24 results on '"Naito, K"'
Search Results
2. Birth of Normal Calves Resulting from Bovine Oocytes Matured, Fertilized, and Cultured with Cumulus Cells in Vitro up to the Blastocyst Stage1
- Author
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Fukuda, Y., primary, Ichikawa, M., additional, Naito, K., additional, and Toyoda, Y., additional
- Published
- 1990
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3. Oocytes suppress FOXL2 expression in cumulus cells in mice†.
- Author
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Emori C, Ito H, Fujii W, Naito K, and Sugiura K
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- Animals, Cells, Cultured, Female, Follicle Stimulating Hormone pharmacology, Gene Expression drug effects, Granulosa Cells physiology, Mice, Mice, Inbred C57BL, Mice, Inbred DBA, RNA, Messenger analysis, Transcriptome, Cumulus Cells metabolism, Forkhead Box Protein L2 genetics, Gene Expression Regulation physiology, Granulosa Cells metabolism, Oocytes physiology
- Abstract
Cumulus cells and mural granulosa cells (MGCs) play distinct roles during follicular development, and normal development of these cell lineages is critical for the female fertility. Transcriptomic diversification between the two cell lineages is obviously a critical mechanism for their functional diversification; however, the transcriptional regulators responsible for this event have not been fully defined. In this study, we sought to identify key transcriptional regulators responsible for the differential gene expression between the two cell lineages. In silico analysis of transcriptomic comparison between cumulus cells and MGCs identified several candidate regulators responsible for the diversification of the two cell lineages. Among them, we herein focused on forkhead box L2 (FOXL2) and showed that expressions of FOXL2 as well as its target transcripts were differentially regulated between cumulus cells and MGCs. The lower expression of FOXL2 in cumulus cells seemed to be due to the suppression by oocyte-derived paracrine signals. These results suggest that FOXL2 is one of the critical transcription factors that determine cumulus cell and MGC lineages under the control of oocytes., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...
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- 2020
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4. Effects of exportin 1 on nuclear transport and meiotic resumption in porcine full-grown and growing oocytes.
- Author
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Onuma A, Fujioka YA, Fujii W, Sugiura K, and Naito K
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- Active Transport, Cell Nucleus drug effects, Animals, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Fatty Acids, Unsaturated pharmacology, Female, Gene Expression drug effects, Karyopherins antagonists & inhibitors, Karyopherins genetics, Meiosis drug effects, Oocytes drug effects, Protein-Tyrosine Kinases genetics, Protein-Tyrosine Kinases metabolism, RNA Cap-Binding Proteins genetics, RNA Cap-Binding Proteins metabolism, Receptors, Cytoplasmic and Nuclear antagonists & inhibitors, Receptors, Cytoplasmic and Nuclear genetics, Swine, Exportin 1 Protein, Active Transport, Cell Nucleus physiology, Karyopherins metabolism, Meiosis physiology, Oocytes metabolism, Receptors, Cytoplasmic and Nuclear metabolism
- Abstract
Exportin 1 (XPO1) is a nuclear transport receptor involved in the nuclear export of majority proteins in somatic cells. In mammalian oocytes, however, only the presence of XPO1 has been reported at mRNA and protein levels, and the definitive functions of XPO1 and its effects on the meiotic maturation of oocytes have never been directly examined. In the present study, the expression state and the nuclear-export function of porcine XPO1 were analyzed in porcine oocytes. In addition, we investigated the effects of the overexpression and inhibition of XPO1 on meiotic regulation in full-grown and growing oocytes by mRNA injection and inhibitor treatment. Endogenous XPO1 was stably expressed in porcine oocytes during the germinal vesicle (GV) stage, and the expression of exogenous XPO1 significantly decreased the nuclear localization of XPO1 cargos, snurportin 1, and WEE1B. Inhibition of XPO1 by a specific inhibitor, leptomycin B, delayed the GV breakdown (GVBD), whereas the overexpression of XPO1 by mRNA injection accelerated the GVBD. XPO1 overexpression overcame the meiotic arrest induced by WEE1B expression in full-grown oocytes. Surprisingly, the GVBD of porcine growing oocytes, which could not resume meiosis by the maturation culture in vitro, was induced by the expression of exogenous XPO1. These results showed the presence of XPO1 and its function as a nuclear export receptor in mammalian oocytes, including growing oocytes, and they suggest that the regulation of nuclear transport has a large influence on the GV maintenance and meiotic resumption of oocytes. more...
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- 2018
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5. Cytoplasmic anchoring of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs) is required for meiotic arrest of porcine full-grown and growing oocytes.
