Abstract: 【Objective】The aim of this study was to verify the expression of epidermal growth factor (EGF) and its receptor (EGFR) on cumulus-oocyte complexes (COCs) during in vitro maturation, and the effect of EGF on development of COCs and possible molecular mechanisms were also investigated. 【Method】Yak ovaries were collected and COCs were aspirated, the COCs with multilayered compact cumulus and uniformly dark, evenly granulated cytoplasm were selected and cultured in vitro; The expression of EGF and EGFR in immaturate and mature COCs was detected by the methods of Real-time PCR and immunofluorescence at mRNA and protein levels. The 0, 50, 100 and 200 ng·mL^-1 EGF and the optimal concentration of EGFR inhibitor (Gefitinib) were supplemented to the basic medium during in vitro maturation, the rate of maturation, cleavage and blastocyst of oocyte were compared among different groups. Moreover, the genes related to apoptosis (Bax and Baxi) in COCs treated with EGF and Gefitinib were determined by the method of Real-time PCR. 【Result】 Both levels of EGF and EGFR gene were higher in maturation COCs than in immaturate COCs, The relative quantitative expression of EGF in maturation COCs was 2.17±0.36 fold when compared with the levels in immaturate COCs, and the relative quantitative expression of EGFR in maturation COCs was 6.82±0.21 fold compared with the levels in immaturate COCs. Both in immaturate COCs and maturation COCs, the level of EGFR gene was higher than EGF. The results of immunofluorescence showed that EGF and EGFR proteins could be detected on immaturate COCs and maturation COCs, they was mainly in cumulus granulosa cells (CGCs), and the expression of EGF and EGFR protein was lower in oocyte. The rates of maturation (73.45±2.09)%, cleavage (59.46±1.41)% and blastocyst (26.23±1.08)% were significantly higher in the groups with 100 ng·mL^-1 EGF, the rates of maturation, cleavage and blastocyst were (54.16±3.25)%, (48.33±1.93)% and (15.34±0.43)% in control group (0 EGF). In the groups with 50 ng·mL^-1 EGF, the rates of maturation, cleavage and blastocyst were (61.79±1.04)%, (51.76±0.61)% and (18.62±1.13)%, and in 200 ng·mL^-1 EGF groups the rates of maturation, cleavage and blastocyst were (71.26±4.18)%, (57.13±2.06)% and (23.96±0.53)%. However the rates of maturation, cleavage and blastocyst were lower in the groups with EGFR inhibitor (Gefitinib), they were (43.63±1.46)%, (41.79±2.81)% and (12.65±0.67)%, respectively. The expression of Bax gene in maturation COCs could be inhibited by EGF, the levels of Bax gene were 0.83±0.12, 0.21±0.02 and 0.27±0.03 fold in maturation COCs treated with 50 ng·mL^-1, 100 ng·mL^-1 and 200 ng·mL^-1 EGF druring in vitro maturation when compared with control group (0 ng·mL^{- 1} EGF), it was the lowest in the group with 200 ng·mL^-1 EGF, however, the expression of Bax gene was enhanced by Gefitinib, which was 4.24±0.10 fold of levels in control group. The expression of Baxi gene in maturation COCs could be enhanced by EGF, the levels of Baxi gene were 2.18±0.12, 7.06±0.59 and 6.73±0.31 fold in groups with 50 ng·mL^-1, 100 ng·mL^-1 and 200 ng·mL^-1 EGF when compared with control group (0 EGF), however the expression of Bax gene was inhibited by Gefitinib, which was 0.32±0.04 fold of levels in control group.【Conclusion】 EGF and EGF were important autocrine growth factors during yak COCs in vitro maturation, and the rates of maturation, cleavage and blastocyst could be enhanced by exogenous EGF, the optimal concentration was 100 ng·mL^-1, and the functional mechanism might be related to the expression of Bax and Baxi genes was regulated by EGF.

Key words:  yak, oocyte, in vitro maturation, EGF, apoptosis