The Role Of XBP1 In Endometrium And Ovarian Granulosa Cell

The function of ovarian granulosa cells and the regulation of endometrial cells are the key to follicular development and embryo implantation.Decades of research have found that endoplasmic reticulum stress was widely involved in follicle development and atresia,embryonic growth and development,implantation and decidualization,and other female reproductive processes.X-box binding protein1?XBP1?,a basic region-leucine zipper?bZIP?protein,is a stress-inducible transcription factor of CREB/ATF family.After being activated by IRE1?cleavage in endoplasmic reticulum stress,XBP1 restored endoplasmic reticulum homeostasis through different degradation pathways.It not only plays a critical role in unfolded protein response,but also has wide biology effect as a transcriptional activator in proliferation,differentiation,migration and apoptosis.In this study,Immunohistochemistry,RNA interference,flow cytometry,Western blot,real-time PCR and ELISA were used to investigate?1?The regulated expression of XBP1 by steroid hormone estrogen and progesterone in mice uterus;?2?The regulation of XBP1and ERS by phytoestrogen molecular ZEA?3?the effect of XBP1 on steroidogenesis,apoptosis and proliferation of mouse granulosa cells.1.During the estrous cycle,XBP1 was clearly present in endometrial luminal epithelium and glandular epithelial cells.And in estrus and diestrus,levels of XBP1 expression were especially high.In vivo,XBP1 was upregulated in endometrial luminal epithelium and glandular epithelial cells from ovariectomized?OVX?mice exposed to 17?-estradiol?E₂?.The expression of XBP1 was rapidly increased from 2 h to 12h after the injection of E₂.P₄treatment also enhanced XBP1 expression in uterine stromal cells and endometrial luminal epithelium cells.The results showed that the expression of XBP1 was upregulated in the primary endometrial stromal cells of mice in a time-dependent pattern after progesterone treatment.2.To suppress the expression of XBP1,three recombinant plasmids named pCD513B-U6-XBP1-shRNA-1,-2,and-3 were constructed,which were validated by PCR and DNA sequencing.A well-by-dilution titer assay was used to determine the titer of recombinant lentivirus after lentivirus package.The titer of 5-10×10⁷ IU/m L was fit for using in subsequent tests.Then,NIH 3T3 cells were transducted with the lentivirus particle.The real-time PCR results showed that XBP1 was knocked down the most by pCD513B-U6-XBP1-shRNA-3 compared with other plasmids,which was also confirmed by Western blotting.Thus,pCD513B-U6-XBP1-shRNA-3 was used for further experiments.3.The expression of Grp78 in granulosa cells reduced to 20%after delivery of lentiviral vector carrying shRNA sequence.ELISA analysis showed that XBP1 depletion significantly decreased the concentrations of estradiol but not progesterone.Additionally,the expression of estrogen synthesis enzyme Cyp19a1 was sharply downregulated.Moreover,flow cytometry showed that knockdown of XBP1 increased the apoptosis rate and arrests the cell cycle in S-phase in granulosa cells.Further study confirmed these results.The expression of CHOP,caspase-3,cleaved caspase-3,and Cyclin E was upregulated,while that of Bcl-2,Cyclin A1,and Cyclin B1 was downregulated.Simultaneously,CCK8 analysis indicated that XBP1disruption inhibited cell proliferation.In addition,XBP1 knockdown also alters the expression of Has2 and Ptgs2,two essential genesfor folliculogenesis.4.After zearalenone treatment,flow-cytometry and western blot results showed that zearalenone significantly induced endometrial epithelial cell apoptosis accompanied with increased expression of Cleaved caspase-3 and AIF,and decreased expression of Bcl-2.The results of ELISA and real-time quantitative PCR showed that zearalenone significantly enhanced the expression and secretion of tumor necrosis factor?TNF?-?,interleukin?IL?-1?,IL-6,but did not alter that of IL-10.Meanwhile,the expression of NF-?B was upregulated in zearalenone stimulated endometrial epithelial cell.Treatment of endometrial epithelial cell with zearalenone elevated the expression of the Grp78,CHOP,XBP1 and three key proteins of the unfolded protein response pathways.The endoplasmic reticulum stress inhibitor,4-phenylbutyrate?PBA?,reduced the zearalenone induced apoptosis rate and expression of Cleaved caspase-3,AIF in endometrial epithelial cell with zearalenone treatment.Furthermore,our results showed that 4-PBA pre-treatment inhibited the expression and release of TNF-?,IL-1?and IL-6 induced by zearalenone.Moreover,zearalenone-induced increases of NF-?B activity were lowered by inhibition of endoplasmic reticulum stress.In conclusion,the above results show that XBP1 plays an important role in female mammal reproductive activity of uterus and ovaries,providing new insights for further study revealing the relationship between endoplasmic reticulum stress and mammalian reproduction.