The Expressions Of GABA Signal And Its Potential Role In The Mouse Placenta Formation In Mice

The placentation of the mammalian is depended on the trophoblasticproliferation、differentiation、invasion to the myometrium and the uterinespiral artery vascular recasting, which is to ensure the success of thepregnant. In the case of excessive invasion of the trophoblast cells occurs inhydatidiform mole and chorionic carcinoma, insufficient shallow invasionleads to various clinical obstetrics disease such as spontaneous abortion andintrauterine growth retardation (IUGR).Polar trophoblast cells adjacent to inner cell mass differentiate intoecto-placental cone (Epc) and chorionic during mouse embryonicimplantation. Subsequently, Epc invade into uterus decidual stromatemperately and regularly and differentiate into Spongiotrophoblast (Sp).Chorionic villi and chorioallantoic fusion into chorionic, Trophoblast cellsdifferentiation and fused into syncytiotrophoblast trophoblast cells andmonocytes trophoblast cells. which constituted to the fetal placentalvascular into labyrinth region (Lab). The maturity placenta of mice is not fully formed until the14.5day (D14.5) of pregnancy which consist of threecompartments: maternal Decidua basalis (Dec), spongiotrophoblasts (Sp)and fetal labyrinth (Lab). Though a series of hormone、the transcriptionfactors、growth factors and cytokines participated in the formation of theplacenta by autocrine or paracrine pathway, the factors of regulationplacentation has not clarified nowadays.γ-Amino butyric Acid (GABA) is a kind of nonprotein amino acidand as an inhibitory neurotransmitter with extensive study initially and itsmain function is transmission the inhibitory neurotransmitters in vertebrate,which is synthetized from glutamic acid through glutamate decarboxylase(GAD65/GAD67). The receptors of GABA can be divided into A, B and Ctype based on different pharmacological characteristics. The receptors ofGABARA and GABARC are ligand-gated Cl-channel receptors, GABA Breceptors are G-protein-coupled receptors which accompanied by K+andCa+2channels. GABA signal is suggested to be involved in pancreas、Liver、uterus、placenta and ovaries, Which participate in a variety ofphysiological functions and regulate the transmission of informationbetween cells, such as endocrine outside the CNS in recent several yearsstudies. Studies have shown that progesterone and its metabolitesallopregnanolone maintain uterine muscle resting state during pregnancythrough intermediate type A receptor in rats. a new GABA A receptorsubunit, named π (GABAP) was significantly upregulated in human embryo implantation window period and late secretory endometrium, it issuggestion that GABA signal has participated in the decidualization of theendometrium. In addition, activation of GABA receptor can promote orinhibit the invasion and metastasis of the tumor cells, there is no GABAsignal is involved in placental development reported so far.In previous study we found that GABA and GABA A receptorsmaybe involved in regulating the decidualization of mouse uterine stromalcells by down-regulation of Cyclin D3. The outgrowth of Epc can beobserved in vitro model of Epc via stimulation of GABA B receptor, whichpromote that GABA signal is involved in placental development. In thisstudy we evaluated the expressions of GABA signal from the ninth day tofourteenth day (D9-D14) during placentation for the first time. Toinvestigate the physiological role of GABA and its receptor in theplacentation through intervention of specific receptors. This will furtherunderstanding the regulation of the formation of the placenta and themolecular mechanism of the pathogenesis of diseases caused by abnormalplacental obstetric during placentation.Part I: The expression of GABA signal in the mouse placentaformation in miceObjective: To study the expressions of GABA signal in mouse placentation. Methods: The expressions levels of GABRP、GB1mRNA and proteinswere analysised by RT-PCR and Western Blot from D10to D14placenta;Immunohistochemistry was applied to detect the expressions of the GABAsignal in placentation during mouse pregnant. Results: GABRP、GB1mRNA and their proteins were expressed in the D10-D14mouseplacenta. The expressions of GABRP、GB1and GABA, Which werelocated on cytoplasm, GABRP is only expressed in decidua basalis ofuterus from ninth day to fourteenth (D9-D14), Which is strong expressed inD10and D13. Interestingly, similar to the expressions of GB1and GABA,Which were were strongly expressed in the Epc on D9, the Chorionic plates(Cp) and