ABP AND THE REGULATION OF GERM CELL APOPTOSIS IN TESTIS

ABP与睾丸生殖细胞凋亡的调控

• 批准号: 6233419
• 负责人: PETER PETRUSZ
• 金额: $ 1.69万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-03-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233419
• 关键词: androgen binding protein; androgen receptor; apoptosis; cell growth regulation; cell proliferation; dihydrotestosterone; estradiol; estrogen receptors; flow cytometry; gene expression; genetically modified animals; germ cells; immunocytochemistry; in situ hybridization; laboratory mouse; northern blottings; polymerase chain reaction; radioimmunoassay; spermatogenesis; testis; testosterone; western blottings

Spermatogenesis is an androgen-dependent process and androgen-binding protein (ABP), a secretory product of Sertoli cells, is considered a mediator of androgens action on the developing germ cells. However, ABP's precise mode of action is not known. In order to understand the functions of ABP and, in a broader sense, the role of androgens in the control of spermatogenesis, we have studied transgenic mice expressing high amounts of ABP. These animals show progressive abnormalities of spermatogenesis and an eventual loss of fertility. In order to explain this finding, we hypothesize that the chronic overproduction of ABP changes the balance between proliferation and degeneration (apoptosis) of germ cells by changing the steroid hormone milieu within the seminiferous tubules. This may involve not only testosterone, but also is biologically active intratesticular metabolites dihydrotestosterone and estradiol. To test the above hypothesis, we propose to determine the effects of excess ABP produced in the transgenic animals on (1)-- testicular levels of testosterone, dihydrotestosterone, estradiol, and the androgen and estrogen receptors. Specific radioimmunoassays, immunocytochemistry, and in situ hybridization will be used to achieve this goal. (2)--the kinetics of germ cell proliferation and apoptosis preceding and during the decline in spermatogenesis. Flow cytometry and morphometric analysis will be used after specific labeling of both proliferating and apoptotic cells. Apoptosis will be detected by in situ end labeling of fragmented DNA and by gel electrophoresis. (3)-- the expression of genes regulating germ cell proliferation and apoptosis. Gene expression will be analyzed before and during the decline in spermatogenesis by immunocytochemistry, in situ hybridization, in situ PCR, and Northern blot analysis. These studies will lead to a better understanding of the regulation of spermatogenesis and of some forms of male infertility; they may also reveal potential new targets for male contraception.

精子发生是一个雄激素依赖性过程，并且与雄激素结合 蛋白质（ABP）是支持细胞的分泌产物，被认为是 雄激素对发育中生殖细胞作用的调节剂。 然而， ABP 的精确作用方式尚不清楚。 为了了解 ABP 的功能，以及更广泛意义上的雄激素在 控制精子发生，我们研究了表达 大量 ABP。 这些动物表现出进行性异常 精子发生并最终丧失生育能力。 为了解释 这一发现，我们假设 ABP 的长期过量产生 改变增殖和退化（细胞凋亡）之间的平衡 通过改变生殖细胞内的类固醇激素环境 生精小管。 这可能不仅涉及睾酮，还涉及 是具有生物活性的睾丸内代谢物二氢睾酮 和雌二醇。 为了检验上述假设，我们建议确定 转基因动物中产生的过量 ABP 对 (1)-- 睾丸激素、二氢睾酮、雌二醇和 雄激素和雌激素受体。 特异性放射免疫测定， 免疫细胞化学和原位杂交将用于实现 这个目标。 (2)--生殖细胞增殖和凋亡的动力学 在精子发生衰退之前和期间。 流式细胞仪和 在对两者进行特定标记后将使用形态测量分析 增殖和凋亡的细胞。 细胞凋亡将被检测到 片段化 DNA 的原位末端标记和凝胶电泳。 (3)-- 调节生殖细胞增殖的基因的表达和 细胞凋亡。 基因表达将在实验之前和过程中进行分析 免疫细胞化学原位精子发生下降 杂交、原位 PCR 和 Northern 印迹分析。 这些研究将有助于更好地理解监管 精子发生和某些形式的男性不育症；他们也可能 揭示男性避孕的潜在新目标。