Reproductive Biochemistry of Testis Specific LDH-X

描述(申请人提供)：本研究的主要目的是确定和了解乳酸脱氢酶C4(LCH-C4)在哺乳动物睾丸中的功能。实现这一目标应该会增加我们对精子发生的分子基础的基础知识。已经描述了许多生殖细胞蛋白质和酶，但很少有像LDH-C4这样被定义的功能，LDH-C4是一种关键的代谢酶，在精子发生和精子中具有重要的催化功能。过去几年的许多研究已经证实，精母细胞的代谢更喜欢支持细胞中的乳酸作为主要能源。糖酵解是精子利用底物的首选途径。现在有令人惊讶的证据表明，LDH-C4在精子成熟中的功能可能超出了酶的作用，在精子中起到了结构性作用，并在生发上皮起到了调节作用。这些发现支持了我的假设，即LDH-C4是完成精子发生和精子功能所必需的。我的实验室对这种同工酶的结构、生化和分子特征做出了贡献，最近还描述了调节初级精母细胞中同源基因表达的顺式和反式元件。这种深入的分析为实验该蛋白质本身的功能提供了平台，并作为精子发生的分子分析的模型。我们的理论基础是，了解IDHC表达的调控与其作为细胞特异性蛋白质的特殊功能有关，而且调控机制可能反映了那些负责激活通常对精子发生至关重要的基因的人。该建议的具体目的是：1)鉴定和鉴定负责LDH-C4生殖细胞特异性表达的转录因子(S)；2)靶向阻断小鼠LDH-C4基因；3)描述LDH-C4在睾丸和精子中的催化和非催化功能，鉴定将LDH-C4拴在纤维鞘上的蛋白质间的相互作用，以及可能调节睾丸基因翻译的蛋白质-核酸之间的相互作用。这些特定目标的完成将进一步加深我们对LDH-C4功能、睾丸特异性基因调控以及正常精子发生和精子功能所必需的大分子相互作用的理解。

DESCRIPTION (provided by applicant): The primary goal of my research is to determine and understand the function of lactate dehydrogenase C4 (LCH- C4) in the mammalian testis. Achieving this goal should increase our fundamental knowledge about the molecular basis of spermatogenesis. Many germ cell proteins and enzymes have been described, but few are as well defined functionally as LDH- C4, which is a key metabolic enzyme with an important catalytic function during spermatogenesis and in spermatozoa. Many studies over the past several years have confirmed that spermatocyte metabolism prefers lactate from Sertoli Cells as a primary energy source. Glycolysis is the preferred route of substrate utilization by sperm. There is now surprising evidence that the function of LDH-C4 in sperm maturation may go beyond the enzymatic to a structural role in the sperm and a regulatory role in the germinal epithelium. These findings support my hypothesis that LDH-C4 is required for both the completion of spermatogenesis and for sperm function. My laboratory has contributed to the structural, biochemical and molecular characterization of this isozyme and more recently to a description of cis and trans elements regulating expression of the cognate gene in primary spermatocytes. This in depth analysis provides the platform for experimentation on the function of this protein itself and as a model in the molecular analysis of spermatogenesis. Our rationale is that understanding the regulation of Idhc expression relates to its specialized function as a cell specific protein and further that the regulatory mechanisms are likely to reflect those responsible for activating genes generally essential to spermatogenesis. The specific aims of this proposal are: 1) identify and characterize the transcription factor(s) responsible for germ cell specific expression of ldhc; 2) to target disruption of the ldhc gene in mice; 3) to describe both catalytic and non-catalytic functions of LDH-C4 in testis and sperm, identifying protein-protein interactions that tether LDH-C4 to the fibrous sheath, and protein-nucleic acid interactions that may regulate testis gene translation. Completion of these specific aims will further our understanding of LDH-C4 function, of testis specific gene regulation and of macromolecular interactions necessary for normal spermatogenesis and sperm function.