REPRODUCTIVE BIOCHEMISTRY OF TESTIS-SPECIFIC LDH-X

为了理解精子发生的复杂性，有必要 检查基因表达的细节，并最终将它们关联起来 随着形态和生化变化的多样性， 发生在精原细胞分化为精子时。基因 睾丸特异性乳酸脱氢酶(LDH-c)的编码使用启动子 在体细胞中转录不活跃的。LDH-c表达开始 在第一次减数分裂的前期。由转基因组成的 在100bpLDH-c核心启动子(鼠)和LacZ的基础上，表达了β- 仅半乳糖苷酶的雄性后代的粗线期精母细胞 创始老鼠。小鼠LDH-c启动子具有双重功能 胚芽转录起始所需的回文序列 细胞和体细胞组织中基因表达的抑制。确实有 几个普遍存在的顺式调控元件以及睾丸特异性 蛋白质-DNA结合。我们可以确定睾丸所扮演的角色 调节该基因的特异性和非特异性转录因子。 这反过来将允许我们选择转录因子作为靶标 在反向遗传学实验中，这应该会增进我们对 从干细胞到细胞的过程中所涉及的分子事件 精子。为了实现这一目标，我们提出了以下具体建议 目的：(1)研究LDH-c缺失对转录的调节作用 转基因构建物中上游序列的分析。已选择 小鼠LDH-c启动子的蛋白结合序列将被用作 转录因子“酵母单杂交”系统中的“诱饵” 捕获、克隆、鉴定和分离；(2)利用基因 基因工程小鼠作为转录研究的模型系统 以转基因的形式表达调控基序；(3)干扰 内源性LDH-c基因座通过同源重组，从而 消除转基因小鼠的LDH-C4合成；以及(4)下调 转录因子在含核酶的初级精母细胞中的表达 在LDH-c启动子控制下表达的转基因。这些目标 对于理解特定的基因表达很重要。他们会 带我们到下一个层次来分析信号转导途径 参与精子细胞：支持细胞相互作用。最终，这些 数据将使我们能够充分认识到基因治疗的潜力 缓解男性不育症和控制生育能力。

To understand the complexity of spermatogenesis it is necessary to examine the details of gene expression and ultimately correlate these with the multiplicity of morphological and biochemical changes which occur as spermatogonia differentiate into spermatozoa. The gene encoding testis-specific lactate dehydrogenase (ldh-c) uses a promoter not transcriptionally active in somatic cells. Ldh-c expression begins during prophase of the first meiotic division. A transgene consisting of an 100 bp ldh-c core promoter (murine) and lacZ, expressed beta- galactosidase only n pachytene spermatocytes from male offspring of founder mice. The murine ldh-c promoter contains a dual function palindrome sequence required for transcriptional initiation in germ cells and repression of gene expression in somatic tissues. There are several ubiquitous cis-regulatory elements as well as testis specific protein -DNA binding. We can determine the roles played by testis specific and non-specific transcription factors in regulating this gene. This in turn will allow us to select transcription factors as targets in reverse genetics experiments that should enhance our understanding of the molecular events involved on the path from stem cell to spermatozoan. To accomplish this we propose the following specific aims: (1) to study transcriptional regulation of ldh-c by deletion analysis of upstream sequences in transgene constructs. Selected protein binding sequences of the murine ldh-c promoter will be used as "bait" in the "yeast one-hybrid" system for transcription factor capture, cloning, identification and isolation; (2) to use genetically engineered mice as model systems for the study of transcription by expressing regulatory motifs as transgenes; (3) to disrupt the endogenous ldh-c gene locus by homologous recombination and thereby eliminate LDH-C4 synthesis in transgenic mice; and (4) to down-regulate transcription factor expression in primary spermatocytes with ribozyme transgenes expressed under control of the ldh-c promoter. These goals are important for understanding specific gene expression. They will take us to the next level to analyze signal transduction pathways involved in sperm cell: supporting cell interactions. Ultimately, these data will permit us to fully realize the potential of gene therapy in alleviating male infertility and for fertility control.