Most of our understanding of oocyte development and maturation was discovered in amphibians or invertebrates. The substantial differences in oocyte physiology and biochemistry, even among mammals, dictates that direct analysis of human tissues is necessary to understand development of human oocytes. In addition, activin, and its related family members can regulate cell-cycle events such as proliferation and differentiation, yet the role of these hormone/growth factors in oocyte maturation is virtually unexplored. Thus, the goal of this proposal is to explore biochemical events that characterize human oocyte maturation, and test the hypothesis that members of the activin/inhibin superfamily have a role in regulating this process. In addition, we hypothesize that MOS, an serine/threonine kinase that activates Maturation Promoting Factor, is a key regulatory protein in the maturational process and further, is regulated by members of the activin/inhibin superfamily.
In Specific Aim 1, we will explore biosynthesis of members of the activin/inhibin superfamily, follistatin, and MOS by human oocytes as they mature in vitro. Steady state message levels will be explored by RT-PCR while protein production will be explored by immunoprecipitation. The role of these hormones will also be investigated directly by culturing immature oocytes in the presence of these hormones.
Specific Aim 2 will concentrate on biosynthesis of the same group of hormones by mural and cumulus granulosa cells using similar techniques. However, this Aim will also include examination of granulosa cell secretion using a novel set of sensitive immunoassays for inhibin, activin, follistatin and free alpha-inhibin subunit.
Specific Aim 3 will use the same immunoassays to measure these hormones in hFF samples from IVF patients whose oocytes were retrieved, fertilized and transferred.
For Specific Aims 2 and 3 then, changes in biosynthesis and secretion of activin/inhibin family members by granulosa cells, and their concentration in hFF from the same follicles, will be correlated with outcome of fertilization, embryo quality, pregnancy, and delivery of viable offspring. In addition, tissues from natural cycle IVF patients will be compared with hyperstimulated-cycle IVF patients, both of which will be compared to samples from preovulatory follicles from normal cycling women. Thus, this proposal is designed to examine human oocyte development in an integrated genetic, protein biosynthetic, and physiological approach. This information will potentially lead to novel, non-destructive methods for assessing quality of individual oocytes in the context of their potential for fertilization and development into viable offspring through use of sensitive and specific immunoassays. In addition, this proposal will provide novel information concerning the mechanisms regulating normal human oocyte maturation and fertilization as well as insights into causes of human infertility relating to pathophysiology of oocyte development.
