Involvement of steroid hormones on in vitro maturation of pig oocytes

Abstract

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus–oocyte complexes (COCs), collected from follicles of 2–5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17β (0–3000 ng/ml), progesterone (0–5000 ng/ml) and testosterone (0–300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect ( P>0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17β or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly ( P>0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17β and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.