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Shail K Chaube - One of the best experts on this subject based on the ideXlab platform.

  • carbenoxolone reduces cyclic nucleotides level destabilizes Maturation Promoting Factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro
    Biomedicine & Pharmacotherapy, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Abstract Background Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing Maturation Promoting Factor (MPF) in rat oocytes cultured in vitro . Methods Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3 h in vitro . The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25 B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. Results CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro . Conclusions Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic Maturation under in vitro culture conditions during assisted reproductive technology (ART) programs.

  • Maturation Promoting Factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes
    Development Growth & Differentiation, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic Maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase-Promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin-dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in Maturation Promoting Factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M-II arrest in rat oocytes.

  • reduction of nitric oxide level results in Maturation Promoting Factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro
    Development Growth & Differentiation, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream Factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to Maturation Promoting Factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.

  • Role of Mitogen Activated Protein Kinase and Maturation Promoting Factor During the Achievement of Meiotic Competency in Mammalian Oocytes.
    Journal of cellular biochemistry, 2017
    Co-Authors: Meenakshi Tiwari, Shilpa Prasad, Ashutosh N Pandey, Anumegha Gupta, Alka Sharma, Pramod K. Yadav, Ajai K. Pandey, Tulsidas G. Shrivastav, Shail K Chaube
    Abstract:

    The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic Maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of Maturation Promoting Factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription Factors in cumulus cells or cytostatic Factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.

  • Maturation Promoting Factor destabilization facilitates postovulatory aging mediated abortive spontaneous egg activation in rat
    Development Growth & Differentiation, 2016
    Co-Authors: Shilpa Prasad, Biplob Koch, Shail K Chaube
    Abstract:

    The present study was designed to investigate whether destabilization of Maturation Promoting Factor (MPF) leads to postovulatory aging-mediated abortive spontaneous egg activation (SEA). If so, we wished to determine whether changes in Wee-1 as well as Emi2 levels are associated with MPF destabilization during postovulatory aging-mediated abortive SEA in rats eggs aged in vivo. For this purpose, sexually immature female rats were given a single injection (20 IU IM) of pregnant mare serum gonadotropin for 48 h followed by single injection of human chorionic gonadotropin (20 IU). Ovulated eggs were collected after 14, 17, 19 and 21 h post-hCG surge to induce postovulatory aging in vivo. The morphological changes, Wee1, phosphorylation status of cyclin dependent kinase 1(Cdk1), early mitotic inhibitor 2 (Emi2), anaphase Promoting complex/cyclosome (APC/C), cyclin B1, mitotic arrest deficient protein (MAD2) levels and Cdk1 activity were analyzed. The increased Wee 1 level triggered phosphorylation of Thr-14/Tyr-15 and dephosphorylation of Thr-161 residues of Cdk1. The decrease of Emi2 level was associated with increased APC/C level and decreased cyclin B1 level. Changes in phosphorylation status of Cdk1 and reduced cyclin B1 level resulted in destabilization of MPF. The destabilized MPF finally led to postovulatory aging-mediated abortive SEA in rat eggs. It was concluded that the increase of Wee 1 but decrease of Emi2 level triggers MPF destabilization and thereby postovulatory aging-mediated abortive SEA in rat eggs.

Meenakshi Tiwari - One of the best experts on this subject based on the ideXlab platform.

  • carbenoxolone reduces cyclic nucleotides level destabilizes Maturation Promoting Factor and induces meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro
    Biomedicine & Pharmacotherapy, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Abstract Background Disruption of gap junction and transfer of cyclic nucleotides to the oocyte lead to meiotic exit from diplotene arrest (EDA) in mammals. In the present study, we examined whether a gap junction blocker, carbenoxolone (CBX) could induce EDA by reducing cyclic nucleotides level and destabilizing Maturation Promoting Factor (MPF) in rat oocytes cultured in vitro . Methods Diplotene-arrested cumulus oocyte complexes (COCs) were collected from ovary of immature female rats after 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. These diplotene-arrested COCs were incubated with various concentration of CBX for 3 h in vitro . The morphological changes, meiotic status of oocyte, inducible nitric oxide synthase (iNOS), total nitric oxide (NO), adenosine 3′,5′-cyclic monophosphate (cAMP), guanosine 3′,5′-cyclic monophosphate (cGMP), cell division cycle 25 B (Cdc25B), changes in specific phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and cyclin B1 levels were analyzed. Results CBX induced EDA in a concentration-dependent manner. The iNOS expression, total NO and cyclic nucleotides level were significantly decreased. The reduced cyclic nucleotides level resulted in the decrease of Cdc25B expression level. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated Cdk1 level. However, Thr161 phosphorylated Cdk1 as well as cyclin B1 levels were significantly reduced leading to MPF destabilization. The destabilized MPF finally induced EDA in rat COCs cultured in vitro . Conclusions Our results suggest that CBX blocked gap junctions interrupted the transfer of cyclic nucleotides to the oocyte. Reduction of cyclic nucleotides level destabilized MPF and induced EDA in vitro. Thus, CBX could be used to induce meiotic Maturation under in vitro culture conditions during assisted reproductive technology (ART) programs.

