portion of IV-FAM and in the lumen of central lymph vessel of IV-FAM in response to the bacterial colonies in the intervillous spaces. These findings revealed that the expansion of bacterial colonies into the intervillous spaces accompanies the change of histological localization of the lymphocyte lineage in both the ordinary mucosa and FAM.
2019年, The Journal of Veterinary Medical Science, 81 (4), 555 - 566, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2018年12月, 環境ホルモン学会研究発表会要旨集, 21回, 76 - 76, 日本語無毒性量のネオニコチノイド系農薬曝露による行動影響の性差
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2018年12月, 環境ホルモン学会研究発表会要旨集, 21回, 80 - 80, 日本語ネオニコチノイド系農薬が老年動物に及ぼす影響
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2018年12月, 環境ホルモン学会研究発表会要旨集, 21回, 102 - 102, 日本語ネオニコチノイド系農薬クロチアニジンはヒト神経芽細胞腫においてカルシウムシグナルのかく乱を引き起こす
Stable reference genes are important for gene expression analyses such as quantitative PCR. The stability of 15 candidate reference genes that can be used to developing mouse gonads was thoroughly verified using combinations of multiple algorithms. The expression of these genes fluctuated greatly depending on the analysis period and/or gender. Peptidylprolyl isomerase A (Ppia) and polymerase (RNA) II (DNA directed) polypeptide A (Polr2a) were the reference genes that were used stably for a wide analysis period in developing mouse gonads. Furthermore, the stable reference genes corresponding to the analysis period and/or gender were ranked. These results are useful for the selection of the optimal reference gene required for high-precision measurements.
2018年11月01日, The Journal of veterinary medical science, 80 (10), 1534 - 1539, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2018年08月, 日本獣医学会学術集会講演要旨集, 161回, 467 - 467, 日本語ネオニコチノイド系農薬が老齢動物に及ぼす影響
- (公社)日本獣医学会, 2018年08月, 日本獣医学会学術集会講演要旨集, 161回, 467 - 467, 日本語ネオニコチノイド系農薬における哺乳動物への免疫毒性の検証
- (公社)日本獣医学会, 2018年08月, 日本獣医学会学術集会講演要旨集, 161回, 468 - 468, 日本語浸透性農薬の行動影響の性差
Paneth cells secrete bactericidal substances in response to bacterial proliferation on the mucosal surface without directly contacting bacteria. However, the induction mechanism of this transient secretion has not been clarified, although nervous system and/or immunocompetent cells in the lamina propria (LP) might be involved. In this study, we ultrastructurally and immunohistochemically investigated which LP cells are localized beneath Paneth cells and examined the relationship between the Paneth cell-derived cellular processes which extended into the LP and the LP cells. The results showed that various cells-including blood capillary, subepithelial stromal cell, and nerve fiber-were present in the LP beneath Paneth cells. Endothelial cells of blood capillary were the cells most frequently found in this location; they were situated within 1 μm of the Paneth cells and possessed fenestration on the surfaces adjacent to Paneth cells. The Paneth cells rarely extended the cellular processes toward the LP across the basal lamina. Most of the cellular processes of Paneth cells contacted the subepithelial stromal cells. Immunohistochemistry revealed that the CD34+ CD31- αSMA- stromal cells preferentially localized in the LP beneath the intestinal crypt base, while PDGFRαhi αSMA+ stromal cells mainly localized around the lateral portions of the intestinal crypt and PDGFRαhi αSMA- stromal cells localized in the intestinal villus. From these findings, the existence of blood capillaries beneath Paneth cells might reflect the active exocrine function of Paneth cells. Furthermore, subepithelial stromal cells, probably with a CD34+ CD31- αSMA- PDGFRα-/lo phenotype, beneath the crypt base might affect Paneth cell activity by interacting with their cellular processes. Anat Rec, 301:1074-1085, 2018. © 2018 Wiley Periodicals, Inc.
2018年06月, Anatomical record (Hoboken, N.J. : 2007), 301 (6), 1074 - 1085, 英語, 国際誌[査読有り]
研究論文（学術雑誌）
Anti-Müllerian hormone (AMH) produced in the developing testis induces the regression of the Müllerian duct, which develops into the oviducts, uterus and upper vagina. In our true hermaphrodite mouse with an ovary on one side and a testis on the other (O/T), the oviduct and uterus are present only on the ovary side, and nothing derived from the Müllerian duct is present on the testis side. Here, we investigate the mechanism underlying the unilateral Müllerian duct regression and the mode of AMH signaling, by performing immunohistology, Western blotting, and organ culture analyses. The histological analysis revealed that during the start of the Müllerian duct regression, the duct in the O/T mice was clearly regressed on the AMH-positive testis side compared to the AMH-negative ovary side. The immunohistochemistry showed a diffuse immunoreaction of AMH in the interstitium surrounding the testis cord and boundary region between the testis and mesonephros, especially in the cranial portion. Western blotting revealed that the amount of AMH in the cranial half of the mesonephros was larger than that in the caudal half. AMH injected into the gonads in organ culture induced the regression of the Müllerian duct via the interstitium of the organ. These results suggest that AMH acts on the Müllerian duct in male mice by exuding into the interstitium surrounding the testis cord and infiltrating through the cranial region from the testis to the mesonephros.
2018年04月18日, The Journal of Veterinary Medical Science, 80 (4), 557 - 567, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
It has been suggested that an increase in the use of pesticides affects neurodevelopment, but there has been no animal experiment showing a causal relation between neonicotinoid pesticides (NNs) and depression. We examined whether dinotefuran (DIN), the most widely used NN in Japan, induces depression. Male mice were administered DIN between 3 and 8 weeks of age, referring to the no-observed-effect level (NOEL). The mice were then subjected to a tail suspension test (TST) and a forced swimming test (FST). After these tests, their brains were dissected for immunohistochemical analyses of serotonin (5-HT). Antidepressant activity in TST and no decrease in 5-HT-positive cells were observed. The subchronic exposure to DIN alone in juvenile male mice may not cause depression-like indication.
Japanese Society of Veterinary Science, 2018年04月01日, Journal of Veterinary Medical Science, 80 (4), 720 - 724, 英語[査読有り]
研究論文（学術雑誌）
Although neonicotinoid pesticides are expected to have harmful influence on mammals, there is little animal experimental data to support the effect and mechanisms. Since acetylcholine causes the release of dopamine, neonicotinoids may confer a risk of developmental disorders via a disturbance in the monoamine systems. Male mice were peripubertally administered dinotefuran (DIN) referring to no observed effect level (NOEL) and performed behavioral and immunohistological analyses. In an open field test, the total locomotor activity was increased in a dose-dependent manner. The immunoreactivity of tyrosine hydroxylase in the substantia nigra was increased in DIN-exposed mice. These results suggest that exposure to DIN in peripubertal male mice causes hyperactivity and a disturbance of dopaminergic signaling.
Japanese Society of Veterinary Science, 2018年04月01日, Journal of Veterinary Medical Science, 80 (4), 634 - 637, 英語[査読有り]
研究論文（学術雑誌）
The host defense system with lysozyme and secretory phospholipase A2 (sPLA2) was immunohistochemically investigated in rat respiratory tract under healthy conditions. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NC) and a small number of goblet cells (GC) were immunopositive for lysozyme and sPLA2. A few acinar cells and almost all epithelial cells of intercalated ducts were immunopositive for both bactericidal substances in the nasal glands. In the laryngeal and tracheal epithelia, few NC and GC were immunopositive for both bactericidal substances. In the laryngeal and tracheal glands, a few acinar cells and most ductal epithelial cells were immunopositive for both bactericidal substances. In extra-pulmonary bronchus, small numbers of NC and GC were immunopositive for lysozyme and sPLA2, whereas few NC and no GC were immunopositive in the intra-pulmonary bronchus. No secretory source of either bactericidal substance was located in the bronchioles. In the alveolus, many glandular epithelial cells and alveolar macrophages were immunopositive for lysozyme but immunonegative for sPLA2. Moreover, lysozyme and sPLA2 were detected in the mucus layer and in the periciliary layer from the nose to the extra-pulmonary bronchus. These findings suggest that secretory sources of lysozyme and sPLA2 are distributed in almost all the respiratory tract. Their secretory products are probably transported to the pharynx and contribute to form the first line of defense against inhaled bacteria throughout the respiratory tract.
Japanese Society of Veterinary Science, 2018年02月01日, Journal of Veterinary Medical Science, 80 (2), 323 - 332, 英語[査読有り]
研究論文（学術雑誌）
Cerebral palsy (CP) is a major neuronal disease and the most common movement disorder in children. Although environmental factors leading to CP have been greatly investigated, the genetic mechanism underlying CP is not well understood. Here we focused on two clinical reports that characterized a deletion involving the KANK1 gene locus in the 9p24.3 region. One report shows spastic CP and the other shows no spastic CP phenotype. Based on the epigenetic status and evolutionary conservation, we first found a functional genomic element at the noncoding region that was deleted only in patients with spastic CP. This element contains the retinoic acid receptor/retinoid X receptor (RAR/RXR) complex-binding motif that is widely conserved among placental mammals. RAR/RXR ChIP-seq data from mouse F9 embryonal carcinoma cells that were treated with trans-retinoic acids showed that the element has a binding ability. In addition, data regarding chromosome conformation capture from mouse neural progenitor and ES cells suggested that the element spatially interacts with the Doublesex and mab-3 related transcription factor 3 (Dmrt3) gene promoter that is located approximately 120 kb downstream of the RAR/RXR-binding site. Dmrt3 is detected in the developing mouse forebrain and in some interneurons in the spinal cord, and it works as a locomotion coordinator in horses and mice. Thus, the deletion of the cis-regulatory element for DMRT3 in humans may cause impaired development of the forebrain and gait abnormalities, resulting in spastic CP. In conclusion, this study provides new mechanistic insights into the genetic basis of CP.
Elsevier B.V., 2018年01月29日, Biochemical and Biophysical Research Communications, 496 (1), 133 - 139, 英語[査読有り]
研究論文（学術雑誌）
Neonicotinoids are novel systemic pesticides acting as agonists on the nicotinic acetylcholine receptors (nAChRs) of insects. Experimental studies have revealed that neonicotinoids pose potential risks for the nervous systems of non-target species, but the brain regions responsible for their behavioral effects remain incompletely understood. This study aimed to assess the neurobehavioral effects of clothianidin (CTD), a later neonicotinoid developed in 2001 and widely used worldwide, and to explore the target regions of neonicotinoids in the mammalian brain. A single-administration of 5 or 50 mg/kg CTD to male C57BL/6N mice at or below the no-observed-adverse-effect level (NOAEL) induced an acute increase in anxiety during the elevated plus-maze test. In addition, mice in the CTD-administered group spontaneously emitted human-audible vocalizations (4-16 kHz), which are behavioral signs of aversive emotions, and showed increased numbers of c-fos immunoreactive cells in the paraventricular thalamic nucleus and dentate gyrus of the hippocampus. In conclusion, mice exposed to NOAEL-dose CTD would be rendered vulnerable to a novel environment via the activation of thalamic and hippocampal regions related to stress responses. These findings should provide critical insight into the neurobehavioral effects of neonicotinoids on mammals.
ELSEVIER IRELAND LTD, 2018年01月, TOXICOLOGY LETTERS, 282, 57 - 63, 英語[査読有り]
研究論文（学術雑誌）
The distributions of β-defensin 1 and 2 in secretory host defense system throughout respiratory tract of healthy rats were immunohistochemically investigated. In the nasal epithelium, a large number of non-ciliated and non-microvillous cells (NCs) were immunopositive for both β-defensin 1 and 2, whereas a small number of goblet cells (GCs) were immunopositive only for β-defensin 1. Beta-defensin 2-immunopositive GCs were few. In the nasal glands, a small number of acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the laryngeal and tracheal epithelia, a very few NCs and GCs were immunopositive for both β-defensins. In laryngeal and tracheal glands, a very few acinar cells and a large number of ductal epithelial cells were immunopositive for both β-defensins. In the extra-pulmonary bronchus, a small number of NCs were immunopositive for both β-defensins. A small number of GCs were immunopositive for β-defensin 1, whereas few GCs were immunopositive for β-defensin 2. From the intra-pulmonary bronchus to alveoli, a very few or no epithelial cells were immunopositive for both β-defensins. In the mucus and periciliary layers, β-defensin 1 was detected from the nose to the extra-pulmonary bronchus, whereas β-defensin 2 was weakly detected only in the nose and the larynx. These findings suggest that the secretory sources of β-defensin 1 and 2 are mainly distributed in the nasal mucosa and gradually decrease toward the caudal airway in healthy rats.
Japanese Society of Veterinary Science, 2018年, The Journal of Veterinary Medical Science, 80 (3), 395 - 404, 英語[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2017年12月, 環境ホルモン学会研究発表会要旨集, 20回, 20 - 20, 日本語リスク研究の新展開 ネオニコチノイド系農薬が哺乳類の脳神経系に及ぼす影響に関する包括的リスク評価
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2017年12月, 環境ホルモン学会研究発表会要旨集, 20回, 69 - 69, 日本語環境ストレスおよびネオニコチノイド系農薬ジノテフランの複合影響評価
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2017年12月, 環境ホルモン学会研究発表会要旨集, 20回, 70 - 70, 日本語発達期におけるネオニコチノイド系農薬曝露により引き起こされたドーパミン神経系の攪乱を伴う多動
The mechanism by which indigenous bacteria on the follicle-associated epithelium (FAE) of lymphatic follicles (LFs) accelerate the differentiation of microvillous columnar epithelial cells (MV) into M-cells was immunohistochemically investigated in rat Peyer's patches. The results showed that the number of Toll-like receptor (TLR) -4(+) M-cells was greater in the FAE with expansion of bacterial colonies (LFs with bacterial colonies on the FAE: b-LF) than the FAE without expansion of bacterial colonies (nb-LF). TLR-4 was also expressed in the striated borders of MV upstream next to M-cells in the FAE of the b-LF. TLR-4(+) vesicles were frequently detected in the cytoplasms of MV with TLR-4(+) striated borders upstream next to TLR-4(+) M-cells in the FAE of b-LF. These findings suggest that TLR-4(+) MV take up TLR-4 ligands and differentiate into M-cells in the b-LF. Neither the distribution of RANK nor that of RANKL was coincident with that of M-cells in the b-LF. Moreover, RANK, but not RANKL, was expressed in intestinal villi, whereas cleaved caspase-3 was immunonegative in the MV and M-cells of the FAE, unlike in villous epithelial cells. Therefore, RANK/RANKL signaling in the LF might contribute to the down-regulation of epithelial apoptosis to facilitate the differentiation of MV into M-cells in rat Peyer's patches.
JAPAN SOC VET SCI, 2017年11月, The Journal of Veterinary Medical Science, 79 (11), 1826 - 1835, 英語[査読有り]
研究論文（学術雑誌）
Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; noobserved- adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life.
JAPAN SOC VET SCI, 2017年07月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 79 (7), 1196 - 1203, 英語[査読有り]
研究論文（学術雑誌）
Background: The left male gonad in the chicken embryo has a thickened cortical layer, but it eventually becomes flattened after the onset of testicular development. Because the destination of the cortical cells migrating from the left gonad remains unclear, we examined this issue herein. Results: The testis-inducing gene doublesex-and mab-3-related transcription factor 1 (DMRT1) was detected in a proportion of the columnar and cubic epithelial cells in the cortex of the left testis as well as Sertoli cells in both testes. Interestingly, some of the DMRT1-expressing cortical cells were contiguous with Sertoli cells in the testis cord. Some cortical cells exhibited a vimentin-positive cytoplasm that was elongated all the way to the medulla. In addition, a desmosome-like structure was observed between the elongated cytoplasm in these cells and the adjacent Sertoli cell. After the organ culture, a few cells labeled with a fluorescent dye that stained only the cortical cells at the beginning of the culture were located in the testis cord of the left testis. Conclusions: Some cortical cells expressing DMRT1 were suggested to contribute to the Sertoli cells in the testis cord only after the onset of testicular development and only in the left testis. (C) 2016 Wiley Periodicals, Inc.
WILEY, 2017年02月, DEVELOPMENTAL DYNAMICS, 246 (2), 148 - 156, 英語[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2016年12月, 環境ホルモン学会研究発表会要旨集, 19回, 63 - 63, 日本語ネオニコチノイド系農薬曝露によりみられた神経行動学的異常および標的脳領域の探索
[査読有り]
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2016年12月, 環境ホルモン学会研究発表会要旨集, 19回, 67 - 67, 日本語ネオニコチノイド系農薬クロチアニジンは胎子型と成獣型、2種類のライディッヒ細胞にそれぞれ異なる影響を及ぼす
[査読有り]
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2016年12月, 環境ホルモン学会研究発表会要旨集, 19回, 101 - 101, 日本語胎子期および発達期におけるネオニコチノイド系農薬曝露がドーパミン神経系および行動に及ぼす影響
[査読有り]
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2016年12月, 環境ホルモン学会研究発表会要旨集, 19回, 102 - 102, 日本語ネオニコチノイド系農薬ジノテフラン曝露はうつ様行動を引き起こすか?
[査読有り]
The expressions of Toll-like receptor (TLR) -2, -4 and -9 were immunohistochemically investigated in the follicle-associated epithelium (FAE), and epithelia of the follicle-associated intestinal villus (FAIV) and ordinary intestinal villus (IV) in rat Peyer's patch regions with no bacterial colonies on the mucous membranes. TLR-2 was expressed in the striated borders of microvillous columnar epithelial cells (MV) in both FAIV and IV except in the apices. However, TLR-2 expression in the striated borders was weaker in the epithelium of the follicular side of FAIV (f-FAIV) than in epithelia of IV and the anti-follicular side of FAIV. TLR-4 and-9 were not expressed in the FAIV and IV. In the FAE, TLR-2,-4 and-9 were not expressed in the striated borders of MV, but the roofs of some typical M-cells were immunopositive for all TLRs. Especially, no TLR-positive MV were found at the FAE sites where M-cells appeared most frequently. In the follicle-associated intestinal crypt (FAIC), immunopositivity for all TLRs was observed in the striated borders of MV and the luminal substances. In conclusion, the lower levels of TLR-2 in both FAE and the epithelium of f-FAIV probably reduce recognition of indigenous bacteria. TLR-2,-4 and-9 appear not to participate directly in differentiation of MV into M-cells, because TLRs were not expressed in any MV in the upstream region of M-cells in FAE with no settlement of indigenous bacteria in the rat Peyer's patches.
JAPAN SOC VET SCI, 2016年12月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 78 (12), 1797 - 1804, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2016年08月, 日本獣医学会学術集会講演要旨集, 159回, 312 - 312, 日本語マウス未分化性腺におけるSry発現制御に関わる新規遺伝子の時空間特異的探索
[査読有り]
- (公社)日本獣医学会, 2016年08月, 日本獣医学会学術集会講演要旨集, 159回, 312 - 312, 日本語マウス真性半陰陽個体におけるAMHの作用様式に関する組織学的解析
[査読有り]
- (公社)日本獣医学会, 2016年08月, 日本獣医学会学術集会講演要旨集, 159回, 314 - 314, 日本語ネオニコチノイド系農薬クロチアニジン急性投与が及ぼす行動学的影響とその作用点
[査読有り]
- (公社)日本獣医学会, 2016年08月, 日本獣医学会学術集会講演要旨集, 159回, 315 - 315, 日本語ネオニコチノイド系農薬ジノテフランによるドーパミン神経系の攪乱および多動性の誘発
[査読有り]
- (公社)日本獣医学会, 2016年08月, 日本獣医学会学術集会講演要旨集, 159回, 315 - 315, 日本語ネオニコチノイド系農薬とうつ発症との関連性の検証
[査読有り]
- (一社)日本毒性学会, 2016年06月, The Journal of Toxicological Sciences, 41 (Suppl.), S339 - S339, 日本語ネオニコチノイド系農薬クロチアニジン単回投与によりみられた異常啼鳴および神経行動学的影響
[査読有り]
- Tohoku University, 2016年, Japanese Journal of Ecology, 66 (1), 69 - 79, 日本語
[査読有り]
研究論文（学術雑誌）
【背景】ネオニコチノイドは1990年代に昆虫型ニコチン性アセチルコリン受容体（nAChRs）を標的として開発された農薬成分である．しかし近年，ネオニコチノイドは哺乳類型nAChRsを介して神経細胞に興奮反応を引き起こすことが示され，昆虫以外の生物に対して不測の影響を与える可能性が懸念されている．我々はこれまでに成熟雄マウスをネオニコチノイドの1種，クロチアニジン（CTD）に4週間曝露すると，新規環境（オープンフィールド）における不安様行動が引き起こされ，その影響は環境ストレス下においてより顕在化することを報告してきた．本研究では，ネオニコチノイドによる行動影響発現に関与する脳領域を明らかにすることを目的とした．
日本毒性学会, 2016年, 日本毒性学会学術年会, 43 (0), P - 252, 日本語
【材料と方法】C57BL/6成熟雄マウスに5または50 mg/kgの CTDを単回経口投与し，投与1時間後に高架式十字迷路試験による行動解析を行った．さらに2時間後に脳を摘出し，c-fos発現を指標とした組織学的解析により投与後誘導された神経活動を評価した．
【結果と考察】行動解析の結果，CTD 5 mg/kg投与群においては溶媒投与対照群と比較してOpen arm滞在時間および侵入回数の減少がみられた．CTD 50 mg/kg投与群においては，さらに総移動距離の減少ならびに迷路探索時における異常啼鳴（Abnormal vocalization）およびすくみ行動（Freezing）が観察された．組織学的解析の結果，情動およびストレス反応に関与する視床下部，海馬においてc-fos陽性細胞数の増加がみられた．
以上の結果から，CTD投与下においては，新規環境ストレスに曝露された際にコリン作動性神経投射を受ける視床下部や海馬における過剰な神経興奮が生じ，不安様行動やストレス応答を濃度依存的に誘発する可能性が示唆された．[査読有り]
- 2016年, 日本生態学会誌, 66 (1), 69 - 79, 日本語ダイオキシン汚染がアカネズミ個体群に及ぼす影響: 遺伝子マーカーを用いて個体群の反応を捉える試み．
[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2015年12月, 環境ホルモン学会研究発表会要旨集, 18回, 109 - 109, 日本語ネオニコチノイド系農薬クロチアニジンの妊娠期及び授乳期曝露がマウス産子の雄性生殖器に与える影響
[査読有り]
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2015年12月, 環境ホルモン学会研究発表会要旨集, 18回, 118 - 118, 日本語環境ストレス下においてネオニコチノイド系農薬クロチアニジンが与える神経行動学的影響
[査読有り]
- (公社)日本生化学会, 2015年12月, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 88回・38回, [2P0993] - [2P0993], 日本語ニワトリ胚の左精巣に特異的なセルトリ細胞の起源
[査読有り]
Mammalian sexual fate is determined by the presence or absence of sex determining region of the Y chromosome (Sly) in the "bipotential" gonads. Recent studies have demonstrated that both male and female sexual development are induced by distinct and active genetic pathways. Breeding the Y chromosome from Mus m. domesticus poschiavinus (POS) strains into C57BL/6J (B6J) mice (B6J-XYPOS) has been shown to induce sex reversal (75%: bilateral ovary, 25%: true hermaphrodites). However, our B6N-XYPOS mice, which were generated by backcrossing of B6J-XYPOS on an inbred B6N-XX, develop as males (36%: bilateral testis with fertility as well as bilateral ovary (34%), and the remainder develop as true hermaphrodites. Here, we investigated in detail the expressions of essential sex-related genes and histological features in B6N-XYPOS mice from the fetal period to adulthood. The onsets of both Sry and SRY-box 9 (Sox9) expressions as determined spatiotemporally by whole-mount immunohistochemistry in the B6N-XYPOS gonads occurred 2-3 tail somites later than those in B6N-XYB6 gonads, but earlier than those in B6J-XYPOS, respectively. It is possible that such a small difference in timing of the Sry expression underlies testicular development in our B6N-XYPOS. Our study is the first to histologically show the expression and ectopic localization of a female-related gene in the XYPOS testes and a male -related gene in the XYPOS ovaries. The results from these and previous experiments indicate that the interplay between genome variants, epigenetics and developmental gene regulation is crucial for testis development.
JAPAN SOC VET SCI, 2015年12月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 77 (12), 1587 - 1597, 英語[査読有り]
研究論文（学術雑誌）
Dioxins are widespread persistent environmental contaminants with adverse impacts on humans and experimental animals. Behavioral and cognitive functions are impaired by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. TCDD exerts its toxicity via the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor. The hippocampus, which plays important roles in episodic memory and spatial function, is considered vulnerable to TCDD-induced neurotoxicity, because it contains the AhR. We herein investigated the effects of TCDD toxicity on hippocampal development in embryonic mice. TCDD was administered to dams at 8.5 days postcoitum with a single dose of 20, 200, 2,000 and 5,000 ng/kg body weight (groups T20, T200, T2000 and T5000, respectively), and the brains were dissected from their pups at embryonic day 18.5. Immunohistochemical analysis demonstrated that the Glial Fibrillary Acidic Protein (GFAP) immunoreactivities in the dentate gyrus (DG) were reduced in the T5000 group. Granular GFAP immunoreactivity was observed in the hippocampal fimbria, and the number of immunoreactive fimbria was significantly decreased in the T5000 group. The number of Proliferating Cell Nuclear Antigen (PCNA)-positive cells was decreased in all TCDD-exposed groups and significantly reduced in the T20, T200 and T5000 groups. Together, these results demonstrate that maternal TCDD exposure has adverse impacts on neural stem cells (NSCs), neural precursor cells (NPCs) and granular cells in the DG and disrupts the NSC maintenance and timing of differentiation in the hippocampal fimbria, which in turn interrupt neuronal development in future generations of mice.
JAPAN SOC VET SCI, 2015年11月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 77 (11), 1355 - 1361, 英語[査読有り]
研究論文（学術雑誌）
Neonicotinoids, some of the most widely used pesticides in the world, act as agonists to the nicotinic acetylcholine receptors (nAChRs) of insects, resulting in death from abnormal excitability. Neonicotinoids unexpectedly became a major topic as a compelling cause of honeybee colony collapse disorder, which is damaging crop production that requires pollination worldwide. Mammal nAChRs appear to have a certain affinity for neonicotinoids with lower levels than those of insects; there is thus rising concern about unpredictable adverse effects of neonicotinoids on vertebrates. We hypothesized that the effects of neonicotinoids would be enhanced under a chronic stressed condition, which is known to alter the expression of targets of neonicotinoids, i.e., neuronal nAChRs. We performed immunohistochemical and behavioral analyses in male mice actively administered a neonicotinoid, clothianidin (CTD; 0, 10, 50 and 250 mg/kg/day), for 4 weeks under an unpredictable chronic stress procedure. Vacuolated seminiferous epithelia and a decrease in the immunoreactivity of the antioxidant enzyme glutathione peroxidase 4 were observed in the testes of the CTD+stress mice. In an open field test, although the locomotor activities were not affected, the anxiety-like behaviors of the mice were elevated by both CTD and stress. The present study demonstrates that the behavioral and reproductive effects of CTD become more serious in combination with environmental stress, which may reflect our actual situation of multiple exposure.
