Published studies from our lab reveal that blockage of Src tyrosine kinase enhances LH stimulated cAMP and androstenedione product in rat thecal- interstitial cells indicating the first potential role of this kinase in ovarian function. Preliminary data presented in this application reveal that Src knockout mice exhibit a significant reduction in the ability to form antral follicles during the prepubertal period, increased primordial follicular development. And enhanced responses to gonadotropins in vivo and in vitro. In addition, in wild type C57BL6 control mice, exogenous gonadotropins induce significant changes in ovarian Src kinase activity during follicular development and the periovulatory period. In the present studies, human and mouse theca-interstitial cells, and Src knockout mice will be used for physiological and a clinically related investigations on the role of Src in ovarian function. Because Src knockout mice exhibit osteopetrosis, stunted growth, post-weaning malnutrition due to the failure of tooth eruption, and early death, it is difficult to fully assess the impact of ovarian Src deficiency. Thus, the first aim proposes to transplant immature Src knockout ovaries (-/-) into age matched wild type litter mates (+/+) in order to study the impact of ovarian specific Src depletion on ovarian function. In order to account for alterations in follicular development in the prepubertal period, the second aim will contrast and compare quantitative aspects of ovarian follicular develop in fetal, neonatal and prepubertal Src knockout mice with wild type littermates.
The third aim will assess the in vitro effects of inhibition and activation of Src tyrosine kinase on LH-mediated stimulation of cAMP and androstenedione secretion in mouse (Src knockout and wild type) and human theca-interstitial cells using pharmacological and genetic approaches. In order to account for the increased gonadotropin responsiveness when the Src gene is deleted or its activity is blocked, the fourth aim will determine whether inhibition or activation of Src tyrosine kinase in mouse thecal interstitial cells and human theca cells alters LH/hCG receptor number and agonist-induced receptor internalization This project will provide new insight into the role of Src tyrosine kinase in regulating follicular development and gonadotropin-stimulate steroidogenesis in the ovary. The results from these studies may indicate new reasons for marked variations in gonadotropin responsiveness in women.
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