Expression of growth and differentiation factor 9 (GDF-9) and its effect on the in vitro culture of caprine preantral ovarian follicles

Abstract

This study examined the expression of growth and differentiation factor 9 (GDF-9) in caprine ovarian follicles, and the effect of GDF-9 with or without FSH on the in vitro culture of preantral follicles. To evaluate the expression of GDF-9 in Experiment 1, follicles were recovered from 32 goat ovaries and the total RNA isolated and transcribed for real-time polymerase chain reaction (PCR). Experiments 2 and 3 each used a further 32 goat ovaries to provide preantral follicles of ≥150μm. These follicles were isolated and cultured individually in 100μL drops. In each experiment at least 45 follicles were used per treatment. Every 6 days, follicles were evaluated for viability, antrum formation and growth rate. At the end of the culture period, oocytes were submitted to in vitro maturation (IVM), viability tests and chromatin evaluation. In Experiment 2, follicles were cultured in a basal medium (control) or this medium supplemented with GDF-9 at a concentration of 100ng/mL (GDF-9 100) or 200ng/mL (GDF-9 200). The same media were used in Experiment 3, supplemented with recombinant FSH at a level of 100ng/mL from day 0, 500ng/mL from day 6 to 12 and 1000ng/mL from day 12 to 18 of culture to form the three treatments: control FSH, GDF-9 (100) plus FSH and GDF-9 (200) plus FSH. Relative GDF-9 expression (Experiment 1) was greater in the secondary (18units) than the primordial (1unit) and the primary (1unit) preantral follicles (P<0.05). In the antral follicles, GDF-9 expression was significantly higher in the cumulus–oocyte complexes COC's<3mm (1.6units) than those of >3mm diameter (1unit; P<0.05), and in COC's<3mm and >3mm (319.2 and 200.1units, respectively), compared to their respective granulosa and theca cells (1unit for each category, P<0.05). In Experiment 2, GDF-9 supplementation significantly improved the survival of the follicles (60.8%, 66.0% and 77.4% for the control, GDF-9 100 and GDF-9 200, respectively; P<0.05), follicular growth rate and antrum formation following 18 days of culture. Oocyte survival was approximately 100% in all treatments. More oocytes were submitted to IVM from GDF-9 100 (78.0%; P<0.05), compared to GDF-9 200 (48.1%), but no suitable oocytes could be retrieved from the control (58.8%). The proportion of oocytes showing a resumption of meiosis, was not significantly different between treatments (41.4%, 35.9% and 36.0% for the control, GDF-9 100 and GDF-9 200, respectively). The addition of GDF-9 to the media supplemented with FSH (Experiment 3) did not significantly affect any of the variables studied. The proportion of oocytes submitted to IVM in Experiment 3 was 53.3%, 56.5% and 63.8% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (no statistical differences). The resumption of meiosis was 75.0%, 60.9% and 60.7% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (NS). The occurrence of metaphase II was very low in both experiments. It was concluded that the supplementation of a basal medium with GDF-9 had a positive effect on the survival and development of caprine preantral follicles, but had no real effect in the presence of FSH.