Rictor/mTORC2 Is Involved In Endometrial Receptivity By Regulating Epithelium Remodeling

Background:Successful embryo implantation requires well-functioning endometrial luminal epithelial to establish uterine receptivity.Inadequate uterine receptivity is responsible for approximately two-thirds of implantation failures in human beings.However,it remains largely unexplored with respect to regulation mechanism governing this functional process.Previous study reveals that the expression of Rictor（the main member of mTORC2）in mice epithelial cells is increased on gestation days（D）4.Here,we speculate that Rictor is involved in the regulation of endometrial receptivity.In this study,Rictor was conditional ablated in the mice endometrium using a progesterone receptor cre(PR^cre)mouse model.Loss of Rictor altered polarity remodeling and Na⁺channel protein of endometrial cells by mediating Rac-1/PAK1（pPAK1）/ERM（pERM）and Sgk1/pSgk1 signaling respectively,ultimately resulting in impaired fertility.In the endometrium of infertility women,the expression of Rictor has been changed,as well as the morphological transformation and Na⁺channel protein of epithelial cells.Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity both in mice and human beings,and the work may improve the molecular regulatory network of endometrial receptivity and provide a new diagnosis and treatment strategy for infertility.Methods:1.Establishment of endometrial conditional knockout Rictor gene mice and model verification.（1）Rictor gene was specifically knocked out in the mouse endometriumby using the Cre-Loxp recombinase system.The Rictor^f/fmice and PR cre^+/-tool mice（C57BL/6J）were carriedinto the same cage to breeding offsprings.After several generations,the rat tail DNA agarose gel electrophoresis identification experiment was performed to screen out the female mice with genotype PR cre^+/-Rictor^f/f,which were the endometrial conditional knockout Rictor mice(Rictor^d/d),and the mice with the genotype PRcre^-/-Rictor^f/f as the control group(Rictor^f/f).（2）The protein expression of Rictor in the endometrium of Rictor^d/dmice and Rictor^f/f mice was detected by Immunohistochemistry and Western blotting.2.Establishment and selection of mouse normal pregnancy model8-week-old sexually mature Rictor^f/f and Rictor^d/d mice（weight20-30g）were mated with wild-type male mice at a ratio of 2:1 at17:30-18:00.Mice with vaginal plug at 08:00-08:30 am on the second day are recorded as the first day of pregnancy（D1）,and so on.Blood,ovaries and uterine tissues was taken from the D4,D4.5,and D5 mice for subsequent experiments.3.The effect ofthe morphology and function of theovaryinconditional knockout endometrium Rictor mouse（1）The ovaries of each group of mice were weighed for statistical analysis.（2）The transcription level andprotein expression level of Rictor in each group of ovaries were detected by using q-PCR and IHC respectively.The serum estradiol and progesterone levels of each group of mice were detected using ELISA.4.The effect of conditional knockout of the endometrium Rictor on mouse uterine morphology and embryo implantation（1）In each group of mice,the wet weight of the uterus was analysised on D4,D4.5 and D5.（2）0.1mL trypan blue solution（0.4%）were injected into the tail vein of D5 mice to observe the embryo implantation sites.The uterus in mice with no implantation sitewas flushed by normal saline to detect blastocysts under the microscope.5.The effect of Rictor-conditional knockout on the receptivity of the mouse endometrium（1）The expression of proliferation marker Ki67 and receptivity marker Lif in the endometrium of D4 and D4.5 Rictor^f/fand Rictor^d/d mice were detected by IHC;（2）The protein expression of estrogen and progesterone receptors ER a/pERa and PR in the endometrial tissue of D4 and D4.5 micewere detected by IHC;the transcription levels of estrogen and progesterone downstream signaling molecules Muc1,Ltf,Lgf,Hsd11b2,Areg,Ihh and Hand2 in D4 Rictor^f/f and Rictor^d/d mice endometrial tissue were detected by q-PCR.6.Detection of mouse endometrial epithelial cell remodeling and related signal pathway（1）The microvilli,tight junctions between cells and morphology of D4.5 Rictor^f/f,Rictor^d/d mouse endometrial epithelial cells were observed by transmission electron microscope.（2）The expression of CK8 was detected by IHC in D4,D4.5Rictor^f/f and Rictor^d/d mouse endometrium to observe the luminal epithelial cell morphology;the expression of E-Cadherin was detected by IHC in D4,D4.5 Rictor^f/f and Rictor^d/d mouse endometrium to observe the tight junction state between epithelial cells;IF were performed to detect the protein expression levels of Occludin,Claudin-7 and Par3 in the D4,D4.5 mouse endometrium of two groups.（3）TUNEL was used to detect cell apoptosis of D4,D4.5 Rictor^f/fand Rictor^d/d mouseendometrium;IHC was used to detect FOXO1 protein in D4,D4.5 Rictor^f/f and Rictor^d/d mouse endometrium.（4）IHC was used to detect the protein expression of TTBK1,a regulatory protein related to microtubule depolymerization,in the endometrium of D4,D4.5 Rictor^f/f and Rictor^d/d mice.