Regulation of Mammalian Oocyte Maturation

哺乳动物卵母细胞成熟的调节

• 批准号: 7031009
• 负责人: STEPHEN M DOWNS
• 金额: $ 15.93万
• 依托单位: MARQUETTE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-02 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031009
• 关键词: cell cycle; cell growth regulation; cyclic AMP; egg /ovum; enzyme activity; gonadotropins; histones; immunocytochemistry; laboratory mouse; mitogen activated protein kinase; phosphodiesterases; protein kinase A; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): A major goal of my research program is to identify molecules and metabolic pathways that participate in the control of oocyte maturation in mammals. In our model for meiotic induction, gonadotropin triggers an increase in cAMP phosphodiesterase (PDE) that induces meiotic resumption by both a loss of cAMP-dependent protein kinase activity and an increase in AMP-activated protein kinase (AMPK) activity. The oocyte-cumulus cell complex, isolated from mice 48 h after hormonal priming, will be used as the principal model system, but oocytes induced to mature in vivo by hCG injection will also be examined. Isoform-specific PDE inhibitors will be used to manipulate meiosis according to the cellular target site, and PDE activity will be measured to test for a cause-and-effect relationship with maturation-promoting factor (MPF; measured as histone H1 kinase activity) and germinal vesicle breakdown (GVB). Western analysis of AMPK (beta, gamma subunits) in complexes will be completed, and the temporal relationship between AMPK activation, MPF activation and GVB during spontaneous maturation or maturation induced by adenosine analogs or hormones will be tested. Oocyte microinjection will determine if active AMPK induces GVB and if antibodies to alpha subunits antagonize meiotic induction. Antisera to alpha subunits of AMPK as well as an anti-phosphoantibody to active AMPK will be employed to localize the enzyme in oocytes by indirect immunofluorescence during meiotic resumption. We will also investigate the potential role of ERK1/2 and p38 mitogen-activated protein kinases in mediating the meiosis-inducing action of AMPK. These results will help delineate specific metabolic pathways involved in meiotic maturation and will have important implications for both fertility and contraception, since each is affected by the ability of the oocyte to successfully initiate and complete meiotic maturation. They may also help in preventing or minimizing erroneous meiotic control that can give rise to aneuploidy and its disastrous consequences. Furthermore, these studies will benefit the development of in vitro systems where either meiotic arrest or completion of meiotic maturation is the desired endpoint.

描述（由申请人提供）：我的研究计划的一个主要目标是确定参与哺乳动物卵母细胞成熟的分子和代谢途径。在我们的减数分裂诱导模型中，促性腺激素触发了CAMP磷酸二酯酶（PDE）的增加，从而通过CAMP依赖性蛋白激酶活性的丧失和AMP激活的蛋白激酶（AMPK）活性诱导减数分裂恢复。激素启动后48小时从小鼠中分离出的卵母细胞 - 肿瘤细胞复合物将被用作主要模型系统，但还将检查由HCG注射在体内诱导成熟的卵母细胞。根据细胞靶位点，将使用同工型特异性PDE抑制剂来操纵减数分裂，并测量PDE活性以测试与成熟促进因子（MPF; MPF;以组蛋白H1激酶活性测量）和生发性囊泡分解（GVB）的因果关系（MPF；测量为组蛋白H1激酶活性）。将完成对复合物中AMPK（β，伽马亚基）的西方分析，并且在自发成熟或由腺苷类似物或激素引起的自发成熟或成熟期间AMPK激活，MPF激活与GVB之间的时间关系将进行测试。卵母细胞显微注射将确定活性AMPK是否诱导GVB和α亚基的抗体是否拮抗减数分裂诱导。对AMPK的α亚基以及对活性AMPK的抗磷酸抗体的抗血清将通过减数分裂恢复期间间接免疫荧光将酶定位在卵母细胞中。我们还将研究ERK1/2和P38促丝分裂原激活蛋白激酶在介导AMPK诱导减数分裂作用中的潜在作用。这些结果将有助于描述与减数分裂成熟有关的特定代谢途径，并将对生育和避孕剂具有重要意义，因为每种途径都受到卵母细胞成功启动和完成减数分裂成熟的能力的影响。它们还可能有助于防止或最小化错误的减数分裂控制，从而导致非整倍性及其灾难性后果。此外，这些研究将使体外系统的发展有益于减数分裂停滞或减数分裂成熟是所需的终点。