Reproductive Biochemistry of Testis Specific LDH-X

描述（由申请人提供）：关于哺乳动物体细胞生长和分化的基因表达调控，我们已经知道了很多，但是由于缺乏适当的细胞培养，以及由于很少有基因被分析在减数分裂细胞中特异性表达，对产生单倍体生殖细胞的这些过程的类似理解受到限制。我们的工作现在已经达到了这样一个阶段：我们有必要的试剂和背景来研究精子细胞分化的遗传调控密码，也许还有卵母细胞发育的遗传调控密码。此外，我们对减数分裂细胞中特异性表达的基因产物乳酸脱氢酶C （LDH-C）的研究提供了令人惊讶的证据，表明该蛋白在精子成熟中的功能可能超出酶的作用，在精子中起结构作用，并在mRNA翻译中起调节作用。我们计划的实验将解决生殖细胞发育中的三个关键假设：1)精子细胞特异性转录因子是减数分裂细胞特异性基因表达所必需的。ldl - c基因为探索这个问题提供了一个理想的系统。2) LDH-C蛋白也将允许我们测试这种酶除了催化之外还有重要功能的想法，包括细胞结构和翻译控制。3)我们提出，我们在精子中的发现将被证明在减数分裂细胞中具有普遍重要性，因此对于LDH-C，基因表达的细胞特异性调控和蛋白质的特定功能都被预测与精子和卵母细胞相关。这项建议有三个具体目标。目的1：表征睾丸特异性Idhc表达所需的TF。目的2：研究LDH-C4的非催化功能。我们将确定将LDH-C4连接到纤维鞘的蛋白质-蛋白质相互作用以及可能调节睾丸基因翻译的蛋白质-核酸相互作用。目的3：检测卵母细胞中Idhc的表达。卵巢中LDH-C4的存在可能暗示它是一个减数分裂因子，而不是一个性别特异性的生殖细胞因子，并为研究卵母细胞中的分子事件提供了一个靶基因。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需的大分子相互作用的理解。这项工作将为认识生殖健康障碍以及解决男性不育和生育调节问题提供基础。

DESCRIPTION (provided by applicant): Much is known about the regulation of gene expression underlying mammalian somatic cell growth and differentiation, but a similar understanding of these processes in generating haploid germ cells has been limited by the lack of appropriate cell cultures and because few genes have been analyzed that are expressed specifically in meiotic cells. Our work has now reached the stage where we have the essential reagents and background to investigate the genetic regulatory code for sperm cell differentiation, and perhaps also for oocyte development. Moreover, our studies of a gene product expressed specifically in meiotic cells, the enzyme lactate dehydrogenase C (LDH-C) have provided surprising evidence that the function of this protein in sperm maturation may go beyond the enzymatic to a structural role in the sperm and a regulatory role in mRNA translation. Our planned experiments will address three critical hypotheses in germ cell development: 1) Sperm cell-specific transcription factors are required for meiotic cell-specific gene expression. The LDH-C gene provides an ideal system in which to explore this question. 2) The LDH-C protein also will allow us to test the idea that this enzyme has important functions other than catalysis, including in cell structure and translational control. 3) We propose that what we find in the sperm will prove to be of general importance in meiotic cells, and thus for LDH-C both the cell-specific regulation of gene expression and the specific functions of the protein are predicted to be relevant to both sperm and oocytes. This proposal has three Specific Aims. Aim 1: To characterize the TF's required for testis specificity of Idhc expression. Aim 2: To study non-catalytic functions of LDH-C4. We will identify protein-protein interactions that tether LDH-C4 to the fibrous sheath and protein-nucleic acid interactions that may regulate testis gene translation. Aim 3: To measure Idhc expression in oocytes. The presence of LDH-C4 in the ovary may implicate it as a meiotic factor rather than a sex-specific germ cell factor and provides a target gene for studying molecular events in the oocyte. 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. This work will provide a foundation for understanding disorders of reproductive health as well as resolving questions of male infertility and fertility regulation.