CYP19A1调控内源性雌激素合成促进牦牛COCs细胞自噬和早期发育能力

CYP19A1 Promotes Autophagy and Early Developmental Ability of Yak Oocytes by Regulating the Levels of Endogenous Estradiol

旨在探索牦牛卵母细胞成熟过程中细胞色素P450芳香化酶(cytochrome P450arom,CYP19A1)对内源性雌激素(17β-estradiol,E_(2))分泌、卵母细胞自噬和后续胚胎发育能力的影响。本研究在牦牛卵丘卵母细胞复合体(cumulus-oocyte complexes,COCs)体外成熟过程中,分别用等体积生理盐水、最佳浓度E_(2)(10^(-7)mol·L^(-1))、CYP19A1诱导剂黄曲霉毒素B1(aflatoxin B1,AFB1)、CYP19A1抑制剂双酚A(bisphenol A,BPA)处理,实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)、Western blot和免疫荧光技术检测各组成熟COCs中CYP19A1表达水平,酶联免疫吸附方法(enzyme-linked immunosorbent assay,ELISA)检测诱导和抑制CYP19A1处理组牦牛成熟COCs培养液中E_(2)水平;分析不同处理组COCs细胞自噬调控关键因子:自噬相关基因5(autophagy related gene 5,ATG5)、微管相关蛋白轻链3(microtubule associated protein light chain 3,LC3)和Bcl-2同源结构域蛋白(Bcl-2 homologous domain protein,BECLIN1)表达,比较不同处理组卵母细胞成熟率、孤雌激活胚胎卵裂率、以卵裂胚胎数为基数的囊胚率。结果显示,AFB1处理组COCs中CYP19A1的表达水平上调,内源性E_(2)分泌增加,自噬相关因子ATG5、BECLIN1和LC3表达水平增加,且与对照组差异均显著(P<0.05);E_(2)处理组CYP19A1水平显著降低,但COCs细胞自噬相关因子表达水平显著升高(P<0.05);BPA处理组CYP19A1的表达显著下调,内源性E_(2)和COCs细胞自噬相关因子均显著降低(P<0.05);与对照组相比,卵母细胞成熟率与胚胎卵裂率在E_(2)和AFB1处理组显著增加(P<0.05),而在BPA处理组显著降低(P<0.05),以卵裂胚胎数为基数的囊胚率在各组间差异不显著(P>0.05)。研究表明,牦牛COCs成熟过程中CYP19A1上调内源性E_(2)水平,其上调的E_(2)与外源性E_(2)生理功能具有相似性,均可诱导细胞自噬发生,提升早期卵母细胞发育能力,结果为深入探索生殖激素调控哺乳动物卵母细胞成熟的分子机制提供了理论依据。

The aim of this study was to investigate the effects of cytochrome P450arom(CYP19A1)on the secretion of endogenous 17β-estradiol(E_(2)),and its effects on autophagy of oocyte and subsequent embryonic development were also evaluated.During the in vitro culture of the yak cumulus-oocyte complexes(COCs),saline,E_(2)(10^(-7)mol·L^(-1)),aflatoxin B1(AFB1)and bisphenol A(BPA)were respectively supplemented to M199 at same volume.The expression and location of CYP19A1 in COCs from different groups were detected by qRT-PCR,Western blot and immunofluorescence,as well as the autophagy related genes 5(ATG5),Bcl-2 homologous domain protein(BECLIN1)and microtubule associated protein light chain 3(LC3).The levels of endogenous E_(2)in mature medium of COCs from the groups treated with saline,AFB1 and BPA were measured by enzyme-linked immunosorbent assay(ELISA).Rates of mature oocyte,cleavage embryo and blastocyst were calculated and compared among different groups.The results showed that both gene and protein levels of CYP19A1 were enhanced significantly in the AFB1 group,along with the increase of the endogenous secretion of E_(2),ATG,BECLIN1 and LC3 levels in mature COCs(P<0.05).The level of CYP19A1 were reduced significantly in the E_(2)group,while the expression of autophagy related factors in mature COCs were enhanced(P<0.05).In BPA treatment group,the CYP19A1 and endogenous E_(2)were significantly reduced(P<0.05),which leading to the lower expressions of ATG5,BECLIN1 and LC3(P<0.05).Comparing to the control group,the rates of mature oocyte and cleavage embryo were enhanced by E_(2)and AFB1(P<0.05),but reduced in BPA group(P<0.05).There was no significant difference about blastocyst rates among 4 groups(P>0.05).It can be concluded that endogenous E_(2)was positive regulated by CYP19A1 during COCs maturation,furthermore both endogenous and exogenous E_(2)can induce autophagy and improved the early developmental capacities of yak oocyte.The results provide a theoretical basis for further exploring the molecular mechanism of reproductive hormones regulating the maturation of oocytes in mammalian.