ROLE OF CAMP AND CALCIUM DEPENDENT PROTEIN KINASES IN SPERMATOGENESIS

CAMP依赖蛋白的调节型(R)和催化型(C)亚型 在发育中的男性中，激酶在特定的时间程序中表达 生殖细胞和支持细胞。生化特性的差异 不同的R亚型表明，这些模式的变化 表达在改变细胞的反应能力方面可能很重要 夏令营。在Sertoli细胞中，调节亚单位(RIIbeta)的一个特定亚型 对FSH的反应是高度诱导的。一个相关的调节亚单位 (RIIpha)在伸长的精子细胞中处于发育状态 在成熟精子中大量表达RIIpha蛋白。 最近，一种新的催化亚基亚基CGamma被克隆出来 并且它的表达似乎仅限于睾丸。 尽管有强有力的证据表明cAMP依赖的蛋白激酶在 在支持细胞和生殖细胞的调节中扮演着重要的角色，许多 这一系统的各个方面仍未得到探索。这个项目的总体目标是 将重点放在特定的基因，RIIpha，RIIbeta和CGamma 要么是高度诱导的，要么是在睾丸中唯一表达的。我们会 调控睾丸特异性表达的启动子元件的特征 以及分子遗传学方法，以深入了解生理 激酶系统在精子发生中的作用。 初步研究将涉及克隆和表征 RIIbeta和RIIpha的推动者。支持细胞的鉴定 RIIbeta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。对RIIpha启动子的分析将需要使用 转基因小鼠，因为没有单倍体精子细胞系存在。鼠标 CGamma催化亚基基因将通过与人类同源克隆 基因，并用于检测CGamma在睾丸细胞中的表达。这个 这些激酶基因的生理作用将通过(1)扰动来测试 支持细胞过表达cAMP依赖的蛋白激酶系统 表达RI显性突变形式的修饰的RII亚基(2) 使用RIIpha和RIIbeta启动子的转基因小鼠中的亚单位和(3) 携带RII和CGamma基因的同源小鼠干扰该基因 颠覆。这些研究将提供有关监管的基本信息 控制睾丸特定基因并将有助于弥合差距的元件 对cAMP依赖的生理作用的理解 蛋白激酶在生殖过程中的异构体。

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.