論文

• Meiotic self-pairing of the Psalidodon (Characiformes, Characidae) iso-B chromosome: A successful perpetuation mechanism
  Duílio Mazzoni Zerbinato de Andrade Silva, Cristian Araya-Jaime, Masakane Yamashita, Mateus Rossetto Vidal, Claudio Oliveira, Fábio Porto-Foresti, Roberto Ferreira Artoni, Fausto Foresti
  Genetics and Molecular Biology, 44, 3, FapUNIFESP (SciELO), 2021年10月, ［査読有り］
  研究論文（学術雑誌）
• Inhibition of medaka ovulation by gap junction blockers due to its disrupting effect on the transcriptional process of LH-induced Mmp15 expression.
  Hagiwara A, Ogiwara K, Sugama N, Yamashita M, Takahashi T
  General and comparative endocrinology, 288, 113373, 2019年12月, ［査読有り］
• Function of leukaemia inhibitory factor in spermatogenesis of a teleost fish, the medaka Oryzias latipes.
  Satoh R, Bando H, Sakai N, Kotani T, Yamashita M
  Zygote (Cambridge, England), 27, 6, 1, 9, 2019年10月, ［査読有り］
• Staufen1, Kinesin1 and microtubule function in cyclin B1 mRNA transport to the animal polar cytoplasm of zebrafish oocytes
  Takahashi K, Ishii K, Yamashita M
  Biochemical and Biophysical Research Communications, 503, 4, 2778, 2783, 2018年09月, ［査読有り］
  英語, 研究論文（学術雑誌）
• ホタテガイ幼生分布調査に有用な免疫染色技術の実用的改善
  清水 洋平, 狩野 俊明, 成田 伝彦, 板倉 祥一, 榎本 洸一郎, 戸田 真志, 川崎 琢真, 高畠 信一, 岩井 俊治, 山下 正兼
  北海道水産試験場研究報告 = Scientific reports of Hokkaido Fisheries Research Institutes, 89, 89, 1, 8, 北海道立総合研究機構水産研究本部, 2016年03月, ［査読有り］
  日本語, 研究論文（学術雑誌）, ホタテガイ幼生の分布調査では，幼生の判別を容易にするため，ホタテガイ幼生のみを染色する免疫染色法が活用されつつある。本研究では，現場でみられた免疫染色の不安定性の原因を試料の取り扱い方法，特に固定方法にあると考え，確実にホタテガイ幼生を染色するための，試料の固定条件を検討した。その結果，0.9%ホルムアルデヒド海水溶液または0.5%グルタールアルデヒド海水溶液で試料を一晩固定することで，良好な免疫染色結果が得られた。また，1%グルタールアルデヒド海水溶液を用いると固定時間を1時間に，また4%グルタールアルデヒド海水溶液を用いると30分に短縮できることが明らかになった。抗体反応時間と発色反応時間をともに30分から20分程度に短縮でき，免疫染色工程が全体で1時間10分程度となった。
• Immunoreactive insulin in diabetes mellitus patient sera detected by ultrasensitive ELISA with thio-NAD cycling
  Etsuro Ito, Mugiho Kaneda, Hiromi Kodama, Mika Morikawa, Momoko Tai, Kana Aoki, Satoshi Watabe, Kazunari Nakaishi, Seiichi Hashida, Satoshi Tada, Noriyuki Kuroda, Hitomi Imachi, Koji Murao, Masakane Yamashita, Teruki Yoshimura, Toshiaki Miura
  BIOTECHNIQUES, 59, 6, 359, 367, BIOTECHNIQUES OFFICE, 2015年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, To minimize patient suffering, the smallest possible volume of blood should be collected for diagnosis and disease monitoring. When estimating insulin secretion capacity and resistance to insulin in diabetes mellitus (DM), increasing insulin assay immunosensitivity would reduce the blood sample volume required for testing. Here we present an ultrasensitive ELISA coupled with thio-NAD cycling to measure immunoreactive insulin in blood serum. Only 5 mL of serum was required for testing, with a limit of detection (LOD) for the assay of 10-16 moles/assay. Additional recovery tests confirmed this method can detect insulin in sera. Comparisons between a commercially available immunoreactive insulin kit and our ultrasensitive ELISA using the same commercially available reference demonstrated good data correlation, providing further evidence of assay accuracy. Together, these results demonstrate our ultrasensitive ELISA could be a powerful tool in the diagnosis and treatment of not only DM but also many other diseases in the future.
• Real-Time Imaging of Actin Filaments in the Zebrafish Oocyte and Embryo
  Yumiko Nukada, Mayu Horie, Akimasa Fukui, Tomoya Kotani, Masakane Yamashita
  CYTOSKELETON, 72, 9, 491, 501, WILEY-BLACKWELL, 2015年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis. (C) 2015 Wiley Periodicals, Inc.
• Detection of HIV-1 p24 at Attomole Level by Ultrasensitive ELISA with Thio-NAD Cycling
  Akira Nakatsuma, Mugiho Kaneda, Hiromi Kodama, Mika Morikawa, Satoshi Watabe, Kazunari Nakaishi, Masakane Yamashita, Teruki Yoshimura, Toshiaki Miura, Masaki Ninomiya, Etsuro Ito
  PLOS ONE, 10, 6, e0131319, PUBLIC LIBRARY SCIENCE, 2015年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, To reduce the window period between HIV-1 infection and the ability to diagnose it, a fourth-generation immunoassay including the detection of HIV-1 p24 antigen has been developed. However, because the commercially available systems for this assay use special, high-cost instruments to measure, for example, chemiluminescence, it is performed only by diagnostics companies and hub hospitals. To overcome this limitation, we applied an ultrasensitive ELISA coupled with a thio-NAD cycling, which is based on a usual enzyme immunoassay without special instruments, to detect HIV-1 p24. The p24 detection limit by our ultrasensitive ELISA was 0.0065 IU/assay (i.e., ca. 10(-18) moles/assay). Because HIV-1 p24 antigen is thought to be present in the virion in much greater numbers than viral RNA copies, the value of 10(-18) moles of the p24/assay corresponds to ca. 10(3) copies of the HIV-1 RNA/assay. That is, our ultrasensitive ELISA is chasing the detection limit (10(2) copies/assay) obtained by PCR-based nucleic acid testing (NAT) with a margin of only one different order. Further, the detection limit by our ultrasensitive ELISA is less than that mandated for a CE-marked HIV antigen/antibody assay. An additional recovery test using blood supported the reliability of our ultrasensitive ELISA.
• ホタテガイ幼生分布調査現場への普及に向けた免疫染色技術の簡易化 (技術報告)
  清水洋平, 川崎琢真, 高畠信一, 岩井俊治, 山下正兼
  北海道水産試験場研究報告, 87, 87, 93, 96, 北海道立総合研究機構水産研究本部, 2015年, ［査読有り］
  日本語, 研究論文（学術雑誌）
• Surface-spreading technique of meiotic cells and immunodetection of synaptonemal complex proteins in teleostean fishes
  Cristian Araya-Jaime, Erica Alves Serrano, Duilio Mazzoni Zerbinato de Andrade Silva, Masakane Yamashita, Toshiharu Iwai, Claudio Oliveira, Fausto Foresti
  MOLECULAR CYTOGENETICS, 8, 1, 4, BIOMED CENTRAL LTD, 2015年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Background: Different moderrn methodologies are presently available to analyze meiotic chromosomes. These methods permit investigation of the behavior of chromosomes in the normal complement and of sex and B chromosomes, two special types of chromosomes that are associated with the A complement and are present in many organisms, including fishes. However, meiotic studies are still scarce in fishes, considering the wide number of species in this group.. Here, we describe a new protocol for the visualization of the synaptonemal complex in spermatocytes and oocytes of fishes and to the sequential use of the technique with other procedures and techniques such as immunodetection of the synaptonemal complex protein with a specific antibody and co-detection of DNA sequences by FISH.
  Results: The meiotic surface-spreading protocol used in the present proposal worked well in representative species of four fish orders and was useful in obtaining good results even in small specimens. Fish-specific antibodies and commercial products worked similarly well to detect synaptonemal complex (SC) proteins. The sequential application of fluorescence in situ hybridization using specific probes showed clear signals associated with the SC structures identified by immunostaining.
  Conclusion: Here, we provide a useful and applicable immunofluorescent protocol for the visualization of synaptonemal complex proteins in the meiotic cells of fishes in surface-spreading preparations. Furthermore, this technique allows for the sequential application of other cytogenetic procedures.
• Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes
  Kazuki Takahashi, Tomoya Kotani, Yoshinao Katsu, Masakane Yamashita
  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 448, 1, 22, 27, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2014年05月, ［査読有り］
  英語, 研究論文（学術雑誌）, In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3' untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3' UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 riaRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumiliol and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation. (C) 2014 Elsevier Inc. All rights reserved.
• ホタテガイ幼生簡易同定に用いる高特異的ポリクローナル抗体の作製
  清水洋平, 岩井俊治, 高畠信一, 川崎琢真, 山下正兼
  水産技術, 7, 1, 31, 36, 水産総合研究センター, 2014年, ［査読有り］
  日本語, 研究論文（学術雑誌）, ほたてがい漁業を支える天然採苗を効率化させるため，関係各機関ではホタテガイ幼生の分布調査を行っている。これまで，ホタテガイの幼生を殻の形態で判別していたが，二枚貝幼生の殻の形態は類似性が高いため判別に経験を要し，また，交板の形状による判別は簡易性に欠け，現場向きではない。そのため，幼生の形態を基にした判別は，作業に従事できる人材を限定し，かつ，時間と労力を要する作業となっていた。そこで本研究では，ホタテガイ幼生に対するポリクローナル抗体を作製した。本抗体はホタテガイ幼生を特異的に染色できた。これによりホタテガイ幼生の判別が容易になり，作業が簡易化されることが期待される。
• A cis-acting element in the coding region of cyclin B1 mRNA couples subcellular localization to translational timing
  Kyota Yasuda, Tomoya Kotani, Masakane Yamashita
  DEVELOPMENTAL BIOLOGY, 382, 2, 517, 529, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2013年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, Subcellular localization of messenger RNAs (mRNAs) to correct sites and translational activation at appropriate timings are crucial for normal progression of various biological events. However, a molecular link between the spatial regulation and temporal regulation remains unresolved. In immature zebrafish oocytes, translationally repressed cyclin B1 mRNA is localized to the animal polar cytoplasm and its temporally regulated translational activation in response to a maturationAnducing hormone is essential to promote oocyte maturation. We previously reported that the coding region of cyclin B1 mRNA is required for the spatio-temporal regulation. Here, we report that a sequence, CAGGAGACC, that is conserved in the coding region of vertebrate cyclin B1 mRNA is involved in the regulation. Like endogenous cyclin B1 mRNA, reporter mRNAs harboring the sequence CAGGAGACC were localized to the animal polar cytoplasm of oocytes, while those carrying mutations in the sequence (with no change in the coding amino acids) were dispersed in the animal hemisphere of oocytes. Furthermore, translational activation of the mutant mRNAs was initiated at a timing earlier than that of endogenous and wild-type reporter mRNAs during oocyte maturation. Interaction of CAGGAGACC with proteins in vitro suggests that this sequence functions in collaboration with a trans-acting protein factor(s) in oocytes. These findings reveal that the sequence in the coding region of cyclin B1 mRNA plays an important role as a cis-acting element in both subcellular localization and translational timing of mRNA, providing a direct molecular link between the spatial and temporal regulation of mRNA translation. (C) 2013 Elsevier Inc. All rights reserved.
• Cyclin b1 mRNA translation is temporally controlled through formation and disassembly of RNA granules
  Tomoya Kotani, Kyota Yasuda, Ryoma Ota, Masakane Yamashita
  Journal of Cell Biology, 202, 7, 1041, 1055, 7, 2013年, ［査読有り］
  英語, 研究論文（学術雑誌）, Temporal control of messenger RNA (mRNA) translation is an important mechanism for regulating cellular, neuronal, and developmental processes. However, mechanisms that coordinate timing of translational activation remain largely unresolved. Full-grown oocytes arrest meiosis at prophase I and deposit dormant mRNAs. Of these, translational control of cyclin B1 mRNA in response to maturation-inducing hormone is important for normal progression of oocyte maturation, through which oocytes acquire fertility. In this study, we found that dormant cyclin B1 mRNA forms granules in the cytoplasm of zebrafish and mouse oocytes. Real-time imaging of translation revealed that the granules disassemble at the time of translational activation during maturation. Formation of cyclin B1 RNA granules requires binding of the mRNA to Pumilio1 protein and depends on actin filaments. Disruption of cyclin B1 RNA granules accelerated the timing of their translational activation after induction of maturation, whereas stabilization hindered translational activation. Thus, our results suggest that RNA granule formation is critical for the regulation of timing of translational activation. © 2013 Shigeoka et al.
• Possible Involvement of Nemo-like Kinase 1 in Xenopus Oocyte Maturation As a Kinase Responsible for Pumilio1, Pumilio2, and CPEB Phosphorylation
  Ryoma Ota, Tomoya Kotani, Masakane Yamashita
  BIOCHEMISTRY, 50, 25, 5648, 5659, AMER CHEMICAL SOC, 2011年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Members of the mitogen-activated protein kinase (MAPK) family play important roles in Xenopus oocyte maturation. Nemo-like kinase (NLK), an atypical MAPK, is known to function in multiple developmental processes in vertebrates and invertebrates, but its involvement in gametogenesis and gamete maturation is unknown. In this study, we biochemically examined NLK1 during Xenopus oocyte maturation. NLK1 is expressed in immature oocytes, and its protein level remains constant during maturation. NLK1 is inactive in immature oocytes but is activated during maturation, depending on Mos protein synthesis but not on p42 MAPK activation. Overexpression of NLK1 by injection of 5 ng of mRNA accelerates progesterone-induced oocyte maturation by enhancing Cyclin B1 protein synthesis through the translational activation of its mRNA, in accordance with precocious phosphorylation of Pumilio1 (Pum1), Pumilio2 (Pum2), and cytoplasmic polyadenylation element-binding protein (CPEB), key regulators of the translational control of mRNAs stored in oocytes. A higher level of NLK1 expression by injection of 50 ng of mRNA induces Pum1/Pum2/CPEB phosphorylation, CPEB degradation, Cyclin B1 protein synthesis, and oocyte maturation in the absence of progesterone. NLK1 phosphorylates Pum1, Pum2, and CPEB in vitro. These findings provide the first evidence for the involvement of NLK1 in Xenopus oocyte maturation. We suggest that NLK1 acts as a kinase downstream of Mos and catalyzes phosphorylation of Pum1, Pum2, and CPEB to regulate the translation of mRNAs, including Cyclin B1 mRNA, stored in oocytes.
• Biochemical Characterization of Pumilio1 and Pumilio2 in Xenopus Oocytes
  Ryoma Ota, Tomoya Kotani, Masakane Yamashita
  JOURNAL OF BIOLOGICAL CHEMISTRY, 286, 4, 2853, 2863, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Precise control of the timing of translational activation of dormant mRNAs stored in oocytes is required for normal progression of oocyte maturation. We previously showed that Pumilio1 (Pum1) is specifically involved in the translational control of cyclin B1 mRNA during Xenopus oocyte maturation, in cooperation with cytoplasmic polyadenylation element-binding protein (CPEB). It was reported that another Pumilio, Pumilio2 (Pum2), exists in Xenopus oocytes and that this protein regulates the translation of RINGO mRNA, together with Deleted in Azoospermia-like protein (DAZL). In this study, we characterized Pum1 and Pum2 biochemically by using newly produced antibodies that discriminate between them. Pum1 and Pum2 are bound to several key proteins involved in translational control of dormant mRNAs, including CPEB and DAZL, in immature oocytes. However, Pum1 and Pum2 themselves have no physical interaction. Injection of anti-Pum1 or anti-Pum2 antibody accelerated CPEB phosphorylation, cyclin B1 translation, and oocyte maturation. Pum1 phosphorylation coincides with the dissociation of CPEB from Pum1 and the translational activation of cyclin B1 mRNA, a target of Pum1, whereas Pum2 phosphorylation occurred at timing earlier than that for Pum1. Some, but not all, of cyclin B1 mRNAs release the deadenylase PARN during oocyte maturation, whereas Pum1 remains associated with the mRNA. On the basis of these findings, we discuss the functions of Pum1 and Pum2 in translational control of mRNAs during oocyte maturation.
• Transgenic zebrafish reveals novel mechanisms of translational control of cyclin B1 mRNA in oocytes
  Kyota Yasuda, Tomoya Kotani, Ryoma Ota, Masakane Yamashita
  DEVELOPMENTAL BIOLOGY, 348, 1, 76, 86, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2010年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, Temporal translation control of localized mRNA is crucial for regulating various cellular and developmental processes. However, little is known about the mechanisms of temporal translation control of localized mRNA due to the limitation in technology. cyclin B1 mRNA at the animal polar cytoplasm of immature zebrafish oocytes is translationally repressed, and its activation is temporally regulated during maturation. Mechanisms of cyclin B1 translation in oocytes were analyzed using transgenic zebrafish in which reporter mRNAs are produced from transgenes introduced into the genome through transcription in the nucleus followed by transport to the cytoplasm, as in endogenous mRNAs. Real-time imaging of the site and timing of translation showed that mRNAs containing the full-length cyclin B1 sequence precisely mimic the localization and translation of endogenous cyclin B1 mRNA. However, mRNAs containing cyclin B1 3' untranslated region but lacking open reading frame (ORF) underwent abnormal localization and precocious translational activation, indicating the significance of the ORF in translational control of cyclin B1 mRNA. Our genetic approach in combination with real-time imaging of the translation site and timing provides a novel insight into the mechanisms of temporal control of translation. (C) 2010 Elsevier Inc. All rights reserved.
• Visualization of spatially and temporally controlled translation by transgenesis
  Tomoya Kotani, Kyota Yasuda, Ryoma Ota, Masakane Yamashita
  GENES & GENETIC SYSTEMS, 85, 6, 416, 416, GENETICS SOC JAPAN, 2010年12月, ［査読有り］
  英語
• Mys Protein Regulates Protein Kinase A Activity by Interacting with Regulatory Type I alpha Subunit during Vertebrate Development
  Tomoya Kotani, Shun-ichiro Iemura, Tohru Natsume, Koichi Kawakami, Masakane Yamashita
  JOURNAL OF BIOLOGICAL CHEMISTRY, 285, 7, 5106, 5116, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, During embryonic development, protein kinase A (PKA) plays a key role in cell fate specification by antagonizing the Hedgehog (Hh) signaling pathway. However, the mechanism by which PKA activity is regulated remains unknown. Here we show that the Misty somites (Mys) protein regulates the level of PKA activity during embryonic development in zebrafish. We isolate PKA regulatory type I alpha subunit (Prkar1a) as a protein interacting with Mys by pulldown assay in HEK293 cells followed by mass spectrometry analysis. We show an interaction between endogenous Mys and Prkar1a in the zebrafish embryo. Mys binds to Prkar1a in its C terminus region, termed PRB domain, and activates PKA in vitro. Conversely, knockdown of Mys in zebrafish embryos results in reduction in PKA activity. We also show that knockdown of Mys induces ectopic activation of Hh target genes in the eyes, neural tube, and somites downstream of Smoothened, a protein essential for transduction of Hh signaling activity. The altered patterning of gene expression is rescued by activation of PKA. Together, our results reveal a molecular mechanism of regulation of PKA activity that is dependent on a protein-protein interaction and demonstrate that PKA activity regulated by Mys is indispensable for negative regulation of the Hh signaling pathway in Hh-responsive cells.
