The Sialyltransferase ST3Gal3 Regulates The Receptivity Of The Uterine Endometrium

Endometrial acceptance is the ability of the endometrium to receive embryos and is regulated by many factors such as hormones,adhesion molecules,growth factors and receptors.Glycosylation is an important way of post-translational modification of proteins and is closely related to many physiological processes and diseases.The receptive uterine endometrium specifically expresses certain glycosyltransferases,and the corresponding oligosaccharides play important roles in accepting the embryo.The sialyltransferase ?-galactoside-?2,3-sialyltransferase III(ST3Gal3)is the key enzyme responsible for sialyl Lewis X(s Le X)oligosaccharide biosynthesis,but the expression and function of ST3Gal3 in the receptive endometrium is still elusive.Here,we found that human endometrial tissues at secretory phase expressed a 4-fold higher ST3Gal3 level relative to the tissues at proliferative phase.Meanwhile,downregulation of ST3Gal3 or s Le X epitope blockage significantly impaired the receptive ability of human endometrial RL95-2 cells to trophoblastic cells in vitro and inhibited implantation in pregnant mice.This study suggests that ST3Gal3 facilitates endometrial receptivity through increasing s Le X oligosaccharide,which gives a better understanding of the glycobiology of implantation.Methods:1.Real-time PCR and Western blot were used to detect the expression levels of ST3Gal3,ST3Gal4 and ST3Gal6 in uterine tissues at proliferative phase,secretory phase and human uterine endometrium cell lines HEC-1A and RL95-2.2.Representative images of immunohistochemical staining showed the expression and localization of ST3Gal3,ST3Gal4 and ST3Gal6 in endometrial lumen epithelium.3.m RNA and protein expression levels of ST3Gal3,ST3Gal4 and ST3Gal6 in human endometrial HEC-1A and RL95-2 cells were detected by q PCR and western blot.4.The effect of ST3Gal3 on cell growth was detected by CCK8 and Ed U.5.The morphological alteration in ST3Gal3 knockdown and Neu-treated RL95-2 cells was assessed by scanning electron microscopy,and RL95-2 cell monolayers were treated with scramble+Ig M,si-ST3Gal3+Ig M,scramble+anti-s Le X and si-ST3Gal3+anti-s Le X before the trophoblastic JAR cells were plated for adhesion.6.Mouse uterine endometrial tissues were collected at NP,PD1,PD2,PD3,PD4 and PD5,and the gene and protein expression levels of ST3Gal3,?2,3-sialylation and s Le X were detected by q PCR,western blot,Lectin blot,Lectin fluorescent staining and Immunofluorescent staining.Results:1.Human endometrium at proliferative phase and secretory phase represent the nonreceptive status and the highly receptive status respectively.Therefore,we first collected the tissues at proliferative and secretory phase to compare the gene and protein expression levels of ST3Gal3,ST3Gal4 and ST3Gal6 by q PCR,western blot and immunohistochemical staining.Results showed that ST3Gal3 and ST3Gal6 expression were higher in endometrial tissues at secretory phase than the tissues at proliferative phase,but there is no difference of ST3Gal4 expression was found between the samples.2.Obviously,immunohistochemical staining showed that luminal epithelial cells of secretory phase expressed abundant levels of ST3Gal3.These data suggest that ST3Gal3 may play a primary role in synthesizing the s Le X oligosaccharide in human endometrium during the �window'.3.Next,we detected the ST3Gal3,ST3Gal4 and ST3Gal6 expression in a pair of endometrial cell lines: HEC-1A and RL95-2,which were generally used as the model to detect the endometrial functions.Consistently,the data showed that ST3Gal3 expression was higher(3.04-fold increased in m RNA level and 2.52-fold increased in protein level,P < 0.05)in RL95-2 cells than HEC-1A cells.4.Adequate growth of uterine endometrial cells is the prerequisite for establishing the receptivity.Therefore,we detected the effect of ST3Gal3 knockdown on regulating the proliferation of endometrial cells.RL95-2 cells were transiently transfected with scramble si RNA or ST3Gal3 si RNA.CCK-8 assay and Ed U incorporation assay showed that ST3Gal3 knockdown inhibited the proliferation of RL95-2 cells.5.The morphological alteration in ST3Gal3 knockdown and Neu-treated RL95-2 cells was assessed by scanning electron microscopy.And the result was samed by adhesion assay.6.To examine the expression of ST3Gal3/s Le X in the mouse endometrium,we collected mouse uterine tissues at nonpregnant(NP)and pregnant days 1�5(PD1,PD2,PD3,PD4 and PD5)stages.q PCR and western blot results showed that the m RNA and protein expression levels of ST3Gal3 were gradually increased from PD1 to PD4,and slightly decreased at PD5.Meanwhile,blots of MAL-II and s Le X epitopes or the fluorescence microscopy detection showed that endometrium at PD4 exhibited higher levels of a2,3-sialylation and s Le X than other time points.7.Subsequently,ST3Gal3 si RNA and s Le X antibody were injected into the uterine lumen of the pregnant mouse at PD3 to evaluate their influences on implantation.These results demonstrated that ST3Gal3/s Le X of mouse endometrium contributed to the successful implantation.Conclusion:1.The expression levels of ST3Gal3 was higher in human endometrium at proliferative phase and human endometrium RL95-2 cell lines.2.Downregulation of ST3Gal3 or s Le X epitope blockage significantly impaired the receptive ability of human endometrial RL95-2 cells to trophoblastic cells in vitro.3.ST3Gal3/s Le X of mouse endometrium contributed to the successful implantation.