Trophoblast giant cell (TGC) on D10and the Sp on D11-D14.The expressions of GAD65/67were the same of GABA, which they havedifferent subcellular localization. GAD65was located on cell nucleus, butGAD67was located on cytoplasm. Conclusion: The spatiotemporalexpressions of GABA signal in the D10-D14placentation in mouse, Weassume that GABA signal may be involved in the placentation in mice.PartⅡ: The influence of GABA signal on mouse placentationSection I: The influence of GABA on mouse placentationObjective：To study the potential role of GABA in mouse placentation. Methods: Pregnant mice were were divided into divided into threedifferent concentrations of GABA groups of three randomly, the controlgroup and blank group. Treatment groups were received GABA atconcentrations of0.04g/kg、0.20g/kg and1.00g/kg respectively fromD7-D13daily intraperitoneal injections. The control injection of salinesolution, blank group is don’t make any deal with. The weight of embryosand placentas was measured and the structure of placenta was examined byHE staining on D14. Besides, the proliferation of placenta was evaluated byexpression level of proliferating cell nuclear antigen (PCNA) usingimmuno-histochemistry. Results: The weight of embodies significantlydecreased in different concentration groups of GABA compared withcontrol group (P<0.001). Surprisingly, the area of placenta decidual zone ismarkedly thickened, the fetal and maternal blood sinusoids were reduced inlabyrinth, and the structure of spongiotrophoblast was abnormal by HEstaining with the increasing of GABA concentration. At the same time, theproliferation of cells in decidua basalis、spongiotrophoblast and labyrinthzone was increased in concentration of GABA with0.20g/kg and1.00g/kggroup compared with control groups abnormally (P<0.001). Conclusion:GABA may play an important role in mouse placentation via regulating theproliferation of placenta cells. SectionⅡ: The influence of GABA receptors on mouseplacentation through agonists and antagonistsObjective: To study the potential role of agonists or antagonists GABAA/B receptor in mouse placentation. Methods: Kunming pregnant ratswere divided into fourteen groups of three randomly, Treatment groupswere treated with A receptor agonists (0.00975mg/k、0.0975mg/kg、0.975mg/kg), A receptor antagonists (0.0184mg/kg、0.184mg/kg、1.84mg/kg),B receptor agonists (0.01068mg/kg、0.1068mg/kg、1.068mg/kg), Breceptor antagonists (0.01329mg/kg、0.1329mg/kg、1.329mg/kg) dailyintraperitoneal injections from D7to D13respectively. The weights ofembryos and placentas were measured and the structure of placenta wasobserved by HE staining on D14. Besides, the expression level vascularendothelial growth factor(VEGF) was evaluated after A receptor treatedwith agonists or antagonists by using immunohistochemistry, the state ofproliferation in placenta was evaluated by expression level of proliferatingcell nuclear antigen (PCNA) using immunohistochemistry and the state ofapoptosis in placenta were evaluated by TUNEL after B receptor treatedwith agonists or antagonists. Results: The blood vessels in placentaldecidual zone and nucleated red blood cells in labyrinth were significantlyincreased in0.975mg/kg GABA A receptor agonist group (P<0.05), inwhich the expressions of VEGF was also significantly increased in placentalabyrinth (P<0.05); Surprisingly, The weights of placentas and embryos were significantly decreased in1.84mg/kg A receptor antagonists groupcompared with control group (P<0.05). At the same time, The expressionsof VEGF was reduced in placenta labyrinth by1.84mg/kg A receptorantagonists group; The area of placental spongiotrophoblast and labyrinthwere significantly increased in0.01068mg/kg GABA B receptor agonistgroup, in which the proliferation、the amount of spongiotrophoblast cells inplacenta is also increased and the apoptosis is decreased. Surprisingly, theproliferation of placenta is decreased and apoptosis is increased by1.068mg/kg GABA B receptor agonist group, In which the weights of placentasand embryos were significantly decreased compared with control groups(P<0.05). At the same time, the proliferation of placenta labyrinthsignificantly reduced by B receptor antagonists groups, in which thenumber in trophoblast cells were reduced, the area of spongiotrophoblast isdecreased and the sponge like structure of spongiotrophoblast is lost.Conclusion: GABA may play an important role in regulating theoccurrence of maternal-fetal blood vessels and the formation of thespongiotrophoblast in mouse placentation via its receptors.