The growth and development of ovarian follicles is dependent upon the population of granulosa cells present in the follicle. A systematic study of the growth and differentiation of granulosa cells has been difficult because of the differences in granulosa cells within the same follicle. Functionally different populations of granulosa cells can be separated using Percoll gradients from the ovaries of immature rats treated with diethyl stilbestrol (DES) to induce follicular growth and pregnant mare's serum gonadotropins (PMSG) to induce follicular growth and differentiation. Two populations of granulosa cells gave been shown to exist by flow cytometry and EM studies. Granulosa cells from DES and PMSG treated rats will be separated by the use of an EPICS 5 flow cytometer and Percoll gradients. The cells will be characterized by light and electron microscopy, presence of FSH and LH receptors and FSH induced estradiol and progesterone production. A combination of the enrichment of ovarian follicles by size and granulosa cell separation is expected to yield populations of granulosa cells in various stages of growth and differentiation. The use of the DES and PMSG primed immature rats is being made to work out experimental procedures in a cost effective manner with minimal animal use and the results obtained will be verified in the adult cycling rat. In order to obtain sufficient numbers of granulosa cells for further studies in different stages of growth and differentiation with use of a minimal number of animals, immortalization by transfection with the large T antigen of the SV40 virus will be attempted to generate stable cell lines. Such transfection studies have been successful in our hands using bovine adrenal cells in which the cells retained their pretransfection characteristics. With purified granulosa cell population with different growth and differentiation characteristics, detailed studies will then be done on the regulation of growth and differentiation by agents such as growth factors, estradiol, cAMP analogues, and FSH singly or in combination in a serum free defined medium. The effect of androgens on the stage dependent stimulation or inhibition of aromatase will also be studied. These studies are expected to provide new insights into the factors that lead to healthy ovulatory follicles or atretic follicles during the process of folliculogenesis.