- Author
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Nishimura T, Fujii W, Sugiura K, and Naito K
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- A Kinase Anchor Proteins genetics, Animals, Blotting, Western, Cloning, Molecular, Cyclic AMP-Dependent Protein Kinases genetics, Epidermal Growth Factor metabolism, Female, Genetic Vectors, Isoenzymes genetics, Isoenzymes physiology, Microinjections, RNA administration & dosage, RNA genetics, RNA, Messenger biosynthesis, RNA, Messenger genetics, Swine, A Kinase Anchor Proteins physiology, Cyclic AMP-Dependent Protein Kinases physiology, Meiosis physiology, Oocytes physiology
- Abstract
Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. We previously hypothesized that the meiotic incompetence of porcine GOs was attributed to complex spatial-temporal regulation of cAMP-dependent protein kinase (PKA) by A-kinase anchor proteins (AKAPs), but found that AKAP1 is not involved in the meiotic incompetence of porcine GOs. In the present study, we cloned porcine cDNAs of AKAP5 and AKAP7alpha, and found that inhibiting the expression of these AKAPs induced PKA translocation into the nucleus and promoted meiotic resumption of porcine GOs without affecting the total PKA activity of GOs, whereas overexpressing these AKAPs had no effect. Because AKAPs regulate PKA localization through binding with regulatory subunits of PKA (PKA-Rs), PKA-R binding with AKAPs was inhibited by AKAP-binding inhibition peptides or PKA-R expression inhibition by antisense RNAs. We found that the expression inhibition and binding inhibition of PRKAR1A, an isoform of mammalian PKA-R, promoted meiotic resumption of porcine GOs, whereas these inhibitions of PRKAR2A, another PKA-R isoform, had no effect. In contrast, the expression inhibition and binding inhibition of PRKAR2A had higher effects than those of PRKAR1A on meiotic resumption of porcine full-grown oocytes. These results suggest that cytoplasmic anchoring of PKA by AKAPs is required for meiotic arrest of oocytes and that the PKA-R isoform working for the maintenance of meiotic arrest changed from PRKAR1A to PRKAR2A during the acquisition of meiotic competence. more...
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- 2014
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6. A-kinase anchor protein 1 (AKAP1) regulates cAMP-dependent protein kinase (PKA) localization and is involved in meiotic maturation of porcine oocytes.
- Author
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Nishimura T, Sugiura K, and Naito K
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- A Kinase Anchor Proteins antagonists & inhibitors, A Kinase Anchor Proteins genetics, A Kinase Anchor Proteins metabolism, Animals, Cells, Cultured, Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit metabolism, Female, In Vitro Oocyte Maturation Techniques veterinary, Oligodeoxyribonucleotides, Antisense pharmacology, Oocytes drug effects, Oocytes metabolism, Oogenesis drug effects, Oogenesis genetics, Oogenesis physiology, Tissue Distribution drug effects, Transfection, A Kinase Anchor Proteins physiology, Cyclic AMP-Dependent Protein Kinases metabolism, Meiosis drug effects, Meiosis genetics, Meiosis physiology, Oocytes physiology, Swine genetics, Swine metabolism, Swine physiology
- Abstract
In mammalian oocytes, cAMP-dependent protein kinase (PKA) has critical functions in meiotic arrest and meiotic maturation. Although subcellular localization of PKA is regulated by A-kinase anchor proteins (AKAPs) and PKA compartmentalization is essential for PKA functions, the role of AKAPs in meiotic regulation has not been fully elucidated. In the present study, we performed far-Western blot analysis using porcine PRKAR2A for detection of AKAPs and found, to our knowledge, several novel signals in porcine oocytes. Among these signals, a 150-kDa AKAP showed the major expression and was the product of porcine AKAP1. Overexpression of AKAP1 changed the PKA localization and promoted meiotic resumption of porcine oocytes even in the presence of a high concentration of cAMP, which inhibits meiotic resumption by inducing high PKA activity. On the contrary, knockdown of AKAP1 showed inhibitory effects on meiotic resumption and oocyte maturation. In addition, the expression level of AKAP1 in porcine growing oocytes, which show meiotic incompetence and PKA mislocalization, was significantly lower than that in fully grown oocytes. However, AKAP1 insufficiency was not the primary cause of the meiotic incompetence of the growing oocytes. These results suggest that the regulation of PKA localization by AKAP1 may be involved in meiotic resumption and oocyte maturation but not in meiotic incompetence of porcine growing oocytes. more...
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- 2013
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7. Analyses of the involvement of PKA regulation mechanism in meiotic incompetence of porcine growing oocytes.
