The research in this proposal will accomplish three objectives, focusing on GTP binding proteins of cultured rat Sertoli cells. The first objective will be to characterize these binding proteins using novel photoactivatable affinity analogs such as, [32P]8-N3GTP, [32P]8-N3Gpp(NH)p and [32P]8-N3TNP-GTP. These binding proteins include the stimulatory (Ns) and inhibitory (Ni) forms of the guanine nucleotide regulation unit (N), which plays a pivotal role in transduction of the hormonal signal (FSH) and generation of biological response. N activation of adenylate cyclase will also be characterized using several GTP analogs including Gpp(NH)p, GTPGammaS, GDPBetaS, and TNP-GTP. The N protein will be photolabeled with [Alpha32P]8-N3GTP and identified using SDS gel electrophoresis with autoradiography. In conjunction with photolabeling, the N protein will be studied by cholera and pertussis toxin induced ADP-ribosylation using [32P]NAD. Both of these approaches will be correlated with nucleotide regulation of adenylate cyclase. The comparison of cholera and pertussis toxin regulation of adenylate cyclase, will demonstrate that both Ng and Ni forms of N exist in the Sertoli cell plasma membrane. [Gamma32P]8-N3GTP will be used to determine the GTPase activity of Sertoli cell membranes associated with the photoaffinity labeled N protein. This GTPase, involved in a potential turn off mechanism, will also be examined by measuring hydrolysis of [Gamma32P]GTP in the presence of various agents. The second objective will be to define the role of FSH in regulating the interaction of GTP with its binding proteins. Potential regulatory mechanisms to be studied include modulation of affinity constants, GTPase activity, and phosphorylation of binding proteins. Examination of these processes will also be incorporated into studies of hormonal desensitization. Experiments aimed at the first two objectives will use membranes isolated from Sertoli cells cultured from testes of 15 day old rats. The third objective will be to examine developmental changes in Sertoli cells cultured from rats 5 to 40 days of age for the correlations among GTP binding sites, adenylate cyclase activity, and FSH receptor binding. The experimental approaches needed for this objective are similar to those used for the first two objectives. Since Sertoli cells play an integral role in spermatogenesis and maintenance of normal fertility, basic knowledge derived from these biochemical investigations will provide significant insight into hormonal regulation of sperm production and development of new techniques to regulate spermatogenes.
| Johnson, G P; Fletcher, P W (1988) Guanosine diphosphate binding, metabolism and regulation of follitropin-sensitive adenylate cyclase activity in Sertoli cell membranes. Biochim Biophys Acta 970:343-54 |
| Schanbacher, B D; Fletcher, P W; Reichert Jr, L E (1987) Testicular compensatory hypertrophy in the hemicastrated calf: effects of exogenous estradiol. Biol Reprod 36:1142-8 |
| Fletcher, P W; Reichert Jr, L E (1986) Guanine triphosphate-binding site regulation by follicle-stimulating hormone and guanine diphosphate in membranes from immature rat Sertoli cells. Endocrinology 119:2221-6 |
