Directory of Researchers

HARAYAMA Hiroshi
Graduate School of Agricultural Science / Department of Bioresource Science
Professor
Veterinary Medicine / Zootechnical Science
Last Updated :2024/02/02

Researcher Profile and Settings

Affiliation

  • <Faculty / Graduate School / Others>

    Graduate School of Agricultural Science / Department of Bioresource Science
  • <Related Faculty / Graduate School / Others>

    Faculty of Agriculture / Department of Bioresource Science , Biosignal Research Center

Teaching

Research Activities

Research Interests

  • 細胞内シグナリング
  • Intracellular signaling
  • Sperm
  • Reproduction

Research Areas

  • Life sciences / Animal production science

Awards

  • Apr. 2021 Japanese Society of Animal Science, The 2021 Award for Excellence in Reviewing for Animal Science Journal in 2020, Animal Science Journal Reviewers Award

    HARAYAMA Hiroshi

  • Mar. 2019 Japanese Society of Animal Science, The 2019 Award for Excellence in Reviewing for Animal Science Journal in 2018, Animal Science Journal Reviewers Award

    HARAYAMA Hiroshi

    Official journal

  • Jul. 2017 JSPS, 日本学術振興会(JSPS)平成28年度 特別研究員等審査会専門委員(書面担当)の表彰, 日本学術振興会(JSPS)平成28年度 特別研究員等審査会専門委員(書面担当)の表彰

    HARAYAMA HIROSHI

    書面審査における有意義な審査意見を付した 専門委員として選ばれたため。

    Others

  • Sep. 2012 The Society for Reproduction and Development (SRD), The 2012 SRD Outstanding Research Award, Study on intracellular cAMP signaling in mammalian spermatozoa

    HARAYAMA HIROSHI

    繁殖生物学に関する基盤研究、応用研究等に顕著な功績があった。

    Japan society

  • Aug. 2006 関西畜産学会, 第56回関西畜産学会優秀発表賞(第1位), ブタ精子の頭部におけるcAMP依存性細胞内カルシウム濃度上昇に及ぼす炭酸水素ナトリウムおよびPKA阻害剤の影響

    TATE Shunsuke, SHIDARA Osamu, HARAYAMA Hiroshi

    Japan society

  • Apr. 2006 American Society of Andrology, 2006 Lalor Foundation Award, Role of protein kinase C in the cyclic adenosine 3',5'-monophosphate-dependent hyperactivation of boar spermatozoa

    HARAYAMA Hiroshi

    International society

Published Papers

  • Comparative characteristics between calyculin A-induced and thimerosal-induced hyperactivation of cryopreserved bovine spermatozoa

    MIYAMOTO Natsuko, OHYA Akihiro, DURITAHALA, SAKASE Mitsuhiro, HARAYAMA Hiroshi

    Jun. 2023, Journal of Reproduction and Development, 69 (3), 170 - 177, English

    [Refereed]

    Scientific journal

  • SAI Seiken, HARAYAMA Hiroshi

    Nov. 2022, Animal Science JOurnal, 93, e13777, English

    [Refereed]

    Scientific journal

  • Involvement of Ca2+-ATPase in suppressing the appearance of bovine helically motile spermatozoa with intense force prior to cryopreservation

    DURITAHALA, SAKASE Mitsuhiro, HARAYAMA Hiroshi

    Jun. 2022, Journal of Reproduction and Development, 68 (3), 181 - 189, English

    [Refereed]

    Scientific journal

  • Calmodulin is involved in the occurrence of extracellular Ca2+-dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs

    WADA Atsushi, HARAYAMA Hiroshi

    Apr. 2021, Animal Science Journal, 92 (e13552), English

    [Refereed]

    Scientific journal

  • Effects of digoxin on full-type hyperactivation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades

    SAHA Soma Rani, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    Sep. 2020, Theriogenology, 154, 100 - 109, English

    [Refereed]

    Scientific journal

  • Characteristics of bull sperm acrosome associated 1 proteins

    MINAMI Kenta, ARAI-ASO Miyuki M, OGURA-KODAMA Yukari, YAMADA Ayano, KISHIDA Kazumi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    Aug. 2020, Animal Reproduction Science, 218 (106479), English

    [Refereed]

    Scientific journal

  • Identification of isoforms of calyculin A-sensitive protein phosphatases which suppress full-type hyperactivation in bull ejaculated spermatozoa

    ARAI Yuka, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    15 Apr. 2019, Theriogenology, 129, 46 - 53, English

    [Refereed]

    Scientific journal

  • Reconsideration of the evaluation criteria for bull ejaculated sperm motility in the context of rotation

    YAMADA Ayano, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    Oct. 2018, Journal of Reproduction and Development, 64 (5), 377 - 384, English

    [Refereed]

    Scientific journal

  • Nagisa Otsuka, Hiroshi Harayama

    Ejaculated boar spermatozoa exhibit two types of hyperactivation: full and non-full. Full-type hyperactivation is characterized by the asymmetrical bending of the entire middle piece-principal piece and a twisting/figure-eight-like trajectory, and can be induced by simple incubation with CaCl2 after preincubation with a cAMP analog (Sp-5,6-dichloro-1--D-ribofuranosyl-benzimidazole-3,5-cyclic monophosphorothioate [cBiMPS]). Here, we compared the sperm flagellar motility after treatments with elevators of [Ca2+](i) (cBiMPS/CaCl2, thimerosal, procaine, and 4-aminopyridine) to characterize the regulatory mechanism of extracellular Ca2+-dependent, full-type hyperactivation in ejaculated boar spermatozoa, and examined the possible involvement of Transient receptor potential cation channel subfamily C member 3 (TRPC3) in this event using the specific inhibitor Pyr3. Full-type hyperactivation was induced by a 60-min incubation with CaCl2 following a 180-min preincubation with cBiMPS but without Ca2+. Thimerosal-treated spermatozoa exhibited full-type hyperactivation in a manner independent of extracellular Ca2+; conversely, this was not observed in procaine- or 4-aminopyridine-treated spermatozoa. A 20-min treatment with Pyr3 between preincubation with cBiMPS and incubation with CaCl2, significantly suppressed the normal phenotype. These observations indicated that mechanisms underlying full-type hyperactivation in spermatozoa incubated with CaCl2 after preincubation with cBiMPS are different from those in the thimerosal-treated spermatozoa. Furthermore, indirect immunofluorescence localized TRPC3 in the upper segment of the middle piece, which bends asymmetrically during full-type hyperactivation but not in non-full-type hyperactivation, suggesting that TRPC3 may be involved in the extracellular Ca2+-dependent full-type hyperactivation in ejaculated boar spermatozoa.

    WILEY, Nov. 2017, MOLECULAR REPRODUCTION AND DEVELOPMENT, 84 (11), 1203 - 1217, English

    [Refereed]

    Scientific journal

  • Miyuki M. Arai, Kenta Minami, Yukari Ogura, Nagisa Otsuka, Shohei Hama, Hiroshi Harayama, Mitsuhiro Sakase, Moriyuki Fukushima

    In Japanese black cattle, AI severely subfertile males have occasionally been found. In order to solve this problem, we previously asserted the need for exact examinations of acrosomal tyrosine-phosphorylated proteins and acrosome morphology in cryopreserved spermatozoa. In the present study, we further investigated acrosomal tyrosine-phosphorylated proteins in spermatozoa before cryopreservation and examined possible relationships between these phosphoproteins and acrosome stability. Ejaculated, epididymal and cryopreserved spermatozoa were subjected to examinations of general characteristics (motility, shape and acrosome morphology) and indirect immunofluorescence of acrosomal phosphoproteins. Unlike all general characteristic parameters, the distribution of acrosomal tyrosine-phosphorylated proteins in ejaculated and cauda epididymal spermatozoa varied considerably among bulls and was linked to the maintenance of morphologically normal acrosomes in cryopreserved spermatozoa or ejaculated spermatozoa after 270min incubation. Moreover, the distribution of these phosphoproteins was arranged in the spermatozoa of the proximal epididymides. These findings indicate that acrosomal tyrosine-phosphorylated proteins are distributionally arranged during early process of sperm maturation, that their distribution of cauda epididymal and ejaculated spermatozoa are largely different among bulls, and that varied states of acrosomal phosphoproteins may result in individual differences in acrosome stability among bulls.

    Corresponding, CSIRO, 2017, Reproduction, Fertility and Development, 29 (7), 1297 - 1305, English

    [Refereed]

    Scientific journal

  • Masaki Fukuda, Mitsuhiro Sakase, Moriyuki Fukushima, Hiroshi Harayama

    To obtain basic information of bull IZUMOI (a sperm protein essential for sperm-egg fusion) and disclose possible causes for the impaired fertilizing ability in bull cryopreserved spermatozoa, we investigated this protein in bull spermatozoa collected from various regions of epididymides, freshly ejaculated spermatozoa, acrosome-reacted spermatozoa, and cryopreserved spermatozoa by Western blotting and the triple staining with the anti-IZUMO1 antibody, fluorescein isothiocyanate-peanut agglutinin, and 4',6-diamidino-2-phenylindole. In the cauda epididymal spermatozoa and freshly ejaculated spermatozoa, bull IZUMOI was detected mainly as a 45-kDa major form. This major form was derived probably from a 52-kDa precursor form in the epididymis. Bull IZUMOI was immunolocalized along the border between the principal and equatorial segments of the acrosomal region (pattern P1 of IZUMOI) in the most of epididymal and freshly ejaculated spermatozoa with normal acrosomes. In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMOI in whole equatorial segment (pattern P2 of IZUMOI) significantly increased. These results indicate that bull IZUMOI undergoes maturation-related changes during sperm transit through the epididymis and that it is translocated to the equatorial segment of acrosomal region during the acrosome reaction. On the other hand, severe damages were observed in the acrosomes of 60% of the cryopreserved spermatozoa. Localization of IZUMOI in these spermatozoa was pattern P2 (IZUMOI in whole equatorial segment), P3 (IZUMOI in whole acrosomal region), or P4 (IZUMO was lost). Moreover, after the incubation to compare the stability of acrosomes and IZUMOI localization between cryopreserved spermatozoa and freshly ejaculated spermatozoa, much more spermatozoa lost acrosomes and IZUM01 in the cryopreserved samples compared with freshly ejaculated samples. These findings indicate that impaired fertilizing ability of bull cryopreserved spermatozoa with damaged acrosomes is related partially to the aberrant translocation of IZUMO1 which may be followed by the loss of intact IZUMOI. (C) 2016 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, Dec. 2016, THERIOGENOLOGY, 86 (9), 2179 - 2188, English

    [Refereed]

    Scientific journal

  • K. Kishida, H. Harayama, F. Kimura, T. Murakami

    The aims of this study were to show the existence of individual differences in the distribution of sperm acrosome-associated 1 (SPACA1) among male patients of infertile couples and to examine their possible impact on the outcomes of conventional in vitro fertilization (IVF). The spermatozoa were collected from male patients of infertile couples, washed by centrifugation, collected by the swim-up method, and then used for clinical treatments of conventional IVF. The surplus sperm samples were fixed and stained with an anti-SPACA1 polyclonal antibody for the immunocytochemistry. In the clinical IVF treatments, fertilization rates and blastocyst development rates were evaluated. The immunocytochemical observations revealed that SPACA1 were localized definitely in the acrosomal equatorial segment and variedly in the acrosomal principal segment. Specifically, the detection patterns of SPACA1 in the acrosomal principal segment could be classified into three categories: (A) strong, (B) intermediate or faint, and (C) almost no immunofluorescence. The SPACA1 indexes were largely different among male patients with the wide range from 13 to 199 points. The SPACA1 indexes were significantly correlated with developmental rates of embryos to blastocysts (r = 0.829, P = 0.00162), although they were barely associated with fertilization rates at 19 h after insemination (r = 0.289, P = 0.389). These results suggest that the distribution of SPACA1 in sperm affects the outcomes of conventional IVF. In conclusion, this study provides initial data to promote large-scale clinical investigation to demonstrate that the SPACA1 indexes are valid as molecular biomarkers that can predict the effectiveness of conventional IVF of infertile couples.

    CAMBRIDGE UNIV PRESS, Oct. 2016, ZYGOTE, 24 (5), 654 - 661, English

    [Refereed]

    Scientific journal

  • Yukari Ogura, Yuki Takagishi, Hiroshi Harayama

    The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca2+-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca2+-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42 kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28 kDa) increased in extracellular Ca2+-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions. (C) 2016 Elsevier B.V. All rights reserved.

    ELSEVIER SCIENCE BV, Sep. 2016, ANIMAL REPRODUCTION SCIENCE, 172 (1), 94 - 104, English

    [Refereed]

    Scientific journal

  • Ayane Isono, Shunsuke Tate, Kazumi Nakamura-Mori, Taichi Noda, Sho Ishikawa, Hiroshi Harayama

    We previously suggested that protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins may be necessary for the occurrence of acrosome reaction in livestock spermatozoa (Adachi et al., J Reprod Dev 54, 171-176, 2008; Mizuno et al., Mol Reprod Dev 82, 232-250, 2015). The aim of this study was to examine the involvement of the intracellular cAMP signaling cascades in the regulation of the decrease of postacrosomal phosphorylated proteins in boar spermatozoa. Boar ejaculated spermatozoa were incubated with CAMP analogs and then used for the immunodetection of serine/threonine-phosphorylated proteins and assessment of acrosome morphology. The protein phosphatase-dependent decrease of postacrosomal phosphorylated proteins was greatly promoted by the incubation with a cAMP analog Sp-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate (cBiMPS). This decrease was induced before the initiation of acrosome reaction and did not require the millimolar concentration of extracellular Ca2+ which was necessary for the initiation of acrosome reaction. Moreover, suppression of protein kinase A activity with an inhibitor (H89) had almost no influence on both decrease of phosphorylated proteins and occurrence of acrosome reaction in the spermatozoa incubated with cBiMPS. In addition, the prolonged incubation with a potentially exchange protein directly activated by cAMP-selective cAMP analog (8pM) could only partially mimic effects of cBiMPS on these events. These results indicate that the CAMP-dependent signaling cascades which are less dependent on protein kinase A may regulate the decrease of postacrosomal phosphorylated proteins in boar spermatozoa before the extracellular Ca2+-triggered initiation of acrosome reaction. (C) 2016 Elsevier Inc. All rights reserved.

    ELSEVIER SCIENCE INC, Apr. 2016, THERIOGENOLOGY, 85 (6), 1152 - 1160, English

    [Refereed]

    Scientific journal

  • Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle

    Kazumi Kishida, Mitsuhiro Sakase, Kenta Minami, Miyuki M. Arai, Reiko Syoji, Namiko Kohama, Takayuki Akiyama, Akio Oka, Hiroshi Harayama, Moriyuki Fukushima

    The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AT using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.

    Corresponding, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Dec. 2015, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 61 (6), 519 - 524, English

    [Refereed]

    Scientific journal

  • A. Kojima, Y. Matsushita, Y. Ogura, S. Ishikawa, T. Noda, T. Murase, H. Harayama

    There are species differences in the regulatory system for sperm capacitation and subsequent hyperactivation between livestock and laboratory animals. In livestock spermatozoa, it is poorly understood when and how extracellular Ca2+ is necessary for hyperactivation, although it has been demonstrated that the [Ca2+](i) increase is indispensable to occurrence of hyperactivation. In this study, we examined necessity of extracellular Ca2+ for the initiation and maintenance of hyperactivation and then sought possible target molecule of Ca2+ that was involved in hyperactivation of boar spermatozoa. Boar ejaculated spermatozoa were pre-incubated with a cell-permeable cyclic adenosine monophosphate (cAMP) analog cBiMPS' and without CaCl2 to induce the cAMP-triggered events including capacitation-associated changes. Subsequently, they were incubated with CaCl2 to induce hyperactivation and then used for motility assessment. Many of the spermatozoa after the incubation exhibited full-type hyperactivation which was characterized by high-amplitude and extremely asymmetrical beating of whole middle piece and principal piece. The initiation of full-type hyperactivation required the millimolar concentration of CaCl2 in the medium. However, CaCl2 of the medium was less necessary for maintenance than initiation of full-type hyperactivation, as hyperactivated spermatozoa were barely affected by the incubation with the Ca2+-chelating reagent. On the other hand, the pre-treatment with the inhibitor for Ca2+-dependent protease calpain 1 and 2' clearly suppressed the occurrence of CaCl2-induced hyperactivation without influences on the percentages of motile spermatozoa. Western blotting and indirect immunofluorescence showed distribution of calpain 2 in the middle and principal pieces in which full-type hyperactivated spermatozoa exhibited extremely asymmetrical beating. On the basis of these results, we conclude that the millimolar concentration of extracellular Ca2+ is necessary for the initiation, but not for the maintenance of full-type hyperactivation in boar spermatozoa that beforehand undergo the cAMP-triggered events including capacitation-associated changes. Moreover, we suggest possible involvement of calpain 2 in the intracellular Ca2+ signal transduction leading to full-type hyperactivation.

    WILEY, Mar. 2015, ANDROLOGY, 3 (2), 321 - 331, English

    [Refereed]

    Scientific journal

  • Yohei Mizuno, Ayane Isono, Aya Kojima, Miyuki M. Arai, Taichi Noda, Mitsuhiro Sakase, Moriyuki Fukushima, Hiroshi Harayama

    Livestock spermatozoa possess more tenacious suppressors of cAMP-triggered eventsincluding capacitation-associated changesthan laboratory animal spermatozoa, leading to flagellar hyperactivation. In order to identify the suppressors, we examined effects of an inhibitor of serine/threonine protein phosphatases (calyculin A) on cAMP-triggered changes in the protein phosphorylation state, and subsequent occurrence of hyperactivation and acrosome reaction in ejaculated bull spermatozoa. Ejaculated spermatozoa were incubated in cAMP-supplemented medium, then assessed for motility, acrosome morphology, and phosphorylated protein localization. The addition of calyculin A greatly enhanced cAMP-triggered protein phosphorylation at serine/threonine and tyrosine residues in the connecting piece and induction of flagellar hyperactivation. Most hyperactivated spermatozoa exhibited extremely asymmetrical bends at the middle piece, which produced intensive twisting or figure-eight movements. In the sperm head, however, cAMP-triggered dephosphorylation of serine/threonine-phosphorylated proteins and subsequent acrosome reaction were abolished by the addition of calyculin A. Based on these results, we suggest that calyculin A-sensitive protein phosphatases in the connecting piece are suppressors of cAMP-triggered events leading to hyperactivation. By contrast, similar protein phosphatases in the sperm head accelerate cAMP-triggered events leading to the acrosome reaction. These findings are consistent with the indication that calyculin A-sensitive protein phosphatases have distinct functions in the regulation of cAMP-triggered events in different regions of ejaculated bull spermatozoa. Mol. Reprod. Dev. 82: 232-250, 2015. (c) 2015 Wiley Periodicals, Inc.

    WILEY, Mar. 2015, MOLECULAR REPRODUCTION AND DEVELOPMENT, 82 (3), 232 - 250, English

    [Refereed]

    Scientific journal

  • Taichi Noda, Kenta Minami, Aya Kojima, Yohei Mizuno, Ayane Isono, Mitsuhiro Sakase, Moriyuki Fukushima, Hiroshi Harayama

    The characterization and quantitative analyses of the key transcription factors for spermiogenesis are necessary in the identification of causal factors for the production of the seemingly normal sperm with dysfunctions in Japanese Black bulls and further elucidation of whole aspect of molecular mechanisms for spermiogenesis in livestock. The objective of this study was to obtain the information regarding the characterization and individual changes of an activator cAMP-responsive element modulator (CREM), which is necessary to the normal progress of spermiogenesis and is required for the transcriptional activity of genes coding essential factors for the sperm fertilization ability in rodents, using testes from 21 Japanese Black bulls with the ability to produce sperm indicating the normal motility and morphology. The bull CREM Tay (one of activator variants) was detected in testes more strongly than livers by reverse transcription-polymerase chain reaction and Northern blotting. This variant was localized in the nuclei of spermatids as shown by indirect immunofluorescence with the homemade mouse antiserum. The motility and morphology of the cauda epididymal sperm from 16 Japanese Black bulls were examined before the quantitative analyses of testicular activator CREM to confirm the ability to produce sperm with normal motility and morphology in these males. The percentages of the motile sperm, those of the sperm with the normal acrosomes, and those of morphologically normal sperm were 60.0% to 90.0%, 88.0% to 100%, and 83.0% to 97.9%, respectively. The quantitative analyses with real-time polymerase chain reaction using the testicular RNA from the same bulls revealed that the relative expression levels of activator CREM variants in testes varied significantly among these bulls in the range from 0.56 to 1.64 (P < 0.05). These results are consistent with the suggestions that CREM Tay are involved in the spermiogenesis in the testes of Japanese Black bulls and that the expression levels of the activator CREM variant mRNAs in the testes are varied significantly among individual bulls that have the ability to produce sperm with the normal motility and morphology. (c) 2014 Elsevier Inc. All rights reserved.

    Last, ELSEVIER SCIENCE INC, May 2014, THERIOGENOLOGY, 81 (8), 1012 - 1020, English

    [Refereed]

    Scientific journal

  • Taichi Noda, Mitsuhiro Sakase, Moriyuki Fukushima, Hiroshi Harayama

    There is a serious problem with the reduction of male reproductive performance of the livestock in the world. We have a hypothesis that the splicing error-caused derivation of aberrant sperm motility-related proteins may be one of its causal factors. It is thought that fresh testicular tissues are necessary for the detection of splicing errors of the mRNA. However, it is difficult to obtain testicular tissues from a number of agriculturally important bulls by surgical methods, because such procedures may have deleterious effects on bulls' reproductive performance. The aim of this study was to examine the usefulness of mRNA fragments collected from ejaculated spermatozoa as alternative analytical samples for detection of the splicing errors. In the first experiment, we characterized the alternative splicing and splicing error of bull testicular ADCY10 mRNA which coded the synthase of the regulatory molecule for sperm motility "cAMP". In testes, the exon 11-lacking variant coding the truncated ADCY10 was derived by alternative splicing. However, splicing errors, which accompanied the frame shift in the second cyclase domain, were occasionally observed in the exon 11-lacking variant. This aberrant variant retained intronic nucleotides (4 bases, CCAG) connecting the initial part of exon 10 due to splicing errors and consequently yielded the cleavage site for a restriction enzyme (Cac8I) which recognized the nucleotide sequences (GCNNGC). In the second experiment, we recovered residual testicular mRNA fragments from ejaculated spermatozoa and observed the splicing error-caused derivation of the aberrant variant of ADCY 10. Ejaculated spermatozoa conserved mRNA fragments of the exon 11-lacking variant coding exons 9, 10, 12 and 13. Moreover, the above-mentioned aberrant variant of ADCY10 mRNA fragment was detectable by Cac8I digestion treatment using the sperm mRNAs. These results indicate the utility of sperm mRNA fragments for the detection of splicing errors in bull testicular mRNAs.

