REGULATION OF MEIOSIS IN SPERMATOGENESIS

这项研究的长期焦点是男性生殖系中细胞分裂的调节，以及细胞周期与精母细胞发育和分化的协调。在果蝇中，分子遗传学分析已经确定BULEE基因是雄性减数分裂的关键调节基因。BUBLE突变体的精子发生在减数分裂前是正常的，但减数分裂I的G2/M转换未能发生，雄性不育。分子水平的研究表明，BULEE属于一个保守的生育因子家族，其中包括DAZ(在无精子症中缺失)基因，该基因是人类无精子因子(AZF)的候选基因。虽然其脊椎动物同源物在精子发生中的分子作用尚未确定，但Boule已被证明对CDC25磷酸酶Twin的翻译是必不可少的，CDC25磷酸酶Twin是细胞周期机制的关键组成部分。这项提案的四个具体目标是确定Boule蛋白的靶标、功能结构域、配对伙伴和调节因子。转基因方法将被用来分析一种RNA结合蛋白Boule在Twin调节中的功能。缺失衍生物和定点突变将用于分析Boule的结构和功能，包括体内和体外。将进行酵母双杂交筛选，以确定与Boule蛋白直接相互作用的蛋白质。最后，将对一组雄性不育突变体进行筛选，以寻找Boule表达和活性的调节因子。这些研究应该提供对配子发生过程中控制细胞分裂的基本调控机制的洞察，并对调查和治疗男性不育的重要遗传原因具有实质性的相关性。

The long term focus of this research is the regulation of cell division in the male germline and the coordination of the cell cycle with spermatocyte development and differentiation. In Drosophila, molecular genetic analyses have identified the boule gene as a key regulatory of meiosis in males. Spermatogenesis in boule mutants is normal up to meiosis, but the G2/M transition of meiosis I fails to occur and males are sterile. Molecular studies have revealed that boule belongs to a conserved family of fertility factors that includes the Daz (Deleted in Azoospermia) gene, a candidate locus for the human Azoospermia Factor (AZF). Whereas the molecular role of its vertebrate homologs in spermatogenesis has not been defined, Boule has been shown to be essential for translation of the cdc25 phosphatase Twine, a key component of the cell cycle machinery. The four specific aims of this proposal are directed at identifying the targets, functional domains, pairing partners, and regulators of the Boule protein. A transgenic approach will be used to analyze the function of Boule, an RNA-binding protein, in Twine regulation. Deletion derivatives and site-directed points mutants will be employed to analyze Boule structure and function both in vitro and in vivo. A yeast two-hybrid screen will be carried out to identify proteins that interact directly with the Boule protein. Lastly, a collection of male-sterile mutants will be screened for regulators for Boule expression and activity. These studies should provide insight into the basic regulatory mechanisms governing cell division in gametogenesis, as well as be of substantial relevance to the investigation and treatment of a significant genetic cause of infertility in men.