EGF、EGFR在牦牛卵母细胞中的表达及对胚胎发育能力的作用

The Expression of EGF and EGFR in Yak Oocyte and Its Function on Development Competence of Embryo

【目的】探明表皮生长因子(epidermal growth factor,EGF)及其受体(EGFR)在牦牛卵母细胞成熟过程中的表达动态,并且研究EGF对牦牛卵母细胞成熟的影响及可能的分子作用机制。【方法】采集牦牛卵巢,捡取质量较好的牦牛卵丘-卵母细胞复合体(cumulus-oocyte complexes,COCs),进行体外成熟培养;分别处理未成熟和成熟的牦牛COCs,采用Real-time PCR和间接免疫荧光方法检测COCs成熟过程中EGF及EGFR在基因和蛋白水平的表达动态;牦牛COCs成熟培养时在基础培养基中分别添加0、50、100和200 ng·m L-1 EGF及最佳作用浓度的EGFR抑制因子Gefitinib,比较作用前后卵母细胞成熟率、受精后卵裂率及囊胚率;Real-time PCR方法分析不同浓度EGF和Gefitinib作用后成熟COCs中凋亡相关基因Bax和Baxi的相对表达量。【结果】EGF,EGFR基因在成熟COCs的表达显著高于未成熟COCs,成熟COCs中EGF基因的相对表达量为未成熟COCs的2.17±0.36倍,EGFR基因在成熟COCs的表达量为未成熟COCs 6.82±0.21倍,且在未成熟和成熟COCs中EGFR基因表达量均显著高于EGF。免疫荧光检测显示未成熟COCs和成熟COCs均表达EGF和EGFR蛋白,EGF和EGFR蛋白的标记荧光主要集中在卵丘细胞,卵母细胞表达较弱。100 ng·m L-1 EGF可以显著提高COCs成熟率(73.45±2.09)%及其受精后的卵裂率(59.46±1.41)%和囊胚率(26.23±1.08)%;对照组中(0 EGF)COCs成熟率为(54.16±3.25)%,卵裂率为(48.33±1.93)%,囊胚率为(15.34±0.43)%;EGF浓度为50 ng·m L-1时成熟率、卵裂率及囊胚率分别为(61.79±1.04)%、(51.76±0.61)%和(18.62±1.13)%;200 ng·m L-1成熟率、卵裂率及囊胚率分别为(71.26±4.18)%、(57.13±2.06)%和(23.96±0.53)%;而加入EGFR抑制因子Gefitinib显著的降低了COCs的成熟率及其受精后的卵裂率及囊胚率,分别为(43.63±1.46)%、(41.79±2.81)%和(12.65±0.67)%。COCs成熟过程中EGF的作用可以显著抑制促凋亡基因Bax的表达,在50 ng·m L-1、100 ng·m L-1、200 ng·m L-1EGF作用后成熟COCs中Bax基因的相对表达量分别仅为对照组(0 EGF)的0.83±0.12,0.21±0.02,0.27±0.03倍,EGF浓度为100 ng·m L-1时Bax基因表达量最低,而Gefitinib作用后可以显著促进成熟COCs中Bax基因的表达,其相对表达量为对照组的4.24±0.10倍;EGF在COCs成熟过程中可以显著促进抗凋亡基因Baxi的表达,其相对表达量在EGF浓度为50、100、200 ng·m L-1EGF分别为对照组的2.18±0.12、7.06±0.59和6.73±0.31倍,EGF浓度为100 ng·m L-1时Baxi基因表达量最高,而Gefitinib作用后可以显著降低成熟COCs中Baxi基因的表达,其相对表达量为对照组的0.32±0.04倍。【结论】EGF和EGFR作为牦牛COCs体外成熟过程中重要的自分泌因子,且外源的EGF可以显著提高卵母细胞成熟率、卵裂率及囊胚率,最佳作用浓度为100 ng·m L-1,其作用机制可能与调控凋亡相关基因Bax和Baxi表达有关。

【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 m RNA and protein levels. The 0, 50, 100 and 200 ng·m L-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·m L-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·m L-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·m L-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·m L-1, 100 ng·m L-1 and 200 ng·m L-1 EGF druring in vitro maturation when compared with control group（0 EGF）, it was the lowest in the group with 200 ng·m L-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·m L-1, 100 ng·m L-1 and 200 ng·m L-1 EGF when compared with control group（0 ng·m L- 1 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·m L-1, and the functional mechanism might be related to the expression of Bax and Baxi genes was regulated by EGF.