TRANSCRIPTIONAL REGULATION DURING SPERMIOGENESIS

转录调节机制通过以下方式决定细胞表型： 指导时间基因表达的精确模式。 中央 这一假设的基础是，一个或多个关键睾丸- 特异性转录因子介导末端信号， 通过激活精子细胞特异性 基因. 这项研究计划的目标是分离和表征 睾丸转录因子参与减数分裂后基因 调控这项研究的重点是一个睾丸特异性基因， 顶体蛋白SP-10是一种研究后- 减数分裂，睾丸特异性基因表达。 该基因1）表现出 基因表达的时间模式，仅限于高尔基体， 精子发生的帽状期; 2）在人类和 小鼠;和3）在圆形精子细胞中唯一表达，并且存在于 作为一个单拷贝基因。 有证据表明，SP-10并不属于 CREM的转录控制， 睾丸中的转录因子。 目前的建议使用SP-10 捕获新的睾丸特异性转录因子的基因启动子， 为了检验这些因素出现在特定阶段的假设， 在精子发生过程中的方式，使协调表达 精子细胞特异性基因 识别和描述 调节SP-10基因表达的转录因子;转基因 使用SP-10启动子+GFP报道基因构建体产生小鼠， 将进行凝胶迁移试验和DNA酶I足迹分析， 识别顺式作用元件。 确定的职能作用 体外SP-10基因表达中的假定转录因子 将使用睾丸核提取物开发转录测定 和SP-10启动子驱动的无G盒。3)为了检验这一假设 SP-10转录因子出现在发育阶段 睾丸特异性方式，睾丸切片原位杂交， 青春期前睾丸RNA的北方分析和RT-PCR 以及成年小鼠。 识别 控制SP-10基因表达的转录因子将为 今后研究这些因素在 在精子发生过程中协调基因调控。 这些目标的实现将大大增加对以下方面的了解： 男性生殖细胞在分子水平上的分化。 拟议 研究是临床相关的，因为减数分裂停滞在 精子发生是男性不育症的一个有文献记载的原因。的因素 控制精子发生的基因调控可能提供新的方法 男性避孕

Transcriptional regulatory mechanisms determine cellular phenotypes by directing precise patterns of temporal gene expression. The central hypothesis underlying this proposal is that one or more key testis- specific transcription factors mediate the signal for terminal differentiation of male germ cells by activation of spermatid-specific genes. The goal of this research program is to isolate and characterize testicular transcription factor(s) involved in postmeiotic gene regulation. The study focuses on a testis-specific gene encoding the acrosomal protein SP-10, a model gene for studying regulation of post- meiotic, testis-specific gene expression. This gene 1) Exhibits a temporal pattern of gene expression, being restricted to the golgi and cap phases of spermiogenesis; 2) Is well characterized in both human and mouse; and 3) Is expressed uniquely in round spermatids, and is present as a single copy gene. Evidence indicates that SP-10 is not under the transcriptional control of CREM, the only well characterized transcription factor in testis. The current proposal utilizes the SP-10 gene promoter to capture novel testis-specific transcription factors and to test the hypotheses that such factors arise in a stage-specific manner during spermatogenesis to bring about coordinate expression of spermatid-specific genes. To identify and characterize the transcription factors which regulate SP-10 gene expression; transgenic mice will be generated using SP-10 promoter +GFP reporter constructs, and gel shift assays and Dnase I footprinting will be performed to identify the cis-acting elements. To determine the functional role of the putative transcription factor(s) in SP-10 gene expression, in vitro transcription assay will be developed using testicular nuclear extracts and SP-10 promoter driven G-less cassette. 3) To test the hypothesis that SP-10 transcription factor(s) appear in a developmental-stage specific manner in the testis, in situ hybridization of testis sections, Northern analysis and RT-PCR of testicular RNA obtained from prepuberal as well as adult mice will be performed. Identification of the transcription factor(s) governing SP-10 gene expression will pave the way for future studies concerning the role of these factor(s) in coordinate gene regulation during spermiogenesis. Achievement of these aims will add significantly to the knowledge of male germ cell differentiation at the molecular level. The proposed studies are clinically relevant because meiotic arrest in spermatogenesis is a documented cause of infertility in men. The factors governing gene regulation of spermiogenesis may provide new approaches to male contraception.