The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm.

Sperm capacitation is a complex process that takes place in the female reproductive tract and empowers mammalian sperm with the competence to fertilize an egg. It consists of an intricate cascade of events that can be mimicked in vitro through incubation in a medium containing essential components such as bicarbonate, albumin, Ca2+ and energy substrates, among others. Genetic and pharmacological studies have underscored the unique significance of the K+ channel SLO3 in membrane potential hyperpolarization, as evidenced by the infertility of mice lacking its expression. Notably, two key molecular events, sperm hyperpolarization and intracellular alkalinization, are central to the capacitation process. SLO3 is activated by alkalinization. However, the molecular mechanisms responsible for intracellular alkalization and activation of SLO3 are not completely understood. In this study, we examined the impact of Na+/H+ exchangers on mouse sperm membrane hyperpolarization during capacitation. Pharmacological inhibition of the NHE1 exchanger blocked membrane hyperpolarization. A similar effect was observed in sperm deficient of the Ca2+ channel CatSper, because of NHE1 not being activated by Ca2+. In addition, the sperm specific NHE (sNHE) KO, did not show membrane hyperpolarization upon capacitation or induction with cAMP analogues. Our results show that sNHE is dually modulated by cAMP and membrane hyperpolarization probably through its cyclic nucleotide binding domain and the voltage-sensor motif respectively. Together, sNHE and NHE1provide the alkalinization need for SLO3 activation during capacitation.

Human sperm rotate with a conserved direction during free swimming in four dimensions.

Head rotation in human spermatozoa is essential for different swimming modes and fertilisation, as it links the molecular workings of the flagellar beat with sperm motion in three-dimensional (3D) space over time. Determining the direction of head rotation has been hindered by the symmetry and translucent nature of the sperm head, and by the fast 3D motion driven by the helical flagellar beat. Analysis has been mostly restricted to two-dimensional (2D) single focal plane image analysis, which enables tracking of head centre position but not tracking of head rotation. Despite the conserved helical beating of the human sperm flagellum, human sperm head rotation has been reported to be uni- or bi-directional, and even to intermittently change direction in a given cell. Here, we directly measure the head rotation of freely swimming human sperm using multi-plane 4D (3D+t) microscopy and show that: (1) 2D microscopy is unable to distinguish head rotation direction in human spermatozoa; (2) head rotation direction in non-capacitating and capacitating solutions, for both aqueous and viscous media, is counterclockwise (CCW), as seen from head to tail, in all rotating spermatozoa, regardless of the experimental conditions; and (3) head rotation is suppressed in 36% of spermatozoa swimming in non-capacitating viscous medium, although CCW rotation is recovered after incubation in capacitating conditions within the same viscous medium, possibly unveiling an unexplored aspect of the essential need of capacitation for fertilisation. Our observations show that the CCW head rotation in human sperm is conserved. It constitutes a robust and persistent helical driving mechanism that influences sperm navigation in 3D space over time, and thus is of critical importance in cell motility, propulsion of flagellated microorganisms, sperm motility assessments, human reproduction research, and self-organisation of flagellar beating patterns and swimming in 3D space.

C2CD6 regulates targeting and organization of the CatSper calcium channel complex in sperm flagella.

The CatSper cation channel is essential for sperm capacitation and male fertility. The multi-subunit CatSper complexes form highly organized calcium signaling nanodomains on flagellar membranes. Here, we report identification of an uncharacterized protein, C2CD6, as a subunit of the mouse CatSper complex. C2CD6 contains a calcium-dependent, membrane-targeting C2 domain. C2CD6 associates with the CatSper calcium-selective, core-forming subunits. Deficiency of C2CD6 depletes the CatSper nanodomains from the flagellum and results in male sterility. C2CD6-deficient sperm are defective in hyperactivation and fail to fertilize oocytes both in vitro and in vivo. CatSper currents are present but at a significantly lower level in C2CD6-deficient sperm. Transient treatments with either Ca2+ ionophore, starvation, or a combination of both restore the fertilization capacity of C2CD6-deficient sperm. C2CD6 interacts with EFCAB9, a pH-dependent calcium sensor in the CatSper complex. We postulate that C2CD6 facilitates incorporation of the CatSper complex into the flagellar plasma membrane and may function as a calcium sensor. The identification of C2CD6 may enable the long-sought reconstitution of the CatSper ion channel complex in a heterologous system for male contraceptive development.

Differential role of bovine serum albumin and HCO3- in the regulation of GSK3 alpha during mouse sperm capacitation.

