Expression Of SGLT1 In The Mouse Endometrial Epithelium And Its Role In Embryo Implantation

The morphology and function of EECs are periodically changed under the regulation of estrogen and progesterone.The function of endometrial epithelium is very important to maintain the suitable intrauterine environment for early embryo development and establish implantation.Glucose is the main energy source of EECs and one of the main nutrients in uterine fluid.The transmembrane transport of glucose in EECs plays an important role in maintaining the glucose level in uterine fluid for early embryo development and glucose metabolism related to the energy consumption of cells.There is a close functional relationship between EECs and ESCs.In the process of embryo implantation,EECs can further promote the synchronous decidualization of ESCs through a paracrine manner,which provides nutrients and protection for the embryos.Glucose transporters are important carriers that mediate the transmembrane transport of glucose,which can be divided into two types:facilitated diffusion glucose transporters(GLUTs)and sodium-glucose linked transporters(SGLTs).SGLTs transport glucose via a secondary active transport mechanism based on the sodium concentration gradient established by the Na~+/K~+-ATPase pump.The SGLTs family contains 12 identified members,of which SGLT1 and SGLT2 have been confirmed to act as glucose transporters.SGLT3a can work as a sensor molecule to induce cellular depolarization,further affecting cellular function.Our previous works have shown that SGLT1 was expressed in EECs.According to the functional characteristics of SGLTs,we speculate that SGLT1 plays an important role in the maintenance of glucose level in uterine fluid,implantation related energy consumption,and decidualization of stromal cells.In this research,the main research results include the following aspects.1.The spatiotemporal characteristics of SGLT1 expression in the mouse endometrium epithelium and its regulationUterine tissues were collected from the mice on days 1 to 4 of pregnancy,IHC and immunofluorescence confocal microscopy were used to detect the expression and localization of SGLT1,q RT-PCR and western blotting were used to check the level of SGLT1 in EECs,respectively.The results showed that on day 1 and 2 of pregnancy,when the uterus was primarily affected by ovarian E2,only low expression of SGLT1in the endometrial luminal epithelium was detected.Its expression increased on pregnant day 3 and 4,when the uterus was affected both by increasing P4 and E2from newly formed corpora lutea and it reached a peak on day 4.To explore the regulatory effects of ovarian hormones on the expression of SGLT1 in vitro,cultured primary EECs of mouse were randomly divided into control,E2 or P4 and E2 plus P4(E2+P4)treatment groups.After treatment with 24h,the SGLT1 level was detected by western blotting.To evaluate the effects in vivo,6-week-old female mice were ovariectomised and rested for 2 weeks prior to subcutaneous injection with E2 alone or P4 alone or E2 in combination with P4 or with sesame oil as a control.Uterine tissues from mice in the control,E2,P4 and E2+P4 groups were collected and subjected to IHC to detect SGLT1 expression in the endometrial epithelium.The results showed that a significantly higher level of SGLT1was observed in the E2 and P4 group than in the control group,whereas higher level was observed in the E2+P4 group relative to the E2 and P4 group.Cultured primary EECs were randomly divided into control,AMP,and ATP treatment groups.After treatment with 24h,EECs were collected and subjected to IHC to detect SGLT1,APMK,p-AMPK expression.The results showed that the level of SGLT1 increased with the increase of energy requirement of EECs.Cultured primary EECs were randomly divided into control,AICAR,an AMPK agonist and Compound C(Cc),an AMPK inhibitor treatment groups.After treatment with 24h,EECs were collected and subjected to western blotting to detect SGLT1expression.Five mice on day 2 of pregnancy received a direct injection of AICAR into one uterine horn,an equal volume of control solution was injected into the contralateral horn.Five mice on day 2 of pregnancy received a direct injection of Cc into one uterine horn,an equal volume of control solution was injected into the contralateral horn.On day 4,the uterine tissues were collected and subjected to IHC to detect SGLT1 expression in the endometrial epithelium.The results showed that compare with the control group,AICAR up-regulated the level of SGLT1 in EECs,while Cc down-regulated the level of SGLT1 in EECs.2.SGLT1 in the Endometrial Epithelium Helps to Maintain the Glucose Concentration in the Uterine FluidMice on day 2 of pregnancy received a direct injection of phlorizin or SGLT1-si RNA into one uterine horn.An equal volume of control solution or NC-si RNA was injected into the contralateral horn.On day 4,the uterine fluid was collected.The embryos were collected on day 2-4 and subjected to a morphological evaluation.The results showed that higher glucose concentrations in uterine fluid samples from the phlorizin-or SGLT1-si RNA-treated horns than in the control horns.The embryos were almost normal on pregnant day 2 and 3 before entering