In vitro capacitating effect of gamma-aminobutyric acid in ram spermatozoa

We have evaluated the capacitating effect of gamma-aminobutyric acid (GABA) in ram spermatozoa in vitro, in a chemically defined medium, by means of the chlortetracycline (CTC) binding assay. Semen from adult Australian Merino rams was collected in an artificial vagina; spermatozoa were washed once in modified Biggers, Whitten, and Wittingham medium (m-BWW), without BSA or serum, and incubated in m-BWW alone or in m-BWW containing GABA, GABA agonists, or antagonists for 2 h at 38.5 degrees C under 5% CO2 in air. Samples were taken for assessment of CTC binding pattern or were further incubated for 15 min in the presence of 5 mu M calcium ionophore A23187. Acrosomal exocytosis was evaluated by Pisum sativum agglutinin binding, Addition of GABA to the incubation medium resulted in a concentration-dependent increase in the percentage of CTC forms II and III, corresponding to mid-capacitated and capacitated spermatozoa, respectively. The effect was marginally significant at 1 mu M and maximal at 20 mu M. The action of 20 mu M GABA was mimicked by the GABA(A)-receptor agonist, muscimol, but not by the GABA(B)-receptor agonist, baclofen, and completely blocked by the GABA(A)-receptor antagonists, bicuculline and picrotoxin, which lacked effect per se. In a separate set of experiments, incubation of spermatozoa with GABA at a concentration of 1 mu M, which was insufficient to stimulate sperm capacitation, together with the neuroactive steroid allopregnanolone (1 mu M) provoked a capacitating effect similar to that achieved by 20 mu M GABA alone. These results show that GABA has a capacitating action on ram spermatozoa through a GABA(A) receptor-mediated mechanism.