JAPAN SOC VET SCI, 2015年10月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 77 (10), 1207 - 1215, 英語[査読有り]
研究論文（学術雑誌）
Indigenous bacteria in the alimentary tract are exposed to various bactericidal peptides and digestive enzymes, but the viability status and morphological changes of indigenous bacteria are unclear. Therefore, the present study aimed to ultrastructurally clarify the degeneration and viability status of indigenous bacteria in the rat intestine. The majority of indigenous bacteria in the ileal mucous layer possessed intact cytoplasm, but the cytoplasm of a few bacteria contained vacuoles. The vacuoles were more frequently found in bacteria of ileal chyme than in those of ileal mucous layer and were found in a large majority of bacteria in both the mucous layer and chyme throughout the large intestine. In the dividing bacteria of the mucous layer and chyme throughout the intestine, the ratio of area occupied by vacuoles was almost always less than 10%. Lysis or detachment of the cell wall in the indigenous bacteria was more frequently found in the large intestine than in the ileum, whereas bacterial remnants, such as cell walls, were distributed almost evenly throughout the intestine. In an experimental control of long-time-cultured Staphylococcus epidermidis on agar, similar vacuoles were also found, but cell-wall degeneration was never observed. From these findings, indigenous bacteria in the mucous layer were ultrastructurally confirmed to be the source of indigenous bacteria in the chyme. Furthermore, the results suggested that indigenous bacteria were more severely degenerated toward the large intestine and were probably degraded in the intestine.
JAPAN SOC VET SCI, 2015年09月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 77 (9), 1121 - 1128, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2015年08月, 日本獣医学会学術集会講演要旨集, 158回, 280 - 280, 日本語環境ストレス下において浸透性農薬クロチアニジンが行動及び生殖機能に与える影響
[査読有り]
A portion of the minute chylomicrons less than 75 nm in diameter are transcytosed from the extravascular tissue into the subepithelial blood capillaries (sBC) in the villous apices of the rat jejunum. However, the details of the transportation mechanism have not been clarified. In this study, the endothelial receptor involved in the transportation of minute chylomicrons into the sBC's lumina was immunohistochemically and histoplanimetrically examined in intestinal villi of the rat jejunum. Immunopositivity for very low density lipoprotein (VLDL) receptor was detected on the luminal and basal surfaces of the endothelial cells of sBC in approximately 68% of those apices of jejunal villi that possessed numerous chylomicrons in the lamina propria, while VLDL receptor was detected on the endothelial cells of sBC in only approximately 8% of intestinal villi that possessed few or no chylomicrons in the lamina propria. No immunopositivity for VLDL receptor was detected in the sBC of all intestinal villi. These findings suggest that VLDL receptor is expressed by the endothelial cells of the sBC in conjunction with the filling of the lamina propria of jejunal villi with many chylomicrons produced by the villous columnar epithelial cells and that the VLDL receptor mediates the transportation of minute chylomicrons, maybe VLDL, into the subepithelial portal blood from the extravascular tissue of the rat jejunal villi.
JAPAN SOC VET SCI, 2015年04月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 77 (4), 387 - 393, 英語[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2014年12月, 環境ホルモン学会研究発表会要旨集, 17回, 132 - 132, 日本語ネオニコチノイド系農薬クロチアニジン及び環境ストレス複合曝露が生殖器及び行動に与える影響
[査読有り]
We investigated the toxicity of bisphenol A (BPA) by determining the gene expression of nerve growth factor (Ngf) in the embryonic mouse cell line mHypoE-N44 derived from the hypothalamus exposed to BPA dose range between 0.02 and 200 μmol L-1 for 3 h. Ngf mRNA levels decreased in a dose-dependent manner, with significant reductions observed in the 2 to 50 μmol L-1 BPA treatment groups compared to controls. However, at 100 to 200 μmol L-1 the Ngf mRNA gradually increased and was significantly higher than control, while the expression of the apoptosis-related genes Caspase 3 and transformation-related protein 73 decreased significantly. These results suggest that in an embryonic hypothalamic cell line the higher doses of BPA induce a unique pattern of Ngf gene expression and that BPA has the potential to suppress apoptosis essential for early-stage brain development.
Institute for Medical Research and Occupational Health, 2014年09月01日, Arhiv za Higijenu Rada i Toksikologiju, 65 (3), 293 - 299, 英語[査読有り]
研究論文（学術雑誌）
Environmental stress affects various parts of mammals typically through the circulation of stress hormones. It has been identified as one of the possible reasons for male reproductive difficulties, but the complex mechanisms responsible for stress-induced reproductive suppression are poorly understood. Here, we examined the relationship between chronic environmental stress and hypothalamic kisspeptin, a recently discovered upstream regulator of the reproductive endocrine feedback system. We studied male mice under an unpredictable chronic stress procedure to replicate the situation of animals under chronic stress. Histological and immunohistochemical analyses were performed focusing on kisspeptin neurons in the arcuate hypothalamic nucleus (ARC) and DNA fragmented cells in seminiferous tubules. Although the ARC was not morphologically altered in either the stressed or non-stressed group, granular kisspeptin immunoreactivities decreased slightly in the stress group. In the testes of the stress group, several signs of testicular degeneration were observed, including increased numbers of ssDNA-positive cells per seminiferous tubule, thinning, vacuoled seminiferous epithelia and multinucleated giant cells. The decreases in kisspeptin in the stress group might be due to other hypothalamic peptides, such as corticotropin-releasing hormone and leptin, whose receptors are known to coexpress in the ARC. In addition, environmental stress directly and indirectly affects testicular function through stress hormones and gonadotropins. In summary, our findings enhance the understanding of stress-induced reproductive suppression possibly mediated by kisspeptin in the ARC.
JAPAN SOC VET SCI, 2014年09月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 76 (9), 1201 - 1208, 英語[査読有り]
研究論文（学術雑誌）
- Insight into the Mechanism of Reproductive Dysfunction Caused by Neonicotinoid Pesticides
Neonicotinoids, which were developed in the 1990s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.
PHARMACEUTICAL SOC JAPAN, 2014年09月, BIOLOGICAL & PHARMACEUTICAL BULLETIN, 37 (9), 1439 - 1443, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2014年08月, 日本獣医学会学術集会講演要旨集, 157回, 318 - 318, 日本語マウス胎子生殖腺における性分化関連遺伝子発現の左右差に関する分子形態学的研究
[査読有り]
- (公社)日本獣医学会, 2014年08月, 日本獣医学会学術集会講演要旨集, 157回, 318 - 318, 日本語ニワトリ胚における性腺の形成メカニズムと左右差
[査読有り]
- (公社)日本獣医学会, 2014年08月, 日本獣医学会学術集会講演要旨集, 157回, 319 - 319, 日本語発生中のマウス胎子生殖腺における内部標準遺伝子の検討
[査読有り]
- (公社)日本獣医学会, 2014年08月, 日本獣医学会学術集会講演要旨集, 157回, 331 - 331, 日本語ネオニコチノイド系農薬クロチアニジンの低用量曝露とストレスの複合影響
[査読有り]
Paneth cells (PCs) contribute to the host defense against indigenous bacteria in the small intestine. We found Paneth cell-like cells (PLCs) in the rat ascending colon, but the nature of PLCs is never clarified. Therefore, the present study aimed to clarify the cytological characteristics of PLCs and discuss their cellular differentiation. PLCs were localized in the bases of intestinal crypts, especially follicle-associated intestinal crypts in proximal colonic lymphoid tissue, but were very seldom found in the ordinary intestinal crypts of the ascending colon. PLCs possessed specific granules with highly electron-dense cores and haloes, as well as PCs in the small intestine. The secretory granules of PLCs were positive for PAS reaction, lysozyme and soluble phospholipase A2, but negative for Alcian blue staining, beta-defensin-1 and -2, as well as the ones of PCs. Furthermore, intermediate cells possessing both the PLC-specific granules and the mucus granules similar to those of goblet cells (GCs) were occasionally found in the vicinity of PLCs. Intermediate cells ranged from goblet cell-like cells rich in mucus granules to PLC-like cells with few mucus granules. The cellular condensation and fragmentation were exclusively found in PLCs but never seen in intermediate cells or GCs. The PLCs, which were identified as PC, were suggested to be transformed from GCs through intermediate cells and finally to die by apoptosis in intestinal crypts of proximal colonic lymphoid tissue in the rat ascending colon. (C) 2014 Wiley Periodicals, Inc.
WILEY-BLACKWELL, 2014年08月, ANATOMICAL RECORD-ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, 297 (8), 1462 - 1471, 英語[査読有り]
研究論文（学術雑誌）
The epithelial cell composition was investigated in the follicle-associated intestinal crypt (FAIC) of rat Peyer's patches. The epithelium of the FAIC mainly consisted of columnar epithelial cells, goblet cells and Paneth cells. The characteristics of secretory granules in Paneth cells and goblet cells of both the FAIC and ordinary intestinal crypts (IC) were almost the same in periodic acid-Schiff (PAS) reaction, Alcian blue (AB) staining and the immunohistochemical detection of lysozymes and soluble phospholipase A2. Both goblet cells and Paneth cells were markedly less frequent on the follicular sides than on the anti-follicular sides of the FAIC. Goblet cells were also markedly less frequent in the follicle-associated epithelium (FAR) than in the ordinary intestinal villi (IV). Indigenous bacteria were more frequently adhered to FAR than to follicle-associated intestinal villi or IV. These findings suggest that the host defense against indigenous bacteria is inhibited on the follicular sides of FAIC, which might contribute to the preferential settlement of indigenous bacteria on the FAE; they also suggest that differentiation into secretory cells is inhibited in the epithelium of the follicular sides of FAIC, so that differentiation into M cells might be admitted in the FAR of rat Peyer's patches. Furthermore, intermediate cells possessing characteristics of both Paneth cells and goblet cells were rarely found in the FAIC, but not in the IC. This finding suggests that the manner of differentiation into Paneth cells in the FAIC differs from that in the IC.
JAPAN SOC VET SCI, 2014年06月, The Journal of Veterinary Medical Science, 76 (6), 833 - 838, 英語[査読有り]
研究論文（学術雑誌）
Sex of birds is genetically determined by the inheritance of sex chromosomes (ZZ for male and ZW for female), and the Z-linked gene named doublesex and mab-3 related transcription factor 1 (DMRT1) is a candidate sex- determining gene in avian species. However, the mechanisms underlying sex determination in birds are not yet understood, and the expression patterns of the DMRT1 protein in urogenital tissues have not been identified. In the current study, we used immunohistochemistry to investigate the detailed expression patterns of the DMRT1 protein in the urogenital systems (including Mullerian ducts) in male and female chicken embryos throughout embryonic development. Gonadal somatic cells in the male indifferent gonads showed stronger expressions of DMRT1 compared with those in the female indifferent gonads well before the presumptive period of the sex determination, and Sertoli cells forming testicular cords expressed DMRT1 in the testes after sex determination. Germ cells expressed DMRT1 equally in males and females after sex determination. The expression was continuous in males, but in females it gradually disappeared from the germ cells in the central part of the cortex of the left ovary toward both edges. The DMRT1 was also detected in the tubal ridge, which is a precursor of the Mullerian duct, and at the mesenchyme and outermost coelomic epithelium of the Mullerian duct in both sexes. Strong expression was observed in the males, but it was restricted to coelomic epithelium after the regression of the duct started. Thus, we observed the detailed spatiotemporal expression patterns of DMRT1 in the developing chicken urogenital systems throughout embryonic development, suggesting its various roles in the development of urogenital tissues in the chicken embryo.
OXFORD UNIV PRESS, 2014年04月, POULTRY SCIENCE, 93 (4), 953 - 958, 英語[査読有り]
研究論文（学術雑誌）
- 2014年04月, International Journal of Molecular Science, 15, 8959 - 8978, 英語Genes and gene networks involved in sodium fluoride-elicited apoptosis accompanying endoplasmic reticulum stress in oral epithelial cells.
[査読有り]
研究論文（学術雑誌）
- (公社)日本薬学会, 2014年03月, 日本薬学会年会要旨集, 134年会 (1), 283 - 283, 日本語環境化学物質による内分泌かく乱研究の新展開 ネオニコチノイド系農薬による生殖機能障害とそのメカニズム
Fetal exposure to dioxins and related compounds is known to disrupt normal development of the midbrain dopaminergic system, which regulates behavior, cognition and emotion. The toxicity of these chemicals is mediated mainly by aryl hydrocarbon receptor (AhR) signaling. Previously, we identified a novel binding motif of AhR, the AhR-responsive element III (AHRE-III), in vitro. This motif is located upstream from the gene encoding tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. To provide in vivo evidence, we investigated whether 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could regulate AHRE-III transcriptional activity in midbrain dopaminergic neurons. We produced transgenic mice with inserted constructs of the AHRE-III enhancers, TH gene promoter and the c-myc-tagged luciferase gene. Single oral administrations of TCDD (0-2000ngkg(-1) body weight) to the transgenic dams markedly enhanced TH-immunoreactive (ir) intensity in the A9, A10 and A8 areas of their offspring at 3days and 8weeks of age. The offspring of dams treated with 200ngkg(-1) TCDD exhibited significant increases in the numbers of TH- and double (TH and c-myc)-ir neurons in area A9 compared with controls at 8weeks. These results show that fetal exposure to TCDD upregulates TH expression and increases TH-ir neurons in the midbrain. Moreover, the results suggest that TCDD directly transactivates the TH promoter via the AhR-AHRE-III-mediated pathway in area A9. Fetal exposure to TCDD caused stable upregulation of TH via the AhR-AHRE-III signaling pathway and overgrowth of TH-ir neurons in the midbrain, implying possible involvement in the etiology of neurodevelopmental disorders such as attention-deficit/hyperactivity disorder (ADHD). Copyright (c) 2013 John Wiley & Sons, Ltd.
WILEY, 2014年02月, JOURNAL OF APPLIED TOXICOLOGY, 34 (2), 117 - 126, 英語[査読有り]
研究論文（学術雑誌）
- Development of Oral Epithelial Cell Line ROE2 with Differentiation Potential from Transgenic Rats Harboring Temperature-Sensitive Simian Virus 40 Large T-Antigen Gene
We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33 degrees C or an intermediate temperature of 37 degrees C. At the nonpermissive temperature of 39 degrees C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochennical analyses revealed that ROE2 cells at 37 degrees C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive nnicroarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.
INT PRESS EDITING CENTRE INC, 2014年01月, EXPERIMENTAL ANIMALS, 63 (1), 31 - 44, 英語[査読有り]
研究論文（学術雑誌）
- 2014年01月, Animal Eye Research, 32, 15 - 21, 英語Ferret animal model of corneal endothelial dysfunction for evaluation of drug effect on corneal endothelial wound healing
[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2013年12月, 環境ホルモン学会研究発表会要旨集, 16回, 150 - 150, 日本語マウス海馬の神経新生に及ぼすTCDD毒性の時空間的影響
[査読有り]
Chylomicrons from villous columnar epithelial cells are generally known to be transported only by central lymph vessels (CLV), whereas antigenic particulates derived from the intestinal lumen can also be transported by subepithelial blood capillaries (sBCs) in rat intestinal villi. The possibility of chylomicron absorption by sBCs was histoplanimetrically studied in the rat jejunum under a transmission electron microscope. The chylomicrons more abundantly presented in villous venules than in arterioles. The most frequent size (MFS) of chylomicrons was 75 to 90 nm in diameter in the areas near sBCs, while it was 45 to 60 nm in the epithelial intercellular spaces just above sBCs or the intermediate areas between sBCs. The MFS of chylomicrons was 45 to 60 nm in the intermediate areas between sBCs and in the epithelial intercellular spaces just above these areas. The MFS of chylomicrons in CLV was intermediate between that in the area adjacent to sBCs and that in the intermediate areas between sBCs. Chylomicrons were found in small vesicles in the endothelial cytoplasms of sBCs. No chylomicrons larger than 600 nm were observed in the lamina propria. These findings suggest that some of the chylomicrons smaller than 75 nm, which are probable intestinal very low-density lipoproteins (VLDL), are directly transported to the liver by hepatic portal blood in addition to CLV and that epithelial fat droplets larger than 600 nm are not discharged into lamina propria in rat jejunum under physiological conditions.
JAPAN SOC VET SCI, 2013年12月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 75 (12), 1563 - 1569, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2013年08月, 日本獣医学会学術集会講演要旨集, 156回, 197 - 197, 日本語性腺分化とその破綻メカニズムに関する分子形態学的研究
[査読有り]
- (公社)日本獣医学会, 2013年08月, 日本獣医学会学術集会講演要旨集, 156回, 201 - 201, 日本語環境ストレスが成熟雄マウスの生殖機能に与える影響
[査読有り]
- (公社)日本獣医学会, 2013年08月, 日本獣医学会学術集会講演要旨集, 156回, 201 - 201, 日本語ネオニコチノイド系農薬クロチアニジンが鳥類の繁殖能力に及ぼす影響とその作用機序
[査読有り]
- 2013年07月, The Journal of Veterinary Medical Science, 75 (5), 939 - 947, 英語Histoplanimetrical study on the relationship between invasion of indigenous bacteria into intestinal crypts and proliferation of epithelial cells in rat ascending colon.
[査読有り]
研究論文（学術雑誌）
- 2013年07月, Environmental Toxicology, 英語Downregulation of cytochrome P450scc as an initial adverse effect of adult exposure to diethylstilbestrol on testicular steroidogenesis
[査読有り]
研究論文（学術雑誌）
Clothianidin (CTD) is a neonicotinoid developed in the 1990s as an insecticide having selective toxicity, but it was later found to cause reproductive abnormalities in rats through oxidative stress. There is an attempt to preserve endangered animals, including the Japanese crested ibis, in Japan. However, there is a concern that neonicotinoid affects the reproduction of this bird, since it is used in its habitat. CTD toxicity in the birds is poorly understood, so we investigated whether or not the daily oral administration of CTD has any deleterious effects on the reproductive functions of mature male quails as experimental animals. The animals were randomly divided into four groups of 6 or 7 quails each, treated orally with 0, 0.02, 1 or 50 mg CTD/kg body weight (Control, CTD0.02, CTD1 and CTD50). After that the males bred with untreated females to estimate the egg weights, and rates of fertilization and normal development, the testes, liver and spleen were examined histologically. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, and the number and size of vacuoles in hepatocytes increased dose-dependently. There were no significant differences in egg weights and fertilization rates between the groups, but some eggs of the CTD1 and CTD50 groups failed to develop, and embryonic length decreased dose-dependently. Thus, it was found that CTD affected the reproduction of the male quail through the fragmentation of germ cells and the inhibition or delay of embryonic development.
JAPAN SOC VET SCI, 2013年06月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 75 (6), 755 - 760, 英語[査読有り]
研究論文（学術雑誌）
- 2013年05月, Japanese Journal of Zoology and Wildlife Medecine, 18 (3), 107 - 114, 英語Study on the ejaculate characteristics and liquid storage of semen in the common bottlenose dolphin (Tursiops truncatus)
[査読有り]
研究論文（学術雑誌）
- 2013年05月, Reproductive Biology, 13 (1), 92 - 95, 英語Immunohistochemical analysis of steroidogenic acute regulatory protein (StAR) and StAR-binding protein (SBP) expressions in the testes of mice during fetal development
[査読有り][招待有り]
研究論文（学術雑誌）
- Gene expression of epigenetic regulatory factors related to primary silencing mechanism is less susceptible to lower doses of bisphenol A in embryonic hypothalamic cells
DNA methyltransferases (DNMTs) are associated with epigenetic regulation of gene expression, and methyl-CpG binding protein 2 (MECP2) acts as a long-range regulator of methylated genes. We evaluated the effects of bisphenol A (BPA) on embryonic mouse hypothalamic cells, with particular emphasis on the gene expression of Dnmts (Dnmt1, Dnmt3a, and Dnmt3b) and Mecp2 isoforms. In a dose-dependent (0.02-200 mu M BPA) 3-hr experiment, real-time reverse transcription polymerase chain reaction revealed that gene expression of both Dnmts and Mecp2_e2 was affected at 200 mu M and that of Mecp2_e1 was affected at > 20 mu M. These results suggest that gene expression of Dnmts and Mecp2 are less susceptible to lower doses of BPA in developing hypothalamic cells. However, as BPA concentration increases, this agent has the potential to alter gene expression of key players that provide stability and flexibility of epigenetic gene regulation, which could disrupt the normal development of hypothalamic functions.
JAPANESE SOC TOXICOLOGICAL SCIENCES, 2013年04月, JOURNAL OF TOXICOLOGICAL SCIENCES, 38 (2), 285 - 289, 英語[査読有り][招待有り]
研究論文（学術雑誌）
角膜内皮は角膜の透明性を維持するために重要な役割を果たしている。我々は、角膜内皮研究のための新たな実験動物モデルを開発することを目的とし、フェレットの角膜内皮の形態学的特徴を調べ、実験動物として用いることができるか否かを評価した。さらに、フェレット角膜を組織学的に解析し、ウサギおよびカニクイザルの角膜と比較した。フェレット角膜組織のフラットマウント標本を用いた免疫組織学的検討の結果、フェレットの角膜内皮細胞は角膜内皮機能に関連するマーカーであるZO-1、N-カドヘリンおよびNa+/K+-ATPアーゼを発現していることが確認された。また、フェレット角膜において、臨床的に重要な角膜内皮細胞のパラメーターである角膜内皮密度、六角形細胞率および変動係数を、臨床診療で用いられている分析ソフトウェアを用いて解析することができた。フェレット角膜内皮細胞の初代培養も可能であり、初代培養された角膜内皮細胞は生体内の角膜内皮細胞にきわめて類似した六角形細胞からなる単層構造を示し、機能関連マーカーであるZO-1およびNa+/K+-ATPアーゼを発現していた。
Japanese Society of Comparative and Veterinary Ophthalmology, 2013年04月, Animal Eye Research, 31, 3 - 11, 英語
これらの結果より、フェレットは眼科領域、特に角膜内皮研究における有用な実験動物となる可能性が示唆された。[査読有り]
研究論文（学術雑誌）
We evaluated the effects of bisphenol A (BPA) on embryonic mouse hypothalamic cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) indicated that gonadotropin-releasing hormone 1 (Gnrh1) expression in 0.02-20μM BPA-treated cells did not differ from that in control cells but decreased significantly in 200μM BPA-treated cells. The mRNA level for brain-derived neurotrophic factor (Bdnf), which participates in GNRH1 secretory system development, decreased significantly in 200μM BPA-treated cells, but that for neurotrophic tyrosine kinase, receptor, type 2 (Ntrk2), did not change. This indicates that Gnrh1 gene expression in mice fetuses is not affected by exposure to < 20μM BPA and that the adverse effects of BPA on the BDNF-NTRK2 neurotrophin system are induced by decrease in the mRNA level of the ligand, not of its receptor. © 2012 Japanese Teratology Society.
2013年03月, Congenital Anomalies, 53 (1), 42 - 45, 英語[査読有り]
研究論文（学術雑誌）
The expression patterns of steroidogenic acute regulatory protein (StAR) and StAR-binding protein (SSP) in fetal Leydig cells were compared by using immunohistochemistry. While StAR immunoreactivity was detected during the first steps of testis differentiation, SBP expression was detected slightly later. The timing of SBP expression closely correlated with that of the testosterone surge, an event which is known to induce masculinization. Our results suggest that SBP plays an important role in male sexual development via interactions with StAR. (C) 2013 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
INST ANIMAL REPRODUCTION FOOD RESEARCH, 2013年03月, REPRODUCTIVE BIOLOGY, 13 (1), 92 - 95, 英語[査読有り]
研究論文（学術雑誌）
The relationship between the invasion of indigenous bacteria into intestinal crypts and the proliferation of epithelial cells was histoplanimetrically investigated in the rat ascending colon. Indigenous bacteria preferentially adhered to the intestinal superficial epithelial cells in the mesenterium-attached mucosa (MAM) compared to those in the mesenterium-non-attached mucosa (MNM). Intestinal crypts with indigenous bacteria were also significantly more frequently found in MAM than in MNM. Total epithelial cells, columnar epithelial cells and goblet cells were significantly more abundant in the intestinal crypts with no-indigenous bacteria in MAM (MAM-C) than those in MNM (MNM-C), whereas the columnar epithelial cells were less abundant in MAM-C than in the intestinal crypts with indigenous bacteria in MAM (MAM-C-B). Columnar epithelial cells and goblet cells immuno-positive for proliferating cell nuclear antigen (PCNA) in MAM-C were more abundant than those in MNM-C, but less abundant than those in MAM-C-B. Toll-like receptor (TLR)-2, -4 and -9 were immuno-positive in the striated borders of the intestinal superficial epithelial cells, but their positive intensities were weaker in MAM than in MNM. From these findings, indigenous bacteria were confirmed to preferentially settle on the intestinal superficial epithelium of MAM in the rat ascending colon, and low TLRs-expression might contribute to the preferential settlement of indigenous bacteria in MAM. The increase of proliferating epithelial cells is probably induced by the invasion of indigenous bacteria into the intestinal crypts of MAM. © 2013 The Japanese Society of Veterinary Science.
2013年, Journal of Veterinary Medical Science, 75 (7), 939 - 947, 英語[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2012年12月, 環境ホルモン学会研究発表会要旨集, 15回, 60 - 60, 日本語マウス胎子海馬の神経発達に及ぼすTCDD毒性の基礎的評価
[査読有り]
Previously, the specific antibody-mediated persorption of antigenic molecules and particulates from the small-intestinal lumen into the peripheral blood was clarified in rats, but the intermediation of the receptor for the specific antibodies was not. In this study, the existence of receptor for the specific antibody was experimentally examined in the rat small intestine. Glutaraldehyde-fixed sheep erythrocytes (SEs) coated by Fc-fragments of IgG (IgG-Fc), (Fab')(2)-fragments of IgG (IgG-Fab) or bovine serum albumin (BSA), were injected into 3 jejunal loops each 2 cm in length in non-orally pre-immunized rats, respectively. Thirty minutes after the injection, IgG-Fc-coated SEs were significantly more engulfed by villous columnar epithelial cells than Fab- or BSA-coated SEs. The most frequent absorption sites were the intestinal villous apices. The IgG-Fc-coated SEs were adhered to the striated borders and were engulfed by villous columnar epithelial cells. IgG-Fc-coated SEs passing through the epithelial cells were also detected in the subepithelial blood capillaries just beneath the villous epithelium, but not in the connective tissue and the lymph vessels. These findings suggest that the absorption of luminal antigenic particulates is probably mediated by the Fc-receptor, and that the absorbed antigenic particulates are directly transferred to the hepatic portal blood by passing through the endothelium of the subepithelial blood capillaries.
JAPAN SOC VET SCI, 2012年11月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 74 (11), 1447 - 1452, 英語[査読有り]
研究論文（学術雑誌）
Localization of Toll-like receptors (TLRs) in the exocrine glands associated with the rat alimentary tract was immunohistochemically studied using anti-TLR antibodies. TLR-2, -4 and -9 were detected in the secretory granules of acinar cells or the luminal substances of the gustatory gland, extraorbital lacrimal gland, Harderian gland, proper gastric gland and pancreas. TLR-2 and -9 were also detected in the mucous acinar cells of the sublingual gland. Positivity for all TLRs was found in the striated borders of columnar epithelial cells and the luminal substances of the intestinal crypts throughout the small intestine, and also in the goblet cells throughout the large intestine. Only TLR-4 was detected in the secretory granules of Paneth cells. A reduction of TLR-4-positive secretory granules and the formation of TLR-4-positive vacuoles were found in the ileal Paneth cells under the hyper-proliferation of indigenous bacteria. In the apical to middle intervillous portions of the ileum, Gram-positive bacterial colonies were significantly more abundant than Gram-negative bacterial colonies, whereas this difference disappeared in the basal intervillous portions. These findings suggest that there are distribution differences in the secretory sources of soluble TLRs that possibly neutralize their luminal ligands, in the rat alimentary tract. Therefore, the bacterial ligand-recognition system composed of the membranous TLRs of villous columnar epithelial cells and soluble TLRs from crypt epithelial cells might contribute to host defense mechanisms for the selective elimination of Gram-positive bacteria rather than Gram-negative bacteria in the rat small intestine.