（5）WB was used to detect Rictor and related signaling pathway proteinexpressionlevels（pAKT/AKT,Rac-1,ERM/pERM,PAK1/pPAK1 and b-actin）in the endometrium of D4,D4.5,D5 Rictor^f/f and Rictor^d/d mice.7.Detection of Na⁺channel function of mouse endometrial epithelial cells and related signal pathways（1）Primary endometrial epithelial cellsof Rictor^f/f and Rictor^d/dmouse were isolated,and epithelial cell marker（CK8）was detected using IF to identify cells.Trypsin were used to simulate blastocyst stimulation signals.（2）Fluorescent dye DiBAC（4）3 was used to indicate the changes in cell membrane potential,images were collected using the laser confocal microscope.（3）WB was used to detect the protein expression of Na⁺channels（ENa C a）and related signaling pathway proteins（Sgk1/pSgk1）in the primary endometrial epithelial cells of Rictor^f/fand Rictor^d/d mice.8.The detection of regulatory function of Rictor on human endometrial receptivity and related mechanismThe secretory endometrial tissue of the normal control group and infertile patients were obtained by hysteroscopy;IHC was used to detect the protein expression level of Rictor in the endometrial tissues of the two groups.（1）The detection of the role of Rictor in the remodeling of human endometrial epithelial cells and related signal pathways.IF were used to detect the protein expression levels of Occludin and Claudin-7 in human endometrial,the cell morphology and tight junction state were observed under a microscope.Humanendometrial cancer cells（Ishikawa,receptive endometrial epithelial cell line）were transfected with shRNA-Rictor to down regulate the Rictor.Then,the protein expression levels of Rictor,pAKT/AKT,Rac-1,ERM/pERM,PAK1/pPAK1 and b-actin were detected by WB.（2）The detection of human endometrial epithelial Na⁺channel function and related signal pathway regulated by Rictor.Ishikawa cells were transfected with shRNA-Rictor to downregulate the expression of Rictor and treated with trypsin to simulate the blastocyst stimulation signal.Then,the fluorescent dye DiBAC（4）3 was used to indicate the changes in cell membrane potential.Images were collected by laser confocal microscope.WB was used to detect the protein expression levels of ENaCa,Sgk1/pSgk1 of Ishikawa cells before and after transfection with shRNA-Rictor.9.RNA sequencing and bioinformatics analysis were used to deeply explore the mechanism of Rictor’s regulation of mouse endometrial function during the receptive phase（1）Fresh endometrial tissue of D4 Rictor^f/f and Rictor^d/d mouse were collected for RNA-Secquencing;（2）Statistical analysis of differential genes on sequencing data were performed to draw a Venn diagram.Functionalclustering（GO）analysis and KEGG signal pathway analysis were performed to deeply explore differential genes related to molecular events（cell proliferation,apoptosis and cytoskeleton）of epithelial cells that are closely related to the establishment of endometrial receptivity.Results:1.The mouse model of the conditional deletion of the Rictor in endometrium was successfully establishedRictor^f/f and Rictor^d/d mice were screened out by agarose gel electrophoresis identification experiment on mouse tail DNA.IHC and WB showed that the level of Rictor was significantly reducedin D4,D4.5,and D5 Rictor^d/d mice endometrium,and the mouse model of the conditional deletion of the Rictor gene in the endometrium was successfully established.2.The effect on the morphological and function of mice ovary in Rictor-deleted endometrium（1）There was no difference intranscription level of Rictor in each group of ovaries detected by q-PCR,and the results of IHC showed that the Rictor expression in the ovarian granulosa cells of Rictor^d/d micewas decreased;（2）The wet weight of Rictor^d/d mice ovaries on D4 was lower than Rictor^f/f,but the difference between D4.5 and D5 was not statistically significant;（3）The results of ELISA showed the levels of serum E₂ and P₄ were up-regulated in D4 Rictor^d/dmice.3.Loss of Rictor expression in the mouse endometrium leaded to embryo implantation failure（1）No implantation sites were observedin D5 uterus of Rictor^d/dmice after injecting trypan blue intail vein,which is statistically significant compared with Rictor^f/fmice;（2）The mouse uterus with no implantation sites were flushed with normal saline,a certain number of normal blastocysts wereobserved under the microscope in the flushing fluid.4.The endometrial receptivity of Rictor-deleted mice was impaired（1）Ki67 and Lif staining found that the endometrial epithelial proliferation of Rictor^d/d mice was increased,the proliferation of stromal cells was decreased,the expression of Lif was decreased,and the receptivity was impaired;（2）ER was widely expressed in cytoplasm and nuclear in both epithelial cells and stromal cells in mice.p-ER was high expressed in nuclear in Rictor^f/fmicestromal cells.ERand p-ER were strong positive in epithelial cells and decreased in stromal cells in Rictor^d/d mice.PR was widely expressed in cytoplasm and nuclear in stromal cells in mice.But in Rictor^d/dmice,the expression of PR protein in stromal cells was decreased.The transcription level of endometrial receptivity markers in D4 Rictor^f/fand Rictor^d/d mice was detected by q-PCR:Muc1,Ltf were up-regulated,Lgf,Hsd11b2,Areg,Ihh and Hand were down-regulated,and receptivity was impaired.5.Rictor regulatedepithelial cell remodeling of mouse endometriumthrough pAKT/Rac-1（1）Transmission electron microscope showed the microvilli of the epithelial cells of the Rictor^f/fmice were shorten and some of the tight connection opened.But,the microvilli of Rictor^d/d mice disappeared,the perinuclear space was widened,and vacuole-like cells appeared.