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Nishimura T, Fujii W, Kano K, Sugiura K, and Naito K
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- Amino Acid Sequence, Animals, Cells, Cultured, Cyclic AMP analysis, Cyclic AMP metabolism, Cyclic AMP-Dependent Protein Kinases analysis, Cyclic AMP-Dependent Protein Kinases genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Enzyme Activation genetics, Enzyme Assays methods, Female, Gene Expression Regulation, Enzymologic physiology, In Vitro Oocyte Maturation Techniques, Molecular Sequence Data, Oocytes enzymology, Oocytes metabolism, Oogenesis physiology, Protein Subunits analysis, Protein Subunits genetics, Protein Subunits metabolism, Cyclic AMP-Dependent Protein Kinases physiology, Meiosis genetics, Oocytes physiology, Oogenesis genetics, Swine genetics, Swine metabolism, Swine physiology
- Abstract
Mammalian growing oocytes (GOs) lack the ability to resume meiosis, although the molecular mechanism of this limitation is not fully understood. In the present study, we cloned cDNAs of cAMP-dependent protein-kinase (PKA) subunits from porcine oocytes and analyzed the involvement of the PKA regulation mechanism in the meiotic incompetence of GOs at the molecular level. We found a cAMP-independent high PKA activity in GOs throughout the in vitro culture using a porcine PKA assay system we established, and inhibition of the activity by injection of the antisense RNA of the PKA catalytic subunit (PKA-C) induced meiotic resumption in GOs. Then we examined the possibility that the amount of the PKA regulatory subunit (PKA-R), which can bind and inhibit PKA-C, was insufficient to suppress PKA activity in GOs because of the overexpression of two PKA-Rs, PRKAR1A and PRKAR2A. We found that neither of them affected PKA activity and induced meiotic resumption in GO although PRKAR2A could inhibit PKA activity and induce meiosis in cAMP-treated full-grown oocytes (FGOs). Finally, we analyzed the subcellular localization of PKA subunits and found that all the subunits were localized in the cytoplasm during meiotic arrest and that PKA-C and PRKAR2A, but not PRKAR1A, entered into the nucleus just before meiotic resumption in FGOs, whereas all of them remained in the cytoplasm in GOs throughout the culture period. Our findings suggest that the continuous high PKA activity is a primary cause of the meiotic incompetence of porcine GOs and that this PKA activity is not simply caused by an insufficient expression level of PKA-R, but can be attributed to more complex spatial-temporal regulation mechanisms. more...
- Published
- 2012
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8. CDK7 and CCNH are components of CDK-activating kinase and are required for meiotic progression of pig oocytes.
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Fujii W, Nishimura T, Kano K, Sugiura K, and Naito K
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- Animals, Carrier Proteins metabolism, Cloning, Molecular, Female, Gene Expression, Microinjections, Phosphorylation, RNA, Messenger, Swine, Cyclin-Dependent Kinase-Activating Kinase, CDC2 Protein Kinase metabolism, Cyclin H metabolism, Cyclin-Dependent Kinases metabolism, Meiosis, Oocytes enzymology
- Abstract
CDK-activating kinase (CAK) phosphorylates threonine 161 (T161) of CDC2, a catalytic subunit of maturation/M-phase promoting factor (MPF), and is essential for MPF activation in mitosis. CAK has been thought to consist of a catalytic subunit, a regulatory subunit and an assembly factor: CDK7, CCNH (also known as cyclin H), and MNAT1 (also known as MAT1), respectively. Although it is known that the meiotic progression of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in the meiotic progression of porcine immature oocytes. The T161 phosphorylation of CDC2 was found around germinal vesicle breakdown (GVBD) and thereafter from 18 to 48 h of culture. The GVBD rate at 18 h was increased by the overexpression of CDC2 but not mutated CDC2 (T161 replaced by alanine). Transcripts of CDK7, CCNH, and MNAT1 were detectable throughout the culture period, and their protein distribution patterns during oocyte maturation were the same as those reported in mitotic somatic cells. Overexpression of CDK7 or CCNH accelerated the meiotic events, such as meiotic resumption, T161 phosphorylation of CDC2, CCNB (also known as Cyclin B) synthesis, and MPF activation. On the contrary, knockdown of CDK7 or CCNH caused the inhibition of these meiotic events. In contrast, overexpression and antisense RNA injection of MNAT1 had no influence on meiotic resumption, the status of T161 phosphorylation of CDC2, or MPF activity. These results suggest that CDK7 and CCNH activate CDC2 by T161 phosphorylation and make up CAK, which is required for normal meiotic progression during porcine oocyte maturation. more...
- Published
- 2011
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9. Functions of FZR1 and CDC20, activators of the anaphase-promoting complex, during meiotic maturation of swine oocytes.
- Author
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Yamamuro T, Kano K, and Naito K
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- Anaphase-Promoting Complex-Cyclosome, Animals, Blotting, Western, Cell Cycle Proteins biosynthesis, Cloning, Molecular, Cyclin B biosynthesis, Cyclin B genetics, Cyclin B1, DNA, Complementary biosynthesis, DNA, Complementary genetics, Female, Maturation-Promoting Factor biosynthesis, Maturation-Promoting Factor genetics, Meiosis drug effects, Microinjections, RNA, Antisense genetics, RNA, Antisense pharmacology, RNA, Messenger biosynthesis, RNA, Messenger genetics, Swine, Anaphase physiology, Cell Cycle Proteins genetics, Meiosis physiology, Oocytes physiology, Ubiquitin-Protein Ligase Complexes physiology
- Abstract
Cell division cycle 20 (CDC20) and fizzy/cell division cycle 20 related 1 (FZR1) are activators of the anaphase-promoting complex (APC), which ubiquitinates M-phase regulating proteins, such as cyclin B and securin, and induces their degradation. In the present study, porcine CDC20 and FZR1 were cloned by reverse transcriptase-polymerase chain reaction, and their functions in the meiotic maturation of porcine oocytes were analyzed. FZR1 was readily detected in porcine immature oocytes by immunoblotting, but its levels decreased substantially during maturation. In contrast, CDC20 levels rose during oocyte maturation and were highest by the second meiotic metaphase. The inhibition of CDC20 expression by the injection of CDC20 antisense RNA induced the meiotic arrest at the first meiotic metaphase (M1) and the accumulation of a large amount of cyclin B. On the other hand, the inhibition of FZR1 expression accelerated cyclin B accumulation and the start of germinal vesicle breakdown (GVBD), but did not affect the exit from M1. Conversely, the overexpression of FZR1 by the injection of FZR1 mRNA suppressed the cyclin B accumulation and retarded GVBD. Surprisingly, the injection of CDC20 mRNA into the immature oocytes could not increase CDC20 expression, but increased cyclin B accumulation and accelerated the meiotic progression. As CDC20 is a substrate of APC (FZR1), CDC20 might have competed with cyclin B and inhibited the FZR1 function. These results suggest that porcine FZR1 and CDC20 work on the maintenance of meiotic arrest at the first meiotic prophase and on the exit from M1, respectively, and that their functional phases are strictly distinguished during porcine oocyte maturation. more...