To become fertile, mammalian sperm are required to undergo capacitation in the female tract or in vitro in defined media containing ions (e.g. HCO3 -, Ca2+, Na+, and Cl-), energy sources (e.g. glucose, pyruvate) and serum albumin (e.g. bovine serum albumin (BSA)). These different molecules initiate sequential and concomitant signaling pathways, leading to capacitation. Physiologically, capacitation induces changes in the sperm motility pattern (e.g. hyperactivation) and prepares sperm for the acrosomal reaction (AR), two events required for fertilization. Molecularly, HCO3 - activates the atypical adenylyl cyclase Adcy10 (aka sAC), increasing cAMP and downstream cAMP-dependent pathways. BSA, on the other hand, induces sperm cholesterol release as well as other signaling pathways. How these signaling events, occurring in different sperm compartments and with different kinetics, coordinate among themselves is not well established. Regarding the AR, recent work has proposed a role for glycogen synthase kinases (GSK3α and GSK3ß). GSK3α and GSK3ß are inactivated by phosphorylation of residues Ser21 and Ser9, respectively, in their N-terminal domain. Here, we present evidence that GSK3α (but not GSK3ß) is present in the anterior head and that it is regulated during capacitation. Interestingly, BSA and HCO3 - regulate GSK3α in opposite directions. While BSA induces a fast GSK3α Ser21 phosphorylation, HCO3 - and cAMP-dependent pathways dephosphorylate this residue. We also show that the HCO3--induced Ser21 dephosphorylation is mediated by hyperpolarization of the sperm plasma membrane potential (Em) and by intracellular pH alkalinization. Previous reports indicate that GSK3 kinases mediate the progesterone-induced AR. Here, we show that GSK3 inhibition also blocks the Ca2+ ionophore ionomycin-induced AR, suggesting a role for GSK3 kinases downstream of the increase in intracellular Ca2+ needed for this exocytotic event. Altogether, our data indicate a temporal and biphasic GSK3α regulation with opposite actions of BSA and HCO3 -. Our results also suggest that this regulation is needed to orchestrate the AR during sperm capacitation.

Reviewing mathematical models of sperm signaling networks.

Dave Garbers' work significantly contributed to our understanding of sperm's regulated motility, capacitation, and the acrosome reaction. These key sperm functions involve complex multistep signaling pathways engaging numerous finely orchestrated elements. Despite significant progress, many parameters and interactions among these elements remain elusive. Mathematical modeling emerges as a potent tool to study sperm physiology, providing a framework to integrate experimental results and capture functional dynamics considering biochemical, biophysical, and cellular elements. Depending on research objectives, different modeling strategies, broadly categorized into continuous and discrete approaches, reveal valuable insights into cell function. These models allow the exploration of hypotheses regarding molecules, conditions, and pathways, whenever they become challenging to evaluate experimentally. This review presents an overview of current theoretical and experimental efforts to understand sperm motility regulation, capacitation, and the acrosome reaction. We discuss the strengths and weaknesses of different modeling strategies and highlight key findings and unresolved questions. Notable discoveries include the importance of specific ion channels, the role of intracellular molecular heterogeneity in capacitation and the acrosome reaction, and the impact of pH changes on acrosomal exocytosis. Ultimately, this review underscores the crucial importance of mathematical frameworks in advancing our understanding of sperm physiology and guiding future experimental investigations.

Two-pore channel 1 and Ca2+ release-activated Ca2+ channels contribute to the acrosomal pH-dependent intracellular Ca2+ increase in mouse sperm.

The acrosome is a lysosome-related vesicular organelle located in the sperm head. The acrosomal reaction (AR) is an exocytic process mediated by Ca2+ and essential for mammalian fertilization. Recent findings support the importance of acrosomal alkalinization for the AR. Mibefradil (Mib) and NNC 55-0396 (NNC) are two amphipathic weak bases that block the sperm-specific Ca2+ channel (CatSper) and induce acrosomal pH (pHa ) increase by accumulating in the acrosomal lumen of mammalian sperm. This accumulation and pHa elevation increase the intracellular Ca2+ concentration ([Ca2+ ]i ) and trigger the AR by unknown mechanisms of Ca2+ transport. Here, we investigated the pathways associated with the pHa increase-induced Ca2+ signals using mouse sperm as a model. To address these questions, we used single-cell Ca2+ imaging, the lysosomotropic agent Gly-Phe-ß-naphthylamide (GPN) and pharmacological tools. Our findings show that Mib and NNC increase pHa and release acrosomal Ca2+ without compromising acrosomal membrane integrity. Our GPN results indicate that the osmotic component does not significantly contribute to acrosomal Ca2+ release caused by pHa rise. Inhibition of two-pore channel 1 (TPC1) channels reduced the [Ca2+ ]i increase stimulated by acrosomal alkalinization. In addition, blockage of Ca2+ release-activated Ca2+ (CRAC) channels diminished Ca2+ uptake triggered by pHa alkalinization. Finally, our findings contribute to understanding how pHa controls acrosomal Ca2+ efflux and extracellular Ca2+ entry during AR in mouse sperm. KEY POINTS: The acrosomal vesicle is a lysosome-related organelle located in the sperm head. The acrosome reaction (AR) is a highly regulated exocytic process mediated by Ca2+ , which is essential for fertilization. However, the molecular identity of Ca2+ transporters involved in the AR and their mechanisms to regulate Ca2+ fluxes are not fully understood. In mammalian sperm, acrosomal alkalinization induces intracellular Ca2+ concentration ([Ca2+ ]i ) increase and triggers the AR by unknown molecular mechanisms of Ca2+ transport. In this study, we explored the molecular mechanisms underlying Ca2+ signals caused by acrosomal alkalinization using mouse sperm as a model. TPC1 and CRAC channels contribute to [Ca2+ ]i elevation during acrosomal alkalinization. Our findings expand our understanding of how the acrosomal pH participates in the physiological induction of the AR.