JAPAN SOC VET SCI, 2012年11月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 74 (11), 1429 - 1438, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2012年08月, 日本獣医学会学術集会講演要旨集, 154回, 176 - 176, 日本語ターナーモザイク患者の性腺分化とその破綻機構の分子基盤の解明
[査読有り]
- (公社)日本獣医学会, 2012年08月, 日本獣医学会学術集会講演要旨集, 154回, 180 - 180, 日本語マウス胎子の海馬におけるTCDDの神経発達毒性作用
[査読有り]
角膜内皮は角膜の透明性を維持するために重要な役割を果たしている。我々は、角膜内皮研究のための新たな実験動物モデルを開発することを目的とし、フェレットの角膜内皮の形態学的特徴を調べ、実験動物として用いることができるか否かを評価した。さらに、フェレット角膜を組織学的に解析し、ウサギおよびカニクイザルの角膜と比較した。フェレット角膜組織のフラットマウント標本を用いた免疫組織学的検討の結果、フェレットの角膜内皮細胞は角膜内皮機能に関連するマーカーであるZO-1、N-カドヘリンおよびNa+/K+-ATPアーゼを発現していることが確認された。また、フェレット角膜において、臨床的に重要な角膜内皮細胞のパラメーターである角膜内皮密度、六角形細胞率および変動係数を、臨床診療で用いられている分析ソフトウェアを用いて解析することができた。フェレット角膜内皮細胞の初代培養も可能であり、初代培養された角膜内皮細胞は生体内の角膜内皮細胞にきわめて類似した六角形細胞からなる単層構造を示し、機能関連マーカーであるZO-1およびNa+/K+-ATPアーゼを発現していた。
Japanese Society of Comparative and Veterinary Ophthalmology, 2012年06月, Animal Eye Research, 31, 3 - 11, 英語
これらの結果より、フェレットは眼科領域、特に角膜内皮研究における有用な実験動物となる可能性が示唆された。[査読有り]
研究論文（学術雑誌）
The apoptosis process in rat esophageal epithelium was investigated using enzyme-immunohistochemistry and transmission electron microscopy. As a result, Fas and Fas-L were expressed in the epithelial cell membrane and cytoplasm from the stratum spinosum (SS) to the stratum granulosum (SG). No TNF-R1 show immunopositivity in the cell membranes. TNF-alpha and caspase-8 were not observed in any layer. Caspase-10, cleaved caspase-3, XIAP and DNase-1 were found in the epithelial cytoplasm from the SS to the SG, whereas Bid, Apaf-1 and cleaved caspase-9 were detected only in the SG. Cytochrome c was observed as cytoplasmic granular positivity from the stratum basale (SB) and altered into homogeneous immunopositivity in the SG. Bcl-2 and Bcl-X immunopositivity was detected in cytoplasm from the SB to the SG. Immunoreactions of Bak in the cytoplasm and Bax beneath the cell membrane were observed from the upper portion of the SS with increasing intensity toward the SG. In the sites with the hyperproliferation of indigenous bacteria, TNF-R1, TNF-alpha and caspase-8 were detected in the SG and the immunopositive intensities of Bid, Apaf-1 and cleaved caspase-9 were altered to be strong. Prominently swollen cells and decreased mitochondria were ultrastructurally confirmed in the uppermost layers of stratum corneum. These findings suggest that the Fas-Fas-L-interaction initially induces apoptosis through a mitochondria-independent pathway and secondarily through a mitochondria-dependent pathway, leading to eventual epithelial cell death in the rat esophageal epithelium. The bacterial stimuli probably enhance the mitochondria-dependent pathway through the TNF-R1-TNF-alpha interaction.
JAPAN SOC VET SCI, 2012年05月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 74 (5), 597 - 605, 英語[査読有り]
研究論文（学術雑誌）
- InTech, 2012年02月, Immunosuppression - Role in Health and Diseases
[査読有り]
- Microarray and gene ontology analyses reveal downregulation of DNA repair and apoptotic pathways in diethylstilbestrol-exposed testicular Leydig cells.
This study investigated the deleterious effects of the synthetic non-steroidal estrogen diethylstilbestrol (DES) on testicular Leydig cells and compared these effects with those of the natural estrogen 17β-estradiol (E2). For that purpose, we performed microarray analysis of a mouse Leydig cell line (TTE1) treated with these estrogens, followed by Gene Ontology (GO) analysis and parametric analysis of gene set enrichment (PAGE). Most notably, GO analysis revealed a significant decrease in the biological processes of the GO categories "DNA repair" and "apoptotic program" in DES-exposed cells. PAGE showed that "cell death," which is a superior GO category including apoptosis in the GO tree structure, significantly decreased in DES-exposed cells but significantly increased in E2-exposed cells. Interestingly, only 2 genes (Tia1 and Gas1) with altered expression patterns in the "cell death" category were common between DES- and E2-treated cells. The downregulation of apoptotic cell death pathways and DNA repair capability of DES-exposed cells implies that DES promotes carcinogenic processes more strongly than E2 does. These findings suggest that molecular events that occur following DES and E2 treatments differ substantially in Leydig cells, and that the effects of synthetic estrogen and natural estrogen differ more substantially than previously suspected.
2012年02月, Journal Toxicology Science, 37 (2), 287 - 295, 英語, 国内誌[査読有り]
研究論文（学術雑誌）
- Site differences of Toll-like receptor expression in the mucous epithelium of rat small intestine
Toll-like receptors (TLRs) are known to recognize pathogen-associated molecular patterns and might function as receptors to detect microbes. In this study, the distribution of TLR-2, -4 and -9 were immunohistochemically investigated in the rat small intestine. As a result, TLR-2 was detected in the striated borders of villous columnar epithelial cells throughout the small intestine, except for the apices of a small number of intestinal villi. TLR-4 and -9 were detected in the striated borders of the villous columnar epithelial cells only in the duodenum. TLR-4-immunopositive minute granules were found in the apical cytoplasms of epithelial cells, subepithelial spaces and blood capillary lumina. TLR-2 and -4 were detected in the striated borders of undifferentiated epithelial cells and in the luminal substances of the intestinal crypts throughout the small intestine, but TLR-9 was not detected in the crypts throughout the small intestine. Only TLR-4 was detected in the secretory granules of Paneth cells in both the jejunal and ileal intestinal crypts. These findings suggest that duodenal TLRs might monitor indigenous bacteria proliferation in the upper alimentary tract, that TLR-2 might also monitor the proliferation of colonized indigenous bacteria throughout the small intestine, that the lack of TLR-2 at the villous apices might contribute to the settlement of indigenous bacteria, and that TLR-2 and -4 are secreted from intestinal crypts.
F HERNANDEZ, 2011年10月, HISTOLOGY AND HISTOPATHOLOGY, 26 (10), 1295 - 1303, 英語[査読有り]
研究論文（学術雑誌）
- Immunohistochemical and Histoplanimetrical Study on the Spatial Relationship between the Settlement of Indigenous Bacteria and the Secretion of Bactericidal Peptides in Rat Alimentary Tract
To clarify the regulatory mechanism by bactericidal peptides secretion, the secretion of bactericidal peptides was immunohistochemically and histoplanimetrically compared with the degree of Gram-positive/negative bacterial colonization throughout the rat alimentary tract. In the associated exocrine glands from the oral cavity to the stomach, no comparable differences were observed under the changes of development of indigenous bacterial colonies. In the small intestine, immunopositive granules for lysozyme and secretory phospholipase A2 (sPLA2) were markedly decreased, whereas immunopositive vacuoles in the Paneth cells were more increased at sites with hyper-development of indigenous bacterial colonies in the intervillous spaces than at sites with no or less development. No changes in exocrine glands were observed in the large intestine because of the constant existence of large quantities of bacteria. Gram-positive bacterial colonies on the mucosal surfaces were dominant from the oral cavity to the stomach. Gram-negative bacteria were dominant in the large intestine, and the distributions of both Gram-positive and negative bacteria were intermediate in the small intestine. These findings suggest that lysozyme and sPLA2 secreted from the Paneth cells contribute to the regulation of the proliferation of indigenous bacteria in the intervillous spaces of the small intestine, and that the inversion of distributions of Gram-positive and -negative bacteria in the alimentary tract might be caused by the secretion of lysozyme and sPLA2 in the small intestine.
JAPAN SOC VET SCI, 2011年08月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 73 (8), 1043 - 1050, 英語[査読有り]
研究論文（学術雑誌）
- Immunohistochemical study of the apoptosis process in epidermal epithelial cells of rats under a physiological condition
Epidermal homeostasis is maintained by both epithelial proliferation in the stratum basale (SB) and the apoptosis of epithelial cells under physiological conditions. In this study, the induction and regulation mechanisms of epidermal apoptosis were immunohistochemically investigated in the epidermis from Wistar rat's palm and foot pad by using several apoptotic related proteins under a physiological condition. The results showed that Fas and Fas-L were expressed in cellular membranes of the stratum spinosum (SS), whereas TNF-R1 did not show any membranous expression in any epidermal layers. TNF-alpha was not observed in the epidermis. Caspase-10, cleaved caspase-3 and DNase-1 were found in the epithelial cytoplasms from the SS to stratum granulosum (SG), whereas caspase-8 was not detected in the epidermis. XIAP and Bak were found in the cytoplasm from the SS to SG, and the intensity of Bak-positivity was stronger in the SG than the SS, whereas Bid, Apaf-1 and cleaved caspase-9 were restricted in the SG. Homogenous cytoplasmic immunoreactivity of Bcl-2 was found in the SB and the intensity was gradually decreased from the SB to the SG. The granular-cytoplasmic immuno-positivity of cytochrome C gradually altered into homogenous cytoplasmic expression in the upper half of the SG. Single-stranded DNA was rarely detected in the upper portion of the SG. These results suggest that epidermal apoptosis is induced by the interaction between Fas and Fas-L and the activation of caspase-10, and might initially proceed through a mitochondrial-independent pathway, and that a mitochondrial-dependent pathway finally accelerated under physiological conditions.
F HERNANDEZ, 2011年07月, HISTOLOGY AND HISTOPATHOLOGY, 26 (7), 811 - 820, 英語[査読有り]
研究論文（学術雑誌）
The hypothesis that apoptotic factors play some roles in the denucleation of erythroblasts has been confirmed by the immunohistological detection of both phosphatidylserine and thrombospondin as phagocytosis-inducing factors in general apoptotic events. Both phosphatidylserine and thrombospondin were detected on the surface of cell membrane of mature erythroblasts, while thrombospondin was also detected in more immature erythroblasts. The intensities of their immune reactions increased as the erythroids matured. During denucleation, the positivities of both phosphatidylserine and thrombospondin were restricted on the surface of the cell membrane surrounding the protruding nuclei. Thus, the apoptotic process involves denucleation of erythroblasts and phosphatidylserine, and thrombospondin acts as phagocytosis-inducing factors in the denucleation event.
JAPAN SOC VET SCI, 2011年07月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 73 (7), 949 - 952, 英語[査読有り]
研究論文（学術雑誌）
To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. β -defensin1 was detected in the gustatory and extraorbital lacrimal glands. β -defensin2 was detected in the Harderian glands. In the stomach, β -defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and β -defensins were detected in the ascending colon, whereas only β -defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.
2011年02月, Journal of Veterinary Medical Science, 73 (2), 217 - 225, 英語[査読有り]
研究論文（学術雑誌）
- Lectin Histochemical Detection of Special Sugars on the Mucosal Surfaces of the Rat Alimentary Tract
Surfaces of the most luminal positions of mucosae are fundamental settlement sites of indigenous bacteria throughout the rat alimentary tract. In these positions, also epithelial cell-shedding sites, the special sugar expression in the glycocalyx is very important as it provides possible ligands of bacterial lectins for attachment to epithelial cells. Therefore, the sugar expression in glycocalyx of epithelial cells was lectin-histochemically surveyed using 21 lectins throughout the rat alimentary tract. From the tongue to the nonglandular part of the stomach, alpha-D-Man, alpha-D-Glc and alpha-D-GalNAc were detected on the surface of the keratinized stratified squamous epithelium. In the glandular part of the stomach, alpha-D-Man, beta-D-Gal-4GlcNAc, D-Gal, D-GalNAc, D-GlcNAc, alpha-L-Fuc-alpha-D-Gal-beta(1-4)GlcNAc and bisected triantennary N-glycans were detected on the surface of gastric superficial epithelial cells. From the duodenum to the ileum, (GlcNAc)(2-4) was expressed exclusively on the epithelial cells in the apical portions of the intestinal villi. From the cecum to the rectum, alpha-D-Man, beta-D-Gal-4GlcNAc, D-Gal, D-GalNAc, alpha-D-Gal(1-3)D-GalNAc, (GalNAc)(n) and NeuNAc were expressed on the intestinal superficial epithelial cells. These results suggest that special sugars are expressed on the most luminal portions of mucosae as exclusive epithelial cell-shedding sites, and that sugar expression differs among the various segments of the alimentary tract. These site differences might reflect differences in resident bacterial species in the rat alimentary tract.
JAPAN SOC VET SCI, 2010年09月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 72 (9), 1119 - 1127, 英語[査読有り]
研究論文（学術雑誌）
Aims: To investigate the precise mechanisms underlying the action of estrogenic endocrine disruptors, we evaluated the direct effects of synthetic estrogen diethylstilbestrol (DES) on steroidogenesis in Leydig cells, with particular emphasis on the expression of the cholesterol side-chain cleavage enzyme P450scc. Furthermore, the mechanism underlying the action of DES was compared with that of endogenous estrogen 17 beta-estradiol (E2), which has a potency equivalent to that of DES.Main methods: TTE1 Leydig cells were treated with 5 x 10(-8) mu M to 5 mu M DES or E2 for 24 h, and P450scc gene expression and the histone modifications underlying their transcriptional activation were examined using reverse transcription-polymerase chain reaction (RT-PCR) and chromatin immunoprecipitation (ChIP), respectively.Key findings: P450scc mRNA expression in the DES-treated and E2-treated cells reduced in inverse proportion to the dose of DES and E2, respectively; however, cAMP stimulation induced a recovery in the expression to a level approximately equal to those in the controls. In the DES-treated cells, ChIP assay revealed histone deacetylation in the P450scc promoter region. Interestingly, E2 did not cause histone deacetylation.Significance: In the early stages of steroidogenesis, DES and E2 directly induced a reduction in P450scc mRNA expression in inverse proportion to their doses, and treatment with CAMP restored the decreased P450scc mRNA expression. Furthermore, DES can induce alterations in the histone modification of the P450scc gene, and natural estrogen and synthetic estrogenic compounds such as DES may induce reproductive disorders through different molecular mechanisms. (C) 2010 Elsevier Inc. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2010年08月, LIFE SCIENCES, 87 (9-10), 281 - 285, 英語[査読有り]
研究論文（学術雑誌）
In normal ontogenetic development, the expression of the sex-determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex-determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17-year-old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex-determining gene expressions in the patient-derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex-determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex-determining genes. Collectively, alterations in the sex-determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.
WILEY, 2010年06月, CONGENITAL ANOMALIES, 50 (2), 85 - 94, 英語[査読有り]
研究論文（学術雑誌）
XY females are rare individuals who carry a Y chromosome but are phenotypically female. In approximately 80-90% of these cases, there are no mutations in the SRY gene, a testis-determining gene on the short arm of the Y chromosome, and the pathophysiology of XY females without SRY mutation remains unclear. In the present study, we used a molecular data mining technique to analyze the pathophysiology of an XY female with functional SRY and pericentric inversion of the Y chromosome, and compared the results with those of a normal male. Interestingly, upregulations of numerous genes included in the development category of the Biological Process ontology, including genes associated with sex determination and organ morphogenesis, were seen in the patient. Additionally, the transforming growth factor-beta (TGF-beta) signaling pathway and Wnt signaling pathway, in which most cell-cell interactions during embryonic development are involved, were altered. Alterations in the expression of numerous genes at the developmental stage, including alterations at both the gene and pathway levels, may persist as a vestige of anomalies of sex differentiation that presumably began in the fetal period. The present study indicates that a data mining technique using bioinformatics contributes to identification of not only genes responsible for birth defects, but also disorders of sex development (DSD)-specific pathways, and that this kind of analysis is an important tool for clarifying the pathophysiology of human idiopathic XY gonadal dysgenesis. Our findings could serve as one of the basic datasets which will be used for future follow-up investigations.
WILEY, 2010年03月, CONGENITAL ANOMALIES, 50 (1), 40 - 51, 英語[査読有り]
研究論文（学術雑誌）
Recent animal experiments confirmed that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases the sex ratio of offspring at birth without altering litter size. However, the timing of this decrease remained unclear. Male mice were administered TCDD at 7-12 weeks of age and mated with non-treated females. The Y-bearing/X-bearing sperm ratio was examined by real-time PCR and FISH methods, and the sex ratio of the 2-cell embryos collected from non-treated females that had been mated with TCDD-exposed males were investigated by nested PCR. The Y-bearing/X-bearing sperm ratio was not significantly decreased in the TCDD group. However, the sex ratio of the 2-cell embryos of the TCDD group was significantly lower than that of the control group. These results may have resulted from a decrease in fertility of Y-bearing sperm. Thus, the results of this study suggested that the sex ratio of the offspring was decreased at fertilization and not during the spermatozoa stage. (C) 2009 Elsevier Inc. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2010年01月, REPRODUCTIVE TOXICOLOGY, 29 (1), 68 - 73, 英語[査読有り]
研究論文（学術雑誌）
A major question is whether exposure to mixtures of low-dose endocrine disruptors (EDs) having different action mechanisms affects neurodevelopment differently than exposure to EDs individually. We therefore investigated the effects of fetal and neonatal exposure to three typical EDs - bisphenol A (BPA), di-(2-ethylhexyl)-phthalate (DEHP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) - on the midbrain dopaminergic system associated with functions - including motor activity, emotion, and cognition affected by neuropsychiatric diseases such as attention-deficit/hyperactivity disorder. ICR mouse dams and their pups were orally treated with BPA (5 mg/(kgday)), DEHP (1 mg/(kgday)), or TCDD (8ng/kg) individually, or with mixtures thereof, to compare the effects between sole and mixed administration. We analyzed tyrosine hydroxylase (TH)- and Fos-immunoreactive (it) neurons as markers of dopamine and neuronal activation, respectively. The numbers of TH- and/or Fos-ir neurons and the intensity of TH-immunoreactivity within midbrain dopaminergic nuclei (A9, A10, and A8) of each sole administration group significantly differed from controls at 2, 4, and 6 weeks of age. In contrast, no significant differences were detected in the mixture groups, suggesting counteractions among those chemicals. These results indicate that ED mixtures as pollution have unique and elusive effects. Thyroid hormones and/or aryl hydrocarbon receptor-related mechanisms may be responsible for this counteraction. (C) 2009 Published by Elsevier Ireland Ltd.
ELSEVIER IRELAND LTD, 2009年08月, Toxicology Letters, 189 (1), 40 - 47, 英語[査読有り]
研究論文（学術雑誌）
- Morphological Analyses of Sex Differences and Age-Related Changes in C3H Mouse Midbrain
While it is commonly hypothesized that sexual differentiation in the mammalian brain is initiated mainly by gonadal sex steroids, recent evidence has suggested that dopaminergic (DA) neurons within the rodent midbrain have sex differences independent of gonadal secretions. More recently, it has been reported that Sty (the sex-determining region of the Y chromosome) is directly involved in this difference. The possibility of sexual dimorphism in the mouse midbrain needs to be elucidated. In the present Study, the midbrain of C3H mice, which is little understood, was investigated histologically and immuno-histoplanimetrically to reveal sexual and developmental differences. The female ventral tegmental area appeared to contain higher immunoreactivity to tyrosine hydroxylase (TH) than that of males at 11 weeks of age, whereas general histological differences between the sexes were not clearly found. The TH-immunoreactive (TH-ir) neurons within the A8, A9, and A10 mesencephalic areas were examined separately. There was the sex difference in the time period when TH-ir cell numbers significantly increased, indicating that the growth rate of midbrain DA nuclei also differs and that the midbrain DA system may trace different processes of sexual maturation between the sexes. These differences between female and male may reflect the direct regulation by Sry or the multiple effects of both Sty and sex steroids. Further experiments are needed to determine which factor forms this difference in the growth pattern in the numbers of TH-ir neurons.
JAPAN SOC VET SCI, 2009年07月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 71 (7), 855 - 863, 英語[査読有り]
研究論文（学術雑誌）
Cytogenetic amniocentesis (CA) has been performed as a reliable prenatal diagnostic method for decades. The aims of the present study were to reveal the frequency of fetal chromosome abnormalities according to medical indications of CA, and to assess the risks of specific abnormal ultrasound findings. Data on chromosome karyotypes of fetuses from 5043 Japanese mothers were collected. Group I comprised 4626 fetuses whose mothers underwent CA due to a variety of parental reasons. Group II comprised 417 fetuses whose mothers underwent CA due to fetal abnormality, abnormality of amniotic fluid volume and fetal growth restriction. The frequency of chromosome abnormalities in Group II (17.7%) was significantly higher than in Group I (1.8%). The frequencies of chromosome abnormalities in Group II singleton fetuses with fetal abnormality, polyhydramnios and fetal growth restriction were 21.5, 22.9 and 19.6%, respectively. By multivariate analyses, we found that cystic hygroma (odds ratio 5.6, 95% CI 2.7-11.6), abnormal extremity (5.0, 1.7-14.4) and cardiovascular abnormality (3.3, 1.1-10.1) were significant variants associated with fetal chromosomal abnormalities. Information revealed in the present study constitutes a beneficial reference for genetic counseling. © 2009 Japanese Teratology Society.
2009年06月, Congenital Anomalies, 49 (2), 61 - 65, 英語[査読有り]
研究論文（学術雑誌）
- Histoplanimetrical Study on the Relationship between Cellular Kinetics of Epithelial Cells and Proliferation of Indigenous Bacteria in the Rat Colon
The aim of this study was to clarify the regulatory effects of epithelial kinetics on indigenous bacterial proliferation in the large intestine. The lifespan, migration speed and proliferation rate of crypt epithelial cells in the initial 20% of the colon (proximal colon) and the 50% of the colon (middle colon) in bromodeoxyuridine-administrated rats were histoplanimetrically and chronologically compared. The proximal colon possessed well-developed mucosal folds and a large amount of indigenous bacteria which filled the crypt lumen, whereas no folds or bacteria were found to occupy the crypt lumen in the middle colon. The cell lifespans were 32.2, 42.5 and 33.6 hr in the apical and the basal parts of the mucosal folds of the proximal colon, and in the middle colon, respectively. The migration speeds were 4.2, 2.1 and 3.3 mu m/hr, respectively, while the appearance frequencies of proliferating cell nuclear antigen (PCNA)-positive crypt epithelial cells were 35.0, 24.6 and 33.8%. These findings suggest that the lifespan was shortened and the migration speed increased in the most luminal mucosa of colon, contributing to the elimination of the adhered bacteria from the most luminal mucosa. By contrast, the elongation of the lifespan and deceleration of the migration of epithelial cells in the basal parts of the mucosal folds might contribute to reliable settlement of indigenous bacteria, resulting in the maintenance of a large amount of indigenous bacteria in the lumen of the proximal colon.
JAPAN SOC VET SCI, 2009年06月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 71 (6), 745 - 752, 英語[査読有り]
研究論文（学術雑誌）
- Histoplanimetrical Study on the Spatial Relationship of Distribution of Indigenous Bacteria with Mucosal Lymphatic Follicles in Alimentary Tract of Rat
The spatial relationship between the distribution of indigenous bacteria (IB) and the Situation of mucosal lymphatic follicles (LF) is histoplanimetrically studied in the rat alimentary tract. From the oral cavity to the nonglandular part of the stomach, IB adhered to the corneal layer of the most luminal mucosa. In the glandular part of the stomach, IB adhered only to the most luminal mucosa but not in the gastric pits. In the small intestine, IB consistently adhered around the apices of both intestinal villi and the doilies, and their amounts decreased toward their basal portions. No IB entered the intestinal crypts. In the large intestine, IB consistently adhered to the most luminal mucosa. Numerous IB were Suspended in the intestinal crypts of both the cecum and the proximal colon, whereas there were no IB in the crypts of the distal colon and the rectum. When IB spread over the basal portions of the intestinal villi, IB with the same morphology were detected on the neighboring LF, whereas no bacteria were detected on the neighboring LF, when IB were located in the apical to middle portions of the intestinal villi. This close relationship between the distribution of IB and mucosal LF was also observed in the large intestine. These results Suggest that the most luminal mucosae are a fundamental settlement site of IB throughout the alimentary tract and that the hyperproliferation of IB's colonies might be detected by neighboring LF in the rat intestine.
JAPAN SOC VET SCI, 2009年05月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 71 (5), 621 - 630, 英語[査読有り]
研究論文（学術雑誌）
- Histoplanimetrical Study on the Relationship between the Cell Kinetics of Villous Columnar Epithelial Cells and the Proliferation of Indigenous Bacteria in Rat Small Intestine
The relationship between the kinetics of villous columnar epithelial cells and the expansion of colonies of indigenous bacteria from the narrow apical portions of intestinal villi was immunohistochemically and histoplanimetrically investigated in the small intestine of bromodeoxyuridine administred Wistar rats. As a result, the lifespan Of villous columnar epithelial cells was slightly shorter in the distal ileum than in other portions of small intestine, accompanying the minimum height of the intestinal villi of the distal ileum in the small intestine. The migration speed of villous columnar epithelial cells was significantly decreased toward the distal small intestine. The migration speed in the distal ileum was about one-fourth of that in the duodenum. The migration speed of the villous columnar epithelial cells was greater and their lifespans were shorter in the sites with Wide expansion of the indigenous bacterial colony from the narrow apical portions of the intestinal villi than that in sites with no or less expansion. Additionally, the expansion of the indigenous bacterial colony from narrow villous apices also immediately shortened the heights of the intestinal villi. These findings suggest that the migration speed of villous columnar epithelial cells might contribute to the regulation of the settlement of bacteria at the villous apices and the inevitable proliferation of indigenous bacteria at the intervillous spaces in the rat small intestine.
JAPAN SOC VET SCI, 2009年04月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 71 (4), 463 - 470, 英語[査読有り]
研究論文（学術雑誌）
The mechanism of physical elimination of indigenous bacteria was ultrastructurally and immunohistochemically investigated in microvillous columnar epithelial cells of Peyer's patches and intestinal villi of the rat jejunoileum. From ultrastructural observation, the microfilaments accumulated to form several electron-dense layers beneath the bacteria adhering to the cell membrane, which was slightly invaginated in the epithelial cells of Peyer's patches and intestinal villi. As the microfilamentous layers were forming, the end portions of invaginations were deformed into a cone-shape and were finally collapsed. At the same time, the end portions of the adhered bacteria were also deformed into cone-shapes. The bacterial cells were moved back toward the invagination orifices with no morphological change in their inner structure. From immunohistochemical observation, beta-actin and nonmuscle-type myosin were detected at the thin layer just beneath the invaginated cell membrane. These findings suggest that indigenous bacteria which adhere to epithelia] cells are removed by only a physical action of actin and myosin filaments, but are not killed. This bacterial cell removal system might lead to the establishment of a settlement of indigenous bacteria on host cells.
JAPAN SOC VET SCI, 2008年11月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 70 (11), 1153 - 1158, 英語[査読有り]
研究論文（学術雑誌）
The ultrastructure of epithelial responses against the membrane adhesion of indigenous bacteria was investigated in the follicle-associated epithelium (FAE) of rat small intestine. The most frequent adherence of the various morphological types of bacteria to the epithelial membranes was found at the apex of the FAE. The attachment sites were deeply invaginated, and their bottoms were deformed into a sharp cone shape. Four layers with different electron densities were formed just beneath the apical membranes by microfilaments which surrounded the invaginations. The electron density of each layer was gradually decreased as being apart from the invaginations. The extremities of some bacteria in the invaginations were deformed into sharpened shapes. The cell walls of the extremities of the bacteria were occasionally dissolved in the invaginations, and their cytoplasms were slightly swollen with low electron densities. In some invaginations, the attached bacteria were eliminated to leave their fragments such as filamentous debris and a part of cell walls. Finally these remnants disappeared completely. When the bacterial colonies existed in the middle region of the FAE, the attachment of bacteria resulted in the engulfment of bacteria by M cells. The degenerated bacteria whose cytoplasmic matrices were separated into high electron dense materials and cleared materials were occasionally engulfed by ordinary microvillous columnar epithelial cells or goblet cells throughout the FAE. These findings suggest that the epithelial cells reject the attachment of live indigenous bacteria and that the M cells absorb indigenous bacteria in rat Peyer's patches.