• Introduction of a Foreign Gene into Zebrafish and Medaka Cells Using Adenoviral Vectors
  Toshihiro Kawasaki, Kenji Saito, Kaoru Mitsui, Masahito Ikawa, Masakane Yamashita, Yoshihito Taniguchi, Shunichi Takeda, Kohnosuke Mitani, Noriyoshi Sakai
  ZEBRAFISH, 6, 3, 253, 258, MARY ANN LIEBERT INC, 2009年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Viral vectors represent a tractable system that can efficiently introduce an exogenous gene into different target cells and are thus a potentially powerful genetic manipulation tool. In our current study, we investigated the infection efficiency of mammalian virus vectors, adenoviruses ( Ads), adeno-associated viruses, and lentiviruses to the Sertoli cell line and the newly established cell line from a single embryo in zebrafish. Among the viral vectors tested, Ads showed the highest infection efficiency of 10(7)-10(8) green fluorescent protein-transducing units (gtu)/mL in zebrafish cells. In addition, the adenoviral vector was also infected at 10(5) gtu/mL in the medaka testicular somatic cell line that was established from the testes of p53-deficient mutant. Further, we found that Ads could successfully infect cultured male zebrafish germ cells. Our results thus indicate that the adenoviral vector could be used as a chromosomally nonintegrating vector system in zebrafish.
• Mys protein antagonizes Hedgehog signaling through activation of PKA during embryonic development
  Tomoya Kotani, Shun-ichiro Iemura, Tohru Natsume, Koichi Kawakami, Masakane Yamashita
  MECHANISMS OF DEVELOPMENT, 126, S324, S324, ELSEVIER SCIENCE BV, 2009年08月, ［査読有り］
  英語
• Artificial Fertilization by Intracytoplasmic Sperm Injection in a Teleost Fish, the Medaka (Oryzias latipes)
  Satoshi Otani, Toshiharu Iwai, Shingo Nakahata, Chiharu Sakai, Masakane Yamashita
  BIOLOGY OF REPRODUCTION, 80, 1, 175, 183, SOC STUDY REPRODUCTION, 2009年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.
• Production of Transgenic Medaka Fish Carrying Fluorescent Nuclei and Chromosomes
  Toshiharu Iwai, Shinya Inoue, Tomoya Kotani, Masakane Yamashita
  ZOOLOGICAL SCIENCE, 26, 1, 9, 16, ZOOLOGICAL SOC JAPAN, 2009年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, As with zebrafish, attention has focused on the teleost medaka Oryzias latipes as an experimental animal representative of non-mammalian vertebrates in various fields of biological science. To enable real-time analyses of the dynamics of nuclei and chromosomes in living medaka cells, we produced a transgenic medaka expressing a fusion protein between histone H2B and green fluorescent protein (GFP) under the control of a cytomegalovirus (CMV) promoter. Since the nuclei and chromosomes of transgenic medaka cells are labeled with GFP, their morphological changes can be instantly monitored throughout the mitotic cell cycle progression under a fluorescent microscope without any fixation and staining of samples. However, GFP-labeling of nuclei and chromosomes is not successful during early embryonic development until zygotic expression begins and during the meiotic cell cycle progression, because the CMV promoter does not work in these stages. In addition, histone H2B-GFP fusion proteins are expressed in an organ-specific manner; strong and ubiquitous expression occurs in cells comprising the gut and fin, whereas the expression is restricted to certain types of cells in the liver and brain. These findings suggest that the CMV-driven expression of the histone H2B-GFP transgene is modified depending on the integration site of the transgene in the genome. Nevertheless, easy and precise monitoring of cytological changes in nuclei and chromosomes in the majority of mitotic cells by using the transgenic medaka will greatly contribute to a better understanding of control mechanisms of nuclear and chromosomal behaviors in vertebrate cells.
• Possible Involvement of Phosphatidylinositol 3-Kinase, but Not Protein Kinase B or Glycogen Synthase Kinase 3 beta, in Progesterone-Induced Oocyte Maturation in the Japanese Brown Frog, Rana japonica
  Ryoma Ota, Kaori Suwa, Tomoya Kotani, Koich Mita, Masakane Yamashita
  ZOOLOGICAL SCIENCE, 25, 7, 773, 781, ZOOLOGICAL SOC JAPAN, 2008年07月, ［査読有り］
  英語, 研究論文（学術雑誌）, It is known that amphibian oocytes undergo maturation through the formation and activation of maturation-promoting factor (MPF) in response to stimulation by the maturation-inducing hormone progesterone; however, the signal transduction pathway that links the hormonal stimulation on the oocyte surface to the activation of MPF in the oocyte cytoplasm remains a mystery. The aim of this study was to investigate whether the signal transduction mediated by phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB), and glycogen synthase kinase 3 beta (GSK3 beta) is involved in progesterone-induced oocyte maturation in the Japanese brown frog, Rana japonica. Inhibitors of PI3K, wortmannin and LY294002, inhibited progesterone-stimulated germinal vesicle breakdown (GVBD) only when the oocytes were treated at the initial phase of maturation, suggesting that PI3K is involved in the progesterone-induced maturation of Rana oocytes. However, we also obtained results suggesting that PKB and GSK3 beta are not involved in Rana oocyte maturation. A constitutively active PKB expressed in the oocytes failed to induce GVBD in the absence of progesterone despite its high level of kinase activity. A Myc-tagged PKB expressed in the oocytes (used to monitor endogenous PKB activity) was not activated in the process of progesterone-induced oocyte maturation. Overexpression of GSK3 beta, which is reported to retard the progress of Xenopus oocyte maturation, had no effect on Rana oocyte maturation. On the basis of these results, we propose that PI3K is involved in the initiation of Rana oocyte maturation, but that neither PKB nor GSK3 beta is a component of the PI3K signal transduction pathway.
• Reproductive role of attaching filaments on the egg envelope in Xenopoecilus sarasinorum (Adrianichthidae, Teleostei)
  Takashi Iwamatsu, Hirokuni Kobayashi, Masahiro Sato, Masakane Yamashita
  JOURNAL OF MORPHOLOGY, 269, 6, 745, 750, WILEY-BLACKWELL, 2008年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Eggs of Xenopoecilus sarasinorum possess two distinct types of filaments on the surface of the egg envelope (chorion), long, attaching filaments restricted to the vegetal pole and weak, nonattaching filaments around the animal pole (micropyle). Both types are formed during oogenesis. After mature eggs were spawned through the urogenital pore, they were fertilized and hung in an abdominal concavity of the female. Oviposition never took place in the presence of embryos in the concavity because of the retardation of oogenesis. The loosely tangled tips of the attaching filaments that are retained within the ovarian cavity plug the urogenital pore by forming a hard complex with the epithelial cells. Into this plug structure that fuses with the inner wall of the urogenital pore, capillaries are provided. Within 5 days after the initiation of hatching, this plug degenerates and is released from the urogenital pore. Thus, in female X. sarasinorum, the reproductive cycle seems to be regulated by the physiological function of the plug structure formed by the attaching filaments in response to the presence of developing embryos.
• Regulation of oocyte maturation in fish
  Yoshitaka Nagahama, Masakane Yamashita
  DEVELOPMENT GROWTH & DIFFERENTIATION, 50, S195, S219, BLACKWELL PUBLISHING, 2008年06月, ［査読有り］
  英語, A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three-step induction process involving gonadotropin (LH), maturation-inducing hormone (MIH), and maturation-promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17 alpha, 20 beta-dihydroxy-4-pregnen-3-one, 17 alpha, 20 beta-DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two-cell type model), is required for the synthesis of 17 alpha,20 beta-DR The dramatic increase in the capacity of postvitellogenic follicles to produce 17 alpha,20 beta-DP in response to LH is correlated with decreases in P450c17 (P450c17-1) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17(P450c17-II) and 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) mRNA. Transcription factors such as Ad4BP/SF-1, Fox12, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G-protein-coupled membrane-bound MIH receptors has been shown to mediate non-genomic actions of 17 alpha, 20 beta-DR The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin-dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH2 terminus at lysine 57.
• Identification of an olfactory imprinting-related gene in the lacustrine sockeye salmon, Oncorhynchus nerka
  Hiroshi Hino, Toshiharu Iwai, Masakane Yamashita, Hiroshi Ueda
  AQUACULTURE, 273, 2-3, 200, 208, ELSEVIER SCIENCE BV, 2007年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, The olfactory system of salmonids is essential for imprinting and the subsequent homing migration. Olfactory imprinting-related genes were identified in the olfactory system of 1- and 3-year-old lacustrine sockeye salmon (Oncorhynchus nerka) using a subtractive hybridization technique of representational difference analysis (cDNA-RDA). We have obtained a partial clone from a subtractive cDNA library of 1-year-old fish that contains a 756 bp open reading frame coding for a putative protein with 252 amino acid residues named the Sockeye salmon Olfactory system Imprinting related Gene (SOIG). By northern hybridization, the SOIG mRNA was only expressed in the olfactory epithelium and not in other tissues. In situ hybridization showed that the expression of SOIG mRNA was observed in the olfactory receptor cells and basal cells of the olfactory epithelium. This suggests that SOIG could have specific and important roles in olfactory system. (C) 2007 Elsevier B.V. All rights reserved.
• Mechanism of degradation of CPEB during Xenopus oocyte maturation
  Daiki Setoyama, Masakane Yamashita, Noriyuki Sagata
  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 104, 46, 18001, 18006, NATL ACAD SCIENCES, 2007年11月, ［査読有り］
  英語, 研究論文（学術雑誌）, CPEB, a cytoplasmic polyadenylation element-binding protein, plays an important role in translational control of maternal mRNAs in early animal development. During Xenopus oocyte maturation, CPEB undergoes a Cdc2-mediated phosphorylation- and ubiquitin-dependent degradation that is required for proper entry into meiosis II. However, the precise mechanism of CPEB degradation, including the identity of the responsible E3 ubiquitin ligase, is not known. Here, we show that the SCF beta-TrCP E3 ubiquitin ligase complex targets CPEB for degradation during Xenopus oocyte maturation. beta-TrCP, the F-box protein of SCF beta-TrCP, specifically binds to a sequence (190)TSGFSS(195) (termed here the TSG motif) of CPEB, thereby targeting CPEB for degradation. P-TrCP binding depends on phosphorylation of Thr-190, Ser-191, and Ser-195 in the TSG motif. Among these residues, Ser-191 is phosphorylated by the Polo-like kinase Plx1, which binds CPEB at a specific Thr-125 residue prephosphorylated by Cdc2. Finally, Cdc2-mediated phosphorylation of other multiple Ser residues, previously implicated in CPEB degradation, is required for both Thr-125 phosphorylation and beta-TrCP binding, presumably causing conformational changes of CPEB. We propose that Cdc2 and PIx1 sequentially phosphorylate CPEB and target it for SCF beta-TrCP-dependent degradation in Xenopus oocytes. We suggest that many other proteins carrying the TSG-like motif may be targeted by SCF beta-TrCP.
• Chromosome elimination in the interspecific hybrid medaka between Oryzias latipes and O-hubbsi
  C. Sakai, F. Konno, O. Nakano, T. Iwai, T. Yokota, J. Lee, C. Nishida-Umehara, A. Kuroiwa, Y. Matsuda, M. Yamashita
  CHROMOSOME RESEARCH, 15, 6, 697, 709, SPRINGER, 2007年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, An interspecific hybrid medaka (rice fish) between Oryzias latipes and O. hubbsi is embryonically lethal. To gain an insight into the cellular and molecular mechanisms that cause the abnormalities occurring in the hybrid medaka, we investigated the behavior of chromosomes and the expression patterns of proteins responsible for the chromosome behavior. The number of chromosomes in the hybrid embryos gradually decreased to nearly half, since abnormal cell division with lagging chromosomes at anaphase eliminated the chromosomes from the cells. The chromosome lagging occurred at the first cleavage and continued throughout embryogenesis even after the midblastula transition. Fluorescent in-situ hybridization analyses revealed that the chromosomes derived from O. hubbsi are preferentially eliminated in both O. latipes-hubbsi and O. hubbsi-latipes embryos. Whole-mount immunocytochemical analyses using antibodies against alpha-tubulin, gamma-tubulin, inner centromere protein, Cdc20, Mad2, phospho-histone H3 and cohesin subunits (SMC1 alpha, SMC3 and Rad21) showed that the expression patterns of these proteins in the hybrid embryos are similar to those in the wild-type embryos, except for phospho-histone H3. Phospho-histone H3 present on chromosomes at metaphase was lost from normally separated chromosomes at anaphase, whereas it still existed on lagging chromosomes at anaphase, indicating that the lagging chromosomes remain in the metaphase state even when the cell has proceeded to the anaphase state. On the basis of these findings, we discuss the cellular and molecular mechanisms of chromosome elimination in the hybrid medaka.
• Characterization and expression of a maternal axolotl Cyclin B1 during oogenesis and early development
  Helene Pelczar, Stephane Caulet, Catherine Thibier, Genevieve Aubet, Robert Poulhe, Ioanna Vallianou, Masakane Yamashita, Yannick Andeol
  DEVELOPMENT GROWTH & DIFFERENTIATION, 49, 5, 407, 419, BLACKWELL PUBLISHING, 2007年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, The M phase promoting factor (MPF) is a dimer composed of a catalytic Cdk1 subunit and a Cyclin B regulatory subunit. We have characterized a cDNA containing the entire coding sequence of an axolotl Cyclin B1 protein that is able to promote MPF activity when added to a fraction from prophase I oocytes that contains monomeric Cdk1. The axolotl cyclin B1 gene is expressed as a maternal mRNA in oocytes and early embryos. Its poly(A) tail length increases in metaphase II oocytes and then decreases regularly during the first embryonic cell cycles. Endogenous Cyclin B1 protein is first expressed during oocyte meiotic maturation. Its level oscillates after fertilization and is coordinated to the phosphorylation level of tyrosine 15 residue of Cdk1 (pTyr(15)), with both maxima preceding each cell division. As expected, when translated into microinjected oocytes, axolotl Cyclin B1 induces the resumption of meiosis. In electrically activated unfertilized eggs (UFE), Cyclin B1 and pTyr(15) cyclic accumulations are observed with kinetics different from those of the early embryonic cycles, The axolotl embryo and UFE provide interesting in vivo comparative models for studying events controlling Cyclin B1 regulation during development.
• Identification and expression analysis of rainbow trout pumilio-1 and pumilio-2
  Ikuo Kurisaki, Toshiharu Iwai, Masakane Yamashita, Miwako Kobayashi, Etsuro Ito, Ichiro Matsuoka
  CELL AND TISSUE RESEARCH, 327, 1, 33, 42, SPRINGER, 2007年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.
• Regulatory mechanisms of oocyte maturation and ovulation
  Kaori Suwa, Masakane Yamashita
  The Fish Oocyte: From Basic Studies to Biotechnological Applications, 323, 347, Springer Netherlands, 2007年, ［査読有り］
  英語, 論文集(書籍)内論文, Oocytes are produced in the ovaries by the entry of mitotically proliferating oogonia into meiosis. In vertebrates, including fishes, oocytes stop their meiotic cell cycle at prophase I, during which they grow by the accumulation of substances, such as yolk and mRNAs, necessary for early embryonic development (see Chapters 1-3). These prophase I-Arrested oocytes are still immature and unable to be fertilized even when they reach their fully grown stage. Hormonal stimulation allows the oocytes to resume meiosis and proceed to metaphase II, where meiosis stops again. These metaphase II-Arrested oocytes are now mature (called ova or eggs) and can undergo embryonic development when fertilized. The process from prophase I arrest to metaphase II arrest is termed oocyte maturation in the field of biological sciences or final oocyte maturation in the field of fisheries sciences (Figure 1). © 2007 Springer Netherlands.
• Structural components of the synaptonemal complex, SYCP1 and SYCP3, in the medaka fish Oryzias latipes
  Toshiharu Iwai, Atsushi Yoshii, Takehiro Yokota, Chiharu Sakai, Hiroshi Hori, Akira Kanamori, Masakane Yamashita
  EXPERIMENTAL CELL RESEARCH, 312, 13, 2528, 2537, ELSEVIER INC, 2006年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, The synaptonemal complex (SC) is a meiosis-specific structure essential for synapsis of homologous chromosomes. For the first time in any non-mammalian vertebrates, we have isolated cDNA clones encoding two structural components of the SC, SYCP1 and SYCP3, in the medaka, and investigated their protein expression during gametogenesis. As in the case of mammals, medaka SYCP1 and SYCP3 are expressed solely in meiotically dividing cells. in the diplotene stage, SYCP1 is diminished at desynaptic regions of chromosomes and completely lost on the chromosomes at later stages. SYCP3 is localized along the arm and centromeric regions of chromosomes at metaphase 1, and its existence on the whole chromosomes persists up to anaphase I, a situation different from that reported in the mouse, in which SYCP3 is confined to the centromeric regions but lost on the arm regions at metaphase I. Thus, the expression patterns of SC components are different in mammals and fish despite the resemblance in morphological structure of the SC, suggesting divergence in the function of the SC in regulation of meiosis-specific chromosomal behavior. Since the antibody against medaka SYCP3 is cross-reactive to other fishes, it should be generally useful for a meiosis-specific marker in fish germ cells. (c) 2006 Elsevier Inc. All rights reserved.
• Loss of Rec8 from chromosome arm and centromere region is required for homologous chromosome separation and sister chromatid separation, respectively, in mammalian meiosis
  Jibak Lee, Konosuke Okada, Sugako Ogushi, Takashi Miyano, Masashi Miyake, Masakane Yamashita
  CELL CYCLE, 5, 13, 1448, 1455, LANDES BIOSCIENCE, 2006年07月, ［査読有り］
  英語, 研究論文（学術雑誌）, Chromosome separation in meiosis I is different from those in mitosis and meiosis II in that homologs separate from each other in the former while sisters do so in the latter. We show here that meiosis-specific cohesin subunit Rec8 in mouse oocytes shows essentially the same pattern of localization to those reported in yeasts and mammalian spermatocytes; Rec8 along chromosome arm ( armRec8) is lost at the metaphase I-to-anaphase I transition, although centromeric Rec8 (cenRec8) is maintained until the onset of anaphase II. Suppression of the loss of armRec8 by microinjection of anti-Rec8 antibody into the oocytes inhibits homolog separation but not the first polar body emission ( cytokinesis). Similarly, the injection of anti-Rec8 antibody into metaphase II oocytes prevents sister separation in anaphase II after oocyte activation. These data demonstrate that the loss of armRec8 and cenRec8 is required for separation of homologs and sisters, respectively, but both are not required for other late mitotic events such as spindle elongation and cytokinesis in mouse oocytes. Further, by using some inhibitors for spindle assembly, proteasome and Topoisomerase II and overexpression of Securin, we propose that loss of armRec8 ( homolog separation) and cytokinesis are suppressed until anaphase I by Securin whose destruction is regulated by spindle checkpoint-proteasome pathway, and that Topoisomerase II is required for homolog separation independently from such pathway.