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- 2008
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10. Nuclear histone deacetylases are not required for global histone deacetylation during meiotic maturation in porcine oocytes.
- Author
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Endo T, Kano K, and Naito K
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- Acetylation, Animals, Cell Nucleus enzymology, Cytoplasm enzymology, Female, Histone Deacetylases classification, Histones metabolism, In Vitro Techniques, Meiosis physiology, Nuclear Transfer Techniques, Oocytes cytology, Oocytes growth & development, Sus scrofa, Histone Deacetylases metabolism, Oocytes enzymology
- Abstract
Histone acetylation plays an important role in the regulation of chromatin structure and gene function. In mammalian oocytes, histones H3 and H4 are highly acetylated during the germinal vesicle (GV) stage, and global histone deacetylation takes place via a histone deacetylase (HDAC)-dependent mechanism after GV breakdown (GVBD). The presence of HDACs in the GVs of mammalian oocytes in spite of the high acetylation states of nuclear histones indicates that the HDACs in the nucleus are inactive but become activated after GVBD. However, the fluctuation pattern, the localization of HDAC activity during meiotic maturation and, moreover, the responsibility of nuclear HDACs for global histone deacetylation are still unknown. Here, we demonstrated using porcine oocytes that total HDAC activity was maintained throughout meiotic maturation, and high HDAC activity was observed in both the nucleus and the cytoplasm at the GV stage. The experiments with valproic acid (VPA), a specific class I HDAC inhibitor, revealed that the HDACs in GVs were class I, and those in the cytoplasm were other than class I. Interestingly, VPA had no effect on global histone deacetylation after GVBD, indicating that nuclear HDACs were not required for global histone deacetylation. To confirm this possibility, we removed the nuclei from immature oocytes, injected somatic cell nuclei into the enucleated oocytes, and showed that injected somatic cell nuclei were dramatically deacetylated after nuclear envelope breakdown. These results revealed that nuclear contents, including class I HDACs, are not required for the global histone deacetylation during meiosis, and that cytoplasmic HDACs other than class I are responsible for this process. more...
- Published
- 2008
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11. Porcine SPDYA2 (RINGO A2) stimulates CDC2 activity and accelerates meiotic maturation of porcine oocytes.
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Kume S, Endo T, Nishimura Y, Kano K, and Naito K
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- Amino Acid Sequence, Animals, Base Sequence, CDC2 Protein Kinase drug effects, Cell Cycle Proteins metabolism, Cell Cycle Proteins pharmacology, Cells, Cultured, Cloning, Molecular, Female, Gene Expression Regulation, Molecular Sequence Data, Oocytes drug effects, Sequence Homology, Amino Acid, Sus scrofa, Xenopus Proteins metabolism, Xenopus Proteins pharmacology, CDC2 Protein Kinase metabolism, Cell Cycle Proteins genetics, Meiosis, Oocytes physiology
- Abstract
RINGO, a protein with no homology to cyclin B, has been reported to be involved in activation of CDC2 and regulation of meiotic maturation in Xenopus oocytes. Although the presence of homologues of RINGO families, which are known as SPDY families, has been reported in mammals, their roles in meiotic maturation of mammalian oocytes have never been examined. In the present study, the effects of SPDY on meiotic maturation of porcine oocytes were examined. At first, Xenopus RINGO (xRINGO) mRNA was injected into immature porcine oocytes and found to significantly accelerate CDC2 activation and meiotic resumption. The CCNB (also known as cyclin B) synthesis was prematurely started at 12 h of culture, whereas it started at 18 h in normal oocytes. We next cloned RINGO A2 homologue in pig (pigSPDYA2) from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was more than 85% and 40% homologous with mammalian SPDYA2 and xRINGO, respectively. Acceleration effects similar to those by xRINGO were observed in CDC2 activation, meiotic resumption, and the start of CCNB synthesis in pigSPDYA2 mRNA-injected porcine oocytes. In clear contrast with the effects of xRINGO, which was accumulated abnormally in porcine oocytes and arrested them in the first meiotic metaphase (M1), pigSPDYA2 accelerated the meiotic progression, with about half of pigSPDYA2 mRNA-injected oocytes completing meiotic maturation within 30 h. These results suggest that pigSPDYA2 has important roles on meiotic maturation of porcine oocytes and that the rapid degradation of SPDY was necessary for the normal maturation of oocytes. more...