JAPAN SOC VET SCI, 2008年03月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 70 (3), 235 - 241, 英語[査読有り]
研究論文（学術雑誌）
Steroidogenic acute regulatory (SCAR) protein plays a crucial role in the intramitochondrial movement of cholesterol, where P450 side chain cleavage enzyme resides. Cholesterol sulphate (CS), which is present ubiquitously in mammalian tissues, is not only a precursor of sulphated adrenal steroids but also an inhibitor of cholesterol biosynthesis. This study was designed to examine the biological roles of CS in steroidogenesis in adrenocortical cells. Human adrenocortical carcinoma H295R cells were cultured with various amounts of CS. To evaluate steroid hormone synthesis, pregnenolone production in cells was assayed. The amount of pregnenolone produced by H295R cells in culture medium, to which over 50 mu g/ml CS was added, was significantly (P<0.05) decreased compared with that produced by control cells. Western blot analysis was performed to determine StAR protein level using whole cell extracts from cells. StAR protein level decreased when the concentration of CS in the medium was 50 mu g/ml, whereas the level of glyceraldehyde-3-phosphate dehydrogenase did not change. To examine the mechanism by which SCAR gene expression is controlled, we performed RT-PCR and measured promoter activity in cells transfected with pGL(2) StAR reporter constructs. StAR mRNA level and promoter activity were decreased in cells. The decrease in StAR protein level is a result of the low StAR gene expression level. In conclusion, CS affects the production of steroid hormones by reducing StAR protein level in adrenocortical cells.
SOC ENDOCRINOLOGY, 2007年12月, JOURNAL OF ENDOCRINOLOGY, 195 (3), 451 - 458, 英語[査読有り]
研究論文（学術雑誌）
It is well known that some caspases in apoptosis is involved in determinant of terminal differentiation and maturation of various cells. Our previous study ultrastructurally clarified the differentiation into M cells from immature microvillous epithelial cells and the redifferentiation from M cells to microvillous epithelial cells in the follicle-associated epithelium (FAE) of rat Peyer's patch. In this study, the difference of epithelial apoptosis between the FAE of Peyer's patch and intestinal villi was immunohistochemically investigated in rat jejunoileum. As a result, cleaved caspase-3 was limited to several epithelial cells at the tip of FAE, whereas almost all of the epithelial cells were cleaved caspase-3 positive in intestinal villi. Cleaved caspase-9 was detected only in a few exfoliating or exfoliated epithelial cells of both FAE and intestinal villi. Nuclear DNA-fragmentation was detected only in several epithelial cells of the tip of FAE, while it was expressed from the middle regions in the intestinal villi. The DNase I expression of the epithelial cytoplasm was much weaker in FAE than in intestinal villi. Bcl-x expression was restricted in the apical cytoplasms of epithelial cells in the FAE, whereas it was restricted in whole cytoplasms in villous epithelial cells. These findings suggest that the progression of the apoptotic process in the epithelial cells of FAE is later than in the intestinal villi, so that the possibility of epithelial differentiation might be remained in the FAE, unlike in the intestinal villi.
2007年11月, Journal of Veterinary Medical Science, 69 (11), 1123 - 1129, 英語[査読有り]
研究論文（学術雑誌）
The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.
2007年05月, Journal of Veterinary Medical Science, 69 (5), 501 - 508, 英語[査読有り]
研究論文（学術雑誌）
In 1976, men who were exposed to the highest concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) after an explosion at a chemical plant near Seveso, Italy, produced more girls than boys. However, few studies have examined the possibility that the exposure of laboratory animals to TCDD, especially that of males, could lead to a lower male/female sex ratio. The aim of this study was to investigate whether direct paternal exposure to TCDD affects the sex ratio of offspring using a relatively large-scale experimental design. Male ICR mice (n=120) were randomly assigned to three, one of which served as a vehicle control, the other two were administered TCDD orally with an initial loading dose of 2 or 2,000 ng TCDD/kg, followed by a weekly maintenance dose of 0.4 (T2/0.4 group) or 400 (T2000/400 group) ng/kg prior to mating. The major organs of each mouse were weighed and histopathologically and immunohistologically investigated, and the sex ratio of offspring [males/(males + females) x 100] was calculated in each dam. There were no significant effects on organ weights, or on the structure of the testis and epididymis between the control and TCDD-exposed males, but TCDD administration produced a significantly lower proportion of male offspring from T2000/400-exposed sires despite no alteration in litter size (Control: 53.1 ± 1.7 T2/0.4: 48.8 ± 2.5 T2000/400: 46.2 ± 2.1). In addition, we further divided the T2000/400 group into 3 subgroups based on the proportion of CYP1A1- immunoreactive areas in the liver there was a significant correlation between sex ratio and CYP1A1 immunoreactivity. Thus, the present study confirms that direct paternal exposure to TCDD might be associated with an alteration in the sex ratio of offspring. Possible mechanisms through which TCDD might decrease the fertility potential of Ybearing gametes before conception are discussed.
2007年04月, Journal of Veterinary Medical Science, 69 (4), 347 - 352, 英語[査読有り]
研究論文（学術雑誌）
The possibility of persorption of prefixed bovine serum albumin-coated sheep erythrocytes (BSA-SEs) from mucous epithelial cells and its mechanisms were investigated in rats orally immunized by BSA for 14 consecutive days. On the day after the final oral immunization, the rats were duodenally perfused by BSA-SEs or non-coated SEs. BSA-SEs were also duodenally perfused in nonimmunized rats. Thirty min after perfusion, BSA-SEs were significantly more engulfed by late-apoptotic-stage villous columnar epithelial cells in the orally immunized rats than those in other experiments. The specific antibody (SpAb) was detected on the surfaces of BSA-SEs in rats with oral immunization. In Peyer's patches of all animals, no SEs reached the follicle-associated epithelium, because of the close attachment of follicle-associated intestinal villi and the thick mucous layer. BSA-SEs were more frequently persorbed into portal blood in the orally immunized rats than in other rats. Small numbers of BSA-SEs or SEs were detected in the systemic blood of all animals. BSA-SEs were also histologically found in the blood vessels of the liver, but not in mesenteric lymph nodes. These findings suggest that sensitized antigenic particulates are taken up by late-apoptotic-stage villous columnar epithelial cells in the small intestine and are finally persorbed into the systemic blood circulation. The uptake of antigenic particulates might be mediated by its luminal SpAb.
2007年04月, Journal of Veterinary Medical Science, 69 (4), 339 - 346, 英語[査読有り]
研究論文（学術雑誌）
Our previous study clarified that the apical regions of both the follicle-associated epithelium (FAE) of Peyer's patches and the intestinal villi are the only adhesion sites of indigenous bacteria in rat jejuno-ileum. To survey the ligands against bacterial lectins, sugar expression patterns on epithelial cells were lectin-histochemically investigated using 21 lectins in the jejuno-ileal Peyer's patches of rats. As a result, (D-glcNAc)2-4, detected by Solanum tuberosum (STL) and by Lycopersicon esculentum (LEL), and β?D-gal(1-3)-D-galNAc detected by Peanut agglutinin (PNA), were strongly expressed on the brush borders of the apical regions of the FAE and the intestinal villi. On the other hand, neither sugar was expressed on the brush borders of the basal regions of both FAE and intestinal villi. The positive intensities for the lectins correlated with the progression of epithelial apoptosis in the FAE and in the intestinal villi. Moreover, the double staining with lectin histochemical method and the in situ nick end-labeling method could simultaneously detect the strong expression of both sugars and nuclear DNA fragmentation in epithelial cells at the late apoptotic stage. Other sugar expression patterns in the intestinal villi were similar with those in the FAE. There were no lectins specific for M cells in the FAE. From these findings, the possible sugars of ligands against some indigenous bacterial lectins, expressing specially on the apoptotic epithelial cells, might be narrowed down in rat jejuno-ileum.
2007年02月, Journal of Veterinary Medical Science, 69 (2), 193 - 199, 英語[査読有り]
研究論文（学術雑誌）
For the purpose of investigation of working mechanisms in endocrine disruptors, we evaluated the dose-related effects of fetal and/or neonatal exposure to an estrogenic compound on the male reproductive organs in adult mice, particularly with respect to gene expression of steroidogenic acute regulatory protein (StAR). The pregnant ICR mice were given subcutaneous injections of 10 μg/day/animal of diethylstilbestrol (DES) to subject the fetal mice to in utero exposure (IUE). Subsequently, the newborn male mice were subjected to neonatal exposure (NE) by treatment with vehicle or 0.1-10 μg/day/animal of DES. Fertility rates of each group were as follows: control, 100% IUE only, 60% IUE+NE 0.1 μg, 25% IUE+NE 1 μg, 0% IUE+NE 10 μg, 0%. In general histology, germ cell layers in the seminiferous tubules were thinned in the group of IUE+NE 10 μg. Hypoplasia of the Leydig cells, in which the staining intensity of eosin was diminished, was also observed in the groups of IUE+NE 0.1-10 μg. The androgen receptor (AR) and estrogen receptor alpha (ERα) immunoexpression in the Leydig cells of IUE+NE 1-10 μg was slightly lower than that in the controls. Longterm dysfunction of the hypothalamo-pituitary-testicular axis, including sustained hypoproduction of gonadotropin and testosterone, and altered expressions of steroid hormone receptors and StAR genes were observed. The hypothalamo-pituitary control of gonadotropin secretion may be affected by the smaller doses of estrogenic agents than the reproductive organs. Furthermore, the fertility rate in the male mice exposed to this estrogenic agent was closely correlated with the testosterone levels, and even more so with the rate-limiting factor of steroidogenesis, StAR. This finding suggests that endocrine disruptors have an important pronounced effect on StAR gene expression.
2006年12月, Journal of Veterinary Medical Science, 68 (12), 1257 - 1267, 英語[査読有り]
研究論文（学術雑誌）
- Persorption of luminal antigenic molecules and its specific antibody via apoptotic epithelial cells of intestinal villi and Peyer’s patches into peripheral blood in rats
The possibility of persorption of bovine serum albumin (BSA) molecules from mucous epithelial cells and its mechanism were investigated in rats orally pre-immunized by BSA for 14 consecutive days. In the small and large intestines, both the BSA antigen (BSA-Ag) and its specific antibody (SpAb) were absorbed by the epithelial cells at the late apoptotic stage (ApoEp), and were subsequently transcytosed by membranes of the small vesicles. The basal cytoplasms containing highly-concentrated BSA-Ag and SpAb were occasionally fragmented into small cytoplasmic droplets that were secreted into the lamina propria. In Peyer's patches, both BSA-Ag and SpAb were more actively absorbed and transcytosed toward the dome area by the ApoEp of the dome apex than by the M cells. BSA-Ag and SpAb were finally persorbed into the portal blood and lymph, but were never secreted into the bile. They were also engulfed by macrophage-like cells in the villous lamina propria, mesenteric lymph node and spleen, and by hepatocytes in the liver. These findings suggest that sensitized soluble luminal antigens are taken up by ApoEp in the small intestine and are finally persorbed into the peripheral blood. The uptake of luminal antigen might be mediated by its luminal SpAb.
JAPAN SOC VET SCI, 2006年12月, The Journal of Veterinary Medical Science, 68 (12), 1297 - 1305, 英語[査読有り]
研究論文（学術雑誌）
- Differentiation of epithelial cells to M cells in response to bacterial colonization on the follicle-associated epithelium of Peyer's patch in rat small intestine
To clarify the relationship between M cells and intestinal microflora, histoplanimetrical investigation into the bacterial colonization and the differentiation to M cells was carried out in rat Peyer's patch under physiological conditions. The follicle-associated epithelium (FAE), except for the narrow area of apical region, was closely covered with both neighboring intestinal villi and a thick mucous layer, the latter of which also filled the intervillous spaces as well as the space between the FAE and the neighboring intestinal villi. Indigenous bacteria adhered almost constantly to the narrow areas of apical regions of both intestinal villi and the FAE. Bacterial colonies were occasionally located on the basal to middle region of FAE, where M cells also appeared, forming large pockets. When bacterial colonies were located on the basal to middle region of FAE, bacteria with the same morphological characteristics also proliferated in the intervillous spaces neighboring the Peyer's patch. In cases with no bacterial colonies on the basal to middle region of FAE, however, M cells were rare in the FAE. Histoplanimetrical analysis showed the similar distribution pattern of bacterial colonies on the FAE and M cells in the FAE. M cells ultrastructurally engulfed indigenous bacteria, which were then transported to the pockets. These results suggest that indigenous bacterial colonization on the FAE stimulates the differentiation of M cells in the FAE under physiological conditions. The uptake of bacteria by M cells might contribute the regulation of the development of indigenous bacterial colonies in the small intestine.
JAPAN SOC VET SCI, 2006年10月, JOURNAL OF VETERINARY MEDICAL SCIENCE, 68 (10), 1023 - 1028, 英語[査読有り]
研究論文（学術雑誌）
- (公社)日本獣医学会, 2006年08月, 日本獣医学会学術集会講演要旨集, 142回, 32 - 32, 日本語マウス性腺の発生におけるStARタンパクの局在と性差
Steroid sulfatase (STS) is localized in the endoplasmic reticulum and catalyzes desulfation of 3 beta-hydroxysteroid sulfates. X-linked ichthyosis (XLI) is an inherited skill disorder caused by deficiency of STS enzyme activity. We previously reported a case in which XLI with a one-base change in the STS gene and variation in amino acid Q560P developed. In this study, we performed molecular analysis to determine the importance of terminal regions of STS and the effect of mutant STS oil STS enzyme activity. To examine the effect of terminal truncated STS on the enzyme activity, N- and C-terminal truncated STS expression vectors were transfected into COS-1 cells. The activity of truncated STS lacking the N-terminal regions declined, and the activity of C-terminal-truncated STS declined with extension of the truncated C-terminal region. Although the results of pulse-chase experiments showed that a one-base mutant STS (Q560P) and C-terminal-truncated STS (Delta C2 (1-559)) had no effects on protein synthesis and degradation, the mutant STS and C-terminal-truncated STS have dominant negative effect oil STS enzyme activity when the STS mutant or truncated STS protein and a wild-type STS protein coexist in cells. Results of coprecipitation of the truncated STS with all STS-FLAG fusion protein showed that STS formed a dimer conformation in cells. In this study, we have shown that both the N-terminal region and C-terminal region are important for STS enzyme activity. The C-terminal mutant has a dominant negative effect on wild-type STS.
SOC ENDOCRINOLOGY, 2006年02月, JOURNAL OF ENDOCRINOLOGY, 188 (2), 365 - 374, 英語[査読有り]
研究論文（学術雑誌）
- 2006年, Reproduction of Toxicology (accepted), 英語Evaluation of dose-response effects on offspring of the male mouse exposed to diethylstilbestrol as endocrine disrupting chemicals
[査読有り]
研究論文（学術雑誌）
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2005年09月, 環境ホルモン学会研究発表会要旨集, 8回, 259 - 259, 日本語
The present study examined the effects of aging and sex differences on the suprachiasmatic nucleus (SCN) of F344/N rats. In juveniles (1.6-1.9 months of age), adults (11.7-16.3 months of age), and old (29.2-34 months of age) rats, the volume, size of neuronal nucleus and neuronal cell number of the SCN were determined with cresyl fast violet-stained sections. In addition, immunohistochemical analysis was performed for glial fibrillary acidic protein (GFAP). There was no significant effect of aging and sex differences on the SCN volume. The number of neurons in the SCN gradually decreased from juvenile to old age in females. However, in males, the number was significantly decreased in adult and old age rats. The size of neuronal nuclei in the SCN was significantly decreased by increasing age in both sexes, except for the ventrolateral part of the SCN of males. In the dorsomedial part of the SCN of females, the density of GFAP-immunoreactive components was significantly higher in adult age rats than in rats of other ages. However, there was no significant increase in the density of the SCN in adult males. These results suggest that morphological changes in neuronal and astroglial cells occur in the SCN with aging in a sex-specific manner. (c) 2004 Elsevier Inc. All rights reserved.
PERGAMON-ELSEVIER SCIENCE LTD, 2005年03月, EXPERIMENTAL GERONTOLOGY, 40 (3), 147 - 155, 英語研究論文（学術雑誌）
- Familial 15p tetrasomy due to extra supernumerary marker 15 chromosome associated with Robertsonian translocation between chromosomes 13 and 14.
We report a cytogenetic and molecular cytogenetic study of a novel familial case of 15p tetrasomy due to an extra supernumerary marker chromosome (SMC) (15), which is associated with der (13 ; 14) (q10 ; q10). The proband is a 36-year-old married male with severe oligozoospermia and normal intelligence. He and his pregnant wife attended our department to receive prenatal diagnosis based on the indications of intra-cytoplasmic sperm injection (ICSI) pregnancy and advanced maternal age (39 years). The chromosome analysis of cultured amniocytes showed a Robertsonian translocation between chromosomes 13 and 14 : 45, XX, der (13 ; 14) (q10 ; q10). A cytogenetic examination of the couple revealed that the fetal translocated chromosome is paternally derived, and an extra small bisatellited psudodicentric marker chromosome was detected as a new finding in the husband (proband). Karyotyping of the proband's parents showed that his mother, who is of normal intelligence, carried the same translocation and SMC (15). Although fluorescence in situ hybridization (FISH) with DYZ3, D13/21Z1, D14/22Z1, and SNRPN probes showed no signal on the marker, two strong D15Z1 hybridization signals were found on either side of the centromeric heterochromatin. Thus, the marker chromosome was identified as familial inv dup (15) (pter→q11∷q11→pter) without loci such as the Prader-Willi syndrome (PWS) /Angelman syndrome (AS) critical regions, and we determined that there was a low risk of it affecting the baby's phenotype. Following these results, the pregnancy was continued, and a baby girl was born and developed without any clinically abnormal findings during 3 years after delivery. The coexistence of the two different chrmosome abnormalities in this family might be merely fortuitous.
Society of Chromosome Research, 2004年, Chromosome Science, 7,pp.7-13 (1), 7 - 13, 英語研究論文（学術雑誌）
Steroidogenic acute regulatory ( StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 10(6) clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003年10月, JOURNAL OF BIOLOGICAL CHEMISTRY, 278 (43), 42487 - 42494, 英語研究論文（学術雑誌）
Objective: The aim of this study was to elucidate the clinical characteristics of uterine leiomyomas having abnormal chromosome karyotype. Study design: A total of 394 myomas were obtained from 213 patients for cytogenetic analysis. The size (number of nodules = 144), histopathology (n = 302), and gonadotropin-releasing hormone analogue (GnRHa)-response (it = 58) were investigated in relation to chromosome karyotype in myomas. Results: 302 myomas from 166 patients were successfully karyotyped. A total of 21 myomas from 21 patients showed abnormal chromosome karyotype. The high frequencies of involved chromosomes 12, 14, 1, 7 were observed. The diameters of myomas with abnormal karyotype were significantly larger than those of myomas with normal karyotype. The frequency of the degeneration in myomas with abnormal karyotype was significantly higher than that with normal karyotype. The reduction rate in size of myomas by GnRHa treatments did not differ between the two types (karyotype normal versus abnormal) of nodules. Conclusions: Chromosomally abnormal myomas were larger in diameter and showed a higher frequency of degeneration, suggesting that the cytogenetic background in uterine leiomyoma affects a tumor's growth potential. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.
ELSEVIER SCI IRELAND LTD, 2003年09月, EUROPEAN JOURNAL OF OBSTETRICS GYNECOLOGY AND REPRODUCTIVE BIOLOGY, 110 (1), 58 - 62, 英語研究論文（学術雑誌）
- 2003年, The Hokkaido Journal of Medical Science., 78,pp.139-145, 英語Maternal serum markers in pregnancies associated with or without fetal morphological abnormalities.
研究論文（学術雑誌）
We cloned human activin βE subunit cDNA from a liver cDNA library using the polymerase chain reaction (PCR) technique. The deduced amino acid sequence was 97 and 96% homologous to the mouse and rat activin βE subunits. Human activin βE subunit tagged with Myc and polyhistidine residues at the COOH terminus was expressed in mammalian cells and secreted into the medium as a disulphide-linked homodimer protein. We also found that the human activin βE protein could bind to follistatin, an activin-binding protein. Northern blot analysis showed that this gene was expressed as a major transcript of 2.7 kb predominantly in human liver. These findings suggest that activin E (dimeric protein) may play a role in humans. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
2002年08月30日, Molecular and Cellular Endocrinology, 194 (1-2), 117 - 122, 英語[査読有り]
研究論文（学術雑誌）
Background: Three serum tests, α-fetoprotein (AFP), human chorionic gonadotrophin and unconjugated oestriol, are now widely used for screening for Down's syndrome. Lens culinaris agglutinin-reactive α-fetoprotein (AFP-L3) is a variant of α-fetoprotein with α1→6 fucose appended to the reducing terminal N-acetylglucosamine. It is the most prominent AFP detected in the serum of patients with hepatocellular carcinoma. Methods: We investigated microheterogeneities of the carbohydrate chain on AFP in fetal liver tissues, amniotic fluids and maternal sera obtained from pregnancies with Down's syndrome using lectin affinity electrophoresis with four lectins. The percentages of AFP-L3 in maternal sera from 22 Down's syndrome and 227 unaffected pregnancies were determined. Results: Unlike the case with AFP concentration, the percentage of AFP-L3 in maternal serum and amniotic fluid was similar, and apparently not influenced by membrane permeability. Knowing the percentage of AFP-L3 in maternal serum was effective for discriminating between Down's syndrome-affected pregnancies and unaffected pregnancies. The percentage of AFP-L3 in maternal serum identified 55% of Down's syndrome cases with a 5% false-positive rate. Conclusions: AFP-L3 should be an effective replacement for AFP in prenatal Down's syndrome screening.
Oxford University Press, 2001年, Human Reproduction, 16 (11), 2438 - 2444, 英語[査読有り]
研究論文（学術雑誌）
- Novel mutation (E113X) of antithrombin III gene (AT3) in a woman with gestational recurrent thrombosis
A 35-year-old Japanese woman with a low level (42-54%) of blood antithrombin (AT) III, experienced two induced abortions due to deep venous thrombosis at 8 weeks of gestation (GW) and cerebral thrombosis at 10 GW, The present pregnancy was successfully managed with intravenous administration of AT III (6,000-8,000 U/wk), Analysis of polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) for exons 3A and 4 of the AT III gene (AT3) using her DNA revealed extra expansion bands with altered migration. The DNA sequencing demonstrated novel mutations in exon 3A of AT3: a G to T substitution at nucleotide position 5333 in codon GAG for Glu 113, causing a stop codon (E113X), and an A to T substitution at position 5338 in codon AAA for Lys 114, forming Asn (K114N). These novel mutations, especially E113X, in AT3 may be related to recurrent thrombosis in the pregnancy. (C) 2000 Wiley-Liss, Inc.
WILEY-LISS, 2000年04月, AMERICAN JOURNAL OF MEDICAL GENETICS, 91 (5), 348 - 350, 英語[査読有り]
研究論文（学術雑誌）
Objective: This prospective study assesses the efficacy of maternal serum screening for use in prenatal diagnosis of fetal anomaly and chromosome imbalance in Japanese women. Methods: Maternal serum α-fetoprotein, human chorionic gonadotropin, and unconjugated estriol were measured in 1055 singleton pregnant women between 14 and 20 weeks of gestation. A calculated risk for trisomy 21 of ≥ 1/299 or α-fetoprotein ≥ 2.5 multiples of the median was adopted as positive. Results: Three hundred and seventy-eight of the 1055 women screened (35.8%) were identified as positive. Sensitivity, false-positive rate, and positive predictive value in women aged < 35 years were 60.0, 10.6, and 6.8%, respectively, and these values were 87.5, 49.3, and 4.2%, respectively, in women aged ≥ 35 years. The false-positive rate in women aged < 35 years was significantly lower than that for women aged ≥ 35 years (p < 0.001). Chromosomal abnormalities were identified in 21 cases, including 10 with trisomy 21, 5 with trisomy 18, 2 with trisomy 13, and 4 with other chromosomal disorders. Seventeen of the 21 cases (81.0%) showed screen-positive results, and among these all 10 cases with trisomy 21 were detectable. Two cases with trisomy 18, 1 with trisomy 13 and 1 with isochromosome X showed extremely low human chorionic gonadotropin levels (0.4 ± 0.1 multiples of the median, mean ± SE), although they were screen negative. Of the 264 women who did not undergo amniocentesis, none had any clinical findings consistent with aneuploidy after birth. Conclusions: Our results suggest that the evaluation of each serum marker, as well as of the calculated risk, was significantly important in the prenatal detection of fetal aneuploidy. Copyright (C) 2000 S. Karger AG, Basel.
S. Karger AG, 2000年, Fetal Diagnosis and Therapy, 15 (2), 112 - 117, 英語[査読有り]
研究論文（学術雑誌）
- Factor XII deficiency in women with recurrent miscarriage
Congenital thrombophilia is known to cause significant maternal complications, and possibly has an adverse effect on normal feta I development, The aim of this study was to assess the prevalence of factor XII (FXII) deficiency in women with a history of recurrent miscarriage. Two hundred and forty-one consecutive Japanese women with a history of two or more recurrent miscarriages were prospectively assessed for their etiology by conventional screening methods. Seven women were found to have reduced FXII activity (19.2-46.1%) and prolonged activated partial thromboplastin time (33.3-51.3 s). Of these 7 women, 6 had experienced early pregnancy losses, while 1 woman had experienced repeated midtrimester fetal losses with coincidental gestational thrombocytopenia, In 241 women with a history of recurrent miscarriage, the prevalence of FXII deficiency was 2.9%. Copyright (C) 2000 S. Karger AG, Basel.
KARGER, 2000年, GYNECOLOGIC AND OBSTETRIC INVESTIGATION, 49 (2), 80 - 83, 英語[査読有り]
研究論文（学術雑誌）
- Clinicopathological comparisons of familial and sporadic cases in 219 consecutive Japanese epithelial ovarian cancer patients
Background: It is estimated that approximately 5-10% of epithelial ovarian cancer patients in Western countries are associated with an autosomal dominant inheritance with variable penetrance. There are a few reports of familial ovarian cancer in Japan and considerable uncertainties remain regarding Japanese familial ovarian cancer. The aim of this study was to clarify the clinicopathological features of Japanese familial ovarian cancer. Methods: We studied clinicopathological findings for 219 consecutive epithelial ovarian cancer patients treated at our institution from April 1987 to September 1997. Results: Eleven patients in nine families were diagnosed as familial ovarian cancer and the incidence of familial cases was 5.0%. Most women (90.9%) with familiar cases were diagnosed as the breast ovarian cancer syndrome, whereas ovarian cancer associated with hereditary nonpolyposis colorectal cancer was relatively rare (9.1%). Serous adenocarcinoma, high histological grade, advanced FIGO stage and breast cancer as multiple primary cancer were significantly more common in familial cases compared with sporadic cases (p < 0.001, p < 0.05, p < 0.005 and p < 0.005, respectively). Earlier age of onset was thought to be a characteristic of familial ovarian cancer in Western countries; however, we did not find any difference in age at diagnosis between familial and sporadic Gases (53.4 vs 51.3 years). The prognosis of familiar ovarian cancer remains controversial and our data did not show a significant difference (p = 0.45) in prognosis between these two groups. Conclusion: These findings, except for age at diagnosis, in Japanese familial ovarian cancer are in accordance with the features of familial ovarian cancer in Western countries.