• Requirement of new protein synthesis of a transcription factor for memory consolidation: Paradoxical changes in mRNA and protein levels of C/EBP
  D Hatakeyama, H Sadamoto, T Watanabe, A Wagatsuma, S Kobayashi, Y Fujito, M Yamashita, M Sakakibara, G Kemenes, E Ito
  JOURNAL OF MOLECULAR BIOLOGY, 356, 3, 569, 577, ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD, 2006年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, Some specific transcription factors are essential for memory consolidation across species. However, it is still unclear whether only the activation of constitutively expressed forms of these conserved transcription factors is involved in memory consolidation or their de novo synthesis also occurs after learning. This question has remained unanswered partly because of the lack of an efficient method for the determination of copy numbers of particular mRNAs in single neurons, which allows the detection of new transcription at the cellular level. Here we applied a newly developed protocol of single-cell quantitative real-time polymerase chain reaction (qRT-PCR) to single neurons playing an important role in associative learning. Specifically, we examined the changes in the mRNA and protein expression levels of a highly conserved transcription factor, CCAAT/enhancer binding protein (C/EBP), in the paired B2 motoneurons of the pond snail Lymnaea stagnalis. These buccal neurons are involved in the motor control of feeding behavior, with a potentially important role in conditioned taste aversion (CTA). Single-cell qRT-PCR revealed a significant decrease in LymC/EBP mRNA copy numbers in the B2 motoneurons during memory consolidation after CTA training. By contrast, isoelectric focusing and immunoblotting of extracts of the buccal ganglia showed that translation and phosphorylation levels of LymC/EBP significantly increased during memory consolidation. The C/EBP-like immunoreactivity in the B2 motoneurons, which are the major immunopositive component in the buccal ganglia, also significantly increased during memory consolidation, suggesting that the main source of increase in the level of protein in the buccal ganglia are the B2 motoneurons. Thus, early memory consolidation after CTA learning in L. stagnalis involves both the rapid synthesis and phosphorylation of LymC/EBP as well as the rapid breakdown of LymC/EBP mRNA in the neural network controlling feeding, suggesting that all of these processes play a role in the function of C/EBP in memory consolidation. (c) 2005 Elsevier Ltd. All rights reserved.
• Sex reversal in medaka treated in vitro with 17 alpha-methyldihydrotestosterone during oocyte maturation
  T Iwamatsu, H Kobayashi, M Yamashita
  DEVELOPMENT GROWTH & DIFFERENTIATION, 48, 1, 59, 64, BLACKWELL PUBLISHING, 2006年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Using the S-rR strain of the medaka Oryzias latipes, we examined the effect of a non-aromatizable androgen on sex determination. Intrafollicular immature oocytes isolated before breakdown of the germinal vesicle were incubated in the presence of 17 alpha-methyldihydrotestosterone (MDHT) for about 10 h during their maturational period. At the end of incubation, mature oocytes were rinsed and then artificially inseminated in regular saline. The fertilized eggs were then allowed to develop in tap water, and the fry were reared on a regular powdered diet until adulthood. Sex reversal of female to male was observed in a manner dependent on the dose of MDHT. In the solvent control group in which intrafollicular oocytes were matured in medium containing no exogenous androgen, no sex reversal was observed. The present finding, that the sex of medakas can be reversed by a single in vitro exposure of immature oocytes to androgen during the preovulatory period, suggests the existence in the oocyte of a sex determinant sensitive to sex steroids. This method for controlling the sex of eggs before fertilization may establish sex-determined eggs as potent material for investigating the mechanism of sex determination in the medaka.
• Inhibition of mitogen activated protein kinase activity induces parthenogenetic activation and increases cyclin B accumulation during porcine oocyte maturation
  Takakura, I, K Naito, N Iwamori, M Yamashita, S Kume, H Tojo
  JOURNAL OF REPRODUCTION AND DEVELOPMENT, 51, 5, 617, 626, SOCIETY REPRODUCTION & DEVELOPMENT-SRD, 2005年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.
• Overexpression of truncated gamma-tubulins disrupts mitotic aster formation in Xenopus oocyte extracts
  T Kotani, M Yamashita
  BIOCHEMICAL JOURNAL, 389, 3, 611, 617, PORTLAND PRESS LTD, 2005年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Mechanisms of spindle pole formation rely on minus-end-directed motor proteins. gamma-Tubulin is present at the centre of poles, but its function during pole formation is completely unknown. To address the role of gamma-tubulin in spindle pole formation, we over-expressed GFP (green fluorescent protein)-fused gamma-tubulin (gamma-TuGFP) in Xenopus oocytes and produced self-assembled mitotic asters in the oocyte extracts. gamma-Tu-GFP associated with endogenous alpha-, beta- and gamma-tubulin, suggesting that it acts in the same manner as that of endogenous gamma-tubulin. During the process of aster formation, gamma-Tu-GFP aggregated as dots on microtubutes, and then the dots were translocated to the centre of the aster along microtubules in a manner dependent on cytoplasmic dynein activity. Inhibition of the function of gamma-tubulin by an anti-gamma-tubulin antibody resulted in failure of microtubule organization into asters. This defect was restored by overexpression of gamma-Tu-GFP, confirming the necessity of gamma-tubulin in microtubule recruitment for aster formation. We also examined the effects of truncated gamma-tubulin mutants, which are difficult to solubly express in other systems, on aster formation. The middle part of gamma-tubulin caused abnormal organization of microtubulles in which minus ends of microtubules were not tethered, but dispersed. An N-terminus-deleted mutant prevented recruitment of microtubules into asters, similar to the effect of the anti-gamma-tubulin antibody. The results indicate possible roles of gamma-tubutin in spindle pole formation and show that the system developed in the present study could be useful for analysing roles of many proteins that are difficult to solubly express.
• Behavior of gamma-tubulin during spindle formation in Xenopus oocytes: requirement of cytoplasmic dynein-dependent translocation
  T Kotani, M Yamashita
  ZYGOTE, 13, 3, 219, 226, CAMBRIDGE UNIV PRESS, 2005年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. gamma-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of gamma-tubulin during spindle formation in oocytes. We first observed sequential localization of gamma-tubulin during spindle formation in Xenopus oocytes. gamma-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and gamma-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, gamma-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of gamma-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with gamma-tubulin for the assembly of microtubules, but not gamma-tubulin, was phosphorylated during oocyte maturation. These results suggest that gamma-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with gamma-tubulin, but not gamma-tubulin itself, is important for microtubule reorganization.
• Changes in the expression and localization of cohesin subunits during meiosis in a non-mammalian vertebrate, the medaka fish
  T Iwai, J Lee, A Yoshii, T Yokota, K Mita, M Yamashita
  GENE EXPRESSION PATTERNS, 4, 5, 495, 504, ELSEVIER SCIENCE BV, 2004年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Until the onset of anaphase, sister chromatids are bound to each other by a multi-subunit protein complex called cohesin. Since chromosomes in meiosis behave differently from those in mitosis, the cohesion and separation of homologous chromosomes and sister chromatids in meiosis are thought to be regulated by meiosis-specific cohesin subunits. Actually, several meiosis-specific cohesin subunits, including Rec8, STAG3 and SMC1beta, are known to exist in mammals; however, there are no reports of meiosis-specific cohesin subunits in other vertebrates. To investigate the protein expression and localization of cohesin subunits during meiosis in non-mammalian species, we isolated cDNA clones encoding SMC1alpha, SMC1beta, SMC3 and Rad21 in the medaka and produced antibodies against recombinant proteins. Medaka SMC1beta was expressed solely in gonads, while SMC1alpha, SMC3 and Rad21 were also expressed in other organs and in cultured cells. SMC1beta forms a complex with SMC3 but not with Rad21, in contrast to SMC1alpha, which forms complexes with both SMC3 and Rad21. SMC1alpha and Rad21 were mainly expressed in mitotically dividing cells in the testis (somatic cells and spermatogonia), although their weak expression was detected in pre-leptotene spermatocytes. SMC1beta was expressed in spermatogonia and spermatocytes. SMC1beta was localized along the chromosomal arms as well as on the centromeres in meiotic prophase 1, and its existence on the chromosomes persisted up to metaphase H, a situation different from that reported in the mouse, in which SMC1beta is lost from the chromosome arms in late pachytene despite its universal presence in vertebrates. (C) 2004 Elsevier B.V. All rights reserved.
• Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection
  T Kuroda, K Naito, K Sugiura, M Yamashita, Takakura, I, H Tojo
  BIOLOGY OF REPRODUCTION, 70, 1, 154, 159, SOC STUDY REPRODUCTION, 2004年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CR1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.
• Pyrenoid formation associated with the cell cycle in the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)
  C Nagasato, S Yoshikawa, M Yamashita, H Kawai, T Motomura
  JOURNAL OF PHYCOLOGY, 39, 6, 1172, 1180, BLACKWELL PUBLISHING INC, 2003年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti-RUBISCO antibody in vegetative cells of gametophytes. From mid-S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.
• Experimental hybridization among Oryzias species. II. Karyogamy and abnormality of chromosome separation in the cleavage of interspecific hybrids between Oryzias latipes and O-javanicus
  T Iwamatsu, H Kobayashi, M Yamashita, Y Shibata, A Yusa
  ZOOLOGICAL SCIENCE, 20, 11, 1381, 1387, ZOOLOGICAL SOC JAPAN, 2003年11月, ［査読有り］
  英語, 研究論文（学術雑誌）, In interspecific hybridization between Oryzias latipes and O. javanicus, all hybrid embryos failed to develop and died before hatching. Cytological examination of fertilization and early development was performed to discover the cause of lethal development. When O. latipes eggs were inseminated by sperm of O. javanicus, the cortical reaction was induced normally. Chromosomal material in the fertilized eggs was visualized using the DNA-specific fluorochrome Hoechst. The spermatozoon was capable of penetrating into the egg cytoplasm through the micropyle, and the sperm nucleus transformed to the male pronucleus. The female pronucleus that formed after extrusion of the second polar body migrated towards the male pronucleus. The female and the male pronuclei underwent DNA synthesis and encountered each other in the center of the blastodisc, fused with one another and formed a zygote nucleus before breakdown of the nuclear envelope. Metaphase chromosomes with electron dense chromatin regions were abnormally divided into each blastomere in cleavage. The abnormally separating chromatin masses were also labeled by BrdU. The abnormal separation resulting in partial loss of fragmented chromatin might be a cause of abortive development in the interspecific hybrids between O. latipes and O. javanicus.
• Involvement of Xenopus Pumilio in the translational regulation that is specific to cyclin B1 mRNA during oocyte maturation
  S Nakahata, T Kotani, K Mita, T Kawasaki, Y Katsu, Y Nagahama, M Yamashita
  MECHANISMS OF DEVELOPMENT, 120, 8, 865, 880, ELSEVIER SCIENCE BV, 2003年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation. (C) 2003 Elsevier Ireland Ltd. All rights reserved.
• Temporally and spatially selective loss of Rec8 protein from meiotic chromosomes during mammalian meiosis
  J Lee, T Iwai, T Yokota, M Yamashita
  JOURNAL OF CELL SCIENCE, 116, 13, 2781, 2790, COMPANY OF BIOLOGISTS LTD, 2003年07月, ［査読有り］
  英語, 研究論文（学術雑誌）, Sister chromatid cohesion is maintained from DNA replication to metaphase-to-anaphase transition by multisubunit protein complexes called cohesin, which include at least four proteins, SMC1alpha, SMC3, Rad21. and either SA1 or SA2, in mammalian somatic cells. We report here the first evidence of the involvement of Rec8 protein, a mammalian homolog of yeast Rec8p, in meiosis-specific chromosome behavior in mammals. In immunoblotting and immunohistochemical analysis using specific antibodies against mouse Rec8, we found that Rec8 was expressed in the testis but not in the kidney or liver; more precisely, it was expressed in spermatocytes and spermatids but not in spermatogonia or other somatic cells. We also found that Rec8 is present in both phosphorylated and dephosphorylated states in vivo. Immunoprecipitation analyses revealed that Rec8 associates with other cohesin proteins, SMC1beta (meiosis-specific protein) and SMC3 and with a component of synaptonemal complexes, SCP3, but not with SMC1alpha. In meiotic chromosome spreads, Rec8 was localized along the axial/lateral elements of the synaptonemal complexes in meiotic prophase from the leptotene to diplotene stages. At later stages, diakinesis and metaphase I, Rec8 was localized along the interstitial axes of chromosomes, including both centromere and arm regions of chromosomes. However, concomitantly with separation of homologous chromosomes in anaphase I, Rec8 was no longer detected along the arm regions, while it persisted on centromere regions up to metaphase II. In anaphase II, the centromeric signals were diminished. We propose from these results that mammalian Rec8 protein, in association with SMC3 and SMC1beta but not SMC1alpha, is involved in meiosis-specific chromosome behavior, and that homologous chromosome separation is triggered by selective loss of Rec8 from chromosome arms in meiosis I, while sister chromatid cohesion is maintained until metaphase II/anaphase II transition by centromeric Rec8 during mammalian meiosis.
• Maturational factors as indicators of egg quality in Japanese eel, Anguilla japonica
  D. H.S. Shin, H. Matsubara, S. Kaneko, T. Kotani, M. Yamashita, S. Adachi, K. Yamauchi
  Fish Physiology and Biochemistry, 28, 1-4, 519, 520, 2003年, ［査読有り］
  英語, 研究論文（学術雑誌）, In order to identify maturational factors that can serve as indicators of egg quality in artificially matured Japanese eel, Anguilla japonica maturation-promoting factor (MPF
  consists of cdc2 and cyclin B) related proteins and chromosomes were examined. Mitogen-activated protein kinase (MAPK
  44 kDa band) and cdc2 (35 kDa and 34 kDa band) were detected. Chromosomes aligned on a vertical spindle appeared normal
  however, chromosomes on a horizontal spindle and dispersed chromosomes seemed unusual. © 2004 Kluwer Academic Publishers.
• Discrimination of the roles of MPF and MAP kinase in morphological changes that occur during oocyte maturation
  T Kotani, M Yamashita
  DEVELOPMENTAL BIOLOGY, 252, 2, 271, 286, ACADEMIC PRESS INC ELSEVIER SCIENCE, 2002年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, Maturing amphibian oocytes undergo drastic morphological changes, including germinal vesicle breakdown (GVBD), chromosome condensation, and spindle formation in response to progesterone. Two kinases, maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK), are involved in these changes, but their precise roles are unknown. Unlike in Xenopus oocytes, discrimination of the functions of MAPK and MPF in Rana oocytes is easy owing to the lack of pre-MPF. We investigated the roles of these kinases by careful observations of chromosomes and microtubules in Rana oocytes. MPF and MAPK activities were manipulated by treatment with progesterone, c-mos mRNA, or cyclin B mRNA in combination with MAPK kinase inhibitors. Activation of one kinase without activation of the other induced only limited events; GVBD was induced by MPF without MAPK, and reorganization of microtubules at GVBD was induced by MAPK without MPF, but other events were not induced. In contrast, coactivation of MPF and MARK by injection of c-mos and cyclin B mRNA promoted almost all of the morphological changes that occur during maturation without progesterone, indicating that these are controlled by cooperation of MPF and MAPK. The results revealed the functions of MAPK and MPF in each process of sequential morphological changes during oocyte maturation. (C) 2002 Elsevier Science (USA).
• Localization of gamma-tubulin and cyclin B during early cleavage in physiologically polyspermic newt eggs
  Y Iwao, T Murakawa, J Yamaguchi, M Yamashita
  DEVELOPMENT GROWTH & DIFFERENTIATION, 44, 6, 489, 499, BLACKWELL PUBLISHING ASIA, 2002年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, To understand the mechanism of the very slow block to polyspermy in physiologically polyspermic eggs of the newt Cynops pyrrhogaster, we used confocal laser microscopy to determine the distribution of gamma-tubulin and cyclin B1 in fertilized eggs. More gamma-tubulin was localized in the animal hemisphere than in the vegetal. The centrosomes of the principal sperm nucleus and the zygote nucleus had much accumulated gamma-tubulin, but little gamma-tubulin was associated with the centrosomes of the accessory sperm nuclei. These results are consistent with observations that the largest sperm aster is associated with the principal sperm nucleus. More cyclin B1 appeared in the animal hemisphere than in the vegetal at the end of interphase. The zygote nucleus had much accumulated cyclin B1, but little cyclin B1 was associated with the accessory sperm nuclei. Cyclin B1 disappeared earlier around the zygote nucleus at metaphase than around the accessory sperm nuclei. These findings correspond well with the earlier entry and exit into metaphase in the zygote nucleus than in the accessory sperm nuclei in newt eggs, supporting our maturation-promoting factor (MPF) model that accounts for the mechanism of nuclear degeneration in physiologically polyspermic eggs. Cyclin B1 began to accumulate in the nucleus during interphase in synchronous cleavage, and its greatest expression was in the centrosomes and the nucleus at prometaphase.
• Zebrafish DAZ-like protein controls translation via the sequence 'GUUC'
  S Maegawa, M Yamashita, K Yasuda, K Inoue
  GENES TO CELLS, 7, 9, 971, 984, BLACKWELL PUBLISHING LTD, 2002年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Background: In many species, DAZ homologous genes encode RNA-binding proteins containing two conserved motifs, namely the RNA-recognition motif (RRM) and the DAZ motif. Genetic analysis and gene disruption studies have demonstrated that DAZ family proteins play important roles in gametogenesis. However, little is known about the biochemical functions of DAZ family proteins.
  Results: Using in vitro selection and UV-crosslinking experiments, we identified the sequence 'GUUC' as the target RNA sequence of zebrafish DAZ-like protein (zDAZL). In transfection experiments, zDAZL protein activated translation in a manner dependent on the binding sequence in the 3'UTR of the Drosophila twine gene or zDazl gene. Moreover, it is highly likely that the zDAZL protein associates with polysomes through the DAZ motif in vivo , and that the association with polysomes is indispensable for translational activation.
  Conclusions: This is the first report that the DAZ family protein directly promotes the translation of the target mRNAs in vertebrates. This study provides important insights into the molecular mechanisms underlying the post-transcriptional regulation of DAZ family proteins in gametogenesis.
• Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs
  T Iwamatsu, Y Shibata, O Hara, M Yamashita, S Ikegami
  DEVELOPMENT GROWTH & DIFFERENTIATION, 44, 4, 293, 302, BLACKWELL PUBLISHING ASIA, 2002年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes . Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.