    Last, PUBLIC LIBRARY SCIENCE, Feb. 2013, PLOS ONE, 8 (2), e57296, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Taichi Noda, Shou Ishikawa, Osamu Shidara

    In mammalian spermatozoa, the state of protein tyrosine phosphorylation is modulated by protein tyrosine kinases and protein tyrosine phosphatases that are controlled via cyclic AMP (cAMP)-protein kinase A (PKA) signaling cascades. The aims of this study were to examine the involvement of cAMP-induced protein tyrosine phosphorylation in response to extracellular calcium and to characterize effects of pharmacological modulation of the cAMP-induced protein phosphorylation state and calmodulin activity during hyperactivation in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog) and CaCl2 at 38.5 degrees C to induce hyperactivation, and then used for Western blotting and indirect immunofluorescence of phosphorylated proteins and for the assessment of motility. Both cBiMPS and CaCl2 were necessary for hyperactivation. The increase in hyperactivated spermatozoa exhibited a dependence on the state of cBiMPS-induced protein tyrosine phosphorylation in the connecting and principal pieces. The addition of calyculin A (an inhibitor for protein phosphatases 1/2A (PP1/PP2A), 50100?nM) coincidently promoted hyperactivation and cAMP-induced protein tyrosine phosphorylation in the presence of cBiMPS and CaCl2. Moreover, the addition of W-7 (a calmodulin antagonist, 24 mu M) enhanced the percentages of hyperactivated spermatozoa after incubation with cBiMPS and CaCl2, independently of protein tyrosine phosphorylation. These findings indicate that cAMP-induced protein tyrosine phosphorylation in the connecting and principal pieces is involved in hyperactivation in response to extracellular calcium, and that calmodulin may suppress hyperactivation via the signaling cascades that are independent of cAMP-induced protein tyrosine phosphorylation. Mol. Reprod. Dev. 79: 727739, 2012. (C) 2012 Wiley Periodicals, Inc.

    WILEY-BLACKWELL, Oct. 2012, MOLECULAR REPRODUCTION AND DEVELOPMENT, 79 (10), 727 - 739, English

    [Refereed]

    Scientific journal

  • T. Noda, O. Shidara, H. Harayama

    It is necessary to obtain basic information on transcription factors expressed in the spermatids of livestock to determine mechanisms of defective spermiogenesis. In this study, we characterized the activator cAMP responsive element modulator (CREM) ortholog isoforms in porcine testicular germ cells and ejaculated sperm. At least two kinds of porcine activator CREM T family ortholog mRNAs were more strongly expressed in the testis than in kidney or liver. The activator CREM isoform ortholog proteins were localized in nuclei of round spermatids and around nuclei of elongated spermatids. Furthermore, approximately 34% of ejaculated sperm had the activator CREM isoform ortholog proteins at their connecting piece. This is apparently the first report demonstrating the expression and localization of the activator CREM isoform ortholog proteins in spermatids and ejaculated sperm of a livestock species. (C) 2012 Elsevier Inc. All rights reserved.

    Last, ELSEVIER SCIENCE INC, Apr. 2012, THERIOGENOLOGY, 77 (7), 1360 - 1368, English

    [Refereed]

    Scientific journal

  • Ilse Silvia Cayo-Colca, Hiroshi Harayama, Takashi Miyano

    Purpose: We studied the effect of H89, an inhibitor of protein kinase A (PKA), on the meiotic resumption of pig oocytes. Methods: Pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were cultured for 27 h to induce meiotic resumption. COCs and DOs were exposed to H89 for different periods. Oocyte PKA activity was assessed by in vitro kinase assay and immunocytochemistry using an antibody against fully active PKA catalytic subunits. Oocyte serine/threonine (Ser/Thr)-phosphorylated proteins were detected by Western blotting and immunocytochemistry using an anti-pSer/pThr PKA substrate antibody. Results: H89 suppressed germinal vesicle break down (GVBD) in COCs and DOs. To determine whether the suppression was due to inhibition of oocyte PKA, we analyzed oocyte PKA. Kinase assay showed that both types of oocytes possessed PKA activity throughout the culture period. Immunocytochemistry showed that fully active PKA catalytic subunits and Ser/Thr phosphorylated proteins were present in the oocytes at the GV stage and after GVBD. Western blotting indicated that both types of oocytes contained Ser/Thr phosphorylated proteins at the GV stage, and that several proteins became phosphorylated after GVBD. Conclusions: Pig oocytes contain active PKA during the occurrence of GVBD, and H89 suppresses the GVBD. © 2011 Japan Society for Reproductive Medicine.

    John Wiley and Sons Ltd, 2011, Reproductive Medicine and Biology, 10 (2), 89 - 96, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Kazuhiro Nishijima, Tetsuma Murase, Mitsuhiro Sakase, Moriyuki Fukushima

    The aim of this study was to elucidate the relationship between protein tyrosine phosphorylation state and sperm characteristics in frozen-stored spermatozoa of Japanese Black bulls. The spermatozoa were washed with PBS containing polyvinyl alcohol and then incubated with cell-permeable cAMP analog cBiMPS to induce flagellar hyperactivation. Before and after incubation, the spermatozoa were used for immunodetection of tyrosine-phosphorylated proteins, assessment of morphological acrosome condition and evaluation of motility. In bulls whose frozen-stored spermatozoa were classified as having a high-grade acrosome condition before incubation, sperm tyrosine-phosphorylated proteins, including the 33-kDa tyrosine-phosphorylated SPACA1 protein, were localized in the anterior region of the acrosome and equatorial subsegment. The immunodetection level of the 41- and 33-kDa sperm tyrosine-phosphorylated proteins in the Western blots and the immunofluorescence of tyrosine-phosphorylated proteins and SPACA1 proteins in the anterior region of the sperm acrosome were lower in bulls whose frozen-stored sperm were classified as having a low-grade acrosome condition. On the other hand, after incubation with cBiMPS, immunodetection levels of at least 10 tyrosine-phosphorylated proteins increased in the connecting and principal pieces of spermatozoa, coincident with the induction of flagellar hyperactivation. Many of the spermatozoa also exhibited detection patterns similar to those of boar hyperactivated spermatozoa. These results are consistent with the suggestion that immunodetection levels of tyrosine-phosphorylated proteins are valid markers that can predict the level of tolerance to frozen storage and the potential to undergo cAMP-dependent hyperactivation for the spermatozoa of individual Japanese Black bulls.

    WILEY-LISS, Oct. 2010, MOLECULAR REPRODUCTION AND DEVELOPMENT, 77 (10), 910 - 921, English

    [Refereed]

    Scientific journal

  • Evidence of the Existence of Adenylyl Cyclase 10 (ADCY10) Ortholog Proteins in the Heads and Connecting Pieces of Boar Spermatozoa

    Shunsuke Tate, Kazumi Nakamura, Chihiro Suzuki, Taichi Noda, Jibak Lee, Hiroshi Harayama

    The aim of this study is to provide evidence of the existence of the adenylyl cyclase 10 (ADCY-10) ortholog proteins in boar spermatozoa. Experiments with RT-PCR techniques, nucleotide sequence analyses and Northern blot analyses revealed that boar testes exclusively express approximately 5.1-kbp RNA, the nucleotide sequence of which is highly similar to that of human A DCY10. Database analyses with CDART suggested that pig ADCY10 ortholog proteins conserve two catalytic domains of adenylyl cyclase. Western blot techniques and indirect immunofluorescence with a specific antiserum to pig recombinant ADCY10 ortholog proteins showed that 48-kDa and 70-kDa truncated forms of pig ADCY10 ortholog proteins are localized in the equatorial segments and connecting pieces of boar ejaculated spermatozoa. Finally, cell imaging techniques with fluo-3/AM indicated that incubation with sodium bicarbonate (an ADCY10 activator) can initiate the calcium influx in the boar sperm heads that is controlled via the cyclic AMP signaling cascades. These results are consistent with the suggestion that functional ADCY10 ortholog proteins exist in the heads of boar spermatozoa. This is the first direct evidence of the existence of ADCY10 proteins in the heads of mammalian spermatozoa.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Apr. 2010, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 56 (2), 271 - 278, English

    [Refereed]

    Scientific journal

  • Hyperactivated Motility of Frozen-Thawed Spermatozoa from Fertile and Subfertile Japanese Black Bulls Induced by Cyclic Adenosine 3 ',5 '-Monophosphate Analogue, cBiMPS

    Tetsuma Murase, Ismail El-Kon, Hiroshi Harayama, Koushi Mukoujima, Masaki Takasu, Kenji Sakai

    This study investigated whether a cyclic adenosine 3',5'-monopliosphate (cAMP) analogue, cBiMPS, could induce hyperactivated motility in frozen-thawed Japanese Black bull spermatozoa and compared the ability of spermatozoa to undergo hyperactivation between fertile and subfertile bulls. Frozen-thawed spermatozoa from 3 fertile and 2 subfertile bulls were washed, suspended in BO-Hepes medium and incubated in the presence of 0.1 mM cBiMPS for up to 4 h. At 1-h intervals, the spermatozoa were examined for hyperactivated motility. The proportions of spermatozoa showing a circular swimming pattern with asymmetrical flagellar beating and those showing whiplash beating of flagella to the total number of motile spermatozoa were expressed as C% and W%, respectively. The motile spermatozoa % was barely affected by treatment with cBiMPS or the fertility status of the sperm donor, although it gradually decreased in all sperm samples during the 4-h incubation. In the fertile bulls, the C% was 0% at 0 h of incubation but rapidly increased during the 1-h incubation with cBiMPS. It then decreased slightly towards 4 h concomitantly with a gradual increase in W% towards 4 h. In the subfertile bulls, however, the cBiMPS-induced increase of C%. was delayed for 1-3 h, although the incubation time-related changes in mean W% were similar between the fertile and subfertile bulls. In the vehicle controls for cBiMPS, the C% and W% were 0% throughout incubation for all the samples examined. The results suggest that hyperactivation of the flagellum can be induced by the cAMP analogue, cBiMPS, in frozen-thawed Japanese Black bull spermatozoa and that induction of hyperactivation may serve as a useful tool for detection of functional abnormality of spermatozoa from subfertile Japanese Black bulls.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Feb. 2010, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 56 (1), 36 - 40, English

    [Refereed]

    Scientific journal

  • Kana Ishihara, Seiichiroh Ohsako, Ken Tasaka, Hiroshi Harayama, Masashi Miyake, Katsuhiko Warita, Takashi Tanida, Tomoko Mitsuhashi, Takashi Nanmori, Yoshiaki Tabuchi, Toshifumi Yokoyama, Hiroshi Kitagawa, Nobuhiko Hoshi

    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, Jan. 2010, REPRODUCTIVE TOXICOLOGY, 29 (1), 68 - 73, English

    [Refereed]

    Scientific journal

  • Calyculin A-Sensitive Protein Phosphatases are Involved in Maintenance of Progressive Movement in Mouse Spermatozoa In Vitro by Suppression of Autophosphorylation of Protein Kinase A

    Namiko Goto, Hiroshi Harayama

    Protein serine/threonine phosphorylation in mammalian sperm flagella has been considered to play important roles in regulation of motility. Protein phosphorylation state reflects balance of enzymatic activities between protein phosphatases and protein kinases [predominantly protein kinase A (PKA)]. The aims of this study were to disclose roles of protein phosphatases in the regulation of sperm motility and to provide evidence for suppression of PKA full activation by protein phosphatases in sperm flagella. Mouse epididymal spermatozoa were incubated with a cell-permeable protein phosphatase 1. (PP1)/protein phosphatase 2A (PP2A) inhibitor (calyculin A: 25-125 nM) at 37.5 C. After incubation, they were used for immunodetection of phosphorylated proteins, PKA and PP1 gamma 2, assessment for motility and co-immunoprecipitation of PP1 gamma 2 with PKA. Incubation with calyculin A enhanced the phosphorylation states of several proteins (>250 kDa, 1.70 kDa, 155 kDa, 140 kDa and 42 kDa for serine/threonine phosphorylation and 70 kDa for tyrosine phosphorylation) and PKA catalytic subunits [at the autophosphorylation residue (Thr-197) for its full enzymatic activation] in the flagella. Coincidently, this incubation induced changes of sperm flagellar movement from the progressive type to the hyperactivation-like type. Indirect immunofluorescence and co-immunoprecipitation showed that PKA was co-localized with PP1 gamma 2 in the principal pieces of sperm flagella. These findings suggest that calyculin A-sensitive protein phosphatases (PP1/PP2A) suppress full activation of PKA as well as enhancement of the phosphorylation states of other flagellar proteins in sperm flagella in order to prevent precocious changes of flagellar movement from the progressive type to hyperactivation.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Jun. 2009, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 55 (3), 327 - 334, English

    [Refereed]

    Scientific journal

  • A 32-kDa Tyrosine-phosphorylated Protein Shows a Protease-dependent Increase in Dead Boar Spermatozoa

    Tomohito Tabuchi, Osamu Shidara, Hiroshi Harayama

    Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is Still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including crvocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TvrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 call show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Dec. 2008, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 54 (6), 502 - 507, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Kazumi Nakamura

    A cAMP-induced protein tyrosine phosphorylation and flagellar hyperactivation are controlled via complicated signaling cascades in mammalian spermatozoa. For instance, these events seem to be regulated positively by the PKA-mediated signaling and negatively by the PI3K/PDK1 -mediated signaling. In this article, we have shown molecular changes of PKA and PDK1 in cAMP analog (cBiMPS)-treated boar spermatozoa in order to disclose possible roles of these kinases in protein tyrosine phosphorylation and hyperactivation. Ejaculated spermatozoa were incubated with cBiMPS, and then they were used for biochemical analyses of sperm kinases by Western blotting and indirect immunofluorescence and for assessment of flagellar movement. The first 30-min incubation with cBiMPS highly activated PKA of the principal piece to the accompaniment of autophosphorylation on Thr-197 of catalytic subunits. However, protein tyrosine phosphorylation and hyperactivation were fully induced in the sperm samples after the 180-min incubation. A potentially active form of PDK1 (54/55-kDa phospho-PDK1) was detected in the principal piece of the spermatozoa during the 90-min incubation. Another potentially active form (59-kDa phospho-PDK1) gradually increased during the same incubation period. However, the PDK1 suddenly became inactive by the dephosphorylation after the 180-min incubation, namely coincidently with full induction of protein tyrosine phosphorylation and hyperactivation. Additionally, existence of POK-dependently suppressing mechanisms for protein tyrosine phosphorylation was confirmed in the principal piece by pharmacological experiments with LY294002 and biochemical analyses with anti-PI3K p85 antibodies. These findings suggest that dephosphorylation of PDK1 may be a molecular switch for enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa.

    WILEY-BLACKWELL, Sep. 2008, MOLECULAR REPRODUCTION AND DEVELOPMENT, 75 (9), 1396 - 1407, English

    [Refereed]

    Scientific journal

  • Effects of protein phosphatase inhibitor calyculin a on the postacrosomal protein serine/threonine phosphorylation state and acrosome reaction in bear spermatozoa incubated with a cAMP analog

    Jun Adachi, Shunsuke Tate, Masashi Miyake, Hiroshi Harayama

    In order to reveal the involvement of the sperm postacrosomal region in the acrosome reaction, we examined the effects of the protein phosphatase inhibitor calyculin A on the postacrosomal protein serine/threonine phosphorylation state and acrosorne morphology in boar spermatozoa incubated with a cAMP analog. Proteins were highly phosphorylated on the serine/threonine residues only in the postacrosomal region before incubation. After 90-min incubation. without calyculin A, the protein phosphorylation state declined in the postacrosomal region irrespective of the capacitation state while it remained under the detectable level in the other regions of the sperm head. However, addition of calyculin A effectively suppressed the decline in protein phosphorylation state and increased an. inactive form of protein phosphatase I in the postacrosomal region. On the other hand, this inhibitor had no influence on the protein phosphorylation state in the acrosome and equatorial segment. After incubation without calyculin A for 180 or 360 min, many spermatozoa exhibited acrosomal changes and loss that indicated occurrence of the acrosome reaction. However, addition of calyculin A significantly blocked these events. These results are consistent with our suggestion that postacrosomal serine/threonine-phosphorylated proteins are involved in suppression of the acrosome reaction in boar spermatozoa in vitro.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Jun. 2008, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 54 (3), 171 - 176, English

    [Refereed]

    Scientific journal

  • Murase Tetsuma, Ismail El-kon, Mukohjima Kohji, Harayama Hiroshi, Takasu Masaki, Sakai Kenji

    【目的】黒毛和種種雄牛において,一般精液性状に異常が認められないにも関わらず人工授精後の受胎成績が低下した例が見られ,本種の繁殖に支障を来している。このため一般検査では発見できない異常を調べる新規検査法の開発を目的として,鞭毛の超活性化運動の誘起能力が指標として使用できるか否かを検討した。【方法】受胎率が正常な個体 3 頭のそれぞれ3射出精液(合計9射出)及び低下した2頭(ウシ A および B)の凍結融解精液(ウシ A の3射出およびウシ B の 2 射出)を使用した。精子を洗浄後,0.1 mM cAMP アナログ(cBiMPS)を添加あるいは無添加(対照)の 0.1% ポリビニルアルコール含有 HEPES-緩衝 BO 液に再浮遊し,38.5°Cにて 0~4 時間培養した。1 時間おきに一部を取り運動性を位相差顕微鏡下で観察した。100~200 倍で円を描いて運動する精子で,400 倍にて尾部の振動が左右非対称性である精子を円運動とした。また,移動をほとんどせずその場で激しく尾部を振動させる精子を鞭打ち運動と判定した。運動する全精子に対する円運動及び鞭打ち運動する精子の割合を超活性化運動(HA)率とした。【結果】正常な個体の 9 射出精液においては,cBiMPS 存在下の培養 1 時間後に HA 率が急激に上昇し(平均 82%),4 時間(平均 88%)まで持続した。培養 1 時間における HA 精子のうちほとんどが円運動精子であり,培養 3 時間まで徐々に鞭打ち運動する精子の割合が増加した。一方,低受胎率を示す個体においては,培養1時間後において,ウシ A の1射出精液の場合(17%)を除き,HA 率は 0% であった。また,ウシ A においては,培養 2 時間後に円運動を含む HA 率が上昇し(38~73%),ウシ B においては,培養 2 時間までに円運動がほとんど見られないにも関わらず,2 あるいは 3 時間後に HA 率が上昇する(11~100%)特異な変化が見られた。対照においてはいずれも HA 率は 0% であった。【考察】cBiMPSにより誘起される凍結融解精子の HA を指標として低受胎率を示す精液サンプルを検出できる可能性が示された。

    The Society for Reproduction and Development, 2008, The Journal of Reproduction and Development Supplement, 101, 204 - 204, Japanese

  • Premature Capacitation of Frozen-Thawed Spermatozoa from Subfertile Japanese Black Cattle

    KURODA Ken, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    Nov. 2007, J. Reprod. Dev., 53(5), pp.1079-1086, English

    [Refereed]

    Scientific journal

  • Evidence for existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa

    Reiko Amano, Jibak Lee, Namiko Goto, Hiroshi Harayama

    Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeCPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Feb. 2007, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 53 (1), 127 - 133, English

    [Refereed]

    Scientific journal

  • A cyclic adenosine 3',5'-monophosphate-dependent protein kinase C activation is involved in the hyperactivation of boar spermatozoa.

    HARAYAMA Hiroshi, MIYAKE Masashi

    Sep. 2006, Mol. Reprod. Dev., 73(9), pp.1169-1178, 1169 - 1178, English

    [Refereed]

    Scientific journal

  • M Isaji, H Iwata, H Harayama, M Miyake

    We have shown that the assembly of lamin-associated polypeptide (LAP) 2 beta was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2 beta assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2 beta assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2 beta assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2P assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2 beta did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2 beta assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2 beta assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2 beta assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2 beta assembly around oCh but not histone H3 dephosphorylation.

    CAMBRIDGE UNIV PRESS, May 2006, ZYGOTE, 14 (2), 157 - 167, English

    [Refereed]

    Scientific journal

  • H Harayama, T Murase, M Miyake

    The aim of this study was to reveal a downstream part of the intracellular signaling that is mediated by cyclic adenosine monophosphate (cAMP)-dependent tyrosine kinases, including spleen tyrosine (Y) kinase (SYK), in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog; 0.1 mM) at 38.5 degrees C for 180 minutes and then used for Western blot and indirect immunofluorescence. Incubation of spermatozoa with cBiMPS induced tyrosine phosphorylation at the linker region of SYK (which was essential to binding to phospholipase C [PLC]gamma l) in the connecting and principal pieces, but the tyrosine phosphorylation was abolished by the addition of H-89 (a protein kinase A [PKA] inhibitor; 0.01-0.1 mM). Moreover, the cAMP-dependent tyrosine phosphorylation was also induced at the key regulatory residue of PLC gamma 1 in the same segments of spermatozoa, but it was inhibited by the addition of herbimycin A (a tyrosine kinase inhibitor; 5 mu M). These results suggest that the sperm cAMP-dependent tyrosine kinases, including SYK, are linked to the activation of PLC gamma 1. Indirect immunofluorescence clearly detected both inositol 1, 4,5-trisphosphate (IP3) receptor and calreticulin in the connecting piece, indicating the presence of internal calcium store. Cell imaging with fluo-3/AM (a cell-permeable Ca2+ indicator) showed that incubation of spermatozoa with cBiMPS increased intracellular free calcium in the middle piece, but that it was reduced by the addition of U-73122 (a PLC inhibitor; 0.02 mM). Based on our findings, we conclude that the connecting piece of boar spermatozoa possesses the PLC gamma 1-IP3 receptor-calcium signaling that is triggered by cAMP and mediated by PKA and herbimycin A-sensitive tyrosine kinases, including SYK.

    AMER SOC ANDROLOGY, INC, Nov. 2005, JOURNAL OF ANDROLOGY, 26 (6), 732 - 740, English

    [Refereed]

    Scientific journal

  • Exogenous hyaluronic acid enhances porcine parthenogenetic embryo development in vitro possibly mediated by CD44

    Toyokaw K, Harayama H, Miyake M

    Jan. 2005, Theriogenology, 64 (1), 378 - 392, English

    [Refereed]

    Scientific journal

  • H Harayama, M Muroga, M Miyake

    A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5degreesC for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the antiphospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+ -independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa. (C) 2004 Wiley-Liss, Inc.

    WILEY-LISS, Dec. 2004, MOLECULAR REPRODUCTION AND DEVELOPMENT, 69 (4), 436 - 447, English

    [Refereed]

    Scientific journal

  • H Harayama, K Sasaki, M Miyake

    A cAMP-induced increase of tyrosine-phosphorylated proteins is involved in the expression of fertilizing ability in mammalian spermatozoa. We (Harayama, 2003: J Androl 24:831-842) reported that incubation of boar spermatozoa with a cell-permeable cAMP analog (cBiMPS) increased a 32-kDa tyrosine-phosphorylated protein (TyrP32). The purpose of this study is to characterize the signaling cascades that regulate the cAMP-induced increase of TyrP32. We examined effects of tyrosine kinase inhibitor (lavendustin A), tyrosine phosphatase inhibitor (Na3VO4), cell-permeable calcium chelator (BAPTA-AM), and cholesterol acceptor (methyl-beta-cycloclextrin: MBC) on the increase of TyrP32 and the change and loss of acrosomes in boar spermatozoa. The spermatozoa were used for detection of tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence and for examination of acrosomal integrity by Giemsa staining. At least eight tyrosine-phosphorylated proteins including TyrP32 exhibited the cAMP-dependent increase during incubation with cBiMPS. In many proteins of them, this increase was reduced by lavendustin A but was enhanced by Na3VO4. In contrast, the cAMP-induced increase of TyrP32 was abolished by Na3VO4 but was hardly affected by lavendustin A. Giemsa staining showed that the increase of spermatozoa with weakly Giemsa-stained acrosomes (severely damaged acrosomes) or without acrosomes was correlative to the cAMP-induced increase of TyrP32. Moreover, the lack of calcium chloride in the incubation medium or pretreatment of spermatozoa with BAPTA-AM blocked the change and loss of acrosomes and the increase of TyrP32, suggesting these events are dependent on the extracellular and intracellular calcium. On the other hand, incubation of spermatozoa with MBC in the absence of cBiMPS could mimic the change and loss of acrosomes and increase of TyrP32 without increase of other tyrosine-phosphorylated proteins. Based on these results, we conclude that the cAMP-induced increase of TyrP32 is regulated by a unique mechanism that may be linked to the calcium-dependent change and loss of acrosomes. (C) 2004 Wiley-Liss, Inc.

