In cattle breeding, co-culture with granulosa cells (GCs) is one of the strategies to improve oocyte maturation and fertilization potential, but yields are still suboptimal due to GC apoptosis. We previously set up an in vitro co-culture system of cumulus-oocyte-complexes (COCs) anchored to GC multilayers adhering to the basal lamina (COCGs), in which GC apoptosis was inhibited by FSH supplementation. Here, we assessed the antiapoptotic effect of EGF (5 ng/ml-EGF5) alone or in synergism to FSH (50mU/ml-FSH50) on pig COCGs. COCG morphology, apoptotic rate, procaspase-8 and-9 expression levels and surface ultrastructure were determined. Results showed an increased % of apoptotic GCs in control and EGF5 (≈80%) respect to sampling (≈3%) and caspase-8 and -9 activation. In contrast, apoptotic cells were significantly reduced by FSH50 (≈35%) supplementation, with inactive Procaspase-8 and -9 highly expressed. The pro-survival effect of FSH was strengthened by EGF (EGF5+FSH50), as evidenced by a significant reduction of apoptosis (≈15%) and high expression levels of Procaspase-8 and -9. Ultrastructural analysis revealed that GC multilayers were characterized by round-to-ovoid cells connected each other and to the basal lamina by cytoplasmic projections. Microvilli shortening/thickening/reduction, cytoplasmic projection rarefaction, blebbing of apoptotic bodies and degenerating/atresic GCs were observed in control and EGF5 groups. FSH50 induced the formation of an abundant mucinous matrix, due to granulosa expansion. Blebs and atresic areas were rarely observed. In EGF5+FSH50 group, GCs were well-preserved, richly covered by microvilli and connected by numerous cytoplasmic projections. Degenerative phenomena were rarely observed. In conclusion, EGF in synergism with FSH seems to better counteract GC apoptosis in a co-culture of pig GC multilayers.
Keywords: Apoptosis; Co-culture system; Granulosa cells; Pig; Scanning electron microscopy.
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