Host: Society for Reproduction and Development
Objective: Our previous studies have revealed that follicle stimulating hormone (FSH) inhibited apoptosis in pig granulosa cells. FSH stimulated hyaluronan (HA) synthesis. Furthermore the synthesized HA could decrease apoptosis, indicating that one of FSH inhibitory mechanism is mediated through HA. The present study was aimed to elucidate the apoptosis inhibitory action of HA in pig granulosa cell by analyzed the binding of HA and CD44, the receptor of HA. Materials and Methods: Pig follicles having 3-5 mm in diameter were isolated from ovaries. Cumulus-oocyte complexes with a granulosa layer which is designated as COCG, were dissected from follicles, and then cultured for 48 hours in DMEM/F-12 supplemented with FSH 50mU and various concentration of 4-MU (HA synthase inhibitor) or IM7 (anti-CD44 antibody) which reduced the binds of HA and CD44. The control group had only the medium. The protein expressions were detected using western blotting. We examined the expression of CD44 at COCG cell. Furthermore we analyzed the effect of HA-CD44 bind on apoptosis. Results: HA synthesis could be stimulated by FSH and inhibited by 4-MU. In this study, CD44 expressed after cultured for 48 h with FSH, decreased in addition of 0.1 mM/ml 4-MU, whereas no expression detected in high concentration of 4-MU, indicating that when the synthesis HA is inhibited, the amount of CD44 also decreased. Addition of IM7 decreased the expression of precursor caspase-3,8,9 indicating that the perturbation of HA-CD44 bind leads to activation of caspase-8,9 the initiator of apoptosis, also caspase-3 the executer of apoptosis. In conclusion, HA-CD44 interaction has been implicated in granulosa cells apoptosis inhibition.