• Analyses of mRNA expression patterns of cohesin Subunits rad21 and rec8 in mice: Germ cell-specific expression of rec8 mRNA in both male and female mice
  J Lee, T Yokota, M Yamashita
  ZOOLOGICAL SCIENCE, 19, 5, 539, 544, ZOOLOGICAL SOC JAPAN, 2002年05月, ［査読有り］
  英語, 研究論文（学術雑誌）, A multisubunit protein complex called cohesin is required for the cohesion between sister chromatids in both mitosis and meiosis in yeast. We investigate here the mRNA expression patterns of mouse homologues of the yeast mitotic cohesin rad21 and the meiotic cohesin rec8 in various organs, with special attention to their expression in gonads. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that, in contrast to the ubiquitous expression of rad21 mRNA in all of the organs examined, rec8 was expressed only in the gonads. We conducted in situ hybridization analysis to identify the cells that express rad21 and rec8 mRNAs in the gonads. In the testis, rad21 mRNA was expressed in somatic cells and spermatogonia but not in spermatocytes, and conversely, rec8 mRNA was expressed in spermatocytes but not in spermatogonia or somatic cells. Spermatids expressed rad21 and rec8 mRNAs simultaneously. In the ovary, rad21 rnRNA was detected in all of the ovarian cells including germ cells and somatic cells, whereas rec8 mRNA was detected only in oocytes. Unlike the widespread expression of rad21 gene, therefore, the gene expression of rec8 is strictly confined to spermatocytes and spermatids in male mouse and oocytes in female mouse. The restricted expression pattern of rec8 mRNA implies its essential role in meiosis in both sexes of mammals, as has been reported in yeast. We also discuss the cooperative functions of Rad21 and Rec8 on the basis of the finding that their mRNAs are coexpressed in oocytes and spermatids.
• PCNA protein expression during spermatogenesis of the Japanese eel (Anguilla japonica)
  C Miura, T Miura, M Yamashita
  ZOOLOGICAL SCIENCE, 19, 1, 87, 91, ZOOLOGICAL SOC JAPAN, 2002年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Spermatogenesis can be initiated by a single injection of human chorionic gonadotropin (hCG) into the cultivated Japanese eel, which produces only spermatogonia in the testis. To isolate the genes responsible for regulating spermatogenesis, we performed a differential mRNA display using poly (A) RNA extracted from the testes at different time points after hCG injection. Among several cDNA clones, the expression of which was initiated before the onset of meiosis, one clone has high homology with the proliferating cell nuclear antigen (PCNA). In this study, we investigated the protein expression of eel PCNA and found for the first time in any species that two forms (32-kDa and 36-kDa) of PCNA are present in the testis. Although the 36-kDa form existed in both the testis and spleen, the 32-kDa form was specifically expressed in the testis. In contrast to the appearance of 36-kDa PCNA 1 day after the hCG treatment, the 32-kDa PCNA appeared only 9 days after the hCG treatment, at which time active spermatogonial proliferation occurred in the testis. Both the 32- and 36-kDa forms were recognized by antibodies raised against different epitopes of PCNA, and their N-terminal amino acid sequences were identical. The 36-kDa form, but not the 32-kDa form, was recognized by antibodies against phosphoamino acids. These results suggest that the two PCNA proteins are the same molecule with different chemical modifications, including phosphorylation. We discuss the roles of these two forms of PCNA in the spermatogenesis of the Japanese eel.
• Requirement of cyclin B2, but not cyclin B1, for bipolar spindle formation in frog (Rana japonica) oocytes
  T Kotani, N Yoshida, K Mita, M Yamashita
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 59, 2, 199, 208, WILEY-LISS, 2001年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cyclin B, the regulatory subunit of maturation-promoting factor (MPF), com prises several subtypes that are presumed to confer different functions on MPF although no direct evidence has been provided to date. To clarify the difference in the roles of cyclins B1 and B2, we used frog (Rana japonica) oocytes in which MPF is formed only after progesterone stimulation because it is possible to produce oocytes containing either cyclin B1-MPF or cyclin B2-MPF by antisense RNA-mediated translational inhibition of each mRNA. Using this advantage, we investigated the functions of cyclins B1 and B2 and obtained the following results: (a) oocytes synthesizing cyclin B2-MPF underwent meiosis I and II with formation of a bipolar spindle at each metaphase; (b) oocytes synthesizing cyclin B1-MPF formed a monopolar spindle at metaphase I and extruded an abnormal polar body; and (c) both oocytes underwent germinal vesicle breakdown (GVBD) and chromosome condensation. Immunocytochemical observations also revealed continuous localization of cyclin B2 on the spindle during meiosis. These results provide evidence of the requirement of cyclin B2, but not cyclin B1, for organizing the bipolar spindle, though either cyclin B1 or B2 is redundant for inducing GVBD and chromosome condensation. Mol. Reprod. Dev. 59:199-208, 2001. (C) 2001 Wiley-Liss, Inc.
• Biochemical identification of Xenopus pumilio as a sequence-specific cyclin B1 mRNA-binding protein that physically interacts with a nanos homolog, Xcat-2, and a cytoplasmic polyadenylation element-binding protein
  S Nakahata, Y Katsu, K Mita, K Inoue, Y Nagahama, M Yamashita
  JOURNAL OF BIOLOGICAL CHEMISTRY, 276, 24, 20945, 20953, AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2001年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes, This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stared in oocytes, This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.
• Immunological detection and characterization of poly(A) polymerase, poly(A)-binding protein and cytoplasmic polyadenylation element-binding protein in goldfish and Xenopus oocytes
  S Nakahata, K Mita, Y Katsu, Y Nagahama, M Yamashita
  ZOOLOGICAL SCIENCE, 18, 3, 337, 343, ZOOLOGICAL SOC JAPAN, 2001年04月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cytoplasmic polyadenylation regulates translational activation of dormant cyclin B1 mRNA stored in immature oocytes, a process required for the initiation of oocyte maturation in goldfish and Xenopus. As a first step towards understanding the molecular mechanisms of translational activation of cyclin B1 during oocyte maturation, we have isolated cDNA clones encoding proteins involved in cytoplasmic polyadenylation and produced specific antibodies against recombinant proteins. These include poly(A) polymerase (PAP), poly(A)-binding protein (PABP) and cytoplasmic polyadenylation element-binding protein (CPEB). Monoclonal antibodies raised against goldfish PAP recognized several forms of PAP in goldfish and Xenopus oocytes. Besides ordinary PAPs with high molecular weight (ca. 100 kDa), the antibodies also detected those with low molecular weight (ca. 40 kDa) that are present specifically in the cytoplasm, raising new players that might be responsible for cytoplasmic polyadenylation. An antibody against goldfish PABP showed for the first time in Xenopus oocytes the protein expression of PABPII, another PABP distinct from the well-characterized PABPI. Monoclonal antibodies raised against Xenopus CPEB recognized both unphosphorylated 62-kDa and phosphorylated 64-kDa forms but did not cross-react with goldfish CPEB, which was specifically detected by anti-goldfish CPEB monoclonal antibodies produced previously. The cDNAs, recombinant proteins and antibodies produced in this study are expected to provide useful tools for investigating the regulatory mechanisms of cyclin B1 translation during oocyte maturation in goldfish and Xenopus.
• Identification and molecular cloning of germinal vesicle lamin B3 in goldfish (Carassius auratus) oocytes
  A Yamaguchi, M Yamashita, M Yoshikuni, Y Nagahama
  EUROPEAN JOURNAL OF BIOCHEMISTRY, 268, 4, 932, 939, BLACKWELL SCIENCE LTD, 2001年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, A bulk isolation method was developed to collect a large number of germinal vesicles (GV) from postvitellogenic oocytes of goldfish (Carassius auratus). Using this method, we obtained GV lamina which are resistant to high salt and nonionic detergent. 2D PACE revealed that the goldfish GV lamina contained several spots with similar molecular masses (67 kDa) and slightly different neutral isoelectro-focusing values (pI 5.8-6.2). After trypsin digestion and extraction of a major spot (pI 6.1), the peptide was subjected to RP-HPLC and sequenced. A homology search identified this spot as a nuclear lamin. A cDNA encoding goldfish GV lamin was isolated by RT-PCR using degenerate primers designed from the GV lamin tryptic peptide sequence. The goldfish GV lamin cDNA encodes a predicted molecular mass of 67 455 Da with a pI of 5.84. Phylogenetic analysis indicates that the amino-acid sequence is most similar to Xenopus oocyte-specific GV lamin B3, but differs from somatic lamins (A, B-1 or B-2). In contrast to somatic lamins, neither goldfish nor Xenopus GV lamin contain conserved phosphorylation sites for nuclear transport, except the nuclear localization sequence. Therefore, we conclude that the goldfish oocyte GV is mainly comprised of GV-type lamin (the homolog of Xenopus lamin B3).
• Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation
  T Kondo, T Kotani, M Yamashita
  DEVELOPMENTAL BIOLOGY, 229, 2, 421, 431, ACADEMIC PRESS INC, 2001年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17 alpha ,20 beta -dihydroxy-4-pregnen-3-one (17 alpha ,20 beta -DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17 alpha ,20 beta -DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17 alpha ,20 beta -DP, cytochalasin B (10 mug/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17 alpha ,20 beta -DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 mug/ml) of cytochalasin B inhibited 17 alpha ,20 beta -DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation. (C) 2001 Academic Press.
• Toward modeling of a general mechanism of MPF formation during oocyte maturation in vertebrates
  M Yamashita
  ZOOLOGICAL SCIENCE, 17, 7, 841, 851, ZOOLOGICAL SOC JAPAN, 2000年09月, ［査読有り］
  英語, Vertebrate oocytes arrested at meiotic prophase I are still immature even when they reach their fully grown stage. For the acquisition of fertilizability, the fully grown oocytes must undergo oocyte maturation, during which the meiosis is released from prophase I arrest and stops again at metaphase II until inseminated. The resumption of meiosis from prophase 1 to metaphase II is induced by the action of maturation-promoting factor (MPF). The molecular structure of MPF is common to all eukaryotes, but the mechanisms of its formation and activation vary in cell types and in species. In this review, I summarize the mechanisms of MPF formation during oocyte maturation in two amphibian species, Xenopus laevis and Rana japonica. In Xenopus, immature oocytes are equipped with inactive MPF (pre-MPF) sufficient for completing oocyte maturation, and therefore only its activation is required after hormonal stimulation. In contrast, immature Rana oocytes contain no pre-MPF. Therefore, MPF must be newly formed during oocyte maturation, as is the case in fishes and other amphibians (toads and newts). The mechanism of MPF formation in Xenopus therefore seems to be different to that in other lower vertebrates. However, 1 wish to propose a new mechanism of MPF formation that might be appropriate for all species of lower vertebrates, inclusive of Xenopus, based on the novel concept that pre-MPF is an artifact produced under unnatural conditions and is not an essential molecule for initiating oocyte maturation, in contradiction to the generally believed notion that pre-MPF is actively stocked for the initiation of oocyte maturation. The standpoint that pre-MPF is not indispensable for initiating oocyte maturation might provide a new insight into the mechanism of MPF formation during oocyte maturation, allowing us to model a comprehensive mechanism applicable to all vertebrates, including mammals.
• The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish
  H Kajiura-Kobayashi, N Yoshida, N Sagata, M Yamashita, Y Nagahama
  DEVELOPMENT GENES AND EVOLUTION, 210, 8-9, 416, 425, SPRINGER-VERLAG, 2000年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF. The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17 alpha,20 beta-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential For initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes.
• Production of diploid eggs through premeiotic endomitosis in the hybrid medaka between Oryzias latipes and O-curvinotus
  Y Shimizu, N Shibata, M Sakaizumi, M Yamashita
  ZOOLOGICAL SCIENCE, 17, 7, 951, 958, ZOOLOGICAL SOC JAPAN, 2000年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, A hybrid medaka between Oryzias latipes and O. curvinotus spawns diploid eggs. We examined the cytological mechanisms of diploid egg formation in this hybrid. Oocytes in the hybrid passed through the first and second meiotic divisions and excluded two polar bodies, associated with reduction of the DNA content in oocytes. Each germinal vesicle in vitellogenic oocytes of the hybrid had 48 chromosomes with bivalent chiasmata, precisely twice the number of chromosomes in normal oocytes. These results suggest that before meiosis the chromosomes are doubled by mitosis without cytokinesis, that is, endomitosis, and that the resulting tetraploid oogonia undergo normal meiosis to produce diploid eggs. Except for a few vitellogenic oocytes that are probably derived from endomitotic oogonia, most oocytes were arrested at the zygotene stage in the hybrid ovary, suggesting the existence of checkpoint control that ensures the pairing of homologous chromosomes at prophase I, a situation differing from that in the male in which the checkpoint is at metaphase I. A sac-like structure was characteristic of the hybrid ovary. Although this structure was observed only in the hybrid, it may be a native component of the medaka ovary but difficult to observe because of its deformed outer structure due to enlargement of the inside oocytes. The present study confirms that this hybrid medaka provides a useful experimental system for investigations into the mechanisms of oogenesis and basic architecture of the ovary, which are difficult to analyze by using normal medaka.
• Maturation/M-phase promoting factor: A regulator of aging in porcine oocytes
  K Kikuchi, K Naito, J Noguchi, A Shimada, H Kaneko, M Yamashita, F Aoki, H Tojo, Y Toyoda
  BIOLOGY OF REPRODUCTION, 63, 3, 715, 722, SOC STUDY REPRODUCTION, 2000年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Deterioration in the quality of mammalian oocytes during the metaphase-II arrest period is well known as "oocyte aging." Oocytes in which aging has occurred are called aged oocytes, and these oocytes show enhanced activation and higher fragmentation rates after parthenogenetic activation. Previously we showed that porcine aged oocytes had low maturation/M-phase promoting factor (MPF) activity, and we suggested that this low MPF activity contributed at least in part to the aging phenomena. In the present study, we examined the relationship between MPF activity and these aging phenomena by artificially regulating MPF activity in porcine metaphase-II-arrested oocytes. Since we have shown recently that aged porcine oocytes contain abundant phosphorylated inactive MPF, so-called pre-MPF, we used vanadate and caffeine, which affect the phosphorylation status of MPF, to regulate MPF activity. Incubation of 48-h-matured oocytes with vanadate for 1 h increased the phosphorylation of MPF and decreased MPF activity. The parthenogenetic activation and fragmentation rates were significantly increased compared with those of control oocytes. Conversely, treatment of 72-h-cultured aged oocytes with caffeine (last 10 h of culture) decreased the level of pre-MPF and elevated MPF activity. These oocytes revealed significantly lower parthenogenetic activation rates and a lower percentage of fragmentation than did untreated aged oocytes. These results indicate that not only the increased ability for parthenogenetic activation but also the increased fragmentation rate observed in porcine aged oocytes may be attributable in part to the gradual decrease in MPF activity during prolonged culture. Control of MPF phosphorylation with these agents may allow for some degree of manipulation of oocyte aging.
• Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica
  N Yoshida, K Mita, M Yamashita
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 57, 1, 88, 98, WILEY-LISS, 2000年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation. (C) 2000 Wiley-Liss, Inc.
• Expression of gelatinases A and B in the ovary of the medaka fish Oryzias latipes
  H Matsui, K Ogiwara, R Ohkura, M Yamashita, T Takahashi
  EUROPEAN JOURNAL OF BIOCHEMISTRY, 267, 15, 4658, 4667, WILEY-BLACKWELL, 2000年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, 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.
• Non-dependence of cyclin E/Cdk2 kinase activity on the initiation of oocyte maturation in goldfish
  N Yoshida, M Yamashita
  DEVELOPMENT GROWTH & DIFFERENTIATION, 42, 3, 285, 294, BLACKWELL SCIENCE ASIA, 2000年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cdk2 kinase activity increases during oocyte maturation but neither cyclin A nor B is associated with Cdk2 in mature oocytes in goldfish. As a potential Cdk2 partner in meiosis, a cyclin E homolog was isolated from a goldfish oocyte cDNA library. A monoclonal antibody was raised against bacterially produced full-length goldfish cyclin E. Both cyclin E and Cdk2 were already present in immature oocytes and their protein levels did not change remarkably during oocyte maturation. Cyclin E formed a complex mainly with Cdk2 just at the time of germinal vesicle breakdown (GVBD) in association with the increase in Cdk2 kinase activity, although a fraction of cyclin E bound to Cdk(s) other than Cdk2 and Cdc2. Ectopic activation of cyclin E/Cdk2 by the injection of cyclin E messenger RNA (mRNA) into immature oocytes did not induce maturation-promoting factor (MPF) activation and GVBD. Furthermore, inhibition of cyclin E/Cdk2 kinase activity by the injection of p21(SDI1) into the oocytes treated with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one had no effect on MPF activation and GVBD. These results indicate that cyclin E/Cdk2 kinase activity is insufficient and unnecessary for initiating goldfish oocyte maturation.
• Expression of Xenopus Daz-like protein during gametogenesis and embryogenesis
  K Mita, M Yamashita
  MECHANISMS OF DEVELOPMENT, 94, 1-2, 251, 255, ELSEVIER SCIENCE BV, 2000年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Using a monoclonal antibody raised against Xenopus Daz-like protein (Xdazl), we showed that Xdazl is present in all stages of male and female germ cells except mature spermatozoa. Xdazl is not localized to any specific regions in early-stage embryos, in contrast to the strict localization of its mRNA in the germ plasm. Xdazl disappears after gastrulation but reappears in the primordial germ cells situated at the genital ridge. This is the first detailed report on the protein expression of a Daz-like gene during gametogenesis and embryogenesis in Xenopus, showing the difference in expression patterns of its mRNA and protein. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
• Comparative study of the molecular mechanisms of oocyte maturation in amphibians
  N Yoshida, K Mita, M Yamashita
  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 126, 2, 189, 197, PERGAMON-ELSEVIER SCIENCE LTD, 2000年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, Maturation-promoting factor (MPF), a complex of Cdc2 and cyclin B, is the final inducer of oocyte maturation. Its activity is controlled by inhibitory phosphorylation of Cdc2 on Tyr15/Thr14 and activating phosphorylation on Thr161. Full-grown immature oocytes of the African clawed frog Xenopus laevis contain inactive MPF (pre-MPF) that comprises cyclin B-bound Cdc2 phosphorylated on Tyr15/Thr14 and Thr161. The synthesis of Mos, but not cyclin B, after stimulation by the maturation-inducing steroid progesterone, is believed to be necessary for initiating Xenopus oocyte maturation through Tyr15/Thr14 dephosphorylation of pre-MPF. In contrast, amphibians other than Xenopus (and also fishes) employ a different mechanism. Full-grown immature oocytes of these species contain monomeric Cdc2 but not cyclin B. MPF is formed after hormonal stimulation by binding of the newly produced cyclin B to the pre-existing Cdc2 and is immediately activated through Thr161 phosphorylation. Mos/MAP kinase is neither necessary nor sufficient for initiating maturation in fishes and amphibians except for Xenopus. We propose a new model of MPF formation and activation during oocyte maturation that is applicable to all amphibians (as well as fishes), based on a novel concept that pre-MPF is an artificial molecule that is not essential for inducing oocyte maturation. (C) 2000 Elsevier Science Inc. All rights reserved.
