Effect of GDF-9 on in vitro culture of equine preantral follicles: preliminary results

The aim of this study was to evaluate the effects of different concentrations of GDF-9 on in vitro culture of equine ovarian fragments. Ovaries (n=5) from mares in the anovulatory season were collected at local slaughterhouse and washed in 70o ethanol and PBS, and transported for one hour in PBS plus penicillin (200 IU/mL) and streptomycin (200 mg/mL) at 4oC to the laboratory, where the internal portion of the ovary was divided into 9 pieces of approximately 5 x 5 x 1mm. One fragment of each ovary was immediately fixed in Bouin (control group, D0). The other 8 fragments were cultured individually in culture dishes containing 1mL of MEM (MEM control) or base medium supplemented with GDF-9 (0, 50, 100 or 200 μg/mL). MEM+ was MEM (Gibco BRL, Rockville, MD, USA – 300 mOsm/L osmolarity, pH 7.2) supplemented with pyruvate (500 μL), glutamine (500 μL), hypoxanthine (500 μL), ITS, BSA (1.25 mg/mL-Gibco BRL, Rockville, MD, USA), penicillin (500 μL) and streptomycin (500 µL). Culture was performed at 39°C in an atmosphere with 5% CO2 in air and saturated humidity for 2 or 6 days. Medium was replaced every 2 days. After culture, fragments were fixed in Bouin and processed for histology. Follicles were classified according to their stage of development as primordial, primary, secondary and antral follicles, and according to their morphology as normal or degenerated. Data were compared by nonparametric analysis of variance (Kruskal Wallis test), considering significant differences with P<5%. Overall, 675 slides were evaluated with an average of 4 ovarian fragments in each. We found 552 follicles, of which 68 (12.3%) were developing follicles in the control group (MEM+), 355 (64.3%) were primordial, 197 (35.7%) were developing and 379 (68.6%) were morphologically normal. Fragments cultured with 50 μg/mL of GDF-9 for two days of had higher proportion of developing follicles (9%; 50/552). We concluded that MEM supplemented with 50 µg/mL of GDF-9 provided better follicle development during in vitro culture of equine ovarian fragments for two days.