Gamma-aminobutyric acid (GABA) can affect physiological processes in preimplantation embryos via GABAA and GABAB receptors

Abstract

Purpose: Several widely used substances (e.g., some therapeutics or food supplements) can act on gamma-aminobutyric acid (GABA) receptors, and we investigated whether the activation of these receptors could affect the preimplantation embryo.

Methods: Transcripts of all GABA receptor subunits and selected proteins were examined using quantitative RT-PCR and immunohistochemistry. To analyze the effects of receptor activation, in vitro culture of mouse preimplantation embryos with natural and synthetic GABA receptor ligands was used.

Results: We detected nine GABA receptor transcripts in mouse blastocysts and 14 GABA receptor transcripts in ovulated oocytes. The results of this study indicate that ionotropic GABA_A receptors can be formed from α5, β3, and γ3 (or δ, π) subunits, GABA_A-ρ receptors can be formed from ρ2 subunits and metabotropic GABA receptors can be formed from GABA_B1b and GABA_B2 subunits in mouse blastocysts. Supplementing the culture medium with GABA at concentrations of 2-10 mM or with specific GABA_A and GABA_B receptor agonists (at concentrations of 10-100 μM) significantly increased the proportion of dead cells in blastocysts. The GABA-induced effects were prevented by pretreatment of embryos with GABA_A and GABA_B receptor antagonists.

Conclusion: The results of this study indicate that GABA and synthetic GABA receptor ligands can negatively affect preimplantation embryos via GABA_A and GABA_B receptors.

Keywords: GABA receptors; early pregnancy; oocytes; preimplantation embryos.

FIGURE 1

RT–PCR analysis of GABA_B receptor 1 transcript variants in mouse blastocysts and oocytes. (A) Representative agarose gels with separated PCR products are shown. Lanes: MW, molecular weight markers; Ooc, oocytes; Blast, blastocysts; PC, positive control tissue (brain); B, blank reaction. PCR templates: +, cDNA synthesis with reverse transcriptase; −, corresponding reverse transcriptase negative controls. The MWs and the predicted sizes of the PCR products in base pairs (bp) are indicated to the left and right of the panels, respectively. (B) Alignment of the 277‐bp PCR product nucleotide sequence (amplified with GABA_B1b primers in mouse oocyte, blastocyst and brain cDNA samples) with the published mouse GABA_B1b N‐terminal nucleotide sequence (GenBank acc.no. AF120255 ²⁹ ). Asterisks indicate positions with identical nucleotides in the compared sequences. CDS‐Ex1, the nucleotide sequence of GABA_B1b coding exon 1; CDS‐Ex2, the nucleotide sequence of GABA_B1b coding exon 2 (corresponding to exon 7 in the GABA_B1a transcript variant, GenBank acc.no. NM_019439, see Figure S1). Primer sequences are shown in boxes (RP primer sequence is reverse complementary).

FIGURE 2

Expression of GABA receptor proteins in mouse blastocysts. GABA receptor subunits were detected by immunofluorescence. Representative images are shown. Optical sections were observed via CLSM. Cell nuclei were stained with Hoechst 33342 (blue staining, A columns). Embryos were incubated with primary antibodies against the GABA receptor subunits and with a secondary antibody labeled with Alexa Fluor 488 (green staining, B columns); C columns, merged images. For negative controls, Figure S3. Scale bars, 30 μm.

FIGURE 3

Effects of GABA and GABA receptor agonists on cell number and proportion of dead cells. (A) Cell numbers and proportions of dead cells in blastocysts incubated with the indicated concentrations of GABA. Numbers of blastocysts in the groups (n): Control, n = 38; GABA 2 mM, n = 24; GABA 5 mM, n = 35; GABA 10 mM, n = 42. (B) Cell numbers and proportions of dead cells in blastocysts incubated with indicated concentrations of muscimol. Numbers of blastocysts in the groups (n): Control, n = 42; muscimol 10 μM, n = 36; muscimol 50 μM, n = 43; muscimol 100 μM, n = 37. (C) Cell numbers and proportions of dead cells in blastocysts incubated with indicated concentrations of (R)‐baclofen. Numbers of blastocysts in the groups (n): Control, n = 42; (R)‐baclofen 10 μM, n = 32; (R)‐baclofen 50 μM, n = 31; (R)‐baclofen 100 μM, n = 32. (D) Cell numbers and proportions of dead cells in embryos pretreated with the mixture of GABA receptor antagonists (mixture of bicuculline, CGP 35348, and TPMPA at final concentration of 50 μM) prior to the addition of GABA. Numbers of blastocysts in the groups (n): Control, n = 38; GABA 10 mM, n = 45; GABA 10 mM + antagonists (bicuculline+CGP35348 + TPMPA), n = 48. The values are arithmetical means + SEMs. Statistical significance of differences: *p < 0.05, **p < 0.01, ***p < 0.001.