In preliminary studies, we have developed a simple method for isolating a population highly purified undifferentiated ovarian interstitial cells which are free of granulosa cell contamination. Using this model, we have made the novel discovery that IGF-I can directly stimulate the undifferentiated cells to express their fully differentiated state in serum-free medium. These findings suggest the exciting hypothesis that a cAMP independent differentiation mechanism exists which may be mediated by a tyrosine kinase. In the proposed studies, we will use specific antisera and cDNA probes for key steroidogenic enzymes to probe the biochemical events that occur during differentiation stimulated by IGF-I in vitro. These events will be correlated with interstitial cell function at selected stages during folliculogenesis Experiments will be performed to test the hypothesis that IGF-I stimulates a tyrosine kinase which phosphorylates unique proteins that correlate with increased steroidogenic enzyme synthesis and message transcription. The mechanism of interaction between the IGF-I/tyrosine kinase pathway and the LH/cAMP pathway will also be examined. It will be determined if IGF-I action is totally independent of cAMP, or alternatively, if the IGF-I and LH pathways converge into a final common differentiation mechanism. Finally, the modulation of IGF-I action by estrogens, glucocorticoids prolactin, and epidermal growth factor will be examined. Thus, we will determine the role of IGF-I in regulating the differentiated state of the ovarian interstitial cells and examine how IGF-I fits into the current concept of multifactorial regulation of follicle development and atresia. We anticipate that by completing our research goals we will describe a new regulatory pathway for differentiation of ovarian interstitial cells which may be equally important to ovarian physiology as LH. These studies will provide new and important information regarding ovarian function which may open the door to novel therapies that exploit presently unknown regulatory mechanisms in the ovary.
