研究者データベース

研究者情報

マスター

アカウント(マスター)

  • 氏名

    荻原 克益(オギワラ カツエキ), オギワラ カツエキ

所属(マスター)

  • 理学研究院 生物科学部門 生殖発生生物学分野

所属(マスター)

  • 理学研究院 生物科学部門 生殖発生生物学分野

独自項目

syllabus

  • 2021, 生殖発生機構学特論, Reproductive and Developmental Sciences, 修士課程, 生命科学院, 幹細胞,クローン技術,始原生殖細胞,性ステロイド,性ホルモン受容体,配偶子形成,配偶子成熟,排卵,組織修復,生殖医療,受精,胚発生,性分化,母性因子
  • 2021, 生命システム科学基礎論, Biosystems Science, 修士課程, 生命科学院, 生命システム, 生命機能, 研究方法論, 研究技術論
  • 2021, 大学院共通授業科目(一般科目):自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, 生命システム, 生命機能, 研究方法論, 研究技術論
  • 2021, 細胞生物学概論, Introduction to Cell Biology, 学士課程, 理学部, 現代生物科学,21世紀に生物科学が解決しなければならない課題,生体高分子,細胞の構造と機能,エネルギー代謝,細胞の成長と分裂,遺伝現象と遺伝子発現制御
  • 2021, 科学・技術の世界(1単位), The World of Science and Technology, 学士課程, 全学教育, 現代生物科学,21世紀に生物科学が解決しなければならない課題,生物の多様性,系統,進化,生物の形態,生命活動の多様性
  • 2021, 一般教育演習(フレッシュマンセミナー), Freshman Seminar, 学士課程, 全学教育, 卵巣、精巣、配偶子形成、幹細胞、分化、性ホルモン、形態形成
  • 2021, 生物学Ⅱ, Biology II, 学士課程, 全学教育, 生物の多様性,生物の形態,生命活動の多様性
  • 2021, 基礎生化学実習, Laboratory Course in Basic Biochemistry, 学士課程, 理学部, 生体物質の定性と定量、細菌の取扱い方、酵素、電気泳動、リコンビナントタンパク質、タンパク質精製
  • 2021, 基礎生化学実習, Laboratory Course in Basic Biochemistry, 学士課程, 理学部
  • 2021, 基礎生物学実習, Laboratory Course in Basic Biology, 学士課程, 理学部, 脊椎動物の形態、染色体、細胞、植物、昆虫、土壌動物、ショウジョウバエ、唾腺染色体、博物館標本
  • 2021, 生殖発生生物学Ⅱ, Reproductive and Developmental Biology II, 学士課程, 理学部, 卵巣、精巣、卵(子)形成、精子形成、排卵、濾胞選択、胚軸、オーガナイザー、形態形成、母性因子、幹細胞、noncoding RNA、エピジェネティクス
  • 2021, 教科教育法(理科II), Teaching Method of School Subjects(Science Ⅱ), 学士課程, 教育学部, 教員養成、理科教育法、指導法

researchmap

プロフィール情報

学位

  • 博士(理学)(北海道大学)

プロフィール情報

  • 荻原
  • 克益
  • ID各種

    201301000922869333

対象リソース

業績リスト

研究キーワード

  • 排卵   卵巣   マウス   プロテアーゼ   メダカ   トランスジェニック   生殖   RNAi   

研究分野

  • ライフサイエンス / 形態、構造

経歴

  • 2017年10月 - 現在 北海道大学 理学研究院 准教授
  • 2008年04月 - 2017年09月 北海道大学 理学(系)研究科(研究院) 助教

論文

  • Katsueki Ogiwara, Chika Fujimori, Takayuki Takahashi
    Molecular and cellular endocrinology 111816 - 111816 2022年11月18日 
    We have previously shown that the prostaglandin E2/Ptger4b receptor system is involved in ovulation in teleost medaka and induces intracellular actin cytoskeleton rearrangement in the granulosa cells of preovulatory follicles. In this study, we investigated the signaling pathways through which prostaglandin E2 induces a change in the actin cytoskeleton. Treating preovulatory follicles with GW627368X (Ptger4b antagonist), a Rho inhibitor, or Y-27632 [Rho-associated protein kinase (Rock) inhibitor] inhibited not only in vitro follicle ovulation but also intracellular actin cytoskeleton rearrangement. Active Rhoa-c and Rock1 were detected in follicles immediately before ovulation. GW627368X also inhibited Rhoa-c activation and cytoskeleton rearrangement. PGE2-induced actin cytoskeleton rearrangement was not observed in the Ptger4b-, Rhoa-c-, or Rock1-deficient OLHNI-2 cells. These results indicate that the PGE2/Ptger4b pathway regulates intracellular actin cytoskeleton rearrangement via the Rho/Rock pathway in the granulosa cells of preovulatory follicles during medaka ovulation.
  • Katsueki Ogiwara, Miyuki Hoyagi, Takayuki Takahashi
    Biology of Reproduction 105 2 413 - 426 2021年08月03日 [査読有り][通常論文]
     
    Abstract Nuclear progestin receptor (PGR) is a ligand-activated transcription factor that has been identified as a pivotal mediator of many processes associated with ovarian and uterine function, and aberrant control of PGR activity causes infertility and disease including cancer. The essential role of PGR in vertebrate ovulation is well recognized, but the mechanisms by which PGR is rapidly and transiently induced in preovulatory follicles after the ovulatory LH surge are not known in lower vertebrates. To address this issue, we utilized the small freshwater teleost medaka Oryzias latipes, which serves as a good model system for studying vertebrate ovulation. In the in vitro ovulation system using preovulatory follicles dissected from the fish ovaries, we found that inhibitors of EPAC (brefeldin A), RAP (GGTI298), PI3K (Wortmannin), AKT (AKT inhibitor IV), and CREB (KG-501) inhibited LH-induced follicle ovulation, while the PKA inhibitor H-89 had no effect on follicle ovulation. The inhibitors capable of inhibiting follicle ovulation also inhibited follicular expression of Pgr and matrix metalloproteinase-15 (Mmp15), the latter of which was previously shown to not only be a downstream effector of Pgr but also a proteolytic enzyme indispensable for follicle rupture in medaka ovulation. Further detailed analysis revealed for the first time that the cAMP/EPAC/RAP/PI3K/AKT/CREB signaling pathway mediates the LH signal to induce Pgr expression in preovulatory follicles. Our data also showed that phosphorylated Creb1 is a transcription factor essential for pgr expression and that Creb1 phosphorylated by Akt1, rather than PKA, may be preferably used to induce pgr expression.
  • Takayuki Takahashi, Katsueki Ogiwara
    Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 254 110907 - 110907 2021年04月 [査読有り][通常論文]
  • Hagiwara A, Ogiwara K, Sugama N, Yamashita M, Takahashi T
    General and comparative endocrinology 288 113373  2019年12月 [査読有り][通常論文]
  • Katsueki Ogiwara, Takayuki Takahashi
    Cells 8 3 215 - 215 2019年03月04日 [査読有り][通常論文]
     
