Abstract

Mammalian sperm must undergo a complex process called capacitation in order to fertilize the egg. During this process, hyperpolarization of the sperm plasma membrane has been mostly studied in mouse, and associated to its importance in the preparation to undergo the acrosome reaction (AR). However, despite the increasing evidence of membrane hyperpolarization in human sperm capacitation, no reliable techniques have been set up for its determination. In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination. In addition, we have conducted parallel measurements of the same human sperm samples by flow cytometry and population fluorimetry, before and after capacitation, to conclusively address their reliability. This integrative analysis sets the basis for the study of Em in human sperm allowing future work aiming to understand its role in human sperm capacitation.

Highlights

  • During fertilization, the sperm cell has a simple and fundamental goal: to merge with the oocyte and deliver its genetic material

  • In this report we describe human sperm membrane potential (Em) measurements by a fluorimetric population assay, establishing optimal conditions for Em determination

  • Em measurements were performed with a positively charged carbocyanine probe, DiSC3(5). This dye partitions into sperm cells according to their membrane potential but independently on the nature of ionic fluxes, rendering it suitable for potential measurements of the plasma membrane

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Summary

Introduction

The sperm cell has a simple and fundamental goal: to merge with the oocyte and deliver its genetic material. Mammalian sperm are not able to fertilize an oocyte shortly after ejaculation. They must go through a process called sperm capacitation. Capacitation includes a complex series of molecular events that normally take place in the female genital tract but can be mimicked in vitro in defined media. This process prepares sperm to acquire hyperactivated motility (HA) and to undergo the acrosome reaction (AR) upon stimulation (Chang, 1951; Austin, 1952). Data regarding capacitation has been acquired in different mammalian species. Due to the complexity of the capacitation process, each of these events has been studied independently

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