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Master

Affiliation (Master)

  • Faculty of Science　Biological Sciences　Reproductive and Developmental Biology

Affiliation (Master)

  • Faculty of Science　Biological Sciences　Reproductive and Developmental Biology

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Profile and Settings

Profile and Settings

  • Name (Japanese)

    Ogiwara

  • Name (Kana)

    Katsueki

  • Name

    201301000922869333

Alternate Names

Achievement

Research Interests

  • ovulation   ovary   mouse   protease   medaka   transgenic   reproduction   RNAi   

Research Areas

  • Life sciences / Morphology, anatomy

Research Experience

  • 2017/10 - Today Hokkaido University Faculty of science Associate professor
  • 2008/04 - 2017/09 Graduate school of science, Hokkaido university Assistant Professor

Published Papers

  • Katsueki Ogiwara, Chika Fujimori, Takayuki Takahashi
    Molecular and cellular endocrinology 111816 - 111816 2022/11/18 
    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 0006-3363 2021/08/03 [Refereed][Not invited]
     
    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 1095-6433 2021/04 [Refereed][Not invited]
  • Hagiwara A, Ogiwara K, Sugama N, Yamashita M, Takahashi T
    General and comparative endocrinology 288 113373  0016-6480 2019/12 [Refereed][Not invited]
  • Katsueki Ogiwara, Takayuki Takahashi
    Cells 8 (3) 215 - 215 2019/03/04 [Refereed][Not invited]
     
    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) 1470-1626 2018/10 [Refereed][Not invited]
  • Takahashi T, Hagiwara A, Ogiwara K
    Global Journal of Reproductive Medicine 4 (3) 2018/04/30 [Refereed][Not invited]
  • Takayuki Takahashi, Akane Hagiwara, Katsueki Ogiwara
    Molecular and Cellular Endocrinology 461 236 - 247 1872-8057 2018/02/05 [Refereed][Not invited]
     
    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 0303-7207 2017/07 [Refereed][Not invited]
     
    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  0006-3363 2016/03 [Refereed][Not invited]
     
    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 0289-0003 2016/02 [Refereed][Not invited]
     
    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  0006-3363 2015/01 [Refereed][Not invited]
     
    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 0289-0003 2014/12 [Refereed][Not invited]
     
    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  0006-3363 2014/06 [Refereed][Not invited]
     
    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 The Society for the Study of Reproduction 88 (5) 118 - 118 0006-3363 2013/05 [Refereed][Not invited]
     
    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 0289-0003 2013/04 [Refereed][Not invited]
     
    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  1932-6203 2013/01 [Refereed][Not invited]
     
    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 0303-7207 2012/10 [Refereed][Not invited]
     
    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  0006-3363 2012/04 [Refereed][Not invited]
     
    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 Japan Society for Comparative Endocrinology 37 (142) 168 - 170 1882-6636 2011/08/31
  • Kenji Moriyama, Atsuko Hanai, Kazuyuki Mekada, Atsushi Yoshiki, Katsueki Ogiwara, Atsushi Kimura, Takayuki Takahashi
    JOURNAL OF BIOMEDICAL SCIENCE 18 60  1021-7770 2011/08 [Refereed][Not invited]
     
    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 0303-7207 2011/01 [Refereed][Not invited]
     
    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 0289-0003 2010/09 [Refereed][Not invited]
     
    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 0302-766X 2010/06 [Refereed][Not invited]
     
    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 0289-0003 2008/09 [Refereed][Not invited]
     
    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 1040-452X 2007/08 [Refereed][Not invited]
     
    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 0027-8424 2007/04 [Refereed][Not invited]
     
    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 0289-0003 2006/11 [Refereed][Not invited]
     
    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 0014-5793 2005/12 [Refereed][Not invited]
     
    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 0027-8424 2005/06 [Refereed][Not invited]
     
    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 0378-1119 2004/08 [Refereed][Not invited]
     
    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.
  • K Ogiwara, M Shinohara, T Takahashi
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-COMPARATIVE EXPERIMENTAL BIOLOGY 5 301A (5) 449 - 459 0022-104X 2004/05 [Refereed][Not invited]
     
    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 日本比較内分泌学会 (111) 52 - 55 0913-9044 2003/12/01
  • R Ohkura, A Kimura, T Kihara, K Ogiwara, T Takahashi
    ZOOLOGICAL SCIENCE 20 (7) 847 - 854 0289-0003 2003/07 [Refereed][Not invited]
     
    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 1040-452X 2002/01 [Refereed][Not invited]
     
    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 0006-3363 2001/11 [Refereed][Not invited]
     
    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 0014-2956 2000/08 [Refereed][Not invited]
     
    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

Research Projects

  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2021/04 -2024/03 
    Author : 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。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : OGIWARA Katsueki
     
