The Effect And Regulation Of Estrogen To Autophagy In Porcine Oocyte

Oocytes make provision for maturation and subsequent embryonic development by accumulating the necessary nutrients in the process of meiosis.As an important reproductive hormone,estrogen plays an irreplaceable role in the maturation and development of oocytes.Previous evidences have shown that autophagy,as a cell survival mechanism,plays an important role in oxidative stress cell apoptosis and senescence of somatic cells and granulosa cells.However,fewer reports have been conducted on autophagy and oocyte development,and the relationship between estrogen and autophagy in this process is even less clear.In this study,the inhibitor,immunohistology,cellular immunofluorescence,western blot and other techniques and methods were employed to analyze the the role of autophagy in estrogen-regulated porcine oocytes maturation and embryo development;reveal the effects of estrogen on autophagy of oocytes;determine the roles of estrogen-mediated autophagy on oxidative stress and early apoptosis of mature oocytes;investigate the effect of estrogen in autophagy-regulated gap junction intercellular communication,explore the molecular pathway of estrogen receptor on autophagy.This study attempted to systematically elucidate the mechanism of the combined regulation of estrogen and autophagy on the maturation and development of porcine oocytes,and might provide scientific basis for improving the quality of porcine oocytes and the development ability of embryo.The main results and conclusions of this study are as follows:1.Autophagy was highly expressed in porcine ovarian oocytes,cumulus cells and granulosa cells.During in vitro maturation,0.1 and 1?M of 17?-estradiol significantly increased the rate of the first polar body,and the rate increased in a time-dependent manner after 1?M treatment with 17?-estradiol.After treatment with autophagy inhibition Autophinib,the rate of the first polar body decreased in a dose-dependent manner,and the decreasing trend of rate could be mitigated by adding an appropriate concentration of17?-estradiol.Autophagy inhibition with Autophinib decreased the rate of cleavage in a dose-dependent manner,adding an appropriate concentration of 17?-estradiol alleviated the decreasing trend the rate of cleavage rate and blastocyst.These results suggest that estrogen may be mediated by autophagy involvement in oocyte maturation as well as embryo development.2.Confocal scanning and quantitative analysis revealed that treatment with17?-estradiol decreased autophagy degradation of SQSTM1.At the early stage of in vitro maturation,17?-estradiol increased the expression of autophagy marker protein LC3-II,and the expression of SQSTM1 was at a low level,suggesting that estrogen promotes autophagy in oocytes.After inhibiton autophagy by Autophinib,LC3-II protein expression decreased in a dose-dependent manner in mature oocytes,which proved that Autophinib could effectively inhibited autophagy in porcine oocytes.Interestingly,17?-estradiol significantly enhanced the autophagy inhibited by Autophinib.These findings demonstrate that estrogen is involved in autophagy during oocyte maturation.3.Inhibition of autophagy activity resulted in higher ROS levels and abnormal mitochondrial distribution in mature oocytes.While treatment with 17?-estradiol not only had lower ROS levels,also the mitochondria were more evenly distributed.Further analysis showed that added 17?-estradiol into Autophinib group significantly reduced ROS levels and repaired the normal distribution of mitochondria.More importantly,the level of intracellular Ca²⁺in oocytes treated with 17?-estradiol was lower than treated with autophagy inhibitor Autophinib,but the level of intracellular Ca²⁺increased significantly when treated together.These evidences suggest that the effect of estrogen on oxidative stress induced by autophagy inhibition in mature oocytes may depend on the up-regulation of normal mitochondrial distribution and the level of intracellular Ca²⁺.Annexin-V staining revealed that the autophagy was accompanied with apoptosis,and autophagy inhibition with Autophinib increased the apoptosis rate in mature oocytes,this effect was significantly down-regulated by 17?-estradiol.Meanwhile,autophagy inhibition resulted in low mitochondrial membrane potential,thereby causing mitochondrial dysfunction,17?-estradiol enhanced mitochondrial function and significantly reduced mitochondrial dysfunction caused by autophagy inhibition.Further study revealed that apoptosis increased by autophagy inhibition might be caused by the activation of caspase-8and then the increase of the cleavage of caspase-3,while 17?-estradiol also activated caspase-8,and significantly inhibited the cleavage of caspase-3,thereby further reducing the autophagy inhibition of caspase-3 activity.These results demonstrated that17?-estradiol reduced autophagy-inhibition-induced apoptosis in oocytes by mediating apoptosis-related proteins and repairing mitochondrial dysfunction.4.During the maturation of porcine oocytes,the gap junction communication between cumulus and oocytes decreased in a time-dependent manner.17?-estradiol significantly reduced gap junction communication in the early stage of in vitro maturation,and the inhibition of autophagy can significantly improve gap junctions.In addition,17?-estradiol downregulated the gap junction communication mediated by autophagy inhibition.Analysised of the COCs subcellular structure found that the results were consistent with the interstitial junctions,suggesting that inhibition of autophagy significantly increased the number of TZPs,which is down-regulated by 17?-estradiol.Further study found that17?-estradiol not only down-regulated the phosphorylation level of Cx43,but also decreased the phosphorylation modification of autophagy inhibited Cx43.These results suggested that 17?-estradiol down-regulated autophagy-inhibition-induced gap junction communication depended on reduced the Cx43 phosphorylation in porcine COCs.5.GPR30,a novel receptor of estrogen located on the plasma membrane,decreased in a time-dependent manner during in vitro maturation of porcine oocytes,and 17?-estradiol maintain the high expression of GPR30 during in vitro maturation.Treated with G15completely eliminated the promoting effect of 17?-estradiol on nuclear maturation.Inactivation of GPR30 down-regulated MEK1/2 and ERK1/2 phosphorylation,suggesting that GPR30 regulates porcine oocytes through the MEK/ERK pathway.Further analysis showed that the inactivation of MEK1/2 and ERK1/2 promoted the expression of SQSTM1and reduced the level of LC3II to inhibited autophagy.These results suggested that GPR30induced by 17?-estradiol promoted autophagy through the MEK/ERK signaling pathway,thereby promoted porcine oocyte maturation.In summary,17?-estradiol and autophagy of porcine oocyte closely related to the occurrence.17?-estradiol promoted the autophagy in porcine oocyte,added the appropriate concentration of 17?-estradiol improved the oocyte maturation and early embryonic development ability.Furthermore,17?-estradiol reduced ROS levels and early apoptosis caused by autophagy inhibitor Autophinib,and down-regulated gap junction communication in early stage of oocyte maturation.The effect of 17?-estradiol on the autophagy of porcine oocytes may be through GPR30/MEK/ERK pathway,thus promoted the maturation of porcine oocytes.This study attempted to provied a new theoretical basis of the regulation mechanism in porcine follicular atresia.