- Published
- 2007
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12. Study of germinal vesicle requirement for the normal kinetics of maturation/M-phase-promoting factor activity during porcine oocyte maturation.
- Author
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Sugiura K, Naito K, Endo T, and Tojo H
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- Animals, Cell Division physiology, Cell Nucleus, Cells, Cultured, Female, MAP Kinase Signaling System physiology, Sus scrofa, Cyclin B1 metabolism, Maturation-Promoting Factor metabolism, Meiosis physiology, Oocytes cytology, Oocytes metabolism, Oogenesis physiology
- Abstract
Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation. more...
- Published
- 2006
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13. Analysis of the roles of cyclin B1 and cyclin B2 in porcine oocyte maturation by inhibiting synthesis with antisense RNA injection.
- Author
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Kuroda T, Naito K, Sugiura K, Yamashita M, Takakura I, and Tojo H
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- Animals, Base Sequence, Cyclin B genetics, Cyclin B1, Cyclin B2, Female, Meiosis physiology, Molecular Sequence Data, RNA, Antisense pharmacology, Cyclin B metabolism, Oocytes cytology, Oocytes physiology
- Abstract
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 CB1 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. more...
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- 2004
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14. Heat shock transcription factor 1 is involved in quality-control mechanisms in male germ cells.
- Author
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Izu H, Inouye S, Fujimoto M, Shiraishi K, Naito K, and Nakai A
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- Animals, Cell Death, Cell Survival, Cryptorchidism pathology, Cryptorchidism physiopathology, Gene Expression, Heat Shock Transcription Factors, Heat Stress Disorders pathology, Heat Stress Disorders physiopathology, Male, Mice, Mice, Mutant Strains, Spermatids cytology, Spermatogonia cytology, Transcription Factors, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Spermatids physiology, Spermatogenesis physiology, Spermatogonia physiology
- Abstract
Quality-control mechanisms in spermatogenesis are important to eliminate injured or abnormal cells, thereby protecting the organism from abnormal development in the next generation. The processes of spermatogenesis are highly sensitive to high temperatures; however, the mechanisms by which injured germ cells are eliminated remain unclear. Here, we found that heat shock proteins are not induced in male germ cells in response to thermal stress, although heat shock transcription factor 1 (HSF1) is activated. Using HSF1-null mice, we showed that apoptosis of pachytene spermatocytes was markedly inhibited in testes with a single exposure to heat and in the cryptorchid testes, indicating that HSF1 promotes apoptotic cell death of pachytene spermatocytes exposed to thermal stress. In marked contrast, HSF1 acts as a cell-survival factor of more immature germ cells, probably including spermatogonia, in testes exposed to high temperatures. These results demonstrate that HSF1 has two opposite roles in male germ cells independent of the activation of heat shock genes. more...
- Published
- 2004
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15. Infertility observed in reproductive toxicity study of N-acetyl-L-cysteine in rats.
- Author
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Harada M, Kishimoto K, Furuhashi T, Naito K, Nakashima Y, Kawaguchi Y, and Hiraoka I
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- Acetylcysteine administration & dosage, Animals, Embryonic and Fetal Development drug effects, Female, Male, Oocytes drug effects, Oocytes pathology, Pregnancy, Rats, Rats, Sprague-Dawley, Zona Pellucida drug effects, Zona Pellucida pathology, Acetylcysteine toxicity, Infertility, Female chemically induced, Reproduction drug effects
- Abstract
The toxic effects of i.v. administration of N-acetyl-l-cysteine (NAC), a component of parenteral nutrition solutions, on fertility and embryonic development were investigated in SD male and female rats at doses of 100, 300, and 1000 mg kg-1 day-1. Infertility was observed in females in the 1000-mg/kg group throughout the period from before mating to embryogenesis. No effect of NAC on the reproductive ability of the male rats was seen. The oocytes and embryos were assessed morphologically to clarify the cause of the effects of NAC. The unfertilized oocytes (UO) recovered from the ampullae of the uterine tubes and Gestational Day (GD) 1 and 2 embryos recovered from the oviducts or uterus of the rats that received NAC i.v. at a dosage of 1000 mg kg-1 day-1 for more than 1 wk before mating were assessed morphologically by stereomicroscopy. In addition, the thickness of the zona pellucida (ZP) was calculated by morphometric evaluation of the UO. Fewer UO were collected in the NAC group than in the control (nontreatment) group. Interestingly, ZP-lacking or partially ZP-lacking oocytes were observed in the NAC group, and the morphometric evaluation of the UO showed thinning of the ZP. The number of embryos in each animal was markedly decreased on GD1, and no embryos were recovered on GD2 in the NAC group. The oocytes that had ZP affected by NAC treatment were abnormal or nonviable. The findings of the present study suggest that changes in the ZP are related to the infertility associated with NAC. more...
- Published
- 2003
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16. Analyses of mitogen-activated protein kinase function in the maturation of porcine oocytes.