• Differential expression of cyclins B1 and B2 during medaka (Oryzias latipes) spermatogenesis
  K Mita, T Ohbayashi, K Tomita, Y Shimizu, T Kondo, M Yamashita
  ZOOLOGICAL SCIENCE, 17, 3, 365, 374, ZOOLOGICAL SOC JAPAN, 2000年04月, ［査読有り］
  英語, 研究論文（学術雑誌）, The Cdc2-cyclin B complex (named the M-phase-promoting factor, MPF) is well known to be a key regulator of G2-M transition in both mitosis and meiosis. However, MPF may have functions other than the cell cycle regulation, since its activity is detectable in post-mitotic (or post-meiotic) non-dividing cells. Cyclin B comprises several subtypes, but their functional differences are still unknown. Despite the established function of MPF during oocyte maturation, its role during spermatogenesis, where spermatogenic cells undergo drastic morphological changes after meiosis, remains to be elucidated. To address these issues, we have isolated cDNA clones encoding cyclins B1 and B2 from medaka testis and raised polyclonal antibodies against their products. Using these as probes, we examined the expression patterns of cyclins B1 and B2 in medaka testis at both mRNA and protein levels. Cyclin B1 and B2 mRNAs were expressed in all stages of spermatogenic cells except for spermatozoa, although the expression levels varied according to the spermatogenic stages. Cyclin B1 protein was expressed only in spermatogonia and spermatocytes at prophase and metaphase with a transient disappearance at anaphase. On the other hand, cyclin B2 protein was continuously expressed throughout spermatogenesis, even in spermatogonia and spermatocytes at anaphase and in post-meiotic spermatids and spermatozoa. The difference in their expression patterns suggests that cyclins B1 and B2 have distinct roles in medaka spermatogenesis; i.e., cyclin B1 controls the meiotic cell cycle, whereas cyclin B2 is involved in process(es) other than meiosis.
• Developmental study of anatomical substrate for conditioned taste aversion in Lymnaea stagnalis
  H Sadamoto, M Yamanaka, D Hatakeyama, H Nakamura, S Kojima, M Yamashita, E Ito
  ZOOLOGICAL SCIENCE, 17, 2, 141, 148, ZOOLOGICAL SOC JAPAN, 2000年03月, ［査読有り］
  英語, 研究論文（学術雑誌）, The pond snail, Lymnaea stagnalis, is a useful preparation for analyzing the commonality between development and learning. To promote this analysis, the anatomical substrate should be provided upon which learning is superposed during development. Because we previously demonstrated that L. stagnalis change their ability of conditioned taste aversion (CTA) as a long-term memory from veliconcha embryos to immatures, we examined in the present study the numbers of cells and the volume of the buccal and cerebral ganglia in the snails at the critical developmental stages. The buccal and cerebral ganglia include the majority of neurons involved in the CTA. We found that the numbers of cells in these ganglia are almost saturated in the immatures, but the volumes of these ganglia still increase from the immatures to the adults. These results suggested that most of the cells indispensable to the CTA emerge at the immature stage, but that individual cells in the ganglia continue to enlarge even in adulthood. Furthermore, the key neuron for the CTA was found to mature at the immature stage. The present study provided the anatomical substrate upon the long-term CTA, by which snails can eat safe food in a wide territory.
• Requirement of protamine for maintaining nuclear condensation of medaka (Oryzias latipes) spermatozoa shed into water but not for promoting nuclear condensation during spermatogenesis
  Y Shimizu, K Mita, M Tamura, K Onitake, M Yamashita
  INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY, 44, 2, 195, 199, UNIV BASQUE COUNTRY PRESS, 2000年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, Protamine is an arginine-rich basic protein found in the sperm nuclei of many vertebrates, but its actual roles in spermatozoa remain to be elucidated. In this study, we investigated the physiological roles of protamine by examining protamine-less spermatozoa produced in vitro in the presence of the transcriptional inhibitor actinomycin D. Even under inhibited transcription, medaka spermatocytes underwent meiosis and differentiated into spermatozoa with a condensed nucleus and an elongated flagellum. Using a newly produced anti-medaka protamine antibody, we confirmed the absence of protamine protein in the spermatozoa differentiated in the presence of actinomycin D. These findings clearly indicate that sperm nuclear condensation in medaka is independent of protamine. Since medaka spermatozoa are shed into water upon natural fertilization, we also investigated the roles of protamine by comparing the differences between the nuclear morphology of protamine-equipped and protamine-less spermatozoa immersed in water. The nuclei without protamine more rapidly swelled than did those with protamine and completely broke down within 10 min, whereas more than 80% of the sperm nuclei with protamine resisted the disruption under similar conditions. These findings strongly suggest that a physiological role of protamine in medaka spermatozoa is to protect the ejaculated spermatozoa against the disruption by low osmotic pressure until arrival at the eggs for successful fertilization.
• Phosphorylation of mitogen-activated protein kinase cascade during early embryo development in the mouse
  N Iwamori, K Naito, K Sugiura, H Kagii, M Yamashita, S Ohashi, S Goto, K Yamanouchi, H Tojo
  REPRODUCTION FERTILITY AND DEVELOPMENT, 12, 3-4, 209, 214, C S I R O PUBLISHING, 2000年, ［査読有り］
  英語, 研究論文（学術雑誌）, The mitogen-activated protein kinase (MAPK) cascade is one of the most important signal transduction pathways that regulate the cell cycle in somatic cells. The present study examined the phosphorylation states of components in the MAPK cascade, Raf-1, MEK-1, and extracellular signal regulated kinases (ERKs), which are activated by mitogens, throughout early mouse embryo development and in cultured somatic cells generally. In somatic cells, Raf-1 and MEK-1 were phosphorylated at M-phase and dephosphorylated during interphase. ERKs were not phosphorylated at any stage during the cell cycle. These results were similar to previous findings for the first and second cell cycles of early mouse embryos. In contrast, after the four-cell stage, not only ERKs, but also Raf-1 and MEK-1, were not phosphorylated at any stage during the cell cycle in mouse early embryos. These results suggest that the MAPK cascade in mouse embryos is regulated by the same mechanism as in somatic cells before the two-cell stage, and that regulation is changed to an embryo-specific mechanism after the four-cell stage.
• Translational regulation of cyclin B mRNA by 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone) during oocyte maturation in a teleost fish, the goldfish (Carassius auratus)
  Y Katsu, M Yamashita, Y Nagahama
  MOLECULAR AND CELLULAR ENDOCRINOLOGY, 158, 1-2, 79, 85, ELSEVIER SCI IRELAND LTD, 1999年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, 17 alpha,20 beta-Dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP) was identified as maturation-inducing hormone (MIH) in several teleost fishes. In goldfish (Carassius auratus), 17 alpha,20 beta-DP induces oocyte maturation by stimulating the de novo synthesis of cyclin B, a regulatory subunit of maturation-promoting factor (MPF). In this study, we examined the control mechanisms of 17 alpha,20 beta-DP-induced de novo synthesis of cyclin B protein in oocytes, which is a prerequisite step for MPF activation during oocyte maturation in goldfish. Cycloheximide-treated oocytes failed to undergo meiotic maturation in response to 17 alpha,20 beta-DP; in this group neither cyclin B nor 34-kDa active cdc2 was detectable in oocytes. In contrast, oocytes exposed to actinomycin D plus 17 alpha,20 beta-DP or 17 alpha,20 beta-DP underwent maturation; in these groups both cyclin B and 34-kDa cdc were present. Northern blotting showed that cyclin B mRNA is present in both immature and mature oocytes.-Sequence analysis revealed that goldfish cyclin B mRNA contains four copies of cytoplasmic polyadenylation element (CPE)-like motifs in the 3' noncoding region, suggesting that the initiation-of cyclin B synthesis during oocyte maturation may be controlled by the elongation of poly (A) tail. We then examined the polyadenylation state of cyclin B mRNA during 17 alpha,20 beta-DP-induced oocyte maturation by means of a PCR poly (A) test, and found that cyclin B mRNA is polyadenylated during oocyte maturation. Polyadenylation of cyclin B mRNA occurred at the same time of germinal vesicle breakdown. Furthermore, cordycepin, an inhibitor of poly (A) addition of mRNA, prevented 17 alpha,20 beta-DP-induced oocyte maturation. These findings suggest that,in goldfish oocytes, the synthesis of cyclin B protein is under translational control and that cytoplasmic 3' poly(A) elongation is involved in 17 alpha,20 beta-DP-induced translation of cyclin B mRNA. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
• cDNA cloning of a stage-specific gene expressed during HCG-induced spermatogenesis in the Japanese eel
  C Miura, T Miura, N Kudo, M Yamashita, K Yamauchi
  DEVELOPMENT GROWTH & DIFFERENTIATION, 41, 4, 463, 471, BLACKWELL SCIENCE ASIA, 1999年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)(+) RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin PB subunit, was expressed in the initial phase of spermatogenesis (1-6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames. 3-18 days (two clones), 6-18 days (one clone) and 9-18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full-length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12-15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. it has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG-induced spermatogenesis in the eel.
• Studies on fertilization of the teleost. II. Nuclear behavior and changes in histone H1 kinase
  T Iwamatsu, Y Shibata, M Yamashita
  DEVELOPMENT GROWTH & DIFFERENTIATION, 41, 4, 473, 482, BLACKWELL SCIENCE ASIA, 1999年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, In order to understand the dynamic responses of gamete nuclei upon fertilization in the fish, Oryzias latipes, the relationship between changes in the activity of histone H1 kinase and nuclear behavior was examined during fertilization. Kinase activity rapidly decreased concomitant with the initiation of the propagative exocytosis of cortical alveoli following sperm attachment to the egg plasma membrane post-insemination (PI). Activity again increased 30min PI. Similar changes in kinase activity, migration and syngamy of pronuclei, and subsequent cleavage were observed with aphidicolin or actinomycin D treatment, except that formation of abnormal metaphase chromosomes was retarded in aphidicolin-treated zygotes. Pretreatment of unfertilized eggs with cycloheximide or 6-dimethylaminopurine (6-DMAP) caused no nuclear changes. The activity of histone H1 kinase in these eggs rapidly declined following sperm penetration and exocytosis, but did not undergo subsequent increase in the presence of these inhibitors. In these eggs with low histone H1 kinase activity, the fertilization process from sperm penetration to syngamy occurred normally, but the pronuclear membrane did not break down and the chromosomes did not condense. The present data suggest that in fish eggs, DNA replication as well as the synthesis and phosphorylation of proteins, especially cyclin B, are required for normal formation of metaphase chromosomes at the first cleavage, but not for fertilization events from sperm penetration through to nuclear migration resulting in syngamy.
• Rise of intracellular Ca2+ level causes the decrease of cyclin B1 and Mos in the newt eggs at fertilization
  S Yamamoto, M Yamashita, Y Iwao
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 53, 3, 341, 349, WILEY-LISS, 1999年07月, ［査読有り］
  英語, 研究論文（学術雑誌）, Unfertilized eggs of the newt, Cynops pyrrhogaster, are arrested at the second meiotic metaphase, with activity of the M-phase promoting factor (MPF) maintained at a high level. After fertilization, the eggs resume the cell cycle, and emit the second polar body. When the change in [Ca2+](i) in the fertilized eggs was monitored by aequorin, an early increase in [Ca2+](i) was observed 5-10 min after insemination and continued for about 30 sec. A late increase in [Ca2+](i) then occurred 10-15 min after fertilization and continued for 30-40 min. The injection of 1,2-Bis (2 aminophenoxy) ethane-N,N,N',N',-tetraacetic acid (BAPTA) into unfertilized eggs inhibited reinitiation of the cell cycle after fertilization. Western blot analysis with antibodies against cyclin B1 or Mos indicated that both cycljn B1 and Mos were present in unfertilized eggs, but both disappeared within 30 min after fertilization. Treatment with Ca2+-ionophore decreased both cyclin B1 and Mos. Chymotryptjc activity in Cynops egg extracts was not significantly increased after fertilization or activation by treatment with the Ca2+-ionophore. No change in [Ca2+](i) was observed following treatment with cycloheximide, but the amount of both cyclin B1 and Mos rapidly decreased. These results indicate that resumption of meiosis in Cynops eggs is induced by an increase in [Ca2+](i) at fertilization, which causes degradation of both cyclin B1 and Mos by inhibition of de novo synthesis of those proteins. Mel. Reprod. Dev. 53:341-349, 1999. (C) 1999 Wiley-Liss, Inc.
• Inactivation of p34(cdc2) kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro
  K Kikuchi, K Naito, J Noguchi, A Shimada, H Kaneko, M Yamashita, H Tojo, Y Toyoda
  ZYGOTE, 7, 2, 173, 179, CAMBRIDGE UNIV PRESS, 1999年05月, ［査読有り］
  英語, 研究論文（学術雑誌）, Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34(cdc2), a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34(cdc2) with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34(cdc2) and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34(cdc2), whereas that of oocyte activation is based on the degradation of cyclin B.
• Developmental changes in conditioned taste aversion in Lymnaea stagnalis
  M Yamanaka, H Sadamoto, D Hatakeyama, H Nakamura, S Kojima, T Kimura, M Yamashita, A Urano, E Ito
  ZOOLOGICAL SCIENCE, 16, 1, 9, 16, ZOOLOGICAL SOC JAPAN, 1999年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, As the first step to study relationships between development and learning in the molluscan central nervous system,we examined developmental changes in acquisition and retention of a conditioned taste aversion (CTA) in the pond snail, Lymnaea stagnalis. We found that snails developed ability of a CTA as a long-term memory through three critical stages. Embryos in veliconcha started to respond to appetitive sucrose at the first critical stage. This response was in good agreement with morphological observations that embryos at this developmental stage seemed to be physically ready to eat. However, they could not associate this appetitive stimulus (conditioned stimulus: CS) with an aversive stimulus of KCI (unconditioned stimulus: UCS). At the second critical stage, embryos just before hatching acquired the CTA, but the conditioned response did not persist. Through this stage, they may acquire learning ability to safely seek out food in an external environment. At the third critical stage, immature snails with a 10 mm shell could use a long-term memory to maintain the conditioned response. This memory persisted for at least a month, showing that now they are able to maintain a long-term memory so that they can safely eat a variety of food when they cover wide territory to search for a mate. The present findings indicate that the development of learning ability in snails, which secures acquisition of better survival ability, is coincident with the major changes in their life cycle.
• Tyrosine phosphorylation of p34(cdc2) in metaphase II-arrested pig oocytes results in pronucleus formation without chromosome segregation
  J Lee, K Hata, T Miyano, M Yamashita, YF Dai, RM Moor
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 52, 1, 107, 116, WILEY-LISS, 1999年01月, ［査読有り］
  英語, 研究論文（学術雑誌）, At the G2-M boundary, maturation-promoting factor (MPF) activation is usually induced in one or both of two ways; tyrosine dephosphorylation of p34(cdc2) Or synthesis of cyclin B according to cell type and species. At the end of M-phase, however, MPF inactivation is normally triggered only by cyclin degradation. We investigated whether tyrosine phosphorylation of p34(cdc2) can inactivate MPF and what kinds of events are induced in pig metaphase II (MII)-arrested oocytes. First, cyclin B1 in MII-arrested oocytes is degraded upon fertilization. Second, when MII oocytes were treated with vanadate, an inhibitor of tyrosine phosphatases, they were released from MII arrest, but MPF was inactivated by further tyrosine phosphorylation of p34(cdc2) rather than cyclin B1 degradation. The vanadate-induced exit from M-phase is distinct from normal M-phase exit, which is accompanied by cyclin B1 degradation; the former lacks both sister chromatid separation and second polar body emission. Vanadate itself has no inhibitory effect on chromosome segregation since calcium ionophore induced chromosome segregation in the presence of vanadate. Furthermore, when MII oocytes were treated with olomoucine, an inhibitor of cyclin-dependent kinases, they exited from MII arrest in a manner similar to vanadate-treated MII oocytes. Finally, we propose that MPF inactivation by tyrosine phosphorylation of p34(cdc2) enables MII oocytes to form an interphase nucleus, but not to segregate sister chromatid due to the absence of the mechanisms required to trigger sister chromatid separation such as anaphase-promoting complex (APC)-mediated proteolysis, on the signaling pathway from intracellular Ca2+ increase to MPF inactivation, Mol. Reprod. Dev. 52:107-116, 1999. (C) 1999 Wiley-Liss, Inc.
• Studies on fertilization in the teleost. III. The relationship between nuclear behavior and the histone H1 kinase activity in anesthetized medaka eggs
  T Iwamatsu, Y Shibata, M Kikuyama, M Yamashita
  DEVELOPMENTAL GENETICS, 25, 2, 137, 145, WILEY-LISS, 1999年, ［査読有り］
  英語, 研究論文（学術雑誌）, To investigate the mechanisms of fertilization in the teleostean egg, the relationship between the nuclear behavior and the activity of histone H1 kinase was examined in medaka, Oryzias latipes, eggs that were anesthetized at sperm penetration. Inseminated in the anesthetized state, most eggs failed to undergo the propagative waves of increase in cytoplasmic Ca2+ and exocytosis of cortical alveoli (CABD). The sperm-penetrated eggs that exhibited no or partial CABD only around the animal pole underwent a transient contraction of the cortical cytoplasm toward the animal pole region and were designated nonactivated eggs. Temporary compaction of the second meiotic metaphase (MII) chromosomes was accompanied by contractile movement of the cortical cytoplasm, but not by completion of the second meiotic division. The activity of histone H1 kinase in nonactivated eggs remained high, although ii decreased slightly concurrent with sperm penetration. Cyclin B and cdc2 levels remained unchanged as well. The nonactivated eggs began to transform the penetrated sperm nucleus into metaphase chromosomes in the cortical cytoplasm facing the inner end of micropylar canal within 20 min postinsemination (PI). Two figures of typical metaphase chromosomes were found in the animal pole area at less than or equal to 40 min PI. Chromosome condensation in nonactivated eggs was not inhibited by actinomycin D, nor was the high activity of histone H1 kinase reduced. In the presence of cycloheximide or 6-dimethylaminopurine (6-DMAP), how ever, the compact sperm nucleus and the MII chromosomes transformed to interphase nuclei without CABD or extrusion of the polar body, although the activity of histone H1 kinase remained high. These results suggest that in the fish egg, transformation of MII chromosomes to an interphase nucleus may not be caused by loss of MPF activity, but rather than by the loss of activity of a short-lived protein kinase(s), sensitive to 6-DMAP that is independent of CABD in the cascade reactions triggered by increased cytoplasmic calcium. Dev. Genet. 25:137-145, 1999. (C) 1999 Wiley-Liss, Inc.
• Molecular mechanisms of meiotic maturation and arrest in fish and amphibian oocytes
  M Yamashita
  SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY, 9, 5, 569, 579, ACADEMIC PRESS LTD, 1998年10月, ［査読有り］
  英語, Fish and amphibian oocytes provide excellent experimental systems for both biochemical and cytological analyses of regulatory mechanisms of meiotic maturation and arrest. Recent work shows that despite the adoption of common players, such as maturation-promoting factor (MPF), c-mos proto-oncogene product (Mos), and mitogen-activated protein kinase (MAPK), there is clear species-specificity in the mechanisms, probably due to the difference in the states of inactive MPF in immature oocytes. However, it has also been revealed that the mechanisms controlling meiotic maturation and arrest include ubiquitous pathways; The translational activation of mashed mRNAs encoding Mos and cyclin B for initiating maturation and the Mos-MAPK pathway for maintaining metaphase arrest.
