ABSTRACT
Ovarian follicular fluid is widely used for in vitro oocyte maturation, but its in-depth characterization to extract full beneficial effects remains unclear. Here, we performed both shotgun (nanoscale liquid chromatography coupled to tandem mass spectrometry or nanoLC-MS/MS) and gel-based (two dimension-differential in-gel electrophoresis or 2D-DIGE) proteomics, followed by functional bioinformatics to compare the proteomes of follicular fluids collected from small (<4 mm) and large (>6-12 mm) follicles of pig ovaries. A total of 2321 unique spots were detected with the 2D-DIGE across small and large follicles, while 2876 proteins with 88% successful annotations were detected with the shotgun approach. The shotgun and 2D-DIGE approaches revealed about 426 and 300 proteins that were respectively common across samples. Six proteins detected with both technical approaches were significantly differently expressed between small and large follicles. Pathways such as estrogen and PI3K-Akt signaling were significantly enriched in small follicles while the complement and coagulation cascades pathways were significantly represented in large follicles. Up-regulated proteins in small follicles were in favor of oocyte maturation, while those in large follicles were involved in the ovulatory process preparation. Few proteins with potential roles during sperm-oocyte interactions were especially detected in FF of large follicles and supporting the potential role of the ovarian FF on the intrafallopian sperm migration and interaction with the oocyte.
ABSTRACT
Factors that control the onset of folliculogenesis are critical to female gamete production, but poorly understood. The aim of the present study was to investigate the effects of FSH and EGF on the activation and growth of goat primordial follicles in vitro. To this end, pieces of goat ovarian cortex were cultured in vitro for 1, 3 or 5 days, at 39 degrees C in an atmosphere containing 5% CO(2), in minimum essential medium supplemented with insulin, transferrin, selenium, pyruvate, glutamine, hypoxanthine, BSA, penicillin, streptomycin and fungizone and with or without FSH (100 ng/ml) and/or EGF (100 ng/ml). At the end of the culture periods, the relative proportions of primordial, intermediate, primary and secondary follicles were calculated and compared with those in non-cultured tissue. In addition, mitotic activity of granulosa cells was studied by immunohistochemistry for proliferating cell nuclear antigen (PCNA). In brief, it was found that goat primordial follicles activate spontaneously during culture in vitro and, while neither FSH nor EGF affected the proportion of primordial follicles that entered the growth phase, both stimulated an increase in oocyte and follicle diameter, especially in intermediate and primary follicles cultured for 5 days. On the other hand, there was no significant effect of culture or either growth factor on the proportion of PCNA-stained growing follicles. Contrary to expectations, neither FSH nor EGF affected follicle viability or integrity during culture, since the percentages of intact follicles did not differ between control, FSH and/or EGF containing medium. In conclusion, this study demonstrated that goat primordial follicles activate spontaneously in vitro, and that both FSH and EGF stimulate an increase in follicle size by promoting oocyte growth.