    Ovulation denotes the discharge of fertilizable oocytes from ovarian follicles. Follicle rupture during ovulation requires extracellular matrix (ECM) degradation at the apex of the follicle. In the teleost medaka, an excellent model for vertebrate ovulation studies, LH-inducible matrix metalloproteinase 15 (Mmp15) plays a critical role during rupture. In this study, we found that follicle ovulation was inhibited not only by roscovitine, the cyclin-dependent protein kinase (CDK) inhibitor, but also by CDK9-inhibitor II, a specific CDK9 inhibitor. Inhibition of follicle ovulation by the inhibitors was accompanied by the suppression of Mmp15 expression in the follicle. In follicles treated with the inhibitors, the formation of the phosphorylated nuclear progestin receptor (Pgr) was inhibited. Roscovitine treatment caused a reduction in the binding of Pgr to the promoter region of mmp15. The expression of Cdk9 and cyclin I (Ccni), and their association in the follicle was demonstrated, suggesting that Cdk9 and Ccni may be involved in the phosphorylation of Pgr in vivo. LH-induced follicular expression of ccni/Ccni was also shown. This study is the first to report the involvement of CDK in ECM degradation during ovulation in a vertebrate species.
  • Takahashi T, Hagiwara A, Ogiwara K
    Reproduction (Cambridge, England) 2018年10月 [査読有り][通常論文]
  • Takahashi T, Hagiwara A, Ogiwara K
    Global Journal of Reproductive Medicine 4 3 2018年04月30日 [査読有り][通常論文]
  • Takayuki Takahashi, Akane Hagiwara, Katsueki Ogiwara
    Molecular and Cellular Endocrinology 461 236 - 247 2018年02月05日 [査読有り][通常論文]
     
    Prostaglandins are well known to be central regulators of vertebrate ovulation. Studies addressing the role of prostaglandins in mammalian ovulation have established that they are involved in the processes of oocyte maturation and cumulus oocyte complex expansion. In contrast, despite the first indication of the role of prostaglandins in teleost ovulation appearing 40 years ago, the mechanistic background of their role has long been unknown. However, studies conducted on medaka over the past decade have provided valuable information. Emerging evidence indicates an indispensable role of prostaglandin E2 and its receptor subtype Ptger4b in the process of follicle rupture. In this review, we summarize studies addressing the role of prostaglandins in teleost ovulation and describe recent advances. To help understand differences from and similarities to ovulation in mammalian species, the findings on the roles of prostaglandins in mammalian ovulation are discussed in parallel.
  • Katsueki Ogiwara, Takayuki Takahashi
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 450 C 54 - 63 2017年07月 [査読有り][通常論文]
     
    Hormonal regulation of the expression of Mmp15, a proteolytic enzyme indispensable for ovulation in the teleost medaka, was investigated. In an in vitro culture system using preovulatory follicles, Mmp15 expression and ovulation were induced in the presence of recombinant luteinizing hormone (rLh). Both rLh-induced Mmp15 expression and ovulation were 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one-dependent, suggesting the involvement of a nuclear progestin receptor (Pgr). In vitro follicle ovulation and Mmp15 expression were reduced by treatment with the Pgr antagonist RU-486. Like Pgr, the transcription factor CCAAT/enhancer-binding protein beta (Cebpb) was induced by rLh. ChIP analyses indicated that Pgr and Cebpb bound to the mmpl5 promoter region. These results indicate that the rLh-induced expression of Mmp15 is mediated by Pgr and Cebpb. A differential timing of expression of Pgr and Cebpb in the preovulatory follicles appears to explain the considerably long time-lag from the pgr gene activation to mmpl5 gene expression. (C) 2017 Elsevier B.V. All rights reserved.
  • Katsueki Ogiwara, Takayuki Takahashi
    BIOLOGY OF REPRODUCTION 94 3 64  2016年03月 [査読有り][通常論文]
     
    Understanding the direct effects of melatonin on vertebrate ovulation remains a challenge. The present study provides the first characterization of the role of melatonin in ovulation using the teleost medaka. The melatonin receptor antagonist luzindole inhibited in vitro follicle ovulation. In the preovulatory follicles, arylalkylamine N-acetyltransferase 1a and hydroxyindole-O-methyltransferase 2, the enzymes responsible for melatonin synthesis, were expressed in the granulosa cells throughout the 24 h spawning cycle. The granulosa cells of the follicle also expressed the melatonin receptor 1a-a. An in vitro characterization study using medaka OLHNI-2 cells revealed that melatonin and luzindole act as an agonist and an antagonist, respectively, of the melatonin receptor. The intracellular cAMP levels in these cells were reduced after melatonin treatment. The expression of cytosolic phospholipase A2 group 4a (Pla2g4a), the enzyme producing arachidonic acid (cyclooxygenase-2 substrate), was inhibited in the granulosa cells in luzindole-treated follicles. Follicular prostaglandin E-2 levels and in vitro follicle ovulation were suppressed in follicles isolated at 12 h prior to ovulation and incubated with the Pla2g4a inhibitor AACOCF3. The G-actin: F-actin ratios in follicular cells increased with approaching ovulation, but this increase was suppressed after luzindole treatment. The phosphorylation of moesin, an ezrin-radixin-moesin protein, was inhibited in the follicular cells in luzindole-treated follicles. These results indicate a dual role for melatonin in medaka ovulation: melatonin ensures prostaglandin E-2 synthesis throughout the spawning cycle and induces actin cytoskeleton rearrangement in the follicular cells at ovulation.
  • Akane Hagiwara, Katsueki Ogiwara, Takayuki Takahashi
    ZOOLOGICAL SCIENCE 33 1 98 - 105 2016年02月 [査読有り][通常論文]
     
