TRANSCRIPTIONAL REGULATION DURING SPERMIOGENESIS

精子发生过程中的转录调控

基本信息

批准号：
6125593
负责人：
PRABHAKARA P REDDI
金额：
$ 10.3万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-12-08 至 2003-11-30
项目状态：
已结题

项目摘要

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.

转录调控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。中央这一提议的假设是一个或多个关键睾丸—— 特定的转录因子介导末端信号通过激活精子细胞特异性来分化雄性生殖细胞基因。该研究计划的目标是分离和表征参与减数分裂后基因的睾丸转录因子规定。该研究的重点是编码睾丸特异性基因顶体蛋白 SP-10，用于研究顶体后调节的模型基因减数分裂，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，仅限于高尔基体和精子发生的帽期； 2）在人类和老鼠; 3) 在圆形精子细胞中独特表达，并且存在作为单拷贝基因。有证据表明 SP-10 不属于 CREM 的转录控制，唯一得到充分表征的睾丸中的转录因子。目前的提案使用 SP-10 基因启动子捕获新的睾丸特异性转录因子和检验这些因素出现在特定阶段的假设精子发生过程中的方式来协调表达精子细胞特异性基因。识别和表征调节 SP-10 基因表达的转录因子；转基因的将使用 SP-10 启动子 + GFP 报告构建体生成小鼠，将进行凝胶位移测定和 DNA 酶 I 足迹分析识别顺式作用元件。确定功能角色体外 SP-10 基因表达中假定的转录因子将使用睾丸核提取物开发转录测定法和SP-10启动子驱动的无G盒。 3）检验假设 SP-10 转录因子出现在发育阶段睾丸中的特定方式，睾丸切片的原位杂交，青春期前睾丸 RNA 的 Northern 分析和 RT-PCR 以及成年小鼠也将进行。鉴定控制 SP-10 基因表达的转录因子将为未来研究这些因素的作用的方法协调精子发生过程中的基因调控。这些目标的实现将大大增加我们的知识男性生殖细胞在分子水平上分化。拟议的研究具有临床相关性，因为减数分裂停滞精子发生是男性不育的一个有记录的原因。因素精子发生的基因调控可能提供新方法到男性避孕。