Fig 2: GABA expression in mouse uterus at each designated time point following pregnancy. (A) Quantitative RT-PCR analyses revealed expression level of the GABAB1 subunit. Each mRNA expression level was normalized to GAPDH. The experiments were repeated three times and all the data were shown as a mean plus or minus the standard error of the mean. *P < 0.05,***P < 0.001 (B) Western blotting analyses of the GABAB1 subunit in mouse uterus on days 1, 4, 5, 6 and 8. (C) The relative expression of GABAB1 subunit by gray scanning analyses. *P < 0.05,***P < 0.001 (D–E) Analyses of uterine GABAB1 expression via in situ hybridization and immunostaining on days 1, 4, 5, 6 and 8 following pregnancy. Scale bars, 200 µm e, embryo; bl, blastocyst; le, luminal epithelium; ge, glandular epithelium; m, mesometrial pole; am, antimesometrial pole; pdz, primary decidual zone; sdz, secondary decidual zone.

Fig 3: Minimal function of the GABAB receptor for proliferation and differentiation of primary stromal cells. (A) GABAB1 expression in isolated endometrial stromal cells shown by a combination of vimentin-positive and cytokeratin-negative signals via immunofluorescence analyses. Scale bars, 50 µm. (B) 100 µM saclofen or 100 µM baclofen exhibited a weak influence on proliferation of stromal cells. (C) Quantitative RT-PCR analyses revealed that Prl8a2 mRNA levels were not significantly changed in experimental groups compared to the control group at all time points, and all the mRNA expression were normalized to GAPDH. Experiments were repeated three times, and all the data were shown as a mean plus or minus the standard error of the mean.

Fig 4: GABAB1 upregulation following embryo implantation in embryo delayed implantation models. (A) Obvious expression of GABAB1 subunit in stromal cells following embryo implantation; (B) Undetectable expression levels of GABAB1 subunit in stromal cells in the non-implantation group. The right panel is a close-up of the left panel. Scale Bars, 50 µm. (C) Quantitative RT-PCR analyses revealed that GABBR1 mRNA was higher in the implantation group compared to that in the control group, both of which were normalized to GAPDH expression. Experiments were repeated three times and all the data was shown as a mean plus or mine the standard error of the mean. ***P < 0.001.

Fig 5: GABAB1 is not respond to E2 and P4. (A) Quantitative RT-PCR analyses revealed a similar level of GABAB1 expression in uteri of ovariectomized mice after injected with E2, P4, and a combination of E2 and P4 respectively for 24 h. The mRNA expression levels were normalized to GAPDH. Experiments were repeated three times and all the data was shown as a mean plus or mine the standard error of the mean. (B) Analyses of uterine GABAB1 expression via immunostaining in ovariectomized adult mice injected with E2, P4, and a combination of E2 and P4 respectively for 6 h and 24 h. Scale bars, 50 µm le, luminal epithelium; ge, glandular epithelium.