  • Maturation Promoting Factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes
    Development Growth & Differentiation, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic Maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase-Promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin-dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase-II (M-II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in Maturation Promoting Factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M-II arrest in rat oocytes.

  • reduction of nitric oxide level results in Maturation Promoting Factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro
    Development Growth & Differentiation, 2017
    Co-Authors: Meenakshi Tiwari, Shail K Chaube
    Abstract:

    Nitric oxides (NO) act as one of the major signal molecules and modulate various cell functions including oocyte meiosis in mammals. The present study was designed to investigate the mechanism of NO action during spontaneous meiotic exit from diplotene arrest (EDA) in rat cumulus oocytes complexes (COCs) cultured in vitro. Diplotene-arrested COCs collected from ovary of immature female rats after 20 IU pregnant mare's serum gonadotropins (PMSG) for 48 h were exposed to various concentrations of NO donor, S-nitroso-N-acetyl penicillamine (SNAP) and inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG) for 3 h in vitro and downstream Factors were analyzed. Our results suggest that SNAP inhibited, while AG induced EDA in a concentration-dependent manner. The iNOS-mediated total NO, cyclic nucleotides and cell division cycle 25B (Cdc25B) levels were reduced significantly. The decreased Cdc25B was associated with the increased Thr14/Tyr15 phosphorylated cyclin-dependent kinase 1 (Cdk1) level and decreased Thr161 phosphorylated Cdk1 as well as cyclin B1 levels leading to Maturation Promoting Factor (MPF) destabilization. The destabilized MPF finally induced spontaneous EDA. Taken together, these results suggest that reduction of iNOS-mediated NO level destabilizes MPF during spontaneous EDA in rat COCs cultured in vitro.

  • Role of Mitogen Activated Protein Kinase and Maturation Promoting Factor During the Achievement of Meiotic Competency in Mammalian Oocytes.
    Journal of cellular biochemistry, 2017
    Co-Authors: Meenakshi Tiwari, Shilpa Prasad, Ashutosh N Pandey, Anumegha Gupta, Alka Sharma, Pramod K. Yadav, Ajai K. Pandey, Tulsidas G. Shrivastav, Shail K Chaube
    Abstract:

    The oocyte quality remains as one of the major problems associated with poor in vitro fertilization (IVF) rate and assisted reproductive technology (ART) failure worldwide. The oocyte quality is dependent on its meiotic Maturation that begins inside the follicular microenvironment and gets completed at the time of ovulation in most of the mammalian species. Follicular oocytes are arrested at diplotene stage of first meiotic prophase. The resumption of meiosis from diplotene arrest, progression through metaphase-I (M-I) and further arrest at metaphase-II (M-II) are important physiological requirements for the achievement of meiotic competency in mammalian oocytes. The achievement of meiotic competency is dependent upon cyclic stabilization/destabilization of Maturation Promoting Factor (MPF). The mitogen-activated protein kinase3/1 (MAPK3/1) modulates stabilization/destabilization of MPF in oocyte by interacting either with signal molecules, transcription and post-transcription Factors in cumulus cells or cytostatic Factors (CSFs) in oocyte. MPF regulates meiotic cell cycle progression from diplotene arrest to M-II arrest and directly impacts oocyte quality. The MAPK3/1 activity is not reported during spontaneous meiotic resumption but its activity in cumulus cells is required for gonadotropin-induced oocyte meiotic resumption. Although high MAPK3/1 activity is required for the maintenance of M-II arrest in several mammalian species, its cross-talk with MPF remains to be elucidated. Further studies are required to find out the MAPK3/1 activity and its impact on MPF destabilization/stabilization during achievement of meiotic competency, an important period that decides oocyte quality and directly impacts ARTs outcome in several mammalian species including human. J. Cell. Biochem. 119: 123-129, 2018. © 2017 Wiley Periodicals, Inc.

