Effect Of EECs’ SGLT3a Activation Inducing Paracrine Associated With Decidualization And Its Mechanisms

BackgroundThere are close functional links between endometrial epithelial cells（EECs）and stromal cells.The decidual signal from the uterine cavity firstly acts on EECs to induce stroma cells decidualization through paracrine pathway.However,few studies have identified the key"sensor"molecules in EECs whose activation causes the release of paracrine factors.There have been evidences that sodium-glucose linked transporter 3a（SGLT3a）can work as a sensor molecule to induce cellular depolarization,further affecting cellular function.In our previous study,we have found that SGLT3a was highly expressed in the EECs of mice during the period of peri-implantation,and the down-regulation of its expression led to the failure of artificial decidualization induction in pseudopregnant mice.Therefore,we speculate that SGLT3a,as a sensor molecule,might play an important role in decidualization of stromal cells.To prove our deduction,the techniques of cellular and molecular biology and reproductive biology,as well as laser confocal microscopy were used to detect the temporal and spatial expression of SGLT3a in EECs of mice and prove that the role of EECs’SGLT3a of in embryo implantation and decidualization of stromal cells.Then,the paracrine induced by EECs’SGLT3a activation and its role in decidualization of stroma cells were studied.Finally,the sensor molecular characteristics of SGLT3a in EECs and the molecular mechanism of its activation-inducing decidualization-related paracrine effect were studied.The aim was to further reveal the functional relationship between EECs and stromal cells in decidualization,which might provide a new clue for the diagnosis and treatment of female infertility.1.The spatio-temporal expression of SGLT3a in peri-implantation period and regulation of estrogen and progesteroneThe spatio-temporal expression of SGLT3a in EECs was observed by immunohistochemical staining and RT-PCR on pregnant day 1-4 of 12 mice.Nine ovariectomized mice were divided into three groups（n=3 per group）,which were respectively treated with vehicle、estrogen or co-treated with estrogen and progesterone.In vitro,EECs were respectively treated with vehicle、estrogen or co-treated with estrogen and progesterone.The expression of SGLT3a was detected by RT-PCR and immunohistochemical staining.The results of RT-PCR and Immunohistochemical staining showed that SGLT3a mainly located at the top of endometrial epithelium and increased gradually from pregnant day 1 to day 4.The expression of SGLT3a in endometrial epithelium enhanced in the both ovariectomized mice and the cultured EECs treated with estrogen,and it was further increased in combination with progesterone and estrogen.These results suggest that SGLT3a is highly expressed in peri-implantation EECs,E₂ promotes SGLT3a expression,and P₄ further co-promotes SGLT3a expression.2.Study the effect of activation of EECs’SGLT3a on stromal cell decidualization and embryo implantationSi RNA-SGLT3a was injected into one side of uterine horn of a mouse,while nonsense si RNA was injected into the other side as control on pregnant day 2.Then the number and shape of blastocysts of 5 mice were observed on pregnant day 4.The implantation sites of embryos of 5 mice were observed on pregnant day 5,and decidualization of stromal cells and the development of embryos of 5 mice were detected on pregnant day7.Si RNA-SGLT3a and nonsense si RNA were respectively injected into one side of the uterus of each pseudopregnant mice（n=5）,the blastocysts from normal donor mice were transplanted into the uterus of pseudopregnant mice on pregnant day 4.Then the development of embryos after implantation was compared between two sides of uterine horns on pseudopregnant day 7.SGLT3a activator,1-deoxynojirimycin（DNJ）was injected into one side of uterine horns of pseudopregnant mice（n=5）on pregnant day 4,the decidualization of stromal cells was observed,and expression of the decidualization marker molecules prl8a2 and IGFBP1 in stromal cells was detected by RT-PCR on pregnant day 7.Si RNA-SGLT3a was injected into one side of uterine horn of pseudopregnant mice（n=6）and the decidualization was induced by injection of sesame oil into two sides of uterine horns,the decidualization of stroma cells was observed and the expression of prl8a2 and IGFBP1 in stromal cells was detected on pseudopregnant day 7.Compared with the control side,the number and shape of blastocysts on pregnant day 4 were normal,and the number of implantation sites did not decrease significantly on pregnant day 5.However,the decidualization of stromal cells was blocked on pregnant day 7.Meanwhile,the number of implanted embryos decreased,and the average weight of embryos significantly reduced on pregnant day 7.In allogeneic embryo transfer mice,the number of embryos implanted and the average weight of embryos significantly reduced in the side of uterus with low expression of EECs’SGLT3a on pregnant day 7.DNJ could induce decidualization of stromal cells in pseudopregnant mice,and the number of decidua cells and the weight of uterus increased significantly.The expression of prl8a2 and IGFBP1 in stromal cells significantly enhanced accordingly.While in pseudopregnant mice,the number of decidua cells and the weight of uterus decreased significantly in the side of uterus with low expression of SGLT3a in EECs.The expression of prl8a2and IGFBP1 in stromal cells also reduced significantly.These results suggest that EECs’SGLT3a activation can induce decidualization of stroma cells and play an important role in the decidualization of stromal cells and the development of embryos after implantation.3.Study the role of paracrine effect induced by EECs’SGLT3a activation in decidualization of stromal cellsIn vitro experiment,primary EECs and stromal cells of pregnant mice were respectively isolated