The Expression And Regulation Of Talin1 On Endometrium During Peri-implantation

Embryo implantation is a complex physiological process of interaction between activated blastocyst and receptive endometrium,is the result of the bidirectional regulation between embryo and mother.In this process,the maternal-fetal interface expresses a series of key molecules which interacts with each other,establishes dialogues and transmits signals to synchronize the development of embryos and endometrium.The endometrium allows implantation of the embryo only for a short period of time,this period with spatio-temporal constraints known as the "window of implantation".In this period,uterine fluid provides the microenvironment for blastocyst development and embryo implantation.And under the precise control of ovarian hormones,specific morphological,structural and molecular changes occurred in the endometrium to facilitate embryo implantation.At the same time,embryo releases a variety of factors that interact with maternal endometrium to promotethe formation of receptive endometrium.Talin1 is a ubiquitous,cytoplasmic adapter protein providing the connections between the intracellular network and the extracellular matrix(ECM).It is direct activator of integrins and an important component of focal adhesions,it plays a key role in a series of physiological and pathological processes related to cell adhesion and migration.Previous studies of talin1 mainly focused on malignant tumors,the process of tumor growth,invasion and metastasis is similar to that of embryonic implantation.However,it is not known whether Talin1 also plays a role in embryo implantation.In this study,we first analyzed and identified the uterine fluid protein in implantation window by trace ultrahigh resolution multi-stage mass spectrometry,and screened out the protein Talin1 related to the endometrium receptivity by bioinformatics.Secondly,immunohistochemistry and Western blotting were used to study the expression of Talin1 in peri-implantation mouse endometrium.Using bilateral ovariectomized mouse model,pseudopregnancy model,delayed implantation and activation models,HCG intrauterine perfusion model to study the regulatory factors of Talin1 on endometrium.The study has three parts as the followings.PART Ⅰ Expression and validation of Talin1 in uterine fluid during implantation windowObjective: Uterine fluid provides the microenvironment for blastocyst development and embryo implantation.Proteomic analysis of uterine fluid was performed to obtain information related to endometrial receptivity.Methods:The differences of uterine fluid protein during implantation window stage in patients with recurrent implantation failure(RIF)and patients with natural cycle were analyzed by the trace ultrahigh resolution multi-stage mass spectrometry.Bioinformatics was used to screen for proteins associated with endometrial receptivity,and immunohistochemistry and Western blotting on the mouse endometrium were further used to verify the result.Results: After trace ultrahigh resolution multi-stage mass spectrometry and database search of peptide mass fingerprint,38 matched proteins were screened out in the RIF group,and 45 matched proteins were screened out in the natural cycle group.By comparing the difference of uterine fluid protein in patients with RIF and patients with natural cycle during implantation window stage,it was found that transthyretin,thymosin beta-4 and macrophage migration inhibitory factor were highly expressed in RIF group,and α-1 antitrypsin and talin1 were lower in RIF group.Through detailed bioinformatics analysis of these differential proteins,we found that Talin1,a protein associated with cell adhesion and migration,may be related to endometrial receptivity.The results of immunohistochemistry and Western blotting showed that the expression of Talin1 in mouse endometrium during the implantation window was significantly higher than that of the non-pregnant mice.Conclusion: The differences of uterine fluid protein during implantation window stage in patients with recurrent implantation failure(RIF)and patients with natural cycle were analyzed by the trace ultrahigh resolution multi-stage mass spectrometry,Talin1 level was lower in patients with RIF.Functional verification revealed that Talin1 is associated with endometrial receptivity.PART Ⅱ Expression of Talin1 in mouse endometrium during peri-implantation period and it regulated by ovarian hormonesObjective: To investigate the expression of Talin1 in mouse endometrium during peri-implantation period and the effects of ovarian hormones on Talin1.Methods: The distribution and expression of talin1 in mouse endometrium during early pregnancy was analyzed by immunohistochemistry and Western blotting,regulation of exogenous ovarian hormones on talin1 was analyzed in ovariectomized mice.Results: Talin1 underwent dynamic changes in mouse endometrium during early pregnancy.Its expression increased after pregnancy,the peak appeared on day 4 of pregnancy,especially along the apical side of luminal epithelium.On day 5 of pregnancy,Talin1 decreased obviously,especially in luminal epithelium cells of implantation sites.Up to day 7 and day 8,it increased again in the decidua.The corresponding statistical significances were demonstrated by Western blotting(P < 0.05,versus day 1).Compared with inter-implantation sites,the level of Talin1 at implantation sites was lower on day 5 of pregnancy(P < 0.05).In the ovariectomized mice,Talin1 was enhanced response to estrogen treatment,decreased response to progesterone or progesterone in combination with estrogen.The corresponding statistical significances were demonstrated by Western blotting(P < 0.05).Conclusion: Talin1 may play an important role in the establishment of endometrial receptivity and ovarian hormones can regulate its expression.PART Ⅲ Regulation of embryonic signal on Talin1 in mouse endometriumObjective: To investigate the effect of embryonic signals,especially HCG talin1 in mouse endometrium.Methods: Pseudopregnancy model,delayed implantation and activation models,HCG treatment models were made,immunohistochemistry and Western blotting were used to examine the expression of Talin1 in mouse endometrium of these models.Results: Talin1 showed scattered cytoplasmic staining on the endometrium of pseudopregnancy mice.Its expression increased on day 3–4 of pseudopregnancy,and did not decrease on day 5 of pseudopregnancy.Compared with day 3 and day 4,there was no significant difference in talin1 expression on day 5(P > 0.05).During delayed implantation,Talin1 showed strong cytoplasmic staining,especially in stromal cells.Its expression decreased obviously in activated implantation(P < 0.05).Talin1 at the implantation sites of the activated model was significantly lower than that at the inter-implantation sites(P < 0.05).There is almost no expression of Talin1 in luminal epithelial cells of the implantation sites.After intrauterine perfusion of 10 u HCG in pseudopregnant mice,the expression of Talin1 was significantly reduced in both stromal and epithelial cells(P < 0.05).Afterwards,increasing the concentration had no effect on the expression of Talin1 in mouse endometrium.Conclusion: The presence of embryos can affect the expression of Talin1 in mouse endometrium.A certain concentration of HCG can regulate the expression of Talin1 in mouse endometrium.