REPRODUCTIVE BIOCHEMISTRY OF TESTIS-SPECIFIC LDH-X

为了了解精子发生的复杂性， 检查基因表达的细节，并最终将这些 随着形态和生化变化的多样性， 当精原细胞分化成精子时发生。 基因 编码睾丸特异性乳酸脱氢酶（LDH-C）使用启动子 在体细胞中没有转录活性。 Ldh-c表达式开始 在第一次减数分裂前期。 一种转基因， 的100 bp ldh-c核心启动子（鼠）和lacZ，表达β- 半乳糖苷酶只在粗线期精母细胞从男性后代的 创始人老鼠 小鼠ldh-c启动子具有双重功能 芽中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。 有 几种普遍存在的顺式调节元件以及睾丸特异性 蛋白质-DNA结合 我们可以确定睾丸所起的作用 特异性和非特异性转录因子在调节该基因中的作用。 这反过来将使我们能够选择转录因子作为目标 在反向遗传学实验中， 在从干细胞到 精子 为此，我们提出以下具体建议： 目的：（1）研究ldh-c基因缺失对ldh-c基因转录的调控 分析转基因构建体中的上游序列。 选择 鼠LDH-C启动子的蛋白结合序列将用作 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）利用遗传学 基因工程小鼠作为研究转录的模型系统， 表达调控基序作为转基因;（3）破坏 内源LDH-C基因座，从而 消除转基因小鼠中LDH-C4的合成;以及（4）下调 利用核酶技术研究初级精母细胞转录因子的表达 在LDH-C启动子控制下表达的转基因。 这些目标 对于理解特定基因的表达很重要。 他们将 带我们进入下一个层次来分析信号传导途径 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。

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.