Flow cytometry analysis reveals a decrease in intracellular sodium during sperm capacitation

Jessica Escoffier 2 Dario Krapf 1 Felipe Navarrete 2 Alberto Darszon ( 0 Pablo E. Visconti ) 2 0 Department of Developmental Genetics and Molecular Physiology, IBT-UNAM , Cuernavaca, M e xico 1 Institute of Molecular and Cell Biology of Rosario (CONICET-UNR) , Argentina 2 Department of Veterinary and Animal Science, Integrated Sciences Building, University of Massachusetts , Amherst, MA , USA - Introduction After ejaculation, sperm are still unable to fertilize. They gain their fertilizing ability in the female genital tract (Yanagimachi, 1994). The physiological and molecular events that render the sperm able to fertilize are collectively known as capacitation. Although this process was discovered 60 years ago (Austin, 1952; Chang, 1951), the molecular mechanisms of sperm capacitation are still poorly understood. In mouse sperm, capacitation depends on lipid remodeling of the plasma membrane (Trevino et al., 2001; Visconti et al., 1999a; Visconti et al., 1999b), increase of intracellular pH (Nakanishi et al., 2001; Zeng et al., 1996), increase in protein tyrosine phosphorylation (Arcelay et al., 2008; Krapf et al., 2010; Visconti et al., 1995a; Visconti et al., 1995b) and changes in fluxes of ions such as Ca2+, HCO32, K+ and Na+ (Arnoult et al., 1997; Demarco et al., 2003; Felix et al., 2002; Munoz-Garay et al., 2001). These events are associated with functional sperm parameters such as hyperactivated motility (Yanagimachi, 1994), the ability to undergo a physiological stimulated acrosome reaction (Buffone et al., 2008; Escoffier et al., 2010b; Tomes, 2007) and the ability to penetrate the egg. Among ion fluxes occurring during capacitation, transport of HCO32 (Demarco et al., 2003) constitutes one essential step in the initiation of capacitation. Influx of HCO32 into sperm promotes cAMP synthesis through activation of the atypical soluble adenylyl cyclase (Esposito et al., 2004; Hess et al., 2005) and subsequent protein kinase A (PKA) activation. This activation of a cAMP-dependent pathway is upstream of the capacitationassociated increase in tyrosine phosphorylation (Visconti, 2009). It has been shown that capacitation in mouse sperm is also accompanied by hyperpolarization of the sperm plasma membrane potential (Em) (Arnoult et al., 1999). The molecular mechanism of this hyperpolarization is not clear. Previous studies by our group indicate that Na+ permeability is involved in the establishment of the sperm resting Em and that this Na+ permeability is reduced during sperm capacitation (Demarco et al., 2003; HernandezGonzalez et al., 2006). These results are consistent with the hypothesis that electrogenic Na+ transport is reduced during sperm capacitation, resulting in hyperpolarization. In this work, it is shown that in a Na+-free medium, addition of Na+ induced a fast depolarization of the sperm Em. Interestingly, this depolarization was blocked by both amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA), an amiloride analogue, suggesting that epithelial Na+ channels (ENaCs) were involved in this Na+ permeability. ENaCs are made up of four subunits: a, b, c and d, where a and d can replace each other and are directly involved in forming the channel pore (Canessa et al., 1994). Using antibodies against ENaC subunits, we had previously demonstrated the presence of ENaC-a and ENaC-d in mouse sperm (HernandezGonzalez et al., 2006). By contrast, patch-clamp current recordings in testicular sperm detected an amiloride-sensitive component consistent with the presence of ENaCs in these cells (MartinezLopez et al., 2009). However, how the ENaC is regulated during sperm capacitation is not known. In other cell types, it has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) inactivates ENaC, either through direct stimulation involving the first nucleotide binding domain of CFTR (Schreiber et al., 1999) or through increase of intracellular Cl2 concentration (Konig et al., 2001). More recently, work by our group as well as others have demonstrated the presence of this Cl2 channel in mammalian sperm (Hernandez-Gonzalez et al., 2007), opening the possibility of a similar regulatory pathway in these cells. Regarding signaling pathways associated with sperm capacitation, it is necessary to consider that only a fraction of the sperm population undergoes capacitation. Owing to difficulties in experimental conditions, most studies on the molecular basis of capacitation have been done with complete sperm suspensions containing sperm in different stages of capacitation, as well as subpopulations of deteriorated sperm cells. To solve problems related to sperm heterogeneity and compartmentalization during capacitation, recent studies have used flow cytometry (Martinez-Pastor et al., 2010; Nakanishi et al., 2001; Piehler et al., 2006; Tao et al., 1993), single cell analysis (Arnoult et al., 1999; Escoffier et al., 2007; Fukami et al., 2003) and combinations of both, that is, microscopy visualization e of signaling pathways after sorting of sperm subpopulations by c flow cytometry (de Vries et al., 2003; Flesch et al., 2001). ine Altogether, these experimental approaches are vital for c understanding how sperm capacitation occurs and to lS discriminate certain features between live and degenerating le sperm that can skew the conclusions of a particular experiment. C In this work, we used flow cytometry to qualitatively analyze fo changes in intracellular Na+ concentration ([Na+]i). For this la purpose, we double stained sperm suspensions with the Na+ dye rn CoroNa Red and propidium iodide to analyze changes in Na+ in u individual live sperm. These experiments show that capacitated Jo sperm are a cell population characterized by lower Na+ concentrations. This decrease in Na+ can be blocked by PKA inhibitors and induced by genistein in conditions that do not support sperm capacitation. Moreover, ENaC inhibitors are able to induce a similar Na+ decrease when added to medium that does not support capacitation. Overall, our observations are consistent with a model in which PKA activation of CFTR inactivates ENaC and consequently induces the capacitation-associated hyperpolarization of the sperm Em. Results Regulation of [Na+]i during capacitation Previously, we presented evidence that Na+ is involved in determining the sperm plasma membrane resting Em (Hernandez-Gonzalez et al., 2006). We hypothesized that the contribution of Na+ to the resting Em is reduced during capacitation because of downregulation of Na+ channels such as ENaCs. This possibility was supported by experiments demonstrating that incubation of sperm in non-capacitating medium supplemented with ENaC inhibitors such as amiloride or EIPA was conducive to hyperpolarization. To further investigate this model, [Na+]i was measured in individual cells using a flow cytometry approach. Towards this goal, sperm were loaded with the Na+ probe CoroNa Red, stained with propidium iodide (PI) and analyze (...truncated)