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.