The Roles Of Aquaporin 3 In Embryo Implantation

I.ObjectiveSuccessful implantation involves three distinct processes,namely the embryo apposition,attachment,penetration through the luminal epithelium of the endometrium.Estrogen,progestin,cytokines,growth factor and so on are known to regulate the endometrium receptive to require both temporal and spatial synchronization of the uterine endometrium and the embryo.It has long been known that a highly regulated uterine environment is required for successful implantation of the blastocyst.This is evident from observations that the embryo can only attach to the uterine epithelium for a brief period of time known as the ?window of implantation�.Stabilizing the blastocyst in the uterine lumen to establish a close contact with the endometrium is believed to be a prerequisite for implantation.The uterine lumen contains fluid,the volume of which is a significant reduction in uterine luminal fluid when the endometrium is receptive for implantation.Water channel protein(Aquaporins,AQPs)widely expressed on the cell membrane,promoting the various epithelial and endothelial tissue fluid transport in order to maintain the stability of the internal environment and is involved in angiogenesis,tumor cell migration and invasion and tissue damage repair,and maintenance of steady state of cell and the immune function,and other important life activities.In this study,to determine the presence and distribution of AQP3 during the estrous cycle and early pregnancy,experimental manipulation of AQP3 activities was performed in in vivo in the mice.In the human,we research the expression and regulation of AQP3 in the menstrual cycle and differential receptive endometrial epithelium cells.E2 and P4 regulate the expression of AQP3,and regulate abilities of adhesion,migration and invasion in differential receptive endometrial epithelium cells through AQP3.The study will promote the further elucidation of the mechanism in embryo implantation.It has a certain significance on reproductive diseases treatment and improving the success rate of assisted reproduction.II.Methods1.The expression of AQP3 in the mice endometrium and decidual tissue during the estrous cycle and early pregnancy1)We took a vaginal smear to determine the stage of the estrous cycle;Gross anatomic of uteri washed by 37?PBS during estrous cycle;The expression and location of AQP3 in mice endometrium during the estrous cycle were detected by immunohistochemistry,and detect the expression of AQP3 by Real-time PCR and Western Blot;2)The expression and location of AQP3 in mice endometrium of D1 and D5 were detected by immunohistochemistry,and detect the expression of AQP3 by Real-time PCR and Western Blot;3)The expression and location of AQP3 in the uterine stromal cells around the attachment site and non-attachment site of embryo implantation,and decidual tissue on D8 were detected by immunohistochemistry;2.AQP3 expression alters with human endometrial cycles and study on the mechanism of AQP3 in the embryo implantation1)The expression and location of AQP3 in human endometrium during the menstrual cycle were detected by immunohistochemistry;2)Detect the expression of AQP3 in the differential receptive endometrial epithelium cells by Real-time PCR and Western Blot(RL95-2 and HEC-1A cell lines represent the high-and low-receptive endometrial epithelium,respectively);Indirect immunofluorescence staining was used to detect the expression and location of AQP3;3)The recombinant vector was transfected into cells.Over-AQP3 was transfected into HEC-1A cells;AQP3-si RNA was transfected into RL95-2 cells;4)The invasion and migration abilities were evaluated using transwell assay and Wound healing assay effected by AQP3;Detect the percent adhesion of JAR cell adhesion to RL95-2 or HEC-1A cell monolayer;5)Treat RL95-2 and HEC-1A cells with E2,P4 and E2P4,extract the m RNA and protein,and detect the expression of AQP3 by Real-time PCR and Western Blot;6)The expression of several MET-related factors changed after RL95-2 treated with E2P4 and/or AQP3-si RNA by Western Blot,the invasion and migration abilities were evaluated using transwell assay and Wound healing assay after RL95-2 treated with E2P4 