    WILEY, Oct. 2004, MOLECULAR REPRODUCTION AND DEVELOPMENT, 69 (2), 194 - 204, English

    [Refereed]

    Scientific journal

  • M Isaji, H Iwata, H Harayama, M Miyake

    The present study was designed to clarify the localization of LAP2beta and to compare it with those of lamins A/C and B in bovine oocytes after activation and in vitro fertilization (IVF). After fertilization, LAP2beta was not found until telophase II, and was observed around condensed chromatin after the extrusion of the second polar body, but not in activated oocytes. Although the reaction of LAP2beta was temporally negative or weak on the membrane of the growing small pronuclei, it became strong on the fully grown pronuclei of both activated and fertilized oocytes. Examination of the timing of DNA synthesis using bromodeoxyuridine revealed that the expression of LAP2beta on the pronuclear membrane became strong around the end of the DNA synthesis in both activated and fertilized oocytes. Both male and female pronuclei exhibited the same reactivity to all nuclear proteins examined. It was also shown that LAP2beta first assembled around condensed chromatin, followed by the integration of lamins B and A/C as in somatic cells. LAP2beta staining was maintained on the nuclear membrane of the embryonic cells at interphase until the later stage of preimplantational development. There were no differences between parthenogenetic and fertilized embryos in the expression and localization of LAP2beta from the PN-stage oocyte to the blastocyst. The assembly of LAP2beta was observed around the telophase chromatin of both blastocyst and cumulus cells. Thus, it was shown that the timing of the aggregation of LAP2beta at the second meiosis was different from that in the mitosis of blastocyst and somatic cells. LAP2beta was constantly expressed in the nuclear membrane in iii vitro fertilized and parthenogenetic embryos as was lamin B, and lamin A/C was expressed stage-dependently in both types of embryos. Lamin A/C was positive in some inner cell mass cells of parthenogenetic blastocysts, but not those of iii vitro fertilized embryos.

    CAMBRIDGE UNIV PRESS, Feb. 2004, ZYGOTE, 12 (1), 81 - 93, English

    [Refereed]

    Scientific journal

  • Viability and protein phosphorylation patterns of boar spermatozoa agglutinated by treatment with a cell-permeable cyclic adenosine 3 ',5 '-monophosphate analog

    H Harayama

    Boar spermatozoa become agglutinated with one another at the head when their intracellular cyclic adenosine 3',5'-monophosphate (cAMP)-signaling cascades are activated in the head. The aim of the present study is to examine viability and protein phosphorylation patterns of cAMP-dependently agglutinated boar spermatozoa. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium containing polyvinyl alcohol (mKRH-PVA) plus 0.1 mM Sp-5,6-clichloro-1-beta-D-ribofuranosyl-benzimidazole-3',5'-monophosphorothioate (cBiMPS, a cell-permeable cAMP analog) at 38.5degreesC up to 180 minutes. Aliquots of the sperm suspensions were recovered after various incubation periods and then used to examine the state of agglutination, the viability by SYBR14-PI staining and motility assay, and the state of protein phosphorylation by Western blotting and indirect immunofluorescence. In the control samples incubated without cBiMPS for 180 minutes, less than 30% of the total spermatozoa were agglutinated with one another at the heads, and more than 70% of the agglutinated spermatozoa were propidium iodide (PI)-positive (dead). However, the incubation with cBiMPS rapidly increased the percentages of head-to-head agglutinated spermatozoa to approximately 60% within 30 minutes, but did not significantly change them thereafter. In the samples incubated with cBiMPS for 180 minutes, moreover, the percentages of PI-positive cells of the agglutinated spermatozoa (approximately 30%) were significantly lower than those obtained in the control samples (more than 70%). This result was supported by the observation that the percentages of motile cells of the agglutinated spermatozoa were much higher in the samples incubated with cBiMPS for 180 minutes than in the control samples incubated without cBiMPS. As revealed by Western blotting and indirect immunofluorescence, cBiMPS-induced serine/threonine phosphorylation of the proteins (eg, >220 kd, 220 kd, 180 kd, 84 kd, and 54 kd) appeared mainly in the connecting and principal pieces of both agglutinated and free spermatozoa within 30 minutes, and additional phosphorylation occurred in the middle piece later than 30 minutes. Moreover, tyrosine phosphorylation of the proteins (eg, >220 kc, 190 kc, 93 kd, 59 kd, 54 kc, and 32 kd) was induced intensely in the connecting and principal pieces and moderately in the middle piece of almost one half of the agglutinated spermatozoa after incubation with cBiMPS for more than 30 minutes, but rarely in those of the free spermatozoa. These findings are consistent with the following suggestions: activation of the cAMP-signaling cascades leads to rapid (within 30 minutes) head-to-head agglutination in live spermatozoa; rapid (within 30 minutes) protein serine/threonine phosphorylation in the connecting and principal pieces of both cAMP-dependently agglutinated and free spermatozoa and subsequent (later than 30 minutes) phosphorylation in the middle piece of them; and slow (later than 30 minutes) protein tyrosine phosphorylation in the connecting, middle, and principal pieces of the cAMP-dependently agglutinated spermatozoa. Based on these suggestions, we conclude that many of cAMP-dependently agglutinated spermatozoa are live cells in which cAMP-signaling cascades leading to protein serine/threonine and tyrosine phosphorylation are activated in the whole flagellum.

    AMER SOC ANDROLOGY, INC, Nov. 2003, JOURNAL OF ANDROLOGY, 24 (6), 831 - 842, English

    [Refereed]

    Scientific journal

  • Effects of season on concentrations of seminal plasma acidic proteins from Japanese Black cattle

    西本 尚美, 中村 弘毅, 岩木 史之, 岡 章生, 福島 護之, 太田垣 進, 原山 洋

    Sep. 2003, Japanese Journal of Embryo Transfer, 25,pp.101-107 (3), 101 - 107, Japanese

    [Refereed]

    Scientific journal

  • Stage-specific effects of the osmolarity of a culture medium on the development of parthenogenetic diploids in the pig

    VT Nguyen, S Kure-bayashi, H Harayama, T Nagai, M Miyake

    The objective of this study was to investigate the effects of osmolarity of culture media on the development of porcine parthenogenetic diploids. Oocyte-cumulus-granulosa cell complexes were collected from ovaries and then in vitro-cultured for 48 h. The mature oocytes were subjected to a single electro-stimulation (El-St; 100 mus, 1500 V/cm), treated with 5.0 mug/ml Cytochalasin B for 4 h and then cultured under various conditions as described below. In Experiment 1, the diploids were cultured for 168 h after El-St in modified Whitten's medium with 256 mOsmol (mWM256), mKRB with 309 mOsmol, and mWM with 309 mOsmol (mWM309), in which the osmolarity was adjusted by addition of NaCl or mannitol, or by reduction of distilled water. In Experiment 2, the diploids were cultured in the five media used in Experiment I for the first 48 h, and then in mWM256 until 168 h after El-St. In Experiment 3, the diploids were cultured for the first 48 h in mWM with osmolarity adjusted from 256 to 330 mOsmol by addition of NaCl for the first 48 h and then in mWM256 until 168 It after EI-St. In Experiment 4, the diploids were cultured in mWM with 290 mOsmol (mWM290) for the first period of 24, 48, or 72 h, and then in mWM256 untDil 168 h after El-St. In Experiment 5, after diploids were cultured in mWM290 for the first 48 h, the obtained 4-cell diploids were transferred to mWM with osmolarity adjusted from 200 to 310 mOsmol by addition of NaCl, then cultured until 168 h after EI-St. All media were supplemented with 0.5 mg/ml hyaluronic acid and 4.0 mg/ml bovine serum albumin. The results obtained in Experiments 1-5 indicate that the osmolarity of a medium, but not the Na+/K+ ratio, exerts effects on the development of diploids to the blastocyst stage. The change of osmolarity of the culture media after the 4-cell stage increased the rate of expanded blastocyst formation in porcine diploids. The optimal osmolarities of culture medium for the first 48 h after El-St (before the 4-cell stage) were 290 and 280-320 mOsmol, and those for the later period (after the 4-cell stage) were 256 and 220-270 mOsmol, respectively. (C) 2002 Elsevier Science Inc. All rights reserved.

    ELSEVIER SCIENCE INC, Feb. 2003, THERIOGENOLOGY, 59 (3-4), 719 - 734, English

    [Refereed]

    Scientific journal

  • Involvement of cytoplasmic free calcium in boar sperm: head-to-head agglutination induced by a cell-permeable cyclic adenosine monophosphate analog

    HARAYAMA Hiroshi, OKADA K, MIYAKE Masashi

    Feb. 2003, J. Androl., 24,pp.91-99, 91 - 99, English

    [Refereed]

    Scientific journal

  • Localization of Lap2B in oocytes and embryos during early development in cattle

    Isaji M, Iwata H, Ohota M, Harayama H, Miyake M

    LAP2βはラミンの裏打ち蛋白であるがその機能については不明な点が多い。ウシ胚においてこのLAP2βの局在と発現について観察した。LAP2βの発現や局在は体外受精卵や単為発生胚で差がなく発生が進むにつれて前核期よりも発現が強くなることを示した。

    Jan. 2003, Theriogenology 第59巻 474 (abstr), English

    [Refereed]

    Scientific journal

  • Capacitation-like alterations in cooled boar spermatozoa: assessment by the chlortetracycline staining assay and immunodetection of tyrosine-phosphorylated sperm proteins

    M Kaneto, H Harayama, M Miyake, S Kato

    This study was undertaken in order to characterize alterations occurring in cooled boar spermatozoa by a chlortetracycline (CTC) staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Spermatozoa were collected from 10 mature boars, washed and then resuspended in a Tris-citric acid-glucose (TCG) solution. The sperm suspensions were slowly cooled to 4 degreesC over 5 h and held for 2 days. Aliquots of the sperm suspensions were recovered before and after the cooling treatment and then used for the CTC staining assay and immunodetection of tyrosine-phosphorylated sperm proteins. Before the cooling treatment, almost all of the spermatozoa stained with CTC were characterized by uniform fluorescence over the whole head (an F pattern: uncapacitated spermatozoa). After the cooling treatment, however, significant higher percentages of spermatozoa exhibited a B pattern with a dark band of diminished fluorescence in the post acrosomal region and a relatively bright fluorescence in the acrosomal region (the pattern of capacitated spermatozoa). Coincidently, a 32 kDa tyrosine-phosphorylated protein appeared in the spermatozoa. However, these alterations occurring in the cooled spermatozoa were attenuated by the supplementation to the sperm suspensions with seminal plasma (20% (v/v)). Additionally, the same alterations were observed in the spermatozoa incubated in a capacitation-supporting medium (a modified Krebs-Ringer bicarbonate; mKRB) for 5 h. These results suggest that cooling could induce capacitation-like alterations in boar spermatozoa that were associated with the tyrosine phosphorylation of the 32 kDa sperm protein. (C) 2002 Elsevier Science B.V. All rights reserved.

    Corresponding, ELSEVIER SCIENCE BV, Oct. 2002, ANIMAL REPRODUCTION SCIENCE, 73 (3-4), 197 - 209, English

    [Refereed]

    Scientific journal

  • Relationship between bicarbonate and cyclic nucleotide in the promoting effects on head-to-head agglutination in boar spermatozoa

    H Harayama, S Kato

    Aim: To clarify the relationship between bicarbonate and cAMP in the promoting effects on the sperm agglutination. Methods: Spermatozoa were collected from mature boars, washed and resuspended in a modified Krebs-Ringer HEPES lacking calcium chloride (mKRH). The sperm suspensions were incubated in a water bath (38.5 degreesC) for 60 min and then the percentage of head-to-head agglutinated spermatozoa was determined. Results: Supplementation of the mKRH with sodium bicarbonate (5-10 mM) significantly raised the percentage of head-to-head agglutinated spermatozoa in the samples. The addition of selective inhibitors for calcium/calmodulin-dependent phosphodiesterases (type 1: 8-methoxymethyl-IBMX and vinpocetine, 25-50 muM) or for cAMP-specific phosphodiesterases (type 4: Ro20-1724 and rolipram, 25-50 muM) enhanced the effect of bicarbonate on sperm agglutination as highly as did the addition of non-selective inhibitors for phosphodiesterases (IBMX and papaverine, 25-50 muM). A calmodulin antagonist (W-7, 2 muM), that potentially blocks the stimulator of the calcium/calmodulin-dependent phosphodiesterases, significantly enhanced the effect of bicarbonate on sperm agglutination. Moreover, a phosphodiesterase-resistant cAMP analogue (cBiMPS, 0.1 mM) markedly induced agglutination in more spermatozoa (76%) after the incubation without bicarbonate and phosphodiesterase inhibitors than did a less potent cAMP analogue (dibutyryl cAMP, 1 mM) (21%), while three kinds of cGMP analogues (0.1-1 mM) had no effect on sperm agglutination. In addition, a cAMP antagonist (Rp-cAMPS, 1 mM) significantly reduced the sperm agglutination resulting from the actions of bicarbonate and IBMX. On the other hand, the effect of bicarbonate was abolished by a change of incubation temperature from 38.5degreesC to 25degreesC. Conclusion: These findings demonstrate that the bicarbonate-induced agglutination of boar spermatozoa is controlled via the cAMP-mediated, temperature-dependent signaling cascade. This cascade is suppressed by the action of the phosphodiesterase (at least types 1 and 4).

    SCIENCE CHINA PRESS, Jun. 2002, ASIAN JOURNAL OF ANDROLOGY, 4 (2), 87 - 96, English

    [Refereed]

    Scientific journal

  • Characteristics of preimplantational development of porcine parthenogenetic diploids relative to the existence of amino acids in vitro

    Van Thuan N, Hiroshi HARAYAMA, Masashi MIYAKE

    Jun. 2002, Biol Reprod., 67(6), pp. 1688-1698, 1688 - 1698, English

    [Refereed]

    Scientific journal

  • The development of porcine parthenogenetic diploid oocytes with homogeneous genomic components in vitro

    NV Thuan, H Harayama, M Miyake

    The aim of this study was to determine the influence of the homogeneity of genomic components and ploidy on the in vitro development of porcine parthenogenetic oocytes to the blastocyst stage. In vitro matured oocytes were subjected to a single pulse of electro-stimulation (El-St; 100 musec, 1,500 V/cm) for activation. First, the activated oocytes were cultured for 6, 16, 18, 20 and 22 h after El-St, and examined for the timing of the first cleavage of parthenogenetic haploids. Next, the effects of the timing and duration of cytochalasin B (CB) treatment on inhibition of the first cleavage of haploids were examined in order to produce homogenous parthenogenetic diploids. Then the developmental ability to the blastocyst stage was compared among activated oocytes with genomic components of haploid (without CB treatment), homogeneous diploid (nn-diploid, CB treatment for 6 h from 20 h after El-St), and heterogeneous diploid (nn'-diploid, CB treatment for 4 h immediately after El-St). Most haploids were at the prophase to telophase of the first cleavage between 16 and 22 h after El-St. When the haploids were treated with 5.0 mug/ml CB for 6 h from 20 h after El-St, their first cleavage was efficiently suppressed, and most of them (84%) became diploids. The frequency of parthenogenetic development to the blastocyst stage was significantly lower in haploids (5%) than in nn-diploids (48%, P<0.01) or nn'-diploids (57%, P<0.01). These results shows that ploidy of activated oocytes, but not the homology of genomic components, affects the development of porcine parthenogenetic oocytes to the blastocyst stage.

    SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Apr. 2002, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 48 (2), 157 - 166, English

    [Refereed]

    Scientific journal

  • Detection of a fertility-associated protein osteopontin in seminal plasma from Japanese Black cattle

    FURUTSUKA Masayuki, HARAYAMA Hiroshi, MIYAKE Masashi, FUKUSHIMA Moriyuki, OHTAGAKI Susumu, KATO Seishiro

    Corresponding, Jan. 2002, Japanese Journal of Embryo Transfer, 24(1), pp.1-8 (1), 1 - 8, Japanese

    [Refereed]

    Scientific journal

  • Biochemical characterization of sialoprotein "anti-agglutinin" purified from boar epididymal and seminal plasma

    H Harayama, PC Liao, DA Gage, M Miyake, S Kato, RH Hammerstedt

    Sialoprotein "anti-agglutinin," previously shown to inhibit sperm head-to-head agglutination, is found in both boar epididymal and seminal plasma. The present report characterizes anti-agglutinin by mass spectrometry, by N-terminal amino acid sequence analysis, and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting techniques to assess phosphate content of the molecule. Anti-agglutinin had the SDS-PAGE mobility of approximately 25 kDa. By electrospray ionization-mass spectrometry, however, mass spectra of anti-agglutinin were characterized by two major peaks (19,379-19,382 Da and 19,395-19,397 Da) and several minor peaks. Mass spectrometry of tryptic peptide fragments of deglycosylated anti-agglutinin and amino acid sequence analysis revealed that the protein has a unique peptide-mass fingerprinting of fragments (12,668 Da, 5,209 Da, 1,226 Da, and 1,168 Da) and a novel N-terminal amino acid sequence (KTDDY AISGA KEEEF YDYME ELYAV), respectively. Additionally Western blot techniques, using commercially available monoclonal antibodies, were used to detect presence of phosphothreonine and phosphoserine substituents, but two different monoclonal antibodies did not detect phosphotyrosine. Moreover, treatment with two different alkaline phosphotases converted the molecule, as assessed by SDS-PAGE and detection by silver stain, from the parent form of about 25 kDa to forms of approximately 19 kDa (similar to that assigned by mass spectrometry) and/or 15 kDa. Original antiserum generated toward, and reacting with native anti-agglutinin, reacted only with 19 kDa form. These results are consistent with the conclusion that the native anti-agglutinin may be a novel protein that is phosphorylated at serine and/or threonine residues. (C) 2000 Wiley-Liss, Inc.

    WILEY-LISS, Jan. 2000, MOLECULAR REPRODUCTION AND DEVELOPMENT, 55 (1), 96 - 103, English

    [Refereed]

    Scientific journal

  • Role of cyclic adenosine 3 ',5 '-monophosphate and serum albumin in head-to-head agglutination of boar spermatozoa

    H Harayama, M Miyake, S Kato

    It has previously been shown that when boar spermatozoa are incubated in a modified Krebs-Ringer bicarbonate (mKRB), head-to-head agglutination occurs in many cells. The aim of the present study was to investigate the effects of cyclic adenosine 3',5'-monophosphate (cAMP) and serum albumin on sperm agglutination and to discuss a possible mechanism for sperm agglutination. Spermatozoa were collected from four mature boars, washed and incubated in mKRB. After a 1-h incubation, a sample of each sperm suspension was smeared gently on a separate glass slide, dried and stained in a phosphate-buffered solution of Giemsa to assess the percentage of head-to-head agglutinated cells in each suspension. In the samples incubated in mKRB, approximately 50% of the spermatozoa were agglutinated with one another at the acrosome. However, the percentages of head-to-head agglutinated spermatozoa were greatly reduced by a lack of calcium chloride in mKRB, but were recovered by the addition of dibutyryl cAMP (dbcAMP, a cAMP analogue) in a dose-dependent manner between 1 and 1000 muM. Addition of 3-isobutyl-1-methylxanthine (IBMX, 100 and 500 muM) instead of dbcAMP also significantly increased the percentages of head-to-head agglutinated spermatozoa. Moreover, the effects of adding dbcAMP were attenuated by treatment with Rp-adenosine 3',5'-cyclic monophosphorothioate triethylamine salt (0.25-1.0 mM, a cAMP antagonist) or H-89 (5 muM, a protein kinase-A inhibitor), but were enhanced by treatment with okadaic acid (500 nM) and calyculin A (500 nM) (inhibitors of protein serine/threonine phosphatase). In sperm samples incubated in mKRB containing 0.1% polyvinyl alcohol (mKRB-P) or mKRB-P lacking calcium chloride and supplemented with 1 mM dbcAMP, a lack of bovine serum albumin (BSA) resulted in a significant decrease in the percentages of head-to-head agglutinated spermatozoa. Addition of porcine serum albumin (PSA, 1-4 mg mL(-1)) or methyl-beta -cyclodextrin (MBC, 5-10 mg mL(-1)) instead of BSA was as effective as BSA (4 mg mL(-1)) in enhancing sperm agglutination. However, the effects of BSA (4 mg mL(-1)) or MBC (5 mg mL(-1)) were reduced by pre-mixing these reagents with cholesterol 3-sulfate (a cholesterol analogue, 5 mug mL(-1) for BSA and 375 mug mL(-1) for MBC). In addition, a protein 'anti-agglutinin' inhibiting sperm agglutination, was extracted from spermatozoa incubated with serum albumin or MBC and detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. The obtained Western blots revealed that sperm-bound anti-agglutinin was detected less in the samples incubated with either BSA (4 mg mL(-1)) or MBC (5-10 mg mL(-1)), compared with control samples. Moreover, pre-mixing MBC (5 mg mL(-1)) with cholesterol 3-sulfate (375 mug mL(-1)) reduced this reagent's effects on the loss of sperm-bound anti-agglutinin. Additionally, the assay of sperm agglutination and a chlortetracycline staining assay revealed that the percentages of head-to-head agglutinated spermatozoa were positively correlated with those of spermatozoa classified into B pattern (capacitated spermatozoa). These results are consistent with the following suggestions: (i) an adenylyl cyclase-cAMP-protein kinase system mediates a signalling pathway leading to head-to-head agglutination; and (ii) loss of anti-agglutinin from the spermatozoa may be modulated by changes in the plasma membrane induced by actions of serum albumin or MBC contained in a medium.

    C S I R O PUBLISHING, 2000, REPRODUCTION FERTILITY AND DEVELOPMENT, 12 (5-6), 307 - 318, English

    [Refereed]

    Scientific journal

  • Immunolocalization of anti-agglutinin for spermatozoa in boars

    H Harayama, M Miyake, S Kato

    A boar "anti-agglutinin," which inhibits head-to-head agglutination of spermatozoa, has been identified as a 25-kDa sialoprotein contained in epididymal and seminal plasma. This study was conducted to determine the location of the anti-agglutinin on spermatozoa and in various organs, including epididymides, by indirect immunofluorescence and Western blotting techniques. Ejaculated boar spermatozoa were washed and subjected to immunocytochemical observation. Epididymal plasma was recovered from three different regions of epididymides and subjected to sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting. Twelve kinds of organs (testis, epididymis, seminal vesicle, prostate, heart, liver, kidney, spleen, stomach, small intestine, lung, and muscle) were recovered from boars, The unilateral epididymides were fixed, cut into 10-mu m frozen sections, and subjected to immunohistochemical observation. The other organs were homogenized and used for SDS-PAGE and Western blotting. Immunocytochemical observations revealed that the antiserum strongly recognized the acrosomal region and equatorial segment on unfixed and methanol fixed spermatozoa. Immunohistochemical observations revealed that the epithelia of the epididymal ducts were recognized by the antiserum mainly in the corpus epididymides. Moreover, the antiserum reacted with the luminal contents of the corpus and cauda epididymides, However, no specific reaction was detected in the caput epididymides. Western blotting showed that the antiserum selectively recognized a band of the anti-agglutinin in the corpus and cauda epididymal plasma, although no band was detected in the caput epididymal plasma. In the extracts from various organs, the single band was detected in the corpus and cauda epididymides at the same mobility as the anti-agglutinin, but not in the other organs. Based on these results, the following matters concerning the anti-agglutinin are discussed: (1) the importance of its association with the acrosome of spermatozoa in inhibiting sperm head-to-head agglutination; (2) its origin in the epididymis; and (3) its tissue specificity. Mel. Reprod. Dev. 52:269-276, 1999. (C) 1999 Wiley-Liss, Inc.

