Human Sperm Capacitation Requires the Inhibition of the Activity of the Serine/Threonine Phosphatase PP2A.

Abstract

Capacitation is a complex process that involves a series of morphological and biochemical changes in the sperm and that allow them to acquire fertilizing capacity. Some of the changes described that take place during the early stages of capacitation includes increase in the concentration of intracellular calcium, the generation of reactive oxygen species, changes in the membrane potential, and increase in the AMPc intracellular concentration. It has also been reported that during capacitation the phosphorylation pattern in tyrosine and threonine proteins residues changes. The role of protein kinases, including tyrosine kinases and PKA, in this process is well documented. However, little is known about the role of protein phosphatases (PPs) in this event. PP2A is serine threonine phosphatase that has been detected in human and primate sperm extracts, but its role during sperm capacitation is not known. The aim of this work was to study the role of PP2A during human sperm capacitation.To this end, we have used inhibitors and measured the activity of PP2A during this process. Semen samples were obtained from normal donors according to the WHO guidelines. Highly motile sperm were selected by a Percoll gradient using a modified Tyrode's medium (without BSA and bicarbonate). Subsequently, sperm aliquots were resuspended in the same medium and incubated at 37 °C and 5% CO2 in the presence or absence of PP2A inhibitors (okadaic acid and Endothal with IC50= 0.1 nM and IC50= 90 nM respectively). Other sperm aliquots were treated by adding 2.6% BSA and 25 nM bicarbonate to the culture medium. The capacitation process was evaluated at different times (0, 15, 30, 60, and 300 min) by using the chlortetracycline (CTC) fluorescence assay. The activity of PP2A was determined in human sperm extracts by using the "RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit" of Invitrogen.The results indicate that treatment with the inhibitors blocked the activity of PP2A and rapidly increased the percentage of capacitated sperm. Moreover, upon addition of BSA and bicarbonate to the culture medium the sperm became capacitated, as evidenced by CTC assay, and the activity of PP2A rapidly decreased and remained low thereafter.In conclusion, the onset of capacitation in human sperm is associated with a rapid decrease in the activity of PP2A and inhibition of this phosphatase rapidly triggers capacitation. Fondecyt 1080028. (poster)