The glycoprotein hormones comprise a structurally related family of proteins produced in the pituitary gland (TSH, FSH, LH) and in the placenta (CG). The hormones are heterodimers, each containing a common alphasubunit and different beta-subunits, which confer distinct biological activities to the hormones. The overall goals of this project have been to understand the mechanisms that control the expression and actions of the glycoprotein hormones. During earlier phases of the project we found that the cAMP-response transcription factors, cAMP-response element-binding protein (CREB) and cAMP-response element modulator (CREM), are expressed at high levels in the testis, that the expression of the genes encoding these factors is autoregulated during the 12-day spermatogenic cycles in the rat, and that alternative exon splicing, internal translation, and alternative promoter usage cyclically interconverts transcriptional activators to repressors. In particular, we have discovered that the splicing of exon W into CREB mRNA during stages II-VIII of spermatogenesis prematurely terminates translation and activates two cryptic internal translation sites, resulting in the synthesis of inhibitor CREB isoforms (I-CREBs). We also have hypothesized that the pituitary gonadotropin FSH acts on receptors in Sertoli cells to generate cyclical fluctuations of cAMP in the seminiferous tubule, resulting in the regulation of the activities of CREB and CREM and other target genes essential for the development of germ cells. However, recent rather definitive evidence questions the essential requirement of FSH for male fertility. These findings lead us to propose that in the absence of FSH signaling, the hormone PACAP (pituitary adenylyl cyclase activating peptide), produced locally in the testis in round spermatids, acts on its receptors in Sertoli cells to produce the cyclical waves of cAMP signaling.
The Aims are to: (1) Prepare mice with a targeted deletion of exon W to test the functional importance in vivo of the proposed cyclical expression of I-CREBs during spermatogenesis; (2) Prepare mice with a targeted disruption of the testis-specific PACAP promoter, create transgenic mice expressing the LacZ transcriptional reporter under control of the testis-specific PACAP promoter, and to examine the role of the SRY-related homeobox factor Sox5 in the regulation of the testis-specific PACAP promoter; (3) To examine the role of the novel alternatively spliced (inserted) exon in the extracellular domain of the PACAP type 1 receptor, expressed on Sertoli cells, on the binding affinities of PACAP isopeptides and receptor coupling to signal transduction pathways. These studies have potential relevance to understanding the molecular mechanisms controlling spermatogenesis and fertility.
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