（2）CK8 was found that widely expressed in cytoplasm of Rictor^d/dmouse epithelial cells.And the cells were still columnar when uterine receptivity was established,some cells were multilayered and vacuolated instead of the structure of single layer of epithelial cells in Rictor^f/fmice.E-Cadherin is localized in the cytoplasm and cell membrane of epithelial cells and stromal cells.In Rictor^f/f mouse luminal epithelial cells,E-Cadherin protein was gathered at the top of the cell,and the E-Cadherin of side adhesion molecules is reduced.But,the positive signaling of E-Cadherin in Rictor^d/d mouse luminal epithelial cells still strong.（3）Tight junction proteins Occludin,Claudin-7 were mainly located in the cytoplasm and nuclear in both luminal and grandular epithelium of mouse.Par3 was mainly located in thecytoplasm and nuclear in luminal epithelial cells.IF was used to detect Occludin,Claudin-7,and Par3 and the results showed that the expression levels of it in Rictor^d/d mouse luminal epithelial cells were increased.（4）TTBK1 protein was detected by using IHC and the result showed it mainly located in cytoplasm of luminal and glandular epithelium.It was decreased obviously in Rictor^d/dmice.（5）TUNEL revealed that theapoptosis ofluminal epithelial cell in Rictor^d/d mouse was increased,and the nuclear localization of FOXO1protein detected by IHC was increased.（6）WB showed that the expression of pAKT,Rac-1 in the endometrium of D4,D4.5and D5 Rictor^d/d mice was downregulated.6.Rictor regulated the Na⁺channel function of mouse endometrial epithelium via Sgk1/pSgk1（1）Detection of membrane potential of epithelial cells showed that the membrane potential signal of epithelial cells in Rictor^d/d miceafter trypsin treatment to simulate blastocyst stimulation signal was less obvious than that of the control group;（2）WB showed that the expression of pSgk1 was downregulated in the endometrium of Rictor^d/d mice,and the expression of ENaCa was not significantly different.7.Rictor regulated human endometrial receptivity.（1）Rictor staining showed that the expression of Rictor in the endometrium of infertile patients was significantly lower than that of the normal control group;（2）The expression level of tight junction proteins Occludin and Claudin-7 detected by IF was found to be located in the cytoplasm and nuclear in both luminal and grandular epithelium of human tissues.It also elevated in infertile patients,especially near the uterine luminal.WB showed that the expression of pAKT,Rac-1,pERM,and pPAK1 in Ishikawa cells after transfected with shRNA-Rictor were down-regulated compared with the control group.（3）Detection of membrane potential of epithelial cells found that the signal did not change significantly in Ishikawa cells（shRNA-Rictor treated）compared with the control group after treating with the trypsin to simulate the blastocyst stimulation signal.WBshowed the expression of pSgk1,ENaCa was downregulated compared with the control groupafter transfected with shRNA-Rictor.8.RNA-Secquence anddata analysis（1）RNA sequencing of the endometrium in D4 Rictor^f/f and Rictor^d/dmicerevealed that there were 1677 differential genes related to Rictor downregulation when the receptivity process was established.883 genes were upregulated and 794 were downregulated.（2）The GO Analysis and KEGG signal pathway analysis of these1677 genes revealed 36 differential genes closely related to cell proliferation,25genes were up-regulated and 11 were down-regulated;12differential genes closely related to cell apoptosis,11 genes were up-regulated and 1 wasdown-regulated;12 differential genes closely related to the cytoskeleton,7genes were up-regulated and 5 were down-regulated.Conclusion:（1）Loss of Rictor in the endometrium impaired endometrial receptivity in mice,which in turn leaded to infertility;（2）Rictor regulated endometrial epithelial cell remodeling and Na⁺channel function via pAKT/Rac-1 and Sgk1/pSgk1 respectively,thereby affecting the endometrial receptivity;（3）Rictor regulated human endometrial epithelial cell remodeling and Na⁺channel function,which may further affect the success of pregnancy.（4）Bioinformatics analysis showed that Rictor participated inestablishment of endometrial receptivity by regulating theproliferation,apoptosis and the stability of the cytoskeletonof epithelial cells.But,the mechanism need further explored.