    WILEY-LISS, Mar. 1999, MOLECULAR REPRODUCTION AND DEVELOPMENT, 52 (3), 269 - 276, English

    [Refereed]

    Scientific journal

  • Changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during capacitation in vitro

    H Harayama, SF Magargee, E Kunze, O Shidara, E Iwamoto, S Arikawa, M Miyake, S Kato, RH Hammerstedt

    This study is a detailed investigation of changes in epididymal protein anti-agglutinin on ejaculated boar spermatozoa during an incubation designed to promote capacitation in vitro. Ejaculated spermatozoa were collected from six mature boars, washed, and incubated to promote capacitation. Sperm samples were subjected to Western blotting-densitometric analyses, flow cytometry after immunostaining and immunocytochemical observation by indirect immunofluorescence. An antiserum to anti-agglutinin was raised in a rabbit by subcutaneous injection of a purified antigen, as described previously (Harayama et al. 1999). Western blotting-densitometric analyses revealed an approximate halving of the amount of sperm-bound anti-agglutinin during the first 45-min incubation, followed by a gradual decrease thereafter. Comparison between immunostained sperm samples by flow cytometry before and after incubation confirmed this decrease in sperm-bound anti-agglutinin during the incubation. Microscopic characterization established that this decrease occurred mainly on the acrosome. Supplementation with seminal plasma (5% or 10%, v/v) attenuated the decrease. These findings are consistent with the conclusion that a large portion of the anti-agglutinin bound to sperm acrosomes is released at an early stage of the capacitation process in vitro.

    CSIRO PUBLISHING, 1999, REPRODUCTION FERTILITY AND DEVELOPMENT, 11 (4-5), 193 - 199, English

    [Refereed]

    Scientific journal

  • Effects of seminal plasma components on motility and acrosomal integrity of Meishan boar spermatozoa after cooling treatments

    Hiroshi HARAYAMA, Akira IMANO, Seishiro KATO

    Aug. 1998, Anim. Sci. Technol. (Jpn), 69(8), pp. 720-727 (8), 720 - 727, English

    [Refereed]

    Scientific journal

  • Yasuyuki KANNAN, Hiroshi HARAYAMA, Seishiro KATO

    The effect of hypervitaminosis A on bone histomorphology was studied in the tibiae of young growing chicks. Male White Leghorn chicks were orally administered excessive vitamin A (300 or 600IU per g body weight per day) for 10 consecutive days. For histological examination, middle diaphyses were excised and cross sections were stained with toluidine blue. Morphological differences were examined by a computerlized histomorphometry. Radiographic and histological observations indicated the hypervitaminotic chicks to have short tibial length, narrow diaphyseal width and abnormal bone morphology characterized by flattened spindle-shaped osteoblasts. Histomorphometric analysis indicated excessive vitamin A intake to cause severe retardation of bone formation. Cortical bone thickness, measured at middle diaphysis, was less in hypervitaminotic than control chicks receiving the vehicle alone. Osteoblast area and number significantly decreased in the hypervitaminotic chicks. Changes in these osteoblast parameters are associated with marked decrease in bone volume, expressed as percentage of mineralized bone matrix area. Excessive vitamin A intake did not affect all histomorphometric indices of bone resorption. Hypervitaminosis A would thus appear to decrease osteoblastic activity and inhibit bone formation in chick tibia.

    Japan Poultry Science Association, Mar. 1998, Japanese Poultry Science, 35(2), pp. 108-116 (2), 108 - 116, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Shin-Ya Watanabe, Hiroshi Masuda, Yasuyuki Kannan, Masashi Miyake, Seishiro Kato

    Glycosylated components of boar spermatozoa and epididymal plasma from the central caput, distal corpus and distal cauda epididymides were compared by electrophoretic and lectin blotting techniques. The four lectin reagents used in this study were Concanavalin A (Con A), Lens Culinaris Agglutinin (LCA), Ricinus Communis Agglutinin I (RCA I) and Peanut Agglutinin (PNA), which were labeled with peroxidase. The affinity of Con A and of LCA increased for three bands and one smear (A: > 86.8 kDa, B: 80 kDa, C: 65-75 kDa and D: 45 kDa) and for two bands and one smear (A, B and C) of extracts from spermatozoa, respectively, as they passed through the epididymis. RCA I apparently recognized one band and one smear (G: > 86.8 kDa and H: 46-58 kDa) of extracts from the distal cauda spermatozoa, though it hardly or faintly reacted with those from the central caput and distal corpus spermatozoa. PNA showed a similar affinity for band "G" to RCA I. Moreover, bands and smears with similar mobility (A, B, C, D, G and H) were also detected in epididymal plasma by lectin blotting techniques. On the other hand, several sperm components were detected more visibly with Con A (E: 37-41 kDa and F: 32-37 kDa), RCA I (I: 41 kDa) and PNA (I: 41 kDa and J: 47-51 kDa) in sperm extracts from the distal cauda than in those from the central caput, though no corresponding components were observed in epididymal plasma. These epididymal maturation-dependent modifications related to glycosylation in boar spermatozoa are discussed.

    The Japanese Society of Animal Reproduction (JSAR), 1998, Journal of Reproduction and Development, 44 (1), 21 - 27, English

    [Refereed]

    Scientific journal

  • Effects of calcium and bicarbonate on head-to-head agglutination in ejaculated boar spermatozoa

    H Harayama, M Miyake, O Shidara, E Iwamoto, S Kato

    The present study was conducted to reveal the effects of calcium and bicarbonate on the occurrence of head-to-head agglutination in ejaculated boar spermatozoa in vitro. Boar spermatozoa were washed and incubated in a modified Krebs-Ringer bicarbonate (mKRB) in a 37 degrees C CO2 incubator (5% CO2 in air) for 1-5 h. Before and after the incubation, aliquots of each sperm sample were fixed, smeared on glass slides, and stained with a phosphate-buffered solution of Giemsa to assess the percentages of head-to-head agglutinated spermatozoa. Before the incubation, only 5-12% of the spermatozoa were agglutinated. After the 1-h incubation, however, the percentage of head-to-head agglutinated spermatozoa rose to approximately 50%, followed by only minor increases thereafter. This rise was dependent on the concentrations of calcium chloride contained in the mKRB and was attenuated by the addition of 2 mM [ethylene-bis(oxyethylenenitrilo)]tetra-acetic acid (EGTA) to the medium. Moreover, the replacement of sodium bicarbonate with 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (Hepes) in the medium and treatment with ruthenium red, which have both been shown previously to inhibit calcium uptake by boar spermatozoa, significantly reduced the rise. Based on these findings, it was concluded that extracellular calcium and bicarbonate are key factors regulating head-to-head agglutination in boar spermatozoa. The possible relationship between agglutinability and the fertilizing ability of boar spermatozoa is also discussed.

    C S I R O PUBLISHING, 1998, REPRODUCTION FERTILITY AND DEVELOPMENT, 10 (5), 445 - 450, English

    [Refereed]

    Scientific journal

  • Effects of dietary calcium levels on the histomorphology of proximal tibia in vitamin D-deficient chicks

    Yasuyuki KANNAN, Hiroshi HARAYAMA, Seishiro KATO

    Mar. 1997, Japanese Poultry Science, 34(2), pp. 124-131, 107 - 116, English

    [Refereed]

    Scientific journal

  • Electrophoretic characterization of boar epididymal antiagglutinin

    H Harayama, S Kato, RH Hammerstedt

    Boar epididymal antiagglutinin, previously shown to inhibit sperm head-to-head agglutination, was purified from cauda epididymal plasma by precipitation with ammonium sulfate, anion-exchange chromatography, and reverse-phase HPLC, and was characterized by electrophoretic and membrane blotting techniques. Blotting techniques, using the ECL Glycoprotein Detection System (Amersham Life Science, Buckinghamshire, UK) and wheat germ agglutinin (WCA)-peroxidase, established the presence of sialic acid residues on purified antiagglutinin. Removal of sialic acid residues from antiagglutinin greatly reduced its immunoreactivity with the specific antiserum. Further purification by two-dimensional PAGE established the presence of one major and two minor forms that cross-reacted with the antiserum, with only the major form reacting with WGA-peroxidase. Extracts of washed epididymal spermatozoa contained a polypeptide with the same electrophoretic mobility as the major form. Additionally, the antiserum detected cross-reacting material in seminal plasma and in extracts from ejaculated spermatozoa. When spermatozoa were incubated under conditions shown to promote capacitation, the cross-reacting material could not be detected in sperm extracts. These results are consistent with the following conclusions: 1) antiagglutinin contains sialic acid residues that may be related to its immunoreactivity and molecular heterogeneity, and 2) either sperm-bound antiagglutinin is released or its epitope recognized by the antiserum is altered after ejaculation and in vitro capacitation.

    SOC STUDY REPRODUCTION, Aug. 1996, BIOLOGY OF REPRODUCTION, 55 (2), 325 - 332, English

    [Refereed]

    Scientific journal

  • Seishiro Kato, Toshitaka Shibukawa, Hiroshi Harayama, Yasuyuki Kannan

    The timing of shedding and disintegration of cytoplasmic droplets from boar and goat spermatozoa was determined. More than 90% of spermatozoa from the cauda epididymidis and ductus deferens had a droplet each at the distal region of the middle piece in both species. In boars, the droplets were still observed in 97% of spermatozoa immediately after ejaculation. After 1 min of ejaculation, however, the percentage of spermatozoa without the droplet greatly increased to 65%, with no significant changes thereafter. Moreover, most of the free droplets shed from spermatozoa had already disappeared after 1 min of ejaculation. In goats, most spermatozoa (90%) had no droplet even immediately after ejaculation, and the rate of free droplets remaining in the semen was 63%. However, within 2 min of ejaculation, the rate of free droplets remaining in the semen dropped to 8%. These results indicate that shedding of cytoplasmic droplets from boar spermatozoa and their disintegration occur within 1 min after ejaculation. They also suggest that droplets are shed from most of goat spermatozoa during their transit through the urethra and/or immediately after ejaculation, and that their disintegration occurs within a few minutes after ejaculation.

    The Japanese Society of Animal Reproduction (JSAR), 1996, Journal of Reproduction and Development, 42 (4), 237 - 241, English

    [Refereed]

    Scientific journal

  • Fructose stimulates shedding of cytoplasmic droplets from epididymal boar spermatozoa

    H Harayama, M Miyake, T Shibukawa, Y Kannan, S Kato

    The aim of the present study was to establish the presence of an inducer(s) for the shedding of cytoplasmic droplets from boar spermatozoa after ejaculation. Cauda epididymal spermatozoa were incubated with seminal plasma, seminal vesicular fluid (SVF) or chemical agents at 39 degrees C for 30 min. After fixation and staining, percentages of spermatozoa without a droplet were determined. In the samples incubated with seminal plasma, SVF and a filtrate of SVF obtained after passage through an ultrafilter (molecular weight cut-off, 10 000), 43%, 60-69% and 43% of the spermatozoa were without a droplet respectively. The percentage of spermatozoa without a droplet after incubation with D-fructose (1.0 mM), which was one of the energy substrates included in SVF, was 76%. Furthermore, percentages increased to 93% and 90% with the addition of caffeine (2.0 mM) and N-6,2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate (1.0 mM), respectively, but decreased to 48% with the addition of imidazole (2.0 mM). Based on these results, it is suggested that the shedding of cytoplasmic droplets from boar spermatozoa is induced by fructose originating from SVF. It also appears that this event is mediated by increasing the concentration of intracellular cyclic adenosine 3',5'-monophosphate.

    C S I R O PUBLICATIONS, 1996, REPRODUCTION FERTILITY AND DEVELOPMENT, 8 (7), 1039 - 1043, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Takashi Miyano, Seishiro Kato, Hiroshi Masuda, Masashi Miyake

    Distribution of the 25-kDa epididymal anti-agglutinin for spermatozoa was examined by the Western blotting technique in fluid collected from boar rete testes and epididymides. Fluid was collected from the rete testes and various regions of the epididymis, and then plasma and sperm extracts were prepared. Antiserum was raised against the anti-agglutinin, and specific antibody was purified from it. In the plasma, a reaction between the anti-agglutinin and purified antibody was first observed in the proximal corpus epididymidis and became stronger in the more distal regions. In extracts from washed spermatozoa using the original sample buffer, anti-agglutinin was detected between the proximal corpus and the distal cauda and more strongly in the distal corpus. A thorough washing of spermatozoa on a discontinuous Percoli gradient brought about only minor changes in the detection pattern of anti-agglutinin. However, in samples extracted from washed spermatozoa using a modified sample buffer containing twofold detergent and twofold 2-mercaptoethanol, a stronger reaction was observed between the anti-agglutinin and purified antibody in the distal cauda than in the distal corpus. These results indicate that free or loosely sperm-bound anti-agglutinin increases in the corpus and cauda epididymides. It is also suggested that solubility of the anti-agglutinin associated with spermatozoa in the original sample buffer decreases in the cauda epididymidis. © 1995, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.

    1995, Journal of Reproduction and Development, 41 (2), 113 - 121, English

    [Refereed]

    Scientific journal

  • IDENTIFICATION OF ANTI-AGGLUTININ FOR SPERMATOZOA IN EPIDIDYMAL BOAR PLASMA

    H HARAYAMA, T MIYANO, M MIYAKE, H KUSUNOKI, S KATO

    The present report identifies epididymal boar anti-agglutinin and examines its effect on sperm motility. Boar spermatozoa from the cauda epididymidis were washed and incubated in modified Krebs-Ringer bicarbonate at 37-degrees-C (5% CO2 in air). In the samples washed three or five times and then incubated for 3-5 h, higher rates (72-79%) of spermatozoa were associated with one another at the acrosomal region, mainly in groups of 2-5 cells (head-to-head agglutination), and many cells exhibited intensively flagellant and/or circular types of movement but rarely progressive motility. The addition of epididymal plasma or 25 kDa protein purified from it markedly inhibited the occurrence of head-to-head agglutination in washed spermatozoa, whereas heat treatment and subsequent removal of insoluble materials reduced the anti-agglutination activity of epididymal plasma. The percentages of progressively motile cells in the samples incubated with epididymal plasma or 25 kDa epididymal protein rose coincident with the reduction of sperm agglutination. These findings demonstrate that the 25 kDa epididymal protein is an anti-agglutinin for the cauda spermatozoa and that it effectively functions to maintain progressive motility of the cells in vitro. (C) 1994 Wiley-Liss, Inc.

    WILEY-LISS, Apr. 1994, MOLECULAR REPRODUCTION AND DEVELOPMENT, 37 (4), 436 - 445, English

    [Refereed]

    Scientific journal

  • CAPACITY OF RETE TESTICULAR AND CAUDA EPIDIDYMAL BOAR SPERMATOZOA TO UNDERGO THE ACROSOME REACTION AND SUBSEQUENT FUSION WITH EGG PLASMA-MEMBRANE

    H HARAYAMA, H KUSUNOKI, S KATO

    The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis.

    WILEY-LISS, May 1993, MOLECULAR REPRODUCTION AND DEVELOPMENT, 35 (1), 62 - 68, English

    [Refereed]

    Scientific journal

  • Hiroshi HARAYAMA, Seishiro KATO

    The age at which ejaculatory ability is achieved and age-related changes in the characteristics of ejaculates were examined in Meishan boars. All of the 12 boars examined showed detachment of the penis from the prepuce by 64 days of age. The first ejaculate could be collected at 73 to 78 days of age. The volume of the liquid portion, the weight of the gelatinous material, the total number of spermatozoa per ejaculate and the sperm concentration increased significantly until 11, 10, 9 and 5 months of age, respectively. Percentages of progressively motile spermatozoa and morphologically abnormal spermatozoa in the ejaculates altered greatly until 3 months of age, and thereafter stayed at a good level. Percentages of live spermatozoa with normal acrosomes after storage at 4°C for 5 and 10 days were higher in ejaculates collected from boars older than 9 months, and percentages of progressively motile spermatozoa and live spermatozoa with normal acrosomes after freeze-thawing in boars older than 8 months. These results suggest that Meishan boars acquire ejaculatory ability at approximately 2 months of age, but should not be used in service until later ages. It is also suggested and that ejaculates from boars younger than 9 months are less suitable for storage at 4°C-196°C.

    Japanese Society of Animal Science, Apr. 1993, Anim. Reprod. Technol. (Jpn.), 64(4), pp. 333-339 (4), 333 - 339, English

    [Refereed]

    Scientific journal

  • Hiroshi Harayama, Hiroshi Kusunoki, Seishiro Kato

    The objectives of the present study are to characterize the motility and penetrability into zona-free hamster eggs of boar spermatozoa from 7 regions of the epididymis. Spermatozoa from the caput epididymidis were immotile or exhibited a flagellant or circular type of movement. However, 80% of the cells from the proximal cauda showed an intensive, forward movement. When the spermatozoa from each region of the epididymis were coincubated with zona-free hamster eggs after preincubation for inducing the acrosome reaction, they exhibited high penetration rates (95-100%) and average number of spermatozoa (3.4-5.8) in penetrated eggs irrespective of their origin. These findings indicate that most of the spermatozoa acquire the ability to exhibit progressive motility during their transit through the corpus, and that at least some of the cells located in the proximal caput have the ability to fuse with egg plasma membrane. © 1993, THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT. All rights reserved.

    1993, Journal of Reproduction and Development, 39 (1), 41 - 45, English

    [Refereed]

    Scientific journal

  • CAPACITY OF GOAT EPIDIDYMAL SPERMATOZOA TO UNDERGO THE ACROSOME REACTION AND SUBSEQUENT FUSION WITH THE EGG PLASMA-MEMBRANE

    H HARAYAMA, H KUSUNOKI, S KATO

    The capacity to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane was examined in goat epididymal spermatozoa. Spermatozoa from the proximal and distal caput and distal cauda were preincubated in a sealed glass tube for induction of the acrosome reaction, and their viability, acrosome morphology and penetrability into zona-free hamster eggs were determined. A simplified triple-stain technique revealed that most of the preincubated live spermatozoa in the samples from the distal caput and distal cauda epididymides underwent morphological changes that indicated the occurrence of the acrosome reaction. Electron microscopic examination revealed that the outer acrosomal membrane of many spermatozoa in these samples showed fusion at multiple sites to the plasma membrane. However, the rates of acrosome-reacted cells in the proximal caput spermatozoa were still lower. The sperm penetration assay demonstrated that the penetration rates of distal caput and distal cauda spermatozoa preincubated for 2 h were 93% and 74% respectively, whereas proximal caput spermatozoa scarcely penetrated into eggs. These results indicate that increasing numbers of goat spermatozoa improve in the functions related to the acrosome reaction and subsequent fusion with the egg plasma membrane during their transit through the caput epididymidis.

    C S I R O PUBLICATIONS, 1993, REPRODUCTION FERTILITY AND DEVELOPMENT, 5 (3), 239 - 246, English

    [Refereed]

    Scientific journal

  • Hiroshi HARAYAMA, Sunao KANDA, Seishiro KATO

    The developmental process of the seminal vesicle, corpus prostatae and bulbourethral gland was morphologically and histologically investigated in Meishan boars after birth. All of the glands remarkably increased in weight after 30-45 days of age. In the seminal vesicle and corpus prostatae at 1 day of age, the glandular tubules were characterized by an undeveloped epithelium and a narrow lumen. At 45-60 days of age, the terminal portions of the tubules of these glands were partially ramified and the lumen contained PAS-positive secretions. At 75-90 days of age, the portions presented a fully-developed structure. On the other hand, in the bulbourethral gland at 1 day of age, the terminal portions of the glandular tubules were already ramified and contained PAS-positive substances. At 30-45 days of age, they showed rapid growth and more ramifications and were filled with the secretions. After this age, their structural aspects presented no changes. These results indicate that the secrectory function of the seminal vesicle, corpus prostatae and bulbourethral gland in Meishan boars in well developed at 75-90, 75-90 and 30-45 days of age, respectively.

    Japanese Society of Animal Science, Nov. 1992, Anim. Sci. Technol. (Jpn.), 63(11), pp. 1115-1122 (11), 1115 - 1122, English

    [Refereed]

    Scientific journal

  • INFLUENCE OF SEASON ON CHARACTERISTICS OF EPIDIDYMAL AND EJACULATED SEMEN IN MEISHAN BOARS

    H HARAYAMA, S KANDA, S KATO

    Most of the epididymal spermatozoa collected in all the seasons examined maintained an ability to move progressively, had a cytoplasmic droplet in the distal site of the middle piece, and were morphologically normal. Reduced desire to mount a dummy was not observed during the experimental period. Characteristics of ejaculated semen were not significantly altered throughout the year. However, progressive motility and acrosomal integrity of spermatozoa ejaculated between July and September were more susceptible to storage at 4-degrees-C than spermatozoa ejaculated during the other months and acrosomal integrity of spermatozoa ejaculated during the 3 months was to freezing-thawing. These results indicate that the reproductivity activity of Meishan boars in Japan is only slightly influenced by season, but semen ejaculated during the summer is less suitable for storage than that ejaculated during the other seasons of the year.

    BUTTERWORTH-HEINEMANN, Sep. 1992, THERIOGENOLOGY, 38 (3), 491 - 500, English

    [Refereed]

    Scientific journal

  • Seasonal changes of reproductive performance in Meishan boars

    Hiroshi HARAYAMA, Masashi MIYAKE, Seishiro KATO

    Lead, Aug. 1992, Proceedings of the International Symposium on Chinese Pig Breeds., pp. 162-165, English

    [Refereed]

    International conference proceedings

  • In vitro storage of Meishan boar spermatozoa

    Seishiro KATO, Koichiro KANO, Hirioshi HARAYAMA, Hiroshi KUSUNOKI, Iwao NANJO

    Aug. 1992, Proceedings of the International Symposium on Chinese Pig Breeds, pp. 582-585, English

    [Refereed]

    International conference proceedings

  • Hiroshi HARAYAMA, Seishiro KATO

    Changes in motility and morphology of spermatozoa during their transit through the epididymis were investigated in Meishan boars at various ages. At 75 days of age, only 26% of spermatozoa from the cauda epididymidis were progressively motile, 59% presented a morphological abnormality, mainly an abnormal head, and 62% had a cytoplasmic droplet at the proximal region of the middle piece. At 105-120 days of age, however, 89% of spermatozoa showed intensive, forward movement and 98% had a distal droplet after epididymal transit. At more than 90 days of age, the proportion of spermatozoa with an abnormal head significantly decreased during their transit through the caput epididymidis. These results indicate that epididymal functions associated with sperm maturation and selective exclusion of abnormal spermatozoa in Meishan boars are fully developed by approximately 120 days of age.

