Gaba Signal Expression And Possible Function In Periimplantation Mouse Utrine

Objective: Mammalian embryo implantation is a key step insuccessful pregnancy. In mouse, embryo implantation and placentaformation include these steps: the blastocyst trophectoderm adhesion toendometrial luminal epithelium; trophoblast proliferation and then throughthe luminal epithelium; endometrial stromal cells surrounding theblastocyst adhesion around the decidual cells; trophoblast differentiationinto the invasive phenotype through decidual tissues into the maternalblood vessels; at last forming a functional placenta. Endometrialdecidualization and trophoblast moderately invasion decidual are ssentialfor establishment and maintenance of pregnancy and become a researchhotspot for these reason.gamma-aminobutyric acid as an inhibitory neurotransmitter withextensive study initially and its main function is transmission the inhibitoryneurotransmitter and have physiological effects such as sedative, hypnotic,anticonvulsant and lowering blood pressure. Now GABA have developed asynthetic oral preparations for medical and health, agriculture, etc and show some production value. GABA can be used as a gastrointestinalpeptide, promote gastric hormone secretion and animals growth forstimulating animal feed central to increase the feed intake and also canreduce the animal unconscious movement, to reduce energy consumption.In recent years, the regulatory role in the GABA in the peripheraltissues with more attention especially in the hot issues: cancer research, theGABA receptor specificity expression in the peripheral variety ofendocrine tissues and organstumors, and demonstrate the regulation oftumor cell proliferation, invasion and metastasis. Earlier studies reportedthat GABA A receptor π subunit also played an important role inreproductive research field. Progestogen and their metabolic intermediatesof allopregnanolone inhibit uterine smooth muscle contraction, maintainingfull-term pregnancy through containing π subunits. In rats, π subunit hadbeen maintaining a high level before starting labor while sharp decreasingin D19.5and the delivery process for diminishing sensitivity toallopregnanolone, thus lifting the allopregnanolone resting state of theuterus to maintain and promote the delivery.In previous study we found that GABA A receptor π subunit and Breceptor B1subunit were dynamically expressed in the uterine luminal,glandular epithelium in pre-implantation (D1-D4) in mice and participatedin hatching of blastocysts, attachment and trophoblasts outgrowthsuggesting their participation in the process of implantation in the maternal -embryonic dialogue. In this experiment, we studied the expression ofGABA signaling in the mouse uterus D5-D8, and examined the GABAsignaling in the process of decidualization and early trophoblast invasionwhich will add new datas to clarify the mammalian embryoimplantationwith molecular mechanism, at the same time which willprovide a new visio for study central neurotransmitter in reproductiveareas.Part Ⅰ: Expression of GABA signal in periimplantation mouse utrinePurpose: To detection the expression of GABRP, GB1and the content ofGABA dynamic changes in the periimplantation mouse uterus. Methods:extraction of total RNA, GABRP, and GB1mRNA expression level wereanalysised by two step RT-PCR from D4to D8;making paraffin sections,GABA,GABRP and GB1expression were analysised byimmunohistochemistry from D4to D8;extraction of total protein, GABRP,and GB1protein expression level were analysised by western blot from D4to D8;content of GABA were analysised by chromatometry from D1to D8.Results:GABRP and GB1mRNA expression in the D4-D8mouse utrineand the level of GABRP were increased significantly in D5and D7implantation sites, compared with D4(P <0.05) while there were nosignificant differences betweeen inner-implantation sites(P＞0.05). The mRNA expression level of GABRP were higher in D6and D8implantationsites compared with correspondence inner-implantation sites expressionlevel. In implantation sites,compared with D4, the expression level of GB1was only increased in D5(P <0.05).GABA, GABRP and GB1wereexpressed in cytoplasm. In D4, GABA, GABRP and GB1were located inmouse uterine luminal and glandular epithelium and GABA and GABRPwere also expressed in stromal cells by immunohistochemistrymeasurement. In D5, GABA, GABRP and GB1were located in mouseuterine remaining luminal and glandular epithelium. From D6toD8GABA, GABRP and GB1were expressed in primary decidualizationzone or second decidualization zone. GB1was also located in EPCs inD8.Western blot analysis showed that GABRP and GB1protein wereexpressed in the D4-D8mouse utrine. In the D4-D8, the protein expressionof GABRP was highest in D5with significant difference (P <0.01)followed by a downward trend in implantation sites or inner-implantationsites. No significant differences between implantation sites andinner-implantation sites in GABRP protein expression. The peak proteinexpression of GB1was appeared in D7implantation sites andcorrespondence inner-implantation sites (P <0.05). Chromatometry resultsshowed that in early priimplantation, compared with D1, the content ofGABA was increased in D3(P＜0.05), but declined in D4with peak valuein D5implantation sites from D5to D8(P＜0.01) followed by a downward trend. The contents of GABA from D6to D8were higher in implantationsites than which in correspondence inner-implantation sites (P＜0.01).Conclusion:GABRP and GB1mRNA and protein expressions andGABA content were dynamic expressed in in the periimplantation mouseuterus with peak value in D5. GABA and GABRP was mainly located instromal cell and decidual cell from D4to D8while GB1mainly locatedindecidual zone from D5to D8and chorionic cones parts in D8.Part Ⅱ: Functional study GABA signal on mouse uterinedecidua-lization processSection Ⅰ: Functional study GABA signal on mouse uterinedecidua-lization process in vitroPurpose:establishment of mouse uterine stromal cells induced artificialdecidualization model in vitro successfully;detection GBARP