Effective dosage of GDF9 in folliculogenesis and angiogenesis in the sheep ovarian tissues grafted onto chick embryo chorioallantoic membrane

Background: Many researchers have tried to culture and transplant the ovarian tissues, to preserve fertility in cancer patients. However, one of the main limitation to the applicability of this technique is the folliculogenesis disruption after transplantation.Objective: Due to the roles exerted by Growth Differentiation Factor-9 (GDF-9), we decided to determine the most effective dose of GDF9 on promotion of folliculogenesis and angiogenesis in sheep OTs grafted onto the Chick chorioallantoic membrane (CAM).Materials and Methods: Fresh sheep ovarian tissues were grafted onto the CAM for 5 days, and divided into four groups based on the addition of increasing doses of GDF9 (0, 150, 200, and 250 ng/ml). Following 5 days of culture, the tissues were retrieved and histological (H&E staining) and immunohistological studies (with Ki-67) were done. Fibrotic and necrotic surface were measured using MICROVISIBLE software. For comparing follicle percentage between groups, as well as differences in Ki67-positive follicles, ANOVA was applied.Results: In the 200 and 250 ng/ml GDF9 groups, significantly higher rates of intermediary and primary follicles were observed, accompanied by a decrease in proportion of primordial follicles. The numbers of good quality follicles increased in aforementioned groups. In respect to the 0 (control) and 150 ng/ml GDF9 groups, the presence of fibrotic and necrotic areas decreased. Moreover, in the 200 and 250 ng/ml GDF9 groups, the number of capillaries and the proliferative activity increased. These results showed that higher dosages of GDF9 (200 and 250 ng/ml) were able to improve follicular development, Transplantation features, in ovarian tissue grafted onto the CAM due to improvement in angiogenesis.Conclusion: Folliculogenesis didn t take place in ovarian grafted onto the CAM. addition of high dosages of GDF9 to the OTs, grafted onto the CAM, activated folliculogenesis in vitro, leading to higher rates of growing follicles, better blood supply, and granulosa cells proliferation.