REPRODUCTIVE BIOCHEMISTRY OF TESTES-SPECIFIC LDH-X

这项研究的目的是阐明功能作用和 描述调节儿童发育外观的因素 与哺乳动物精子发生有关的独特蛋白质。在此期间 第一次减数分裂前期的一些基因产物是 表达的基因是阶段和/或细胞特有的。其中之一是 乳酸脱氢酶(LDH)同工酶可能发挥重要作用 精子发生过程中的代谢作用。LDH-C4一直很好 在结构、生物化学和遗传学方面具有其特点。一个 对LDH-c基因调控的认识对相关问题的影响 从发育过程和精子产生的角度 不孕不育以及生育控制。小鼠和人类LDH-c基因 在表达上表现出组织和时间的特异性，但物种 转录速率和信使核糖核酸稳定性的差异。这些属性 将被用来识别对基因调控重要的DNA序列 以及DNA结合蛋白和转录因子参与 精子发生过程中的基因表达。具体目标有三个： (1)鉴定调控乳酸脱氢酶-c表达的DNA序列 生精细胞。一种生殖细胞培养系统，在该系统中，该基因可以 已经建立了转基因小鼠，并将用于 确认睾丸特异的表达。甲基化模式将是 在表达和非表达组织中进行比较；缺失分析将 用于确定调控所需的序列范围 转染法中的活性；确定精确的调控序列 通过蛋白质-DNA相互作用将被用作筛查睾丸的探针 克隆DNA结合因子的表达文库；(2) 检测LDH-c基因的稳定性和3‘端未翻译基因的作用 调节翻译的区域；(3)干扰内源性LDH-c 与编码可选基因的DNA同源重组的基因座 新霉素抗性标记，从而消除LDH-C4蛋白 在这些靶向转基因小鼠中进行生产。这将允许我们询问 LDH-C4在精子发生过程中和/或在 精子。这些目标对于获得理解很重要 精子发生过程中特定基因的表达。它们还提供 对其他系统中类似发展过程的潜在洞察， 例如，造血、肿瘤发生和器官发生。

The goal of this research is to elucidate the functional role and describe the factors that regulate the developmental appearance of a unique protein associated with mammalian spermatogenesis. during the prophase of the first meiotic division a number of gene products are expressed that are stage and/or cell specific. One of these is an isozyme of lactate dehydrogenase (LDH) that may play an important metabolic role during spermatogenesis. LDH-C4 has been well characterized in terms of its structure, biochemistry and genetics. An understanding of ldh-c gene regulation impinges on questions concerning sperm production from the standpoint of developmental processes and infertility as well as fertility control. Mouse and human ldh-c genes show tissue and temporal specificity in expression but species differences in transcription rates and mRNA stability. These properties will be exploited to identify DNA sequences important for gene regulation as well as DNA binding proteins and transcription factors involved in gene expression during spermatogenesis. there are three specific aims: (1) to identify DNA sequences that regulate ldh-c expression in spermatogenic cells. A germ cell culture system in which this gene can be expressed, has been established and transgenic mice will be used to confirm testis-specific expression. Methylation patterns will be compared in expressing and non-expressing tissues; deletion analysis will be used to establish the extent of sequence necessary for regulatory activity in transfection assays; precise regulatory sequences identified by protein-DNA interactions will be used as probes to screen testis expression libraries in order to clone DNA binding factors; (2) to examine the role of ldh-c mRNA stability and of the 3' untranslated region in regulating translation; (3) to disrupt the endogenous ldh-c gene locus by homologous recombination with DNA encoding a selectable neomycin resistance marker and thereby eliminate LDH-C4 protein production in these targeted transgenic mice. This will permit us to ask whether LDH-C4 is absolutely required during spermatogenesis and/or in the spermatozoan. These goals are important to obtain an understanding of specific gene expression during spermatogenesis. They also provide potential insights into similar developmental processes in other systems, e.g. haematopoiesis, oncogenesis and organogenesis.