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目的:探讨γ-氨基丁酸(γ-aminobutyric acid,GABA)对小鼠植入前胚胎发育的影响及其机制。方法:(1)收集不同发育阶段的小鼠植入前胚胎,通过激光共聚焦检测GABA及其B受体B1亚基(B receptor B1 subunit,GB1)、A受体pi亚基(A receptor pi subunit,GABRP)以及GABA合成酶(L-glutamate decarboxylase,GAD67)的表达。(2)不同发育阶段的胚胎分别培养在含10,20,30,40,50μg/μL GABA和40μmol/L B型受体抑制剂(3-Amino-2-(4-chlorophenyl)-2-hydroxypropanesulfonic acid,2-hydroxysaclofen)的培养液中,48~72 h时观察囊胚的形成并计算囊胚形成率。结果:(1)GABA与其受体GB1蛋白在小鼠胚胎中的表达模式一致,在2-细胞和4-细胞阶段,主要表达在细胞膜上,而在8-细胞至囊胚阶段,则转移到细胞核中表达;而另一受体GABRP在植入前胚胎的各个阶段均不表达。GAD67在胚胎发育的各个阶段均有表达。(2)各发育阶段胚胎的囊胚形成率均随着GABA浓度的增加而下降,且呈剂量依赖性;10μg/μL GABA可显著降低2-细胞胚胎的囊胚形成率(92%vs.99%,χ2=5.70,P=0.035)。(3)40μmol/L 2-hydroxysaclofen能够有效逆转30μg/μL和40μg/μL GABA对囊胚形成的抑制作用(99%vs.45%,χ2=72.32,P=0.000;99%vs.4%,χ2=180.66,P=0.000)。结论:GABA通过其B受体抑制小鼠植入前胚胎的发育。
Objective: To investigate the effect of γ-aminobutyric acid (GABA) on the preimplantation embryo development and its mechanism in mice. Methods: (1) Preimplantation embryos were collected from mice of different developmental stages. The expression of GABA and B receptor B1 subunit (GB1), A receptor pi subunit (A receptor pi subunit, GABRP) and GABA synthase (GAD67). (2) Embryos at different developmental stages were cultured in the medium containing 10, 20, 30, 40 and 50μg / μL GABA and 40μmol / LB receptor inhibitor (3-Amino-2- , 2-hydroxysaclofen), 48 ~ 72 h observation of blastocyst formation and calculation of blastocyst formation rate. Results: (1) The expression pattern of GABA and its receptor GB1 protein in mouse embryos was consistent, mainly in the cell membrane in the 2-cell and 4-cell phases, while in the 8-cell to blastocyst stage, While the other receptor GABRP was not expressed in all stages of the preimplantation embryo. GAD67 is expressed at all stages of embryonic development. (2) The blastocyst formation rate of embryos at all developmental stages decreased in a dose-dependent manner with the increase of GABA concentration; 10μg / μL GABA significantly reduced the blastocyst formation rate of 2-cell embryos (92% vs.99 %, χ2 = 5.70, P = 0.035). (3) 40μmol / L 2-hydroxysaclofen can effectively inhibit the inhibition of blastocyst formation by 30μg / μL and 40μg / μL GABA (99% vs.45%, χ2 = 72.32, P = 0.000; 99% vs.4% χ2 = 180.66, P = 0.000). Conclusion: GABA inhibits the development of preimplantation embryos in mice via its B receptor.