FSH and androgens act on Sertoli cells to regulate the secretion of proteins that function in the control of spermatogenesis, however, the mechanisms by which these hormones regulate Sertoli gene expression is not known. The expression of one of the major secretory proteins, androgen- binding protein (ABP), parallels the regulation of spermatogenesis. During the two years of funding we have developed the rat ABP gene as a model system to study the molecular mechanisms of hormone-mediated gene regulation of the Sertoli cell. Our accomplishments include isolation and characterization of testicular ABP cDNA and the ABP gene. Furthermore, as a collaborative effort we have cloned and characterized the rat androgen receptor (AR) cDNA and generated specific antibodies to AR. With ABP cDNA as probe we have begun elucidation of the mechanisms of ABP hormonal regulation. With the developed tools described above, we propose to extend these studies with three specific aims: 1. Locate ABP gene regulatory elements, including promoters, Sertoli cell specific enhancer elements and hormone regulatory elements, utilizing in vitro transfection of CAT vector constructs on primary Sertoli cells and transgenic animals. 2. Delineate the effects of FSH and androgen on ABP gene expression. In vitro studies with Sertoli cell cultures will be used to determine if these hormones have direct or indirect effects on ABP gene expression. We will also determine modulatory effects of other hormones, paracrine and autocrine factors. 3. Help decipher the function(s) and mode of action of ABP in the testis and epididymis. These studies include cellular localization of the ABP membrane receptor in testis and epididymis and mapping of the steroid- binding domain of ABP by in vitro mutagenesis. These studies also include a search for ABP mutants by linkage analysis of the mouse ABP gene. Studies of ABP gene expression in brain may provide new insights into ABP function. Our studies will contribute greatly to the knowledge of Sertoli cell gene regulation and the function(s) of ABP, a major Sertoli cell secretory protein. This maintenance of spermatogenesis, and will aid in the identification and possible treatment of fertility defects.
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