PARENT SESSION
Capacitation & Acrosome Reaction

SPECIFIC STRYCHNINE BINDING SITES INCREASE DURING CAPACITATION OF GOLDEN HAMSTER SPERMATOZOA.

Llanos, Miguel¹, Aguirre, Marķa ¹, Ronco, Ana ¹, ¹

ABSTRACT- We have recently shown that hamster spermatozoa posses specific strychnine binding sites as an evidence for the presence of glycine receptors (GlyR) in these cells. In addition, activation of this receptor via its agonists leads to the development of the acrosome reaction (AR) in several mammalian sperm cells. The aim of this work was to evaluate specific [³H]-strychnine [³H]-stry; the classical GlyR antagonist) binding sites and some kinetics parameters of these sites in both, capacitated (cap) and non-capacitated (non-cap) hamster sperm preparations. Highly motile, washed cauda epididymal spermatozoa selected through a glass beads column were incubated in a modified Tyrode^,s buffer pH 7.4, containing 12 mg/ml fatty acid free-BSA, 0.25 mM hypotaurine and 25 M penicillamine. Incubations were performed from 0-6 h in a humidified atmosphere of 5% CO₂/95 % air at 37°C. After each incubation time, cells were evaluated for motility, AR and [³H]-stry binding. Specific [³H]-stry binding was similar from 0-3 h incubation (910 ±133 cpm/10⁶ cells; n=4, Mean ±SEM) but increased by threefold from 4-6 h. Percentages of AR were 0-15 % from 0-3 h and increased to 40-80% from 4-6 h, indicating that from 4-6 h most of the sperm population was capacitated. Incubation in low Ca²⁺ (20 M) or in the presence of Sr²⁺ instead Ca²⁺, no capacitation occurred (0-5 % AR), and [³H]-stry binding was always low (for instance at 6h: 1,150 ± 242 cpm/10⁶ cells; n=4, Mean ±SEM). Sperm membrane preparations partially purified by ultracentrifugation (110,000 x g, 2 h) from cap and non-cap spermatozoa were also evaluated for [³H]-stry binding. Scatchard and Hill plots analysis showed that both preparations had same Hill coefficient (1.0) and K_D(120-124 nM), Nevertheless, maximum number of binding sites (B_max) was 48 and 142 fmol/10⁶ spermatozoa in those non-cap and cap membrane preparations respectively. Results indicate that [³H]-stry binding sites do not change their affinity for the agonist during capacitation, but the increased [³H]-stry binding to cap spermatozoa and the increased B_max observed in those cap membrane preparations suggest that these sites are uncovered or exposed during this process. Changes in [³H]-stry binding sites number may reflect changes of the GlyR population, reinforcing the role of this receptor in the mammalian sperm AR.

KEY WORDS: spermatozoa, capacitation, exocytosis, strychnine