• Either cyclin B1 or B2 is necessary and sufficient for inducing germinal vesicle breakdown during frog (Rana japonica) oocyte maturation
  J Ihara, N Yoshida, T Tanaka, T Mita, M Yamashita
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 50, 4, 499, 509, WILEY-LISS, 1998年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Oocyte maturation is finally triggered by the maturation-promoting factor (MPF), which consists of Cdc2 and cyclin B. We have cloned cDNAs encoding frog (Rana japonica) cyclins B1 and B2 and produced antibodies against their products. Using the antibodies, we investigated changes in protein states and levels of Cdc2 and cyclins B1 and B2 during oocyte maturation. In immature oocytes, all Cdc2 was a monomeric unphosphorylated inactive 35 kDa form and neither cyclin B1 nor cyclin B2 was present. Mature oocytes contained the MPF complex consisting of an active 34 kDa Cdc2 phosphorylated on threonine161 and a 49 kDa cyclin B1 or a 51 kDa cyclin B2. After progesterone stimulation, both cyclins B1 and B2 were synthesized from their stored mRNAs and bound to the preexisting 35 kDa Cdc2. The binding of Cdc2 with cyclin B and its activation probably through the phosphorylation on threonine161 occurred at almost the same time, in accordance with an electrophoretic mobility shift of Cdc2 from 35 to 34 kDa. Microinjection into immature oocytes of cyclin B1 or B2 mRNA alone, or a mixture of them, induced germinal vesicle breakdown (GVBD) with similar dose-dependence. When the translation of endogenous mRNAs of both cyclins B1 and B2 was inhibited with antisense RNAs, progesterone failed to induce GVBD in the oocytes, but the inhibition of only one of the two was unable to inhibit the puogesterone-induced GVBD. These results indicate that either cyclin BI or B2 is necessary and sufficient for inducing GVBD during Rana oocyte maturation. Mol. Reprod. Dev. 50:499-509, 1998. (C) 1998 Wiley-Liss, Inc.
• Cytogenetic mechanisms for triploid and haploid egg formation in the triploid loach Misgurnus anguillicaudatus
  QQ Zhang, K Arai, M Yamashita
  JOURNAL OF EXPERIMENTAL ZOOLOGY, 281, 6, 608, 619, WILEY-LISS, 1998年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, The triploid (diploid x tetraploid) loach Misgurnus anguillicaudatus lays large, triploid eggs and small, haploid eggs simultaneously.We conducted cytogenetic examination of gonadal cells to determine the mechanisms responsible for this unusual oogenesis. In immature ovaries of triploids, both triploid (3n = 75) and hexaploid (6n = 150) metaphases were detected. Oocytes at the pachytene stage were categorized into two types: small and large. The small oocytes gave about 50 chromosome elements, comprising 25 thick, densely stained bivalents and 25 thin, faintly stained univalents, that is, bivalent-univalent complexes. The large ones showed no fewer than 60 thick elements, considered to be 75 bivalents. Cytological examination of full-grown oocytes cultured in vitro revealed that both large and small oocytes undergo two conventional successive meiotic cycles: formation of a bipolar spindle in meiosis I and equal segregation of homologues, extrusion of the first polar body, and the appearance of a bipolar spindle in meiosis II. In the small, full-grown oocytes, some chromosomes were observed to detach from the spindle and remain in the surrounding cytoplasm, and then these chromosomes (presumably unpaired univalents) were eliminated from the meiotic event. We conclude that triploid eggs are formed by a quasinormal meiosis of the hexaploid oocytes of large size, which are generated by premeiotic endomitosis, while the haploid eggs are produced from bivalents after eliminating univalents in the meiosis of triploid oocytes of small size. (C) 1998 Wiley-Liss, Inc.
• Changes in cyclin B during oocyte maturation and early embryonic cell cycle in the newt, Cynops pyrrhogaster: Requirement of germinal vesicle for MPF activation
  Sakamoto, I, K Takahara, M Yamashita, Y Iwao
  DEVELOPMENTAL BIOLOGY, 195, 1, 60, 69, ACADEMIC PRESS INC, 1998年03月, ［査読有り］
  英語, 研究論文（学術雑誌）, When full-grown oocytes of the newt Cynops pyrrhogaster were treated with progesterone in O-R2 solution containing antibiotics, approximately 85% of the oocytes completed meiosis synchronously. Maturation-promoting factor (MTF) activity appeared just. before germinal vesicle breakdown (GVBD) and the oocytes maintained high MPF activity throughout metaphase I and metaphase II of meiosis, A slight decrease of MPF activity was observed at the first polar body Emission. The distribution of cyclin El was investigated with anti-cyclin B1 antibody. No cyclin B1 was found in the oocytes before progesterone treatment. Cyclin B1 appeared in the cortex of animal hemispheres, especially around and inside germinal vesicle just before GVBD. A large amount of cyclin B1 accumulated at metaphase I, approximately half disappeared at the first polar body emission, and then cyclin BI accumulated again at metaphase II. An inactive form of cdc2 kinase was observed in both the germinal vesicles and the oocyte cytoplasm, while an active form appeared at the M phase. No MPF was observed in the oocytes from which the germinal vesicle had been removed. A cdk7-like molecule was localized in the germinal vesicle, but nut in oocyte cytoplasm, indicating that inactive cdc2 kinase associated with cyclin El derived from cytoplasm is activated by phosphorylation In the germinal vesicle. The changes in the amount of cyclin El were synchronous with the first cell cycle after fertilization. Cyclin B1 was primarily localized in the cortex of the animal hemisphere. A shift in band mobility upon electrophoresis of cyclin B1 was observed from samples taken during the cell cycle; this shift was probably due to the protein's phosphorylation state. (C) 1998 Academic Press.
• 魚類生殖細胞の形成と成熟の制御機構
  山下 正兼, 長濱 嘉孝
  生化学, 69, 11, 1246, 1260, 日本生化学会, 1997年11月, ［査読有り］
  日本語
• Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA-binding protein
  Y Katsu, M Yamashita, Y Nagahama
  EUROPEAN JOURNAL OF BIOCHEMISTRY, 249, 3, 854, 861, SPRINGER VERLAG, 1997年11月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cyclin B is a regulatory subunit of maturation-promoting factor. In goldfish (Carassius auratus) oocytes, cyclin B is synthesized de novo after stimulation by 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (maturation-inducing hormone). In this study, we examined goldfish oocyte proteins bound to cyclin B mRNA. Using oligo(dT)-cellulose affinity chromatography and northwestern blotting analysis, we identified a 54-kDa cyclin B mRNA-binding protein (p54). Southwestern blotting analysis showed the binding of p54 to the Y box DNA element (CTGATTGGCCAA), suggesting that p54 is a Y box protein in goldfish. We isolated two cDNA clones, GFYP1 and GFYP2, the latter of which encodes a germ-cell-specific Y box protein. An antibody against a GFYP2 protein recognized p54, suggesting that p54 is identical or highly similar to GFYP2 protein. This is also supported by the finding that a recombinant GFYP2 expressed in bacteria bound to both the Y box DNA element and the goldfish cyclin B mRNA synthesized in vitro. These results suggest that p54 is a germ-cell-specific Y box protein and is a potential masking protein of cyclin B mRNA in goldfish oocytes.
• Introduction of cyclin B induces activation of the maturation-promoting factor and breakdown of germinal vesicle in growing zebrafish oocytes unresponsive to the maturation-inducing hormone
  T Kondo, T Yanagawa, N Yoshida, M Yamashita
  DEVELOPMENTAL BIOLOGY, 190, 1, 142, 152, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1997年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, When treated with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP), a natural maturation-inducing hormone in fishes, fully grown zebrafish oocytes are induced to mature via the activation of the maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). In contrast, 17 alpha,20 beta-DP is unable to induce growing (previtellogenic and vitellogenic) oocytes to mature. To know the reason growing oocytes fail to mature upon 17 alpha,20 beta-DP treatment, we investigated changes in the components of machinery responsible for MPF activation during zebrafish oogenesis. Immunoblotting experiments using monoclonal antibodies against cdc2, cyclin B, and cdk7 (an activator of cdc2) have revealed that the concentrations of cdc2 and cdk7 are almost constant during oogenesis. Cyclin B was present in mature oocytes but absent in growing and fully grown immature oocytes. These results, which are identical to those in goldfish, strongly suggest that cyclin B is synthesized from stored (masked) mRNA after 17 alpha,20 beta-DP stimulation and that its binding to the preexisting cdc2 allows cdk7 to activate MPF. Microinjection of cyclin B protein induced MPF activation and germinal vesicle breakdown in growing oocytes, as well as in fully grown oocytes, indicating that cdk7 present in growing oocytes is already active. Northern blot analysis revealed the presence of cyclin B mRNA in both previtellogenic and fully grown oocytes. These results indicate that, as in fully grown oocytes, growing oocytes are already equipped with the catalytic subunit of MPF (cdc2) and its activator (cdk7) and that the appearance of the regulatory subunit of MPF (cyclin B) is sufficient for initiating maturation. Therefore, the unresponsiveness of growing oocytes to 17 alpha,20 beta-DP is attributable to a deficiency in the processes leading to cyclin B synthesis, which include 17 alpha,20 beta-DP reception on the oocyte surface, subsequent signal transduction pathways, and unmasking the stored cyclin B mRNA. (C) 1997 Academic Press.
• Spermiogenesis without preceding meiosis in the hybrid medaka between Oryzias latipes and O-curvinotus
  Y Shimizu, N Shibata, M Yamashita
  JOURNAL OF EXPERIMENTAL ZOOLOGY, 279, 1, 102, 112, WILEY-LISS, 1997年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, In the hybrid medaka between Oryzias latipes and O. curvinotus, the testicular structure, including an efferent duct, seminiferous tubules, and testicular cysts, was similar to that of the parent fishes, indicating that gonadal development during embryogenesis proceeds normally. The hybrid fish had spermatogonia, spermatocytes, and spermatid-like cells in the cyst. Sperm-like cells with a condensed nucleus and elongated flagellum were released into an efferent duct. However, measurement of DNA contents in spermatogenic cells demonstrated that no haploid cells were produced in the hybrid testis. Cytological observations revealed that the meiotic cell cycle is arrested before reaching metaphase I. The absence of protamine mRNA, of which expression starts from the later phase of meiosis, confirms the abnormality of meiosis in the hybrid testes. Direct evidence for the absence of meiotic division in the hybrid medaka was obtained from a cell culture study of primary spermatocytes. One spermatocyte isolated from the parent medaka produced 4 spermatozoa after meiotic division, whereas one spermatocyte isolated from the hybrid differentiated into one sperm-bike cell without meiotic division on a similar time schedule to that of the parent medaka. These results indicate that despite the absence of normal meiosis, spermiogenesis proceeds an schedule, suggesting that spermiogenesis is intrinsically independent of the preceding meiosis. (C) 1997 Wiley-Liss, Inc.
• Initiation of cyclin B degradation by the 26S proteasome upon egg activation
  T Tokumoto, M Yamashita, M Tokumoto, Y Katsu, R Horiguchi, H Kajiura, Y Nagahama
  JOURNAL OF CELL BIOLOGY, 138, 6, 1313, 1322, ROCKEFELLER UNIV PRESS, 1997年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH2 terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.
• Involvement of 26S proteasome in oocyte maturation of goldfish Carassius auratus
  M Tokumoto, R Horiguchi, M Yamashita, Y Nagahama, T Tokumoto
  ZOOLOGICAL SCIENCE, 14, 2, 347, 351, ZOOLOGICAL SOC JAPAN, 1997年04月, ［査読有り］
  英語, 研究論文（学術雑誌）, Diisopropyl fluorophosphate (DFP), a serine protease inhibitor, inhibited the activity of 26S proteasome in goldfish oocytes and arrested the oocytes at two distinct stages of maturation. One was prior to the migration of germinal vesicle (GV) toward the animal pole, and the other was between the attachment of GV to the oocyte plasma membrane and GV breakdown (GVBD). The first DFP-sensitive period corresponded to the period during which the activity of proteasomes increased, but maturation-promoting factor (MPF) was still inactive. During the second DFP-sensitive period, MPF was abruptly activated, although the proteasome activity detectable in the oocyte cytosol extracted by ultracentrifugation reached the lowest level during this period. These results suggest the requirement of 26S proteasome for at least two stages of oocyte maturation, the early stage before GV migration and the later stage, including MPF activation immediately before GVBD.
• Change in intracellular Ca2+ is not involved in serotonin-induced meiosis reinitiation from the first prophase in oocytes of the marine bivalve Crassostrea gigas
  K Kyozuka, R Deguchi, N Yoshida, M Yamashita
  DEVELOPMENTAL BIOLOGY, 182, 1, 33, 41, ACADEMIC PRESS INC ELSEVIER SCIENCE, 1997年02月, ［査読有り］
  英語, 研究論文（学術雑誌）, In response to the neurohormone serotonin (5-hydroxytryptamine, 5-HT), prophase-arrested oocytes of the marine bivalve Crassostrea gigas (oyster) reinitiate meiosis, undergo germinal vesicle breakdown (GVBD), and are arrested again at metaphase I. We examined the pharmacological characteristics of 5-HT receptors and the signal transduction pathway following 5-HT stimulation in oyster oocytes. Among 5-HT agonists tested, only alpha-methyl 5-HT, a 5-HT2 agonist, induced GVBD, although it was 1000 times less sensitive than 5-HT. The rank order of the potency of 5-HT antagonists to inhibit GVBD was propranolol, cyproheptadine > metoclopramide > mianserin. These results are quite different from those reported for other mollusks, suggesting the presence of unique 5-HT receptors on oyster oocytes. Using the fluorescent Ca2+ dyes fura-2 and calcium green and the pH indicator 1-hydroxypyrene-3,6,8-trisulfonic acid, we examined changes in intracellular Ca2+ ([Ca2+](i)) and intracellular pH (pH(i)) during 5-HT-induced meiosis reinitiation. 5-HT did not trigger any changes in [Ca2+](i). However, an increase in pH(i) was observed during the 5-HT-induced meiosis reinitiation. The increased pH(i) level was rather small before GVBD and not necessary for GVBD, because lowering pH(i) by sodium acetate seawater (pH 7.0) did not prevent 5-HT-induced GVBD. Measurement of the kinase activity toward a peptide substrate specific to cdc2 demonstrated that maturation-promoting factor (MPF) was activated in accordance with the occurrence of GVBD in response to 5-HT. Therefore, it is likely that in oyster oocytes the signal transduction pathways and intracellular effecters participating in 5-HT-induced meiosis reinitiation via the activation of MPF are insensitive to [Ca2+](i) and pH(i). (C) 1997 Academic Press.
• Organization of cytoplasmic microtubules during maturation of goldfish oocytes
  JQ Jiang, M Yamashita, M Yoshikuni, S Fukada, Y Nagahama
  ZOOLOGICAL SCIENCE, 13, 6, 899, 907, ZOOLOGICAL SOC JAPAN, 1996年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, The organization of cytoplasmic microtubules during hormone-induced meiotic maturation of goldfish oocytes in vitro was examined by confocal immunofluorescence microscopy using an anti-tubulin antibody. The microtubule network was well distributed in fully grown immature oocytes. Once goldfish oocytes resumed meiotic maturation by a proposed maturation-inducing hormone of this species (17 alpha, 20 beta-dihydroxy-4-pregnen-3-one, 17 alpha, 20 beta-DP), cytoplasmic microtubules serially re-organized. Soon after the onset of the germinal vesicle (GV) migration towards the animal pole, the former microtubule network disappeared, followed by the appearance of a long perinuclear tail with high ordered microtubules extending from the vegetal surface of the GV. Incubation of fully grown immature follilces in colcemid, an inhibitor of tubulin polymerization, caused the disappearance of microtubules. However, this treatment did not prevent either the 17 alpha, 20 beta-DP-induced migration of the GV or GVBD. Coincident with the breakdown of the GV (GVBD), numerous microtubules intruded into the GV from its vegetal surface. Soon after GVBD, a disk-shaped ring consisting of microtubule asters and a small ring with a radial array of microtubules in its center was observed at the animal pole region. In mature oocytes with meiotic spindles at the animal pole surface, cytoplasmic microtubules were concentrated in a small region around the animal pole showing complicated microtubule arrays. The results presented define distinct changes in microtubule organization during the 17 alpha, 20 beta-DP-induced meiotic maturation of goldfish oocytes.
• Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states
  S Haraguchi, K Naito, S Azuma, E Sato, Y Nagahama, M Yamashita, Y Toyoda
  BIOLOGY OF REPRODUCTION, 55, 3, 598, 603, SOC STUDY REPRODUCTION, 1996年09月, ［査読有り］
  英語, 研究論文（学術雑誌）, This study demonstrated the effects of phosphate on the 2-cell block of AKR/N mouse embryos at the molecular level and focused on changes in the kinase activity and the phosphorylation state of cdc2, which is shown to regulate the cell division cycle. Removal of phosphate from the culture medium dramatically increased developmental rates to the 4-cell (91.8%) and blastocyst (42.6%) stages compared with those of embryos cultured in 1.17 mM phosphate (3.3% and 0%, respectively). The rate of development to the 4-cell stage was significantly inhibited by 0.001 mM phosphate (p < 0.05), and no morula formation was observed at 1.0 mM. The patterns of cdc2 kinase activity during the first cell cycle in AKR/N embryos were similar to those of control MCH embryos, showing the highest activity at M phase and low activity during the interphase. The phosphorylated form of cdc2 increased during the interphase, indicating that the synthesis of cyclin B and accumulation of inactive pre-maturation-promoting factor (pre-MPF) as well as abrupt dephosphorylation of cdc2 at the first cleavage correlated with the activation of cdc2 kinase. When phosphate was absent, the activation pattern of cdc2 kinase during the second cell cycle in AKR/N embryos was similar to that in the first cell cycle. On the other hand, no dephosphorylation of cdc2 was observed and the kinase activity remained at a low level until 56 h after insemination in the presence of phosphate, although an increase in phosphorylated cdc2 was observed as in the phosphate-free group. Treatment of AKR/N embryos arrested at the 2-cell stage with okadaic acid resulted in the dephosphorylation and activation of cdc2, confirming the presence of a sufficient amount of pre-MPF. These results show that phosphate has a deteriorative effect on the in vitro development of AKR/N embryos and suggest that this effect was not on the synthesis of cyclin B but on the dephosphorylation of phosphorylated cdc2.
• Hormonal induction of all stages of spermatogenesis in germ-somatic cell coculture from immature Japanese eel testis
  C Miura, T Miura, M Yamashita, K Yamauchi, Y Nagahama
  DEVELOPMENT GROWTH & DIFFERENTIATION, 38, 3, 257, 262, BLACKWELL SCIENCE, 1996年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, In cultivated male eel, spermatogonia are the only germ cells present in testis. Our previous studies using an organ culture system have shown that gonadotropin and 11-ketotestosterone (11-KT, a potent androgen in teleost fishes) can induce all stages of spermatogenesis in vitro. For detailed investigation of the control mechanisms of spermatogenesis, especially of the interaction between germ cells and testicular somatic cells during 11-KT-induced spermatogenesis in vitro, we have established a new culture system in which germ cells and somatic cells are cocultured after they are aggregated into pellets by centrifugation. Germ cells (spermatogonia) and somatic cells (mainly Sertoli cells) were isolated from immature eel testis. Coculture of the isolated germ cells and somatic cells without forming aggregation did not induce spermatogenesis, even in the presence of 11-KT. In contrast, when isolated germ cells and somatic cells were formed into pellets by centrifugation and were then cultured with 11-KT for 30 days, the entire process of spermatogenesis from premitotic spermatogonia to spermatozoa was induced. However, in the absence of 11-KT in the culture medium spermatogenesis was not induced, even when germ cell and somatic cells were aggregated. These results demonstrate that physical contact of germ cells to Sertoli cells is required for inducing spermatogenesis in response to 11-KT.