    Japanese Society of Animal Science, May 1992, Anim. Sci. Technol. (Jpn.), 63(5), pp. 462-467 (5), 462 - 467, English

    [Refereed]

    Scientific journal

  • Hiroshi HARAYAMA, Akio Tsutsui, Seishiro KATO

    The developmental process of the reproductive organs was morphologically and histologically investigated in Jinhua boars after birth. The testis greatly developed after 45 days of age. Spermatozoa were first found in the seminiferous tubules at approximately 75 days of age, when the histological composition of the seminiferous epithelium improved to a mature type. Epididymis grew rapidly after 45 days of age, and revealed a well-developed structure of the ductal epithelium in any region by 60-75 days of age. On the other hand, the weight of each accessory genital gland increased rapidly after 60 days of age. In the seminal vesicle, corpus prostatae and blubourethral gland, the terminal portions were well ramified and filled with PAS-positive secretions by 75-90, 90 and 30-45 days of age, respectively. These results indicate that Jinhua boars reach puberty as early as 75 days of age. It also appears that development of the reproductive organs occurs at an earlier age in Jinhua boars than in European and American breeds.

    The Japanese Society of Swine Science, Feb. 1992, Jpn. J. Swine Sci, 29(3), pp. 174-183 (3), 174 - 182, Japanese

    [Refereed]

    Scientific journal

  • Development of the epididymis in Meishan boars

    Hiroshi HARAYAMA, Iwao NANJO, Sunao KANDA, Seishiro KATO

    Feb. 1992, Asian-Aust. J. Anim. Sci., 5(1), pp. 165-171, English

    [Refereed]

    Scientific journal

  • TESTICULAR DEVELOPMENT IN CHINESE MEISHAN BOARS

    H HARAYAMA, NANJO, I, S KANDA, S KATO

    The developmental process of the testis and age-related changes in the morphology of rete testicular spermatozoa were investigated in Meishan boars at 1 to 364 days of age. Testicular weight and the diameter of seminiferous tubules increased rapidly until 150 to 180 days of age. Leptotene stage spermatocytes, round spermatids and spermatozoa were first found in the section of seminiferous tubules at 30 to 45, 60 and 75 days of age, respectively. However, after 105 to 120 days of age, most rete testicular spermatozoa were morphologically normal. These results indicate that Meishan boars reach puberty as early as 75 days of age, though the testes acquire the ability to produce morphologically normal spermatozoa at about 120 days.

    BUTTERWORTH-HEINEMANN, Oct. 1991, THERIOGENOLOGY, 36 (4), 637 - 643, English

    [Refereed]

    Scientific journal

  • Effect of imidazole in diluent on motility and acrosomal morphology of Meishan boar spermatozoa stored at 4 C

    Koichiro KANO, Hiroshi KUSUNOKI, Hiroshi HARAYAMA, Seishiro KATO

    Sep. 1991, Jpn. J. Anim. Reprod. Technol., 13(3), pp. 158-164, Japanese

    [Refereed]

    Scientific journal

  • Takashi MIYANO, Kiyoshi YOSHIKAWA, Seishiro KATO, Hiroshi HARAYAMA, Sunao KANDA

    Using prepubertal Meishan gilts, at 70-71 days of age, weighting 20-30 kg, we examined the details of the fertilization of in vitro and in vivo matured oocytes. At 39-40 h after hCG injection, follicles larger than 7mm in diameter were observed on the surface of ovaries(18.4±11.3, n=4). At 42-43h, all of the ovaries had recently ovulated follicles (6.5±3.7, n=4)in addition to follicles larger than 7mm in diameter. Immature oocytes were collected from hormone-nontreated gilts, and matured in a maturation medium. The cumuli surrounding follicular oocytes, recovered from the hormone-nontreated gilts, expanded after culture in a maturation medium. Follicular oocytes and ovulated eggs from hormone-treated gilts also had expanded cumuli. At 12h after insemination of the in vitro and in vivo matured follicular oocytes and of the ovulated eggs with the same sperm suspension, 72.3, 72.3% of oocytes and 84.4% of eggs reached the metaphase II, respectively. Formation of a male pronucleus (ei) was delayed in the oocytes matured in vitro, although the percentages of penetrated oocytes were not different among those three groups. The mean number of spermatozoa penetrating an ovulated egg was 2.1± 1.0, while the number was significantly higher in oocytes matured in vitro (3.6±2.8, p<0.05). These results show that mature oocytes and eggs recovered from prepubertal Meishan gilts as early as 70 days of age can be fertilized, we suggest that the Meishan gilt is a promising donor of oocytes and eggs for study of pig reproduction.

    Japanese Society of Animal Science, Nov. 1990, Jpn. J. Zootech. Sci., 61(11), pp. 1011-1016 (11), 1011 - 1016, English

    [Refereed]

    Scientific journal

  • Hiroshi KUSUNOKI, Morito SAKAUE, Hiroshi HARAYAMA, Seishiro KATO, Sunao KANDA

    The retention of the motility and viability of acrosome-reacted goat spermatozoa and their ability to penetrate zona-free hamster eggs was investigated. Effects of caffeine and imidazole on these parameters were also examined. Ejaculated goat spermatozoa were washed and preincubated in K-3 medium in sealed glass tubes at 39.5°C for 2h to induce the acrosome reaction. This was fo11owed by resuspension in BO medium BO medium with caffeine and BO medium with imidazole and incubated at 37°C for 5 h. During incubation, sperm motility and viability were assessed at one hour intervals. The acrosome-reacted cells after resuspension in BO medium or BO medium with imidazole and those incubated for 3h in these media were further incubated with zonafree hamster eggs at 37°C. During incubation with the eggs, time-related changes in egg penetration were examined. The following results were obtained: 1) The motility patterns of goat spermatozoa before and after the acrosome reaction were different. Acrosome-reacted goat spermatozoa exhibited 'whiplash' motility. 2) The motility and viability of goat cells after the acrosome reaction were remarkably lower than those before the reaction. 3) The ability of acrosome-reacted goat spermatozoa topenetrate zona-free hamster eggs was retained for as much as 3h. 4) The addition of imidazole to sperm suspension promoted the maintenance of motility and viability of acrosome-reacted goat spermatozoa and prolonged the retention of their ability to penetrate zona-free hamster eggs. The addition of caffeine was effective for the temporary activation of the motility but made difficult the maintenance of motility and viability.

    Japanese Society of Animal Science, Jul. 1990, Jpn. J. Zootech. Sci., 61(7), pp. 640-647 (7), 640 - 647, English

    [Refereed]

    Scientific journal

  • Hiroshi KUSUNOKI, Morito SAKAUE, Hiroshi HARAYAMA, Seishiro KATO, SUNAO KANDA

    The purpose of this study was to develop a new technique for identifying acrosomereacted goat spermatozoa. Ejaculated spermatozoa were washed and incubated in an airtight glass tube at 39.5°C for 1, 2 and 3hrs for inducing the acrosome reaction, and washed and frozen-thawed three times. Sperm samples were incubated with trypan blue for 15min, smeared on a slide, fixed in ORTH solution for 45min, and finally stained in Giemsa solution (trypan blue-Giemsa method). Stained smears had spermatozoa with the following four staining patterns: a) unstained or light blue postacrosomal regions with reddish purple acrosomes (live sperm with normal acrosomes); b) unstained or light blue postacrosomal regions with unstained acrosomal regions or damaged acrosomes (live sperm without normal acrosomes); c) dark blue postacrosomal regions with dark reddish purple acrosomes (dead sperm with normal acrosomes); d) dark blue postacrosomal regions with dark blue acrosomal regions or damaged acrosomes (dead sperm without normal acrosomes). There was a significant positive partial correlation between the percentage of live sperm without normal acrosomes (category b) and that of zona-free hamster eggs penetrated by spermatozoa (r=0.4179, P〈0.01, n=44). Additionally, there was no difference between the results obtained by means of the present method and a triple-stain technique, while the staining procedure and solution preparation were much simpler in the former method than in the latter. These results indicate that the trypan blue-Giemsa method can be used to identify acrosome-reacted goat spermatozoa.

    Japanese Society of Animal Science, Mar. 1988, JPN. J. Zootech. Sci., 59(3), pp. 235-240 (3), 235 - 240, Japanese

    [Refereed]

    Scientific journal

  • Storage of Meishan pig spermatozoa

    Hiroshi HARAYAMA, Seishiro KATO, Iwao NANJO, Sunao KANDA, Tetsushi TOKUMARU

    Lead, Jan. 1988, Jpn. J. Anim. Reprod. Technol., 10(1), pp. 10-14, Japanese

    [Refereed]

    Scientific journal

  • Hiroshi HARAYAMA, Morito SAKAUE, Hiroshi KUSUNOKI, Seishiro KATO, Sunao KANDA

    雄ヤギ5頭を用い, 精巣網カテーテル装着手術後の精巣および精巣上体頭の組織構造を観察するとともに精巣静脈血中のテストステロン濃度を調べた。カテーテル装着手術後の精巣は, いずれも萎縮し, 正常な精子形成過程が観察される精細管は少なかった。また, ライディヒ細胞の形態や染色性には異常は認められなかった。一方, 精巣上体頭では, 精巣上体管が萎縮し, 主細胞の高さは無傷のものよりも有意に減少した。同様の組織構造の変化は, 精巣輸出管を切除した場合の精巣および精巣上体頭でも認められた。精巣静脈血中のテストステロ

    Lead, 神戸大学農学部, Jan. 1988, Sci. Rept. Fac. Agr. Kobe Univ., 18(1), pp. 125-132 (1), 125 - 132, Japanese

    Scientific journal

  • Morito SAKAUE, Osamu Doi, Hiroshi KUSUNOKI, Hiroshi HARAYAMA, Seishiro KATO, Sunao KANDA

    4頭の成熟雄ヤギ(日本在来種)から精巣網カテーテル法によって採取した精巣網液および頚静脈血を用い, ラジオイムノアッセイによりテストステロン, エストラジオール-17βおよびプロジェステロン濃度を測定した。精巣網漿液中のホルモンの平均濃度は, テストステロンで67.8ng/ml, エストラジオール-17βで43.9pg/ml, プロジェステロンでは517.1pg/mlであった。どの個体においても昼間(0900-2100)と夜間(2100-0900)に採取した試料のテストステロン濃度に有意な差は認められなか

    神戸大学農学部, Jan. 1988, Sci. Rept. Fac. Agr. Kobe Univ., 18, pp. 133-136 (1), 133 - 136, English

    Scientific journal

MISC

  • 家畜精子での受精制御分子について

    HARAYAMA Hiroshi

    Oct. 2019, 日本アンドロロジー学会ニュースレター, 19, 3 - 4, Japanese

    [Invited]

    Others

  • Flagellar hyperactivation of bull and boar spermatozoa [Invited review article]

    HARAYAMA Hiroshi

    Lead, Oct. 2018, Reproductive Medicine and Biology, 17 (4), 442 - 448, English

    [Refereed][Invited]

    Introduction scientific journal

  • 特集 哺乳類の精子研究における新展開 -基礎と応用― 序

    HARAYAMA Hiroshi

    日本胚移植研究会, Sep. 2017, 日本胚移植学雑誌, 39 (3), 141, Japanese

    [Invited]

    Others

  • 家畜精子鞭毛の超活性化運動に関する研究の現状 [招待総説論文]

    HARAYAMA Hiroshi, MIZUNO Yohei

    Lead, 日本胚移植研究会, Sep. 2017, 日本胚移植学雑誌, 39 (3), 159 - 167, Japanese

    [Refereed][Invited]

    Introduction scientific journal

  • Hiroshi Harayama, Kenta Minami, Kazumi Kishida, Taichi Noda

    Background: Although artificial insemination (AI) technique is an established biotechnology for bovine reproduction, the results of AI (conception rates) have a tendency to decline gradually. To our annoyance, moreover, AI-subfertile bulls have been occasionally found in the AI centers. To resolve these serious problems, it is necessary to control the sperm quality more strictly by the examinations of sperm molecules. Methods: We reviewed a number of recent articles regarding potentials of bovine sperm proteins as the biomarkers for bull AI-subfertility and also showed our unpublished supplemental data on the bull AI-subfertility associated proteins. Main findings: Bull AI-subfertility is caused by the deficiency or dysfunctions of various molecules including regulatory proteins of ATP synthesis, acrosomal proteins, nuclear proteins, capacitation-related proteins and seminal plasma proteins. Conclusion: In order to control the bovine sperm quality more strictly by the molecular examinations, it is necessary to select suitable sperm protein biomarkers for the male reproductive problems which happen in the AI centers.

    Lead, John Wiley and Sons Ltd, 01 Apr. 2017, Reproductive Medicine and Biology, 16 (2), 89 - 98, English

    [Refereed][Invited]

    Book review

  • Roles of Intracellular Cyclic AMP Signal Transduction in the Capacitation and Subsequent Hyperactivation of Mouse and Boar Spermatozoa

    Hiroshi Harayama

    It is not until accomplishment of a variety of molecular changes during the transit through the female reproductive tract that mammalian spermatozoa are capable of exhibiting highly activated motility with asymmetric whiplash beating of the flagella (hyperactivation) and undergoing acrosomal exocytosis in the head (acrosome reaction). These molecular changes of the spermatozoa are collectively termed capacitation and promoted by bicarbonate, calcium and cholesterol acceptors. Such capacitation-promoting factors can stimulate intracellular cyclic AMP (cAMP) signal transduction in the spermatozoa. Meanwhile, hyperactivation and the acrosome reaction are essential to sperm fertilization with oocytes and are apparently triggered by a sufficient increase of intracellular Ca2+ in the sperm flagellum and head, respectively. Thus, it is necessary to investigate the relationship between cAMP signal transduction and calcium signaling cascades in the spermatozoa for the purpose of understanding the molecular basis of capacitation. In this review, I cover updated insights regarding intracellular cAMP signal transduction, the acrosome reaction and flagellar motility in mammalian spermatozoa and then account for possible roles of intracellular cAMP signal transduction in the capacitation and subsequent hyperactivation of mouse and boar spermatozoa.

    Lead, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, Oct. 2013, JOURNAL OF REPRODUCTION AND DEVELOPMENT, 59 (5), 421 - 430, English

    [Refereed][Invited]

    Introduction scientific journal

  • DETECTION OF SOLUBLE ADENYLYL CYCLASE (ADCY10) HOMOLOG PROTEINS IN BOAR SPERMATOZOA

    Kazumi Nakamura, Chihiro Suzuki, Shunsuke Tate, Jibak Lee, Hiroshi Harayama

    AMER SOC ANDROLOGY, INC, Mar. 2009, JOURNAL OF ANDROLOGY, 37 - 37, English

    Summary international conference

  • Fertility Suppression of Feral Animal to Reconstitute Environmental Relationship with Human Society

    HARAYAMA Hiroshi, GOTO Namiko, TATE Shunsuke

    Apr. 2008, Annual Report of Interdisciplinary Research Institute of Environmental Sciences, 26, pp. 45-52, Japanese

    Others

  • Cyclic amp-induced inactivation of PDK1 is linked to the enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa

    Hiroshi Harayama

    AMER SOC ANDROLOGY, INC, Mar. 2008, JOURNAL OF ANDROLOGY, 48 - 48, English

    Summary international conference

  • 精子鞭毛のハイパーアクチベーションを指標とする黒毛和種雄ウシの繁殖能力評価法の開発

    HARAYAMA Hiroshi

    Dec. 2007, 財)三島海雲記念財団平成18年度受贈者研究報告書, 44, pp. 19-23, Japanese

    Others

  • Localization of Claudin family proteins in pig embryos during pre-implantation development

    S. Xu, J. Lee, H. Harayama, M. Miyake

    CSIRO PUBLISHING, 2007, REPRODUCTION FERTILITY AND DEVELOPMENT, 19 (1), 197 - 197, English

    Summary international conference

  • Fertilization-related parameters of frozen-thawed spermatozoa from subfertile Japanese Black cattle

    K. Kuroda, M. Fukushima, M. Miyake, H. Harayama

    CSIRO PUBLISHING, 2007, REPRODUCTION FERTILITY AND DEVELOPMENT, 19 (1), 266 - 267, English

    Summary international conference

  • Establishment of simplified assay for the reproductive performance of male Japanese Black cattle: additional application of sperm CTC staining technique

    Hiroshi HARAYAMA, Ken KURODA

    Dec. 2006, 食肉に関する助成研究調査成果報告書, 24, pp. 52-56, Japanese

    Others

  • Role of protein kinase C in the cyclic adenosine 3 ',5 '-mono phosphate-dependent hyperactivation of boar spermatozoa

    H Harayama, M Miyake

    AMER SOC ANDROLOGY, INC, Mar. 2006, JOURNAL OF ANDROLOGY, 46 - 46, English

    Summary international conference

  • Simplified assay for the reproductive performance of male Japanese Black cattle by quantitative analysis of sperm phosphorylated proteins

    Hiroshi HARAYAMA, Ken KURODA

    Dec. 2005, 食肉に関する助成研究調査成果報告書, 23, pp. 39-44, Japanese

    [Refereed]

    Others

  • cAMP-induced increase of 32-kDa tyrosine-phosphorylated protein (TyrP32) is related to the calcium-dependent disintegration of the acrosome in boar sperm

    H Harayama, K Sasaki, M Miyake

    AMER SOC ANDROLOGY, INC, Mar. 2004, JOURNAL OF ANDROLOGY, 44 - 44, English

    Summary international conference

  • Behavior of chromatins in pronuclei in the cattle and mouse

    M Isaji, H Iwata, M Ohta, H Harayama, M Miyake

    SOC STUDY REPRODUCTION, 2003, BIOLOGY OF REPRODUCTION, 68, 160 - 160, English

    Summary international conference

  • Hiroshi Harayama, Seishiro Kato

    In boar spermatozoa, the head-to-head agglutinability changes in parallel with the development of the fertilizing ability. Namely, both abilities gradually increase in the distal caput and corpus epididymides, but are subsequently suppressed in the cauda epididymidis. It has been postulated that these changes of the agglutinability are controlled via sperm interaction with specific epididymal plasma factors including agglutination mediators (agglutinins) and inhibitors (anti-agglutinins). Expression of these abilities (sperm agglutination and capacitation) is hardly observed in spermatozoa immediately after ejaculation, but it occurs during incubation in a capacitation medium. Recently, we have purified and characterized epididymal plasma anti-agglutinin for boar spermatozoa. Moreover, we have conducted a series of experiments to reveal biological significance and mechanism of the head-to-head agglutination and have accumulated data indicating that boar sperm agglutination is mediated by capacitation-supporting factors including calcium, bicarbonate and sterol acceptors. This review introduces our recent data and discusses a possible mechanism for suppression of the agglutinability in the distal epididymidis and relationship between agglutinability and fertilizing ability.

    Asian-Australasian Association of Animal Production Societies, 2001, Asian-Australasian Journal of Animal Sciences, 14 (8), 1196 - 1202, English

    [Refereed]

    Book review

  • Signal transduction system regulating head-to-head agglutination in capacitating boar spermatozoa

    Hiroshi hARAYAMA, Masashi MIYAKE, Seishiro KATO

    Dec. 2000, 食肉に関する助成研究調査成果報告書, 18, pp. 38-42, Japanese

    Others

  • Molecules regulating maturational changes in mammalian spermatozoa

    Hiroshi HARAYAMA

    Lead, 2000, J. Reprod. Dev., 46(Suppl.), pp. j43-j50, Japanese

    [Refereed]

    Introduction scientific journal

  • Changes of boar sperm-bound anti-agglutinin during capacitation process

    Hiroshi HARAYAMA, Masashi MIYAKE, Seishiro KATO

    Dec. 1999, 食肉に関する助成研究調査成果報告書, 17, pp. 29-33, Japanese

    Others

  • Purification of seminal plasma proteins enhancing cold tolerance of boar spermatozoa and their use for sperm frozen storage

    Hiroshi HARAYAMA, Akira IMANO, Masashi MIYAKE, Seishiro KATO

    Dec. 1998, 食肉に関する助成研究調査成果報告書, 16, pp. 79-83, Japanese

    Others

  • Effects of Seminal Plasma Components on Motility and Acrosomal Integrity of Meishan Boar Spermatozoa after Cooling Treatments

    HARAYAMA Hiroshi, IMANO Akira, MIYAKE Masashi, KATO Seishiro

    日本畜産学会, 25 Aug. 1998, Animal science journal, 69 (8), 720 - 727, English

  • 雄梅山豚における副生殖腺の発達〔英文〕

    原山 洋, 苅田 淳, 加藤 征史郎

    日本畜産学会, Nov. 1992, 日本畜産学会報, 63 (11), p1115 - 1122, Japanese

Books etc

  • 繁殖生物学 改訂版(第2版)

    原山 洋;内藤邦彦

    Joint work, 第5章3 受精 pp. 228~237, 株式会社インターズ―, 28 Mar. 2020

  • 発生生物学 「哺乳類の受精」

    原山 洋

    Joint work, 第5章 受精ー個体発生のはじまりー 5.3 哺乳類の受精 pp. 44~47, 株式会社培風館, 10 Apr. 2019, Japanese

    Scholarly book

  • Capacitation-related events in boar spermatozoa, Animal Frontier Sciences(pp. 37-43)

    Hiroshi HARAYAMA, Makiko KANETO, Masashi MIYAKE, Seishiro KATO

    Joint work, Hokuto Shobo, 2003, English

    Scholarly book

  • Regulation of head-to-head agglutination in boar spermatozoa by capacitation-related factors. Reproductive Biotechnology: Reproductive biotechnology update and its related physiology (pp. 203-209)

    Hiroshi HARAYAMA, Takami MURATE, Masashi MIYAKE, Seishiro KATO

    Joint work, Hokuto Shobo, 2001, English

    Scholarly book

  • Early development of porcine parthenogenetic diploids in-vitro (Reproductive Biotechnology: Reproductive biotechnology update and its related physiology. pp.285-295)

    Masashi MIYAKE, Thuan Van NGUYEN, Konosuke OKADA, Mika KATAYAMA, Statoshi KURE-BAYASHI, Hiroshi HARAYAMA

    Joint work, Hokuto Shobo, 2001, English

    Scholarly book

  • Purification and characterization of boar epididymal plasma protein Anti-Agglutinin. (Reproductive Biology Update: Novel tools for assessment of environmental toxicity. pp. 207-217)

    Hiroshi HARAYAMA, Takashi MIYANO, Masashi MIYAKE, Seishiro KATO

    Joint work, Nakanishi Pub. Co., Dec. 1998, English

    Scholarly book

  • Electro-activation of in vitro-matured porcine oocytes and their development in vitro and in vivo (Reproductive Biology Update: Novel tools for assessment of environmental toxicity, pp. 117-127)

    Masashi MIYAKE, Satoshi KUREBAYASHI, Takashi MIYANO, Hiroshi HARAYAMA, Seishiro KATO

    Joint work, Nakanishi Pub. Co., 1998, English

    Scholarly book

Presentations

  • ウシ凍結保存精子でのCalyculin-A 誘発性Full-type ハイパーアクチベーションの発生に及ぼすストア作動性チャネル非選択的阻害剤の影響

    吉田 早紀, 坂瀬 充洋, 原山 洋

    第73 回関西畜産学会大会, 11 Nov. 2023, Japanese

    Oral presentation

  • ウシ凍結保存精子でのCalyculin-A誘発性Full-type Hyperactivationの発生におけるストア作動性チャネルの関与

    吉田 早紀, 坂瀬 充洋, 原山 洋

    第116回日本繁殖生物学会大会, 25 Sep. 2023, Japanese

    Oral presentation

  • お父さん牛の精子がお母さんの卵子と どのように受精するのか?