    The present study aims to elucidate a molecular mechanism of mouse follicle selection. Most primordial follicles that have started growing die by apoptosis, and only the survived follicles are ovulated. Many investigators have studied about it, but the detail is still unclear. The present study was carried out using a hormone substance that is suggested to suppress follicle selection (apoptosis). The results suggest that production and secretion of estradiol 17b (E2) are regulated by Activin-A induced by FSH and that E2 is involved in apoptosis. Based on the results, we propose a model that apoptosis is controlled by E2.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2016/04 -2019/03 
    Author : Takahashi Takayuki
     
    The purpose of this project was to examine whether ovualtion and oocyte maturation, which are both triggered by the ovulatory surge of LH in vertebrates, would be closely linked, and if any, what would be the mechanism by which the two processes occur in harmony? To this end, we used an in vitro ovulation system established for medaka ovarian follicles. Assuming that gap junctional communication between follicle cells and the oocyte and/or between two neighboring follicle cells might play an important role in ovulating follicles, we examine the effect of gap junction blockers on ovulation and oocyte maturation. In vitro follicle ovulation, but not oocyte maturation, was strongly inhibited by the blockers. The inhibition of follicle ovulation by gap junction blockers was demonstrated to be accompanied by the inhibition of MT2-MMP expression. In addition, we suggest that several connexin species may be involved in the gap junctional communication for successful ovulation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2015/04 -2018/03 
    Author : OGIWARA Katsueki
     
    Ovulation is the process that fertilizable oocytes are released into the ovarian sac.The precess is involved in a breakdown of the follicular wall,which are composed of folicular tissues and ovarian surface tissues.In medaka, which ovulates every day, the tissue repair of the follicle wall starts just after ovulation, and follicular tissues that had released an oocyte are degraded rapidly in the ovary. The aim of the present study is to elucidate the molecular mechanism of the process.Cadherin protein was suggested to be involved in the tissue repair of the follicle wall.Gelatinase B and plasmin, both of which were expressed in follicular tissues that had released an oocyte, were implied to be implicated in the rapid degradation of the follicle tissues in the ovary. We will continue to study to elucidate the molecular mechanism of the rapid tissue repair, based on the results abtained from the present study.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
    Date (from‐to) : 2012/05 -2016/03 
    Author : Takahashi Takayuki, OGIWARA KATSUEKI
     
    Using in vitro ovulation system established for medaka ovarian follicles, we investigated the mechanism by which ovulation-related genes/proteins, membrane type-2 matrix metalloproteinase (MT2-MMP) and prostaglandin E2 receptor subtype (EP4b) were induced at ovulation. Expression of both genes/proteins was under the control of LH. Our data indicated that LH induced the expression of a transcription factor nuclear progestin receptor (nPR) in the granulosa cells and that nPR, together with other transcription factors, played roles in the subsequent expression of MT2-MMP and EP4b. We next examined if a communication between granulosa cells and the oocyte in the follicles may be important for successful ovulation. Preovulatory follicles were treated with carbenoxolone, a gap-junction inhibitor, using in vitro ovulation system. We found strong inhibition of MT2-MMP expression by the treatment, indicating that ovulation would be influence by oocyte maturation event occurring in the oocyte.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
    Date (from‐to) : 2011/04 -2014/03 
    Author : OGIWARA Katsueki
     
    The aim of the present study is to elucidate the endocrine regulatory mechanism of ovulation in the medaka. The study disclosed a mechanism for inducing nuclear progestin receptor (nPR), which was the essential transcription factor for induction of ovulation-associated genes. It was revealed that nPR was involved in the induction of MT2-MMP and PAI-1, both which played an important role on ovulation. An expression pattern of the synthesizing enzymes for a maturation inducing hormone (17a, 20b-DHP), a ligand of nPR, was elucidated. Based on the results, we propose the novel model of the endocrine regulatory mechanism for inducing the ovulation-related enzymes responsible for degradation of follicle layer during ovulation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2009 -2011 
    Author : TAKAHASHI Takayuki, OGIWARA Katsueki
     
    In in vitro ovulation system using medaka ovarian preovulatory follicles, we confirmed that prostaglandin E2 (PGE2) played a role in the fish ovulation. Activity levels of cyclooxygenase-2, the enzyme responsible for PGE2 synthesis, and intrafollicular levels of PGE2 were both relatively constant, while the transcript levels of EP4b, a PGE2 receptor subtype, were drastically induced at ovulation, indicating the importance of induction of the receptor for PGE2's effect on medaka ovulation. Transcription of EP4b gene was demonstrated to be regulated by luteininzing hormone and nuclear progesterone receptor.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2009 -2010 
    Author : OGIWARA Katsueki
     
    The aim of the present study were to identify the mouse ovulation enzymes responsible for degradation of the follicle wall during ovulation, and to elucidate the molecular mechanism of degradation of the follicle wall during ovulation. It was also attempted to improve the culture condition by optimizing the buffer system and by changing the supplements added. As results, it was found that two MMPs were changed their expression between before and after ovulation. However, it was suggested that they were not involved in ovulation. Active form of MT2-MMP was found to be increased before ovulation, implying that it was involved in ovulation.
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
    Date (from‐to) : 2009 -2010 
    Author : 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。
  • Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2007 -2008 
    Author : OGIWARA Katsueki
     
    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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