OXFORD UNIV PRESS, 1999年11月, JAPANESE JOURNAL OF CLINICAL ONCOLOGY, 29 (11), 556 - 561, 英語[査読有り]
研究論文（学術雑誌）
- BRCA1 mutation testing for Japanese patients with ovarian cancer in breast cancer screening
From February 1996 to April 1998, 2967 women received screening for breast cancer in the gynecologic ambulatory practice ofthe Hokkaido University Hospital. In 116 Japanese women with epithelial ovarian cancer, mutation analysis of BRCA1 exon 11 in genomic DNA was performed by the stop codon (SC) assay and DNA sequence analysis. Clinicopathological factors were also investigated in these patients. The aim of this study was to examine the advantages of performing BRCA1 mutation testing for ovarian cancer patients during breast cancer screening. We achieved a high detection rate (6.0) of patients with germline mutations in BRCA1. The high frequencies of breast ovarian cancer syndrome, serous adenocarcinoma, high histological grades, advanced FIGO stages, and breast cancer as double cancer were found to be characteristic of ovarian cancer with germline mutations in BRCA1. These characteristics may assist physicians in selecting BRCA1 mutation testing for ovarian cancer patients. The mean age at diagnosis of ovarian cancer was 51.0 and 51.2 years in the groups with and without mutation, respectively, and no difference was found in age at diagnosis. All of the nine living female mutation carriers were offered the options of increased surveillance or prophylactic surgery, and all chose the former. We have performed breast cancer screening and/or ovarian cancer screening every 6 months for these carriers. This may allow another advantage in establishing a relationship of mutual trust with a patient from a series of responsible follow-ups.
KLUWER ACADEMIC PUBL, 1999年11月, BREAST CANCER RESEARCH AND TREATMENT, 58 (1), 11 - 17, 英語[査読有り]
研究論文（学術雑誌）
Objective: To report a case of tubal pregnancy in a unicornuate uterus with rudimentary horn on the side of the rudiment and its pathology. Design: Case report. Setting: University hospital. Patient: An 18-year-old woman, primigravida, with tubal pregnancy. Intervention(s): Systemic administration of methotrexate, salpingectomy by laparotomy, and laparoscopic surgery for resection of rudimentary horn. Main Outcome Measure(s): Emergent laparotomy revealed that the intraperitoneal hemorrhage was caused by the rupture of the tubal pregnancy on the same side as the rudimentary horn of the unicornuate uterus. A corpus luteum was found at the ipsilateral ovary of the rudimentary horn. Result(s): Image diagnosis and pathological examination of the rudimentary horn revealed that this uterine malformation was a unicornuate uterus with a noncommunicated, noncavitary rudimentary horn, corresponding to class IIc of the American Fertility Society classification of mullerian anomalies. Conclusion(s): This is the first report of a tubal pregnancy on the side of the noncommunicating rudimentary horn with the ipsilateral ovary carrying a corpus luteum in a unicornuate uterus.
1999年08月, Fertility and Sterility, 72 (2), 354 - 356, 英語[査読有り]
研究論文（学術雑誌）
The aims of the present study were to determine recent trends in the prevalence of Down syndrome (DS) in Japan, and to determine whether recent changes in demographic and social habits and access to prenatal diagnosis have influenced the livebirth rates of DS. Livebirth statistics indicate that the birth rate in Japan has decreased for women in their 20s and has increased for those in their 30s and 40s. During an 18-year period between 1980 and 1997, 1,299 consecutive DS infants were born among a total of 2,232,694 births, a rate corresponding to approximately 10% of all births in Japan over the same period. The increasing risk of DS with advancing maternal age was confirmed. The overall prevalence was 5.82 DS births per 10,000 livebirths (8.3-9.7 per 10,000 after correction according to the estimated ascertainment ratio: 60-70%). The prevalence rate by year of child birth represents a statistically significant increase (P = 0.001). In conclusion, recent trends in the prevalence of DS in Japan from 1980 to 1997 failed to show a consistent tendency to decrease, probably because of the concomitant increase in pregnancy in advanced maternal age.
Wiley-Liss Inc., 1999年06月04日, American Journal of Medical Genetics, 84 (4), 340 - 345, 英語[査読有り]
研究論文（学術雑誌）
- (一社)日本周産期・新生児医学会, 1999年06月, 日本新生児学会雑誌, 35 (2), 198 - 198, 日本語ヒツジ胎仔における障害時期とPVL発症
[査読有り]
The human deleted in azoospermia-like autosomal (DAZLA) gene is thought to be a candidate gene for azoospermia. cDNA encoding the C-terminal 94 amino acid residues of human DAZLA was used to express a bacterial fusion protein which was then used to raise polyclonal antibodies in rabbits. Immunohistochemical analyses with the human DAZLA antiserum showed that the DAZLA protein is expressed at a cytoplasmic location in female germ cells. Available evidence suggests that the DAZLA gene is a participant in human oogenesis.
1999年06月, Molecular Human Reproduction, 5 (6), 495 - 497, 英語[査読有り]
研究論文（学術雑誌）
Objective: To evaluate the risk of recurrence of fetal chromosomal aberrations in women who had offspring with numeric chromosomal abnormalities. Subjects and Methods: This collaborative study consisted of 1076 Japanese women with a history of offspring with trisomy-21, -18, -13, or 45,X. Second-trimester amniocenteses were performed, resulting in 1248 fetal karyotypes that were analyzed with reference to prior offspring karyotypes and maternal age. Results: Of the 842 women with trisomy-21 offspring, 10 conceived another such fetus. In 2 women with 3 or more such offspring, parental mosaicism of trisomy-21 was suspected. The incidence of recurrence of trisomy-21 increased with age, and significantly exceeded the incidence of trisomy-21 fetuses in the general population. None of the 170 women with trisomy-18 offspring, and none of the 46 women with trisomy-13 offspring, had another such fetus. Of the 18 women with 45,X offspring, 1 with mos 45,X/46,XX had another such fetus. Conclusions: The risk of recurrence of trisomy-21 is affected by maternal age and parental germline mosaicism. The risk of recurrence of trisomy-18 or -13 appears to be much lower than that of trisomy-21. Women who give birth to more than 1 offspring with 45,X should be examined for mos 45,X/46,XX.
University of Tokyo Press, 1999年, Journal of Obstetrics and Gynaecology Research, 25 (6), 373 - 379, 英語[査読有り]
研究論文（学術雑誌）
OBJECTIVES: Our purpose was to determine whether systemic hypotension induced by rapid withdrawal of fetal blood would induce periventricular leukomalacia in the brain of premature fetal sheep. STUDY DESIGN: At 113 days' gestation, systemic hypotension (mean blood pressure < 30 mm Hg) was induced by withdrawing approximately 35% of the fetoplacental blood volume in the hemorrhage group (n = 6), whereas in the control group (n = 4) isovolemic exchange transfusion was carried out. Six days after the insult, fetal brains were removed and processed for histologic analysis. RESULTS: Five of the 6 fetuses in the hemorrhage group exhibited periventricular white-matter lesions, consisting of nodular coagulation necrosis or diffuse axonal swellings or both. No abnormal findings other than these lesions were detected in the fetal brains in either experimental group. CONCLUSION: Hemorrhagic hypotension antenatally induced brain lesions similar to those of periventricular leukomalacia, suggesting that it is an essential element in the pathogenesis of periventricular leukomalacia in premature fetuses.
Mosby Inc., 1999年, American Journal of Obstetrics and Gynecology, 181 (3), 725 - 730, 英語[査読有り]
研究論文（学術雑誌）
Ectopia cordis is a rare congenital anomaly. We present 4 cases of ectopia cordis, 1 of which is the first report of an affected fetus in a triplet pregnancy. The morphological relationship between the types of ectopia cordis and their outcomes were investigated in all 4 cases. In addition, the literature on ectopia cordis in Japan was reviewed and discussed.
University of Tokyo Press, 1999年, Journal of Obstetrics and Gynaecology Research, 25 (4), 237 - 243, 英語[査読有り]
研究論文（学術雑誌）
- 1998年01月, Acta Obstetrica et Gynaecologica Japonica, 50 (1), 35 - 36Periventricular leukomalacia after acute hemorrhagic hypotension in the chronically instrumented fetal sheep
研究論文（学術雑誌）
- Seminoma in a postmenopausal woman with a Y;15 translocation in peripheral blood lymphocytes and a t(Y;15)/45,X Turner mosaic pattern in skin fibroblasts
We report an unusual case of a 55 year old Japanese woman with a seminoma but relatively normal menses. The patient was a phenotypic female with late onset menarche (18 years of age), who was amenorrhoeic for the first year, followed by menses of one to three days' slight flow with dysmenorrhoea, but an otherwise normal menstrual history. A typical seminoma was removed from the left adnexal region and an immature testis was identified separately as an associated right adnexal mass. Repeated karyotypic studies on peripheral 770, Japan blood lymphocyte cultures showed only Y Nal ahori 46,X,-Y,t(Y 15)(q12 p13). Cytogenetic examination of the patient's younger brother, who had fathered three healthy children, showed an identical karyotype. Mosaicism of 46,X,-Y,t(Y 15)(q12 p13)/45,X cell lines was found in skin samples from the patient's elbow and genital regions, although there were no clinical stigmata of Turner syndrome. An androgen receptor binding assay of cultured genital skin fibroblasts was negative. Molecular analysis using Southern blot hybridisation, PCR, and direct DNA sequencing showed that neither the patient nor her brother had a detectable deletion or other abnormalities of Y chromosome sequences, including the SRY (sex determining region of the Y chromosome) gene sequence. These findings suggest that Turner mosaicism of the 45,X cell line may have contributed to this atypical presentation in an XY female, although we cannot exclude abnormalities of other genes related to sex differentiation.
1998年, Journal of Medical Genetics, 35 (10), 852 - 856, 英語[査読有り]
研究論文（学術雑誌）
We report a case of mos 45,X/46,X,+mar, diagnosed prenatally by amniocentesis, whose physical examination, including external and internal organs, along with serum testosterone values were normal five years after delivery. The mosaic karyotype was seen in 146 of 240 cells examined (amniotic fluid cells, 110/65 placental chorionic vilii: 5/4 cord blood, 21/81 cultured skin fibroblasts, 10/90) from 386 metaphases, and the marker chromosome appeared as a small non-fluorescent acrocentric chromosome. All autosomes appeared normal, and no normal Y chromosome could be demonstrated. Analysis of 26 Y-chromosome loci by molecular techniques such as PCR, Southern analysis using multiple Y-specific DNA probes, and Hae III restriction endonuclease assessment of male-specific repeated DNA in the heterochromatic region of the Y chromosome, and fluorescence in situ hybridization (FISH), revealed the marker was derived from a Y chromosome including p terminal to q11.23, and paracentric inversion in the remaining Y long arm. The formation of testes can be considered as existence of SRY (sex-determining region of Y) as a testis-determining factor. The present report illustrates the importance of FISH and molecular techniques as a complement to cytogenetic methods for accurate identification and characterization of chromosome rearrangements in prenatal diagnosis.
1998年, Prenatal Diagnosis, 18 (12), 1316 - 1322, 英語[査読有り]
研究論文（学術雑誌）
Fetal anemia causes hydrops fetalis and fetal ascites/hydrothorax, and in severe cases the prognosis is poor. Little other than alloimmunity and viral infections are known as mechanisms causing fetal anemia. The aim of this study was to elucidate any pathogenesis in fetal anemia due to otherwise idiopathic etiology. The levels of three hematopoietic cytokines, IL-3, erythropoietin (EPO), and granulocyte colony-stimulating factor (G-CSF) were measured in blood samples obtained by cordocentesis from six fetuses with anemia (Hb < 10.0 g/dl) and 34 fetuses without anemia. Cordocentesis was performed prior to the onset of labor or uterine contractions in all pregnant women. The concentration of IL-3 in fetuses with anemia [M ± (SD), 8.3 (10.1) pg/ml] was significantly lower than that in fetuses without anemia [59.9 (71.0) pg/ml]. EPO and G-CSF levels were not different between the two groups. In addition, through in vitro colony formation assay, using blood stem cells from two fetuses with severe anemia and three fetuses without anemia, it was found that colony forming unit-erythroid, burst forming unit- erythroid and granulocyte macrophage-colony forming unit were significantly suppressed in blood stem cells from the two fetuses with severe anemia. Thus, the malfunction of differentiation and proliferation of blood stem cells and the decrease of hematopoietic cytokine levels in the fetal circulation may be responsible for the occurrence of fetal anemia.
Thieme Medical Publishers, Inc., 1998年, Seminars in Thrombosis and Hemostasis, 24 (5), 485 - 490, 英語[査読有り]
研究論文（国際会議プロシーディングス）
The aim of this trial was to investigate the efficacy of massive i.v. immunoglobulin (MIVIg) treatment for women with a history of recurrent spontaneous abortion (RSA) due to unexplained aetiology. The study included nine women (11 pregnancies) with a history of four or more consecutive RSA with unexplained aetiology and no live births. The mean number of fetal losses was 4.5 (range 4-6 abortions). Over the course of 5 days, immunoglobulin (20 g/day) was infused i.v. at gestational weeks 4-7. No additional infusions were carried out. Two pregnancies out of the 11 conceptions resulted in missed abortions at gestational weeks 6 and 7 respectively. Mosaicism (46XX/ 48XX, + 16, + 20), and tetraploidy (92XXXX) were found by chromosome analyses of the two aborti. Eight out of the other nine pregnancies resulted in full term deliveries of healthy neonates. One pregnancy developed intrauterine growth retardation and fetal distress, resulting in a premature delivery (30 gestational weeks) by Caesarean section. Thus, excluding the two abortions with chromosome aberrations, the MIVIg treatment was effective in all nine pregnancies of RSA women with unexplained aetiology. This MIVIg treatment (100 g administered in early gestation) may be a beneficial alternative to previous IVIg infusion methods, and should be further evaluated in a multicentric, placebo-controlled study, employing a larger number of homogeneous patients who fall into a high risk catagory of first trimester abortions.
1998年, Human Reproduction, 13 (9), 2620 - 2623, 英語[査読有り]
研究論文（学術雑誌）
Objective: To clarify the prenatal sex ratio, and types and frequencies of chromosome anomalies in relation to gestational weeks in women of advanced maternal age. Methods: Chromosomal analysis was performed in 894 induced abortuses derived from elderly pregnant women 35 or more years of age. Results: The embryonic sex ratio in the chromosomally normal (abnormal) gave a preponderance of females over males in lower gestational weeks [366/458 (53/73) 79.9 to 100 (86.3 to 100) in 4-10 weeks] the reversal of dominancy in those from females to males occurred in the 11th gestational week [41/29 (5/4) 141.4 to 100 (125.0 to 100) in 11-20 weeks]. The frequency of trisomy in females at 10 weeks of gestation was significantly high (7/28 25%). Conclusions: This study showed that the existence of a difference of and a reversal of the sex ratio in gestational weeks. It was considered that there might be a lower viability of genetically handicapped males as compared to such females. However, the reversal of the sex ratio, such as that observed between 9th and 10th gestational weeks, might reflect events occurring after implantation.
Blackwell Publishing, 1997年, Journal of Obstetrics and Gynaecology Research, 23 (1), 1 - 7, 英語[査読有り]
研究論文（学術雑誌）
A total of 934 induced abortuses of middle‐aged pregnant women (35–48 years old) were chromosomally surveyed. The mean gestational age was 7.6±2.0 (S.D.) weeks after the first day of the last menstrual period, and the mean maternal age was 39.6±2.7 (S.D.) years. There was a high incidence of chromosomally abnormal fetuses in induced abortions, which markedly increased with the advancing age of the women: 9.6% (40/418) for the 35–39‐year‐old age group, 20.7% (100/484) for the 40–44‐year‐old age group, and 43.8% (14/32) for the 45–48‐year‐old age group, averaging 16.5%. © 1990 Japanese Society of Obstetrics and Gynaecology
1990年, Asia‐Oceania Journal of Obstetrics and Gynaecology, 16 (3), 275 - 281, 英語[査読有り]
研究論文（学術雑誌）
- JAPANESE SOC ELECTRON MICRO BUS CENTER ACAD SOC JAPAN, 1989年, JOURNAL OF ELECTRON MICROSCOPY, 38 (1), 77 - 78, 英語A SCANNING ELECTRON MICROSCOPIC STUDY ON THE ARCHITECTURE OF LYMPH VESSELS AND INTRANODAL LYMPH PATHWAYS OF LYMPH-NODES IN PIG
The architecture of lymphatic microcirculation of pig lymph nodes was studied by scanning electron microscopy using a corrosion cast method. There were two types of lymph vessels for afferent lymph supply as well as those for efferent lymph drainage. Most afferent lymph vessels entered the node at the A-type hilus and penetrated deeply into the node. Others divided from the larger afferents and distributed to the convex surfaces of the node. One type of efferent lymph vessel left from the E-type hilus by several efferent trunks and was formed by confluences of many smaller lymph vessels, and the other type arose from surfaces around the peri-hilar (A-type) region, encompassing the afferent lymph vessels with numerous initial efferent vessels. The afferent lymph vessels were followed by intra-trabecular lymph channels. The peri-trabecular lymph sinuses and the peri-hilar (A-type) sub-capsular lymph sinuses were connected subsequently with the efferent lymph vessels and there was a direct communication between the cortex- and the medulla-like tissue (CT and MT). A marked difference between the casting patterns of the CT and MT was recognized ; that is, the CT was visualized as a dense coral-reef-like shape, whereas the MT exhibited a bead-like structure with numerous larger inter-spaces. These findings suggest that the intranodal lymph pathways not only share reversal in flow but are more minute and complex than those hitherto demonstrated.
北海道大学, 1988年01月30日, Japanese journal of veterinary research, 36 (1), 1 - 14, 英語正常な雌子牛の分娩後に排出された無形無心体について, 血管走行と染色体構成を調べた. 血管の合成樹脂鋳型標本からは, 心臓およびそれに相当する構造は認められなかった. 無形無心体の染色体構成は60,XYで, 正常な雄の核型を示した. 同腹の雌子牛に性染色体のキメラは認められなかった.
JAPANESE SOCIETY OF VETERINARY SCIENCE, 1988年, 日本獸醫學雜誌(The Japanese Journal of Veterinary Science), 50 (5), 1139 - 1141, 英語- HOKKAIDO UNIV, 1988年01月, JAPANESE JOURNAL OF VETERINARY RESEARCH, 36 (1), 15 - 29, 英語BLOOD-SUPPLY AND MICROVASCULATURE OF THE LYMPH-NODES IN PIGS
[査読有り]
研究論文（学術雑誌）
ブタリンパ節の「髄質様組織」とそれに面するリンパ洞の微細構造を電顕的に検索した. 髄質様組織は主に細網細胞と膠原線維束からなり,いわゆる髄洞構造はみとめられなかった. 皮質・髄質様組織境界部の髄質様組織側および被膜下洞付近の髄質様組織には,多数の細長い細胞質突起を相互に,あるいは大食細胞の細胞質突起と複雑迷路状にからめあい,密な網工を形成している樹枝状細胞が多数観察された. 輸出リンパ管の存在しない被膜下領域では,明瞭なリンパ洞の形態を備えた被膜下洞はみとめられなかった. 梁柱周囲洞の梁柱側を構成する内皮細胞はほぼ連続した基底膜で覆われ,その外側にはよく発達した膠原線維束が観察された. 梁柱周囲洞の実質側および被膜下洞の内壁は不連続な洞内皮細胞と膠原線維束の2層構造を呈した. これらの結果から,ブタリンパ節のリンパ洞には他動物のリンパ節にはみられない微細構造が存在するとともにその髄質様組織は強い結合組織性の性格を有し,リンパ節に流入する物質や細胞成分の濾過作用を増強させているものと推察された.
社団法人日本獣医学会, 1988年, Jpn. J. Vet. Sci.,, 50 (1), 83 - 92, 英語- JAPAN SOC VET SCI, 1986年12月, JAPANESE JOURNAL OF VETERINARY SCIENCE, 48 (6), 1097 - 1107, 英語HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDIES ON THE ARCHITECTURE OF LYMPH-NODES IN PIG
[査読有り]
研究論文（学術雑誌）
MISC
【背景・目的】化学物質のリスク評価における課題の一つとして、個々の化学物質を対象とする従来の毒性評価システムによっては複数の化学物質による複合影響のリスクを評価できない点が挙げられる。近年提唱されたAdverse Outcome Pathway（AOP）の概念に基づくと、複合影響に対する効率的な毒性評価を行うためには毒性発現における共通のKey Eventを活用することが重要となる。我々は前回大会にて、ピレスロイド系農薬デルタメトリン曝露時において、マイトファジーの活性化およびプロテアソーム活性低下を介したタンパク質分解系の不均衡状態が関与する新たな神経毒性メカニズムを報告した。そこで本研究では、これらのエンドポイントを神経毒性のKey Eventとすることで農薬類の複合影響評価への応用を検証することを目的とした。 【方法】マウス神経芽細胞腫Neuro-2a細胞に農薬類10種を曝露し、ミトコンドリア膜電位やオートファジーおよびプロテアソーム活性を指標としてミトコンドリアやタンパク質分解系への機能的影響を評価した。またWST-8アッセイにより細胞生存性を評価し、CompuSynソフトウェアにより複合影響の用量反応性を解析した。 【結果・考察】ピレスロイド系農薬ペルメトリンおよびデルタメトリンとフェニルピラゾール系農薬フィプロニルの曝露により、ミトコンドリアの機能低下、オートファジーの分解基質マーカーであるp62の蓄積等の共通したKey Eventの変動がみられた。さらにこれらの複合曝露により、単独曝露時と比べてより低濃度で細胞生存性が低下し、Combination Index < 1を示す相乗効果が認められた。本研究により、神経毒性に関するAOPの共通Key Eventを指標とすることで異種の農薬類の複合曝露が引き起こす相乗的な神経毒性を検出できることが初めて示された。
日本毒性学会, 2022年, 日本毒性学会学術年会, 49.1, P-4E, 日本語- 2021年, 日本先天異常学会学術集会プログラム・抄録集, 61st (CD-ROM)ネオニコチノイド系農薬クロチアニジンのマウス母乳中移行量に関する定量的評価
- 2021年, 日本先天異常学会学術集会プログラム・抄録集, 61st (CD-ROM)ネオニコチノイド系農薬クロチアニジンが次世代マウスの免疫系および腸内細菌叢に及ぼす影響
- 2021年, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM)神経回路形成期におけるステージ別ネオニコチノイド系農薬曝露と発達神経毒性評価
- 2021年, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM)ネオニコチノイド系農薬クロチアニジンの胎子・授乳期曝露が次世代マウスの免疫系および腸内細菌叢に及ぼす影響
- 2021年, 環境ホルモン学会研究発表会プログラム・要旨集, 23rd (Web)低用量クロチアニジンの神経回路形成期におけるステージ別曝露と発達神経毒性評価
- 2021年, 環境ホルモン学会研究発表会プログラム・要旨集, 23rd (Web)ネオニコチノイド系農薬クロチアニジンの胎子・授乳期曝露による次世代マウスの免疫系および腸内細菌叢の変化
- 2021年, 環境ホルモン学会研究発表会プログラム・要旨集, 23rd (Web)タンパク質分解系の不均衡状態を介したピレスロイド系農薬による神経毒性の発現機序
- 2021年, 環境化学討論会要旨集(CD-ROM), 29thニコチン性アセチルコリン受容体アゴニストによるマウスの脳機能攪乱~二光子顕微鏡を用いた検出法の開発~
- 2020年, 日本先天異常学会学術集会プログラム・抄録集, 60th無毒性量ネオニコチノイド系農薬が次世代のマウス雌性生殖機能に及ぼす影響
- 2020年, 日本先天異常学会学術集会プログラム・抄録集, 60th無毒性量の浸透性農薬のマウス胎子・授乳期曝露による神経行動学的影響
- 2020年, Endocrine Disrupter News Letter (Web), 22 (4)低用量クロチアニジンによる情動変容への加齢の影響評価
- 2020年, 日本獣医学会学術集会講演要旨集, 163rd行動毒性試験および二光子イメージングによるアセタミプリドの神経毒性評価
- 2019年, 日本分子生物学会年会プログラム・要旨集(Web), 42nd脆弱なSryを有するYPOSマウスの生殖腺形成に及ぼすC57BL/6マウスの亜系統差の影響
- 2019年, 環境ホルモン学会研究発表会プログラム・要旨集, 22nd (Web)ネオニコチノイド系農薬における野生哺乳動物の曝露状況と都道府県別の出荷量との関連
- 2019年, Endocrine Disrupter News Letter, 21 (4)無毒性量のネオニコチドの系濃薬曝露による行動影響の性差
- 2019年, 環境化学討論会要旨集(CD-ROM), 28thネオニコチノイドの母子間移行の実態と移行メカニズムの解明
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2018年12月, 日本内分泌かく乱化学物質学会研究発表会要旨集, 21st, 79 - 79, 日本語母子間移行における浸透性農薬およびその代謝産物の定量的な解明
- 2018年12月, 日本内分泌かく乱化学物質学会研究発表会要旨集, 21st, 82, 日本語兵庫県における野生哺乳動物のネオニコチノイド系農薬への曝露実態調査
- (公社)日本獣医学会, 2018年08月21日, 日本獣医学会学術集会講演要旨集, 161st, 467 - 467, 日本語浸透性農薬とその代謝産物の胎子移行量の解明
- 2018年, 日本アンドロロジー学会総会記事, 37thニワトリの精巣におけるセルトリ細胞の起源の左右差
- 東京医科大学医学会, 2018年01月, 東京医科大学雑誌, 76 (1), 139 - 140, 英語ニワトリ胚におけるセルトリ細胞の起源(The origin of Sertoli cells in the chicken embryo)
- 東京医科大学医学会, 2018年01月, 東京医科大学雑誌, 76 (1), 139 - 140, 英語ニワトリ胚におけるセルトリ細胞の起源(The origin of Sertoli cells in the chicken embryo)
- 2017年, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 122ndニワトリの発生過程に特徴的な生殖腺の左右差
- 2016年, 日本先天異常学会学術集会プログラム・抄録集, 56th発達期におけるネオニコチノイド系農薬曝露により多動が引き起こされる
- 2016年, 日本先天異常学会学術集会プログラム・抄録集, 56th胎子期から成獣期にかけてのネオニコチノイド系農薬クロチアニジン曝露が及ぼす行動学的影響
- 2016年, 日本解剖学会総会・全国学術集会講演プログラム・抄録集, 121stニワトリ胚の性腺発生における左右差
- 2016年, 日本分子生物学会年会プログラム・要旨集(Web), 39thマウス胎子性腺における性分化関連遺伝子転写調節領域のDNAメチル化状態
- 2016年, 日本先天異常学会学術集会プログラム・抄録集, 56th発達期におけるネオニコチノイド系農薬ジノテフラン曝露とうつ病発症との関係
- 2016年, Endocrine Disrupter News Letter, 19 (2), 3, 日本語ネオニコチノイド系農薬の標的とその作用メカニズム
記事・総説・解説・論説等（学術雑誌）
- 2015年, 日本家禽学会誌, 52ニワトリ性腺の左右差形成メカニズムに関する網羅的遺伝子発現解析
- 2015年, 日本家禽学会誌, 52ニワトリ胚における左精巣特異的な発生メカニズム
- 2015年, 日本実験動物学会総会講演要旨集, 62ndC57BL/6マウスの不可思議さ:その遺伝的背景に潜むSry-HMG box&Q-rich領域と性決定
- 2014年, 日本家禽学会誌, 51ニワトリ胚における性腺の形成機構とその左右差
- 2013年08月30日, 日本獣医学会学術集会講演要旨集, 156th, 191, 日本語ウミガメの総排泄腔傍嚢の形態機能学的特徴
- 2013年03月31日, は虫両棲類学会報, 2013 (1), 66, 日本語カメ類における「膀胱」の形態機能学的研究
- 2013年, 日本野生動物医学会大会・講演要旨集, 19th環境ストレス下動物の生殖機能抑制機序に関する形態機能学的研究
- 2013年, 日本野生動物医学会大会・講演要旨集, 19th, 98, 日本語カメ類における「膀胱」の形態機能学的定義
- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2012年12月, 環境ホルモン学会研究発表会要旨集, 15回, 59 - 59, 日本語生後発達期におけるTCDDによる神経発達障害の分子基盤
- 2012年08月31日, 日本獣医学会学術集会講演要旨集, 154th, 175, 日本語カメ類における「膀胱」の形態機能学的解析
- (一社)日本解剖学会, 2012年06月, 解剖学雑誌, 87 (2), 37 - 37, 日本語胎子・授乳期TCDD曝露と胸腺微小環境の変化
- (一社)日本解剖学会, 2012年06月, 解剖学雑誌, 87 (2), 37 - 37, 日本語胎子・授乳期TCDD曝露による発達中のセロトニン神経系への影響とその作用機序
- (公社)日本獣医学会, 2011年08月, 日本獣医学会学術集会講演要旨集, 152回, 173 - 173, 日本語胎子・授乳期TCDD曝露がマウス胸腺微小環境に及ぼす影響に関する分子形態学的研究
- (公社)日本獣医学会, 2011年08月, 日本獣医学会学術集会講演要旨集, 152回, 177 - 177, 日本語胎子・授乳期TCDD曝露による発達中のセロトニン神経系への影響に関する分子形態学的研究
- (公社)日本獣医学会, 2008年09月, 日本獣医学会学術集会講演要旨集, 146回, 148 - 148, 日本語雄性化破綻メカニズムとエピジェネティクス
- (公社)日本獣医学会, 2008年03月, 日本獣医学会学術集会講演要旨集, 145回, 163 - 163, 日本語外因性エストロゲン様化学物質がLeydig細胞株TTE1のStARおよびP450scc遺伝子発現に与える影響とアセチル化ヒストンの変化
We aimed to elucidate the mechanism of action of estrogenic endocrine disruptors and the rescue of reproductive function, particularly the responsiveness of testes to eCG and/or activin A (ACT) after establishing reproductive disorders. Newborn male mice (n = 29) were randomly divided into an untreated group and three treatment groups that received diethylstilbestrol (DES; 100 mu g per animal) subcutaneously on Postnatal Day 3 to establish reproductive disorders and daily treatment with PBS (controls: DES + PBS), eCG (eCG group: DES + eCG), or eCG + ACT (eCG + ACT group: DES + eCG + ACT) at 6-8 wit of age prior to mating. After treatment, the controls showed diminished Leydig cells in the testes anti thin germ cell layers containing pyknotic germ cells and multinucleated cells. in the eCG and eCG + ACT groups, spermatids and Leydig cells increased markedly. The immunoexpression of androgen receptors in the eCG group and steroidogenic acute regulatory (STAR) protein in the eCG and eCG + ACT groups recovered to approximately the levels in the untreated group; plasma LH and testosterone levels also increased relative to those in the controls. in addition, the cell proliferation index, which is estimated from 5-bromo-2'-deoxyuridine immunoexpression in spermatogonia, increased significantly under eCG treatment, and even more with eCG + ACT. However, the numbers of germ and Leydig cells decreased at 12 wit of age. Thus, ACT and eCG help the! testes to recover from the dysfunction induced by neonatal DES administration. Furthermore, the permanent male reproductive disorder induced by neonatal exposure to estrogenic agents may be more likely to result from dysfunction of the hypothalamic-pituitary axis than from dysfunction of the lower reproductive organs.
