Stage-Specific Germ Cell-Sertoli Cell Interactions

描述(申请人提供)：发育中的雄配子和邻近的支持细胞之间的相互作用对精子发生是必不可少的。Sertoli细胞表达雄性生殖细胞所需的编码细胞黏附分子、生长因子、运输蛋白、蛋白水解酶和蛋白酶抑制物的基因。生殖细胞通过支持细胞调节其中许多基因的表达。因此，随着相邻的生殖细胞在生精上皮周期的各个阶段进行，这些基因的表达会发生变化。这项应用从组织蛋白酶L基因的角度来研究支持细胞阶段特异性基因表达的调节和生物学后果。随着相邻生殖细胞从I期发展到VI和VII期，该基因的转录增加了10倍以上，当生殖细胞达到X期时，转录水平下降到检测不到的水平。这一基因表达周期是对生殖细胞的反应，导致组织蛋白酶L基因转录的顺序抑制、刺激和再抑制。我们在组织蛋白酶L基因中发现了两个可能对这些信号做出反应的结构域：[1]上游的抑制子结构域响应生殖细胞的抑制信号[2]在睾丸成熟达到顶峰时，一个120bp的激活区被激活，这是以第一波精子发生的完成为特征的。这些结构域在体内是有功能的；组织蛋白酶L基因的3kb片段在转基因小鼠中既提供了支持细胞特异性的表达，又提供了阶段特异性的表达。这一应用还建议检测组织蛋白酶L在生精上皮中的功能。这些实验是由我们的观察推动的，即表达非活性组织蛋白L的小鼠表现出更高的生精小管萎缩发生率，并且在非萎缩小管中产生的精子细胞比对照小鼠少30%。在所有这些数据的基础上，这个方案提出了四个问题，这是我们的具体目标：1激活结构域中有哪些特定的顺式作用元件，它们的功能是什么？2.与成熟结构域结合的转录因子的身份是什么？它们的表达是否具有阶段特异性和成熟依赖性？3.哪些顺式作用元件和哪些生精细胞抑制支持细胞中组织蛋白酶L启动子的活性？4.组织蛋白酶L在生精上皮中的作用是什么？

DESCRIPTION (provided by applicant): Interactions between developing male gametes and adjacent Sertoli cells are essential for spermatogenesis. Sertoli cells express genes encoding cell adhesion molecules, growth factors, transport proteins, proteases and protease inhibitors required by male germ cells. Germ cells regulate expression by Sertoli cells of many of these genes. Consequently, expression of these genes changes as the adjacent germ progress through the stages of the cycle of the seminiferous epithelium. This application examines the regulation and biological consequences of stage-specific gene expression by Sertoli cells from the perspective of the cathepsin L gene. Transcription of this gene increases more than 10-fold as adjacent germ cells progress from stage I to stages VI and VII and then decreases to undetectable levels when germ cells reach stage X. This cycle of gene expression is a response to germ cells which causes sequential repression, stimulation and re-repression of transcription of the cathepsin L gene. We have identified 2 domains in the cathepsin L gene that potentially respond to these signals: [1] An upstream repressor domain responds to inhibitory signals from germ cells [2] A 120 bp activation domain is stimulated upon the culmination of testis maturation, which is characterized by the completion of the first wave of spermatogenesis. These domains are functional in vivo; a 3kb fragment of the cathepsin L gene confers both Sertoli cell-specific and stagespecific expression of a reporter in transgenic mice. This application also proposes to examine the function of cathepsin L in the seminiferous epithelium. Those experiments are prompted by our observation that mice which express catalytically inactive cathepsin L exhibit an increased incidence of seminiferous tubule atrophy and produce 30% fewer spermatids in nonatrophic tubules than are produced by control mice. Building on all of these data, this proposal asks four questions which are our specific aims: 1 What are the specific cis-acting elements in the activation domain and what are their functions? 2. What is the identity of the transcription factors that bind to the maturation domain? Is their expression stage-specific amd maturation-dependent? 3. What cis-acting elements and which spermatogenic cells repress cathepsin L promoter activity in Sertoli cells? 4. What is the function of cathepsin L in the seminiferous epithelium?