Role of the Serine/Threonine Phosphatases During the Progesterone-Induced Acrosome Reaction and Increase in Intracellular Free Calcium Concentration in Human Sperm.

Abstract

The acrosome reaction (AR) is an exocytotic event that takes place by fusion of the plasma membrane and the outer acrosomal membrane in sperm previously capacitated. It is widely accepted that progesterone stimulates human sperm AR and that acrosomal exocytosis is accompanied by a transient elevation of intracellular free Ca2+ concentration ([Ca2+]I), which is a consequence of a Ca2+ influx. It is known that during capacitation changes occur in the phosphorylation state of proteins, mediated by protein kinases (PKs) and phosphatase (PPs). However, it is not yet clear whether something similar takes place during the AR. The aim of this work was to study the role of serine/threonine PPs during the progesterone-induced AR and increase of [Ca2+]L, using specific inhibitors. Motile human sperm, free of seminal plasma, were obtained with a Percoll gradient and then incubated in Tyrode medium (2.6% BSA, 37 °C, 5% CO2). After 18 hr, aliquots were treated with different inhibitors of PPs by 15 min. The inhibitors used were 0.1 nM deltamethrin, 10 nM FK-506, and 10 nM cyclosporine A for PP2B; 90 nM endothal and 0.1 nM okadaic acid for PP2A; and 10 nM okadaic acid for PP1. Then, 7 µM progesterone was added and incubated for extra 15 minutes. Acrosomal status and viability were evaluated using PSA-FITC and Hoechst 33258, respectively. To assess the effect of the PPs inhibitors on [Ca2+]I, capacitated sperm were incubated for 30 minutes with 3 µM fura 2AM; after 15 min inhibitors were added. Calcium influx was stimulated with 7 µM progesterone. The fluorescence was monitored with a spectrofluorometer at an excitation wavelength pair of 340/380 nm and emission wavelength of 510 nm. The results indicated that most PPs inhibitors do not affect the induction of the AR by progesterone or the increase in [Ca2+]I. However, two inhibitors of PP2B, FK506 and cyclosporine A, inhibited the progesterone-induced AR but they did not have an effect on the increase of [Ca2+]I. These results suggest that PP2B (calcineurin) may be involved in the exocytosis of the acrosome induced by progesterone but not in the Ca2+ influx that precedes it. The mechanism of this inhibition is still unknown. Fondecyt 1080028. (poster)