and cultured.After EECs were treated with 100μmol/L DNJ for 40 minutes,EECs and stromal cells were cocultured in indirect contact for 72hours.Then the morphological changes of stromal cells were observed.The expression of prl8a2 and IGFBP1 in stromal cells was detected by RT-PCR.Meanwhile,after cultured EECs were treated with DNJ for 3hour,the content of PGE2 and PGF2αin supernatant of EECs was determined by ELISA.In vivo experiment,five pseudopregnant mice were prepared and si RNA-SGLT3a was injected into one side of uterine horn and the decidualization was induced by injection of sesame oil into two sides of uterine horns.Then PGE2 and PGF2αin EECs was detected by ELISA.After cultured EECs were treated with DNJ for3hour,the supernatant was added to the stromal cells,and the cells continued to be cultured for 72 hours.The morphological changes of stromal cells were observed,and the expression of prl8a2 and IGFBP1 in stromal cells was detected.After being cocultured of EECs treated with DNJ,stromal cells differentiated into large polygonal decidual cells,and the expression of prl8a2 and IGFBP1 in stromal cells was significantly higher than that in control group.After the stromal cells were treated with the supernatant of EECs,the PGE2 and PGF2αcontent in the supernatant of EECs significantly increased.However,compared with the control,the content of PGE2 and PGF2αin EECs of the uterus with low expression of EECs’SGLT3a in pseudopregnant mice was significantly lower in vivo.After the stromal cells were treated with the supernatant of EECs,the differentiation of stromal cells into large polygonal decidua cells was also observed.The expression of prl8a2 and IGFBP1 in stromal cells significantly increased.These results suggest that EECs’SGLT3a activation can promote the expression of decidualization-related paracrine factor PGE2 and PGF2α,and induce decidualization by paracrine pathway.4.Study on the characteristics of EECs’SGLT3a as a sensor moleculeIn vitro sodium-containing buffer and sodium-free buffer,the cells were treated with DNJ and the EECs membrane potential was examined by the probe of cell membrane potential.Then the cells were separately treated in Ca²⁺-containing buffer and Ca²⁺-free buffer,and the intracellular[Ca²⁺]changes were examined by Ca²⁺probe.In order to study the effect of depolarization on extracellular Ca²⁺influx induced by SGLT3a activation in EECs,the intracellular[Ca²⁺]changes were examined by Ca²⁺probe.After EECs were separately treated with DNJ in sodium-containing buffer and sodium-free buffer,the intracellular[Ca²⁺]changes were examined by Ca²⁺probe.In Na⁺-containing buffer,the fluorescence intensity of membrane potential in EECs treated with DNJ increased significantly,but the fluorescence intensity in Na⁺-free buffer did not change significantly.In Ca²⁺-containing buffer,the intracellular[Ca²⁺]fluorescence intensity enhanced significantly in EECs treated with DNJ,while the[Ca²⁺]fluorescence intensity in Ca²⁺-free buffer did not significantly change.The fluorescence intensity of[Ca²⁺]in Na⁺-containing solution treated with DNJ significantly increased,while the fluorescence intensity of[Ca²⁺]in Na⁺-free EECs did not significantly change.These results suggest that SGLT3a is a sensor molecule,and SGLT3a agonists can induce the Na⁺-dependent depolarization of EECs,which lead to the influx of extracellular Ca²⁺.5.Study the mechanism of paracrine effect induced by EECs’SGLT3a activationIn order to clarify the effect of SGLT3a activation on the expression of upstream regulatory molecules of PGE2 and PGF2α,the expression of COX-1,COX-2,LIF and i HH in EECs treated with DNJ in vitro was analysed by RT-PCR and western-blot.On the other hand,5 pseudopregnant mice were prepared and si RNA-SGLT3a was injected into one side of uterine horn of a mouse and the decidualization was induced by injection of sesame oil into two sides of uterine horn in vivo.Then the expression of COX-1,COX-2,LIF and i HH in EECs was detected by RT-PCR and western-blot.In order to study the effect of SGLT3a activation on the expression of COX-2 by intracellular calcium signal,EECs cultured in calcium-free medium and calcium-containing medium were separately treated with DNJ.Then the expression of COX-2 in EECs was detected by RT-PCR and western-blot.In order to prove that COX-2 was an upstream molecule of PGE2 and PGF2α,the expression of COX-2 in cultured EECs was specifically interfered with si RNA-COX-2.Then the content of PGE2 and PGF2αin supernatant of EECs treated with DNJ was tested by ELISA.In vitro experiment,the expression of COX-2 in EECs treated with DNJ was significantly higher than that in the control,while the expression of COX-1,LIF and i HH did not significantly change.In pseudopregnant mice,the expression of COX-2 in EECs of uterus with low expression of SGLT3a was significantly less than that in contralateral uterus,while the expression of COX-1,LIF and i HH did not significantly change.The expression of COX-2 in EECs cultured in calcium-free medium was significantly lower than that in calcium-containing medium EECs.After COX-2 expression of EECs in vitro was interfered with si RNA-COX-2,the PGE2 and PGF2αcontent in the supernatant of EECs was significantly lower than that in the control group.These results suggest that EECs’SGLT3a activation can promote the expression of PGE2 and PGF2α.ConclusionDuring the period of peri-implantation,the expression of EECs’SGLT3a which can be co-promoted by estrogen and progestogen is increased.SGLT3a has the characteristics of sensor molecule,and its activation can induce Na⁺-dependent depolarization of cell membrane potential and extracellular Ca²⁺influx.Furthermore,the expression and release of PGE2 and PGF2αcan be promoted by up-regulated COX-2,which can induce decidualization of stromal cells by paracrine pathway.As a result,the development of post-implantation embryos is affected in this way.