• A comparative study on stainability of preprophase bands by the PSTAIR antibody
  Y Mineyuki, H Aioi, M Yamashita, Y Nagahama
  JOURNAL OF PLANT RESEARCH, 109, 1094, 185, 192, BOTANICAL SOC JAPAN, 1996年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, The detectability of preprophase bands (PPBs) by antibody to PSTAIR sequence, which is found in cyclin-dependent kinases and is perfectly conserved in the p34(cdc2) kinase from all known sources, was compared among root tip cells of 12 plant species and cultivars. Although more than 80% of prophase cells in all species examined had PPBs of microtubules (MTs), the detectability of PPBs by anti-PSTAIR varied from 0% to 88% depend on species examined. The detectability of PPBs by the antibody to PSTAIR was as high as that by antibody to tubulin in Allium cepa, A. fistulosum and A. tuberosum. In Triticum and Pisum, the detectability varied greatly among cultivars. Only few faint PPBs could be detectable in Chrysanthemum, and no PPBs were seen in Hibiscus using anti-PSTAIR. The PSTAIR antibody recognized single (Hordeum, Triticum, Zea) or multiple (Allium, Hibiscus, Pisum) bands around 34 kDa on protein blots of root tip exracts. PPBs of anti-PSTAIR cross reactive molecules were detectable in one fourth of the prophase cells of Pisum (cv. Snack) by the conventional method. However, the detectability of PPBs in Pisum increased to 80% when the method for the PSTAIR-fluorescence was modified, suggesting that the low detectability of PPBs by anti-PSTAIR may not be due to genuine differences between species or cultivars, but may be the result of variable staining.
• CHANGES IN PHOSPHORYLATION ACTIVITIES DURING GOLDFISH AND XENOPUS OOCYTE MATURATION
  N YOSHIDA, T TANAKA, M YAMASHITA
  ZOOLOGICAL SCIENCE, 12, 5, 599, 606, ZOOLOGICAL SOC JAPAN, 1995年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, Oocyte maturation is promoted by the sequential actions of several kinases, of which MPF( a histone H1 kinase) and MAP kinase (a myelin basic protein (MBP) kinase) are known to play pivotal roles. However, other kinases responsible for inducing oocyte maturation have yet to be characterized. To identify these kinases, we examined phosphorylation activities toward 44 exogenous substrate proteins during oocyte maturation in goldfish and Xenopus. We found that 4 substrates in goldfish and 6 in Xenopus were phosphorylated and their phosphorylation states changed during oocyte maturation. Among them, only 3 substrates (histone H1, MBP and pepsin) were common to both species. Precipitation of cdc2 showed that, like histone H1, pepsin was also phosphorylated by cdc2 (MPF). These results suggest that different kinase cascades are involved in goldfish and Xenopus oocyte maturation, although MPF and MAP kinases are common to both species. We also found novel phosphorylation activities that precede the activation of MPF and MAP kinases using deoxyribonuclease I, casein, pepsin and protamine as exogenous substrates.
• PURIFICATION AND CHARACTERIZATION OF ACTIVE PROTEASOME (26S PROTEASOME) FROM GOLDFISH OVARIES
  T TOKUMOTO, M YOSHIKUNI, M YAMASHITA, H KAJIURA, Y NAGAHAMA
  BIOMEDICAL RESEARCH-TOKYO, 16, 4, 207, 218, BIOMED RES FOUND, 1995年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, The cytosol fraction (150,000 xg supernatant) of goldfish ovarian homogenate hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA in the absence of SDS. The use of a stepwise gradient and ATP prevented the loss of this activity during purification. Active proteasome was purified to homogeneity from ovarian cytosol using five steps of chromatography. The purified active proteasome had chymotrypsin-, trypsin-, and V8 protease-like activities even in the absence of SDS. The enzyme exhibited two bands on native PAGE. Electrophoresis and Western blot analyses showed that the enzyme consisted of at least 15 protein components ranging in molecular mass from 35.5 to 140 kDa, as well as the multiple subunits of the latent proteasome ranging in molecular mass from 23.5 to 31.5 kDa. The molecular weight and sedimentation coefficient of the active proteasome were estimated to be 1,200 kDa and 29.4S, respectively, both of which are larger than those of the latent proteasome of the same species. In electron micrographs, the active proteasome appeared as a dumbbell-like image. It is concluded that the active proteasome purified from goldfish oocyte cytosol is identical to the 26S proteolytic complex reported in various eukaryotic cells.
• PRE-MPF IS ABSENT IN IMMATURE OOCYTES OF FISHES AND AMPHIBIANS EXCEPT XENOPUS
  T TANAKA, M YAMASHITA
  DEVELOPMENT GROWTH & DIFFERENTIATION, 37, 4, 387, 393, BLACKWELL SCIENCE PUBL AUSTR, 1995年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Maturation-promoting factor (MPF), a final trigger for initiating oocyte maturation, is activated in the oocyte cytoplasm, in response to maturation-inducing hormone (MIH) secreted from follicle cells surrounding the oocyte. MPF consists of cdc2 and cyclin B. We investigated the state of cdc2 and cyclin B in immature and mature oocytes of fishes (carp, catfish and lamprey) and amphibians (Xenopus, frog [Rana] and toad [Bulo]) using monoclonal antibodies raised against mouse cdc2, which also recognize fish and amphibian cdc2, and monoclonal antibodies against goldfish cyclin B1 and polyclonal antibodies against Xenopus cyclins B1 and B2. Anti-cdc2 and anti-cyclin B immunoblotting of oocyte extracts fractjonated by gel filtration chromatography showed that immature oocytes from all of these species with the exception of Xenopus contained only monomeric cdc2. Cyclin B-bound inactive cdc2 (pre-MPF) was present only in immature Xenopus oocytes. Cdc2-cyclin B complex was, however, found in mature oocytes from all the species examined. After the oocyte is induced to mature by MIH, cdc2 should therefore bind to cyclin B in all of these species, except Xenopus. These results suggest that the complex formation of cdc2 and cyclin B in response to MIH stimulation at the oocyte surface is a critical step for initiating oocyte maturation in fishes and amphibians, with the exception of Xenopus, in which pre-MPF already exists in immature oocytes and only its chemical modification is required for MPF activation.
• MOLECULAR-CLONING AND IMMUNOLOGICAL ANALYSIS OF GOLDFISH CYCLIN-A DURING OOCYTE MATURATION
  Y KATSU, M YAMASHITA, T HIRAI, T TOKUMOTO, H KAJIURA, Y NAGAHAMA
  DEVELOPMENTAL BIOLOGY, 170, 2, 616, 625, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1995年08月, ［査読有り］
  英語, 研究論文（学術雑誌）, Cyclin A belongs to a family of proteins involved in the regulation of the eukaryotic cell cycle. Although cyclin A is thought to be involved in the regulation of both S and M phase, its exact role in the cell cycle, especially in the meiotic cycle (oocyte maturation), is uncertain. We isolated cyclin A cDNA clones from a goldfish oocyte cDNA library. Monoclonal antibody raised against bacterially produced goldfish cyclin A recognized a 47-kDa protein that disappeared after egg activation. Unlike goldfish cyclin B, which is absent in immature oocytes, cyclin A was already present in immature oocytes and its protein level did not change remarkably during oocyte maturation. These results differ from the finding in Xenopus, in which cyclin A is absent, but cyclin B is present, in immature oocytes. Goldfish cyclin A was associated with cdc2 kinase in mature oocytes, but not with cdk2. Recombinant cyclin A bound to and activated cdc2 in a cell-free system, but cyclin A and cdk2 binding was not observed. The kinase activity of cyclin A-cdc2 was undetectable in immature oocytes and first appeared at about the time of germinal vesicle breakdown (GVBD). In contrast to the cyclin B-cdc2 activity that corresponded to the occurrence of GVBD, cyclin A-cdc2 activity increased only slightly until GVBD was completed and increased drastically after the completion of the first meiotic division. Furthermore, microinjection of cyclin A mRNA into immature oocytes did not cause GVBD; however, microinjection of cyclin B mRNA did. These results suggest that cyclin A-cdc2 kinase and cyclin B-cdc2 kinase play different roles in controlling oocyte maturation. The roles of cyclin A in the rapid activation of cyclin B-cdc2 kinase at meiosis I and II transition and in the maintenance of high maturation-promoting factor activity in mature unfertilized eggs are discussed. (C) 1995 Academic Press, Inc.
• PURIFICATION OF LATENT PROTEASOME (20S PROTEASOME) AND DEMONSTRATION OF ACTIVE PROTEASOME IN GOLDFISH (CARASSIUS-AURATUS) OOCYTE CYTOSOL
  T TOKUMOTO, M YAMASHITA, M YOSHIKUNI, H KAJIURA, Y NAGAHAMA
  BIOMEDICAL RESEARCH-TOKYO, 16, 3, 173, 186, BIOMED RES FOUND, 1995年06月, ［査読有り］
  英語, 研究論文（学術雑誌）, The cytosol fraction (150,000 g supernatant) of goldfish ovary hydrolyzed a fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, a well-known substrate for proteasome, regardless of the addition of SDS to the reaction mixture. Four steps of column chromatography resulted in a 135-fold purification of proteasome from this supernatant. Both SDS-independent and SDS-dependent hydrolyzing activities co-migrated during purification. However, the SDS-independent activity was markedly reduced during purification. The purified proteasome possessed faint hydrolyzing activity (chymotrypsin-, trypsin; and V8 protease-like activity) even in the absence of SDS; the chymotrypsin- and V8 protease-like activities were significantly increased in the presence of SDS. Its molecular weight and sedimentation coefficient were estimated to be 620 kDa and 19.2 S, respectively. Three kinds of monoclonal antibodies were raised against the purified proteasome. Western blot analyses revealed that these antibodies recognized a single species of protein on native PAGE, but recognized several subunits ranging in molecular mass from 23.5 to 31.5 kDa on SDS-PAGE. Cytosol fractions containing the SDS-independent activity had a band which migrated slower than that of purified proteasome. The SDS-independent protease activity was depleted when the cytosol fraction was immunoprecipitated with the anti-proteasome antibody. From these structural and enzymatical properties, it is concluded that the purified proteasome corresponds to latent proteasomes (20 S proteasomes) reported in other eukaryotic cells. Further, the results demonstrate that goldfish oocytes contain an active form of proteasome which can hydrolyze its substrates in the absence of activators.
• ASSOCIATION OF P34(CDC2) AND CYCLIN B1 DURING MEIOTIC MATURATION PORCINE OOCYTES
  K NAITO, C HAWKINS, M YAMASHITA, Y NAGAHAMA, F AOKI, K KOHMOTO, Y TOYODA, RM MOOR
  DEVELOPMENTAL BIOLOGY, 168, 2, 627, 634, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1995年04月, ［査読有り］
  英語, 研究論文（学術雑誌）, The relative levels and association of p34(cdc2) and cyclin B1 have been determined in pig oocytes during meiotic progression from G2 to metaphase II (MII). Fully grown G2-arrested porcine oocytes contained large amounts of free p34(cdc2) and extremely small amounts of p34(cdc2)-cyclin B1 complex which did not increase in amount during the GV stage. Cyclin B1 is not synthesized in measurable quantities until 23 hr after the start of maturation. During the first metaphase (MI) the amount of both cyclin BI and the p34(cdc)-cyclin B1 complex increased sharply until 35 hr. Thereafter the amount of the p34(cdc2)-cyclin B1 complex increased again to 45 hr, resulting in higher levels of the complex in MII than in MI. Moreover, three distinct migration forms of p34(cdc2) Were detected in pig oocytes by immunoblotting with anti-PSTAIRE antibody, and most of the cyclin B1-associated p34(cdc2) was detected in the lower band in MI oocytes but migrated in the second band during MII. These results suggest that cyclin B1 levels in porcine oocytes differ from those in starfish, clam, and Xenopus oocytes in which considerably higher concentrations of cyclin B1 appear to be present during the GV stage and that the nature of the association between p34(cdc2) and cyclin B1 changes between the first and the second metaphase. (C) 1995 Academic Press, Inc.
• MOLECULAR MECHANISMS OF THE ACTIVATION OF MATURATION-PROMOTING FACTOR DURING GOLDFISH OOCYTE MATURATION
  M YAMASHITA, H KAJIURA, T TANAKA, S ONOE, Y NAGAHAMA
  DEVELOPMENTAL BIOLOGY, 168, 1, 62, 75, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1995年03月, ［査読有り］
  英語, 研究論文（学術雑誌）, Oocyte maturation is triggered by the activation in the oocyte cytoplasm of maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit). Immature goldfish oocytes contain only inactive monomeric 35-kDa cdc2 and do not stockpile cyclin B. In maturing oocytes, activation of cdc2 is associated with its Thr161 phosphorylation and mobility shift on SDS-PAGE from 35 to 34 kDa after binding to cyclin B. Using mutant cdc2, we show that Thr161 phosphorylation is required for both the downward shift and the kinase activation. Since cdc2 Tyr15 is not phosphorylated after binding to cyclin B, it does not require dephosphorylation. This situation is obviously different from that in immature Xenopus oocytes, in which the cdc2-cyclin B complex preexists with cdc2 phosphorylated on both Tyr15 and Thr161, thereby requiring Tyr15 dephosphorylation catalyzed by cdc25 phosphatase for MPF activation. These results indicate that these species employ different mechanisms of MPF activation during oocyte maturation, although the final molecular structure of the active MPF (cdc2 bound to cyclin B and phosphorylated on Thr161) is identical. (C) 1995 Academic Press, Inc.
• 注目されるシグナル伝達因子〈核内因子ハイライト 18〉 CAK(cdk活性化キナーゼ)
  山下正兼
  実験医学, 13, 1, 98-99, 1995年01月
  日本語
• REGULATION OF OOCYTE GROWTH AND MATURATION IN FISH
  Y NAGAHAMA, M YOSHIKUNI, M YAMASHITA, T TOKUMOTO, Y KATSU
  CURRENT TOPICS IN DEVELOPMENTAL BIOLOGY, VOL 30, 30, 103, 145, ACADEMIC PRESS INC, 1995年, ［査読有り］
  英語
• 13 Regulation of Oocyte Maturation in Fish
  Yoshitaka Nagahama, Michiyasu Yoshikuni, Masakane Yamashita, Minoru Tanaka
  Fish Physiology, 13, C, 393, 439, 1994年, ［査読有り］
  英語, 研究論文（学術雑誌）
• ISOLATION AND CHARACTERIZATION OF GOLDFISH CDC2, A CATALYTIC COMPONENT OF MATURATION-PROMOTING FACTOR
  H KAJIURA, M YAMASHITA, Y KATSU, Y NAGAHAMA
  DEVELOPMENT GROWTH & DIFFERENTIATION, 35, 6, 647, 654, BLACKWELL SCIENCE, 1993年12月, ［査読有り］
  英語, 研究論文（学術雑誌）
• A TRIPOLAR SPINDLE FORMED AT MEIOSIS-I ASSURES THE RETENTION OF THE ORIGINAL PLOIDY IN THE GYNOGENETIC TRIPLOID CRUCIAN CARP, GINBUNA CARASSIUS-AURATUS LANGSDORFII
  M YAMASHITA, JQ JIANG, H ONOZATO, T NAKANISHI, Y NAGAHAMA
  DEVELOPMENT GROWTH & DIFFERENTIATION, 35, 6, 631, 636, BLACKWELL SCIENCE, 1993年12月, ［査読有り］
  英語, 研究論文（学術雑誌）
• A FISH HOMOLOG OF THE CDC2-RELATED PROTEIN P40(MO15) - ITS CDNA CLONING AND EXPRESSION IN OOCYTES
  S ONOE, M YAMASHITA, H KAJIURA, Y KATSU, JQ JIANG, Y NAGAHAMA
  BIOMEDICAL RESEARCH-TOKYO, 14, 6, 441, 444, BIOMED RES FOUND, 1993年12月, ［査読有り］
  英語, We have previously shown that threonine (probably Thr-161) phosphorylation of cdc2 kinase (p34(cdc2)), the catalytic subunit of maturation-promoting factor (MPF), is a crucial step for the 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DP, a maturation-inducing hormone)induced activation of MPF in goldfish (Carassius auratus) oocytes. In this study, we have cloned a cDNA that encodes a goldfish homolog of p40(M015), the catalytic subunit of a protein kinase which has been shown to activate cdc2 kinase through phosphorylation of Thr-161, from a goldfish oocyte cDNA library. Reverse transcription PCR using oligonucleotide primers corresponding to highly conserved sequences in cdc2-related genes was used to generate a 144 bp cDNA PCR product from goldfish oocyte RNA. This probe was then used to screen the cDNA library for a full-length cDNA encoding a homolog of p40(M015) in goldfish. The isolated clone of approximately 1.3 kbp contained an open reading frame encoding a 344 amino acid protein that: was 83% and 55% identical to Xenopus p40(M015) and a homolog of p40(M015) in rice, respectively. Using Northern blot techniques, a 1.3 kb M015 mRNA was detected in both full-grown immature oocytes and mature oocytes of ginbuna (Carassius auratus langsdorfii), a species closely related to goldfish. These data suggest that p40(M015) homolog is produced in fish oocytes and plays an important role in the 17 alpha,20 beta-DP-induced activation of MPF through threonine phosphorylation of cdc2 kinase.
• THE EGG NUCLEUS REGULATES THE BEHAVIOR OF SPERM NUCLEI AS WELL AS CYCLING OF MPF IN PHYSIOLOGICALLY POLYSPERMIC NEWT EGGS
  Y IWAO, N SAKAMOTO, K TAKAHARA, M YAMASHITA, Y NAGAHAMA
  DEVELOPMENTAL BIOLOGY, 160, 1, 15, 27, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1993年11月, ［査読有り］
  英語, 研究論文（学術雑誌）
• BEHAVIOR OF THE COMPONENTS OF MATURATION-PROMOTING FACTOR, CDC2 KINASE AND CYCLIN-B, DURING OOCYTE MATURATION OF GOLDFISH
  Y KATSU, M YAMASHITA, H KAJIURA, Y NAGAHAMA
  DEVELOPMENTAL BIOLOGY, 160, 1, 99, 107, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1993年11月, ［査読有り］
  英語, 研究論文（学術雑誌）
• PURIFICATION OF UBIQUITIN FROM GOLDFISH (CARASSIUS-AURATUS) OOCYTE CYTOSOL
  T TOKUMOTO, H KAJIURA, M YOSHIKUNI, M YAMASHITA, Y NAGAHAMA
  BIOMEDICAL RESEARCH-TOKYO, 14, 4, 309, 312, BIOMED RES FOUND, 1993年08月, ［査読有り］
  英語, Ubiquitin was purified from the cytosol fraction (150,000 g supernatant) of goldfish ovaries containing full-grown postvitellogenic oocytes using four steps of column chromatography. The purified goldfish ubiquitin gave a single band with a molecular weight of 5.5 kDa on denaturing polyacrylamide gel electrophoresis and reacted with an anti-bovine ubiquitin antibody on Western blot. The first 40 amino acid residues of the N-terminal sequence of goldfish ubiquitin are identical with those of ubiquitins in other higher eukaryotes. These results indicate that ubiquitin exists and occurs as a free polypeptide in immature oocytes of goldfish.