and/or AQP3-si RNA;7)The interaction of AQP3 and Ezrin was detected by co-immunoprecipitation and immunofluorescence;The expression of Ezrin changed after RL95-2 treated with E2P4 and /or AQP3-si RNA was detected by Real-time PCR and Western Blot;8)The actin cytoskeleton organization in RL95-2 cells was detected by phalloidin;Ezrin organization in RL95-2 cells was detected by immunofluorescence.III.Results1.The expression of AQP3 in the mice endometrium and decidual tissue during the estrous cycle and early pregnancy1)Immunohistochemical results revealed the AQP3 was located in the stromal cells,but not in the glandular epithelium and luminal epithelium,the m RNA and protein steadily increased from the metestrous to proestrous phase;2)Gross anatomic of uteri and immunohistochemistry showed endometrium thickening,uterine edema,and an enlarged uterine cavity gap in the proestrous and estrous of mice.However,in the metestrous and diestrous stages,the endometrium thinned,the uterus narrowed,and the uterine cavity showed little to no gap.3)Low levels of the m RNA and protein of AQP3 were detected at D1 compared with D5,and its localization intensely confined in the cells surrounding implantation site;4)AQP3 showed no obvious expression in the uterine decidual tissue on day 8 of pregnancy,AQP3 showed a specific increase in the uterine stromal cells around the non-attachment site of embryo implantation.2.AQP3 expression alters with human endometrial cycles and study on the Mechanism of AQP3 in the embryo implantation1)In the mid-secretory and late-secretory phase,AQP3 was expressed strongly in cytomembrane both in the basolateral membrane of luminal and glandular epithelium cells,and peaked in mid-secretory phase;2)Both AQP3 m RNA and protein expression were more highly expressed in RL95-2 cells than in HEC-1A cells;3)The expression of AQP3 was dramatically decreased by AQP3-si RNA transfection in comparison to untransfected controls in the RL95-2 cells,the expression of AQP3 was dramatically increased by over-AQP3 plasmid transfection in comparison to untransfected controls in the HEC-1A cells;4)AQP3 promoted cells migration and invasion,but had no effect on embryo adhesion;5)E2 induced up-regulation of AQP3 m RNA and protein expression treated with lower than 1?M E2,but high dose estrogen might induce down-regulation of AQP3 m RNA and protein expression in RL95-2 cells and HEC-1A cells;AQP3 expression at both m RNA and protein levels was stimulated by progesterone in a dose-dependent manner;the function of E2P4 is more evident than E2 or P4 alone;6)E2P4 induced the epithelial-mesenchymal transition(EMT)though AQP3,and E2P4 promoted cells migration and invasion though AQP3;7)AQP3 was colocalized with ezrin,and knockdown of AQP3 had no effect on the expression of ezrin in RL95-2 cells;8)E2P4 induced a marked cytoskeletal reorganization characterized by formation of filopodia and lamellipodia and rearrangement of stress fibers;E2P4 altered the Ezrin organization in RL95-2 cells through AQP3.IV.Conclusions 1.The expression of AQP3 in the mice endometrium and decidual tissue during the estrous cycle and early pregnancy1)AQP3 might be responsible for the two-way water transfer in and out of the uterine cavity during the estrous cycle;2)The expression of AQP3 in the ?window of implantation� phase,AQP3 might affect intrauterine fluid accumulation and uterine receptivity;3)AQP3 had no influence on uterine decidualization,but might be responsible for uterine contraction and ultimately a closing down of the uterine lumen.2.AQP3 expression alters with human endometrial cycles and study on the Mechanism of AQP3 in the embryo implantation1)The highest expression of AQP3 occurred in the window of implantation.Meanwhile,the expression AQP3 on RL95-2 cells is higher than HEC-1A cells.The results indicate that AQP3 might been considered as an endometrial receptivity marker;2)AQP3 promoted cells migration and invasion in the embryo implantation,but had no effect on embryo adhesion;3)E2 and P4 were combinatorial regulation to AQP3;4)E2P4 promoted cells migration and invasion by EMT though AQP3;5)AQP3 was colocalized with ezrin,and altered the actin cytoskeleton and ezrin organization in the EMT.