and GB1mRNA and protein expression levels in the process ofdecidualization;Analysis of GABA A receptor effects on cell proliferation,cycle and apoptosis in decidualization process;analysis of cyclinD3whether involved in the regulation of the GABA A receptor ondecidualization.Methods:extraction of primary mouse uterine stromal cells,the purity of stromal cells was identificated by stromal cell markervimentin and epithelial cell markers cytokeratin antibody and analysis GABA and GABRP expression with indirect immunofluorescence;induction stromal cell decidualization with E2and P4and analysis of theprotein expression of decidualization marker PRL at0h,24h,48h and72hby western blot;extraction total RNA and analysis GABRP and GB1mRNA expression by the two-step RT-PCR at0h,24h,48h and72h;extraction total protein and analysis GABRP and GB1proteinexpression by the western blot at0h,24h,48h and72h;induction stromalcell decidualization and administrated1μM,10μM and100μM GABA;0.5μM,5μM and50μM muscimol; the best effective GABAconcentration plus0.5μM or5μM or50μM S106exogenously andanalysis of stromal cell proliferation by MTS at24h,48h and72h;analysisof stromal cell cycle distribution and apoptosis by flow cytometry at24h,48h and72h with above treatment;analysis of cyclin D3protein expressionby the western blot at24h,48h and72h with abovetreatment.Results:extraction mouse uterine stromal cells successfully andthe cell purity was above95%suggesting that can be used for subsequentexperiments;western blot result showed the expression level of PRL proteinwas increased along with induction time with significant difference (P＜0.01) suggesting that induction stromal cell decidualization successfully;RT-PCR result showed the expression level of GABRP mRNA wasdecreased along with induction time with significant difference (P＜0.05)compared with0h;indirect immunofluorescence result showed GABA and GABRP protein mainly located in primary stromal cell cytoplasm;westernblot result showed the expression level of GABRP protein was decreasedalong with induction time with significant difference (P＜0.05) comparedwith0h;MTS result showed1μM GABA or5μM GABA A receptoragonist Muscimol can inhibit cell proliferation (P <0.05) and50μM S106can be successfully reversed1μM of GABA on the cell proliferationinhibition at48h and72h;Fow cytometry results showed1μM GABA or5μM GABA A receptor agonist Muscimol can promoted the S phase arrest(P <0.05) and50μM S106can be successfully reversed1μM of GABA onthe cell cycle distribution at48h;Fow cytometry result showed1μM and ofGABA and5μM GABA A receptor agonist Muscimol can promotedapoptosis and50μM S106can be successfully reversed1μM of GABA oncell apoptosis at72h;western blot result showed the expression level ofcyclinD3was decreased with1μM and of GABA or5μM GABA Areceptor agonist Muscimol and50μM S106can be successfully reversedsuccessfully1μM of GABA on the expressioncyclinD3.Conclusion:successful induction of mouse uterine stromal celldecidualization reaction; GABA and GABRP was mainly localized in thecytoplasm of stromal cells and the mRNA and protein levels of GABRPwas declined with the decidualization process. GABA may inhibit theproliferation of stromal cells and promote apoptosis, disrupt the cell cycledistribution in order to inhibit the decidualization process in vitro through GABA A receptor. CyclinD3may be involved in regulating these behavior.Section Ⅱ:Functional study GABA signal on mouse uterinedecidualization process in vivoPurpose:to investigate GABA signal on mouse uterine decidualizationprocess in vivo.Methods:from D5, pregnant mouse were injected0.05g/kg0.5g/kg of5g/kg of GABA in intraperitoneal once a day until D8;mousewere killed in D8and removed uterus and calculated the number andweight of the deciduum;decidual tissues were weigh and then madeparaffin section followed by HE staining. Areas of decidual weremeasured by Image J software; the expression of decidualization markerPRL was detected by western blot in D8implantationsites.Results:compared with blank control group,0.05g/kg and5g/kgGABA groups could significantly reduce the number and weight ofdeciduum (P <0.05);compared with blank control group,0.05g/kg and0.5g/kgGABA could significantly reduce nuclear multicore area by theImage J software calculated(P <0.05);compared with blank control group,0.05g/kg,0.5g/kgGABA and5g/kgGABA groups could significantlyreduce the expression of PRL (P <0.05).Conclusion:GABA may beinvolved in inhibiting mouse uterine decidualization process by reducingthe area of decidualization and decidual intensityin vivo. Part Ⅲ: Function of GABA signaling in the early mousetrophoblast invasionPurpose:to investigated GABA and GABA Breceptor effects on decidualcell invasion and EPCs outgrowth.Methods:separation of primary uterinedecidual cell in D8mouse and the expression of GABA and GB1weredetected by indirect immunofluorescence;dcidual cell invasion ability weredetected by transwell chambers with GABA(1μM,10μM and100μM);baclofen (0.5μM,5μM and50μM);0.2μM, the best effective GABAconcentration plus0.2μM,2μM and20μM and2-Hydroxysaclofenintervention; separation of primary EPCs and then planted in Matrigel withabove intervention;adhesion and outgrowth ability were detected at24h,48h and72h by Image J software analyzed. Results:indirectimmunofluorescence result show that GABA and GB1expression inprimary decidual cells cytoplasm;transwell chamber result show that100μM of GABA and5μM baclofen could reduce the lower side of cellnumber(P <0.05) and20μM2-Hydroxysaclofen can be successfullyreversed100μM GABA effects on cell invasio;observed under themicroscope, the analysis shows that all groups of EPCs were more than90%adhesion ratio with no significant differences.100μM GABA and5μM baclofen could significantly promote EPCs outgrowth (P <0.05) and20μM2-Hydroxysaclofen can be successfully reversed100μM GABA effects on EPCs outgrowth at24h and48h by Image J softwareanalyzed.Conclusion:GABA showed to may promote the outgrowth ofEPCs and inhibit the invasion of decidual cell through B receptor.