    Membrane progestin receptor (mPR) alpha on the cell membrane of the oocyte is involved in the meiotic maturation of vertebrates, including teleosts, but little is known about the role of this membrane-bound follicular receptor. We investigated the ovarian expression of membrane progestin receptor (mPR) mRNA in medaka. In follicles that were destined to ovulate, transcripts of mPR alpha and mPR gamma were expressed in the oocytes as well as the granulosa cells. Transcripts of mPR alpha and mPR gamma were expressed at relatively constant levels in the whole ovary and in the preovulatory follicles throughout the 24-h spawning cycle. In vitro incubation of the preovulatory follicles with recombinant medaka luteinizing hormone caused no significant changes in the expression of mPRa alpha and mPR gamma mRNA, suggesting LH-independent follicular expression of these mPR genes. Using HEK293T cells expressing medaka mPRs, forskolin-elevated intracellular cAMP levels were found to be reduced on treatment of the cells with ligand 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), but only in the cells expressing mPRaa. These results indicate that activation of mPRaa and mPR gamma with DHP may cause differential effects on the granulosa cells. Information obtained from the present study may help to elucidate the role of mPR alpha and mPR gamma in the granulosa cells of the follicles.
  • Katsueki Ogiwara, Akane Hagiwara, Sanath Rajapakse, Takayuki Takahashi
    BIOLOGY OF REPRODUCTION 92 1 10  2015年01月 [査読有り][通常論文]
     
    We previously reported that the serine protease plasmin plays a role in follicle rupture during ovulation in the teleost medaka. In this study, we showed that urokinase-type plasminogen activator 1 (Plau1) is a physiological activator of plasminogen. Morphological analyses revealed that in the preovulatory follicle, plau1 mRNA was detected in association with follicle cells, while Plau1 protein was localized in the oocyte egg membrane. Both an inactive precursor and an active form of Plau1 were present at constant levels in the membrane fraction via the latter half of the 24-h spawning cycle. Plasminogen activator inhibitor-1 (Pai1) was detected in the follicle layer of the preovulatory follicle, but the protein level was low at approximately 7 h prior to ovulation. We showed that plasmin hydrolyzed laminin, which is a major component of the basement membrane and is situated between the granulosa and theca cells of the follicle. In vitro ovulation of large follicles was significantly inhibited by anti-Plau1 antibodies and active recombinant Pai1. Levels of Pai1 expression were increased in vivo at approximately 7 h prior to ovulation. Expression of Pai1 was also induced in vitro in the follicle with recombinant medaka luteinizing hormone (Lh). Lh-induced expression of pail mRNA was significantly suppressed by the presence of MDL (an adenylyl cyclase inhibitor), trilostane (a 3beta-hydroxysteroid dehydrogenase inhibitor), and RU486 (a nuclear progestin receptor antagonist). These results support our recent proposal of a sequential two-step ECM protein hydrolysis model for follicle rupture for medaka ovulation.
  • Sanath Rajapakse, Katsueki Ogiwara, Takayki Takahashi
    ZOOLOGICAL SCIENCE 31 12 840 - 848 2014年12月 [査読有り][通常論文]
     
    Previously, we reported that the medaka testis abundantly expresses the mRNA for trypsinogen, which is a well-known pancreatic proenzyme that is secreted into and activated in the intestine. Currently, we report our characterization of the medaka trypsin using a recombinant enzyme and show that this protein is a serine protease that shares properties with trypsins from other species. Two polypeptides (28- and 26-kDa) were detected in the testis extracts by Western blot analysis using antibodies that are specific for medaka trypsinogen. The 28-kDa polypeptide was shown to be trypsinogen (inactive precursor), and the 26-kDa polypeptide was shown to be trypsin (active protease). We did not detect enteropeptidase, which is the specific activator of trypsinogen, in the testis extract. Immunohistochemical analyses using the same trypsinogen-specific antibody produced a strong signal in the spermatogonia and spermatozoa of the mature medaka testis. Substantial staining was found with spermatocytes, whereas extremely weak signals were observed with spermatids. In vitro incubation of testis fragments with the trypsinogen antibody strongly inhibited the release of sperm from the testis into the medium. Trypsin activity was detected in sperm extracts using gelatin zymographic analysis. Immunocytochemistry showed that trypsinogen and trypsin were localized to the cell membranes surrounding the sperm head. Collectively, these results suggest that trypsin plays an important role in the testis function of the medaka.
  • Akane Hagiwara, Katsueki Ogiwara, Yoshinao Katsu, Takayuki Takahashi
    BIOLOGY OF REPRODUCTION 90 6 126  2014年06月 [査読有り][通常論文]
     
    We previously reported that the prostaglandin E-2 receptor subtype Ptger4b plays a role in ovulation in a teleost species, medaka and that ptger4b mRNA is drastically induced in preovulatory follicles prior to ovulation. The present study focuses on the hormonal regulation of ptger4b mRNA expression using this nonmammalian vertebrate model. Preovulatory follicles that had not been exposed to luteinizing hormone (Lh) in vivo were incubated in vitro with medaka recombinant Lh (rLh), which induced the ptger4b mRNA expression. The addition of trilostane, an inhibitor of 3beta-hydroxysteroid dehydrogenase, strongly inhibited rLh-induced ptger4b expression, and trilostane-suppressed ptger4b expression was restored to the level observed in rLh-treated follicles when 17alpha, 20beta-dihydroxy-4-pregnen-3-one was included in the culture. We determined that the expression of the progestin-activated transcription factor nuclear progestin receptor (Pgr) was also induced by medaka rLh in the follicle and that its expression preceded ptger4b expression. Forskolin treatment induced both pgr and ptger4b mRNA expression in the follicle. Follicular ptger4b mRNA expression was drastically suppressed by RU486, which was demonstrated to compete with 17alpha, 20beta-dihydroxy- 4-pregnen-3-one for medaka Pgr in vitro, suggesting a role for Pgr in the expression of ptger4b mRNA. A chromatin immunoprecipitation assay with preovulatory follicles isolated from spawning medaka ovaries demonstrated direct binding of Pgr to the ptger4b promoter. These results indicate that ptger4b expression is regulated by a genomic mechanism involving Pgr.
  • Yoneda R, Takahashi T, Matsui H, Takano N, Hasebe Y, Ogiwara K, Kimura AP
    Biology of reproduction 88 5 118 - 118 The Society for the Study of Reproduction 2013年05月 [査読有り][通常論文]
     
    Spermatogenesis is a complex process that generates spermatozoa; its molecular mechanisms are not completely understood. Here we focused on the functions of three testis-specific serine proteases: Prss42/Tessp-2, Prss43/Tessp-3, and Prss44/Tessp-4. These protease genes, which constitute a gene cluster on chromosome 9F2-F3, were presumed to be paralogs and were expressed only in the testis. By investigating their mRNA distribution, we found that all three genes were expressed in primary and secondary spermatocytes. However, interestingly, the translated proteins were produced at different locations. Prss42/Tessp-2 was found in the membranes and cytoplasm of secondary spermatocytes and spermatids, whereas Prss43/Tessp-3 was present only in the membranes of spermatocytes and spermatids. Prss44/Tessp-4 was detected in the cytoplasm of spermatocytes and spermatids. To assess the roles of these proteases in spermatogenesis, we used organ culture of mouse testis fragments. Adding antibodies against Prss42/Tessp-2 and Prss43/Tessp-3 resulted in meiotic arrest at the stage when each protease was beginning to be translated. Furthermore, the number of apoptotic cells dramatically increased after the addition of these antibodies. These results strongly suggest that the three paralogous Prss/Tessp proteases play different roles in spermatogenesis and that Prss42/Tessp-2 and Prss43/Tessp-3 are required for germ cell survival during meiosis.
  • Takayuki Takahashi, Chika Fujimori, Akane Hagiwara, Katsueki Ogiwara
    ZOOLOGICAL SCIENCE 30 4 239 - 247 2013年04月 [査読有り][通常論文]
     