    原山 洋

    第116回日本繁殖生物学会大会・市民公開講座「黒毛和牛をもっと増やそう!最新の繁殖生物学研究がお父さん牛にできること」, 24 Sep. 2023, Japanese

    [Invited]

    Public discourse

  • 人工授精での受胎率の向上を目指して -ウシ(黒毛和種)精子の新規検査法の開発-

    原山 洋

    日本アンドロロジー学会第42回学術大会 特別講演4, 24 Jun. 2023, Japanese

    [Invited]

    Invited oral presentation

  • ブタ射出精子での体外受精能力発現に及ぼす cAMP シグナリング活性化処理の影響

    原山 洋, 植芝 愛巳, 森川 莉帆, 京極 博久

    日本アンドロロジー学会第42回学術大会, 23 Jun. 2023, Japanese

    Poster presentation

  • Relationship of the SERCA activity with the time courses of occurrence of full-type hyperactivation in bovine sperm

    Duritahala, Mitsuhiro Sakase, Hiroshi Harayama

    第115回日本繁殖生物学会大会, 12 Sep. 2022, English

    Poster presentation

  • Penetration into oocytes in the medium without the PDE inhibitor by boar sperm treated with the cAMP analog and Ca2+

    植芝 愛巳, 森川 莉帆, 京極 博久, 原山 洋

    第115回日本繁殖生物学会大会, 12 Sep. 2022, Japanese

    Poster presentation

  • Involvement of cytoplasmic Ca2+ and SERCAs in the occurrence of helical movement in bovine sperm

    Duritahala, Hiroshi Harayama

    日本アンドロロジー学会第41回学術大会, 03 Jun. 2022, English

    Poster presentation

  • ウシ凍結精子でのFull-typeハイパーアクチベーションの誘起のための処理条件の検討

    原山 洋, 宮本 菜津子

    日本アンドロロジー学会第41回学術大会, 03 Jun. 2022, Japanese

    Poster presentation

  • ウシ精子において化学物質処理により誘起されるHyperactivationの特性解析

    大谷 暁洋, 坂瀬 充洋, 原山 洋

    第10回関西生殖医学集談会, 05 Mar. 2022, Japanese

    Oral presentation

  • A regulatory factor for the occurrence of helical movement in bovine cryopreserved sperm

    Duritahala, Mitsuhiro Sakase, Hiroshi Harayama

    第71回関西畜産学会, 02 Oct. 2021, English

    Oral presentation

  • Involvement of thapsigargin-sensitive Ca2+-ATPase in the occurrence of helical movement with 3-D rotation in cryopreserved bovine sperm

    Duritahala, Sakase M, Harayama H

    第114 回日本繁殖生物学会大会, 23 Sep. 2021, English

    Poster presentation

  • Thimerosal処理によりウシ精子で誘起される細胞内ストアのCa2+依存的なハイパーアクチベーションの運動様式

    大谷暁洋, 坂瀬充洋, 原山 洋

    第114 回日本繁殖生物学会大会, 23 Sep. 2021, Japanese

    Poster presentation

  • ウシ凍結精子でのFull-typeハイパーアクチベーションに及ぼすプロテインホスファターゼ阻害剤Calyculin Aの影響

    宮本菜津子, 坂瀬充洋, 原山 洋

    第114 回日本繁殖生物学会大会, 23 Sep. 2021, Japanese

    Poster presentation

  • ブタ液状保存精子でのFull-typeハイパーアクチベーションの誘起に及ぼすポリビニルアルコールおよびウシ血清アルブミンの影響

    原山 洋, 崔 聖健

    日本アンドロロジー学会第40回学術大会, 13 Jun. 2021, Japanese

    Oral presentation

  • ウシ射出精子の鞭毛運動に及ぼすNa+/K+-ATPase阻害剤Digoxinの影響

    原山 洋, Saha Soma Rani

    日本アンドロロジー学会第39回学術大会, 15 Jan. 2021, Japanese

    Poster presentation

  • ウシ精子における精密性状検査法の開発

    原山 洋

    令和2年度 東海畜産学会 シンポジウム, 04 Dec. 2020, Japanese

    [Invited]

    Keynote oral presentation

  • ブタ精子でのFull-type Hyperactivationの発生制御におけるカルモジュリンの役割

    和田篤士, 原山 洋

    第113回日本繁殖生物学会大会, 25 Sep. 2020, Japanese

    Poster presentation

  • 家畜精子の受精制御分子の探索と利用

    原山 洋

    第46回山梨大学発生工学研究センターセミナー, 13 Dec. 2019, Japanese, Domestic conference

    [Invited]

    Public discourse

  • Involvement of digoxin-sensitive Na+/K+-ATPase in the regulation of bovine sperm motility

    SAHA Soma Rani, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第112回日本繁殖生物学会, 05 Sep. 2019, English, Domestic conference

    Poster presentation

  • Characterization of SPACA1 proteins in the spermatozoa from Japanese Black bulls

    MINAMI Kenta, ASO Miyuki M, YAMADA Ayano, KISHIDA Kazumi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第112回日本繁殖生物学会, 05 Sep. 2019, Japanese, Domestic conference

    Poster presentation

  • 低繁殖症の雄ウシを検出するための精子分子性状検査法のマーカー「SPACA1タンパク質」の解析

    MINAMI Kenta, ASO Miyuki, YAMADA Ayano, KISHIDA Kazumi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    日本アンドロロジー学会第38回学術大会, Jun. 2019, Japanese, 大阪国際会議場(グランキューブ大阪), Domestic conference

    Poster presentation

  • Investigation of movement patterns of bull ejaculated sperm to improve the evaluation method of motility

    YAMADA Ayano, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第111回日本繁殖生物学会大会, Sep. 2018, Japanese, 上田市 信州大学繊維学部, Domestic conference

    Poster presentation

  • cAMP-PKAシグナル伝達活性の違いがウシ精子の運動様式に及ぼす影響 - 精子の活力検査における評価基準の見直し -

    YAMADA Ayano, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    日本アンドロロジー学会第37回学術大会, Jun. 2018, Japanese, ラ・スイート神戸オーシャンガーデン, Domestic conference

    Poster presentation

  • ウシ精子での細胞外Ca2+依存的なFull-typeハイパーアクチベーションの抑制に機能するカリクリンA感受性プロテインホスファターゼアイソフォームの特定

    ARAI Yuka, HARAYAMA Hiroshi

    第6回関西生殖医学集談会, Feb. 2018, Japanese, 大阪市 ハービスPLAZA, Domestic conference

    Oral presentation

  • Mechanism for the extracellular Ca2+-dependent occurrence of full-type hyperactivation in boar sperm treated with a cAMP analog

    OTSUKA Nagisa, HARAYAMA Hiroshi

    Fourth World Congress of Reproductive Biology (WCRB2017), Sep. 2017, English, 沖縄県宜野湾市 沖縄コンベンションセンター, International conference

    Poster presentation

  • Calyculin A-sensitive protein phosphatases which are involved in the suppression for full-type hyperactivation of bovine sperm

    ARAI Yuka, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    Fourth World Congress of Reproductive Biology (WCRB2017), Sep. 2017, English, 沖縄県宜野湾市 沖縄コンベンションセンター, International conference

    Poster presentation

  • ブタ精子での超活性化運動(Full-type HA)の開始におけるTRPC3チャネルの役割

    OTSUKA Nagisa, HARAYAMA HIROSHI

    第5回関西生殖医学集談会, Mar. 2017, Japanese, 大阪市, Domestic conference

    Oral presentation

  • Advanced techniques for the reproduction in the cattle

    HARAYAMA Hiroshi

    UGSVS lecture, Feb. 2017, English, 岐阜大学, Domestic conference

    [Invited]

    Invited oral presentation

  • ブタ精子におけるfull-type hyperactivationの開始制御へのTRPC3チャネルの関与

    OTSUKA Nagisa, HARAYAMA Hiroshi

    第109回日本繁殖生物学会, Sep. 2016, Japanese, 一社 日本繁殖生物学会, 相模原市(麻布大学), Domestic conference

    Poster presentation

  • ウシ精子のハイパーアクチベーションを抑制するカリクリンA感受性プロテインホスファターゼの特定

    OTSUKA Nagisa, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第109回日本繁殖生物学会, Sep. 2016, Japanese, 一社 日本繁殖生物学会, 相模原市(麻布大学), Domestic conference

    Poster presentation

  • 先体タンパク質をマーカーとする哺乳類精子の分子性状検査法

    HARAYAMA HIROSHI

    日本アンドロロジー学会35回学術大会 シンポジウム1:男性不妊症の分子メカニズム, Jun. 2016, Japanese, 前橋市・前橋テルサ, Domestic conference

    [Invited]

    Nominated symposium

  • ブタ精子での先体反応誘起に伴う頭部タンパク質SPACA1の変化

    OGURA YUKARI, HARAYAMA HIROSHI

    日本畜産学会第121回大会, Mar. 2016, Japanese, 日本獣医生命大学, Domestic conference

    Poster presentation

  • ウシ新鮮射出精子における先体チロシンリン酸化タンパク質の分布状態

    ARAI M. MIYUKI, HARAYAMA HIROSHI

    第4回関西生殖医学集談会・第48回関西アンドロロジーカンファレンス合同研究会, Mar. 2016, Japanese, 大阪市, Domestic conference

    Oral presentation

  • ウシ新鮮射出精子の分子性状における個体差 -先体チロシンリン酸化タンパク質と人工授精成績との関係-

    ARAI Miyuki, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第108回日本繁殖生物学会大会, Sep. 2015, Japanese, 日本繁殖生物学会, 宮崎, Domestic conference

    Poster presentation

  • 哺乳類の精子学 ー運動の開始から受精までー

    HARAYAMA HIROSHI

    第35回関西生殖発生毒性フォーラム 基礎教育講演, Apr. 2015, Japanese, 大阪市, Domestic conference

    [Invited]

    Invited oral presentation

  • Mammalian sperm acrosomal protein affecting the artificial insemination results and the in-vitro fertilization results

    HARAYAMA HIROSHI

    第60回日本生殖医学会学術講演会 シンポジウム3 「受精に関する最近の話題」, Apr. 2015, Japanese, 横浜, Domestic conference

    [Invited]

    Nominated symposium

  • ウシ凍結精子での先体損傷・離脱に伴うIZUMO1の変化

    FUKUDA Masaki, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    日本畜産学会第119回大会, Mar. 2015, Japanese, 宇都宮, Domestic conference

    Oral presentation

  • 低繁殖症雄ウシの精子における欠陥型タンパク質の検出および特性解析

    HARAYAMA HIROSHI

    受胎率向上SIG第1回会合, Dec. 2014, Japanese, 東京, Domestic conference

    [Invited]

    Nominated symposium

  • The association between spatial distribution patterns of SPACA1 in human ejaculated sperms and outcomes of conventional IVF

    KISHIDA Kazumi, HARAYAMA Hiroshi, KIMURA Fuminori, MURAKAMI Takashi

    World Congress of Reproductive Biology 2014, Sep. 2014, English, Edinburgh, UK, International conference

    Poster presentation

  • 黒毛和種精子でのチロシンリン酸化タンパク質の分布状態が先体の安定性に及ぼす影響

    ARAI Miyuki, MINAMI Kenta, OGURA Yukari, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第107回日本繁殖生物学会大会, Aug. 2014, Japanese, 日本繁殖生物学会, 帯広, Domestic conference

    Poster presentation

  • ブタ胚盤胞におけるタイトジャンクション関連タンパク質claudin familyの発現性

    MIYAKE Masashi, TOU Tetsu, SHIBUTANI Mihiro, LEE Jibak, HARAYAMA Hiroshi, MIYANO Takashi

    第107回日本繁殖生物学会大会, Aug. 2014, Japanese, 日本繁殖生物学会, 帯広, Domestic conference

    Oral presentation

  • ブタ精子での先体反応に伴う頭部タンパク質「SPACA1」の分布および分子マスの変化

    OGURA Yukari, TAKAGISHI Yuki, KOJIMA Aya, ISHIKAWA Sho, HARAYAMA Hiroshi

    第107回日本繁殖生物学会大会, Aug. 2014, Japanese, 日本繁殖生物学会, 帯広, Domestic conference

    Poster presentation

  • ウシ凍結保存精子における先体の損傷・離脱が頭部でのIZUMO1の分布に及ぼす影響

    FUKUDA Masaki, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第107回日本繁殖生物学会大会, Aug. 2014, Japanese, 日本繁殖生物学会, 帯広, Domestic conference

    Poster presentation

  • ウシ精巣におけるcAMP依存性転写調節因子CREMの発現パターンの解析

    NODA Taichi, MINAMI Kenta, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第107回日本繁殖生物学会大会, Aug. 2014, Japanese, 日本繁殖生物学会, 帯広, Domestic conference

    Poster presentation

  • 家畜精子のハイパーアクチベーションに先立つ部位特異的なキャパシテーション

    HARAYAMA HIROSHI

    日本アンドロロジー学会第33回学術大会 シンポジウム 1 「Capacitation の最前線を極める」, Jun. 2014, Japanese, 軽井沢プリンスホテルウエスト, Domestic conference

    [Invited]

    Nominated symposium

  • ブタ精子の鞭毛に分布するカルパイン2の役割

    KOJIMA Aya, HARAYAMA Hiroshi

    第2回関西生殖医学集談会, Mar. 2014, Japanese, 兵庫医科大学産婦人科学講座, 大阪市, Domestic conference

    Oral presentation

  • ヒト射出精子の頭部におけるSPACA1の分布状態の個体差 -先体主部での分布状態と体外受精成績との関係-

    KISHIDA Kazumi, HARAYAMA Hiroshi, KIMURA Fuminori, MURAKAMI Takashi

    第2回関西生殖医学集談会, Mar. 2014, Japanese, 兵庫医科大学産婦人科学講座, 大阪市, Domestic conference

    Oral presentation

  • Full-type hyperactivationの誘起に伴う家畜精子の頸部および中片部における変化

    HARAYAMA Hiroshi, MIZUNO Yohei, KOJIMA Aya

    日本畜産学会第118回大会, Mar. 2014, Japanese, 日本畜産学会, つくば, Domestic conference

    Oral presentation

  • 人工授精で雌を受胎させにくい精子を産生する黒毛和種雄個体の検出法 -凍結保存後の精子先体の正常性と体内受精由来の移植可能胚率の関係-

    SAKASE Mitsuhiro, KOJI Reiko, KOHAMA Minako, AKIYAMA Yoshitaka, OKA Akio, HARAYAMA Hiroshi, FDUKUSHIMA Moriyuki

    第106回日本繁殖生物学会大会, Sep. 2013, Japanese, 日本繁殖生物学会, 府中市, Domestic conference

    Oral presentation

  • ブタ精子におけるカルパインの検出および機能解析

    KOJIMA AYA, ISHIKAWA Sho, SHIDARA Osamu, HARAYAMA Hiroshi

    第106回日本繁殖生物学会大会, Sep. 2013, Japanese, 日本繁殖生物学会, 府中市, Domestic conference

    Poster presentation

  • ヒト射出精子におけるSPACA1の検出 -先体での検出パターンと体外受精成績との相関性-

    KISHIDA Kazumi, HARAYAMA Hiroshi, KIMURA Fuminori, MURAKAMI Takashi

    第106回日本繁殖生物学会大会, Sep. 2013, Japanese, 日本繁殖生物学会, 府中市, Domestic conference

    Poster presentation

  • ウシ精子の鞭毛におけるタンパク質チロシンリン酸化および超活性化運動の誘起

    MIZUNO Yohei, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    関西畜産学会第63回大会, Sep. 2013, Japanese, 関西畜産学会, 彦根市, Domestic conference

    Oral presentation

  • ブタ精子での後帽部タンパク質の脱リン酸化および先体反応におけるcAMP-EPACシグナリングの役割

    ISONO Ayane, KOJIMA Aya, TAKAGISHI Yuki, ISHIKAWA Sho, SHIDARA Osamu, HARAYAMA Hiroshi

    日本畜産学会第116回大会, Mar. 2013, Japanese, 日本畜産学会, 広島市, Domestic conference

    Oral presentation

  • ウシのハイパーアクチベーション精子における鞭毛タンパク質のリン酸化状態

    MIZUNO Yohei, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    日本畜産学会第116回大会, Mar. 2013, Japanese, 日本畜産学会, 広島市, Domestic conference

    Oral presentation

  • 哺乳類精子の細胞内cAMPシグナル伝達機構に関する研究

    HARAYAMA HIROSHI

    第105回日本繁殖生物学会大会, Sep. 2012, Japanese, 日本繁殖生物学会, つくば市, Domestic conference

    [Invited]

    Invited oral presentation

  • 黒毛和種精子先体前部におけるチロシンリン酸化型SPACA1の解析-サンプル間差および精子成熟に伴う変化について-

    MINAMI Kenta, NODA Taichi, ISONO Ayane, KOJIMA Aya, MIZUNO Yohei, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第105回日本繁殖生物学会大会, Sep. 2012, Japanese, 日本繁殖生物学会, つくば市, Domestic conference

    Poster presentation

  • 黒毛和種精子における先体の耐凍性マーカーの特性解析

    MINAMI Kenta, NODA Taichi, ISONO Ayane, KOJIMA Aya, MIZUNO Yohei, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA HIROSHI

    第62回関西畜産学会大会, Sep. 2012, Japanese, 関西畜産学会, 和歌山市, Domestic conference

    Oral presentation

  • cAMP合成酵素ADCY10の異常型スプライスバリアントの検出によるウシ精子の新規の性状検査法

    NODA Taichi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA HIROSHI

    第105回日本繁殖生物学会大会, Sep. 2012, Japanese, 日本繁殖生物学会, つくば市, Domestic conference

    Oral presentation

  • ブタの精巣および射出精子における活性型CREM(cAMP-responsive element modulator)の分子性状の解析

    NODA Taichi, SHIDARA Osamu, ISHIKAWA Sho, HARAYAMA Hiroshi

    日本アンドロロジー学会第31回学術大会, Jun. 2012, Japanese, 日本アンドロロジー学会, 神戸市, Domestic conference

    Oral presentation

  • 黒毛和種精子における耐凍能マーカーの検出パターン

    MINAMI Kenta, NODA Taichi, ISONO Ayane, KOJIMA Aya, MIZUNO Yohei, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA HIROSHI

    日本畜産学会第115回大会, Mar. 2012, Japanese, 日本畜産学会, 名古屋市, Domestic conference

    Oral presentation

  • ブタ雄性生殖細胞での活性型CREMの検出および分布解析

    NODA Taichi, SHIDARA Osamu, HARAYAMA HIROSHI

    日本畜産学会第115回大会, Mar. 2012, Japanese, 日本畜産学会, 名古屋市, Domestic conference

    Oral presentation

  • ウシ精子における断片型ADCY10の合成メカニズム

    NODA Taichi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA HIROSHI

    第137回日本生殖医学会関西支部集談会, Mar. 2012, Japanese, 大阪市, Domestic conference

    Oral presentation

  • ウシの可溶化型アデニル酸シクラーゼ (ADCY10) の断片型は精巣においてmRNAスプライスバリアントから合成される

    NODA Taichi, WATAI Yusuke, SAKASE Mitsuhiro, KOHAMA Namiko, FUKUSHIMA Moriyuki, HARAYAMA HIROSHI

    第104回日本繁殖生物学会大会, Sep. 2011, Japanese, 日本繁殖生物学会, 盛岡市, Domestic conference

    Poster presentation

  • ブタ精子でのcAMP依存的な鞭毛超活性化運動に及ぼすカルモジュリンアンタゴニストおよびカルパイン阻害剤

    HARAYAMA Hiroshi, NODA Taichi, SHIDARA Osamu

    第136回日本生殖医学会関西支部集談会, Mar. 2011, Japanese, 大阪市, Domestic conference

    Oral presentation

  • 哺乳類精子における部位特異的なcAMPシグナリング

    HARAYAMA HIROSHI

    日本動物学会第80回大会「第8回受精シンポジウム」, Sep. 2010, Japanese, 日本動物学会, 静岡市, Domestic conference

    [Invited]

    Nominated symposium

  • ブタ精子中片部のAMPKはcAMP依存性ハイパーアクチベーションの発現制御に関与する

    HARAYAMA Hiroshi, NODA Taichi, SHIDARA Osamu

    日本アンドロロジー学会第29回学術大会, Jul. 2010, Japanese, 日本アンドロロジー学会, 東京, Domestic conference

    Oral presentation

  • 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

    ISHIHARA KANA, TASAKA KEN, TANIDA TAKASHI, WARITA KATSUHIKO, MITSUHASHI TOMOKO, MIYAKE MASASHI, HARAYAMA HIROSHI, YOKOYAMA TOSHIFUMI, KITAGAWA HIROSHI, HOSHI NOBUHIKO

    11th International Symposium on Spermatology, Jun. 2010, English, 宜野湾市, International conference

    Poster presentation

  • Paternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) affects the sex ratio of offspring at birth by change the sex ratio of fertilized egg

    ISHIHARA K, TASAKA K, TANIDA T, WARITA K, MITSUHASHI T, MIYAKE M, HARAYAMA Hiroshi, YOKOYAMA T, KITAGAWA H, HOSHI N

    11th International Symposium on Spermatology, Jun. 2010, English, Naha, International conference

    Poster presentation

  • Immunodetection patterns of tyrosine-phosphorylated proteins in the head of frozen-thawed spermatozoa of Japanese Black bulls

    HARAYAMA Hiroshi, MURASE Tetsuma, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki

    11th International Symposium on Spermatology, Jun. 2010, English, Naha, International conference

    Public symposium

  • Identification of cAMP responsive element modulator (CREM) isoforms of the porcine testis

    NODA Taichi, SHIDARA Osamu, HARAYAMA Hiroshi

    11th International Symposium on Spermatology, Jun. 2010, English, Naha, International conference

    Poster presentation

  • ブタ精巣におけるcAMP依存性転写調節因子CREMτの発現

    NODA Taichi, SHIDARA Osamu, HARAYAMA Hiroshi

    日本畜産学会第112回大会, Mar. 2010, Japanese, 日本畜産学会, 東京, Domestic conference

    Oral presentation

  • ブタの精子形成関連タンパク質の発現を制御するcAMPシグナリング構成分子の同定

    NODA Taichi, HARAYAMA Hiroshi

    第135回日本生殖医学会関西支部集談会, Mar. 2010, Japanese, 大阪市, Domestic conference

    Oral presentation

  • ウシ精巣におけるアデニル酸シクラーゼ10(ADCY10)mRNAsのスプライスバリアントの検出

    WATAI Yusuke, NODA Taichi, SAKASE Mitsuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第135回日本生殖医学会関西支部集談会, Mar. 2010, Japanese, 大阪市, Domestic conference

    Oral presentation

  • Subfertility in Japanese Black bulls and new methods to detect abnormal functionality of spermatozoa

    MURASE Tetsuma, MUKOUJIMA K, HARAYAMA HIROSHI, HOSHINO Y, KATO T

    International Symposium of Declining Fertility in Dairy Cows in the World; its Causes and Possible Solutions, Feb. 2010, English, Miyazaki, International conference

    Poster presentation

  • Active protein kinase A (PKA) is required for the meiotic resumption of pig oocytes

    CAYO-COLCA I S, HARAYAMA Hiroshi, MIYANO Takashi

    2009 Japan-Taiwan Joint Symposium on Cell Signaling and Gene Regulation, Nov. 2009, English, Kobe, International conference

    Poster presentation

  • ブタ単為発生2倍体の胚盤胞への発生に及ぼすヘキソースの影響

    YONEZAWA Jun-ichi, HARAYAMA Hiroshi, MIYAKE Masashi

    第102回日本繁殖生物学会大会, Sep. 2009, Japanese, 日本繁殖生物学会, 奈良市, Domestic conference

    Poster presentation

  • Changes in protein kinase A (PKA) activity during meiotic resumption of pig oocytes

