ROLE OF CAMP AND CALCIUM DEPENDENT PROTEIN KINASES IN SPERMATOGENESIS

CAMP 和钙依赖性蛋白激酶在精子发生中的作用

• 批准号: 6311614
• 负责人: George STANLEY MCKNIGHT
• 金额: $ 16.65万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6311614
• 关键词: calmodulin dependent protein kinase; developmental genetics; fertility; gene expression; gene induction /repression; gene targeting; genetic regulatory element; genetically modified animals; germ cells; in vitro fertilization; laboratory mouse; male; molecular cloning; nucleic acid hybridization; protein kinase A; protein structure function; reproductive development; sperm motility; spermatogenesis; testis; western blottings

The regulatory(R) and catalytic(C) isoforms of the cAMP-dependent protein kinases are expressed in a specifically timed program in developing male germ cells and in Sertoli cells. Differences in the biochemical properties of the various R isoforms suggest that these changes in the pattern of expression may be important in modifying the ability of cells to react to cAMP. In Sertoli cells, a specific isoform of regulatory subunit (RIIbeta) is highly induced in response to FSH. A related regulatory subunit (RIIalpha) is developmentally switched on in elongating spermatids giving rise to an abundant expression of RIIalpha protein in mature sperm. Recently, a novel isoform of the catalytic subunit, Cgamma, has been cloned from human testis and its expression appears to be restricted to testis. Although there is strong evidence that cAMP dependent protein kinases play an important role in the regulation of Sertoli cells and germ cells, many aspects of this system remain unexplored. The overall goal of this project will be to focus on the specific genes, RIIalpha, RIIbeta, and Cgamma which are either highly induced or uniquely expressed within the testis. We will characterize the promoter elements regulating testis specific expression and the molecular genetic approaches to gain insight into the physiological functions of the kinase system in spermatogenesis. The initial studies will involve the cloning and characterization of the promoters for RIIbeta and RIIalpha. Identification of Sertoli cell specific enhancers and FSH inducible elements on the RIIbeta promoter will be accomplished using fusion genes and transient expression in cultured Sertoli cells. Analysis of the RIIalpha promoter will require the use of transgenic mice since no haploid spermatid cell lines exist. The mouse Cgamma catalytic subunit cDNA will be cloned by homology with the human gene and used to examine expression of Cgamma in testicular cells. The physiological role of these kinase genes will be tested by (1) perturbing the cAMP dependent protein kinase system in Sertoli cells by overexpression of modified RII subunits (2) expressing dominant mutant forms of the RI subunit in transgenic mice using promoters for RIIalpha and RIIbeta and (3) disrupting the RII and Cgamma genes by homologous mice carrying the gene disruption. These studies will provide basic information on regulatory elements that control testis specific genes and will help bridge the gap toward an understanding of the physiological roles of the cAMP-dependent protein kinase isoforms in reproduction.

cAMP依赖性蛋白的调节（R）和催化（C）亚型 激酶在发育中的男性中以特定的时间程序表达， 生殖细胞和支持细胞。 生化特性的差异 各种R亚型的变化表明，这些变化的模式， 表达可能在改变细胞反应的能力方面是重要的， 营 在Sertoli细胞中，调节亚基（RII β）的一种特异性亚型 是对FSH的高度诱导。 一个相关的调节亚单位 （RIIalpha）在延长精子细胞中发育开启， 导致RII α蛋白在成熟精子中大量表达。 最近，一种新的异构体的催化亚基，C γ，已被克隆 其表达似乎仅限于睾丸。 尽管有强有力的证据表明cAMP依赖性蛋白激酶在 在支持细胞和生殖细胞的调节中起重要作用，许多 这一制度的各个方面尚未得到探讨。 本项目的总体目标 将重点关注特定基因RIIalpha、RIIbeta和Cgamma， 在睾丸内高度诱导或独特表达。 我们将 鉴定调控睾丸特异性表达的启动子元件 和分子遗传学的方法来深入了解 激酶系统在精子发生中的作用。 最初的研究将涉及克隆和鉴定 RIIbeta和RIIalpha的启动子。 支持细胞的鉴定 RII β启动子上的特异性增强子和FSH诱导元件将 利用融合基因和瞬时表达在培养的 支持细胞。 RIIalpha启动子的分析需要使用 转基因小鼠，因为不存在单倍体精子细胞系。 鼠标 C γ催化亚基cDNA将通过与人的同源性克隆。 基因，并用于检测睾丸细胞中C γ的表达。 的 这些激酶基因的生理作用将通过以下方式进行测试：（1）干扰 cAMP依赖性蛋白激酶系统在支持细胞中过表达 修饰的RII亚基（2）表达RI的显性突变形式， 使用RII α和RII β的启动子在转基因小鼠中的亚基，和（3） 通过携带基因的同源小鼠破坏RII和C γ基因， 破坏 这些研究将提供有关监管的基本信息， 控制睾丸特异性基因并有助于弥合差距的元件 了解cAMP依赖性的 蛋白激酶亚型在生殖中的作用。