• CHANGES IN THE ACTIVITY AND PROTEIN-LEVELS OF PROTEASOMES DURING OOCYTE MATURATION IN GOLDFISH (CARASSIUS-AURATUS)
  T TOKUMOTO, M YAMASHITA, M YOSHIKUNI, Y NAGAHAMA
  BIOMEDICAL RESEARCH-TOKYO, 14, 4, 305, 308, BIOMED RES FOUND, 1993年08月, ［査読有り］
  英語, Changes in the activity and protein levels of proteasomes were determined in goldfish oocytes during meiotic maturation induced in vitro by 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), a naturally occurring maturation-inducing hormone in this species. Both the activity and protein levels of proteasome exhibited two peaks during the 17alpha,20beta-DP-induced oocyte maturation with a similar pattern of fluctuation. The first peak (a 2-fold increase) occurred before the migration of germinal vesicle (GV) (within 1 h after 17alpha,20beta-DP treatment), followed by a gradual decrease towards GV breakdown (GVBD), reaching the lowest level at 6 h. The second peak occurred immediately after GVBD (7 h after the treatment), followed by a sharp decrease at 8 h. The time of the second peak appears to correspond to the time of the first polar body extrusion, a time when maturation-promoting factor (MPF) activity was shown to decrease transiently in goldfish oocytes during the 17alpha,20beta-DP-induced meiotic maturation. Thus, it is suggested that proteasomes are involved in the decrease in MPF activity during the first polar body extrusion.
• 生命科学を支える新しい生物モデル―発生分化・神経・癌研究への応用 第3章 生物モデルとしての魚類 魚卵‐卵成熟の実験モデル
  高橋美佳, 徳元俊伸, 山下正兼, 長浜嘉孝
  実験医学, 11, 12, 1591-1597, 1993年07月
  日本語
• MOLECULAR ENDOCRINOLOGY OF OOCYTE GROWTH AND MATURATION IN FISH
  Y NAGAHAMA, M YOSHIKUNI, M YAMASHITA, N SAKAI, M TANAKA
  FISH PHYSIOLOGY AND BIOCHEMISTRY, 11, 1-6, 3, 14, KUGLER PUBLICATIONS BV, 1993年07月, ［査読有り］
  英語, 研究論文（学術雑誌）, Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17beta) and maturation (maturation-inducing hormone, MIH; 17alpha,20beta-dihydroxy-4-pregnen-3-one, 17alpha,20beta-DP in salmonid fishes; 17alpha,20beta,21-trihydroxy-4-pregnen-3-one, 20beta-S in sciaenid fishes). It is established that production of estradiol-17beta and 17alpha,20beta-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17beta to 17alpha,20beta-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3beta-hydroxysteroid dehydrogenase (HSD), 17alpha-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAs as well as the pig 20beta-HSD cDNA.
  Estradiol-17beta stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17beta was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17alpha,20beta-DP and 20beta-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17alpha,20beta-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17alpha,20beta-DP-induced oocyte maturation in fish.
• 生体超分子システム生命理解のかなめ II 超分子システム各論 細胞分裂システム ラミンを中心とした核マトリクスとG₂/M期
  山口明彦, 山下正兼, 長浜嘉孝
  蛋白質 核酸 酵素, 38, 7, 1210-1222, 1993年05月
  日本語
• SUPEROXIDE-DISMUTASE AND THIOREDOXIN RESTORE DEFECTIVE P34(CDC2) KINASE ACTIVATION IN MOUSE 2-CELL BLOCK
  S NATSUYAMA, Y NODA, M YAMASHITA, Y NAGAHAMA, T MORI
  BIOCHIMICA ET BIOPHYSICA ACTA, 1176, 1-2, 90, 94, ELSEVIER SCIENCE BV, 1993年03月, ［査読有り］
  英語, 研究論文（学術雑誌）, We recently showed that superoxide dismutase (SOD), a free radical scavenger, and thioredoxin, a potent protein disulfide reductase, release mouse two-cell stage developmental block in vitro. To elucidate the mechanism underlying the two-cell block and the effects of these enzymes, we studied the chronological changes in the kinase activity and the immunoblotting pattern of p34cdc2, a key regulator of the cell cycle, during the first and second cell cycles of the mouse embryonic development. In vivo embryos were freshly collected at fixed times, and in vitro embryos cultured from the pronuclear stage were also sampled with the same time-course. A marked elevation of p34cdc2 kinase activity was observed in vivo at 27-32 and 51 h after an injection of human chorionic gonadotropin. These times coincide with the M-phases of embryo cleavage. In vitro embryos showed high kinase activity during the M-phase of the first cleavage, but this activity was not elevated during the second cell cycle. The addition of recombinant human SOD (200 mug/ml) or thioredoxin from Esherichia coli (500 mug/ml) to the medium enabled kinase activation with a time course similar to that of in vivo embryos. The immunoblotting patterns suggested the dephosphorylation of p34cdc2 at the M-phase of the first and the second cleavages in vivo. Although p34cdc2 was dephosphorylated at the M-phase of the first cleavage and then rephosphorylated for embryos cultured in vitro, the second dephosphorylation was not observed during the second cell cycle. The addition of SOD or thioredoxin permitted the dephosphorylation at the M-phases of both the first and the second cleavage. These results suggest that one of the chief causes of two-cell block in vitro is the impairment in p34cdc2 dephosphorylation, recently shown to be catalyzed by the cdc25 homologue. This impairment is thought to be due to oxidative stress, because both SOD and thioredoxin are known to play a defensive role against it.
• DIRECT ACTIVATION OF P34CDC2-PROTEIN KINASE WITHOUT PRECEDING PHOSPHORYLATION DURING MEIOTIC CELL-CYCLE IN MOUSE OOCYTES
  T CHOI, F AOKI, M YAMASHITA, Y NAGAHAMA, K KOHMOTO
  BIOMEDICAL RESEARCH-TOKYO, 13, 6, 423, 427, BIOMED RES FOUND, 1992年12月, ［査読有り］
  英語, 研究論文（学術雑誌）, During the late phase of the first and the second meiotic cell cycles, the phosphorylated form of p34cdc2 was not detected and sodium orthovanadate, an inhibitor of tyrosine phosphatase, did not inhibit the activation of histone H1 kinase, whereas it inhibited the activation of the kinase and dephosphorylation of p34cdc2 during the early phase of the first meiosis and mitotic cell cycle. These results indicate that the phosphorylation of p34cdc2 is not a prerequisite for its activation. The significance of the absence of phosphorylation in meiotic cell cycle is discussed.
• MEIOSIS-REINITIATION-INDUCING FACTOR OF TETRAHYMENA FUNCTIONS UPSTREAM OF M-PHASE-PROMOTING FACTOR
  M FUJISHIMA, Y KATSU, E OGAWA, M SAKIMURA, M YAMASHITA, Y NAGAHAMA
  JOURNAL OF PROTOZOOLOGY, 39, 6, 683, 690, SOC PROTOZOOLOGISTS, 1992年11月, ［査読有り］
  英語, 研究論文（学術雑誌）, Reinitiation of meiosis (maturation) of amphibian Bufo and Xenopus oocytes can be induced if Tetrahymena extract is injected into them. The activity differed from M-phase-promoting factor, because action of the former factor on the induction of maturation was inhibited by treatment of the oocytes with cycloheximide. Activity of M-phase-promoting factor was not detected in Tetrahymena extract regardless of the presence of cdc2 homologues in the extract. However, cycloheximide-resistant-maturation-inducing activity appeared in the recipients, when the maturation was induced by injection of Tetrahymena extract. Immunoblots using antibodies against cdc2 showed that injection of Tetrahymena extract induced fast mobility of the recipient cdc2 in the presence of the recipient protein synthesis. The same mobility shift of the cdc2 was also induced when M-phase-promoting factor containing Xenopus oocyte extract was injected into immature oocytes or when the immature oocyte extract was treated with alkaline phosphatase. These results indicate that meiosis-reinitiation-inducing factor of Tetrahymena functions upstream of M-phase-promoting factor to induce dephosphorylation of the recipient cdc2. Tetrahymena cdc2 homologues also showed fast mobility when the Tetrahymena extract was treated with alkaline phosphatase. Preliminary experiments showed that the meiosis-reinitiation-inducing factor of Tetrahymena was a soluble protein.
• A DEFICIENCY IN THE MECHANISM FOR P34CDC2 PROTEIN-KINASE ACTIVATION IN MOUSE EMBRYOS ARRESTED AT 2-CELL STAGE
  F AOKI, T CHOI, M MORI, M YAMASHITA, Y NAGAHAMA, K KOHMOTO
  DEVELOPMENTAL BIOLOGY, 154, 1, 66, 72, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1992年11月, ［査読有り］
  英語, 研究論文（学術雑誌）
• CYCLIN-B IN FISH OOCYTES - ITS CDNA AND AMINO-ACID-SEQUENCES, APPEARANCE DURING MATURATION, AND INDUCTION OF P34CDC2-ACTIVATION
  T HIRAI, M YAMASHITA, M YOSHIKUNI, YH LOU, Y NAGAHAMA
  MOLECULAR REPRODUCTION AND DEVELOPMENT, 33, 2, 131, 140, WILEY-LISS, 1992年10月, ［査読有り］
  英語, 研究論文（学術雑誌）, Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17-alpha,20-beta-dihydroxy-4-pregnen-3-one (17-alpha,20-beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [S-35]methionine, indicating its de novo synthesis. Introduction of E coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17-alpha,20-beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.
• TESTICULAR DEVELOPMENT INDUCED BY A RECESSIVE MUTATION DURING GONADAL DIFFERENTIATION OF FEMALE COMMON CARP (CYPRINUS-CARPIO, L)
  J KOMEN, M YAMASHITA, Y NAGAHAMA
  DEVELOPMENT GROWTH & DIFFERENTIATION, 34, 5, 535, 544, BLACKWELL SCIENCE, 1992年10月, ［査読有り］
  英語, 研究論文（学術雑誌）
• ISOLATION AND CHARACTERIZATION OF GOLDFISH CDK2, A COGNATE VARIANT OF THE CELL-CYCLE REGULATOR CDC2
  T HIRAI, M YAMASHITA, M YOSHIKUNI, T TOKUMOTO, H KAJIURA, N SAKAI, Y NAGAHAMA
  DEVELOPMENTAL BIOLOGY, 152, 1, 113, 120, ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS, 1992年07月, ［査読有り］
  英語, 研究論文（学術雑誌）
• A MONOCLONAL-ANTIBODY AGAINST THE PSTAIR SEQUENCE OF P34CDC2, CATALYTIC SUBUNIT OF MATURATION-PROMOTING FACTOR AND KEY REGULATOR OF THE CELL-CYCLE
  M YAMASHITA, M YOSHIKUNI, T HIRAI, S FUKADA, Y NAGAHAMA
  DEVELOPMENT GROWTH & DIFFERENTIATION, 33, 6, 617, 624, BLACKWELL SCIENCE, 1991年12月, ［査読有り］
  英語, 研究論文（学術雑誌）
• ACTIVATION OF P34CDC2 PROTEIN-KINASE ACTIVITY IN MEIOTIC AND MITOTIC CELL-CYCLES IN MOUSE OOCYTES AND EMBRYOS
  T CHOI, F AOKI, M MORI, M YAMASHITA, Y NAGAHAMA, K KOHMOTO
  DEVELOPMENT, 113, 3, 789, 795, COMPANY OF BIOLOGISTS LTD, 1991年11月, ［査読有り］
  英語, 研究論文（学術雑誌）, p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos.
  We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment.
  In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively.
  In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.
• Breakdown of the sperm nuclear envelope is a prerequisite for male pronucleus formation: Direct evidence from the gynogenetic crucian carp Carassius auratus langsdorfii
  M. Yamashita, H. Onozato, T. Nakanishi, Y. Nagahama
  Developmental Biology, 137, 1, 155, 160, 1990年, ［査読有り］
  英語, 研究論文（学術雑誌）, The gynogenetic fish, Carassius auratus langsdorfii (the ginbuna, a crucian carp), provides an interesting model for the study of the mechanisms controlling male pronucleus formation. When the sperm nucleus of a different subspecies (C. a. cuvieri) is incorporated into the gynogenetic egg, the nuclear envelope of the spermatozoon is not broken down, and the pronucleus fails to develop, although dispersion of the sperm chromatin occurs to some extent within the space limited by the nuclear envelope. When spermatozoa without plasma membranes and nuclear envelopes were microinjected into mature activated eggs, the sperm nuclei underwent chromatin dispersion, nuclear envelope formation, DNA synthesis, and transformation into male pronuclei. These results indicate that the failure of the male pronucleus to form in ginbuna is primarily due to the failure of sperm nuclear envelope breakdown. We conclude that sperm nuclear envelope breakdown is an indispensable step for the development of the male pronucleus. © 1990.
• MECHANISMS OF SYNTHESIS AND ACTION OF 17-ALPHA,20-BETA-DIHYDROXY-4-PREGNEN-3-ONE, A TELEOST MATURATION-INDUCING SUBSTANCE
  Y NAGAHAMA, M YAMASHITA
  FISH PHYSIOLOGY AND BIOCHEMISTRY, 7, 1-6, 193, 200, KUGLER PUBLICATIONS BV, 1989年06月, ［査読有り］
  英語, 研究論文（学術雑誌）
• A Fine Structural Study of the Fertilization Process of the Jellyfish Cladonema uchidai jellyfish/fertilization/fine structure/sperm incorporation/zygote nucleus formation
  MASAKANE YAMASHITA
  Development, Growth & Differentiation, 30, 1, 81, 91, 1988年, ［査読有り］
  英語, 研究論文（学術雑誌）, This study described the fertilization process of the jellyfish Cladonema uchidai by means of transmission electron microscopy. Female pronucleus was situated in close vicinity to the animal pole of the spawned egg, where the surface of the egg was flat or slightly depressed. Microvilli were observed except on the surface at the animal pole. The egg was entirely covered with a coat composed of fibrous materials. The spermatozoon was of the primitive type, and the proacrosomal vesicles were found immediately beneath the plasma membrane of the antero‐lateral region of the sperm head. Within 15 sec after insemination, spermatozoa were incorporated in the egg cytoplasm only at the microvilli‐free surface at the animal pole. Neither opening of the proacosomal vesicles nor formation of the acrosomal process was observed. No appreciable changes of cortical cytoplasm could be detected, although the egg became sticky after fertilization. Decondensation of the incorporated sperm nucleus occurred without breakdown of the original nuclear envelope. Within 10 min after insemination, the sperm nucleus still under the process of its decondensation fused with the female pronucleus. These findings were discussed in comparison with the fertilization process of higher metazoans as well as of other cnidarians. Copyright © 1988, Wiley Blackwell. All rights reserved
• Involvement of cAMP in initiating maturation of the brittle-star Amphipholis kochii oocytes: Induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterase and activators of adenylate cyclase
  Masakane Yamashita
  Developmental Biology, 125, 1, 109, 114, 1988年, ［査読有り］
  英語, 研究論文（学術雑誌）, The maturation of brittle-star (Amphipholis kochii) oocytes, i.e., the reinitiation of meiosis accompanied by germinal vesicle breakdown (GVBD) and the acquisition of fertilizability, was induced by acid (pH 3.0) seawater containing 10 mM cAMP. Oocyte maturation was also induced by seawater of normal pH (pH 8.0) that contained either an inhibitor of cyclic nucleotide phosphodiesterase (25 mM theophylline, 25 mM caffeine) or an activator of adenylate cyclase (100 μM forskolin, 0.6 μM cholera toxin). Experiments in which the oocytes were treated with forskolin or theophylline for various periods of time demonstrated that there was a positive correlation between the oocyte cAMP level measured by radioimmunoassay and the extent of GVBD induced in each treatment: both increased as the treatment period became longer and about a threefold increase in cAMP level induced 50% GVBD. These results indicate that an increase in cAMP level initiates maturation of the brittle-star oocytes. © 1988.
• Electron microscopic analysis of the sperm nuclear changes in meiosis inhibited eggs of the brittle‐star Amphipholis kochii
  Masakane Yamashita
  Journal of Experimental Zoology, 235, 1, 105, 117, 1985年, ［査読有り］
  英語, 研究論文（学術雑誌）, This paper describes the sperm nuclear changes in the eggs of the brittle‐star Amphipholis kochii that had been treated with meiosis inhibitors, including the nuclear division inhibitors (colchicine, colcemid, griseofulvin, and vinblastine) and the cytokinesis inhibitor (cytochalasin B), as observed by transmission electron microscopy. In the normal, monospermic fertilization process, the male pronucleus formation consists of two processes: the first dispersion process before formation of the nuclear envelope and the second dispersion process on and after envelope formation. When nuclear division was inhibited, so that the female pronucleus formation was also inhibited, the sperm nucleus incorporated into such eggs decondensed to some extent, in accordance with the first dispersion process, but the nuclear envelope did not form around the nucleus and further decondensation (the second dispersion process) was also inhibited. When cytokinesis was inhibited, the sperm nucleus, as well as the maternal chromosomes, transformed into the pronuclei on schedule. These results suggest that 1) the male pronucleus formation has an intimate relationship with meiosis, especially nuclear division, as well as with the formation of the female pronucleus
  2) the male pronucleus formation consists of at least two different processes, a meiosis‐independent process (the first dispersion process) and a meiosis‐dependent process (the second dispersion process)
  and 3) the factor for the first dispersion process is a soluble component while the factor for the second dispersion process is membrane associated. Copyright © 1985 Wiley‐Liss, Inc., A Wiley Company
• Electron Microscopic Observations on the Cortical Reaction of the Brittle‐Star Amphipholis kochii Lütken, with Special Reference to its Vitelline Coat Modification as Revealed by the Surface Replica Method: cortical reaction/fine structure/ophiuroid egg/surface replica
  MASAKANE YAMASHITA
  Development, Growth & Differentiation, 26, 2, 177, 189, 1984年, ［査読有り］
  英語, 研究論文（学術雑誌）, This paper describes the fine structural changes of the egg of the brittle‐star Amphipholis kochii Lütken during the cortical reaction. The vitelline coat is 20 nm thick, when Ruthenium Red stain is used, and consists of a dense network of fibers. The cortical granules are large, 1.5–2.0 μm in diameter, and exist in several layers in the egg cortex, unlike the monolayer arrangement found in many other animals. The contents of the cortical granules are clearly distinguished into two components: peripheral fibrous (PF) material and central fibrous (CF) material that consists of two components differing in electron density. The PF material is densely stained by periodic acid‐chromic acid‐silver methenamine stain, while the CF material is stained little if at all by this technique. The vitelline coat and some PF materials form the fertilization membrane, which is about 40 nm thick and consists of three layers
  the outer and the inner layer of the fertilization membrane each have a trilaminated structure. The vitelline coat substances are probably located in the upper part of the fertilization membrane. The hyaline layer, 7–8 μm thick, consists mainly of CF materials. These observations on the morphology of the ophiuroid egg are discussed in comparison with those on other echinoderms, especially echinoids and asteroids. Copyright © 1984, Wiley Blackwell. All rights reserved
• Electron microscopic observations during monospermic fertilization process of the brittle‐star Amphipholis kochii Lütken
  Masakane Yamashita
  Journal of Experimental Zoology, 228, 1, 109, 120, 1983年, ［査読有り］
  英語, 研究論文（学術雑誌）