The Role Of Endoplasmic Reticulum Stress In Goat Endometrial Epithelial Cells Function Under Hormone Treatment

The first important step in pregnancy recognition and implantation is interaction between the endometrial luminal epithelium?LE?and the blastocyst.In ruminant,the receptive endometrium and the elongation of the hatched blastocyst are required to complete the process of implantation.In particular,two thirds of failure pregnancy are due to abnormal uterine receptivity.Therefore,the endometrium plays an important role in the establishment and maintenance of pregnancy.During establishment of pregnancy,endometrial epithelium has undergone a dynamic change that is regulated primarily by maternally derived progesterone?P₄?,estradiol?E₂?and conceptus-derived interferon-tau?IFN-??in ruminants.Recent studies clearly support the evidences that P₄ and E₂ stimulates a number of genes in the LE and superficial glandular epithelium?GE?that encode enzymes and secreted proteins.Then,IFN-?increases expression of several P₄-induced genes that are hypothesized to regulate conceptus survival,growth,and adhesion.Although the expression of tens of thousands of genes and many regulatory mediators have been identified during peri-implantation in ruminants,the some mechanisms regulating endometrial function are still unknown.Many studies have confirmed that ER stress induced the unfolded protein response?UPR?involves in several physiological and pathological process.Studies have reported that 78-kDa glucose-regulated protein?GRP78?,which is the main regulator of UPR,and ERS-response-regulated protein CREB3 Regulatory Factor?CREBRF?are up-regulated in implantation site in mice.Our limited knowledge leads us to speculate that ER stress is involved in regulating embryo implantation.In this study,we stimulated EECs with maternally derived P₄,E₂ and conceptus-derived IFN-?to mimic the intrauterine environment during the peri-implantation period of pregnancy.The molecular mechanism of ER stress and related factors in hormone-regulated goat endometrial epithelial cell function was studied by methods of lentiviral interference,Real time PCR,Western Blot,immunofluorescence staining,ELISA,flow cytometry and CCK-8.The main results are as follows:?1?Goat endometrial epithelial cells?EECs?were treated with progesterone,estradiol and IFN-?.We have found that the expression of genes associated with promoting conceptus elongation were increased,significantly up-regualted the spheroid attachment rate and PGE₂/PGF_2?ratio,and activated ER stress.?2?ER stress activator thapsigargin?TG?activated the expression of GRP78,CHOP,ATF6,phosphoIRE1 and phosphoEIF2S1,increased the promoting conceptus elongation and implantation related genes,including ISG15,CXCL10 and RSAD2.TG also increased the PGE₂/PGF_2?ratio and goat trophoblast cells?GTCs?spheroid attachment rate.ER stress inhibitor 4 phenyl butyric acid?4-PBA?inhibited UPR and inhibited promoting conceptus elongation and cellular attachment related genes,but the spheroid attachment rate and PGE₂/PGF_2?ratio were not changed significantly.?3?The ATF6 interference vector was designed and constructed.The packaged shATF6virus titer was determined by using a dilution method.The titer of shATF6 was 5-10?10⁷TU/mL.Then,the lentiviral interference efficiency was detected on EECs.Knockdown of ATF6 via shATF6 promoted the conceptus elongation related genes and the spheroid attachment rate,but increased the dissolution of the corpus luteum.Rapamycin?mTOR inhibitor?pre-treatment inhibited the expression of promoting conceptus elongation and increased PGE₂/PGF_2?ratio.?4?The level of CREBRF and LC3-II were increased under progesterone,estradiol and IFN-?treatment in EECs.The CREBRF interference vector was designed and constructed.The packaged shCREBRF virus titer was determined by using a dilution method.The titer of shATF6 was 5-10?10⁷ TU/mL.Then,the lentiviral interference efficiency was detected on EECs.Knockdown of CREBRF hampered EECs proliferation by S-phase cell cycle arrest,and ignificant down-regulation in mRNA level of CCNA2,CCNB1 and CCND1.The PGE2 to PGF_2?ratio of the shCREBRF group was lower than the shN group,and the expression of endometrial receptivity markers?HOXA10 and HOXA11?were down-regulated in the shCREBRF group compared to the shN group.Then,the attachment of GTCs spheroids to shCREBRF EECs was decreased compared to the shN group,and CREBRF silencing inhibited the expression of SPP1.?5?Inhibition of the mTOR pathway not only partly restores the PGE2 to PGF2?ratio but also increases the expression of endometrial receptivity markers in shCREBRF EECs by activating autophagy.We also noticed that no significant correlation was found between cell attachment and mTOR pathway activity.The PGE2 to PGF2?ratio and the transactivation activity of endometrial receptivity markers were repressed by CQ pretreatment.Then,CQ pretreatment did not stimulate spheroid attachment rate and the mRNA level of SPP1 in shCREBRF EECs.In conclusion,this study found that ER stress-mediated UPR is involved in the regulating endometrial epithelial cells function during early pregnancy.Our finding partially reveals the molecular mechanism of ER stress related factors ATF6 and CREBRF in regulating endometrial epithelial cells function.This study may provide new insight into the mechanistic pathways of regulating endometrial epithelial cells function during early pregnancy in goat.