  • changes in signal molecules and Maturation Promoting Factor levels associate with spontaneous resumption of meiosis in rat oocytes
    Cell Biology International, 2015
    Co-Authors: Shilpa Prasad, Meenakshi Tiwari, Anima Tripathi, Ashutosh N Pandey, Shail K Chaube
    Abstract:

    The present study was aimed to find out changes in signal molecules and Maturation Promoting Factor (MPF) levels during meiotic cell cycle progression from diplotene and metaphase-II (M-II) arrest, a period during which oocyte achieves meiotic competency. Data suggest that high levels of adenosine 3'-5'-cyclic monophosphate (cAMP), guanosine 3'-5'-cyclic monophosphate (cGMP), and nitric oxide (NO) are associated with diplotene arrest, while reduction in their levels correlates with reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increased intracellular NO, calcium (Ca(2+) ) as well as hydrogen peroxide (H2 O2 ) levels correlate with decreased cAMP, Thr-161 phosphorylated cyclin-dependent kinase-1 (Cdk1) as well as cyclin B1 levels. The decreased Thr-161 phosphorylated Cdk1 and cyclin B1 level reduce MPF level leading to exit from M-II arrest in oocytes cultured in vitro. These data suggest that the decrease of cAMP level and increase of cytosolic free Ca(2+) as well as H2 O2 levels associate with the reduced MPF level and meiotic resumption from diplotene arrest. On the other hand, increase of NO, cGMP, Ca(2+) as well as H2 O2 levels are associated with reduced MPF and spontaneous exit from M-II arrest in rat oocytes cultured in vitro.

Qing-yuan Sun - One of the best experts on this subject based on the ideXlab platform.

  • chronic cadmium exposure causes oocyte meiotic arrest by disrupting spindle assembly checkpoint and Maturation Promoting Factor
    Reproductive Toxicology, 2020
    Co-Authors: Feng Dong, Yingchun Ouyang, Zhenbo Wang, Yi Hou, Heide Schatten, Wenlong Lei, Feng Wang, Yue Wang, Qing-yuan Sun
    Abstract:

    Cadmium (Cd) is a bioaccumulative heavy metal element with potential toxicity on the female reproductive system, but the exact molecular mechanisms have not yet been clearly defined. In this study, female mice were exposed to 0.5 mg/kg/day of CdCl2 for 60 consecutive days. We found that chronic Cd exposure significantly decreased the fecundity of female mice by affecting oocyte meiotic progression as indicated by disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, consequently resulting in aneuploid oocytes. Further studies showed that the periodic fluctuations of MPF activity and cyclin B1 expression were disturbed in Cd-exposed oocytes probably by affecting the spindle assembly checkpoint protein Bub3. In addition, Cd exposure induced oxidative stress as indicated by an increased level of reactive oxygen species and apoptosis in oocytes, leading to oocyte quality deterioration. Taken together, these data suggest that Cd exposure causes disrupted molecular events of meiotic progression and deterioration of oocyte quality via oxidative stress, leading to decrease of female fertility.

  • cyclins regulating oocyte meiotic cell cycle progression
    Biology of Reproduction, 2019
    Co-Authors: Weiping Qian, Qing-yuan Sun
    Abstract:

    Oocyte meiotic Maturation is a vital and final process in oogenesis. Unlike somatic cells， the oocyte needs to undergo two continuous meiotic divisions (meiosis I and meiosis II) to become a haploid gamete. Notably, oocyte meiotic progression includes two rounds of unique meiotic arrest and resumption. The first arrest occurs at the G2 (germinal vesicle) stage and meiosis resumption is stimulated by a gonadotropin surge; the second arrest takes place at the metaphase II stage, the stage from which it is released when fertilization takes place. The Maturation-Promoting Factor, which consists of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDK1), is responsible for regulating meiotic resumption and progression, while CDK1 is the unique CDK that acts as the catalytic subunit of Maturation-Promoting Factor. Recent studies showed that except for cyclin B1, multiple cyclins interact with CDK1 to form complexes, which are involved in the regulation of meiotic progression at different stages. Here, we review and discuss the control of oocyte meiotic progression by cyclins A1, A2, B1, B2, B3, and O.

