INHIBITORY ACTION OF FSH IN APOPTOSIS OF SWINE GRANULOSA CELLS IN COCG CULTURE INVOLVES HYALURONAN SYNTHESIS IN AN INTRINSIC PATHWAY

Abstract

Follicular atresia at the antral stage is believed to be initiated by apoptosis of granulosa cells followed by oocyte degeneration. Folliclestimulating hormone (FSH) is known to be a survival factor for preovulatory follicles by inhibiting apoptosis in granulosa cells. The present study aimed to investigate apoptosis-inhibitory action of FSH with regard to a possible link of hyaluronan (HA). Cumulus-oocyte complexes with a granulosa layer (COCG) were mechanically dissected from healthy follicles and then cultured for 48 h in DMEM/F-12, supplemented with FSH or HA. ELISA was used to determine the content of secreted HA; Hoechst staining was used to determine the apoptosis rate of granulosa cells; and RT-PCR and Western blotting were used to detect caspase mRNA and proteins, respectively. FSH treatment increased the content of secreted HA and mRNA expression of hyaluronan synthase (an enzyme for HA synthesis), while the rate of apoptosis increased in granulosa cells treated with beta-methylumbelliferone (4-MU), which is a hyaluronan synthase inhibitor. Addition of HA into the medium inhibited apoptosis of granulosa cells in a dose-dependent manner. These results indicate that in porcine follicles HA has the ability to inhibit apoptosis of granulosa cells, and that the inhibitory action of FSH may be mediated by HA secretion. The second experiment, demonstrating an apoptosissignaling pathway (both intrinsic and extrinsic), revealed that expression of caspase 9 mRNA was inhibited in granulosa cells cultured with the addition of FSH and HA. On the other hand, expression of caspase 8 mRNA was inhibited by FSH treatment, but not by HA treatment. Expression of procaspase 9 and 8 support these findings. These results suggest that FSH can inhibit apoptosis in granulosa cells through both intrinsic and extrinsic pathways, while HA may have an inhibitory action only in the intrinsic pathway. In conclusion, FSH inhibits apoptosis in swine granulosa cells in COCG culture, in at least one case via a mechanism mediated through stimulation of HA synthesis. (poster)