Regulation of apoptosis and steroidogenesis in ovarian preovulatory granulosa cells

Abstract: Only a minute fraction of ovarian follicles present in a foetal ovary will complete the path to ovulation. The majority of the follicles will instead undergo atresia, a hormonally controlled apoptotic process. Apoptosis occurs throughout follicle development, with an extensive reduction in the number of follicles present at birth. Interestingly, this drastic reduction is not present among preovulatory follicles responding to the ovulatory surge of luteinizing hormone (LH), since the number of corpora lutea roughly equals the number of preovulatory follicles. Granulosa cells (surrounding the oocyte in the follicle) exposed to the LH-surge, are characterized by the induction of expression of progesterone receptors (PR) as well as a prominent steroidogenic activity. It was therefore pertinent to study factors involved in PR signalling as well as in regulation of steroidogenesis, to try to elucidate mechanisms regulating ovarian granulosa cell apoptosis. Human as well as rat granulosa cells were isolated after in vivo treatment with hormones to induce luteinization of ovarian follicles. To study apoptosis, internucleosomal DNA fragmentation and caspase-3 activity, two characteristic apoptotic events, were used as endpoints. Cell concentration was shown to be an important factor to take into consideration when interpreting in vitro data, since a cell-concentration-dependent apoptosis was demonstrated in cultured granulosa cells. Internucleosomal DNA fragmentation was shown to be decreased in granulosa cells isolated from rats treated with human chorionic gonadotropin to mimic the endogenous LH-surge. Exposure to the LH-surge is a prerequisite for PR expression in granulosa cells to occur. To be able to interfere with PR signalling, two different PR-antagonists, RU 486 and Org 31710 were used. Addition of PR-antagonists to granulosa cell cultures during 24 h incubation in serum-free medium caused a dose-dependent increase in internucleosomal DNA fragmentation as well as an increase in caspase-3 activity compared to controls. These effects were not present in granulosa cells not exposed to the LH-surge in vivo, consequently not expressing functional PR. LH receptor stimulation of granulosa cells thus makes the granulosa cells less susceptible to apoptosis. This event seems to be at least partly, mediated by PR expression and activation.Leptin is an adipocyte-derived hormone known to affect fertility by interfering with the central regulation of gonadotropin secretion. In addition, direct effects on ovarian cells may be postulated, regulating events in ovarian granulosa cells. Using the reverse transcriptase-polymerase chain reaction, we were able to detect the presence of leptin receptors in the human ovary but no expression of leptin mRNA. However, leptin was detected in follicular fluid, suggesting a possible endocrine action of leptin in the ovary. Stimulation of primary cultures of human luteinizing granulosa cells with leptin suppressed LH-induced estradiol production, indicating a mechanism for leptin to regulate fertility at the ovarian level. Whether this interferes with regulation of granulosa cell apoptosis remains to be determined.In conclusion, this thesis provides insight into important mechanisms involved in regulation of ovarian luteinizing granulosa cell apoptosis, namely signalling via the progesterone receptor and interference with the granulosa cell steroidogenic capacity by leptin.