  • cyclin o regulates germinal vesicle breakdown in mouse oocytes
    Biology of Reproduction, 2013
    Co-Authors: Yingchun Ouyang, Yibo Luo, Zhenbo Wang, Yi Hou, Zhiming Han, Zhonghua Liu, Heide Schatten, Qing-yuan Sun
    Abstract:

    It is well accepted that oocyte meiotic resumption is mainly regulated by the Maturation-Promoting Factor (MPF), which is composed of cyclin B1 (CCNB1) and cyclin-dependent kinase 1 (CDC2). Maturation-Promoting Factor activity is regulated by the expression level of CCNB1, phosphorylation of CDC2, and their germinal vesicle (GV) localization. In addition to CCNB1, cyclin O (CCNO) is highly expressed in oocytes, but its biological functions are still not clear. By employing short interfering RNA microinjection of GV-stage oocytes, we found that Ccno knockdown inhibited CDC2 (Tyr15) dephosphorylation and arrested oocytes at the GV stage. To rescue meiotic resumption, cell division cycle 25 B kinase (Cdc25b) and Ccnb1 were overexpressed in the Ccno knockdown oocytes. Unexpectedly, we found that Ccno knockdown did not affect CDC25B entry into the GV, and overexpression of CDC25B was not able to rescue resumption of oocyte meiosis. However, GV breakdown (GVBD) was significantly increased after overexpression of Ccnb1 in Ccno knockdown oocytes, indicating that GVBD block caused by cyclin O knockdown can be rescued by cyclin B1 overexpression. We thus conclude that cyclin O, as an upstream regulator of MPF, plays an important role in oocyte meiotic resumption in mouse oocytes.

  • function and interaction of Maturation Promoting Factor and mitogen activated protein kinase during meiotic Maturation and fertilization of oocyte
    Progress in Natural Science, 2004
    Co-Authors: Lijun Huo, Hengyu Fan, Dayuan Chen, Qing-yuan Sun
    Abstract:

    Abstract Mitogen-activated protein kinase (MAP kinase) cascade and Maturation-Promoting Factor (MPF) play very important roles during meiotic Maturation and fertilization of oocyte. Interaction between MAP kinase and MPF influences meiotic Maturation and fertilization of oocyte throughout the animal kingdom, including stimulation of germinal vesicle breakdown (GVBD), suppression of DNA replication, control of meiotic chromosome segregation, maintenance of metaphase II arrest, and resumption and completion of second meiosis. This review focuses on the function and interaction of MAP kinase and MPF during meiotic Maturation and fertilization of oocyte.

Yoshitaka Nagahama - One of the best experts on this subject based on the ideXlab platform.

  • molecular mechanisms of the activation of Maturation Promoting Factor during goldfish oocyte Maturation
    Developmental Biology, 1995
    Co-Authors: Masakane Yamashita, Shinji Onoe, Hiroko Kajiura, Toyomi Tanaka, Yoshitaka Nagahama
    Abstract:

    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.

  • a fish homolog of the cdc2 related protein p40 m015 its cdna cloning and expression in oocytes
    Biomedical Research-tokyo, 1993
    Co-Authors: Shinji Onoe, Masakane Yamashita, Hiroko Kajiura, Yoshinao Katsu, Jiang Jianquao, Yoshitaka Nagahama
    Abstract:

    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 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-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 MO15 , 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

  • isolation and characterization of goldfish cdc2 a catalytic component of Maturation Promoting Factor
    Development Growth & Differentiation, 1993
    Co-Authors: Hiroko Kajiura, Masakane Yamashita, Yoshinao Katsu, Yoshitaka Nagahama
    Abstract:

    We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.

  • Behavior of the components of Maturation-Promoting Factor, cdc2 kinase and cyclin B, during oocyte Maturation of goldfish.
    Developmental biology, 1993
    Co-Authors: Yoshinao Katsu, Masakane Yamashita, Hiroko Kajiura, Yoshitaka Nagahama
    Abstract:

    Abstract We examined the changes that occurred in the two components of Maturation-Promoting Factor (MPF), cdc2 kinase and cyclin B, during oocyte Maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13sucl beads and anti-cyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2μg/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte Maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus , an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.

