The maturation of steroidogenic capacity that accompanies the differentiation of ovarian cells during follicular development is of fundamental importance to the regulation of reproduction and fertility. The objective of these studies is to define the relationship between ovarian cell growth and the development of steroidogenic function during follicular maturation. Studies are proposed that will examine the growth, heterogeneity, and steroidogenic function of the granulosa and theca cell populations isolated from ovarian follicles of the domestic hen (Gallus domesticus) at defined stages of follicular development. The research will be accomplished by utilizing multiparameter flow cytometry and cell sorting techniques. Correlated measurements on cell cycle, cell size, cell metabolism, and steroidogenic function will be obtained from individual cells stained with fluorchromes that selectively bind to DNA, RNA, protein, or other cell constituents. The proposed research will address the following Specific Aims: 1. Characterize the granulosa and theca cell populations during follicular maturation in terms of cell cycling, relative cell size, cell metabolism (RNA and protein content), and cell heterogeneity. 2. Identify the steroidogenic ability of theca and granulosa cell subpopulations during follicular maturation based on hormone- responsiveness and steroid metabolism of sorted populations, LH binding, and cytochrome P-450 and mitochondrial activities. Examine the morphometry of cells in each subpopulation by electron microscopy. 3. Determine the influence of hormonal changes during the ovulatory cycle on the distribution, proliferation, and function of granulosa and theca cell subpopulations. The unique features of the hen reproductive system, combined with the high detection capabilities of multiparameter flow cytometry, permit such a thorough investigation of ovarian cell growth, function, and heterogeneity, during maturation of the preovulatory follicles.