    Ovulation is the process of liberating oocytes from the preovulatory follicles, and is observed in the ovaries of virtually all female vertebrate animals. Compared with mammalian species, there have been far fewer studies that address the ovulatory mechanisms of non-mammalian species. We have examined the molecular mechanism of follicle rupture during ovulation using the teleost model, medaka, or Oryzias latipes. Follicle rupture in medaka ovulation involves the cooperation of the tissue inhibitor of metalloproteinase-2b protein with at least three matrix metalloproteinases (MMP): membrane type-1 MMP (MT1-MMP), MT2-MMP, and gelatinase A. Our studies also indicate that the serine protease, i.e., plasmin, participates in the rupture for only a few hours prior to the activation of MMP-mediated hydrolysis at ovulation. The involvement of prostaglandin E-2 (PGE(2)) in medaka ovulation was also demonstrated. Cyclooxygenase-2 and PGE(2) receptor subtype EP4b were respectively shown to be an enzyme responsible for PGE(2) synthesis and a receptor for the generated ligand in the preovulatory follicles. Based on the results obtained from our studies of fish, we discuss the similarities and differences in vertebrate ovulation compared with mammalian species.
  • Katsueki Ogiwara, Chika Fujimori, Sanath Rajapakse, Takayuki Takahashi
    PLOS ONE 8 1 e54482  2013年01月 [査読有り][通常論文]
     
    The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare's serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.
  • Chika Fujimori, Katsueki Ogiwara, Akane Hagiwara, Takayuki Takahashi
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 362 1-2 76 - 84 2012年10月 [査読有り][通常論文]
     
    A cDNA for a prostaglandin E-2 (PGE(2)) receptor subtype 4, EP4b (Ptger4b), was cloned from the medaka ovary. The effect of PGE(2) was examined using COS-7 cells expressing the recombinant Ptger4b protein. An increase in intracellular cAMP levels was observed when the cells were incubated with PGE(2), but the increase in cAMP levels was nullified by the addition of the EP4 antagonist GW627368X. The expression of ptger4b mRNA was drastically induced by the addition of pregnant mare serum gonadotropin to the in vitro culture of large preovulatory follicles. In in vitro ovulation studies of the effect of GW627368X addition on follicle ovulation, the critical timing of the PGE(2)/Ptger4b interaction was suggested to be between -1 and 0 h of ovulation. These results further substantiate that PGE(2)/Ptger4b signaling is involved in follicle rupture during ovulation in the medaka ovary. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Katsueki Ogiwara, Kazuto Minagawa, Naoharu Takano, Takashi Kageyama, Takayuki Takahashi
    BIOLOGY OF REPRODUCTION 86 4 113  2012年04月 [査読有り][通常論文]
     
    Until recently, the role of the proteolytic system involving serine proteases in follicle rupture during ovulation in mammalian species has been a subject of controversy. We undertook the present study to examine whether proteases play a role in follicle rupture using the teleost medaka (Oryzias latipes) model. Various serine protease inhibitors, including a specific plasmin inhibitor, drastically reduced the rate of ovulation, as assessed by an in vitro ovulation assay, which was established for the fish. Biochemical, molecular biological, and immunological analyses demonstrated that plasminogen/plasmin was present in large follicles destined to ovulate. The active protease, plasmin, was detected in follicles approximately 3-7 h before the expected time of ovulation. Specific antibodies against the medaka plasmin light chain suppressed the ovulation rate of the follicles when antibodies were added to the medium during the period in which active plasmin was generated. This finding was an indication that a plasmin-like protease similar if not identical to plasmin plays a role in follicle rupture during ovulation in the medaka. Our data also indicate that this serine protease participates in the rupture for only a few hours prior to the activation of matrix metalloproteinase (Mmp)-mediated hydrolysis at ovulation. Based on our previous and current data, we propose a follicle rupture model involving two different proteolytic enzyme systems, serine protease and Mmp, in medaka ovulation. The current study is the first to provide evidence of the indispensable role of plasmin or a plasmin-like protease in the ovulation of a nonmammalian vertebrate species.
  • 藤森 千加, 荻原 克益, 萩原 茜, 高橋 孝行
    比較内分泌学 = Comparative endocrinology 37 142 168 - 170 Japan Society for Comparative Endocrinology 2011年08月31日
  • Kenji Moriyama, Atsuko Hanai, Kazuyuki Mekada, Atsushi Yoshiki, Katsueki Ogiwara, Atsushi Kimura, Takayuki Takahashi
    JOURNAL OF BIOMEDICAL SCIENCE 18 60  2011年08月 [査読有り][通常論文]
     
    Background: The endopeptidase encoded by Phex (phosphate-regulating gene with homologies to endopeptidases linked to the X chromosome) is critical for regulation of bone matrix mineralization and phosphate homeostasis. PHEX has been identified from analyses of human X-linked hypophosphatemic rickets and Hyp mutant mouse models. We here demonstrated a newly established dwarfism-like Kbus/Idr mouse line to be a novel Hyp model. Methods: Histopathological and X-ray examination with cross experiments were performed to characterize Kbus/Idr. RT-PCR-based and exon-directed PCR screening performed to identify the presence of genetic alteration. Biochemical assays were also performed to evaluate activity of alkaline phosphatase. Results: Kbus/Idr, characterized by bone mineralization defects, was found to be inherited in an X chromosome-linked dominant manner. RT-PCR experiments showed that a novel mutation spanning exon 16 and 18 causing hypophosphatemic rickets. Alkaline phosphatase activity, as an osteoblast marker, demonstrated raised levels in the bone marrow of Kbus/Idr independent of the age. Conclusions: Kbus mice should serve as a useful research tool exploring molecular mechanisms underlying aberrant Phex-associated pathophysiological phenomena.
  • Chika Fujimori, Katsueki Ogiwara, Akane Hagiwara, Sanath Rajapakse, Atsushi Kimura, Takayuki Takahashi
    MOLECULAR AND CELLULAR ENDOCRINOLOGY 332 1-2 67 - 77 2011年01月 [査読有り][通常論文]
     