- Author
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Ohashi S, Naito K, Sugiura K, Iwamori N, Goto S, Naruoka H, and Tojo H
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- Animals, Cells, Cultured, Cellular Senescence, Enzyme Activation physiology, Female, Genes, mos, Injections, Maturation-Promoting Factor physiology, Meiosis drug effects, Oocytes drug effects, Oocytes enzymology, RNA, Antisense pharmacology, RNA, Messenger pharmacology, Swine, Mitogen-Activated Protein Kinases physiology, Oocytes physiology
- Abstract
The function of mitogen-activated protein kinase (MAPK) during porcine oocyte maturation was examined by injecting oocytes with either mRNA or antisense RNA of porcine c-mos protein, an upstream kinase of MAPK. The RNAs were injected into the cytoplasm of porcine immature oocytes immediately after collection from ovaries, then the oocytes were cultured for maturation up to 48 h. The phosphorylation and activation of MAPK were observed at 6 h after injection of the c-mos mRNA injected-oocytes, whereas in control oocytes, MAPK activation was detected at 24 h of culture. The germinal vesicle breakdown (GVBD) rate at 24 h of culture was significantly higher in c-mos mRNA-injected oocytes than in control oocytes. In contrast, although injection of c-mos antisense RNA completely inhibited phosphorylation and activation of MAPK throughout the maturation period, the GVBD rate and its time course were the same in noninjected oocytes. The degree of maturation-promoting factor (MPF) activation was, however, very low in oocytes in the absence of MAPK activation. Most of those oocytes had both abnormal morphology and decondensed chromosomes at 48 h of culture. These results suggest that MAPK activation is not required for GVBD induction in porcine oocytes and that the major roles of MAPK during porcine oocyte maturation are to promote GVBD by increasing MPF activity and to arrest oocytes at the second metaphase. more...
- Published
- 2003
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17. Nitric oxide promotes germ cell necrosis in the delayed phase after experimental testicular torsion of rat.
- Author
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Shiraishi K, Naito K, and Yoshida K
- Subjects
- Animals, Apoptosis drug effects, Calpain metabolism, DNA Fragmentation, Electrophoresis, Polyacrylamide Gel, Enzyme Activation drug effects, Enzyme Inhibitors pharmacology, Guanidines pharmacology, Immunoblotting, Immunohistochemistry, In Situ Nick-End Labeling, Kinetics, Male, Necrosis, Nitric Oxide analysis, Nitric Oxide Synthase analysis, Nitric Oxide Synthase antagonists & inhibitors, Nitric Oxide Synthase Type II, Rats, Rats, Wistar, Spermatogenesis drug effects, Testis chemistry, Nitric Oxide pharmacology, Spermatic Cord Torsion pathology, Spermatozoa pathology
- Abstract
The purpose of this study is to determine whether inducible nitric oxide synthase (iNOS) is involved in the pathogenesis of testicular ischemia-reperfusion (I/R) injury in association with germ cell death, through either necrosis or apoptosis. Western blot analysis showed that iNOS expression was markedly increased 1 h after ischemia, and was accompanied by a huge nitric oxide (NO) production, as measured by the Griess method, with a peak at 48 h of reperfusion. Immunohistochemistry showed that iNOS was expressed predominantly in the macrophage-like cells infiltrated in the interstitial tissues of the testis. Intraperitoneal injection of aminoguanidine (AMG) (400 mg/day), the inhibitor of iNOS, reduced NO production by 57.7% at 96 h of reperfusion. Calpain activation and proteolysis of alpha-fodrin induced by I/R were inhibited by AMG. Germ cell apoptosis was demonstrated by in situ TUNEL and DNA fragmentation on agarose gel electrophoresis. Germ cell apoptosis was maximally induced at 24 h of reperfusion, and was not inhibited by AMG. NO produced by iNOS in the delayed phase of reperfusion promoted alpha-fodrin proteolysis, which is closely associated with necrosis. Inducible NOS inhibition combined with calpain inhibition may improve impaired spermatogenesis after testicular torsion. more...
- Published
- 2001
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18. Inhibition of calpain but not caspase protects the testis against injury after experimental testicular torsion of rat.
