AREG对绵羊小腔卵泡卵母细胞体外成熟的影响

Effect of AREG on in vitro Maturation of Small Antral Follicular Oocytes of Sheep

旨在研究双调蛋白(AREG)对绵羊小腔卵泡卵母细胞体外成熟(IVM)的影响。本研究从屠宰场绵羊卵巢上采集小腔和中腔卵泡的卵母细胞进行试验,利用自发荧光检测卵母细胞的NAD(P)H和FAD^++水平,利用JC-1检测卵母细胞的线粒体膜电位;两种来源的卵母细胞经体外成熟后,比较其卵丘扩展指数(CEI)、第一极体排出率(MII%);比较AREG、AREG+GDF9、AREG+BMP15、AREG+GDF9+BMP15、GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的CEI、MII%及卵母细胞线粒体膜电位的影响;检测了AREG+GDF9+BMP15对小腔卵泡卵母细胞体外成熟后的NAD(P)H和FAD^++水平及其受精后发育能力的影响。结果表明,成熟前,小腔卵泡卵母细胞的线粒体膜电位和FAD^++水平均显著低于中腔卵泡卵母细胞(P<0.05);体外成熟培养后,小腔卵泡卵母细胞的CEI和MII%均显著低于中腔卵泡卵母细胞(P<0.05)。与对照组相比,AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的CEI、MII%和线粒体膜电位(P<0.05),且与中腔卵泡卵母细胞组差异不显著(P>0.05);另外,AREG+GDF9+BMP15显著提高了小腔卵泡卵母细胞体外成熟后的NAD(P)H和FAD^++水平(P<0.05),且与中腔卵泡卵母细胞组差异不显著(P>0.05)。与对照组相比,在成熟液中添加AREG+GDF9+BMP15可以明显提高小腔卵泡卵母细胞体外受精后的卵裂率和囊胚率(分别为(43.79±3.69)%、(28.54±4.31)%和(78.99±1.12)%、(47.46±2.50)%,P<0.05),而且与中腔卵泡卵母细胞组无显著差异(P>0.05)。综上表明,绵羊小腔卵泡卵母细胞的代谢水平及IVM质量较低,AREG在GDF9和BMP15的协同作用下可以显著提高小腔卵泡卵母细胞的代谢水平及IVM质量,并进一步提高小腔卵泡卵母细胞体外受精后的发育能力。

The aim of this study was to investigate the effects of amphiregulin(AREG)on in vitro maturation(IVM)of oocytes from sheep small antral follicles.In this study,oocytes from small and medium antral follicles of sheep ovary from the slaughterhouse were collected.Autofluorescence was used to detect NAD(P)H and FAD^++levels of oocytes,mitochondrial membrane potential of oocytes were detected by JC-1.After IVM,the cumulus expansion index(CEI)and first polar body expulsion rate(MII%)of oocytes from small and medium antral follicles were compared.The effects of AREG,AREG+GDF9,AREG+BMP15,AREG+GDF9+BMP15 and GDF9+BMP15 on CEI,MII%and mitochondrial membrane potential of small antral follicular oocytes were compared after IVM.After IVM,the effects of AREG+GDF9+BMP15 on the levels of NAD(P)H and FAD^++of small antral follicular oocytes and their development ability after in vitro fertilization(IVF)were detected.The results showed that,before IVM,the mitochondrial membrane potential and FAD^++level of small antral follicular oocytes were significantly lower than those of medium antral follicular oocytes(P<0.05).After IVM,the CEI and MII%of the small antral follicular oocytes were significantly lower compared to medium antral follicular oocytes(P<0.05).Compared with the control group,AREG+GDF9+BMP15 treatment significantly increased the CEI,MII%and mitochondrial membrane potential of small antral follicular oocytes after IVM(P<0.05),and there was no significant difference compared with the medium antral follicular oocytes(P>0.05).Moreover,AREG+GDF9+BMP15 treatment significantly increased the levels of NAD(P)H and FAD^++of small antral follicular oocytes after IVM(P<0.05),and there was no significant difference compared with the medium antral follicular oocytes(P>0.05).Compared with the control group,supplemented AREG+GDF9+BMP15 into IVM medium could significantly increase the cleavage rate and blastocyst rate of small antral follicular oocytes after IVF((43.79±3.69)%,(28.54±4.31)%and(78.99±1.12)%,(47.46±2.50)%,respectively,P<0.05),while there was no significant difference compared with the medium antral follicular oocytes(P>0.05).These results indicated that the metabolic level and IVM quality of oocytes from sheep small antral follicles were relatively low.With the synergistic effect of GDF9 and BMP15,AREG can significantly improve the metabolic level and IVM quality of oocytes from small antral follicles,and further improve their development ability after IVF.