Effects of in vitro-growth culture on maturation of pig growing oocytes and proliferation of cells surrounding oocytes.

Abstract

In vitro growth cultrure for growing oocytes from preantral follicles is now still a challengable question for scientists. The objective of this study was to investigation the acquisition of meiotic competence of growing oocyte from preantral follicles (1.5-2mm in diameter) after 5-day in vitro growth and subsequent in vitro culture for maturation. To rescue large number of growing oocytes, dissected 1.5-2mm follicles with clear fluid were collected for in vitro growth culture of oocytes in vitro culture medium consisting of bicarbonate-buffered DMEM (Dulbecco’s Modified Eagle Medium, Sigma) supplemented with 10% fetal bovine serum, 0.1mg/ml sodium pyruvate, 0.1mg/mL Penicillin, 0.05mg/mL Streptomycin, 0.5 mMdbcAMP, supplemented with 0.5mMdbcAMP, 0.05 IU/mL FSH and 1μg/mL estradiol 17β supporting the proliferation of surrounding cells (granulosa-cumulus complexes), which has an effect on the growth of oocytes, was examined. The increase of cells surrounding oocytes were observed after 3 days and 5 days. Then, oocytes were transferred to in vitro maturation condition including TCM 199 supplemented with 10% fetal bovine serum, 0.1mg/ml sodium pyruvate, 0.1mg/mL Penicillin, 0.05mg/mL Streptomycin, 10% porcine follicle fluid and 0.1IU/mL human chronic gonadotrophin (hCG after 5-day in vitro growth. Chromatin configuration of oocytes in vitro cultured for 5 days and after in vitro maturation are investigated by aceto-orcein (1%) staining. The results indicated that oocytes from preantral follicles (1.5-2mm in diameter) were able to become fully grown (25.0%) at germinal vesicle stage I; however, 5-day in vitro grown could progress meiotic competence (40.9% of GVII-IV) and germinal vesicle breakdown (18.2%). In conclusion, it is suggested that in vitro growing oocytes from preantral follicles can become fully grown after 5 days and undergo germinal vesicle breakdown (GVBD) after in vitro culture for maturation; however, no in vitro grown oocytes can reach to metaphase II. This states that the presence of in vitro growth culture medium is insufficient for fully meiotic competence of oocytes. Keywords: preantral follicles, growing oocytes, in vitro growth, fully grown, in vitro maturation.