Test of Oocyte Reversibility
After 24-h culture, isolated COCs were washed twice in dissection medium and transferred into 500 ^l maturation medium (TCM199 with Earle’s salts [Gibco], 75 mg/L kanamycin, 4.75 g/L Hepes, 2.29 g/L NaHCO3 [pH 7.8, osmolarity 280 mOsmol/kg H2O]) supplemented with ovine FSH/LH (0.006 IU/ml), 5% FCS, in 35-mm tissue culture dishes. COCs were then incubated in humidified atmosphere of 5% CO2 in air at 39°C for 24 h. After culture, the oocytes were denuded, fixed, stained with aceto-orcein, and examined under phase contrast.
Use of Okadaic Acid
Antral follicles were immersed in culture medium using 6-well tissue culture dishes without netwell insert membranes for 24 h. Oocytes were recovered and transferred into maturation medium containing gonadotrophic hormone and 0.5 ^M okadaic acid (Sigma). Okadaic acid has been shown to induce a rapid appearance of maturation-promoting factor activity in Xenopus and starfish oocytes and accelerates GVBD in cattle and pig oocytes. buy levaquin online
In Vitro Fertilization
In vitro-matured oocytes were fertilized according to the method described by Vergos et al.. Briefly, COCs were gently pipetted in order to remove adhering granulosa cells and break up aggregated COCs. Disaggregated COCs were then washed once (in oocyte wash medium containing NaCl, 6.8 g/L; KCl, 230 mg/L; NaHCO3, 168 mg/L; Na2HPO4, 47 mg/L; Hepes, 4.8 g/L; kanamycin monosulfate, 75 mg/L; pyruvic acid, 11 mg/L; BSA, 6 g/L; 60% syrup lactic acid, 1.86 ml/L; MgCl2 6H2O, 100 mg/L; CaCl2 2H2O, 840 mg/L; pH 7.4, osmolarity 282 mOsmol/ kg H2O) and transferred into 45-^l microdrops of fertilization medium (5-10 oocytes per drop) containing sperm (1.5 X 106/ml) and cultured for 48 h at 39°C in a humidified incubator of 5% CO2 in air