APOPTOSIS IN GERM CELLS--THE ROLE OF BCL-X

生殖细胞凋亡——BCL-X 的作用

• 批准号: 6108356
• 负责人: PATRICIA L. MORRIS
• 金额: $ 23.88万
• 依托单位: POPULATION COUNCIL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6108356
• 关键词: RNase protection assay; androgens; antisense nucleic acid; apoptosis; cell growth regulation; gel mobility shift assay; gene expression; genetic regulation; genetic regulatory element; genetically modified animals; germ cells; hormone regulation /control mechanism; laboratory mouse; laboratory rat; molecular cloning; nucleic acid biosynthesis; nucleic acid sequence; oncoproteins; protein isoforms; protein structure function; spermatogenesis; testis; tissue /cell culture

The long-term goal of these studies if to determine how the survival of spermatogenic cells is regulated by the Bcl-2 family of onoproteins. Although the potential exists for multiple influences on the regulation of mitotic events in the proliferating germ cells, a significant body of data has established a correlation between the size of the spermatogenic compartment and the number of early germ ells that do not survive to the haploid stage. We recently identified bcl-x as a member of the bcl-2 family of oncogenes preferentially expressed in the rat testis. Studies in this proposal concentrate on the regulation of the expression of two protein isoforms, Bcl-xL and Bcl-xS, and their roles in determining survival versus death of germ cells. Our studies will focus on the unique features of bcl-x expression in the seminiferous tubule. First, the bcl-x gene will be cloned and potential regulatory elements identified in its proximal promoter by DNA sequence analysis. Second, the specific regulation of the isolforms of bcl-x mRNA by androgens and stem cell growth factor will be evaluated using primary culture models of highly purified spermatogonia and early spermatocytes. Third, the stage specificity of bcl-x expression in the seminiferous tubule will be determined and the effects of stem cell factor and androgens on bcl-x expression and DNA synthesis in premeiotic germ cells ascertained. Fourth, the effects of changes in the ratio of the members of the Bcl-2 family of oncoproteins on programmed cell death in spermatogonia will be examined. Specific alterations in the balance of suppression versus induction of cell death will be accomplished by specific inhibition of Bcl-xL mRNA using an antisense oligonucleotide approach and by overexpression of Bcl-2 in transfected germ cells. Characterization of germ cell development and apoptotic processes in the bcl-2 transgenic mouse will facilitate studies to delineate the role of the bcl-2 family of genes in the survival of the male germ cells. Since testicular biopsies rom infertile males often show decreased numbers of proliferating and developing germ cells, we anticipate that this research will lead to significant new data on the relevance of apoptosis and its regulation to human male fertility.

这些研究的长期目标是确定 生精细胞的凋亡受Bcl-2家族的调节。 尽管存在对监管产生多重影响的可能性， 生殖细胞增殖中的有丝分裂事件， 数据已经建立了一个相关性的大小之间的 生精室和早期生殖细胞的数量， 不能存活到单倍体阶段。 我们最近发现bcl-x是一种 癌基因bcl-2家族成员，优先表达于 大鼠睾丸。 本提案中的研究集中于 两种蛋白质亚型Bcl-xL和Bcl-xS的表达，以及 它们在决定生殖细胞的存活与死亡中的作用。 我们 研究将集中在bcl-x表达的独特特征， 生精小管首先，bcl-x基因将被克隆和潜在的 通过DNA鉴定其近端启动子中的调控元件 序列分析 第二，特异性调节的异构体的 bcl-x mRNA通过雄激素和干细胞生长因子的作用， 使用高度纯化的原代培养模型进行评价 精原细胞和早期精母细胞。第三， 将测定生精小管中的bcl-x表达， 干细胞因子和雄激素对bcl-x表达和DNA的影响 确定了减数分裂前生殖细胞的合成。 第四，影响 Bcl-2家族成员比例的变化， 癌蛋白对精原细胞程序性死亡的影响 考察 抑制与抑制之间平衡的具体变化 细胞死亡的诱导将通过特异性抑制 Bcl-xL mRNA的反义寡核苷酸方法， Bcl-2在转染的生殖细胞中过表达。 表征 bcl-2转基因小鼠生殖细胞发育和凋亡过程 小鼠将有助于研究描绘bcl-2家族的作用， 基因在男性生殖细胞存活中的作用。 自从睾丸 不育男性的活组织检查通常显示 生殖细胞的增殖和发育，我们预计， 研究将导致重要的新数据的相关性 细胞凋亡及其对男性生育力的调节。