- Author
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Shiraishi K, Naito K, and Yoshida K
- Subjects
- Animals, Apoptosis drug effects, DNA Fragmentation, Electrophoresis, Polyacrylamide Gel, Flow Cytometry, Immunohistochemistry, In Situ Nick-End Labeling, Male, Necrosis, Organ Size drug effects, Protease Inhibitors pharmacology, Rats, Rats, Wistar, Reperfusion Injury drug therapy, Reperfusion Injury pathology, Spermatogenesis drug effects, Calpain antagonists & inhibitors, Caspase Inhibitors, Enzyme Inhibitors therapeutic use, Spermatic Cord Torsion drug therapy, Spermatic Cord Torsion pathology, Testis pathology
- Abstract
Testicular torsion requires emergent release of the twisted spermatic cord. Ischemia/reperfusion (I/R) plays an important role in its pathogenesis, and recent data suggest that germ cells undergo apoptosis during I/R. In a model of torsion/detorsion (i.e., I/R) of the rat testis, involvement of calpain and caspase in necrotic and apoptotic cell death was examined. After 1 h of ischemia followed by 0, 0.5, 1, 6, or 24 h of reperfusion, the germ cells positively stained with in situ TUNEL, and DNA fragmentation, activation of caspase-3, and proteolysis of caspase substrates increased with time of reperfusion, demonstrating apoptosis. In addition, m-calpain activation and proteolysis of alpha-fodrin were increased during reperfusion, and its activation is thought to be involved in the necrosis. A calpain inhibitor, acety-leucyl-leucyl-norleucinal, inhibited the phenomena associated with apoptosis and necrosis induced by I/R, although a caspase inhibitor, Z-Val-Ala-Asp-fluoromethlyketone, only inhibited apoptotic changes. The inhibition of calpain but not caspase ameliorated the injury after 60 days of reperfusion following 1 h of ischemia. The calpain inhibitor injected just before reperfusion effectively suppressed alpha-fodrin proteolysis, suggesting its usefulness in the treatment of testicular torsion. more...
- Published
- 2000
- Full Text
- View/download PDF
19. Maturation/M-phase promoting factor: a regulator of aging in porcine oocytes.
- Author
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Kikuchi K, Naito K, Noguchi J, Shimada A, Kaneko H, Yamashita M, Aoki F, Tojo H, and Toyoda Y
- Subjects
- Animals, CDC2 Protein Kinase metabolism, Caffeine pharmacology, Cells, Cultured, Female, Histones metabolism, Meiosis, Metaphase, Phosphorylation, Swine, Time Factors, Vanadates pharmacology, Cellular Senescence drug effects, Maturation-Promoting Factor physiology, Oocytes physiology
- Abstract
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. more...
- Published
- 2000
- Full Text
- View/download PDF
20. Mitogen-activated protein kinase translocates into the germinal vesicle and induces germinal vesicle breakdown in porcine oocytes.
- Author
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Inoue M, Naito K, Nakayama T, and Sato E
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinases administration & dosage, Cell Nucleus metabolism, Cytoplasm enzymology, Electrophoresis, Polyacrylamide Gel, Female, Fluorescent Antibody Technique, Microinjections, Oocytes ultrastructure, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Meiosis, Oocytes enzymology, Swine
- Abstract
The role of mitogen-activated protein kinase (MAPK) in the meiotic resumption of porcine oocytes was examined. First, using indirect immunofluorescence staining with a specific antibody against rat MAPK, we monitored the dynamics of the subcellular distribution of MAPK during meiosis initiation. We found that the inactive MAPK was already present in immature oocytes arrested at the G2 stage and that this inactive kinase was localized exclusively in the cytoplasm. At the G2/M transition stage, part of the MAPK moved into the germinal vesicle (GV) before germinal vesicle breakdown (GVBD). In addition, immunoblot analysis showed that the nuclear MAPK existed in an active form. To determine whether this active MAPK could induce GVBD, we microinjected active MAPK into immature porcine oocytes. The active MAPK injected into the cytoplasm was quickly inactivated and could not accelerate GVBD. In contrast, MAPK injection into the GV markedly accelerated GVBD. These results show that in porcine oocytes, 1) inactive MAPK localizes in the cytosol of immature GV oocytes, 2) part of the activated MAPK translocates into the GV just before GVBD, and 3) exogenous MAPK maintains its activity level in the GV and induces GVBD, indicating that MAPK mediates the maturation-inducing signal from the cytoplasm into the nucleus and induces meiosis reinitiation. more...
- Published
- 1998
- Full Text
- View/download PDF
21. Meiotic abnormalities of c-mos knockout mouse oocytes: activation after first meiosis or entrance into third meiotic metaphase.
- Author
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Araki K, Naito K, Haraguchi S, Suzuki R, Yokoyama M, Inoue M, Aizawa S, Toyoda Y, and Sato E
- Subjects
- Animals, Calcium-Calmodulin-Dependent Protein Kinases metabolism, Enzyme Activation, Genotype, Interphase, Kinetics, Mesothelin, Metaphase, Mice, Mice, Knockout, Oocytes physiology, Phosphorylation, Protein Kinases metabolism, Meiosis genetics, Oocytes cytology, Proto-Oncogene Proteins c-mos genetics
- Abstract
In Xenopus oocytes, Mos activates the mitogen-activated protein kinase (MAPK) signal transduction cascade and regulates meiosis. In mammalian oocytes, however, the functions of Mos are still unclear. In the present study, we used c-mos knockout mouse oocytes and examined the roles of Mos in mouse oocyte maturation and fertilization, including whether Mos controls MAPK and maturation promoting factor (MPF) activity. The kinetics of germinal vesicle breakdown (GVBD) and the first polar body emission were similar in wild-type, heterozygous mutant, and homozygous mutant mice. Activities of MPF were also not significantly different among the three genotypes until the first polar body emission. In contrast, MAPK activity in c-mos knockout oocytes did not significantly fluctuate throughout maturation, and the oocytes had abnormal diffused spindles and loosely condensed chromosomes, although a clear increase in MAPK activities was observed after GVBD in wild-type and heterozygous mutant oocytes that had normal spindles and chromosomes. After the first polar body emission, 38% of c-mos knockout oocytes formed a pronucleus instead of undergoing second meiosis, indicating the crucial role of Mos in MPF reactivation after first meiosis. When oocytes that reached second metaphase were fertilized or stimulated by ethanol, many c-mos knockout oocytes emitted a second polar body and progressed into third meiotic metaphase instead of interphase, although all fertilized or activated oocytes in the heterozygote progressed to interphase, indicating that Mos deletion leads to compensatory factors that might not be degraded after fertilization or parthenogenetic activation. These results suggest that Mos is located upstream of MAPK in mouse oocytes as in Xenopus oocytes but is independent of MPF activity, and that Mos/MAPK is not necessary go GVBD and first polar body emission. Our results also suggest that Mos plays a crucial role in normal spindle and chromosome morphology and the reactivation of MPF after first meiosis. more...
