The Regulation Of Endometrial Decidualization By L-type Amino Acid Transporter 1(Lat1) In Pregnant Mice

Background:L-type amino-acid transporter 1(lat1)is responsible for the transportation of large neutral amino acids and is mainly expressed in human fetal liver,placenta,brain,etc.Embryo implantation and decidualization are critical for the establishment of successful pregnancy.Decidualization is characterized by proliferation and differentiation of endometrial stromal cells(ESCs),followed by blastocyst attachment within 22–24 h on Day 4(D4)of pregnancy.Maternal decidu play a multi-functional role in the embryo–uterine dialog,such as feto-maternal immuno-tolerance and placental development.Amino acids have an important role in the pre and post implantation of placenta and embryo development.Few studies have demonstrated the requirement of active transport of amino acids for successful implantation and placentation.In addition,during the process of placentation,lat1 might play a role in the invasive phenotype of trophoblast giant cells(TGCs),but no evidence suggests the role of lat1 in the coordination of ovarian hormone with he embryo–uterine dialog in decidualization.Methods: 1.Immunocytochemistry,RT-PCR,Western Blot were used to evaluate the expression of lat11 in the uterus from Day 4-8 of pregnancy.2.Endometrial stromal cells of a mouse model were etablished,Western Blot was used to evaluate the expresssion of prl of decidualization after 72 h of inducton in vitro followed by transferred Lat1 overexpression and interfering plasmid;morphology of decidualization of ESC extracts was cultured up to 72 h at different concentrations competitive inhibitor of Leucine transport 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid –BCH(0?M?0.05?M?0.1?M?0.5?M?2?M and 4?M)was observed;then using Western Blot assay to detect the expresssion of prl of decidualization.3.H&E was used to evaluate morphology effects of different concentrations BCH(50?g/ml?5?g/ml?1mol/LNH4OH)and the control groups(Blank?1mol/LNH4OH)on D8 implantation site after uterine horns injections on D4.4.Immunocytochemistry was used to evaluate the effect on expression and localization of prl expression on D8 of pregnancy and Western Blot was used to evaluate the effect on expression of prl on D8 of pregnancy with different concentrations BCH(50?g/ml ?5?g/ml)and(Blank ?1mol/LNH4OH)uterine horns injections on D4;.Results: 1.Lat1 was mainly distributed in the luminal and glandular epithelial cells,and ESC cytoplasm on D4 of pregnancy.Increased expression was noted in the decidualizing stromal cells in the cytoplasm throughout the endometrium at the IS on D5.ISs on D6 of pregnancy showed lat1 expression in the cytoplasm of decidual cells in primary decidualization zone(pdz)and embyro.Lat1 mRNA and protein were observed in the D4-8 mouse uteri,but low in the inter-implantation sites(IIS)compared with the implantation sites(IS).Compared with D4 of pregnancy,the levels of lat1 mRNA and protein were increased from D5 to D8 of pregnancy IS,and the peaks levels appeared on D8 of pregnancy(P < 0.05).2.After treatmernt with over-pression of lat1 plasmid,the expression of lat11 on decidualization of ESC extracts was cultured up to 72 h,and the expression of prl was incereased in same time,transferring the effective Lat1-siRNA1 can remarkly inhibit the expression of prl on decidualization of ESC extracts was cultured up to 72 h.3.Western Blot showed that the multinucleation percent of decidualization of ESC extracts was cultured up to 72 h was decreased,and BCH treatment can led to the down-regulation of lat1 and prl protein on decidualization of ESC extracts was cultured up to 72 h and exhibited dose–effect relationship.The expression of lat1 and prl could be significantly inhibited by 4 ?M BCH treatment(P < 0.05).4.H&E assay showed that the 50 ?g/mL and 5 ?g/mL BCH treatments in ISs produced a regression in the tissue with shrinkage of all decidual tissues and the NH4 OH treatment did not interfere with the decidualization process,but have no any impact on the percent of different function decidual area and the number of implanting-embryo(P < 0.05).5.The integral optical density levels of prl positive expression cells on D8 of pregnancy were decreased in the BCH?treated ISs(P < 0.05),Western Blot showed that BCH treatment produced a remarkably reduced lat1 and prl expression on D8 ISs of pregnancy,but the NH4 OH treatment have no effect on IISs(P < 0.05).Conclusion:Lat1 play a positive regulation role in the decidual progression of mouse both in vitro and in vivo by regulating the expression of prl.