    In vitro ovulation of mature medaka ovarian follicles was inhibited by inhibitors of cyclooxygenase (COX) or by an antagonist of the prostaglandin E-2 receptor (EP). Of the three medaka COX genes, ptgs2 was most dominantly expressed in the fish ovary. The ptgs2 transcript was detected in all sizes of growing follicles. In a 24-h spawning cycle, large-sized follicles contained ptgs2 mRNA at a fairly constant level. The levels of COX enzyme activity and prostaglandin E-2 were also constant in the large-sized follicles during the spawning cycle. The expression of prostaglandin E-2 receptor EP4b (ptger419) mRNA was drastically upregulated in the large-sized follicles as the ovulation time approached. The current results indicate that prostaglandin E-2. which might be produced by COX-2, is involved in the ovulation of medaka, and that EP4b is likely the receptor responsible for exerting the action of prostaglandin E-2 in the process. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • Katsueki Ogiwara, Takashi Ikeda, Takayuki Takahashi
    ZOOLOGICAL SCIENCE 27 9 762 - 767 2010年09月 [査読有り][通常論文]
     
    We sought to establish a new in vitro ovulation model using the whole ovaries of the medaka. Ovaries of the fish, which had been acclimated to the usual reproductive conditions (26 degrees C, 14 h light/10 h dark) and which had then been kept at least one day at 30 degrees C, were isolated 2 h before the expected in vivo ovulation time. When the ovaries were cultured in 90% medium 199 solution at 30 degrees C or 36 degrees C, oocytes were liberated with a gradual increase in the ovulation rate at 2 to 5 h of ovulation time. The maximum ovulation rate was similar to 45%. Ovulated oocytes were fertilized and subsequently developed into adults. In vitro ovulation of medaka ovaries was inhibited by the addition of metalloproteinase inhibitors to the culture. In this in vitro ovulation model, the holes formed on the follicle layer upon follicle rupture at ovulation were sealed, strongly suggesting the importance of the germinal epithelium in the process. The present study indicates that our new in vitro ovulation model is useful for investigating the role of germinal epithelial cells in the ovulate process of the medaka fish.
  • Yumiko Kato, Katsueki Ogiwara, Chika Fujimori, Atsushi Kimura, Takayuki Takahashi
    CELL AND TISSUE RESEARCH 340 3 595 - 605 2010年06月 [査読有り][通常論文]
     
    A cDNA clone coding for the collagen type IV alpha 1 chain was obtained from the ovary of the medaka, Oryzias latipes. The clone encoded a protein of 1639 amino acids including a putative 21-residue signal peptide, and the deduced amino acid sequence of the alpha 1 chain was homologous to those of the proteins from other species. The mRNA of the collagen type IV alpha 1 chain was expressed in various tissues of the adult fish. In situ hybridization analysis revealed that the alpha 1 chain mRNA was localized in the follicle layer of all growing follicles. In the post-ovulatory follicle that had released its oocyte during ovulation, the alpha 1 chain transcript was detected in a winding line surrounding the tissue. This localization pattern was different from that of gelatinase B, a marker gene for granulosa cells. A specific antibody was prepared for the medaka collagen type IV alpha 1 chain. Immunohistochemical analysis with this antibody yielded results consistent with those obtained by in situ hybridization. These data indicate that, in the medaka ovary, collagen type IV is synthesized by theca cells and is localized in the basement membrane.
  • Maya Horiguchi, Chika Fujimori, Katsueki Ogiwara, Akihiko Moriyama, Takayuki Takahashi
    ZOOLOGICAL SCIENCE 25 9 937 - 945 2008年09月 [査読有り][通常論文]
     
    Follicle rupture during ovulation is a well-regulated biological process of extracellular matrix degradation in the vertebrate ovary. Although proteolytic enzymes responsible for the rupture have recently been Identified in the medaka, Oryzias latipes, the lack of knowledge about the ovarian expression and distribution of extracellular matrix components in lower vertebrates prevents the understanding of this process's molecular mechanism. To approach the problem, we cloned a cDNA coding for the medaka collagen type-1 alpha 1 chain and examined its mRNA expression in the fish ovary. The deduced amino acid sequence of the collagen type-1 alpha 1 chain was homologous to those of the proteins from other vertebrate species. The alpha 1 chain mRNA was expressed in various tissues of the adult fish. In the ovary sections of mature female fish, this mRNA was detected in a line surrounding ovarian follicles of all sizes. A comparison with the distribution of gelatinase B mRNA In follicles that had just ovulated Indicated that the collagen type-1 alpha 1 gene is expressed in the theca cells. The current results strongly suggest that collagen type I Is synthesized by theca cells and is localized in the same cell layer of the follicles.
  • Sanath Rajapakse, Noriko Yamano, Katsueki Ogiwara, Kensaku Hirata, Sumio Takahashi, Takayuki Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 74 8 1053 - 1063 2007年08月 [査読有り][通常論文]
     
    Tissue kallikrein mK1 is a serine protease involved in the generation of bioactive kinins for normal cardiac and arterial function in the mouse. In the present study, the tissue kallikrein gene Klk1, which codes for mK1, was shown to be one of the most prevalent of the Klk gene species in the uteri of adult mice, and its mRNA level was significantly higher at estrus than at diestrus. Klk1 mRNA expression was enhanced in the uteri of ovariectomized mice receiving estradiol-17 beta treatment. Both endometrial epithelial and stromal cells isolated from the mice exhibited Klk1 expression at detectable levels when cultured in the presence of estradiol-17 beta. mK1 was characterized using the recombinant active enzyme. mK1 had trypsin-like activity with a strong preference for Arg over Lys in the P1 position, and its activity was inhibited by typical serine protease inhibitors. Casein, gelatin, fibronectin, collagen type IV, and high-molecular-weight kininogen were degraded by mK1. The single-chain tissue-type plasminogen activator was converted to the two-chain form by mK1. In addition, mK1 degraded insulin-like growth factor binding protein-3. The present data suggest that mK1 may be implicated in the growth of uterine endometrial tissues during the proliferative phase.
  • Katsueki Ogiwara, Takayuki Takahashi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 17 7021 - 7026 2007年04月 [査読有り][通常論文]
     
    We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the meclaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (DA-cleavage activity. For the cleavage of fusion proteins containing a D4K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D4K cleavage sequence.
  • Sanath Rajapakse, Katsueki Ogiwara, Noriko Yamano, Atsushi Kimura, Kensaku Hirata, Sumio Takahashi, Takayuki Takahashi
    ZOOLOGICAL SCIENCE 23 11 963 - 968 2006年11月 [査読有り][通常論文]
     
