Study On Function Of ATF6 In Mouse Periimplantation Uterus And Granulosa Cell Apoptosis

Activating transcription factor 6(ATF6)is a type II transmembrane protein located on the endoplasmic reticulum and is a member of transcription factors from the CREB family.And ATF6,as a key stress sensor protein in the endoplasmic reticulum stress signaling pathway,can regulate the synthesis and degradation of proteins in cells by binding with the endoplasmic reticulum stress response gene elements when endoplasmic reticulum stress occurs.ATF6 is widely distributed in various tissues,organs and cells,such as placenta,uterine decidual tissue,liver,heart,fetal fibroblasts and RAW264.7 macrophages.The existing evidences have shown that the relevant genes involved in endoplasmic reticulum stress and the endoplasmic reticulum stress signaling pathway play important roles in the reproductive process in mammalian.Such as early embryo implantation,endometrial stromal cells decidualization and ovarian function.Therefore,it has important significance in comprehensively understanding the molecular mechanisms of reproductive regulation in mammals and exploring new targets for regulating animal reproduction by further exploring the roles of ATF6 and the endoplasmic reticulum stress mediated by ATF6 in the reproductive process of female mammals.In this study,the roles of ATF6 in the mouse peri-implantation uterus and granulosa cell apoptosis were explored by using immunohistochemical staining,qRT-PCR,Western blotting,shRNA interference,flow cytometry and TUNEL.And the main results of this research were as follows:1.The expression pattern of ATF6 in ovaries,oviducts and uterus of female mice at different stages of estrus cycle were explored by immunohistochemical staining,qRT-PCR and Western blotting.We found that there are some characteristics in the process of the expression of ATF6 mRNA and protein in the ovaries,oviducts and uterus of female mice at different stages of estrus cycle.The results showed that the ATF6 proteins are mainly distributed in oocytes,granulosa cells and corpus luteum,oviducal epithelial cells,uterine epithelial cells and glandular epithelial cells in female mice.And the expression levels of ATF6 mRNA and protein were relatively low in proestrus and estrus while it gradually increased from metoestrus to dioestrus in these tissues and organs,suggesting that ATF6 may be involved in the reproductive process in female mice,and the expression of ATF6 may be regulated by P4 and E2 secreted by ovaries.2.The expression pattern of ATF6 was explored in the uterus of early pregnancy,pseudopregnancy,delayed-implantation and activated-implantation,artificial decidualization,treatment with steroid hormones and antagonist in mice.The results showed that the expression levels of ATF6 mRNA and protein were significantly up-regulated in the pregnancy uterus on day 5 that was called “implantation window”,and ATF6 immunostaining was intensely expressed in the luminal epithelium and stromal cells nearby the implantation site.But the expression level of ATF6 was significantly down-regulted in the pseudopregnancy uterus on the day 5.The levels of ATF6 mRNA and protein were significantly higher in the activated-implantation uterus than in the delayed-implantation uterus,and ATF6 immunostaining was intensely expressed in stromal cells nearby the implantation site in the activated-implantation uterus,indicating that ATF6 may be involved in the adhesion and implantation of embryo in the early pregnancy uterus.And the expression of ATF6 in the pregnancy uteri may be affected by an actived blastocyst.The expression levels of ATF6 mRNA and protein were gradually increased in the pregnancy uterus from day 6-8.And ATF6 immunostaining was strongly present in the primary decidual zone(PDZ)and secondary decidual zone(SDZ)from the uterus on pregnancy day 7-8.A higher expression level of ATF6 was detected in the artificial decidualization uterus.These results above implied that ATF6 may be involved in the decidualization of the stromal cells and the formation of PDZ and SDZ in the early pregnancy uterus.The ATF6 proteins also were detected in the luminal epithelium and glandular