Effect of intraovarian factors on porcine follicular cells: cumulus expansion, granulosa and cumulus cell progesterone production

The role of granulosa cell conditioned media (CM) containing luteinization stimulator (LS), and the role of EGF in the cumulus expansion of oocyte–cumulus complexes (OCC) isolated from large antral follicles was investigated. The CM were prepared by incubation of granulosa cells isolated from large antral follicles. After 24 h incubation, more than 61 or 64% of OCC expanded to the +3 and +4 stage in the presence of CM (50%) or EGF (10 ng/ml), respectively. The stimulatory effect of LS and EGF on the cumulus expansion was accompanied by the enhanced hyaluronic acid synthesis. Complete suppression of cumulus expansion stimulated by LS and EGF was observed in the presence of 10 μmol/l genistein (tyrosine kinase inhibitor), in the presence of 10 mmol/l LiCl (the inhibitor of inositol 1,4,5-trisphosphate metabolism), and 100 μmol/l gallopamil, verapamil and norverapamil (calcium channel blockers). Stimulatory effect of EGF on the cumulus expansion of OCC isolated from large follicles was accompanied by the increased cumulus cell progesterone production. However, EGF did not affect the progesterone production by OCC isolated from small follicles. To determine whether EGF could modulate the granulosa cell steroidogenesis also, the effect of EGF on granulosa cells isolated from large (LGC) and small (SGC) follicles was compared. EGF (10 ng/ml) failed to affect the progesterone synthesis during 72 h culture of SGC but significantly enhanced the LGC progesterone production. Our results indicate that luteinization factor stimulates the cumulus expansion and hyaluronic acid synthesis by the OCC isolated from large antral follicles. The mechanism of LS- and EGF-induced cumulus expansion may involve tyrosine kinase activation and calcium mobilization. In addition, these results indicate the different response of porcine cumulus and granulosa cells originating from small and large follicles on the stimulatory effect of EGF.