    Mouse tissue kallikreins (Klks) are members of a large, multigene family consisting of 37 genes, 26 of which can code for functional proteins. Mouse tissue kallikrein 5 (KIk5) has long been thought to be one of these functional genes, but the gene product, mK5, has not been isolated and characterized. In the present study, we prepared active recombinant mK5 using an Escherichia coli expression system, followed by column chromatography. We then determined the biochemical and enzymatic properties of purified mK5. mK5 had trypsin-like activity for Arg at the P1 position, and its activity was inhibited by typical serine protease inhibitors. mK5 degraded gelatin, fibronectin, collagen type IV, high-molecular-weight kininogen, and insulin-like growth factor binding protein-3. Our data suggest that mK5 may be implicated in the process of extracellular matrix remodeling.
  • S Rajapakse, K Ogiwara, N Takano, A Moriyama, T Takahashi
    FEBS LETTERS 579 30 6879 - 6884 2005年12月 [査読有り][通常論文]
     
    Human kallikrein 8 (KLK8) is a member of the human kallikrein gene family of serine proteases, and its protein, hK8, has recently been suggested to serve as a new ovarian cancer marker. To gain insights into the physiological role of hK8, the active recombinant enzyme was obtained in a pure state for biochemical and enzymatic characterizations. hK8 had trypsin-like activity with a strong preference for Arg over Lys in the PI position, and its activity was inhibited by typical serine protease inhibitors. The protease degraded casein, fibronectin, gelatin, collagen type IV, fibrinogen, and high-molecular-weight kininogen. hK8 also converted human single-chain tissue-type plasminogen activator (65 kDa) to its two-chain form (32 and 33 kDa) by specifically cleaving the peptide bond Arg(275)-Ile(276). This conversion resulted in a drastic increase in the activity of the activator toward the fluorogenic substrate Pyr-Gly-Arg-MCA and plasminogen in the absence of fibrin. Our findings suggest that hK8 may be implicated in ECM protein degradation in the area surrounding hK8-producing cells. (c) 2005 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
  • K Ogiwara, N Takano, M Shinohara, M Murakami, T Takahashi
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 24 8442 - 8447 2005年06月 [査読有り][通常論文]
     
    Identification of the hydrolytic enzymes involved in follicle rupture during vertebrate ovulation remains a central challenge for research in reproductive biology. Here, we report a previously uncharacterized approach to this problem by using an in vitro ovulation system in the medaka, Oryzias latipes, which is a small freshwater teleost. We found that follicle rupture in the medaka ovary involves the cooperation of at least three matrix metalloproteinases (MMPs), together with the tissue inhibitor of metalloproteinase-2b protein. We determined the discrete roles of each of these proteins during follicle rupture. Our results indicated that gelatinase A induces the hydrolysis of type IV collagen constituting the basement membrane, membrane-type 2 MMP degrades type I collagen present in the theca cell layer, and MT1-MMP and the tissue inhibitor of metalloproteinase-2b are involved in the production and regulation of gelatinase A. These findings will help clarify the mechanism of follicle wall degradation during ovulation in mammalian species.
  • K Ogiwara, M Shinohara, T Takahashi
    GENE 337 79 - 89 2004年08月 [査読有り][通常論文]
     
    Proprotein convertases (PCs) are enzymes responsible for processing the precursors of many bioactive peptides in vertebrates and invertebrates. In the present study, a cDNA for proprotein convertase 2 (PC2) was cloned for the first time from a fish. The clone, which was isolated from the ovary of the medaka, Oryzias latipes, by a combination of RT-PCR cloning and 5'- and 3'-rapid amplification of cDNA ends, codes for a protein of 641 amino acid residues highly homologous to other vertebrate PC2. The medaka preproPC2 consists of a signal sequence, a propeptide with sites for autocatalytic activation, a Kex2-like catalytic domain, and a P-domain. The catalytic triad residues (Asp-169, His-210, and Ser-386) were all conserved. Northern blot analysis revealed that PC2 was expressed in the brain, ovary, and kidney of the fish. The size of PC2 mRNA expressed in the ovary was 2.3 kb, whereas those of the brain and kidney were 2.8 kb. This size difference was attributed to the lack of an approximately 300-bp nucleotide sequence just before the poly(A)(+) tail of the ovarian PC2 mRNA. Ovarian expression of the PC2 gene was found in the medaka but not in the ;mouse, and therefore further analysis was conducted for the fish ovary. The greatest expression of PC2 mRNA in the oocytes of small growing follicles in the mature medaka was demonstrated by Northern blotting, RT-PCR and in situ hybridization analysis. These results suggest that PC2 may play a role in the processing of proproteins and/or pro-hormones expressed in the growing oocytes. (C) 2004 Elsevier B.V All rights reserved.
  • Ogiwara K, Shinohara M, Takahashi T
    Journal of experimental zoology. Part A, Comparative experimental biology 301A 5 449 - 459 5 2004年05月 [査読有り][通常論文]
     
    A cDNA for furin was cloned from the ovary of the medaka, Oryzias latipes, by a combination of cDNA library screening, 5'-rapid amplification of cDNA ends (RACE), and 3'- RACE. The cDNA sequence codes for a protein of 814 amino acid residues highly homologous to other vertebrate furins, Ca2+ -dependent serine proteases belonging to the subtilysin-like proprotein convertase family. The medaka preprofurin consists of a leader sequence, a propeptide with autoactivation sites, a Kex2-like catalytic domain, a P domain, a cysteine-rich domain, a putative transmembrane domain, and a cytoplasmic domain. The catalytic triad residues (Asp-164, His-205, and Ser-379) were all conserved. Furin mRNA was expressed in many tissues of this, including the ovary. In the ovary, the greatest expression of furin mRNA occurred in oocytes of small growing follicles, as demonstrated by Northern blotting, RT-PCR, and in situ hybridization analysis. Temporary and spatial expression patterns of the medaka fish furin were similar to those of stromelysin-3 and MT5-MMP during oocyte growth and postnatal development. (C) 2004 Wiley-Liss, Inc.
  • 荻原 克益, 高橋 孝行
    日本比較内分泌学会ニュース = Newsletter of Japan Society for Comparative Endocrinology 111 52 - 55 日本比較内分泌学会 2003年12月01日
  • R Ohkura, A Kimura, T Kihara, K Ogiwara, T Takahashi
    ZOOLOGICAL SCIENCE 20 7 847 - 854 2003年07月 [査読有り][通常論文]
     
    The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B-2 receptor mRNA was constitutively expressed. Bradykinin B-2 receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B-2 receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B-2 receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B-2 receptor mRNA level. The present data indicate that the level of B-2 receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.
  • K Ogiwara, H Matsui, A Kimura, T Takahashi
    MOLECULAR REPRODUCTION AND DEVELOPMENT 61 1 21 - 31 2002年01月 [査読有り][通常論文]
     
