The overall goal of this project is to determine the molecular mechanisms by which follicle stimulating hormone (FSH) regulates gene expression in the Sertoli cell. Sertoli cell development, differentiated function and the onset of spermatogenesis are all dependent upon FSH. Coordinate cycling of the physiological state including FSH responsiveness of the Sertoli cell with associated germ cells suggests a bidirectional regulatory linkage between the two cell types. Investigations into the mechanisms of FSH action may lead to new understanding of fertility control. Our working hypothesis is that FSH regulates a subset of responsive genes directly by activation of preexisting transcription factors including the cyclic AMP response element binding protein (CREB) as well as a second subset indirectly by first stimulating the transcription of the trans- acting factors, Fos and JunB. The following experiments have been designed to test and develop our hypothesis using both in vitro and in vivo systems.
Specific Aim I is to define the roles of transcriptional regulation and mRNA stability in FSH regulation of gene expression. Experiments are designed to assess whether a single mechanism could mediate FSH stimulation of c-fos, junB, tissue plasminogen activator (tPA) and inhibin alpha transcriptional stimulation. Effects of FSH on the stabilities of these mRNAs will be evaluated. Secreted levels of tPA and inhibin alpha will be determined in the same experiments by radioimmunoassay.
Specific Aim II is to establish the cellular localization and effects of FSH on Fos, JunB and CREB by raising specific antisera to identify immunologically related proteins in the Sertoli cell and their distribution in the seminiferous tubule. Whether the distribution is correlated with developmental stage of associated germ cells will be determined.
Specific Aim III is to obtain more direct evidence for mediation of FSH-induced gene regulation by activation of the trans-acting factors Fos and JunB. Proteins which bind the AP-1 element, their activity and regulation by FSH will be analyzed and functional assessment of the AP-1 sequence to mediate FSH stimulated gene transcription will be determined. Modulation of the activities of CRE and AP-1 enhancer elements by one another will be analyzed.
Specific Aim I V is to identify FSH-stimulated trans-acting factor clones in an FSH-stimulated Sertoli cell cDNA expression library prepared by the subtractive method which removes constitutively transcribed mRNAs. The library will be screened using double stranded oligonucleotides corresponding to the mouse c-fos CRE and AP-1 regulatory elements. These experiments are designed to identify previously unknown mediators of FSH action.
|Raburn, D J; Hamil, K G; Tsuruta, J K et al. (1995) Stage-specific expression of B cell translocation gene 1 in rat testis. Endocrinology 136:5769-77|
|Hamil, K G; Conti, M; Shimasaki, S et al. (1994) Follicle-stimulating hormone regulation of AP-1: inhibition of c-jun and stimulation of jun-B gene transcription in the rat Sertoli cell. Mol Cell Endocrinol 99:269-77|
|Hamil, K G; Hall, S H (1994) Cloning of rat Sertoli cell follicle-stimulating hormone primary response complementary deoxyribonucleic acid: regulation of TSC-22 gene expression. Endocrinology 134:1205-12|