THE SYNERGISTIC ACTION OF EGF AND FSH DECREASES GRANULOSA CELL APOPTOSIS IN AN IN VITRO CO-CULTURE SYSTEM OF PIG CUMULUS-OOCYTE-COMPLEX AND GRANULOSA CELLS (COCG LAYERS).

"INTRODUCTION AND PURPOSE: In porcine ovarian follicles, atresia is mainly due to apoptotic cell death of granulosa cells (GCs). Therefore, there is a high interest to reduce GCs apoptosis in order to improve the developmental rates of oocytes collected from species of zootechnic interest and used for in vitro maturation (IVM). The co-culture of oocytes and GCs, even with supplements, did not provide, up to now, significant protective effect against apoptosis. The purpose of this work was to asses an assumed antiapoptotic action on GCs of Epidermal Growth Factor (EGF) and Follicle Stimulating Hormone (FSH), alone or in combination, by using an in vitro co-culture system.. MATERIALS AND METHODS: cumulus-oocyte complex with GC layers (COCGs) were mechanically dissected from pig ovarian follicles (3-7mm), isolated from prebubertal gilts. Samples were placed in a 30mm dish containing DMEM\/F12, with 1%BSA, streptomycin and penicillin. IVM was done for 48h, at 37°C and 5%CO2. Basic medium (control, group C) was supplemented with: 1) EGF 5ng\/ml (group E), 2) FSH 50mIU\/ml (group F), 3) FSH 50mIU\/ml+EGF 5ng\/ml (group E+F). After co-culture, COCGs were a) fixed in 1% glutaraldehyde\/PBS, stained with Hoechst 33342 and observed by fluorescence microscopy (FM) to determine the apoptotic rate (AR); b) lysed for western blotting (WB) to detect Procaspase-8 and -9 (inactive zymogens of Caspase-8 and -9, initiators of apoptosis); c) fixed in 2.5% glutaraldehyde\/PBS and treated for scanning electron microscopy (SEM) to evaluate the surface morphological and morphometric changes related to the apoptotic damages.. RESULTS: In group C, the AR of GCs was about the 80% as seen by FM. Only a minimal basal expression level of both Procaspase-8 and -9 was detected by WB. The COCGs, as seen by SEM, showed a round or ovoid shape with many microvilli. Several cytoplasmic projections formed a mesh-like structure with numerous intercellular lacunae. Evidence of apoptosis, such as cell shrinkage, plasma membrane blebbing and apoptotic bodies were diffusely observable. In group E, the AR did not significantly differ from controls (~80%, P>0.05) as for the expression of Procaspase-8 and -9. In group F, only the 30% of GCs resulted apoptotic (P<0.05) and the expression of the two zymogens increased respect to controls. In both groups E and F, microvilli were reduced in number and length. In group E+F the AR statistically decreased compared to control group (~18%; P<0.05), while the expression of Procaspase-8 and -9 was higher. By SEM, the apopototic signs decreased respect to controls.. CONCLUSION: EGF and FSH alone did not significantly decrease apoptosis. However, their synergistic action showed a better protective effect against apoptotic cell death. These results may open interesting perspective in the field of IVM to obtain a higher number of oocytes\/embryos with a high potential of implantation.. "