  • Purification and characterization of Maturation-Promoting Factor in fish.
    Developmental biology, 1992
    Co-Authors: Masakane Yamashita, Sachiko Fukada, Michiyasu Yoshikuni, Philippe Bulet, Toshiaki Hirai, Akihiko Yamaguchi, Y.-h. Lou, Z. Zhao, Yoshitaka Nagahama
    Abstract:

    Abstract Maturation-Promoting Factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000 g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13 sucl -affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli -produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32 P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.

Masakane Yamashita - One of the best experts on this subject based on the ideXlab platform.

  • 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
    Developmental Biology, 1997
    Co-Authors: Tomoko Kondo, Toshiharu Yanagawa, Noriyuki Yoshida, Masakane Yamashita
    Abstract:

    When treated with 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-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, 17alpha,20beta-DP is unable to induce growing (previtellogenic and vitellogenic) oocytes to mature. To know the reason growing oocytes fail to mature upon 17alpha,20beta-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 17alpha,20beta-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 17alpha,20beta-DP is attributable to a deficiency in the processes leading to cyclin B synthesis, which include 17alpha,20beta-DP reception on the oocyte surface, subsequent signal transduction pathways, and unmasking the stored cyclin B mRNA.

  • molecular mechanisms of the activation of Maturation Promoting Factor during goldfish oocyte Maturation
    Developmental Biology, 1995
    Co-Authors: Masakane Yamashita, Shinji Onoe, Hiroko Kajiura, Toyomi Tanaka, Yoshitaka Nagahama
    Abstract:

    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.

  • a fish homolog of the cdc2 related protein p40 m015 its cdna cloning and expression in oocytes
    Biomedical Research-tokyo, 1993
    Co-Authors: Shinji Onoe, Masakane Yamashita, Hiroko Kajiura, Yoshinao Katsu, Jiang Jianquao, Yoshitaka Nagahama
    Abstract:

    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 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-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 MO15 , 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

  • isolation and characterization of goldfish cdc2 a catalytic component of Maturation Promoting Factor
    Development Growth & Differentiation, 1993
    Co-Authors: Hiroko Kajiura, Masakane Yamashita, Yoshinao Katsu, Yoshitaka Nagahama
    Abstract:

    We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.

  • Behavior of the components of Maturation-Promoting Factor, cdc2 kinase and cyclin B, during oocyte Maturation of goldfish.
    Developmental biology, 1993
    Co-Authors: Yoshinao Katsu, Masakane Yamashita, Hiroko Kajiura, Yoshitaka Nagahama
    Abstract:

    Abstract We examined the changes that occurred in the two components of Maturation-Promoting Factor (MPF), cdc2 kinase and cyclin B, during oocyte Maturation in goldfish, using monoclonal antibodies against the C-terminal sequence of goldfish cdc2 kinase and Escherichia coli-produced full-length goldfish cyclin B. Immature oocytes contained a 35-kDa inactive cdc2 kinase. In addition to the 35-kDa form, a 34-kDa active cdc2 kinase was detected in oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in immature oocytes and appeared just before GVBD, coinciding exactly with the appearance of the 34-kDa active cdc2 kinase. Precipitation with p13sucl beads and anti-cyclin B antibody revealed that cyclin B formed a complex with cdc2 kinase as soon as it appeared. MPF activation was induced by 1 ng cyclin B after introduction into immature oocytes or oocyte extracts. This corresponds to the amount of cyclin B found in mature oocytes (the concentration in the oocyte is 2μg/ml). These results suggest that MPF activation in fish oocytes is induced by complex formation with preexisting cdc2 kinase and newly synthesized cyclin B during oocyte Maturation, a situation differing from that in Xenopus and starfish, in which the cdc2 kinase-cyclin B complex is already present in immature oocytes. Unlike that in Xenopus , an inhibition of protein synthesis in unfertilized mature goldfish oocytes caused a decrease in the cdc2 kinase activity/cyclin B protein level and led to a progression from meiotic metaphase to meiotic anaphase. This result indicates that the mechanisms of maintaining MPF activity in mature goldfish oocytes differ from those in Xenopus.