    A cDNA clone (2755-bp) for stromelysin-3 was isolated by screening the cDNA library and by 3'- and 5'-rapid amplification of cDNA ends using ovary RNA of the medaka fish Oryzias latipes. The clone encodes a protein of 492 amino acids. Stromelysin-3 mRNA was detected only in the ovary. In situ hybridization analysis revealed that stromelysin-3 mRNA was localized in the oocyte cytoplasm of small growing follicles. RT-PCR analysis of total RNAs isolated from various-sized follicles and ovulated oocytes was conducted in order to determine the mRNA levels during oocyte growth. The stromelysin-3 mRNA level was the highest in the small follicles, and the mRNA levels decreased as the follicles grew. No significant stromelysin-3 mRNA was detected in the ovulated oocytes or immature ovaries. The fish stromelysin-3 cDNA was expressed in COS-1 cells in order to characterize the intracellular localization of the protein. A 56 kDa protein was synthesized and secreted into the culture medium. The secreted stromelysin-3 exhibited gelatin-degrading activity. Mol. Reprod. Dev. 61: 21-31, 2002. (C) 2002 Wiley-Liss, Inc.
  • A Kimura, T Kihara, R Ohkura, K Ogiwara, T Takahashi
    BIOLOGY OF REPRODUCTION 65 5 1462 - 1470 2001年11月 [査読有り][通常論文]
     
    We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B2R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B2R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B2R mRNA. The B2R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B2R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.
  • H Matsui, K Ogiwara, R Ohkura, M Yamashita, T Takahashi
    EUROPEAN JOURNAL OF BIOCHEMISTRY 267 15 4658 - 4667 2000年08月 [査読有り][通常論文]
     
    We cloned cDNAs for gelatinase A and gelatinase B from an ovary cDNA library of the medaka fish Oryzias latipes. The gelatinase A clone encodes a protein of 657 amino acids, whereas the gelatinase B clone encodes a protein of 690 amino acids. Gelatinase A mRNA was expressed in the testis, ovary, intestine, heart, spleen and kidney of the animal. In contrast, gelatinase B mRNA was detected in the ovary. Localization of the respective mRNAs in the ovary was examined using in situ hybridization. Gelatinase A mRNA was found only in the oocytes of small and middle-sized follicles. In contrast, gelatinase B was expressed exclusively in follicular tissues that had ovulated. In situ zymographic analysis revealed that gelatinolytic activity, presumably due to matrix metalloproteinase activity, was detectable in the areas surrounding small and middle-sized follicles, interstitial stromal tissues and the cytoplasm of oocytes. Using extracts of the whole ovary and of ovulated oocytes, several gelatin-degrading enzymes, which probably represent the intermediate and active forms of medaka fish gelatinase A and gelatinase B, were detected by gelatin zymographic analysis. These results clearly indicate that gelatinase A and gelatinase B play a discrete role in the ovary of this lower vertebrate animal.

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 荻原 克益
     
    本研究は、マウス濾胞選択の分子機構解明を目指した研究課題である。何らかの刺激により成長を開始した原始濾胞のうち、大部分は成長途中でアポトーシスにより死滅してしまう。そして、生き残った濾胞のみが成熟し排卵される。この様に死滅する濾胞と生き残り排卵される濾胞を選択する過程が、「濾胞選択」である。濾胞選択に関わる数多くの研究報告があるにもかかわらず、今なお、その詳細な分子機構は解明されていない。一般的な過排卵誘導法(PMSG/hCG等)で20-30個程度の卵が排卵されるが、特殊な薬剤を用いた排卵誘導法では100個程度の卵が排卵されることが知られている。そこで、本研究ではこの薬剤を用いて濾胞選択の研究を展開している。これまでの研究結果から、その薬剤が濾胞選択(アポトーシス)を抑制すること、その薬剤により卵巣内における活性型アクチビン量が有意に増加すること、また、血中および卵巣内エスタジオール量が有意に上昇することが明らかとなっている。そこで今回は、アクチビンと濾胞選択の関係について詳細に解析を行った。その結果、アクチビンを発現する濾胞ではアポトーシスが検出されないこと、卵巣をアクチビン存在下で器官培養するとエストラジオールの産生、分泌が誘導され、また、その受容体の発現も有意に上昇することが明らかとなった。これらの結果は、卵巣内で増加するアクチビンによりエストラジオールの産生、分泌が誘導され、そのエストラジオールによりアポトーシスが抑制されるされるというモデルを強く支持する結果である。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 荻原 克益
     
    マウス卵巣内には、成長を開始する前の濾胞(原始濾胞)が多数存在する。あるホルモンの刺激を受けると、いくつかの原始濾胞が成長を開始するが、その数はマウスの場合、数十個から百個程度と考えられている。成長を開始した濾胞は、途中でアポトーシスにより死滅し、最終的に10-20個程度の卵が排卵される。このように、アポトーシスにより死滅する濾胞とそのまま生き残り排卵まで行きつく濾胞が選別される過程を濾胞選択と呼ぶ。本研究では、マウスの濾胞選択の分子機構に焦点を当て、その解明を目指して研究を行っている。 通常、PMSG/hCG等による過排卵誘導法では20-30個程度の卵が排卵されるが、本研究で用いる薬剤では、100個程度の卵が排卵される。この排卵数は成長を開始した原始濾胞数とほぼ一致することから、この薬剤はアポトーシスを阻害していることが考えられる。この薬剤を用いた研究から、エストラジオールとアロマターゼ(エストラジオール生合成のkey enzyme)が重要であることを昨年度報告した。本年度は、アロマターゼについて、その発現量や酵素活性の変動について詳しく調べた。その結果、薬剤処理とPMSG処理間で、その発現量に有意な増減は見られなかったが、アロマターゼ活性は薬剤処理グループで有意に増加していることが明らかとなった。さらに、PMSG注射後、ある特定の時間にエストラジオールを注射することで、PMSG/hCGによる排卵数と比較して、排卵数が有意に増加することを突き止めた。これらの結果は、この薬剤がどのようにアポトーシスを抑制しているのかを解明する重要なヒントとなり、濾胞選択機構解明に向けた大きな一歩となる。今後は、この薬剤処理により、なぜアロマターゼ活性が増加するのか、その分子機構(作用機構)について研究を進めていく予定である。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 高橋 孝行, 荻原 克益
     
    本研究では,「排卵」と「卵成熟」の関係,特に,「排卵」の実行過程に「卵成熟」の進行がどのように関わるかについて,硬骨魚類のメダカを用いて調べた。「排卵」と「卵成熟」の連携には濾胞細胞と卵細胞の間に存在するギャップ結合の役割の可能性について検討した結果,メダカの排卵に必須の濾胞壁溶解酵素であるMT2-MMPの発現がギャップ結合阻害剤により抑制されることを見出し,さらにギャップ結合阻害剤によるMT2-MMPの発現抑制のメカニズムを明らかにした。加えて,細胞間のコミュニケーションに関わると予想されるギャップ結合構成単位のコネキシン分子の数種を明らかにした。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 荻原 克益
     
