Toxic Effects And Mechanisms Of GABA On Embryo Implantation In Mice

Background: In mammals,following fertilization,the zygote undergoes several rounds of division and morphogenesis to form the blastocyst,which participates in the first physical and physiological interactions with the maternal endometrium to initiate implantation.Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and receptive uterus.An abnormality in this process may cause an adverse outcome to pregnancy.?-Aminobutyrate(GABA)is a major inhibitory neurotransmitter in the central nervous system(CNS),which plays an important role in relieving stress,alleviating anxiety,and improving sleep.In the female reproductive system,previous studies found that the GABA concentration was higher in the fallopian tube than in the brain,in addition,our previous study showed that GABA was dynamically expressed in the mouse uterus from D1 to D4 of pregnancy,and participate in the decidualization and placentation,which suggests GABA may play a key role in the in process.Nowadays,GABA as an additive,which was added to food and health products.But the recommended dose is different in GABA products,ranging from a few hundred milligrams to a few grams.However,the toxi effects of GABA has not been reported in the current study.In the study,firstly,pregnant Kunming mice were treated with different dose GABA,explore the toxic effect and mechanism of GABA on the endometrial receptivity in mice.Subsequently,different concentrations of GABA were added to the medium were used to investigate the toxic effects and mechanisms of GABA on the development of preimplantation embryos,elucidate the toxic effects and possible mechanisms of GABA on embryo implantation in mice.Part 1 Toxic effects of GABA on endometrial receptivity in miceObjective: To investigate the toxic effects of GABA on endometrial receptivity in mice.Methods: 1.pregnant Kunming mice were randomly divided into 4 groups(n = 15 per group)and dosed with GABA at 6.4,10.0,and 12.5 mg/g/day,respectively.Control group were treated with same dose normal saline.On D5 of pregnancy,trypan blue was injected into the tail vein of each mouse,which was used to identify the embryo implantation site.2.Ten mice per group were sacrificed on D4 by cervical dislocation,collected the uteri and the tissue sections(5?m)were stained with hematoxylin and eosin(H&E)for histological evaluation.3.Western blot and qPCR were used to detect the expression of E-cadherin,HOXA10 and LIF protein and mRNA,in addition,the level of IL-6 were detected by enzyme-linked immunosorbent assay.4.The serum levels of E2 and P4 were detected using a radioimmunoassay.Results: 1.Pregnant Kunming mice were treated with GABA,we found that exposure to high concentration GABA(12.5 mg/g)decreased the number of implantation sites on pregnancy D5(10.5±3.5 vs.13±2.2,P <0.05).2.H&E staining showed that uterus structure appeared abnormity and the number of glands were reduced in the 12.5 mg/g GABA treatment group.3.The mRNA and protein expression of receptivity related factors leukemia inhibitory factor(LIF),E-cadherin,homeobox gene 10(HOXA10),and interleukin-6(IL-6)were all significantly downregulated(P < 0.05 vs.control).4.Serum estradiol(E2)and progesterone-4(P4)were remarkably decreased(P < 0.05 vs.control).Conclusion: The high concentration GABA disrupt the endometrial receptivity.Part2 Toxic effects and mechanisms of GABA on the development of preimplantation embryosObjective: To investigate the toxic effects and possible mechanisms of GABA on development of preimplantation embryo in mice.Methods: 1.The embryos of different development stage were collected and the localization of GABA,B-type receptor protein(GB1),the ? subunit of A-type receptor(GABRP),and L-glutamic acid decarboxylase 67(GAD67)was detected by immunofluorescence staining;2.The embryos of different development stage were cultured in vitro in KSOM medium containing 10,20,30,40,or 50 ?g/?l GABA and 40?M 2-hydroxysaclofen(GABA B-type receptor antagonist)for 48-72 h,respectively,and blastocyst formation rate was counted.Results: 1.GABA and GB1 are mainly expressed in the membranes of 2-cell and 4-cell embryos,while in the 8-cell to blastocyst stages,they are localized in the nucleus.However,GABRP was not detected in preimplantation embryos at any stage.2.The blastocyst formation rate of each development stage was significantly declined in a dose-dependent manner.In addition,the blastocyst formation rate of 2-cell embryos stage was remarkably inhibited by 10 ?g / ?l GABA(92%vs.99%,P=0.017).3.40 ?M 2-hydroxysaclofen could effectively reverse the inhibitory effect of 30 ?g / ?l and 40 ?g / ?l GABA on blastocyst formation(45% vs.99%,P=0.000;4% vs.99%,P=0.000).Conclusion: GABA mediated the toxic effect of preimplantation embryo development through its B receptor.