. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acrosomal alkalinization occurs during human sperm capacitation Gabriela Carrasquel Mart  ınez 1 , Andr  es Aldana 1,2 , Jorge Carneiro 3,4 , Claudia Lydia Trevi ~ no 1 , and Alberto Darszon 1, * 1 Departamento de Gen etica del Desarrollo y Fisiolog  ıa Molecular, Instituto de Biotecnolog  ıa, Universidad Nacional Auto ´noma de M exico, Cuernavaca, Morelos, M exico 2 Centro de Ciencias de la Complejidad, Universidad Nacional Auto ´noma de M exico, Ciudad de M exico, M exico 3 Instituto Gulbenkian de Cie ˆncia (IGC), Oeiras, Portugal 4 Instituto de Tecnolog  ıa Qu  ımica e Biolo ´gica Anto ´ nio Xavier, Universida de Nova, Oeiras, Portugal *Correspondence address. Departamento de Gen etica del Desarrollo y Fisiolog  ıa Molecular, Instituto de Biotecnolog  ıa, Universidad Nacional Auto ´ noma de M exico, Cuernavaca, Morelos, 62210 M exico. E-mail: darszon@ibt.unam.mx https://orcid.org/0000-0002- 2502-0505 Submitted on July 02, 2021; resubmitted on January 22, 2022; editorial decision on February 16, 2022 ABSTRACT: Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiologi- cal modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the ac- rosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intra- cellular calcium ([Ca 2þ ] i ) and pH (pH i ). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pH a ) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pH a increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pH a during capacitation and its regulation. We found that pH a progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pH a during capacitation. Acrosome alkalinization was dependent on extracellular HCO  3 and Ca 2þ . Inhibition of the HCO 3  -dependent adenylyl cyclase and protein kinase A induced significant pH a changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR. Key words: human sperm / capacitation / acrosome alkalinization / acrosome reaction / acrosome pH Introduction Fertilization is an essential event for organisms with sexual reproduc- tion. This process requires the highly regulated and synchronized inter- action between the sperm and the oocyte, culminating in their fusion. Human sperm acquire fertilizing capability as they transit through the female reproductive tract, where a maturation process known as ca- pacitation takes place (Austin, 1951; Chang, 1951). Capacitation com- prises a set of biochemical and physiological changes that prepares sperm to acquire hyperactivated motility and undergo the acrosomal reaction (AR), necessary processes to reach and fertilize the oocyte (Thomas and Meizel, 1988; Suarez et al., 1991; Visconti et al., 2011; Leemans et al., 2019). Capacitation entails a reorganization of the plasma membrane, changes in the ionic permeability, including an in- crease in the intracellular pH (pH i ), primarily of the head and flagellar principal piece regions (Nishigaki et al., 2014; Matamoros-Volante and Trevino, 2020), and an increase in the concentration of intracellular calcium ([Ca 2þ ] i ), bicarbonate (HCO 3  ) and chloride (Cl  )(Darszon et al., 2005, 2011; Molina et al., 2018). Additionally, capacitation involves the activation of an HCO 3  -dependent soluble adenylate cy- clase (sAC, SACY) signaling pathway, with a consequential increase in the concentration of cAMP, protein kinase A (PKA) activity and phos- phorylation of several proteins (Darszon et al., 2005; Visconti, 2009; Visconti et al., 2011; Buffone et al., 2014). Membrane potential (Em) hyperpolarization accompanies capacitation in multiple species, includ- ing mouse and human (Arnoult et al., 1999; Lishko and Kirichok, 2010; Visconti et al., 2011; Lo ´pez-Gonza ´lez et al., 2014; Fischer et al., 2020). Only capacitated sperm can undergo the Ca 2þ -dependent and physio- logically triggered AR (Yanagimachi, 2011; Hirohashi and Yanagimachi, 2018). During this process, some of the contents of the acrosome, an intracellular secretory vesicle located at the tip of the head, are liber- ated. This vesicle contains a mix of hydrolytic enzymes that can help sperm to cross the layers surrounding the oocyte, including the zona pellucida matrix (Florman, 1994; Breitbart, 2002; Hirose et al., 2020). The acrosome stores Ca 2þ and is acidic (luminal pH 5.3 6 0.1) (Working and Meizel, 1983; Yanagimachi, 1994; Nakanishi et al., 2001; Hirohashi and Yanagimachi, 2018). In mouse sperm, the acrosomal pH V C The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/ funder_policies/chorus/standard_publication_model) Molecular Human Reproduction, pp. 1–18, 2022 https://doi.org/10.1093/molehr/gaac005 ORIGINAL RESEARCH Downloaded from https://academic.oup.com/molehr/article/28/3/gaac005/6535714 by guest on 28 June 2023