The cellular and molecular mechanisms involved in the growth of human preantral follicles and the subsequent maturation of the oocyte to acquire competence to undergo meiosis remain largely unknown. Numerous studies have contributed to knowledge of these events in several animal species. The clinical application of this knowledge to the human is significant for the treatment of infertility, the improvement of the reproductive potential of women surviving childhood or early adulthood cancers as well as by its contribution to a basic understanding of reproductive events. In order to study the maturation of oocytes in vivo as well as in vitro, oocytes will be retrieved from volunteers (women undergoing tubal ligations) at different times following hCG administration. In addition, a unique aspect of our studies will involve excision of preantral follicles from ovarian tissue in order to construct culture systems that will permit maturation of a competent gamete. A major goal is to develop an informational database by characterizing the nuclear and cytoplasmic changes that occur during human oocyte maturation in vivo and in vitro. This will be evaluated by morphologic criteria including germinal vesicle breakdown as assessed by phase contrast light microscopy and indirect immunofluorescence. Tubulin immunohistochemistry and fluorescent DNA binding stain will determine spindle and nuclear morphology and thus assess the time to MI. Since integrin adhesion molecules have been detected on the plasma membrane of mammalian oocytes and our preliminary results indicate at least one integrin subunit (beta1) being present in human oocytes, we will investigate the expression of the repertoire of integrin subunits during the final stages of human oocyte maturation. This will be achieved through confocal microscopy using specific antibodies to the different integrins and an attempt will be made to assess their expression at the nucleotide level using RT-PCR. Their functional significance with respect to fertilizability of oocytes will then be tested with the use of functional antibodies or antisense mRNA microinjection. In parallel, the secretion of tissue type plasminogen activator by oocytes developing in vivo or in vitro will also be evaluated using substrate zymography. An additional goal is to evaluate culture systems for the in vitro development of preantral follicles that promote oocyte growth, acquisition of competence to resume meiosis, and capable of supporting normal fertilization and embryogenesis. The effects of various growth factors and hormones on follicular and oocyte development will be assessed. Finally, we propose to investigate the biochemical parameters of human egg activation following fertilization of in vivo and in vitro matured oocytes and to examine the potential signaling mechanisms involved in this process. This will be achieved by quantitatively evaluating calcium transients during fertilization, cortical granule exocytosis, recruitment of maternal mRNA, H1 kinase activity, pronuclear formation, and DNA synthesis and cleavage, using standard assay methods. These studies will define the events involved in normal human oocyte activation. Establishment of the optimal conditions for human oocyte culture from preantral follicles can have a tremendous impact in the enhancement of fertility,.
