The absence of K+ and Na+ in extracellular compartment leads to the aberration of gonadotropin- and growth factor-influenced steroidogenic and other biosynthetic functions of ovarian granulosa cells. These observations have been interpreted to reflect the importance of these ions in granulosa cell function. However, very little is known about the regulation and pathways by which these ions enter or leave this endocrine type of cell. The objective of the present proposal is to test the hypothesis suggested by our preliminary data, that the Na+/H+ antiporter is a mediator of EGF action in granulosa cells. We will also test the hypothesis that electrical excitation, K+ and C1- channels and membrane potential play an intimate role in the LH-induced production of progesterone, and in growth factor- (EGF, IGF-1, TGFa and TGFb) influenced steroidogenesis and other biosynthetic functions in granulosa cells.
The specific aims are: (1) Determine if the Na+/H+ antiporter-mediated cytoplasmic alkalinization is an obligatory event in growth factor-activated granulosa cells, and on follicular development using pH sensitive flourochrome. (2) Determine the effect of long term (3-4 days) exposure of immature granulosa cells to growth factor on the Na+/H+ antiporter activity using an in vitro cell culture mode. (3) Study the LH and growth factor regulation (direct or messenger-mediated) of K+ and Cl- channels in granulosa cells isolated from follicles at different stages of maturation using patch clamp techniques. (4) Examine the influence of long term (3-4 days) exposure of immature granulosa cells to LH and growth factor on the occurrence and activity of K+ and Cl- channels. The studies will be accomplished by using patch clamp techniques and spectrofluorometry on granulosa cells isolated from domestic hen (Gallus domesticus) at different stages of follicular development. The distinctive anatomical features of the hen ovary coupled with the powerful patch clamp and spectroflourometric techniques make possible a detailed study of the regulation of ion fluxes in granulosa cells during the maturation of ovarian preovulatory follicles. Our results will explain how the ionic microenvironment necessary for important processes catalysed by enzymes (such as adenylate cyclase and cholesterol side chain cleavage enzyme) are controlled. These studies will also provide information on how gonadotropins and growth factors regulate granulosa cell differentiation during follicular maturation. In addition, they will give knowledge about the mechanism of action growth factors in granulosa cells.