SOC STUDY REPRODUCTION, 2008年01月, BIOLOGY OF REPRODUCTION, 78 (1), 59 - 67, 英語- 環境ホルモン学会(日本内分泌撹乱化学物質学会), 2007年12月, 環境ホルモン学会研究発表会要旨集, 10回, 178 - 178, 日本語外因性エストロゲン様化学物質がLeydig細胞株TTE1のステロイドホルモン産生系遺伝子発現に与える影響とヒストンアセチル化解析
- 日本先天異常学会, 2003年09月, Congenital anomalies, 43 (3), 245 - 245, 英語Effects of activin and PMSG treatment on reproductive dysfunction after neonatal exposure to endocrine disrupting chemicals in male mouse(Poster Session)(Japanese Teratology Society 43rd Annual Meeting)
- Aneuploidy of sex chromosomes in basal cell carcinoma: Its clonality and involvement in the development of carcinogenesis
Although basal cell carcinoma (BCC) is a major skin cancer, the mechanism of carcinogenesis with regard to cytogenetic abnormalities has not been fully investigated. In the present study, we carried out cytogenetic analyses of 18 patients (9 male and 9 female) with BCC. Aneuploidy was seen by Q-banding method in more than half of the cases and was mostly loss of the sex chromosome. We also performed FISH to the interphase nuclei of various tissues and short-term cultured BCC cells. The frequency of sex chromosomal aneuploidy was significantly higher in all samples from BCC patients (peripheral blood lymphocytes, non-lesional tissues, BCC tumor tissues and cultured BCC cells) than in age-matched normal controls. In addition, we analyzed clonality of BCC tissues using a human androgen receptor gene assay and found uniparental pattern of inactive X-chromosomes. This indicates that BCC cells were monoclonal in origin and the development of BCC might be correlated with sex chromosomal aneuploidy, which acquired accumulation of genetic mutations.
PROFESSOR D A SPANDIDOS, 2000年01月, INTERNATIONAL JOURNAL OF ONCOLOGY, 16 (1), 15 - 23, 英語 - 日本産科婦人科学会, 2000年, 日本産科婦人科學會雜誌, 52 (2), "511(S - 435)", 日本語P-416 産婦人科領域における性分化異常症例の表現型/遺伝子型の検討
- PVL 障害時期と発症との関連 ヒツジ胎仔における障害時期とPVL発症
急性脱血を負荷した6例中5例でのみ脳室周囲白質に限局する臨床的PVL(脳室周囲白質軟化)と同様な病変が発現し,対照群には如何なるCNS(中枢神経系)病変も認められなかった.生理学的パラメータの経時的変化の比較では,負荷直後の平均動脈圧以外には両群間で差異が認められなかったので,急性脱血による全身性低血圧はヒツジ未熟胎仔でPVL様病変を発現させることができると結論した.ヒツジ未熟胎仔でも人未熟児と同様に側脳室周囲の深部白質は髄質動脈の終末ならびに境界領域となっており,この部位がPVL様病変の好発部位と一致することが確認された
(一社)日本周産期・新生児医学会, 1999年12月, 日本新生児学会雑誌, 35 (4), 693 - 699, 日本語 - 金原出版(株), 1999年02月, 産婦人科の実際, 48 (2), 285 - 291, 日本語卵巣癌の好発家系とGenetic testing
- 日本産科婦人科学会, 1999年, 日本産科婦人科學會雜誌, 51, "S - 575", 日本語P-433 出生前診断における母体血清マーカー(AFP, hCG, uE3)検査の評価
- 日本産科婦人科学会, 1999年, 日本産科婦人科學會雜誌, 51, "S - 275", 日本語281 子宮筋腫に関する細胞遺伝学的研究
- (有)医学の世界社, 1998年12月, 産婦人科の世界, 50 (12), 971 - 983, 日本語【卵巣癌の臨床の新しい視点】 家族性卵巣癌の臨床像,遺伝子変異と遺伝子検査
- Society of Chromosome Research, 1998年, Chromosome science, 2 (3), 160 - 160, 英語Cytogenetic study on uterine leiomyoma(Abstracts of the 49th Annual Meeting of the Society of Chromosome Research) :
- Five cases of double cancer: breast and uterine cancer
We report 5 cases of double cancer followed by screening breast cancer. [1]A 64-year-old woman was diagnosed as uterine endometrial carcinoma stage Ib and breast cancer stage I. Histologically the breast tumor revealed solid-tubular (ST) and scirrhous carcinoma. [2]A 45-year-old woman was diagnosed as uterine cervical carcinoma in situ (CIS) and breast cancer stage II, The breast tumor was papillotubular (PT) carcinoma. [3]A 48-year-old woman was diagnosed as breast cancer stage II and CIS. The breast tumor revealed PT carcinoma. [4]A 70-year-old woman was diagnosed as uterine cervical adenocarcinoma stage Ib and breast cancer stage I. The breast tumor was diagnosed ST carcinoma. [5]A 55-year-old woman was diagnosed as uterine cervical CIS and breast cancer stage I. The breast tumor was diagnosed ST carcinoma., It is considered that the breast cancer screening is clinically efficient for the patient of gynecologic cancer.
MEDIMOND S R L, 1998年, XVI ASIAN AND OCEANIC CONGRESS OF OBSTETRICS AND GYNAECOLOGY, 83 - 85, 英語 - 北海道産科婦人科学会, 1997年03月, 北海道産科婦人科学会会誌, 41 (1), 47 - 48, 日本語Sclerosing Stromal Tumor of the Ovaryの2例
書籍等出版物
- 分担執筆, pp. 235-247, Springer, 2021年01月, 英語, ISBN: 9789811586880Adverse effects of pesticides on regional biodiversity and their mechanisms. In: Risks and Regulation of New Technologies
- その他, 学窓社, 2017年, 日本語第9章 リンパ器官（リンパ組織，胸腺，リンパ節担当）（獣医組織学 改訂第7版）
教科書・概説・概論
- その他, 学窓社, 2013年04月, 日本語獣医組織学 改訂第6版
教科書・概説・概論
- その他, アドスリー, 2011年10月, 日本語環境学入門
教科書・概説・概論
- 共著, ヘルス出版, 1999年04月, 日本語先天異常
教科書・概説・概論
- 共著, へるす出版, 1999年04月, 日本語性腺・性器の発生
教科書・概説・概論
- 共著, へるす出版, 1999年04月, 日本語性の分化・発育・成熟の異常
教科書・概説・概論
- 共著, Ares Serono Symposia, 1999年04月, 英語Familial ovarian cancer in Japanese women
学術書
- 共著, Monduzzi Editore, 1998年04月, 英語Five cases of double cancer: breast and uterine cancer, XVIth Asian and Oceanic Congress of Obstetrics and Gynaecology
学術書
- 共著, 医歯薬出版, 1997年04月, 日本語高齢出産，別冊医学のあゆみ−リプロダクティブ・ヘルス
一般書・啓蒙書
- 共著, 六法出版社, 1992年04月, 日本語Agingと妊娠・分娩．産婦人科診療
一般書・啓蒙書
講演・口頭発表等
- 第32回モロシヌス研究会, 2019年06月, 日本語, 千葉市, 国内会議C57BL/6マウスの亜系統差による生殖腺表現型の変化
口頭発表（一般）
- 第124回日本解剖学会総会全国学術集会, 2019年03月, 日本語, 新潟市, 国内会議Serial Block face走査型電子顕微鏡を用いたラット回腸粘膜における単核食細胞系列の細胞の三次元的観察
ポスター発表
- International Conference on PharmScience Research & Development 2019, 2019年03月, 英語, Paris, 国際会議Fabrication of a novel culture dish adapter with a small recess structure for flow control in a closed environment.
口頭発表（一般）
- Chemical Hazard Symposium in Hokkaido, 2019年03月, 英語, Sapporo, 国際会議Adverse effects on cognitive-emotional behavior and immune system function in experimental animals administered a NOAEL-dose of neonicotinoids.
[招待有り]
シンポジウム・ワークショップパネル（指名）
- ダイオキシン・環境ホルモン対策国民会議設立20周年記念議演会, 2019年01月, 日本語, 東京, 国内会議ここまで分かったニオニコチノイドの毒性
[招待有り]
口頭発表（招待・特別）
- 環境ホルモン学会第21回研究発表会, 2018年12月, 日本語, 東京都文京区, 国内会議無毒性量のネオニコチノイド系農薬曝露による行動影響の性差
ポスター発表
- 環境ホルモン学会第21回研究発表会, 2018年12月, 日本語, 東京都文京区, 国内会議母子間移行における浸透性農薬およびその代謝産物の定量的な解明
ポスター発表
- 環境ホルモン学会第21回研究発表会, 2018年12月, 日本語, 東京都文京区, 国内会議兵庫県における野生哺乳動物のネオニコチノイド系農薬への曝露実態調査
ポスター発表
- 関西実験動物研究会第140回研究会, 2018年12月, 日本語, 京都市, 国内会議性決定遺伝子発現前のマウス未分化XY性腺の精巣化を誘導する器官培養法
口頭発表（一般）
- 環境ホルモン学会第21回研究発表会, 2018年12月, 日本語, 東京都文京区, 国内会議ラットにおけるネオニコチノイド系農薬の亜急性曝露による免疫毒性の検証
ポスター発表
- 環境ホルモン学会第21回研究発表会, 2018年12月, 日本語, 東京都文京区, 国内会議ネオニコチノイド系農薬が老年動物に及ぼす影響
ポスター発表
- 第94回日本解剖学会近畿支部学術集会, 2018年11月, 日本語, 神戸市, 国内会議性決定前のマウス未分化 XY 性腺の精巣化を誘導する器官培養法の確立とその組織学的評価
口頭発表（一般）
- 第94回日本解剖学会近畿支部学術集会, 2018年11月, 日本語, 神戸市, 国内会議ラット回腸粘膜における好酸球系列の細胞の組織学的研究
口頭発表（一般）
- 第94回日本解剖学会近畿支部学術集会, 2018年11月, 日本語, 神戸市, 国内会議ラット回腸の上皮下線維芽細胞様細胞を制御し得る神経伝達物質の同定
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議生殖腺の表現型に及ぼすマウス亜系統差の影響に関する遺伝学的研究
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議浸透性農薬の行動影響の性差
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議浸透性農薬とその代謝産物の胎子移行量の解明
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ラット小腸粘膜におけるCD11b陽性細胞を中心とした各種免疫担当細胞の組織内配置に関する免疫組織化学的研究
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ラット呼吸器系粘膜におけるSP-AおよびSP-D分泌細胞の分布
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ラット回腸腸絨毛における上皮下および非上皮下線維芽細胞様細胞の三次元的解析
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ラット回腸の上皮直下における線維芽細胞様細胞への神経投射に関する免疫組織化学的研究
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ネオニコチノイド系農薬における哺乳動物への免疫毒性の検証
口頭発表（一般）
- 第161回日本獣医学会学術集会, 2018年09月, 日本語, つくば市, 国内会議ネオニコチノイド系農薬が老齢動物に及ぼす影響
口頭発表（一般）
- 環境ホルモン学会第20回研究発表会, 2017年12月, 日本語, 神戸市, 国内会議発達期におけるネオニコチノイド系農薬曝露により引き起こされたドーパミン神経系の攪乱を伴う多動
ポスター発表
- 環境ホルモン学会第20回研究発表会, 2017年12月, 日本語, 神戸市, 国内会議環境ストレスおよびネオニコチノイド系農薬ジノテフランの複合影響評価
ポスター発表
- 第136回関西実験動物研究会, 2017年12月, 日本語, 京都市, 国内会議マウスの亜系統差が生殖腺に及ぼす影響の遺伝学的研究
口頭発表（一般）
- 第93回日本解剖学会近畿支部学術集会, 2017年11月, 日本語, 大津市, 国内会議ラット大腸の上皮直下における間質細胞の部位差に関する免疫組織化学的研究
口頭発表（一般）
- 第93回日本解剖学会近畿支部学術集会, 2017年11月, 日本語, 大津市, 国内会議ラット回腸における間質細胞を介した細胞間ネットワーク解析へのSerial Block Face走査型電子顕微鏡の有用性
口頭発表（一般）
- 第93回日本解剖学会近畿支部学術集会, 2017年11月, 日本語, 大津市, 国内会議マウスのミュラー管退行におけるAMH作用様式
口頭発表（一般）
- 第160回日本獣医学会学術集会, 2017年09月, 日本語, 鹿児島市, 国内会議生理的条件下のラット呼吸器系粘膜における補体成分C3と補体受容体C3aRおよびCD11bの局在に関する免疫組織化学的研究
口頭発表（一般）
- 第160回日本獣医学会学術集会, 2017年09月, 日本語, 鹿児島市, 国内会議ラットパイエル板におけるlysozyme陽性細胞を介する生体防御機構に関する免疫組織化学的研究
口頭発表（一般）
- 第160回日本獣医学会学術集会, 2017年09月, 日本語, 鹿児島市, 国内会議Serial block face-走査型電子顕微鏡を用いたラット回腸腸陰窩周囲の間質細胞に関する3次元超微形態学的解析
口頭発表（一般）
- 第64回生態学会シンポジウム「ネオニコチノイド系農薬研究の最前線」, 2017年03月, 日本語, 新宿区, 国内会議ネオニコチノイド系農薬の鳥類・哺乳類における標的部位とその作用機序の探索
[招待有り]
口頭発表（招待・特別）
- 第122回日本解剖学会総会・全国学術集会, 2017年03月, 日本語, 長崎市, 国内会議ニワトリの発生過程に特徴的な生殖腺の左右差
[招待有り]
口頭発表（招待・特別）
- 第19回環境ホルモン学会研究発表会, 2016年12月, 日本語, つくば市, 国内会議胎子期および発達期におけるネオニコチノイド系農薬曝露がドーパミン神経系および行動に及ぼす影響
ポスター発表
- 第39回日本分子生物学会年会, 2016年12月, 日本語, 横浜市, 国内会議マウス胎子性腺における性分化関連遺伝子転写調節領域のDNAメチル化状態
ポスター発表
- 第19回環境ホルモン学会研究発表会, 2016年12月, 日本語, つくば市, 国内会議ネオニコチノイド系農薬曝露によりみられた神経行動学的異常および標的脳領域の探索
口頭発表（一般）
- 第19回環境ホルモン学会研究発表会, 2016年12月, 日本語, つくば市, 国内会議ネオニコチノイド系農薬ジノテフラン曝露はうつ様行動を引き起こすか？
口頭発表（一般）
- 第19回環境ホルモン学会研究発表会, 2016年12月, 日本語, つくば市, 国内会議ネオニコチノイド系農薬クロチアニジンは胎子型と成獣型，2種類のライディッヒ細胞にそれぞれ異なる影響を及ぼす
口頭発表（一般）
- 第92回日本解剖学会近畿支部学術集会, 2016年11月, 日本語, 東大阪市, 国内会議ラット呼吸器系粘膜を覆う粘液層における β-defensin 1 および β-defensin 2 の存在および分泌源の同定
口頭発表（一般）
- 第92回日本解剖学会近畿支部学術集会, 2016年11月, 日本語, 東大阪市, 国内会議ラット回腸粘膜における各種間質細胞の局在
口頭発表（一般）
- 第92回日本解剖学会近畿支部学術集会, 2016年11月, 日本語, 東大阪市, 国内会議ラットパイエル板における濾胞被蓋上皮のアポトーシスとRANK/RANKLの発現との関係
口頭発表（一般）
- 第92回日本解剖学会近畿支部学術集会, 2016年11月, 日本語, 東大阪市, 国内会議マウス真性半陰陽個体のミュラー管退行とAMHの作用様式
口頭発表（一般）
- 第92回日本解剖学会近畿支部学術集会, 2016年11月, 日本語, 東大阪市, 国内会議ニワトリ雌右性腺退縮メカニズムの解明
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議生理学的条件下のラットパイエル板におけるRANK/RANKLの発現および常在細菌の増殖による影響
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議ラット呼吸器におけるβ-defensin 1およびβ-defensin 2を介した分泌型の生体防御機構に関する免疫組織化学的研究
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議ラット回腸におけるtelocyteの免疫組織化学的同定
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議マウス真性半陰陽個体におけるAMHの作用様式に関する組織学的解析
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議ネオニコチノイド系農薬曝露とうつ発症との関連性の検証
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議ネオニコチノイド系農薬ジノテフランによるドーパミン神経系の攪乱および多動性の誘発
口頭発表（一般）
- 第159回日本獣医学会学術集会, 2016年09月, 日本語, 藤沢市, 国内会議ネオニコチノイド系農薬クロチアニジン急性投与が及ぼす行動学的影響とその作用点
口頭発表（一般）
- 第56回 先天異常学会学術集会, 2016年07月, 日本語, 姫路市, 国内会議発達期におけるネオニコチノイド系農薬曝露により多動が引き起こされる
ポスター発表
- 第56回 先天異常学会学術集会, 2016年07月, 日本語, 姫路市, 国内会議発達期におけるネオニコチノイド系農薬ジノテフラン曝露とうつ病発症との関係
ポスター発表
- 第56回 先天異常学会学術集会, 2016年07月, 日本語, 姫路市, 国内会議胎子期から成獣期にかけてのネオニコチノイド系農薬クロチアニジン曝露が及ぼす行動学的影響
ポスター発表
- 第30回環境ホルモン学会講演会「ネオニコチノイド系農薬をめぐって」, 2016年06月, 日本語, 文京区, 国内会議ネオニコチノイド系農薬の標的とその作用メカニズム
[招待有り]
口頭発表（招待・特別）
- 第43回日本毒性学会学術年会, 2016年06月, 日本語, 名古屋市, 国内会議ネオニコチノイド系農薬クロチアニジン単回投与によりみられた異常啼鳴および神経行動学的影響
ポスター発表
- Post-Neonics –What Next ? Symposium, 2016年06月, 英語, 新宿区, 国際会議Target and mechanism of neonicotinoid pesticides in birds and mammals.
[招待有り]
口頭発表（招待・特別）
- 第121回日本解剖学会総会・全国学術集会, 2016年03月, 日本語, 郡山市, 国内会議ニワトリ胚の性腺発生における左右差
口頭発表（一般）
- 環境ホルモン学会第18回研究発表会, 2015年12月, 日本語, 下野市, 国内会議環境ストレス下においてネオニコチノイド系農薬クロチアニジンが与える神経行動学的影響
ポスター発表
- 環境ホルモン学会第18回研究発表会, 2015年12月, 日本語, 下野市, 国内会議ネオニコチノイド系農薬クロチアニジンの妊娠期及び授乳期曝露がマウス産子の雄性生殖器に与える影響
ポスター発表
- 第38回日本分子生物学会年会・第88回日本生化学会大会合同大会, 2015年12月, 日本語, 神戸市, 国内会議ニワトリ胚の左精巣に特異的なセルトリ細胞の起源
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議生理学的条件下におけるラットパイエル板濾胞被蓋上皮におけるToll-like receptor-4の発現とM細胞への分化との関係について
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議性逆転系統（B6-XYPOS）マウスにおける性腺分化と遺伝的背景
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議ラット呼吸器系粘膜を覆う液相に含まれるlysozymeおよびsPLA2の分泌源の特定
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議ラット回腸における間質細胞telocyteに関する超微形態学的研究
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議ニワトリ胚の左性腺に特異的な細胞運命の追跡
口頭発表（一般）
- 第91回日本解剖学会・近畿支部学術集会, 2015年11月, 日本語, 京都市, 国内会議ニワトリの性腺形態における左右差形成メカニズムの解明
口頭発表（一般）
- 第158回日本獣医学会, 2015年09月, 日本語, 十和田市, 国内会議環境ストレス下において浸透性農薬クロチアニジンが行動及び生殖機能に与える影響
口頭発表（一般）
- 第158回日本獣医学会, 2015年09月, 日本語, 十和田市, 国内会議ラット小腸のPaneth細胞に連絡する粘膜固有層細胞の超微形態
口頭発表（一般）
- 第158回日本獣医学会, 2015年09月, 日本語, 十和田市, 国内会議ラット呼吸器系におけるlysozymeおよびsPLA2の分泌上皮細胞に関する免疫組織化学的研究
口頭発表（一般）
- 第158回日本獣医学会, 2015年09月, 日本語, 十和田市, 国内会議ラットパイエル板濾胞被蓋上皮におけるToll-like receptor-2, -4および-9の発現に対する常在細菌増殖の影響
口頭発表（一般）
- 日本家禽学会2015年度秋季大会, 2015年09月, 日本語, 江別市, 国内会議ニワトリ胚における左精巣特異的な発生メカニズム
口頭発表（一般）
- 日本家禽学会2015年度秋季大会, 2015年09月, 日本語, 江別市, 国内会議ニワトリ性腺の左右差形成メカニズムに関する網羅的遺伝子発現解析
口頭発表（一般）
- 17th International Congress of Mucosal Immunology, 2015年07月, 英語, Berlin, Germany, 国際会議The short-term effect of peptidoglycan injected into the intestinal lumen against host defense responses.
ポスター発表
- 第62回日本実験動物学会, 2015年05月, 日本語, 京都市, 国内会議C57BL/6マウスの不可思議さ：その遺伝的背景に潜むSry-HMG box & Q-rich領域と性決定
ポスター発表
- 第60回日本生殖医学会学術集会, 2015年04月, 日本語, 横浜市, 国内会議性決定メカニズムの普遍性と多様性
[招待有り]
口頭発表（招待・特別）
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Sex-related genes' asymmetric expression in early mouse gonads
ポスター発表
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Selection of stable reference genes for quantitative RT-PCR analyses in developing mouse gonads
ポスター発表
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Reproductive and behavioral effects of clothianidin in male mice in a chronically stressed condition
ポスター発表
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Light and Electron Microscopical Study on the Particulate Bodies Found in Epithelium of Rat Airway.
ポスター発表
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Left-right asymmetry of testicular formation in the chicken embryo
ポスター発表
- 第120回 日本解剖学会総会･全国学術集会, 2015年03月, 日本語, 神戸市, 国内会議Differential Expression of Toll-like Receptor-2,-4 and -9 in the Various Type of Epithelia Associated with Rat Peyer’s Patches
ポスター発表
- 神戸大学研究基盤センター若手フロンティア研究会2014, 2014年12月, 日本語, 神戸市, 国内会議消化管付属リンパ組織、パイエル板の上皮におけるToll-like receptor-2、-4および-9の発現に関する免疫組織化学的研究
ポスター発表
- 環境ホルモン学会第17回研究発表会, 2014年12月, 日本語, 京都市, 国内会議環境ストレス下動物の生殖機能抑制機序に関する形態機能学的研究
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議発生中のマウス胎子生殖腺における内部標準遺伝子の検討
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議通常飼育下のラットの呼吸器系に吸入された粒子状物の粘膜上皮への取り込みに関する組織学的研究
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議ラット呼吸器系粘膜上皮におけるToll-like receptor -1-9の局在に関する網羅的解析
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議ラットパイエル板濾胞被蓋上皮および濾胞付属腸絨毛の上皮におけるToll-like receptor-2，-4および-9の発現特性
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議マウス胎子生殖腺における性分化関連遺伝子発現の左右差に関する分子形態学的研究
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議ネオニコチノイド系農薬クロチアニジンの低用量曝露とストレスの複合影響
口頭発表（一般）
- 日本家禽学会2014年度秋季大会, 2014年09月, 日本語, 鹿児島市, 国内会議ニワトリ胚における性腺の形成機構とその左右差
口頭発表（一般）
- 第157回日本獣医学会学術集会, 2014年09月, 日本語, 札幌市, 国内会議ニワトリ胚における性腺の形成メカニズムと左右差
口頭発表（一般）
- 日本薬学会第134年会, 2014年03月, 日本語, 国内会議ネオニコチノイド系農薬による生殖機能障害とそのメカニズム
[招待有り]
シンポジウム・ワークショップパネル（指名）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議性腺分化とその破綻メカニズムに関する分子形態学的研究
口頭発表（一般）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ラット上行結腸の集合リンパ小節近傍の腸陰窩に出現するパネート細胞様外分泌細胞に関する超微形態学的および組織化学的研究
口頭発表（一般）
- 第89回日本解剖学会近畿支部学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ラット上行結腸における酸好性顆粒を有する腸陰窩上皮細胞に関する形態学的研究
口頭発表（一般）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ラット空腸の腸絨毛における小型乳ビ球の門脈血への移行の仕組みに関する免疫組織化学的研究
口頭発表（一般）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ラットの消化器系における補体成分C3とC4の産生部位に関する免疫組織化学的研究
口頭発表（一般）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ネオニコチノイド系農薬クロチアニジンが鳥類の繁殖能力に及ぼす影響とその作用機序
口頭発表（一般）
- 第156回日本獣医学会学術集会, 2013年09月, 日本語, 岐阜市, 国内会議ウミガメの総排泄腔傍嚢の形態機能学的特徴
口頭発表（一般）
- 第19回日本野生動物医学会大会, 2013年08月, 日本語, 京都市, 国内会議環境ストレス下動物の生殖機能抑制機序に関する形態機能学的研究
ポスター発表
- 第19回日本野生動物医学会大会, 2013年08月, 日本語, 京都市, 国内会議カメ類における「膀胱」の形態機能学的定義
ポスター発表
- 7th Copenhagen Workshops on Endocrine Disrupters, 2013年05月, 英語, COPENHAGEN, DENMARK, 国際会議Down regulation of cytochrome p450scc as an initial adverse effect of adult exposure to diethylstilbestrol on testicular steroidogenesis.