epithelium in the uterus after treatment with steroid hormones and relevant antagonists of hormones.Compared with untreated uterus,the levels of ATF6 mRNA and protein were significantly changed in these uterus treated with hormones and antagonists,suggesting that the expression of ATF6 in the uterus was affected by P4 and E2.3.The ATF6 shRNA recombinant vectors and lentivirus were successfully obtained by using the technology of shRNA interference and lentivirus package,and the efficiency of ATF6 shRNA recombinant vectors was detected in the RAW264.7 cells.The results showed that the lentivirus concentration was 7 × 107 TU/mL,and the RAW264.7 macrophages were transfected effectively with the ATF6 shRNA lentivirus and the percentage of cells transfected with lentivirus were more than 90%.At the same time,the disruption efficiency of ATF6 shRNA lentivirus was verified at the mRNA and protein level(~80%).And the lentivirus that could deplete ATF6 effectively were selected for the next experiment for exploring the function of ATF6.4.The expression and localization of ATF6 in the ovarian follicles and granulosa cells at different growth phases were explored in the adult female mice.The results showed that ATF6 was present in the ovocyte and granulosa cells of ovarian follicles at different stages of development,and FSH or LH has an effect on ATF6 expression in mouse granulosa cells.The expression of ATF6 was significantly inhibited in the mouse granulosa cells after transfecting with ATF6 shRNA lentivirus and the disruption efficiency of ATF6 shRNA lentivirus was approximately 80%.The results from qRT-PCR,western blotting,flow cytometry and ELISA showed that ATF6 knockdown significantly inhibited the mouse granulosa cells apoptosis by down-regulating the pro-apoptotic genes including p53,Caspase-3,Chop and up-regulating the anti-apoptotic genes Bcl-2 and Grp78.ATF6 depletion obviously disrupted the granulosa cell cycle via affecting the expression of Cyclin A1,CyclinB1 and Cyclin D2 involved in regulating the cell cycle.And ATF6 disruption significantly increased the secretions of P4 and E2 by regulating the expression of key genes that involved in the synthesis and metabolism of steroid hormones including the up-regulation of Cyp11a1,Star and Cyp19a1 and the down-regulation of Cyp1b1.Furthermore,ATF6 knockdown have significant effects on the expression levels of Has2,Ptgs2,Ptgfr and Igfbp4 that played important role in regulating the follicular growth and development.5.The cytotoxic effect of Zearalenone on the mouse granulosa cells was analyzed by CCK-8 kit.And the result indicated that Zearalenone could significantly decrease the cell viability in dose-and time-dependent manners.What’s more,Zearalenone had an important effect on the expression of ATF6 in mouse granulosa cells.The effect of ATF6 on the mouse granulosa cell apoptosis caused by Zearalenone was explored by qRT-PCR,Western blotting,flow cytometry and ELISA.The result suggested that ATF6 knockdown inhibited the mouse granulosa cell apoptosis induced by Zearalenone via down-regulating the levels of the pro-apoptotic genes including p53,Caspase-3 and Bax and up-regulating the levels of the anti-apoptotic gene Bcl-2.And ATF6 depletion obviously alleviated the dysfunction of P4 and E2 secretion caused by Zearalenone.However,ATF6 depletion had no significant effect on the cell cycle arrest caused by Zearalenone.The endoplasmic reticulum stress was activated and the expressions of ATF6,GRP78 and CHOP were increased in the process of mouse granulosa cell apoptosis induced by Zearalenone.ATF6 knockdown decreased the cell apoptosis ratio by up-regulating the level of GRP78 and down-regulating the level of CHOP.Furthermore,the autophagy was activated and ATF6 depletion may play a protective role in the mouse granulosa cells treated with Zearalenone by promoting the activation of autophagy.The results above suggested that ATF6 played important roles in the embryo implantation,uterine decidualization and granulosa cell apoptosis in mouse.Our work may provide a basis for further exploring the roles of ATF6 and the endoplasmic reticulum stress in the reproductive process of mammalian animals.