    Cayo-Colca I S, HARAYAMA Hiroshi, MIYANO Takashi

    第102回日本繁殖生物学会大会, Sep. 2009, English, 日本繁殖生物学会, 奈良市, Domestic conference

    Poster presentation

  • ブタ精巣におけるcAMP依存性転写調節因子(CREM)ファミリーアイソフォームの発現

    NODA Taichi, SHIDARA Osamu, HARAYAMA Hiroshi

    日本アンドロロジー学会第28回学術大会, Jul. 2009, Japanese, 日本アンドロロジー学会, 富山市, Domestic conference

    Oral presentation

  • ブタ精子におけるADCY10の検出

    NAKAMURA Kazumi, SUZUKI Chihiro, TATE Shunsuke, LEE J, HARAYAMA HIROSHI

    日本アンドロロジー学会第28回学術大会, Jul. 2009, Japanese, 日本アンドロロジー学会, 富山市, Domestic conference

    Oral presentation

  • Detection of soluble adenylyl cyclase (ADCY10) homolog proteins in boar spermatozoa

    NAKAMURA Kazumi, SUZUKI Chihiro, TATE Shunsuke, LEE Jibak, HARAYAMA Hiroshi

    34th Annual Meeting of the American Society of Andrology, Apr. 2009, English, The American Society of Andrology, Philadelphia, International conference

    Poster presentation

  • ブタ精子でのハイパーアクチベーションの発現制御における解糖系の役割

    HARAYAMA Hiroshi, NAKAMURA Kazumi, MIYAKE Masashi

    日本畜産学会第110回大会, Mar. 2009, Japanese, 日本畜産学会, 藤沢市, Domestic conference

    Oral presentation

  • cAMPアナログ処理により鞭毛超活性化運動が誘起されたブタ精子でのAMPKサブユニットの分子変化

    HARAYAMA Hiroshi, NODA Taichi, MIYAKE Masashi

    第134回日本生殖医学関西支部集談会, Mar. 2009, Japanese, 大阪市, Domestic conference

    Oral presentation

  • 哺乳類精子の頚部特異的なcAMPシグナリングによるハイパーアクチベーションの制御

    HARAYAMA Hiroshi

    第40回精子研究会シンポシウム, Jan. 2009, Japanese, 精子研究会, 吹田市, Domestic conference

    [Invited]

    Nominated symposium

  • 低受胎率を示した黒毛和種あるいは正常な個体における凍結-融解精子鞭毛の超活性化運動

    MURASE Tetsuma, Ismail EI-kon, MUKAIJIMA Koji, HARAYAMA Hiroshi, TAKASU Masaki, SAKAI Kenji

    第101回日本繁殖生物学会大会, Sep. 2008, Japanese, 日本繁殖生物学会, 福岡市, Domestic conference

    Oral presentation

  • cAMP-Epacシグナリングを介したブタ精子キャパシテーションの新規制御メカニズム

    NAKAMURA Kazumi, TATE Shunsuke, HARAYAMA HIROSHI

    日本アンドロロジー学会第27回学術大会, Jul. 2008, Japanese, 日本アンドロロジー学会, 京都市, Domestic conference

    Oral presentation

  • Cyclic AMP-induced inactivation of PDK1 is linked to the enhancement of protein tyrosine phosphorylation and flagellar hyperactivation in boar spermatozoa

    HARAYAMA Hiroshi

    33rd Annual Meeting of American Society of Andrology, Apr. 2008, English, American Society of Andrology, Albuquerque, International conference

    Poster presentation

  • ウシ精子のハイパーアクチベーション発現制御に関与する候補分子の特定

    NISHIJIMA Kazuhiro, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    第109回日本畜産学会大会, Mar. 2008, Japanese, 日本畜産学会, 水戸市, Domestic conference

    Oral presentation

  • マウス精巣上体尾精子の鞭毛主部においてプロテインホスファターゼはPKA活性を抑制する

    Namiko GOTO, Hiroshi HARAYAMA

    The 100th Annual meeting of the Japanese Society of Animal Reproduction, Oct. 2007, Japanese, 日本繁殖生物学会, Tokyo, Domestic conference

    Oral presentation

  • ブタ精子頭部でのcAMP依存性細胞内カルシウム濃度上昇およびキャパシテーションにおけるEpacの役割

    Kazumi NAKAMURA, Shunsuke TATE, Osamu SHIDARA, Hiroshi HARAYAMA

    The 100th Annual meeting of the Japanese Society of Animal Reproduction, Oct. 2007, Japanese, 日本繁殖生物学会, Tokyo, Domestic conference

    Oral presentation

  • ブタ精子の部位特異的な細胞内cAMPシグナリング:ハイパーアクチベーション誘起に伴う鞭毛主部でのPKAおよびPDK1のcAMP依存的な変化

    HARAYAMA Hiroshi, TATE Shunnsuke, NAKAMURA Kazumi, SHIDARA Osamu, MIYAKE Masashi

    The 100th Annual meeting of the Japanese Society of Animal Reproduction, Oct. 2007, Japanese, 日本繁殖生物学会, Tokyo, Domestic conference

    Poster presentation

  • ウシ,ブタおよびマウスの精子でのタンパク質チロシンリン酸化に及ぼすcAMPアナログとタンパク質チロシンホスファターゼの影響

    NISHIJIMA Kazuhiro, GOTO Namiko, NAKAMURA Kazumi, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    The 100th Annual meeting of the Japanese Society of Animal Reproduction, Oct. 2007, Japanese, Tokyo, Domestic conference

    Poster presentation

  • ブタ精子の部位特異的な細胞内cAMPシグナリング:頚部のcAMP-PKA-PKCシグナリングはハイパーアクチベーションの発現調節に関与する

    Hiroshi HARAYAMA, Masashi MIYAKE, Osamu SHIDARA

    日本アンドロロジー学会第26回学術大会, Jul. 2007, Japanese, 浦安市, Domestic conference

    Oral presentation

  • ウシおよびブタの精子におけるcAMP依存性タンパク質チロシンリン酸化

    Kazuhiro NISHIJIMA, Namiko GOTO, Moriyuki FUKUSHIMA, Hiroshi HARAYAMA

    日本アンドロロジー学会第26回学術大会, Jul. 2007, Japanese, 浦安市, Domestic conference

    Oral presentation

  • 人間との共生環境の回復を目的とした有害野生動物の繁殖抑制

    HARAYAMA Hiroshi, GOTO Namiko, TATE Shunsuke

    環境科学総合研究所成果報告会, May 2007, Japanese, 熱海, Domestic conference

    Public discourse

  • 凍結融解処理されたブタ精子における32 kDaチロシンリン酸化タンパク質Tyr32の出現メカニズム

    Hiroshi HARAYAMA, Tomohito TABUCHI, Osamu SHIDARA

    日本畜産学会第107回大会, Mar. 2007, Japanese, 相模原市, Domestic conference

    Oral presentation

  • ブタ着床前胚におけるタイトジャンクション関連タンパク質―Occludin の発現性

    許 尚丹, HARAYAMA Hiroshi, MIYAKE Masashi

    第100回日本繁殖生物学会大会, Mar. 2007, Japanese, 日本繁殖生物学会, 東京, Domestic conference

    Poster presentation

  • ブタ精子のキャパシテーションにおけるcAMP-Epac-カルシウムシグナリングの役割

    Shun-suke TATE, Kazumi NAKAMURA, Hiroshi HARAYAMA

    日本畜産学会第107回大会, Mar. 2007, Japanese, 相模原市, Domestic conference

    Oral presentation

  • Localization of claudin family proteins in pig embryos during preimplantation development

    S. XU, J. LEE, Hiroshi HARAYAMA, Masashi MIYAKE

    33rd Annual Conference of the International Embryo Transfer Society, Jan. 2007, English, Kyoto, JAPAN, International conference

    Poster presentation

  • Fertilization-related parameters of frozen-thawed spermatozoa from subfertile Japanese Black cattle

    Ken KURODA, Moriyuki FUKUSHIMA, Masashi MIYAKE, Hiroshi HARAYAMA

    33rd Annual Conference of the International Embryo Transfer Society, Jan. 2007, English, Kyoto, Japan, International conference

    Poster presentation

  • Assembly of nuclear membrane proteins in bovine IVF and activated oocytes.

    MIYAKE Masashi, ISAJI Mamiko, HARAYAMA Hiroshi

    The 3rd Asian Reproductive Biotechnology Conference, Nov. 2006, English, Asian Reproductive Biotechnology Conference, Hanoi, Vietnam, International conference

    Invited oral presentation

  • Detection of cAMP-Epac signaling cascades in mouse epididymal spermatozoa

    GOTO Namiko, AMANO Reiko, LEE Jibak, HARAYAMA Hiroshi

    99th Annual meeting of Japanese Society of Animal Reproduction, Sep. 2006, Japanese, 日本繁殖生物学会, Nagoya, Domestic conference

    Poster presentation

  • cAMP-PKA signaling-dependent activation of PKC is involved in regulation of flagellar hyperactivation in boar spermatozoa

    HARAYAMA Hiroshi, MIYAKE Masashi, SHIDARA Osamu

    99th Annual Meeting of Japanese Society of Animal Reproduction, Sep. 2006, Japanese, Japanese Society of Animal Reproduction, Nagoya, Domestic conference

    Oral presentation

  • Localization of proteins related to the tight junction in the pig and mouse preimplantational embryos.

    XU Shangdan, KIMURA Hayato, MATSUMURA Hirokazu, LEE Jibak, HARAYAMA Hiroshi, MIYAKE Masashi

    The 99th Annual meeting of the Japanese Society of Animal Reproduction, Sep. 2006, Japanese, 日本繁殖生物学会, Nagoya, Domestic conference

    Poster presentation

  • Nuclear localization of a tight Junction protein claudin-1 in the preimplantation embryos

    XU Shangdan, KIMURA Hayato, Matsumura Hirokazu, LEE Jibak, HARAYAMA Hiroshi, MIYAKE Masashi

    The 13th Annual Meeting of Japan Embryo Transfer Society, Aug. 2006, Japanese, 日本胚移植研究会, Hiroshima, Domestic conference

    Oral presentation

  • Effects of sodium bicarbonate and PKA inhibitor on cAMP-dependent elevation of intracellular calcium concentration in the boar sperm head

    TATE Shunsuke, SHIDARA Osamu, HARAYAMA Hiroshi

    56th Annual meeting of Kansai Society of Animal Science, Aug. 2006, Japanese, 関西畜産学会, Osaka, Domestic conference

    Oral presentation

  • Role of protein kinase C in the cyclic adenosine 3',5'-monophosphate-dependent hyperactivation of boar spermatozoa

    HARAYAMA Hiroshi, MIYAKE Masashi

    31st Annual meeting of American Society of Andrology, Apr. 2006, English, American Society of Andrology, Chicago, USA, International conference

    Poster presentation

  • Premature capacitation and acrosome reaction in frozen spermatozoa from subfertile Japanese Black cattle

    KURODA Ken, FUKUSHIMA Moriyuki, HARAYAMA Hiroshi

    106th Annual meeting of Japanese Society of Animal Science, Mar. 2006, Japanese, 日本畜産学会, Fukuoka, Domestic conference

    Poster presentation

  • Effects of calyculin A on cAMP-dependent decrease of Ser-/Thr-phosphorylated proteins in the postacrosomal region of boar spermatozoa

    ADACHI Jun, HARAYAMA Hiroshi, MIYAKE Masashi

    106th Annual meeting of Japanese Society of Animal Science, Mar. 2006, Japanese, 日本畜産学会, Fukuoka, Domestic conference

    Poster presentation

  • Detection of Ca2+ store marker proteins in boar and mouse spermatozoa

    ONO Mikiko, HARAYAMA Hiroshi, MIYAKE Masashi

    106th Annual meeting of Japanese Society of Animal Science, Mar. 2006, Japanese, 日本畜産学会, Fukuoka, Domestic conference

    Oral presentation

  • Low progressive motility and premature expression of fertilizing ability of frozen-thawed spermatozoa from subfertile Japanese Black cattle

    KURODA Ken, HARAYAMA Hiroshi

    Kobe University The 21st Century COE Program Symposium: Japan-Taiwan Symposium on Cell Signaling and Gene Expression (Program and Abstract,18), Nov. 2005, English, Kobe University COE Program "Signaling Mechanisms by Protein Modification Reactions", Kobe, International conference

    Oral presentation

  • Low progressive motility and premature capacitation-acrosome reaction in the frozen-thawed spermatozoa from subfertile Japanese Black cattle

    KURODA Ken, FUKUSHIMA Moriyuki, 原山 洋

    98th Annual Meeting of Japanese Society of Animal Reproduction, Sep. 2005, Japanese, 日本繁殖生物学会, Shizuoka, Domestic conference

    Poster presentation

  • Effects of PKA inhibitor on the cAMP-dependent elevation of intracellular calcium concentration in boar sperm head

    TATE Shunsuke, 設楽 修, 原山 洋

    98th Annual Meeting of Japanese Society of Animal Reproduction, Sep. 2005, Japanese, 日本繁殖生物学会, Shizuoka, Domestic conference

    Oral presentation

  • cAMP-dependent changes in glycochains and phosphoproteins in the postacrosomal region of boar spermatozoa

    ADACHI Jun, Tate Shunsuke, 原山 洋, 三宅 正史

    2005 (55th) Annual Meeting of Kansai Society of Animal Science, Sep. 2005, Japanese, 関西畜産学会, Matsuyama, Domestic conference

    Oral presentation

  • Low progressive motility, low PKA activity and premature capacitation of frozen-thawed spermatozoa from subfertile bulls

    KURODA Ken, HARAYAMA Hiroshi

    8th International Congress of Andrology (Int. J. Androl., Vol. 28(Suppl. 1), 79), Jun. 2005, English, International Society of Andrology, Seoul, Korea, International conference

    Poster presentation

  • A cyclic AMP-dependent tyrosine phosphorylation of phospholipase C in boar spermatozoa

    Harayama Hiroshi

    8th International Congress of Andrology (Int. J. Androl., Vol. 28(Suppl. 1), 78), Jun. 2005, English, International Society of Andrology, Seoul, Korea, International conference

    Oral presentation

  • Analysis of the cAMP signaling cascade in mouse spermatozoa

    IWASA Chika, 原山 洋

    104th Annual Meeting of Japanese Society of Animal Science, Mar. 2005, Japanese, 日本畜産学会, Tokyo, Domestic conference

    Oral presentation

  • cAMP-dependent activation of phospholipase Cgamma in boar spermatozoa

    原山 洋, 三宅 正史, Murase Tetsuma

    104th Annual Meeting of Japanese Society of Animal Science, Mar. 2005, Japanese, 日本畜産学会, Tokyo, Domestic conference

    Oral presentation

  • The activation of boar sperm tyrosine kinase SYK is regulated via calcium-independent cAMP signaling

    原山 洋

    82th Annual Meeting of Japanese Society of Swine Science, Oct. 2004, Japanese, 日本養豚学会, National Livestock Breeding Center, Domestic conference

    Oral presentation

  • cAMP-dependent activation of non-receptor tyrosine kinase SYK in boar spermatozoa

    原山 洋, MUROGA Mai, 三宅 正史

    97th Annual Meeting of Japanese Society of Animal Reproduction, Sep. 2004, Japanese, 日本繁殖生物学会, Hiroshima, Domestic conference

    Poster presentation

  • Phosphorylation of PKA substrate proteins in boar capacitating spermatozoa

    KURODA Ken, 原山 洋

    97th Annual Meeting of Japanese Society of Animal Reproduction, Sep. 2004, Japanese, 日本繁殖生物学会, Hiroshima, Domestic conference

    Oral presentation

  • Formation of nuclear envelope and dephosphorylation of histone H3 in the in-vitro fertilized bovine oocytes

    ISAJI Mamiko, IWATA Hisataka, HARAYAMA Hiroshi, Lee Jibak, MIYAKE Masashi

    The 11th Annual Meeting of Japan Embryo Transfer Society, Aug. 2004, Japanese, 日本胚移植研究会, Matsuyama, Domestic conference

    Oral presentation

  • cAMP-induced increase of 32-kDa tyrosine-phosphorylated protein (TyrP32) is related to the calcium-dependent disintegration of the acrosome in boar sperm.

    HARAYAMA Hiroshi, SASAKI Kiyomi, MIYAKE Masashi

    The American Society of Andrology, 29th Annual Meeting., Apr. 2004, English, The American Society of Andrology, USA, Baltimore, International conference

    Poster presentation

  • ウシ受精卵母細胞におけるLys9でメチル化されているヒストンH3の局在性

    伊佐治 麻実子, 岩田 尚孝, 原山 洋, 三宅 正史

    The 103th Annual meeting of Animal Science, Mar. 2004, Japanese, 日本畜産学会, 東京農工大学, Domestic conference

    Oral presentation

  • Role of calcium in the cAMP-induced increase of TyrP32 and acrosomal disintegration

    原山 洋, SASAKI Kiyomi, 三宅 正史

    103th Annual Meeting of Japanese Society of Animal Science, Mar. 2004, Japanese, 日本畜産学会, Tokyo University of Agriculture and Technology, Domestic conference

    Oral presentation

  • ウシ受精ならびに活性化卵母細胞におけるLAP2βの局在とその凝集時期

    伊佐治 麻実子, 岩田 尚孝, 原山 洋, 三宅 正史

    第27回日本分子生物学会年会, 2004, Japanese, 日本分子生物学会, 未記入, Domestic conference

    Oral presentation

  • Viability and protein phosphorylation state of boar sperm undergoing cAMP analogue-induced head-to-head agglutination

    HARAYAMA Hiroshi, SASAKI K, MIYAKE Masashi

    The AmericanSociety of Andrology 28th Annual Meeting,, 2003, English, 未記入, Washington. DC, International conference

    Oral presentation

  • Localization of LAP2beta in oocytes and embryos during early development in cattle, The Annual Conference of the International Embryo Transfer Society

    ISAJI M, IWATA H, OHTA M, HARAYAMA Hiroshi, MIYAKE Masashi

    Theriogenology, 2003, English, 未記入, 未記入, International conference

    Oral presentation

  • Behavior of chromatins in pronuclei in the cattle and mouse

    ISAJI M, IWATA H, OHTA M, HAYARAMA Hiroshi, MIYAKE Masashi

    36th Annual Meeting of the Society for the Study of Reproduction, 2003, English, 未記入, 未記入, International conference

    Oral presentation

Association Memberships

  • 関西アンドロロジーカンファレンス

  • 関西生殖医学集談会

  • 精子研究会

  • 日本生殖医学会

  • The Society for the Study of Reproduction

  • American Society of Andrology

  • 関西畜産学会

  • 日本アンドロロジー学会

  • 日本繁殖生物学会

  • 日本畜産学会

Research Projects

  • Establishment and spread of the new assay for sperm flagellar function to improve artificial insemination results in the cattle

    原山 洋, 中嶋 昭雄

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2022 - 31 Mar. 2025

  • Studies on expressions and roles of INSL3 secretory dynamics and its receptor expression in reproductive organs and germ cells in female cattle

    川手 憲俊, 西村 亮, 原山 洋, 古山 敬祐

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research, Grant-in-Aid for Scientific Research (C), Osaka Prefecture University, 01 Apr. 2021 - 31 Mar. 2025

    2021年度は、1)ウシの発情周期の主席卵胞と黄体および卵胞嚢腫のINSL3分泌能を調べることを目的とし、主席卵胞と嚢腫卵胞の卵胞液および黄体組織のINSL3と性ステロイド(テストステロン、エストラジオール-17β、プロジェステロン)量を解析するとともに、培養黄体細胞からのINSL3とプロジェステロンの分泌能を分析した。同時に、2)正常雌ウシと卵胞嚢腫牛の血中INSL3濃度の動態を解明するために、雌ウシの血中INSL3濃度を測定できるサンドイッチ時間分解蛍光免疫測定(TRFIA)法の開発に取り組んだ。 1)については、食肉処理場において雌ウシの卵巣を採取し、発情周期中の主席卵胞の卵胞液と黄体の黄体組織のINSL3と性ステロイド量を測定した。その結果、主席卵胞のINSL3分泌はその成熟にともなって増加することが示唆された。卵胞のテストステロンとエストラジオール-17β量は卵胞の嚢腫化により低下するが、INSL3量は増加することが示唆された。黄体組織のINSL3とプロジェステロン含量はその開花期に増加することが示された。一方、黄体から黄体細胞を分離して培養し、培養液のINSL3濃度を測定した結果、INSL3分泌能は開花期に上昇するが、その分泌は黄体形成ホルモン(LH)に依存しないことが示された。 2)については、サンドイッチTRFIA法に必要な検出用の抗ウシINSL3ペプチド抗体を作成し、そのビオチン標識を実施した。さらに補足用ウシINSL3抗体、ウシINSL3標準品、ビオチン標識検出用抗体およびユーロピウム標識ストレプトアビジンを用いてサンドイッチTRFIAの測定法の諸条件の検討を行った。その結果、最小検出濃度が0.1 ng/mLの高感度測定法を確立することができた。雌ウシ血漿サンプルを本法で測定したところ、血中INSL3濃度を無抽出で測定できることが判明した。

  • 融合因子SOFの機能解析を通した精子-卵子の細胞膜融合機構の解明

    野田 大地, 原山 洋

    日本学術振興会, 科学研究費助成事業, 基盤研究(B), 01 Apr. 2020 - 31 Mar. 2024

    sperm-oocyte fusion required (SOF)2~3を特異的に認識する抗体を得ることができなかったが、バックアッププランとして作製していたSOF2や3のC末端側にアフィニティータグを融合させたタンパク質を発現するトランスジェニックマウスを用いて、免疫染色法によりSOF2や3が先体反応後の精子頭部に局在することを明らかにできた。さらに、IP/MS解析によりSOF2や3と相互作用する因子の同定にも成功した。そこで、マウスの心臓、肝臓、脾臓、腎臓、脳、精巣、卵巣、および子宮を採取し、得られた各組織由来のcDNAを使ってPCRを行ったところ、これらの遺伝子は精巣や精巣上体で発現することが明らかになった。
    正常および低繁殖症を示す雄ウシの生殖細胞(精巣や成熟精子など)を採取して、ウェスタンブロティングを行ったところ、両者の間で差がある受精関連遺伝子を見出した。
    マウス既知融合関連遺伝子の機能が種を超えて保存されているかを調べる目的で、他動物種の融合関連遺伝子を発現するトランスジェニックマウスの作製に成功した。

  • Analyses of 3-dimensional rotation for the purpose of reconsidering criteria for the evaluation of flagellar functions in bull sperm

    HARAYAMA HIROSHI

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Challenging Research (Exploratory), Challenging Research (Exploratory), Kobe University, Jun. 2019 - Mar. 2021, Principal investigator

    In this study regarding a new candidate "3-dimensional rotation (3D-R)" of the criterion for the evaluation of flagellar functions in bull cryopreserved sperm, we carried out experiments to elucidate the mechanism for its occurrence, to characterize it biologically and to examine its validity. The obtained results were listed below. (1) The cryopreservation-related occurrence of 3D-R is regulated by reduction of the activity of thapsigargin-sensitive Ca2+ pumps in the sperm neck and the consequent increases of the intracellular Ca2+ level. (2) The 3D-R is one of the typical patterns of motility which sperm exhibit during the capacitation process. (3) The 3D-R is a potentially criterial parameter for the evaluation of the flagellar functions in bull cryopreserved sperm.