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議生後発達期におけるTCDDの神経発達障害の分子基盤
口頭発表（一般）
- 第15回環境ホルモン学会研究発表会, 2012年12月, 日本語, 東京，文京区, 国内会議生後発達期におけるTCDDによる神経発達障害の分子基盤
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議性腺分化と性分化カスケードに関する分子形態学的研究
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議ラット上行結腸腸陰窩における常在細菌の増殖に対する生体防御に関する研究
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議ラット十二指腸粘膜上皮におけるMyeloid differentiation factor-2とToll-like receptor-2の発現に関する免疫組織化学的研究
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議ラット空腸腸絨毛の毛細血管内皮における乳ビ球に対する受容体に関する免疫組織化学的研究
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議ラット肝臓における補体C3およびC4の産生に関する免疫組織化学的研究
口頭発表（一般）
- 第15回環境ホルモン学会研究発表会, 2012年12月, 日本語, 東京，文京区, 国内会議マウス胎子海馬の神経発達に及ぼすTCDD毒性の基礎的評価
口頭発表（一般）
- 第15回環境ホルモン学会研究発表会, 2012年12月, 日本語, 東京，文京区, 国内会議ネオニコチノイド系農薬が鳥類の繁殖能力に及ぼす影響の分子形態学的研究
口頭発表（一般）
- 第88回日本解剖学会近畿支部学術集会, 2012年12月, 日本語, 神戸市, 国内会議ネオニコチノイド系農薬が鳥類の繁殖能力に及ぼす影響の基礎的評価
口頭発表（一般）
- 第51回日本爬虫類両棲類学会, 2012年11月, 日本語, 豊田市, 国内会議カメ類における「膀胱」の形態機能学的研究
口頭発表（一般）
- 新学術領域研究「性差構築の分子基盤」若手研究会, 2012年10月, 日本語, 和歌山市, 国内会議遺伝子改変性転換マウス（Ypos）の真性半陰陽個体生殖腺における発生形態学的研究
口頭発表（一般）
- 新学術領域研究「性差構築の分子基盤」若手研究会, 2012年10月, 日本語, 和歌山市, 国内会議ニワトリ胚における性分化関連遺伝子の時空間的発現に関する分子形態学的研究
口頭発表（一般）
- 新学術領域研究「性差構築の分子基盤」若手研究会, 2012年10月, 日本語, 和歌山市, 国内会議ターナーモザイク患者の性腺分化とその破綻機構の分子基盤の解明
口頭発表（一般）
- 新学術領域研究「性差構築の分子基盤」若手研究会, 2012年10月, 日本語, 和歌山市, 国内会議Y染色体腕間逆位を有するXY女性におけるSRY遺伝⼦子のエピジェネティック異常
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議ラット小腸腸陰窩上皮細胞の細胞死に関する超微形態学的および免疫組織化学的研究
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議ラットの上行結腸における常在細菌の定着とその制御に関する定量組織学的研究
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議マウス胎子の海馬におけるTCDDの神経発達毒性
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議ネオニコチノイド系殺虫剤が鳥類の繁殖能力に及ぼす影響の基礎的評価
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議ニワトリ胚における性分化関連遺伝子の時空間的発現に関する分子形態学的研究
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議ターナーモザイク患者の性腺分化とその破綻機構の分子基盤の解明
口頭発表（一般）
- 第154回日本獣医学会学術集会, 2012年09月, 日本語, 盛岡市, 国内会議カメ類における「膀胱」の形態機能学的解析
口頭発表（一般）
- 第87回日本解剖学会近畿支部学術集会, 2011年12月, 日本語, 西宮市, 国内会議胎子・授乳期TCDD曝露による発達中のセロトニン神経系への影響とその作用機序
口頭発表（一般）
- 第87回日本解剖学会近畿支部学術集会, 2011年12月, 日本語, 西宮市, 国内会議胎子・授乳期TCDD曝露と胸腺微小環境の変化
口頭発表（一般）
- 第87回日本解剖学会近畿支部学術集会, 2011年12月, 日本語, 西宮市, 国内会議ラットの大腸粘膜上皮におけるToll-like receptorの発現と分泌
口頭発表（一般）
- 第87回日本解剖学会近畿支部学術集会, 2011年12月, 日本語, 西宮市, 国内会議ラットの呼吸器粘膜におけるToll-like receptorの発現と分泌
口頭発表（一般）
- 第87回日本解剖学会近畿支部学術集会, 2011年12月, 日本語, 西宮市, 国内会議Acceleration of Epithelial Apoptosis through the Secondary Activation of Mitochondria-Dependent Pathway in the Indigenous Bacteria Settlement Sites of Rat Tongue
口頭発表（一般）
- 2011 Mitochondria, Apoptosis and Cancer Conference, 2011年10月, 英語, Singapore, 国際会議Mitochondria-Dependent Pathway as a Final Apoptosis Process Secondarily Induced in Rat Esophageal Epithelium under a Physiological Condition
ポスター発表
- 第17回日本野生動物医学会大会, 2011年09月, 日本語, 東京, 国内会議分娩時におけるカリフォルニアアシカ(Zalophus californianus)の卵巣および胎盤の性ステロイドホルモンの局在
ポスター発表
- 第152回日本獣医学会学術集会, 2011年09月, 日本語, 堺市, 国内会議胎子・授乳期TCDD曝露による発達中のセロトニン神経系への影響に関する分子形態学的研究
口頭発表（一般）
- 第152回日本獣医学会学術集会, 2011年09月, 日本語, 堺市, 国内会議胎子・授乳期TCDD曝露がマウス胸腺微小環境に及ぼす影響に関する分子形態学的研究
口頭発表（一般）
- 第152回日本獣医学会学術集会, 2011年09月, 日本語, 堺市, 国内会議ラット大腸におけるToll-like receptorの局在に関する免疫組織化学的研究
口頭発表（一般）
- 第152回日本獣医学会学術集会, 2011年09月, 日本語, 堺市, 国内会議ラット呼吸器系上皮におけるToll-like receptorの局在に関する免疫組織化学的研究
口頭発表（一般）
- 第17回日本野生動物医学会学術集会, 2011年09月, 日本語, 府中市, 国内会議バンドウイルカにおける精液一般性状および液状保存
ポスター発表
- 第17回日本野生動物医学会学術集会, 2011年09月, 日本語, 府中市, 国内会議カリフォルニアアシカ（Zalophus californianus）の分娩時における卵巣および胎盤の性ステロイドホルモンの局在
ポスター発表
- 第152回日本獣医学会学術集会, 2011年09月, 日本語, 堺市, 国内会議Acceleration of epithelial apoptosis in anterior epithelial column of filiform papilla in rat tongue by indigenous bacterial settlement.
口頭発表（一般）
- 第19回希少動物人工繁殖研究会議, 2011年07月, 日本語, 姫路, 国内会議雄シマヘビの生殖周期に関する一考察
口頭発表（一般）
- 環境ホルモン学会（日本内分泌攪乱化学物質学会）第13回研究発表会, 2010年12月, 日本語, 東京，文京区, 国内会議中脳発達における環境中化学物質のAhR介在性分化制御機構の解明
ポスター発表
- 第86回日本解剖学会近畿支部学術集会, 2010年11月, 日本語, 泉佐野市, 国内会議逆転症系統(Ypos)由来半陰陽マウスの生殖腺における発生形態学的研究
口頭発表（一般）
- 第86回日本解剖学会近畿支部学術集会, 2010年11月, 日本語, 泉佐野市, 国内会議ラット消化管付属分泌腺からのToll-like receptorの分泌に関する免疫組織化学的研究
口頭発表（一般）
- 第86回日本解剖学会近畿支部学術集会, 2010年11月, 日本語, 泉佐野市, 国内会議Changes in apoptosis process of rat esophageal epithelium with hyperproliferation of the indigenous bacteria under a physiological condition
口頭発表（一般）
- 第150回日本獣医学会学術集会, 2010年09月, 日本語, 帯広市, 国内会議遺伝子改変性転換マウス(Ypos)の半陰陽個体生殖腺における発生形態学的研究
口頭発表（一般）
- 第150回日本獣医学会学術集会, 2010年09月, 日本語, 帯広市, 国内会議ラット消化管附属分泌腺からのToll-like receptorの分泌に関する免疫組織化学的研究
口頭発表（一般）
- 第150回日本獣医学会学術集会, 2010年09月, 日本語, 帯広市, 国内会議Immunohistochemical study on the apoptotic process in normal esophageal epithelium under a physiological condition
口頭発表（一般）
- 第50回日本先天異常学会学術集会, 2010年07月, 日本語, 淡路市, 国内会議遺伝子改変-性逆転マウスYposの生殖腺における発生形態学的研究
ポスター発表
- 第50回日本先天異常学会学術集会, 2010年07月, 日本語, 淡路市, 国内会議Sry 導入XXマウスにおける雄性生殖腺の形態学的解析
ポスター発表
- 11th International Symposium on Spermatology, 2010年06月, 英語, 宜野湾市, 国際会議Paternal exposure to 2,3,7,8-tetrachloro- dibenzo-p-dioxin (TCDD) affects the sex ratio of offspring at birth by change the sex ratio of fertilized egg
ポスター発表
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議ラット腸管の常在細菌と寒天平板培養細菌の超微形態学的比較検討
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議ラット腸管の上皮細胞における経時的形態学的変化とアポトーシスの進行に関する免疫組織化学的研究
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議ラット小腸腸絨毛におけるToll-like Receptorの発現
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議ラット小腸および大腸の粘膜リンパ組織における上皮細胞の動態の特性
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議マウス胎子性ライディッヒ細胞に関する発生形態学的研究
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議アカネズミのAhR遺伝子多型におけるダイオキシン曝露の影響
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議TCDDによる次世代出生前性比低下と感受性との関係
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議Sry導入XXマウスにおける雄性生殖腺の生後変化
口頭発表（一般）
- 第85回日本解剖学会近畿支部学術集会, 2009年11月, 日本語, 橿原市, 国内会議Apoptosis Process in Epidermal Epithelial Cells of Rat under a physiological condition
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議投与雄マウスにおける感受性と次世代出生前性比との関係
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議成獣雄マウスにおける性決定遺伝子Sry発現の分子学的解析
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議ラット腸管の濾胞被蓋上皮における上皮細胞の動態に関する計量免疫組織化学的研究
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議ラット小腸の粘膜上皮におけるToll-like receptorの発現に関する免疫組織化学的研究
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議ラットの腸管内腔に存在する常在細菌の超微形態学的観察
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議ラットの腸管上皮におけるアポトーシス発現に関する超微形態学的研究
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議マウスにおける胎子性ライディッヒ細胞の局在に関する発生形態学的研究
口頭発表（一般）
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議アカネズミのAhR遺伝子多型とダイオキシン曝露の影響に関する分子形態学的評価
口頭発表（一般）
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Ultrastructural Study on the Indigenous Bacteria in the Lumen of Rat Intestine.
ポスター発表
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Ultrastructural and immunohistochemical study on the apoptotic process of intestinal epithelial cells in rats.
ポスター発表
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Ultrastructural and histoplanimetrical demonstration of direct transportation of chylomicrons via subepithelial blood capillaries in rat intestinal villi.
ポスター発表
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議Sry導入XXマウスにおける雄性生殖腺の形態学的解析
口頭発表（一般）
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Scanning electron microscopic study on the persorption mechanism of large antigenic particulates from small intestinal lumen in rats.
ポスター発表
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Localization and developmental fate of fetal Leydig cells in mouse testis
ポスター発表
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Immunohistochemical study on the Toll-like receptor expression in villous columnar epithelial cells of rat small intestine
ポスター発表
- 第148回日本獣医学会学術集会, 2009年09月, 日本語, 鳥取市, 国内会議Immunohistochemical study on the apoptosis process in rat epidermis
口頭発表（一般）
- The 6th International Symposium on Electron Microscopy in Medicine and Biology, 2009年09月, 英語, 神戸市, 国際会議Activin A and eCG recover reproductive dysfunction induced by neonatal exposure to an estrogenic endocrine disruptor in adult male mice
ポスター発表
- PT 学会, 2006年02月, 日本語, 不明, 群馬, 国内会議三次元加速度変量の可視化による変形性股関節症の定量的歩様解析
口頭発表（一般）
- 第81 回日本解剖学会近畿支部学術集会, 2005年11月, 日本語, 不明, 京都, 国内会議常在細菌の定着に対するパイエル板濾胞被蓋上皮の応答について
口頭発表（一般）
- 第81回日本解剖学会近畿支部学術集会, 2005年11月, 日本語, 不明, 京都, 国内会議小腸腸絨毛からの粒子状物のパーソープションの仕組みについて
口頭発表（一般）
- 第8 回環境ホルモン学会学術集会講演要旨集、 259, 2005年, 日本語, 不明, 名古屋, 国内会議胎子期・新生子期DES 曝露における成熟雄マウスの用量－影響評価
口頭発表（一般）
- 第139 回日本獣医学会学術集会講演要旨集、182, 2005年, 日本語, 不明, 埼玉, 国内会議人工的に蹄バランスを変えた乳牛の歩様解析
口頭発表（一般）
- 第140 回日本獣医学会学術集会講演要旨集,58, 2005年, 日本語, 不明, 鹿児島, 国内会議ラット小腸絨毛円柱上皮細胞における粒子状物の取り込みへのFc レセプターの関与
口頭発表（一般）
- 第140 回日本獣医学会学術集会講演要旨集,58, 2005年, 日本語, 不明, 鹿児島, 国内会議ラットパイエル板濾胞被蓋上皮への常在細菌の定着に対する防御応答に関する超微形態学的研究，
口頭発表（一般）
- 第140 回日本獣医学会学術集会講演要旨集， 56, 2005年, 日本語, 不明, 鹿児島, 国内会議ブタのアポクリン汗腺における離出分泌へのアポトーシスの関与および細胞動態
口頭発表（一般）
- 第104 回日本畜産学会学術集会, 2005年, 日本語, 不明, 東京, 国内会議3次元加速度センサーによる乳牛歩様解析
口頭発表（一般）
- 4th World Confederation for Physical Therapy-Asia Western Pacific Region & 9th Asia Confederation for Physical Therapy Congress 2005、Proceedings,218-219, 2005年, 日本語, 不明, Korea, 国内会議:Interrelationship between acceleration and walking cycle of human gait obtained with a simultaneously recording
口頭発表（一般）
- 第14回関西生殖発生毒性フォーラム基礎教育講座, 2004年, 日本語, 未記入, 未記入, 国内会議哺乳類の性分化とその異常
口頭発表（一般）
- 第138回日本獣医学会学術集会, 2004年, 日本語, 日本獣医学会, 北海道大学, 国内会議雄ラットのサージ状GnRH分泌発生制御機構における神経活動の経時変化
口頭発表（一般）
- 平成15年度科学研究費補助金（特定領域研究（１））「内分泌攪乱物質の環境リスク」研究成果報告会, 2004年, 日本語, 未記入, 未記入, 国内会議低濃度複合汚染による内分泌攪乱化学物質のマウス雄脳内ステロイドホルモンレセプター遺伝子の発現変化ならびに生殖腺への影響
口頭発表（一般）
- 第138回日本獣医学会学術集会, 2004年, 日本語, 日本獣医学会, 北海道大学, 国内会議低濃度複合汚染による内分泌攪乱化学物質のマウス雄脳内ステロイドホルモンレセプター遺伝子の発現変化ならびに生殖腺への影響
口頭発表（一般）
- 第12回生命科学実験センター学術セミナー, 2004年, 日本語, 未記入, 富山医科薬科大学, 国内会議生物の雄・雌が決まる仕組みはどこまで解明されたか
口頭発表（一般）
- 兵庫医科大学大学院総合セミナー, 2004年, 日本語, 未記入, 兵庫医科大学, 国内会議性の決定・分化機構と性分化異常症の分子細胞遺伝学
口頭発表（一般）
- 第138回日本獣医学会学術集会, 2004年, 日本語, 日本獣医学会, 北海道大学, 国内会議ラット小腸パイエル板の濾胞被蓋上皮におけるM 細胞の分化に関する超微形態学的研究
口頭発表（一般）
- 第80回日本解剖学会近畿支部学術集会, 2004年, 日本語, 日本解剖学会近畿支部, 関西医科大学, 国内会議ラット小腸パイエル板におけるM細胞への分化およびM細胞から微絨毛円柱上皮細胞への再分化に関する超微形態学的メカニズム
口頭発表（一般）
- 第138回日本獣医学会学術集会, 2004年, 日本語, 日本獣医学会, 北海道大学, 国内会議ラット小腸の粘膜上皮における糖の発現と常在細菌の定着との関係
口頭発表（一般）
- 第80回日本解剖学会近畿支部学術集会, 2004年, 日本語, 日本解剖学会近畿支部, 関西医科大学, 国内会議ラット小腸の腸絨毛およびパイエル板上皮への常在最近の定着に関する組織学的研究
口頭発表（一般）
- 第31回日本神経内分泌学会学術集会, 2004年, 日本語, 日本神経内分泌学会, 弘前, 国内会議ラットのGnRHサージ発生制御におけるGnRHニューロン、視索前野前腹側脳室周囲核および視交叉上核のFos発現の経時変化と性差
口頭発表（一般）
- 平成15年度科学研究費補助金（特定領域研究（１））「内分泌攪乱物質の環境リスク」研究成果報告会, 2004年, 日本語, 未記入, 未記入, 国内会議マウス精巣由来培養細胞に対する内分泌攪乱物質の複合作用
口頭発表（一般）
- 第27回日本神経科学大会および第47回日本神経化学学会大会合同大会学術集会, 2004年, 日本語, 日本神経科学学会, 大阪国際会議場, 国内会議GnRHサージ発生機構におけるfos発現の経時的変化とGnRH分泌能の左右差
口頭発表（一般）
- 7th International Symposium on Environmental Endocrine Disrupters 2004, 2004年, 英語, 未記入, Nagoya Congress Center, 国内会議Effects of BPA, DEHP and TCDD with low dose and complex exposure to the brain and reproductive organ in male mouse.
口頭発表（一般）
- 16th International Congress of the IFAA., 2004年, 英語, 未記入, Kyoto International Conference Hall, 国内会議Effects of activin and PMSG treatment on reproductive disrupting chemicals in male mouse.
口頭発表（一般）
- 第7回環境ホルモン学会研究発表会, 2004年, 日本語, 環境ホルモン学会, 未記入, 国内会議BPA, DEHPおよびTCDDの低濃度複合曝露における雄マウス脳および生殖腺への影響
口頭発表（一般）
共同研究・競争的資金等の研究課題
- 日本学術振興会, 科学研究費助成事業 挑戦的研究(開拓), 挑戦的研究(開拓), 北海道大学, 2022年06月 - 2025年03月, 研究分担者毒性試験の高感度化・効率化を目指したMITAシステムの構築と実践
- 日本学術振興会, 科学研究費助成事業部 基盤(B), 基盤研究(B), 神戸大学, 2022年04月 - 2025年03月, 研究代表者発達神経毒性の継世代影響メカニズムの解明-農薬の発生プログラム・ エピゲノム毒性
- 日本学術振興会, 科学研究費助成事業 挑戦的研究(萌芽), 挑戦的研究(萌芽), 神戸大学, 2021年07月 - 2024年03月継世代影響から捉える農薬の発達神経毒性作用と神経前駆細胞・グリアの局所活動
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 神戸大学, 2019年04月 - 2022年03月殺虫剤の「生物学的モニタリングに基づく適応的リスク評価法」の開発と実践
農薬評価書における無毒性量（NOAEL: 雌65.1 mg/kg/day）を参考に，クロチアニジン（CLO）を65 mg/kg/dayの濃度で，母獣マウス（F0世代）に妊娠1日目から離乳（3週齢）まで自由摂取させた．実験①：雄産子に対し幼年期（3週齢）および成年期（10週齢）に行動試験を行い，自発運動量および不安様行動を評価した．試験後に脳を採取し，免疫組織化学および網羅的遺伝子発現解析に供した．実験②：対照群または投与群同士であるF1の雌雄を交配させてF2を作製し，同様にF2の雌雄を交配させてF3（第四世代）を作製した．それぞれ生後3週齢および10週齢に卵巣および血液を採取した．卵巣を一般組織学および抗酸化酵素マーカーによる免疫組織化学ならびに網羅的遺伝子発現解析に，血液をホルモン定量解析に供し，継世代影響ならびにそのAOP（有害発現経路）を検証した． 結果①：無毒性量のCLOの胎子・授乳期曝露は，神経回路形成期の海馬歯状回における神経発達を阻害し，曝露直後の幼年期では，とくにカルシウムシグナリングを低下させ，不安様行動を惹起した．その後曝露が無いにもかかわらず，成年期では，オリゴデンドロサイトおよび脳由来神経栄養因子（BDNF）関連遺伝子群の発現が攪乱され，自発運動量が増加した．結果②：継世代影響では，エストロゲン関連経路の活性化阻害が認められ，「食殺および育子放棄」の頻度が世代を経る毎に増加（F1: 3/9例，F2: 3/6例）した．以上より，CLOに対する神経毒性作用は3週齢と10週齢とで異なること，ならびにCLOの胎子・授乳期曝露が雌マウスにおいて継世代影響を及ぼすことが明らかになった．現行の「無毒性量」は，発達神経毒性的には無毒性ではなく，酸化ストレス→遺伝子発現変化→卵巣（含 形態およびホルモン発現）変化→母獣の母性行動異常，となるAOPを初めて明らかにした．
- 日本学術振興会, 科学研究費助成事業 基盤研究(A), 基盤研究(A), 北海道大学, 2018年04月 - 2022年03月mlAOPコンセプトによる化学物質感受性多様性評価と殺虫剤中毒解明に向けた応用
本年度は昨年度に続き、研究実施計画通り以下の項目について実施した。 ・mlAOPの基礎データ取得に向けた、げっ歯類およびゼブラフィッシュを用いた各種行動試験の実施：個体レベルでの評価として、マウスに加え、ゼブラフィッシュにおける新たな神経毒性評価系を確立する事が出来た。本評価系により、マウスに引き続きゼブラフィッシュにおいても無毒性量(NOAEL)よりも低い曝露量で神経毒性様症状を検出できることが確認できた。 ・組織レベルにおける評価のため、昨年度に引き続き高分解能質量顕微鏡による神経伝達物質のイメージング解析を実施した。また同時にトリプル四重極型LC/MSを用いた超高感度分析系の確立にも成功し、脳の各部位におけるニューロトランスミッターの定量分析系を確立する事が出来た。 ・細胞・分子レベルでの評価のため、昨年度に引き続き、SH-SY5Y細胞をモデル神経細胞として用い、細胞増殖パラメーターなどの各種項目についてデータを取得した。 ・ヒト尿試料を用いたバイオモニタリング研究では、Thiamethoxamに職業的に曝露されている患者に着目したモニタリング研究を実施した。患者に防護具の適切な着用を指示し、尿・血中濃度の低減に伴う、頭痛や微熱などの臨床症状の改善について経時モニタリングを実施する事が出来、Thiamethoxamの臨床症状と曝露濃度との因果関係について報告する事が出来た。
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 富山大学, 2017年04月 - 2020年03月
低出力パルス超音波 (LIPUS) の初期細胞応答を明らかにするために，骨髄間質細胞(BMSCs) の最初期遺伝子 (IEGs) の発現に対するLIPUSの効果を検討した．マウス由来のST2 BMSCsに25 mW/cm2の強度で20分間LIPUSを照射した．LIPUSの1回照射は，有意かつ一過性にFosとEgr1を含むIEGsの発現を誘導した．また，ERK2のリン酸化レベルを有意に上昇させた．MAPKK/ERK経路の阻害剤U0126は，LIPUSで誘導されたFosとEgr1の発現を有意に阻害した．得られた成績は，BMSCsにおけるLIPUSの作用メカニズムを理解するための分子基盤になる．
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 2017年04月 - 2020年03月
Y染色体上に存在する性決定遺伝子Sryの修飾状況並びに性分化関連因子の発現状況について解析を行い，①汎用されるマウスの亜系統差によりSry発現が異なること，②健常雄個体のSry発現にも時空間的な相違や左右差がある事を明らかとした．さらに，③Sryの発現検証に必要な器官培養系および④内部標準遺伝子を確立するとともに，⑤AMHが精巣から中腎方向に組織内を浸潤する様式でミュラー管を退行させることを明らかとした．
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 2016年04月 - 2019年03月
性差の確立は，SRYをマスター遺伝子とした「遺伝的制御」によって未分化性腺の雄(精巣)あるいは雌(卵巣)への運命が決まることにより始まる．その雌雄表現型・機能を維持するために様々な性分化関連因子の時空間特異的な性的二型性発現が必要不可欠である．我々が作出した性逆転系統であるC57BL/6N-XYposマウスの性差構築と破綻機構を詳細に解析した結果，1）C57BL/6のY染色体（SRY遺伝子C末端）の遺伝的背景に性逆転の原因がある可能性，2）常染色体上のノンコーディングDNA領域が関与する可能性，3）新たなエピジェネティック因子が関与する可能性，が示された．
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 神戸大学, 2014年07月 - 2018年03月
農村地域固有の社会文化，自然資源，知識の喪失が危惧されている。本研究では，第一に，「地域固有性」とは何かについて学際的な視点から定義をおこなった。その上で，第二に，農業・農村の現場における地域固有の種や技術の実態や課題，地域固有性に対する社会的な認知などを事例分析を通して明らかにした。さらに，第三に，多様なアクターの相互作用により，地域固有性が発現されたり，継承されたりするプロセスを3つの事例を通して明らかにした。以上の結果をもとに，地域固有性の発現による農村発展のモデルを提示した。
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 2014年04月 - 2017年03月
性分化疾患の発症原因となる性分化機構の検討を行った．性分化疾患系統のマウス（YPOS）の未分化生殖腺は，同一遺伝子型から多様な表現型を産出するが，その原因が性決定遺伝子Sryおよび下流因子のわずかな発現遅延である事を明らかとした．また，哺乳類の性分化過程で機能するDmrt1の調節に対するNon-coding領域の関与および同領域によるシス調節を明らかとし，さらに，同領域が精巣・卵巣ともに非メチル化状態でることも明らかとした．加えて，ニワトリでは，dmrt1の発現に一過性の左右差が認められ，セルトリ細胞の分化過程にも左右差が存在することを明らかとした．
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 富山大学, 2012年04月 - 2016年03月
不死化口腔上皮細胞を用いて，小胞体ストレスを伴うフッ化ナトリウムで誘導される細胞死に関与する遺伝子や遺伝子ネットワークを明らかにした．マウスを用いた動物実験において，ネオニコチノイド系殺虫剤クロチアニジンの行動または生殖に対する毒性作用は環境ストレスを負荷したときにより深刻化した．この成績は，複合汚染の中で生きる我々の社会の実情を映し出しているのかもしれない．今回得られた成績は，哺乳動物において環境化学物質の複合影響の分子メカニズムを理解するための基礎データになると考えられる．
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 2012年04月 - 2015年03月
性差の確立は，SRYをマスター遺伝子とした「遺伝的制御」によって未分化性腺の雄（精巣）あるいは雌（卵巣）への運命が決まり，その雌雄表現型・機能を維持するために様々な性分化関連因子の時空間特異的な性的二型性発現が必要不可欠である．性逆転系統であるC57BL/6N-XYposマウスの性差構築と破綻機構を詳細に解析した結果，①C57BL/6の遺伝的背景に性逆転の原因がある可能性，②新たなエピジェネティック因子が関与する可能性，③常染色体上のノンコーディング領域が関与する可能性が示唆された．
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 2009年 - 2011年
ゲノムDNAの様々な遺伝子座が細胞の種類により特異的にメチル化・アセチル化されていることに関連し,性分化関連遺伝子群とメチル化機構,あるいはX染色体のモザイク発症機構と性分化異常との関係ならびにSRY遺伝子のメチル化・アセチル化プロファイルを明らかにし,性分化の破綻機構およびその多様性の原因を解明する一端を担うことができた.
