Male Fertility & Protein Expression in Spermatogenesis

对精子发生的透彻了解对于男性不育研究和男性避孕药的开发具有重要意义。这项研究的长期目标是了解转录后调控机制如何控制精子蛋白的表达。在精子发生的特定时期，一些mRNAs的翻译抑制是植物、昆虫和哺乳动物使用的一种关键策略。最近对果蝇精子发生的遗传学和分子研究已经确定了翻译抑制途径的三个组成部分。这些是RNA结合蛋白TSR，它调节的SCPR mRNA，以及GTP结合蛋白ARF，它结合TSR并可能调节其功能。有了这些信息，现在就有可能利用遗传、分子和生化方法的组合来解决关于翻译调控机制的基本问题。该建议的第一个具体目标集中在对TSR-SCPR mRNA相互作用的详细描述，以及SCPR mRNA如何在适当的时间被激活以进行翻译。体外结合研究将用于确定相互作用所需的序列，并将在转基因果蝇中测试它们的生物学相关性。第二个目标是在ARF中产生突变，并在体内研究其在精子发生过程中的功能。这些突变对SCPR和TSR的影响也将被检测。最后的具体目标将解决如何调节TSR-ARF蛋白质-蛋白质相互作用的问题，以及这种相互作用对TSR亚细胞定位的影响。酵母双杂交系统将与野生型和突变型蛋白质一起使用，以确定鸟嘌呤核苷酸在相互作用中的作用，以及TSR中对蛋白质结合至关重要的区域。其他实验将使用免疫电子显微镜来检查TSR和ARF在发育中的精母细胞和精子细胞中的定位。鉴于与SCPR相关的人类睾丸蛋白在精子发生中具有重要作用，这项研究的结果不仅将为翻译调控提供基础知识，还将为调节人类精子生成提供新的分子靶点。

A thorough understanding of spermatogenesis is important for advances in male infertility research and male contraceptive development. The long-term goal of this research is to understand how posttranscriptional regulatory mechanisms control the expression of sperm proteins. The translational repression of some mRNAs at specific periods during spermatogenesis is a key strategy that is used in plants, insects and mammals. Recent genetic and molecular studies of spermatogenesis in the fruit fly Drosophila have identified three components of a translational repression pathway. These are the RNA binding protein TSR, the Scpr mRNA that it regulates, and the GTP-binding protein ARF, which binds TSR and may modulate its function. With this information, it is now possible to address fundamental questions about the mechanism of translational regulation, using a combination of genetic, molecular and biochemical approaches. The first specific aim of this proposal focuses on a detailed characterization of the TSR-Scpr mRNA interaction, and how Scpr mRNA becomes activated for translation at the appropriate time. In vitro binding studies will be used to identify the sequences required for the interaction, and their biological relevance will be tested in transgenic flies. The second aim is to generate mutations in ARF and study its function in vivo during spermatogenesis. The effects of these mutations on Scpr and TSR will also be assayed. The final specific aim will address the question of how the TSR-ARF protein-protein interaction is regulated, and the effect of that interaction on the subcellular localization of TSR. The yeast two-hybrid system will be used with wild type and mutant proteins to define the role of guanine nucleotides in the interaction and the regions of TSR that are important for protein binding. Additional experiments will use immunoelectron microscopy to examine the localization of TSR and ARF in developing spermatocytes and spermatids. Given the fact that human testis proteins related to Scpr have important roles in spermatogenesis, the results of the proposed research should not only provide fundamental knowledge regarding translational regulation, but also provide novel molecular targets for modulating sperm production in humans.