- Published
- 1996
- Full Text
- View/download PDF
22. Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states.
- Author
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Haraguchi S, Naito K, Azuma S, Sato E, Nagahama Y, Yamashita M, and Toyoda Y
- Subjects
- Animals, CDC2 Protein Kinase antagonists & inhibitors, Cell Line, Embryo, Mammalian drug effects, Enzyme Inhibitors pharmacology, Female, Fertilization in Vitro, Glucose pharmacology, Histones metabolism, Hybridization, Genetic, Immunoblotting, Maturation-Promoting Factor physiology, Mesothelin, Mice, Mice, Inbred AKR, Okadaic Acid pharmacology, Phosphorylation, Protamine Kinase metabolism, CDC2 Protein Kinase metabolism, Embryo, Mammalian cytology, Phosphates pharmacology
- Abstract
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. more...
- Published
- 1996
- Full Text
- View/download PDF
23. Effect of a partial deletion of Y chromosome on in vitro fertilizing ability of mouse spermatozoa.
- Author
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Xian M, Azuma S, Naito K, Kunieda T, Moriwaki K, and Toyoda Y
- Subjects
- Animals, Cell Separation, Female, Fertilization in Vitro, Hybridization, Genetic, Male, Mice, Mice, Inbred C57BL, Povidone, Silicon Dioxide, Sperm Capacitation, Chromosome Deletion, Spermatozoa physiology, Y Chromosome
- Abstract
The effect of a partial deletion of Y chromosome on sperm fertilizing ability was investigated through an in vitro fertilization technique. Epididymal spermatozoa of a congenic line, B10.BR-Ydel, which is characterized by a high incidence of abnormal spermatozoa, revealed a significantly lower in vitro fertilization rate (22%) than that (79%) of its control strain (B10.BR/SgSn), which has a normal-sized Y chromosome. Incidence of capacitated spermatozoa as determined by chlortetracycline fluorescence assay was significantly lower in B10.BR-Ydel than in B10.BR/SgSn spermatozoa. The fertilization rate was significantly improved when B10.BR-Ydel spermatozoa were separated from the supernatant of sperm suspension by Percoll gradient centrifugation. A reconstitution experiment revealed that the B10.BR-Ydel spermatozoa were more sensitive to the inhibitory effect of the supernatant than B10.BR/SgSn spermatozoa. Spermatozoa from F1 (C57BL/6N male x B10.BR-Ydel female) males showed higher fertilization rates than those from F1 (B10.BR.Ydel male x C57BL/6N female) males. These observations suggest that not only the morphology but also the fertilizing ability of spermatozoa is directly related to partial deletion of Y chromosome. more...
- Published
- 1992
- Full Text
- View/download PDF
24. Comparison of histone H1 kinase activity during meiotic maturation between two types of porcine oocytes matured in different media in vitro.
- Author
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Naito K, Daen FP, and Toyoda Y
- Subjects
- Animals, Cell Cycle physiology, Cells, Cultured, Culture Media chemistry, Female, Follicular Fluid physiology, Isotonic Solutions analysis, Isotonic Solutions pharmacology, Metaphase physiology, Oocytes drug effects, Oocytes physiology, Swine, Time Factors, Culture Media pharmacology, Maturation-Promoting Factor physiology, Meiosis physiology, Oocytes enzymology
- Abstract
Histone H1 kinase (H1K) activity was assayed during meiotic maturation in porcine oocytes matured in a modified Krebs-Ringer bicarbonate solution (KRB) or in porcine follicular fluid (pFF) in vitro. Oocytes matured in KRB displayed lower male pronucleus formation ability, delayed first polar body emission, and a higher spontaneous activation rate than oocytes matured in pFF. In oocytes matured in pFF, H1K activity was low at the germinal vesicle stage and increased about 8-fold at first and second metaphases, with a transient depression at first anaphase and telophase. The H1K activity at second metaphase in oocytes matured in KRB was significantly lower than that in oocytes matured in pFF. These results suggest that the maturation medium used influences the fluctuation pattern of H1K activity and the biological characteristics of porcine oocytes cultured in vitro. more...
- Published
- 1992
- Full Text
- View/download PDF
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