Canine oocyte maturation in culture: Significance of estrogen and EGF receptor gene expression in cumulus cells

Abstract

We examined the role of cumulus cells regarding in vitro maturation of canine oocytes, and investigated estrogen and epidermal growth factor (EGF) receptor gene expression and action on nuclear maturation. Canine cumulus–oocyte complexes (COC) were collected from anestrous and diestrous bitches; only COC with vitelline diameter >100 μm were used. In Experiment 1, expression of estrogen receptor (ER) α, ERβ and EGF-receptor (EGF-R) were determined by reverse transcription-polymerase chain reaction (RT-PCR), using mRNA from the oocyte or cumulus cell. Transcripts for the ERβ and EGF-R were detected in oocytes and cumulus cells, but no message was detected for ERα. In Experiment 2, intact COC and the denuded oocytes were cultured in TCM199 medium supplemented with various concentrations of estradiol-17β (E 2; 0–10 μg/mL) or EGF (0–100 ng/mL) for 72 h; nuclear maturation was then evaluated. In oocytes cultured within intact COC, the rate of germinal vesicle breakdown (GVBD) was higher in the 1 μg/mL E 2 supplemented group ( P < 0.05), and the rate of metaphase I (MI) was higher in the 10 ng/mL EGF supplemented group, than in the non-supplemented group ( P < 0.05). However, supplementation of E 2 or EGF to denuded oocytes failed to promote nuclear maturation. In Experiment 3, intact COC were cultured in TCM199 supplemented with 1 μg/mL E 2, 10 ng/mL EGF, and 10% fetal bovine serum (FBS) for 72 h, and nuclear maturation was evaluated. There was no significant difference in the rate of metaphase II (MII) between the medium only, E 2 + EGF, and FBS supplement groups. When E 2 and EGF in combination with FBS were supplemented, the rate of MII was higher than in other groups ( P < 0.05). We inferred that cumulus cells were involved in mediating the stimulatory effects of E 2 and EGF on nuclear maturation of canine oocytes, and that E 2 and EGF in combination with FBS promoted the completion of oocyte meiotic maturation.