uterine cavity,while the most of them arrested at eight-cell or morula stage,and failed to develop to the blastocyst stage on pregnant day 4 when they had entered the uterine cavity in the uterine horns treated with phlorizin or SGLT1-si RNA.3.The effect of SGLT1 on glucose metabolism of EECs and embryo implantationEECs of mouse on day 4 of pregnancy were isolated and cultured.EECs were treated with phlorizin and/or SGLT1-si RNA,with solvent and/or Nonsense si RNA as a control.After treatment with 48h,EECs were incubated in Hank’s containing the fluorescent glucose analogue 2-NBDG for 2h and the cellular 2-NBDG uptake was measured using a fluorometric assay.The intracellular glycogen,ATP level was measured using a glycogen,ATP assay kit,respectively.The results showed that inhibition of SGLT1 function or low expression of SGLT1 in EECs resulted in decreased 2-NBDG uptake,glycogen,and ATP content in EECs.Mice on day 2 of pregnancy received a direct injection of phlorizin or SGLT1-si RNA into one uterine horn.An equal volume of control solution or NC-si RNA was injected into the contralateral horn.On day 4,uterine tissues were harvested,the levels of glucose and ATP in EECs were detected by specific kits;the level of Lif,integrinαVβ3,Ltf and Muc1 in EECs were detected by western blotting and q RT-PCR;the level of glycogen in endometrial epithelium was detected by PAS staining;the formation and development of pinopodes were detected by scanning electron microscope.On day 5,embryonic implantation sites were observed.Ten pseudopregnant mice were treated with the same method.At 22:00 on day 4,eight high-quality blastocysts from normal donors were transplanted into the uterine horns of each pseudopregnant mouse.The embryo implantation rate was detected on day 5.The results showed that inhibition of SGLT1 function or low expression of SGLT1 in mouse EECs resulted in decreased glucose uptake,glycogen,and ATP content in endometrial epithelium,increased in the level of Muc1 and Ltf,while decreased in the level of integrinαVβ3 and Lif,blocked formation of pinopodes and significantly reduced number of implantation sites on day 5 of pregnancy.The embryos from normal donor mice exhibited a decreased implantation rate on day 5 of pregnancy when they were transferred to uterine horns that had been subjected to SGLT1inhibition or SGLT1-si RNA during the implantation window.The endometrial tissues of women with infertility and recurrent abortion during the proliferative period were collected,and the endometrial tissues of non-endometrial lesions during the same period were used as control.The level of SGLT1 in endometrial epithelium was detected by IHC.The results showed that the level of SGLT1 in the endometrium of women with infertility and recurrent abortion was significantly lower than that in the control.4.SGLT1 of endometrium epithelium mediated decidualization of ESCsEECs and ESCs of mouse on day 4 of pregnancy were isolated and cultured.EECs were treated with SGLT1-si RNA and/or phlorizin,some were co-cultured with ESCs treated with estrogen and progesterone in Transwell,and some were cultured alone,EECs treated with nonsense si RNA and/or solvent as control.After treatment with 48h,the level of PGE2 in the supernatant of EECs cultured alone was detected by ELISA;after treatment with 72h,the morphology of ESCs was observed,and the level of prl8a2 in ESCs was detected by q RT-PCR.The results showed that compared with the control group,the level of PGE2 was significantly decreased,and the ESCs didn’t undergo decidualization,and the level of prl8a2 was also significantly decreased.Mice on day 2 of pregnancy and pseudopregnancy received a direct injection of phlorizin or SGLT1-si RNA into one uterine horn,an equal volume of control solution or NC-si RNA was injected into the contralateral horn.On day 8 of pregnancy,the development of implanted embryos and decidualization of stroma were observed.On day 4 of pseudopregnancy,sesame oil was injected into bilateral uterine horns to induce decidualization.On day 8 of pseudopregnancy,decidualization of stroma was observed,the level of prl8a2 in ESCs was detected by q RT-PCR.The results showed that inhibition of SGLT1 function or low expression of SGLT1 in EECs reduced the mean weight of implanted embryos in pregnant mice,and the decidua was not well developed.The artificial induced decidualization failed in pseudopregnant mice,and the level of prl8a2 was also significantly decreased.In conclusion,the expression of SGLT1 in mouse EECs in spatiotemporal pattern which are regulated by ovarian hormone and endometrial epithelial energy status.SGLT1 mediates glucose transmembrane transport in EECs,which not only maintains a suitable uterine glucose environment for early embryonic development,but also provides energy for the functional activities associated with embryonic implantation.Moreover,EECs’SGLT1 also promotes decidualization of ESCs by inducing EECs themselves paracrine.Therefore,the low expression of SGLT1 may be an important reason for female infertility and abortion.This research elucidates the physiological function of endometrial SGLT1,also provides new ideas for the clinical diagnosis and treatment of female infertility and early abortion.