α-烯醇化酶在籽鹅不同级别卵泡壁的定位及定量

Location and quantification of α-enolase in different grades of follicles wall of Zi geese

目的对籽鹅不同级别卵泡壁中的α-烯醇化酶(α-enolase)进行定位及定量检测。方法分离产蛋期籽鹅等级卵泡(F1、F2、F3、F4、F5)、小黄卵泡(small yellow follicle,SYF)、大白卵泡(large white follicle,LWF)的卵泡壁;应用免疫组化方法检测不同级别卵泡壁的α-烯醇化酶的定位,采用Western blot及实时荧光定量PCR双标准曲线法检测α-烯醇化酶蛋白表达量及mRNA转录水平。结果不同级别卵泡壁均存在α-烯醇化酶表达,颗粒细胞层表达量总体高于膜层,SYF颗粒细胞层α-烯醇化酶表达量较高;F2级卵泡壁α-烯醇化酶mRNA转录水平显著高于LWF、F3及F4级别卵泡,差异有统计学意义(P <0. 01),F1、F5及SYF级卵泡壁α-烯醇化酶m RNA转录水平显著高于其他级别卵泡壁,差异有统计学意义(P <0. 01);SYF级卵泡壁α-烯醇化酶蛋白表达量显著高于其他级别卵泡,差异有统计学意义(P <0. 01)。结论α-烯醇化酶及糖酵解途径可能在卵泡发育中发挥重要功能,为α-烯醇化酶在禽类卵泡选择及生长发育中的功能研究奠定了基础。

Objective To analyze the location and quantification ofα-enolase in different grades of follicles in Zi geese.Methods The follicular walls of various grades(F1,F2,F3,F4,F5),small yellow follicles(SYF)and large white follicles(LWF)in the laying Zi geese were separated.The location ofα-enolase in various grades of follicular walls was determined by immunohistochemical assay.The mRNA transcription and protein expression levels ofα-enolase were determined by real-time fluorescent quantitative PCR with double standard curve method and Western blot respectively.Results The expression ofα-enolase was observed in various grades of follicular walls,and the expression level in granulosa cell layer was higher than that in theca interna.The expression level ofα-enolase in granulosa cell layer of SYF was relatively high.The transcription level ofα-enolase mRNA in F2 follicular walls was significantly higher than that in LWF as well as F3 and F4 follicular wallss(P<0.01).However,the transcription levels ofα-enolase m RNA in F1,F5 and SYF follicular wallss were significantly higher than those in the follicular walls of the other grades(P<0.01).The expression level ofα-enolase in SYF follicular walls was significantly higher than those in follicular walls of the other grades(P<0.01).Conclusion The study indicated thatα-enolase and glycolytic pathway played important roles in follicular development,which laid a foundation of research on the function ofα-enolase in the selection,growth and development of avian follicles.