The principal purpose of the ovary is the periodic production and expulsion of a fertilizable oocyte. Central to this process is the timely and coordinate-differentiation of the theca and granulosa cells. The two-cell, two-gonadotropin concept of follicle estrogen biosynthesis is the fundamental mechanism by which follicle cell differentiation occurs. There are, however many aspects of ovarian physiology which cannot be explained by the gonadotropins alone. Recently, the concepts of autocrine and paracrine regulation in the ovary have received considerable attention with the discovery that a number of peptides originally described as growth factors are produced locally by theca and granulosa cells in the ovary. This concept is particularly attractive because it helps to explain how the theca and granulosa cells can communicate with each other and fine tune the primary signals coming from the brain. In preliminary studies, we have obtained evidence that transforming growth factor-beta (TGF-beta), produced primarily by the theca cells, may be an important regulator of thecal androgen production and may confer FSH responsiveness to granulosa cells in-small follicles. The overall goal of this proposal is to examine the role of TGF-beta in follicle development, selection and atresia. To accomplish this goal we will execute four specific aims. First, the mechanism by which TGF-beta controls thecal steroidogenesis will be studied. Time course and dose-response studies will be performed with LH and TGF-beta alone and in combination to determine the effects of TGF-beta on thecal P450 , 3beta-HSD and P45017alpha mRNA levels using reverse transcriptase to copy cDNA and then the polymerase chain reaction to amplify the signal. We will then determine if the changes in mRNA levels are transcriptional effects using nuclear runoff assays. Second, since nothing is known regarding the regulation of TGF-beta secretion and activation by theca cells, we will test the effects of LH, IGF-I, androgens and estrogens on thecal TGF-beta secretion and activation using a specific TGF-beta RIA and bioassay. Third, our preliminary data show that granulosa cells from a non-estrogenic environment such as in small follicles do not respond to FSH stimulation. TGF-beta treatment renders the granulosa cells responsive to FSH. We will elucidate the mechanism by which TGF-beta exerts this effect by examining the effects of TGF-beta on FSH receptor content and affinity the FSH/cAMP signaling pathway, and the expression of P450scc, 3beta-HSD and P450arom in granulosa cells. Finally, using the techniques of in situ hybridization, immuncytochemistry, autoradiography and immunofluorescence we will correlate the expression of TGF-beta with the expression of steroidogenic enzymes in the theca and granulosa cells in healthy and atretic follicles to try to understand the role of TGF-beta in follicle selection and atresia. The results of our proposed studies will further our understanding of autocrine and paracrine regulation in follicle development, selection and atresia and may lead to an understanding of polycystic ovarian disease.
