Evaluation of ovine pre-antral follicles after in situ culture in medium supplemented with EGF and GDF -9

Ovarian sheep contains thousands of immature oocytes predominantly included in the pre-antral follicles. These follicles are considered a potential source of fertilizable gametes, what is of great interest to ensure the in vitro growth and allow the acquisition of competence of oocytes from these follicles. However, during the female reproductive life, few primordial follicles reach the pre ovulatory follicle stadium, after starting their in vivo growth, because during this process about 99.9 % of follicles undergo atresia. The follicular growth and maturation are controlled by a perfect interaction between endocrine, autocrine and paracrine factors. Many studies demonstrated that growth factors such as EGF, FGF, IGF, TGF- s, GDF -9 and KL are of great importance during this process, which may act in different ways on all stages of folliculogenesis. Therefore, this study aimed to verify the population of pre-antral follicles enclosed in sheep ovarian tissue after in situ culture in medium supplemented with different concentrations of epidermal growth factor (EGF) and growth differentiation factor-9 (GDF-9). Ovaries were obtained from abattoirs (n = 60) and fragmented. A fragment of each pair of ovaries was used for the histology (fresh control), and the others were cultured in vitro for 8 days at 39 °C and 5% CO2. The base medium was α- Minimum Essential Medium added of supplements (α-MEM+), and the fragments were cultured in pure α-MEM+ (culture control) or added with 10, 25, 50, 75 or 100 ng/mL of EGF or GDF-9 (treatment groups). After culture, all fragments were processed for conventional histology and stained with hematoxylin-eosin technique (HE). The number of pre-antral follicles was evaluated in the slides of fresh control, culture control and treatment groups in triplicate. It was counted 30 follicles in each section, which were classified in primordial (PL), primary (PO) and secondary (SE) follicles. Data were subjected to ANOVA (P <0.05). The results showed that medium containing 25 and 50 ng/mL of EGF or GDF-9 showed an increase (p <0.05) in the follicular activation (SE follicles) when compared to fresh control. In addition, treatment with 50 ng/ml of EGF and 25 ng/mL of GDF-9 showed the better results (p <0.05) compared to culture control, after 8 days culture. Given the above, it was concluded that α-MEM+ medium supplemented with reduced concentrations of EGF or GDF -9 is enough to promote the follicular activation of ovine primordial follicles in the in vitro culture. Other studies will be performed in order to test the efficiency of these two growth factors used together in the medium.