The Effect Of Msx1 On Goat Endometrial Epithelial Cell Remodeling Regulated By Hormones

The embryo implantation of mammal is a complex and coordinated process.The successful embryo implantation requires a well-receptive endometrium,a healthy blastocyst and the coordination between endometrium and blastocyst.During the period of goat embryo implantation,the endometrium undergoes dynamic changes to form the best intrauterine environment under combination of maternal-derived progesterone?P₄?,estrogen?E₂?and progesterone-derived interferon-Tau?IFN??.The endometrial epithelial cells undergoes remodeling during receptive endometrium stage,including the shape change from columnar to squareand and the decrease of the microvilli on the top.Along with structural changes,the functions of endometrial epithelial cells including secretion and adhesion also change.Although a large number of genes and regulatory factors involved in the goat embryo implantation process have been discovered,the regulatory mechanism of hormones on endometrial epithelial cell remodeling are still unknown during receptive endometrial stage.The Msx gene family,belongs to one of the oldest and most conserved families of homeobox genes,plays an important role in many complex processes such as embryonic development,organogenesis,and tumorigenesis.Studies have found that Msx1 is involved in regulating the polarity of endometrial epithelial cells during receptive endometrial stage in mice;and abnormal expression of Msx1 has been found in female infertility.During goat embryo implantation,the function and molecular mechanism of Msx1 on endometrial epithelial cell remodeling are unclear.In this study,we stumilated the hormonal environment of uterus in the goat peri-implantation period by P₄?E₂ and IFN?.The role and regulatory mechanism of Msx1 in hormone-regulated endometrial epithelial cell remodeling were investigated by methods of Real time PCR,Western Blot,lentiviral interference,construction of overexpression vectors,immunofluorescence staining,ELISA and adhere of GTCs spheroids.The results are as follows:?1?The expression of Msx1 in goat endometrial epithelial cells is directly or indirectly regulated by E₂ and P₄.When EECs were treated with P₄,E₂ and IFN?,the expression of Msx1 was increased.The expression of CDH2 was increased under hormone treatment,and the transcription levels of epithelial polarity-related genes par3,?-PKC,scribble,Lgl2 and tight junction molecules ZO-1,occludin were decreased.?2?The interference vector p CD513B-U6-sh Msx1 of goat Msx1 gene was constructed.The packaged sh Msx1 virus infected EECs and the lentiviral interference efficiency was detected.Knockdown of Msx1 via sh Msx1 significantly reduced the expression of progesterone elongation genes?ISG15,CXCL10,RSAD2?and the spheroid attachment rate,and increased the PGE₂:PGF_2?ratio.Knockdown of Msx1inhibited the expression of CDH2,and significant up-regulation in m RNA level of par3,?-PKC,scribble,Lgl2,ZO-1 and occludin.Without hormone treatment,up-regulating Msx1 expression via the overexpression vector GV362-Msx1 promotes CDH2expression,inhibits the m RNA levels of epithelial polarity-related genes par3,?-PKC,scribble,Lgl2,and the tight junction molecules ZO-1 and occludin.?3?Knockdown of Msx1 inhibited the expression of the unfolded protein response marker proteins phopho EIF2S1,phopho IRE1 and XBP1 under hormones treatment,but has no effect of Cleaved ATF6.Activation of ER stress not partly restores the expression of CDH2 and decreases the m RNA levels of par3,?-PKC,scribble,Lgl2 and ZO-1,occludin by endoplasmic reticulum stress activator toxic carotene?Tg?pretreatment.But 4 phenyl butyric acid?4-PBA?,ER stress inhibitor,enhances inhibition of CDH2expression and promotes the transcription of scribble,Lgl2 and ZO-1,occludin.In summary,this study found that Msx1,as a hormone-regulated gene,is involved in the regulation of hormone on endometrial epithelial cell remodeling during early pregnancy,further affect the function of the endometrial epithelium.The study provides experimental basis for further study of early pregnancy in goats on endometrial remodeling caused by hormone.