Factors influencing in vitro maturation of pig oocytes

Abstract

The present studies were undertaken to investigate the effect of different medium supplements, the involvement of steroid hormones and the role of estradiol-17$\beta$ on in vitro maturation of pig oocytes. Cumulus oocyte complexes (COC) were obtained by aspiration of 2-5 mm follicles. The oocytes were cultured for 42 hours at 38.5\sp\circC in mTCM-199 supplemented with LH, FSH, follicular fluid, fetal calf serum and estrous gilt serum, alone or in combination. After culture the oocytes were stained with 1% lacmoid. Oocytes showing metaphase II chromosomes were considered nuclear matured. Cytoplasmic maturation was assessed by the presence of one female pronucleus and at least one male pronucleus after in vitro fertilization. Addition of 10% pFF in combination with gonadotropins during maturation provided the best environment for in vitro maturation with the highest percentage of oocytes reaching nuclear and cytoplasmic maturation. Addition of various levels of estrogen, progesterone and testosterone either alone or in combination had no effect on nuclear or cytoplasmic maturation. During culture COC and cumulus cells secreted both estrogen and progesterone. To investigate the role of estradiol in maturation COC were cultured in steroid free medium in the presence of either an antiestrogen (tamoxifen) or an aromatase inhibitor (4-OHA). Before use, the content of estradiol receptors (ER) in CC and oocytes were determined by a binding assay, before, during and after maturation. The highest levels of ER were observed at 24 and 36 hours of maturation on the oocytes and CC respectively. The presence of tamoxifen and 4-HOA in the culture medium did not influence nuclear or cytoplasmic maturation. In conclusion, the optimal environment for in vitro maturation of pig oocytes was found to be culture medium containing gonadotropins and 10% porcine follicular fluid. No steroids need to be added to the maturation medium with estradiol not necessary for pronuclei formation.U of I OnlyETDs are only available to UIUC Users without author permissio