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 株式会社東芝研究開発センター, 2007年 - 2008年
有害環境化学物質による神経発達障害を、ダイオキシン受容体であるアリルハイドロカーボン受容体(AhR)とドーパミン合成酵素であるチロシン水酸化酵素(TH)遺伝子の分子連関から解明することを主眼として、AhR/THシグナルの活性化に対応して発光がモニターできるTH-Tgマウスと、発達初期の多能性神経幹細胞が分化するプロセスにおけるAhR/THシグナルを可視化できるTH-imNSC細胞の構築に成功した。
- 日本学術振興会, 科学研究費助成事業 若手研究(スタートアップ), 若手研究(スタートアップ), 香川大学, 2007年 - 2008年
本研究では, 器官形成期におけるエストロゲン様化学物質の曝露が, 如何にして不可逆的な生殖障害を引き起こすのか, そのメカニズムの一端を解明することを試みた. 本研究により, 視床下部-下垂体-性腺軸一連の機能障害メカニズムにおいては, より中枢側の責任が大きいこと, また, 精巣ライディッヒ細胞における側鎖切断酵素P450scc遺伝子は, ヒストン脱アセチル化を引き起こすことにより遺伝子発現が低下し, アンドロゲン産生能の低下に至ることが推察された. 興味深いことに, 天然エストロゲンではP450scc遺伝子のヒストン脱アセチル化が引き起こされず, エストロゲン様内分泌攪乱物質と天然エストロゲンでは, ヒストン修飾のレベルで作用機序が異なることが示された.
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 新潟大学, 2006年 - 2008年
アカネズミにおけるダイオキシン汚染の影響は、個体レベルではダイオキシンによる毒性発現が認められ、精子数の減少など繁殖機能の低下が起きていることが明らかとなった.一方、ダイオキシン汚染に有利なダイオキシン受容体のアリルを同定し、その頻度を指標とした個体群レベルでの評価を行ったところ、汚染範囲が小規模で周辺からの個体の移出入が頻繁に行われることにより、有利なアリルが集団に固定できないことが明らかになった.
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 北海道大学, 2004年 - 2007年StAR蛋白質結合蛋白質(SBP)のステロイドホルモン産生における役割の解明
ステロイドホルモン合成を調節するSteroidogenic acute regulatory protein (StAR)蛋白質と結合するStAR蛋白質結合蛋白質(SBP)のステロイドホルモン産生における役割を解明した。 1. SBPのプロモーター解析 SBP遺伝子の転写開始点が複数存在することは以前から知られていた。エクソン1では大まかには2種類ある。エクソン1aと1bである。それぞれのプロモーター領域をレポーター遺伝子pGL_4に組み込み、転写活性を測定した。副腎癌細胞H295R細胞では1aのプロモーター活性は1bに比べて100倍以上に高いことが判明した。また、コレステロール硫酸を培養細胞に2μg/ml加えると、転写活性は増加することが判明した。SBP遺伝子とコレステロール硫酸がリガンドであるRORαがSBPの発現に関与することが明らかになった。 2. コレステロール硫酸とSBPの解析 StAR蛋白質のC端側と相同性の高いドメインはコレステロールをはじめ脂質が結合する領域であるため、StAR関連脂質転送(START)ドメインと命名された。SBPはSTARTドメインに結合する。SBP蛋白質、StAR蛋白質およびコレステロール硫酸の関連をステロイドホルモンの産生の観点から解析し、コレステロール硫酸のステロイドホルモンの産生に与える影響について明らかにした。 3. 無精子症とSBP遺伝子の関連 非閉塞性無精子症および乏精子症の患者のSBP遺伝子変異をRT-PCRとダイレクトシークエンス法により解析した。SBP蛋白質構造の変化が無精子症との関連に相関することを明らかにした。
- 日本学術振興会, 科学研究費助成事業 特定領域研究, 特定領域研究, 神戸大学, 2005年 - 2006年性分化異常症における性の決定・分化の分子細胞遺伝機構の解明
遺伝子,環境因子に次いで生物の多様性を位置づける要因としてエピジェネティクスが近年注目されている.ゲノムDNA様々な遣伝子座が,細胞の種類により特異的にメチル化されていることに関連し,これまで我々が明らかにしてきたヒト性分化異常症における精巣化遺伝子SRYならびにX染色体のモザイク発症機構と性分化異常との関係(性染色体遺伝子量補償機構の破綻)の解明を目的とした.北海道大学医学部産婦人科および小児科でfollow-upししている臨床および細胞遣伝学的に性分化異常症が疑われた20症例を対象に,本研究に対するインフォームドコンセントを得た上で,詳細な細胞遺伝学的解析を行い,さらに性分化関連遺伝子および座位の有無の検討を行った.遺伝子の欠失を認めない症例についてはゲノムDNAをPCR法により増幅し,エクソン,イントロン各部位の塩基配列を直接決定し,点突然変異などの異常の有無の有無を解析した.また,臨床的多型性を適伝子レベルでの異常と対応させ検討した.家系解析として,PCR-RFLP, PCR-SSCPおよびPCR-CFLP法を取り入れることにより,迅速かつ大量に遺伝子試料の多型点突然変異の解析を行った.また,Y染色体の構造異常の認められた症例では,患者より性腺および皮膚を採取,培養後,bisulfite sequencing法によるSRY, AR, SLARおよびCox-2のDNAメチル化解析ならびにクロマチン構造とヒストンアセチル化についてSRY座位を含むY染色体短腕末端から長腕末端までのChromatin Immunoprecipitation (ChIP) Assayを行った. その結果,性分化異常症患者の「性の決定・分化機構」の破綻には,SRY遺伝子のCpG islandにおけるDNAメチル化抑制,およびSRYプロモーター＆HMG BOXのヒストンアセチル化によりSRY mRNAの異常発現が関与していることを見いだした.これらの性分化異常症患者ではSRYの細識限定発現部位・時期に異常がみられ,また,高率に45.X核型とのモザイクが関連することから,当初,「モザイクなどの細胞数の単純な減少に伴うSRYの遣伝子量の減少」が関与していると想定していたが,実は,発生初期より『SRY遺伝子の高濃度持続発現』が起こり,その結果,未分化性腺から精巣への発生・分化スイッチに障害が起こり,基本型である雌型に分化(卵巣化)したと考えられた.このことは,性分化異常症患者の染色体異常は,性分化異常の原因ではなく,患者は元々エピジェネティクス(クロマチン)異常等の素因があり,その結果,染色体異常が引き起こされたとも想定され,性分化異常症発症機構に関し,新たな研究の展開が期待される.
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 北里大学, 2003年 - 2005年インヒビンレセプターの構造決定とTGFβファミリーのシグナル伝達系に関する研究
インヒビンファミリーには下垂体からのFSHの分泌を抑制する物質であるインヒビンとフォリスタチン、FSHの分泌を促進するアクチビンが含まれる。これらの生理活性物質は従来のホルモンと異なり多くのサブタイプを持っているしかしながら、インヒビン関連物質の研究は十分な研究材料が得られず、特にインヒビンはレセプターが不明のため未解決の問題が多い。そこで本研究では以下の課題を明らかにすることを目的として行われた。 1.家畜のインヒビンぐアクチビンとフォリスタチンを精製すると共に、ヒトインヒビン類や新しいアクチビンCやEのリコンビナント蛋白質を生産する。 2.全てのインヒビンファミリーの特異的測定法を確立し、血中と各組織中の含量を明らかにする。 3.精製したインヒビン関連物質を標識し、特異的に取り込む組織・細胞を同定する。それらの細胞・組織を器官培養し、細胞増殖・分化に対するインヒビンファミリーの作用を明らかにする。 4.標的器官からインヒビン関連物質の結合タンパク質(BP)とインヒビンレセプターを同定し、それらのタンパク質の構造を決定し、インヒビンの新たなシグナル伝達系を解明する。 5.in vivoにおける役割を解明するために、トランスジェニック(Tg)マウスの作成、活性物質の投与による検討、生体の種々の生理的変化に伴う生理活性物質の濃度・変化を明らかにし、活性物質の生物活性を解明する。 以上のような当初の目的は、インヒビンに特異的なレセプターの構造決定には至らなかったが、ほぼ達成できた。インヒビンのレセプターの代わりに、90kDaのインヒビン結合蛋白質:F-spondinを卵胞液中に発見した。さらに、インヒビン・アクチビンの前駆体部分の蛋白質がインヒビン・アクチビン結合蛋白質であることを初めて明らかにした。これらの結合蛋白質と既知のTGF一βファミリーレセプターあるいはインヒビン・アクチビンとの相互作用の研究はインヒビンのシグナル伝達の研究に新たな展望を開くと確信する。
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 2003年 - 2005年性分化関連遺伝子群の発現とその相互調節機構の解明
遺伝子変異動物hpgマウスを用いてGnRH不在下における性分化関連遺伝子群とStARの発現変化について個体発生学的に解析した.また,胎生期内分泌攪乱化学分子負荷による性分化への影響をStARと性分化および生殖内分泌関連遺伝子群の個体発生学的な生体・分子レベルでの発現パターン解析から遺伝子相互の関係を探ることにより遺伝子ネットワーク全体のしくみと構造を明らかにし,性分化の過程でのステロイドホルモンの産生調節機構の解明を試みた.その結果,アクチビンおよびPMSGの投与は,FSH作用のみならず,強力なLH作用によりライディッヒ細胞の増生を促進してテストステロン合成,さらにセルトリ細胞の活性化により造精機能を回復させた.GnRH分泌の機能的性差はGnRHニューロン活性の性差に起因することが示され,その活性の性差は生後初期ステロイドに影響されることが明らかになった.ゴナドトロピン分泌の制御は,生殖器よりもより低濃度のエストロゲン様物質で影響を受けることが示された.さらに,雄マウスの妊孕能はStAR発現と密接に関わっており,内分泌攪乱物質がStARに与える影響の重要性が示唆された.StAR結合タンパク(SBP)はStARと結合し,ステロイドホルモンの産生を促進することが判明した.SplとSF-1が相互作用して,ヒトStARのプロモーター活性を調整することを明らかにした.発達期の神経系における性的二型性にはアポトーシス制御機構が関与し,その機序に新たな知見を加えるとともに,ヒトを含めた哺乳動物の脳の性分化機構の解明に重要な基礎的知見を提供した.個体発生的にStARタンパクの発現は胎齢13.5〜18.5日において雄に優位であった.以上,StARを中心に生殖腺の分化および性分化制御カスケードの一端を明らかにした.また,エストロゲンの脳の性分化に対する分子メカニズムを示すことができた.
- 日本学術振興会, 科学研究費助成事業 萌芽研究, 萌芽研究, 北里大学, 2002年 - 2004年インヒビンレセプターの同定とシグナル伝達に関する研究
前年度までの研究では、インヒビンの最も代表的な標角器官である下垂体からのインヒビンレセプターの精製を試みたが、組織が極めて小さいために成功しなかった。そこで、アクチビンの標的細細胞のK562と肝臓細胞でのアクチビンの作用について、検討した。 1.コラゲナーゼ灌流法により肝細胞を分散し、プレートへ分注した翌日に各ホルモンを添加して、さらに3日間培養した。各種アクチビンは単独添加では有意なIGF-1分泌抑制が認められなかったが、GH存在下では強力なIGF-1分泌抑制が認められた。 2.XTT法を用い肝細胞に細胞増殖に対するアクチビンの作用を比較したところ、単独添加およびEGF存在下ではアクチビンAとABに強い抑制作用が認められた。しかしHGF存在下ではアクチビンBにも顕著な増殖抑制作用が認められた。以上から、生体内においても様々な因子の影響を受けることにより、アクチビンの各サブタイプのレセプターへの親和性の変化およびレセプターの発現量の変化または未知のレセプターの存在などによりアクチビンの作用が異なって発現すると考えられる。 3.K562細胞は赤芽球系の腫瘍細胞である。アクチビンはこの細胞に作用して、細胞の分化を促進し、ヘモグロビン産生を誘導することが知られている。この現象をアクチビン・インヒビンアッセイ法に開発し、我々の精製した3種類のアクチビンを用いてヘモグロビン産生活性を確認した。さらに、インヒビンの単独、あるはアクチビンの併用添加により、K562細胞にインヒビンが作用することを明らかにした。この細胞を大量培養することにより、インヒビンレセプター単離とアクチビンとのクロストークを明らかにする展望が開けた。 本研究助成金により、インヒビンとアクチビンが作用する培養細胞と機能を発見し、今後の研究の大きな発展のきっかけ得ることが出来た。
- 日本学術振興会, 科学研究費助成事業 特定領域研究, 特定領域研究, 北里大学, 2002年 - 2003年内分泌攪乱物質による次世代生殖器系の分化に関する分子毒性遺伝学的研究
最近,温度感受性Simian Virus40(SV40)大型T抗原遺伝子を導入したトランスジェニックマウスを作製することで,セルトリ細胞,ライディッヒ細胞の本来の性質を持った培養細胞株を得ることができ,個々の細胞への内分泌攪乱物質の作用をみることが可能になった.そのセルトリ細胞株(TTE3)とライディッヒ細胞株(TTET)を用いて,エストラジオール(E_2),ジエチルスチルベステロール(DES),ポリカーボネイト,ポリスチレン樹脂等の原料として用いられ,環境中に多量に排出されているビスフェノールA(BPA),フタル酸エステルの代謝産物で,セルトリ細胞への毒性と催奇形性を起こすといわれているフタル酸モノ-2エチルヘキシル(MEHP),ダイオキシン受容体のリガンドであるβナフトフラボン,さらに,BPA,MEHP,β-ナフトフラボンの3種類の物質を複合した場合の影響をTTE3およびTTE1培養株を用いてステロイドホルモン受容体遺伝子変化ならびにホルモン産生への影響をみることを目的として実験を行った.その結果,5種類の化学物質の中で,ER結合型とされるDES,BPA,E_2で,ARの遺伝子の発現がTTE1で強く抑制され,βナフトフラボンではTTE1のAR発現が増強された.StARではすべての添加物質についてTTE1が強く抑制され,3種複合添加でその抑制が増強された.E_2,DES,BPA,MEHP,βナフトフラボンの5種類とBPA,MEHP,βナフトフラボンの3種を複合添加した結果,セルトリ細胞とライディッヒ細胞は同じ物質を添加しても,ステロイドホルモン受容体の遺伝子発現において異なった反応を示すことが示唆された.また,ERを介して女性ホルモン様作用を持つ物質がセルトリ細胞のARよりライディッヒ細胞のARをより強く抑制し,セルトリ細胞のStARよりライディッヒ細胞のStARの方がより強く抑制されていることが明らかになった.さらに,βナフトフラボンではライディッヒ細胞のARの発現が増強されるという結果が得られ,AhRを介して作用を発現する物質とERを介して作用を発現する物質では細胞に対する影響の与え方に差があることが示唆された.
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 北海道大学, 2000年 - 2003年産婦人科領域におけるステロイドホルモン産生に関する分子生物学的研究
ステロイドホルモンはコレステロールから合成される。ステロイドホルモンの合成に関し、StAR蛋白質はコレステロールのミトコンドリア外膜から内膜の輸送を促進する因子で、ステロイドホルモンの産生の律速段階を形成する。 1.StAR遺伝子の発現の解析 StAR遺伝子の発現の抑制機構に関してプロモーター解析により、StAR遺伝子の発現を制御する因子はRIP140であることが判明した。RIP140はDAX-1と結合し、またSF-1はRIP140およびDAX-1と結合することが判明した。RIP140の発現がStAR蛋白質の発現を制御することにより、ステロイドホルモンの産生を調節することを明らかにした。さらに、StAR遺伝子のプロモーター領域にダイオキシン応答配列を見出し、ダイオキシン受容体を介してStAR遺伝子のプロモーター活性を高めることを明らかにした。 2.腫瘍局所におけるステロイドホルモンの産生 婦人科悪性腫瘍である子宮内膜癌においてステロイドホルモンの産生に関与する遺伝子チトクロームP450遺伝子群とStAR遺伝子の発現を明らかにし、腫瘍局所のステロイドホルモン産生を明らかにした。ステロイドホルモンの産生にはStAR蛋白質およびStAR蛋白質のホモログであるMLN64の蛋白発現がステロイドホルモンの産生に重要であることを明らかにした。 3.StAR蛋白質結合蛋白質の解析 StAR蛋白質と結合する遺伝子をクローニングし、StAR蛋白質結合蛋白質(SBP)と命名した。SBPのステロイドホルモン産生組織における遺伝子の局在をin situハイブリダイゼーション法により同定した。SBPとGFP融合蛋白質を作成し、細胞内の局在を同定した。RNAsi法によりSBP遺伝子発現を抑制し、ステロイドホルモンの産生に与える影響を明らかにした。SBP1はステロイドホルモンの産生に関与することを明らかにした。
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 北里大学, 2000年 - 2001年性の決定・分化と内分泌攪乱物質の胎子・新生子期曝露
一般的に薬物等の感受性が成体と比べて極めて高い胎子,新生子期において内分泌攪乱化学物質がどれぐらいの濃度で,またどのような機序で神経系および生殖器系,内分泌系に影響を及ぼすのか,具体的基礎研究は少ない。そこで本研究では人工的なエストロゲン受容体(ER)結合型内分泌攪乱物質として最も力価の強いdiethylstilbestrol(DES)を代表とし,マウスの胎子,新生子期に曝露させ,生殖内分泌系にどのような影響を与えるかを組織学的,内分泌学的に検討し,その作用機構の一端を解明することを試みた。 本研究では,雄マウスに,scsame oilに溶解させた0.001〜100μgのDES(10μl)を3日齢の単回,3日齢から3日毎の反復,母マウスの膣栓確認後7日目からと出産後3日齢から3日毎の反復,対照群にはsesame oilのみを投与し,25日齢から84日齢(12週齢)の個体を実験に供した。生後単回DES10,100μg投与4週齢群,生後反復DES 1.0μg投与25日齢群,DES 10μg投与6週齢群,胎生期〜生後反復DES 10μg投与6週齢群で,精細管での核萎縮,細胞質内の空胞化および細胞間隙の増大が認められ,細胞密度が低下し,精祖細胞から精子細胞が減少し,精子は観察されなかった。BrdU取り込み実験により精細管の細胞の分化・増殖能抑制が認められた。免疫染色によるERα,Androgen Rcccptor(AR),StAR(steroidogenic acute rcgulatory protein)の検索では,陽性細胞数に顕著な差は認められなかったが,精巣の染色性がDES投与により,ARでは増強し,ERαおよびStARでは減弱した。内分泌学的検索の結果,対照群と比較して,下垂体中のFSH, LHの含量が明らかに減少し,下垂体機能の低下を示した。下垂体中のFSHの減少は,精巣中インヒビンも減少していることから視床下部性の機能低下を示唆した。 これらの結果により,生後単回投与では,DES10μg投与量までの曝露による障害が成長段階で回復し,生後反復投与では,DES 10μg以下の投与量でも同様の障害を受けるもののその障害は回復せず,胎生期〜生後反復投与では,致命的なダメージとなり,生殖器系の分化,発達が著しく阻害されることが示された。その作用機序は視床下部-下垂体性の機能低下が原因となり,精巣の分化・発達が障害されるものと考えられた.
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 1998年 - 1999年性分化関連遺伝子群とその相互調節機構の解明
本研究対象は、産婦人科及び小児科領域で、follow-upされている、臨床及び細胞遺伝子学的に性分化異常症が疑われた40家系、原発性無月経症例104家系(性腺分化異常68例、性管分化異常23例、、男性型外性器4例、その他の機能不全9例であり、そのうちターナー症17例、XY女性11例を含む)、および早発閉経症例20家系で、本研究に対するインフォームドコンセントを得た上で、詳細な細胞遺伝学的検討(星、藤本)、性分化関連遺伝子及び座位の有無の検討を行った(星、藤本)[平成10年度]。 上記結果を踏まえ、平成11年度は、遺伝子の欠失を認めない症例についてはゲノムDNAをPCR法により増幅し、エクソン、イントロン各部位の塩基配列を直接決定し、点突然変異などの異常の有無を解析した。また、臨床的多型性を遺伝子レベルでの異常と対応させ検討した(星)。家系解析として、近年急速に発展している分子生物学的手法、とりわけPCR、PCR-RFLP、PCR-SSCPおよびPCR-CFLP法を取り入れることにより、迅速かつ大量に遺伝子試料の多型点突然変異の解析を行った(星)。また、生殖生理現象を解明する目的でY染色体上の無精子症関連遺伝子DAZ(deleted in azoospermia)および常染色体3p25に我々が同定したDAZLA(DAZ like autosome)遺伝子についても解析を行った。その結果、DAZLA遺伝子が未分化生殖腺細胞の分化に関連していることを初めて明らかにし、公表した。(Mol. Hum. Reprod., 1999)(星、藤本)。またBCC(basel cell carcinoma)において性染色体異数性が高頻度に認められることを明らかにし、それに着目して細胞分子遺伝子解析を行った。その結果、性染色体の異数性、とくにX染色体の不活性化に関連したX monosomyが遺伝子変異を誘発することが示唆された(Int. J. Oncol., 2000)(星、藤本)。 以上の結果、性分化・生殖機能異常症例の臨床像と遺伝子解析結果に詳細な解析を加え、従来不明で終わることの多い性分化異常症患者の遺伝子診断及び家系解析によりヒトの「性の決定・分化機構」は、SRYの組織限定発現部位・時期に依存すること、性分化異常の発症の原因がY染色体以外(とくにX染色体)にも存在し、組織限定モザイシスム、キメリズムが密接に関与することを明らかにした(星、藤本)。
- 日本学術振興会, 科学研究費助成事業 基盤研究(B), 基盤研究(B), 北海道大学, 1996年 - 1997年ヒツジ胎仔をもちいた脳室周囲白質軟化の発症機構の解明
妊娠日数の確定しているヒツジ胎仔をもちいて慢性実験系を作成し,脱血による胎仔全身性低血圧を負荷して,脳室周囲白質軟化(PVL)に相当する病変を誘起しうる条件を検討した.低血圧の負荷(平均動脈圧25mmHg)を妊娠120日(ヒトの妊娠では32週頃に相当する)から開始し,順次妊娠日数を遡ったところ,妊娠113日目の胎仔に約35%の脱血を行った場合にPVL様病変が出現することを確認した.誘起された病変は,肉眼的には線条件ならびに視床レベルの脳室周囲白質に限局する散在性の巣状軟化性病変で,病理組織学的には軸索の膨化,凝固壊死,ミクログリアマクロファージ系細胞の浸潤反応などヒトのPVLと同様の所見を示した.また,免疫組織学的には,神経細胞への障害を反映しPVLの診断に用いられるβ-amyloid precursor proteinも検出された. 対象を妊娠113日目に設定して行った低血圧負荷実験では,7例中5例にPVL様中枢神経系病変が確認され,低血圧負荷をかけなかった対象群3例には,中枢神経系病変は全く認められなかった.また,中等度の低酸素血症のみを負荷した場合にも中枢神経系病変の発生をみなかった.よって低血圧負荷によりPVLが誘起しうることを証明した. 内分泌学的マーカーとして,ACTH,AVP,アドレナリン,ノルアドレナリン,ドーパミン,コーチゾールの変動を,胎児モニターとして,心拍数,末梢血酸素飽和度,脳血流変動を検討しているが,低血圧負荷後に認められる心拍数ならびに脳血流量の特徴的変動が観察されており,現在,波形解析等を用いて各パラメーター間の関連ならびに内分泌変動との連関について解析中である.また,胎仔中枢神経の発達段階とPVL発症との関連に関する成績も蓄積しており,これらを併せてPVLの発症機構を総合的に検討中である.さらに,PVL発症の予知・予防を念頭に置き,本研究の成果を臨床的に応用する可能性も模索中である.
- 日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 北海道大学, 1996年 - 1997年性の決定・分化と性分化異常症の分子遺伝学的研究
臨床および細胞遺伝学的に性分化異常症が疑われた19症例において,症例により末梢血,臍帯血,皮膚,性線,口腔粘膜,骨髄,羊水細胞あるいは胎盤絨毛組織の計40検体(ターナー症3例を含む表現型女性10例25検体,男性型9例15検体)について,必要に応じてFISH法,サザンブロット法(Y染色体17領域),全例に精巣決定遺伝子であるSRYを含むY染色体短腕末端から長腕末端の各部を認識する8座位およびX染色体2座位についてPCR法を用いて性染色体の構造解析を行った.その結果,マーカー染色体はQ,G分染法およびFISH法により,X染色体由来2例,Y染色体由来7例と確認された.これらY由来7症例および45,X/46,XY症例3例,性腺腫瘍2例(XY,XX),外性器異常2例(XX,XY),small Yの計15症例についてY染色体の構造解析を行った結果,46,XXの卵精巣症例の末梢血および左右性腺,およびXX型男性(尿道下裂・停留睾丸)症例の末梢血ではいかなるY成分も認められなかった.一卵性異性双胎症例では両児ともにmos47,X,+mar×2/45,X/46,X,+mar(組織限定性にその構成比率が異なる)であったが,Y長腕11.2から長腕末端までの領域の欠失が確認された.また,二卵性異性双胎では血液キメリズムが明らかとなった.出生前に診断された46,X,+marの2症例では,マーカー染色体はそれぞれY長腕を全欠損しているi(Y)(p10)と,残存Y長腕逆位inv(Y)(q10q11.23),del(Y)(q12)とであった.Y由来細胞を含む46,X,+mar核型の表現型女性5例において,性腺を含む様々な組織の染色体を分析することにより組織特異的ターナーモザイクが検出された.SRY陽性例ではシークエンスの結果,塩基配列に異常は認められなかった。 以上,産婦人科領域における性分化異常症例の多くは,性染色体に関連したモザイク,転座,欠失などが認められ性分化異常症の複雑さが示された.ヒトの「性の決定機構」および「性の分化機構」は,SRYの組織限定発現部位・時期に依存すること,性分化異常症の発症の原因がY染色体以外にも存在することを示すことができた.また,多胎妊娠における性分化異常に性決定遺伝子との関連に組織限定モザイシスム,キメリズムが密接に関与することも明らかにできた.