  • Studies on the mechanisms that cholesterol exerts its therapeutic effects on boar spermatozoa affected with summer infertility

    Murase Tetsuma

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Gifu University, Apr. 2017 - Mar. 2020, Coinvestigator

    cAMP content of boar spermatozoa stored up to 10 days with added cholesterol was not different among storage days. cAMP content in spermatozoa stored and incubated for stimulation of the acrosome reaction with the calcium ionophore A23187 slightly decreased and then rose during stimulation. Magnesium decreased the acrosome reaction triggered by A23187 while polymyxin B enhanced it. When frozen-thawed bull spermatozoa were used as a model, damaged acrosome, hyperactivation and the acrosome reaction were independent parameters from one another, suggesting that this should be taken into account when boar sperm function is investigated. Exposure of immature oocytes to adipose-derived stem cells (ADSC) and/or mechanical vibration during maturation in culture led to the increase in the sperm penetration and male pronuclear formation. A very effective method to collect, isolate and culture ADSC has been established in order to use them for increasing fertilizing ability of boar spermatozoa.

  • Characterization and utilization of SPACA1 proteins for the purpose of establishment of a new method to predict freezability of bull sperm before cryopreservation

    HARAYAMA HIROSHI

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2016 - Mar. 2019, Principal investigator

    The aims of the present study were to develop a new method to predict freezability of bull sperm before cryopreservation and to increase its reliability by disclosing molecular methodology. In this study, we succeeded in the development of the method to predict freezability of bull (Japanese Black bull) sperm before cryopreservation according to the criterion of the distribution of SPACA1 proteins. We also suggested that aberrant distribution of SPACA1 proteins is related to abnormal maturation of the sperm in the epididymis, and that low fertilizing ability of cryopreserved sperm with aberrant distribution of SPACA1 proteins is linked to poor results in the artificial insemination using them.

  • Etiology of boar spermatozoa affected with summer infertility in an attempt to develop a novel therapy

    Murase Tetsuma, SHIMIZU Kenji, +HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Gifu University, Apr. 2014 - Mar. 2017, Coinvestigator

    It was considered that one of the mechanisms underlying the hypersensitive induction of the acrosome reaction may be due to an increased content of cAMP [cAMP]i in boar spermatozoa collected during summer. Addition of cholesterol or removal of bicarbonate in the semen extender did not affect [cAMP]i of spermatozoa. Thus, a mechanism that cholesterol delays the induction of the acrosome reaction may be the effect on the generation of cAMP during the process of the acrosome reaction raher than during storage. Addition of magnesium lowered the occurrence of the acrosome reaction. The staining method of sperm acrosomes with FITC-PNA was modified. Preincubation of boar spermatozoa with adipose-derived stem cells (ADSC) increased the ability of in vitro fertilizing ability. Cholesterol, magnesium and ADSC are prospective in applying to a 'therapy of boar spermatozoa' suffering from summer infertility.

  • Expression of claudin family proteins in preimplantation embryos in the pig

    MIYAKE MASASHI, HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, Apr. 2013 - Mar. 2016, Coinvestigator

    The expression of claudin family, tight junction (TJ) were examined in pig blastocysts derived from activated diploids. Blastocysts expressed both mRNAs and proteins of claudin-1, -2, -4, -6, -7, -8, -9, -12 and -14. While claudin-4, -6 and -7 localized along the cell-boundary TJ structure, the other claudins were cytoplasmic. These results suggest that claudin-4, -6 and -7 are important for the TJ functions in pig blastocysts. Knockdown of claudin-1 and -4 by their siRNA severely suppressed the early cleavage. To examine whether expression patterns of the claudins will be employed as markers for the potential developmental ability of in vitro produced blastocysts in future, features of glucose metabolism was examined in pig preimplantation embryos. It was shown that recommendation of carbohydrates are different depending on the preimplantation developmental stages and hexose biosynthesis pathway and GlcNAcylation have important roles in pig preimplantation development.

  • Development of methods to evaluate molecular characteristics of livestock spermatozoa by the detection of aberrant fertility-regulating factors

    HARAYAMA HIROSHI, MURASE Tetsuma, FUKUSHIMA Moriyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, Apr. 2013 - Mar. 2016, Principal investigator

    The aim of the present study was to develop a new method to evaluate sperm molecular characteristics for identifying AI-subfertile males of livestock. In this study, we succeeded in the development of the method to evaluate molecular characteristics of bull (Japanese Black bull) cryopreserved and freshly ejaculated spermatozoa by the observation of acrosomal tyrosine-phosphorylated proteins. Moreover, we also showed that outcomes of AI and in vitro fertilization can be predicted by this evaluation for the spermatozoa.

  • Enhancement of the tolerance to the storage in boar and mouse spermatozoa by the re-maturation in vitro

    HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research, Grant-in-Aid for Challenging Exploratory Research, Kobe University, 2011 - 2012, Principal investigator

    There are great differences in the freezability of mammalian spermatozoa among species. In cattle, high freezability of the spermatozoa enables us to use frozen semen in the AI program. In this study, we indicated the molecular mechanism for epididymal maturation-related stabilization of the plasma membrane which brings high freezability to bull spermatozoa. In addition, we attempted to develop in vitro incubation system to enhance sperm freezability on the basis of the above-mentioned molecular mechanism.

  • Development of clinical therapeutic methods against failure of fertilizing functions causing summer infertility in boars

    MURASE Tetsuma, HARAYAMA Hiroshi, MIYAKE Masashi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Gifu University, 2009 - 2011, Coinvestigator

    In an attempt to improving boar sperm quality which decreases in summer and stable provision with semen leading to stable production of pork during the year, this study investigated a new diluent for boar semen storage, a new method to evaluate frozen-thawed bull sperm quality and development of a method to improve fertilizing ability of frozen-thawed bull spermatozoa. Bull spermatozoa were used as a reference to boar spermatozoa. Modified diluents for boar semen improved conception rates. In bulls, new method for sperm evaluation was found out and modification of diluent for freezing bull semen enabled improvement of sperm fertilizing ability.

  • Targeted therapy for defect of fertilizing ability in the spermatozoa from subfertile livestock

    HARAYAMA Hiroshi, MIYAKE Masashi, MURASE Tetsuma, FUKUSHIMA Moriyuki

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2008 - 2010, Principal investigator

    The initial step of animal reproduction is accomplishment of in vivo fertilization. For the purpose of successful fertilization, spermatozoa are required to show a precise process in the exertion of their fertilizing ability. In this study, we indicated that pivotal regulators of the intracellular signaling and glucose metabolism are potential targets to suppress precocious fertilization-related events in the spermatozoa. In addition, we also suggested that defective phosphorylation at the tyrosine residues of acrosomal proteins is one of causal factors for unstable structure in the spermatozoa from subfertile male animals.

  • An invention of method to predict reproductive performance of Japanese Black cattle by the evaluation of sperm ability to exhibit flagellar hyperactivation

    HARAYAMA Hiroshi, MIYAKE Masashi, MURASE Tetsuma

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2006 - 2007, Principal investigator

    1.Induction of sperm flagellar hyperactivation: The treatment with a cell-permeable analog cBiMPS (0.1 mM) for 3 h was effective for the induction of sperm flagellar hyperactivation. This reveals that flagellar hyperactivation is regulated via the cAMP singnaling in the sperm from Japanese Black cattle. 2. Characterization of the cAMP signaling: Our previous reports indicate that the cAMP-PKA-SYK signaling cascade is involved in the sperm flagellar hyperactivation in boars. However, this study has suggested that it is regulated via different cAMP signaling cascades in Japanese Black cattle. A possible downstream element of the cAMP signaling has been found as a focal adhesion kinase (FAK)-like protein. 3. Biological markers for the prediction of male reproductive performance: The treatment with cBiMPS rapidly induced transformation of motility pattern to curling movement in the sperm from normal bulls. However delayed transformation was observed in the sperm from subfertile bulls which were produced in Gifu Prefecture. On the other hand, relatively larger reduction of the motility was observed during the cBiMPS treatment in the sperm from subfertile bulls which were produced in Hyogo Prefecture. Namely, different responses of spermatozoa to cBiMPS may be valid as biological markers for prediction of male reproductive performance of Japanese Black cattle. 4. Biochemical markers for the prediction of male reproductive: performance: Detection levels of 130-kDa, 105-kDa and 94-kDa cAMP-dependently tyrosine-phosphorylated proteins were likely correlated with the percentages of hyperactivated sperm after the treatment with cBiMPS. Thus, these proteins are candidates of biochemical markers for the prediction of male reproductive performance of Japanese Black cattle. Additionally the 94-kDa tyrosine-phosphorylated protein has been identified as a FAK-like protein.

  • An invention of method to suppress expression of sperm fertilizing ability for the purpose of establishment of production system of porcine embryos

    HARAYAMA Hiroshi, MIYAKE Masashi, MURASE Tetsuma

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 2004 - 2005, Principal investigator

    1.Identification of cAMP-dependent protein tyrosine kinase : A spleen tyrosine (Y) kinase (SYK) was activated via the cAMP-PKA signaling in the sperm connecting and principal pieces. The mechanism for SYK activation is quite different between spermatozoa and lymphocytes, suggesting that the activation of sperm SYK is controlled by a novel signaling system. 2.Observation of relationship between SYK activation and expression of sperm fertilizing ability : The cAMP analog-treated spermatozoa exhibited flagellar hyperactivation coincidently with the SYK activation. This indicates a role of SYK as a regulator for sperm fertilizing ability. 3.Analyses of cDNA sequence of testicular SYK : The sequence of PCR product (590 bps) amplified from testicular cDNA was same as that of pig spleen syk gene (the C-terminal catalytic domain of SYK), confirming production of SYK in the testis. 4.Identification and functional analyses of sperm SYK substrate : The SYK substrate was a phospholipase Cγ1 (PLCγ1). This enzyme, which was cAMP-dependently phosphorylated at the Y352 residue, bound to and activated PLCγ1 by the phosphorylation at the Y783 residue. Additionally, chemical inhibition of PLCγ1 with U-73122 was valid to the suppression of over-expression of flagellar hyperactivation. 5.Trial to introduce PLCγ1 peptide fragments into spermatozoa : Although two kinds of commercial kits were used in this experiment, we faced problems of methodology that unknown factors included in the reagents and introduction protocol severely damaged sperm motility. A possible method for peptide introduction remains to be improved. 6.Identification and functional analyses of other regulatory factors of sperm fertilizing ability : Sperm PKC was controlled via the cAMP-PKA signaling. The chemical inhibition of sperm PKC with Ro-32-0432 was valid to the suppression of over-expression of flagellar hyperactivation.

  • Changes and functions of Anti-Agglutinin in boar spermatozoa during their capacitation

    KATO Seishiro, HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 1999 - 2001, Coinvestigator

    Three following experiments have been undertaken in this study ; 1. changes of sperm-bound anti-agglutinin during the capacitation, 2 : isolation of anti-agglutinin from seminal plasma and functional characterization of this protein on the regulation of sperm capacitation, and 3 : signaling cascade regulating sperm head-to-head agglutination. Significant results have been obtained in Experiments 1 and 3 and summarized here. Experiment 1 has revealed changes in anti-agglutinin bound to spermatozoa during the incubation designed to promote the capacitation in vitro. The relative amount of sperm-bound anti-agglutinin, assessed by Western blotting-densitometric analyses, was almost halved after the first 45-min incubation and then decreased gradually thereafter. Immunocytocheinical observation showed that the decrease of the sperm-bound anti-agglutinin occurred mainly on acrosomes in many spermatozoa during the incubation. These results indicate that a large portion of the anti-agglutinin bound to sperm acrosomes is released at an early stage of the Capacitation process in vitro. Experiment 3 has been conducted to reveal roles of bicarbonate-adenylyl cyclase (AC)-cAMP-protein kinase (PK) system in sperm head-to-head agglutination. In the samples incubated in Ca-free mKRB supplemented with 100 μM forskolin (an AC activator), significantly higher percentages of the spermatozoa were agglutinated with one another at the acrosome, as compared with control samples. Addition of dibutyryl CAMP (dbcAMP, an activator of CAMP-dependent PK : PKA), instead of forskolin, increased the percentages of head-to-head agglutinated spermatozoa in a dose-dependent manner between 1 and 1,000 μM. However, the effects of the addition of dbcAMP (1,000 μM) were attenuated by treatments with Rp-cAMPS (1,000 μM, a PKA inhibitor) or H-89 (5 μM, a PKA inhibitor). These results indicate that bicarbonate-AC-cAMP-PK system mediates a signaling pathway leading to head-to-head agglutination of spermatozoa.

  • ブタ精子のキャパシテーション過程での頭部間凝集に及ぼすカルシウムの影響

    原山 洋

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A), Grant-in-Aid for Encouragement of Young Scientists (A), Kobe University, 1999 - 2000, Principal investigator

    実験1.カルモジュリン(CaM)の役割:既報(Chan and Dukelow,1985;Leclerc et al.,1990,1992)によると,精子におけるCaM濃度およびCaMの特異タンパク質への結合性はキャパシテーションとともに低下することから,CaMはキャパシテーションに抑制的に作用していると推察されている.そこで,本研究では成分からキャパシテーション誘起因子を除去した媒液中で精子をインキュベートした際のCaM阻害剤の影響について検討した.予備実験においてCalmidazoliumとTrifluoperazineを用いたが,これらの薬剤は精子に対する毒性が強く,本実験での使用には不適切であった.そこで,比較的毒性の低かったW7(5〜10μM)を用いたところ,頭部間凝集精子率は著しく上昇した.この結果から,CaMは精子の頭部間凝集に抑制的に作用していると考えられる. 実験2.電位依存性カルシウムチャンネル(VDCCs)の役割:昨年度の研究において10〜100μMの濃度では精子の頭部間凝集に有意な抑制作用を示さなかったL型VDCCs阻害剤のNifedipineとNitrendipineをより高い濃度(1mM)で添加したところ,頭部間凝集率は有意に低下した.他方,T型VDCCsの役割については,予備実験で精子に対する毒性が比較的少ないことが判明した阻害剤のPimozideとAmiloride(いずれも0.1〜10μM)を用いて検討したが,いずれも凝集精子率に有意な変動は認められなかった.以上の結果から,精子の頭部間凝集の発生においてVDCCsが機能している可能性が示唆されたが,L型阻害剤で抑制効果が認められた濃度は1mMと高く,またT型阻害剤(0.1〜10μM)には効果が認められなかったことから,VDCCsの種類を特定するには至っていない.

  • ブタ活性化卵子の初期発生能ならびに単為発生胚の初期胚特異タンパク質の発現性

    MIYAKE Masashi, KUREBAYASHI Satoshi, HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), Kobe University, 1997 - 1999, Coinvestigator

    This study was designed to establish serum-free culture system for porcine early embryos and to examine the developmental limitation of parthenogenetic porcine diploids. In vitro matured porcine oocytes were electro-stimulated for activation and were treated with cytochalasin B. These oocytes were cultured for 168 hours in various simple media that were based on modified Whitten' medium with 0.5mg/ml hyaluronic acid (mWM). Osmolarity of media, concentration of polyvinyl alcohol (PVA) as substitutes for ovine serum albumin (BSA) and effects of amino acids were examined. These studies clarified following items ; 1.Diploids cultured in isotonic mWM (309mOsmol) throughout culture period resulted in very low frequency (a few %) of development to the blastocyst stage by the failure of compaction and formation of blastocoel. Fourteen to 20% of diploids cultured in hypotonic mWM (254 mOsmol) developed to the blastocyst stage. The cause of the osmotic effect is just osmolarity of a media, but Na+/K+ ratio. 2.Isotonic condition (280 to 320 mOsmol) is more beneficial for the culture of diploids up to 48 hours after activation, and hypotonic condition (220 to 270 mOsmol) is better for the development of diploids from 72 hours after activation. 3.More than 0.5 mg/ml of PVA as substitutes for BSA supports well the development of diploids to the blastocyst stage, but their expansion. The addition of amino acids solution (AA) mixed essential (EA) and nonessential amino acids (NEA) to PVA-mWM from the biginning of culture causes strong 4-cell blocking of diploids. The frequency to the blastocyst stage is significantly lower than AA-free condition. 5.The addition of EA for 0 to 48 hours after activation brings sever 4-cell blocking to the diploids. The blocking effect is weakened by the addition of NEA, and NEA can support the development of diploids to the blastocyst stage as like as BSA. 6.The addition of EA 48 after activation well supports their development to the blastocyst stage as like as BSA. Moreover, most of blastocysts cultured under the condition show the hatching process. To determine the in vivo developmental of parthenogenetic diploids, activated oocytes at the 3- to 4-cell stage 48 hours after activation were transferred to recipients. The animals were sacrificed on 18 to 30 days after activation, and the number of fetuses and existence of corpus lutea was examined. 1.In one of four trails, 30-days fetuses were obtained (35%), but in the other 3 cases no fetuses was obtained. Aborted fetuses were observed on 28- to 29-day after activation in one animal. These results suggest that the developmental limitation of parthenogenetic diploids is around 30 day after activation. 2.Parthenogenetic fetuses on 25-day, at the early stage of post implantation, were obtained in many trials, indicating that the developmental ability of diploids to the stage was relatively high (41% of transferred eggs), however, cyst-like structure of the primitive heart and liver, and acephalus were observed in some of fetuses collected around 20-day. 3.NMR images shows that some of fetuses with normal appearance have abnormal cavity formation in the brain. Expression of some of imprinted genes that have been clarified their imprinting in the mouse and/or human in parthenogenetic porcine diploids was also examined in the present study. 1.As the sequence of most of the imprinted genes in the pig, probes for porcine genes were designed from the sequence of mouse and human genes. PCR products were obtained from normal porcine fetuses using probes for p57KIP2, PEG1/MEST, SNRPN and H19. 2.Expression of candidates of imprinted genes were compared between normal and parthenogenetic fetuses by northern-hybridization using PCR products of IGF2, IGF2R and WT1 in addition to the above PCR products and sub quantitative PCR. The expression of IGF2 is stronger in normal fetuses than parthenogenotes, and that of p57KIP2 was almost same between them.

  • Studies on production of pig embryos from growing oocytes

    KATO Seishiro, MIYANO Takashi, MIYAKE Masashi, KANNAN Yasuyuki, TOMINAGA Keiichiro, HARAYAMA Hiroshi

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B), Grant-in-Aid for Scientific Research (B), KOBE UNIVERSITY, 1997 - 1999, Coinvestigator

    Several hundred thausand primordial follicles are contained in the ovaries of domestic animals. The development of in vitro culture systems for the growth of small oocytes in primordial or small follicles is expected to provide a new source of a large population of oocytes for livestock production. This study was conducted to develop Culture systems for small oocytes from pig ovaries. Preantral follicles containing oocytes with various sizes were isolated from pig ovaries and cultured in collagen gel for up to 16 days in the presence of serum, FSH and oestradiol. Waymouth MB7 52/1 was used as a basic culture medium for oocyte growth. After culture, the oocytes enclosed by one or two layers of cumulus cells were cultured for meiotic maturation and inseminated with spermatozoa. The results obtained were as follows. 1. Pig growing oocytes grew to their final size, acquired meiotic competence du ring in vitro culture, and were penetrated by spcermatozoa in vitro, although there was a very low level of success in fertilization. In contrast to the growing oocytes, no culture methods were available for much smaller oocytes. We tried to culture primordial follicles which contained small oocytes 30 μm in diameter, but the integrity of the follicles was soon broken and the follicles survived for only a few days. 2. Using culture systems, we demonstrated that granulosa cells regulate oocyte growth, and that antrum formation of granulosa cells was induced only by meiotic-competent oocytes, indicating that the oocytes secrete a substance(s) that induces differentiation of granulosa cells. 3. Of four glycosaminoglycans examined, hyalronic acids and chondroitin sulfate A supported the development of in vitro-matured and -fertilized pig oocytes to the blastocyst stage. 4. In addition, degradation of cyclin B1 molecules and MAP kinase deph osphorylation during fertilization, localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II, assembly and transformation of the spindle during the progression through meiotic cell cycle, and the level and localization of CENP-E and kinetochore numaber during maturation were examined.

  • Anti-Agglutinin出現機序の生化学的および免疫組織学的解析

    原山 洋

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A), Grant-in-Aid for Encouragement of Young Scientists (A), Kobe University, 1997 - 1998, Principal investigator

    3頭の成熟雄ブタから採取した精巣上体を頭2部位,体2部位および尾1部位の合計5部位に区分し,パラホルムアルデヒド固定,凍結およびクリオスタットによる薄切の後に各部位でのAnti-Agglutininの分布を免疫組織学的に観察した。その結果,はじめてAnti-Agglutininが検出されたのは精巣上体の体近位部で,精巣上体管の上皮と管腔内容物の両方において認められた。また,精巣上体体遠位部においてもほぼ同様の検出結果が得られた。さらに,精巣上体尾の管腔内容物においてもAnti-Agglutininは検出されたが,管上皮にはほとんど認められなかった。他方,3頭の成熟雄ブタから12種類の臓器(精巣上体,精巣,精のう腺,前立腺,心臓,肝臓,腎臓,脾臓,胃,小腸,肺および筋肉)を採取し,臓器組織の一部を1mMAPMSF,1mMEDTAおよび1%TritonX-100添加リン酸緩衝生理食塩水中でホモジネートにした後,そのホモジネートを用いてSDSポリアクリルアミドゲル電気泳動およびウエスタンブロッティングを行ったところ,Anti-Agglutininが検出されたのは精巣上体のみで,中でも精巣上体体において最も強い反応が得られた。以上の結果から,精巣上体においてAnti-Agglutininを生産および分泌する部位は体であると考えられる。なお,精巣上体管腔内に分泌されたAnti-Agglutininの一部は精子の先体と結合することを細胞免疫学的手法により合わせて明らかにした。

  • 精巣上体液中の精子頭部間凝集抑制タンパク質を修飾する糖鎖の特性解析

    原山 洋

    Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Encouragement of Young Scientists (A), Grant-in-Aid for Encouragement of Young Scientists (A), Kobe University, 1996 - 1996, Principal investigator

    1.研究実績の概要:哺乳動物の精巣上体における精子の成熟変化のひとつである頭部間凝集性低下の分子機構を明らかにする目的で、精子頭部間凝集抑制タンパク質(Anti-Agglutinin:以下AAとする)を修飾する糖鎖の特性を解析し、以下の結果を得た。 2.糖鎖修飾の違いがAAの精子への結合性に及ぼす影響:AAにおいて、分子不均一性が精子への結合性に大きく影響するが、この分子不均一性の原因を明らかにする目的で、精巣上体尾液(30μg)を尿素存在下で2次元電気泳動した後、レクチンブロッティング法に従ってコムギ胚芽凝集素(WGA)との反応性について調べた。その結果、精子との結合性を備える等電点5.8のスポットはWGAと反応したが、精子と結合しない等電点5.6および5.95のスポットは反応しなかった。従って、糖鎖のシアル酸化の有無が分子不均一性の一因であり、また精子への結合性に影響すると考えられる。 3.修飾糖鎖の分子量測定:AAからGlycoFree-Kitを用いて糖鎖を完全に除去し、その際の分子量の変化をMALDI-TOF-Mass Spectrometric Analysisにより測定したところ、無処理のAAは分子量18900-19600においてブロードしたピークとして検出されたが、糖鎖除去後の試料は最高700小さい分子量においてシャープやピークを示した。このことから、総分子量で700以下の糖鎖がAAを修飾しており、その修飾糖鎖は多種類存在すると考えられる。 4.脱シアル酸がAAの生理活性に及ぼす影響:ノイラミニダーゼ(0.5U/ml)を用いて脱シアル酸化したAAを洗浄精子浮遊液に添加し37℃で5時間インキュベートしたところ、AAの精子凝集抑制作用は若干低下する傾向を示した。