    排卵は受精可能となった卵母細胞が卵巣腔へと放出される過程である。この過程は、濾胞組織と卵巣最外層組織の分解を伴う組織破壊現象と捉えることもでき、毎日排卵するメダカの場合、排卵後、直ちに組織修復が開始され、さらに、卵巣内に残留した濾胞組織も迅速に分解され、翌日の排卵に備える必要がある。本研究では、迅速に進行するこの過程の分子機構を解明することを目的に研究を行った。その結果、卵巣開口部の修復にカドヘリンタンパク質の関与を示唆する結果が得られた。また、在留濾胞組織の分解にゼラチナーゼBやプラスミンが関与する結果を得た。今回の研究で得られた成果をもとに、今後も組織修復の分子機構の解明に取り組む。
  • 日本学術振興会:科学研究費助成事業 基盤研究(A)
    研究期間 : 2012年05月 -2016年03月 
    代表者 : 高橋 孝行, 荻原 克益
     
    メダカ排卵濾胞のin vitro実験系を用いて、排卵時に作動する排卵関連遺伝子(MT2-MMPおよびプロスタグランジンE2受容体EP4b)の発現誘導機構を調べた。排卵に伴うMT2-MMPおよびEP4bの発現誘導は、排卵濾胞の顆粒膜細胞がLH刺激に応答して起こる。この一連のプロセスでは、最初に転写因子nPRの発現が起こり、続いてnPRを含む複数の転写因子がこれらの遺伝子の発現に関与することを明らかにした。また、ギャップ結合阻害剤カルベノキソロンを利用した実験結果から、顆粒膜細胞でのMT2-MMPの発現には卵細胞と顆粒膜細胞間のギャップ結合を介した分子的連携が必要であることが示された。
  • 日本学術振興会:科学研究費助成事業 若手研究(A)
    研究期間 : 2011年04月 -2014年03月 
    代表者 : 荻原 克益
     
    本研究は、メダカ排卵の内分泌制御機構の解明を目指して実施された。LHにより誘導される核内プロゲスチン受容体nPR(排卵関連遺伝子の誘導に必要不可欠な転写因子)の発現誘導機構を解明し、メダカ排卵において重要な役割を担うMT2-MMPとPAI-1(共に排卵時に急激に誘導される)の発現誘導にnPRが関与することを突き止めた。さらに、nPRのリガンドである卵成熟誘起ホルモン(17α, 20β-DHP)産生に関与する酵素群の発現パターンを明らかにした。これらの結果から、排卵時に濾胞細胞層の分解に関与する排卵関連酵素の発現誘導機構モデルを提唱するに至った。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2009年 -2011年 
    代表者 : 高橋 孝行, 荻原 克益
     
    メダカの卵巣濾胞のin vitro排卵系を用いて、プロスタグランジE2(PGE2)が排卵過程に関与することを確認した。濾胞におけるPGE2合成酵素cyclooxygenase-2の活性及びPGE2レベルは変化せず、PGE2受容体であるEP4bの発現レベルが排卵時に急激に上昇したことから、EP4b受容体遺伝子の転写が重要なステップであることが明らかになった。EP4b受容体の誘導には黄体形成ホルモンと、その後にプロゲステロン受容体が関与することを明らかにした。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2009年 -2010年 
    代表者 : 荻原 克益
     
    本研究課題は、哺乳類(マウス)の排卵実行に重要な酵素(排卵酵素)の同定と排卵時の濾胞層分解の分子メカニズムの解明を目指して実施された。また、排卵酵素の同定に利用する生体外排卵培養系の改良にも取り組んだ。その結果、2種のMMPが排卵の前後の濾胞で発現変動することを発見したが、これらのMMPは排卵に関与しない可能性が高いことが明らかとなった。一方、MT2-MMPは排卵前にその活性体が増加することから、排卵への関与が示唆された。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2009年 -2010年 
    代表者 : 高橋 孝行, 荻原 克益
     
    昨年度の解析から、哺乳類型siRNAがメダカにおいては効率の良いノックダウン効果を示さないことが示唆されたため、始めにこの点についてより詳細な解析を行った。メダカ培養細胞を用いて強制発現させたMT2-MMPおよびuPAのノックダウン効率を調査したところ、化学合成したsiRNA、miRNA発現ベクターを用いて発現させたsiRNAともにほとんど効果がないことが判明した。これらの結果から,メダカ細胞においては、哺乳類型siRNAが作用し難いことが考えられる。この理由として、メダカで作用するsiRNAの長さが哺乳類型とは異なることが考えられる。そこで、この点に焦点を絞って解析を行った。siRNA産生において重要な因子であるDicerのリコンビンナントタンパク質を用いてdsRNAの切断実験を試みたところ、20-30塩基付近に切断産物が得られたため、現在この産物の配列情報を解析中である。また、RISCの主要構成因子であるArgonaute-2のメダカ特異的抗体を作製、この抗体を用いて免疫沈降を行い、共沈してきたsmall RNA分子の長さを解析中である。これらの解析結果から、メダカで作用するsiRNAの長さを明らかにし、効率良くノックダウン可能な発現ベクター構築へと移行していく予定である。さらに、Droshaに関しても全長配列のクローニングが終了し、リコンビナントタンパク質および抗体作製へと移行した。今後は、こちらに関しても、リコンビナントタンパク質の準備ができ次第、詳細な解析を行う予定である。 マウスではトランスジェニックRNAi技術はすでに確立され、標的とする遺伝子/タンパク質の個体レベルでの解析研究が著しい進展を見せている。本研究は、マウス以外の脊椎動物でも同様の技術を確立することを目指し、メダカを実験材料に2年間の研究計画を提案した。本研究により、マウスとは異なるメカニズムがあることが明らかになったので、これを基盤としてトランスジェニックRNAiメダカの確立に向けてさらに研究を進める予定である。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2007年 -2008年 
    代表者 : 荻原 克益
     
    本研究は、哺乳類(マウス)排卵酵素の探索ならびに同定を目指して実施された。その結果、排卵酵素探索のツールとして重要な生体外排卵培養系について、より排卵数の多い培養条件を突きとめることに成功した。また、メタロプロテアーゼが排卵酵素であることを示唆する結果を得た。さらに、排卵前後で発現変動するいくつかのメタロプロテアーゼを発見した。これらの結果は、哺乳類排卵酵素同定の足がかりとして重要な成果であると考えられる。

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