Molecular Mechanisms of Meiotic Maturation in Drosophila

描述(申请人提供)：减数分裂是与配子发育相关的一个基本过程。卵母细胞停滞在第一次减数分裂的前期，以便生长。前期I的释放发生在减数分裂成熟过程中，但调控这一过程的分子机制尚不完全清楚。减数分裂成熟缺陷可导致不育或染色体分离不准确，导致流产和/或出生缺陷。果蝇提供了复杂的遗传和分子工具，以及描述良好的卵巢细胞生物学，使其成为研究减数分裂成熟控制的强大系统。果蝇卵母细胞在胚系包囊形成后不久进入减数分裂，并在大部分发育的前期I停滞。减数分裂成熟后，卵母细胞在中期I再次停滞，直到排卵。在之前的资助期，我们证明了减数分裂成熟需要1-内硫醚(endosulfinin，Endos)。我们的数据表明，Endos促进细胞周期调节因子CyclinA、Polo和Twin/CDC25(都是后期促进复合体(APC)的靶标)的稳定性，并且它还通过抑制E3泛素连接酶ELGI促进减数分裂成熟而发挥单独的作用。人的1-内磺素ENSA的表达挽救了小鼠卵母细胞的减数分裂缺陷，1-内磺素在小鼠卵母细胞中表达，表明1-内磺素的减数分裂功能是保守的。我们的主要假设是，Endos是减数分裂成熟的中央调节因子，通过两种机制：它抑制APC的活性，允许关键的细胞周期调节因子的积累，以及它拮抗ELGI靶标的泛素化。我们将通过以下具体目标来检验这一假说：1)确定Endos是否是APC的负调控因子；2)确定E3泛素连接酶Elgi如何调控Endos下游的减数分裂成熟；3)阐明Endos的分子作用机制。减数分裂成熟的分子机制的阐明可能导致解决这一不孕原因的治疗方法，以及新的避孕措施。此外，这一知识还可能导致人类卵母细胞体外成熟的技术策略，以补充目前的体外受精方法。 公共卫生相关性：卵母细胞不能进行减数分裂成熟会导致不孕。我们之前的工作已经确定Endos是减数分裂成熟的主要调节因子，我们建议在果蝇中使用强大的研究工具来了解该蛋白的功能。我们的研究可能会带来解决不孕症的其他治疗方法，以及补充当前体外受精方法的技术策略。

DESCRIPTION (provided by applicant): Meiosis is a fundamental process that is coupled to gamete development. Oocytes arrest in prophase of the first meiotic division to allow for growth. Release from prophase I occurs during meiotic maturation, but the molecular mechanisms regulating this process are incompletely understood. Meiotic maturation defects can result in infertility or in inaccurate segregation of chromosomes, leading to miscarriages and/or birth defects. The fruitfly Drosophila melanogaster offers sophisticated genetic and molecular tools, and a well described ovarian cell biology, making it a powerful system to investigate the control of meiotic maturation. Drosophila oocytes enter meiosis shortly after germline cyst formation and arrest in prophase I during most of their development. After meiotic maturation, oocytes arrest again in metaphase I until ovulation. In the prior funding period, we demonstrated that 1-Endosulfine (Endos) is required for meiotic maturation. Our data suggest that Endos promotes the stability of the cell cycle regulators CyclinA, Polo and Twine/Cdc25 (all known to be targets of the Anaphase Promoting Complex, or APC), and that it also has a separate role via inhibition of the E3 ubiquitin ligase Elgi to promote meiotic maturation. Expression of ENSA, the human 1- endosulfine, rescues the endos meiotic defect, and 1-endosulfine is expressed in mouse oocytes, suggesting conservation of the meiotic function of 1-endosulfine. Our overarching hypothesis is that Endos is a central regulator of meiotic maturation via two mechanisms: it inhibits the activity of the APC to allow the accumulation of key cell cycle regulators, and it antagonizes the ubiquitination of Elgi targets. We will test this hypothesis through the following specific aims: 1) to determine if Endos is a negative regulator of the APC; 2) to determine how the E3 ubiquitin ligase Elgi regulates meiotic maturation downstream of Endos; and 3) to elucidate the molecular mechanism of action of Endos. Elucidation of the molecular mechanisms of meiotic maturation could potentially lead to therapies to address this cause of infertility, as well as to new contraceptives. Furthermore, this knowledge could also lead to technological strategies for in vitro maturation of human oocytes to complement current in vitro fertilization methodology. PUBLIC HEALTH RELEVANCE: Failure of the oocyte to undergo meiotic maturation leads to infertility. Our prior work has identified Endos as a major regulator of meiotic maturation, and we propose to use powerful research tools in fruitflies to understand the function of this protein. Our studies could potentially lead to additional therapies to address infertility, as well as to technological strategies to complement current in vitro fertilization methodology.