Long-term segmentation-free assessment of head-flagellum movement and intracellular calcium in swimming human sperm.

Human spermatozoa are the archetype of long-term self-organizing transport in nature and are critical for reproductive success. They utilize coordinated head and flagellar movements to swim long distances within the female reproductive tract in order to find and fertilize the egg. However, to date, long-term analysis of the sperm head-flagellar movements, or indeed those of other flagellated microorganisms, remains elusive due to limitations in microscopy and flagellar-tracking techniques. Here, we present a novel methodology based on local orientation and isotropy of bio-images to obtain long-term kinematic and physiological parameters of individual free-swimming spermatozoa without requiring image segmentation (thresholding). This computer-assisted segmentation-free method evaluates, for the first time, characteristics of the head movement and flagellar beating for up to 9.2 min. We demonstrate its powerful use by showing how releasing Ca2+ from internal stores significantly alters long-term sperm behavior. The method allows for straightforward generalization to other bio-imaging applications, such as studies of bull sperm and Trypanosoma, or indeed of other flagellated microorganisms - appealing to communities other than those investigating sperm biology.

Acrosomal alkalinization occurs during human sperm capacitation.

Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa 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 pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR.

Starvation induces an increase in intracellular calcium and potentiates the progesterone-induced mouse sperm acrosome reaction.

We have recently reported two different methodologies that improve sperm functionality. The first method involved transient exposure to the Ca2+ ionophore A23187 , and the second required sperm incubation in the absence of energy nutrients (starvation). Both methods were associated with an initial loss of motility followed by a rescue step involving ionophore removal or addition of energy metabolites, respectively. In this work, we show that starvation is accompanied by an increase in intracellular Ca2+ ([Ca2+ ]i ). Additionally, the starved cells acquire a significantly enhanced capacity to undergo a progesterone-induced acrosome reaction. Electrophysiological measurements show that CatSper channel remains active in starvation conditions. However, the increase in [Ca2+ ]i was also observed in sperm from CatSper null mice. Upon starvation, addition of energy nutrients reversed the effects on [Ca2+ ]i and decreased the effect of progesterone on the acrosome reaction to control levels. These data indicate that both methods have common molecular features.

Membrane hyperpolarization abolishes calcium oscillations that prevent induced acrosomal exocytosis in human sperm.

Sperm capacitation is essential to gain fertilizing capacity. During this process, a series of biochemical and physiological modifications occur that allow sperm to undergo acrosomal exocytosis (AE). At the molecular level, hyperpolarization of the sperm membrane potential (Em) takes place during capacitation. This study shows that human sperm incubated under conditions that do not support capacitation (NC) can become ready for an agonist stimulated AE by pharmacologically inducing Em hyperpolarization with Valinomycin or Amiloride. To investigate how Em hyperpolarization promotes human sperm's ability to undergo AE, live single-cell imaging experiments were performed to simultaneously monitor changes in [Ca2+ ]i and the occurrence of AE. Em hyperpolarization turned [Ca2+ ]i dynamics in NC sperm from spontaneously oscillating into a sustained slow [Ca2+ ]i increase. The addition of progesterone (P4) or K+ to Valinomycin-treated sperm promoted that a significant number of cells displayed a transitory rise in [Ca2+ ]i which then underwent AE. Altogether, our results demonstrate that Em hyperpolarization is necessary and sufficient to prepare human sperm for the AE. Furthermore, this Em change decreased Ca2+ oscillations that block the occurrence of AE, providing strong experimental evidence of the molecular mechanism that drives the acquisition of acrosomal responsiveness.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Crosstalk between protein kinases A and C regulates sea urchin sperm motility.

Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.

Differences and Similarities: The Richness of Comparative Sperm Physiology.

Species preservation depends on the success of fertilization. Sperm are uniquely equipped to fulfill this task, and, although several mechanisms are conserved among species, striking functional differences have evolved to contend with particular sperm-egg environmental characteristics. This review highlights similarities and differences in sperm strategies, with examples within internal and external fertilizers, pointing out unresolved issues.

Modular analysis of the control of flagellar Ca2+-spike trains produced by CatSper and CaV channels in sea urchin sperm.

Intracellular calcium ([Ca2+]i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca2+]i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pHi), voltage and [Ca2+]i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis.

Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions.This article has an associated First Person interview with the first author of the paper.

Seeing is believing: Current methods to observe sperm acrosomal exocytosis in real time.

Acrosomal exocytosis (AR) is a critical process that sperm need to undergo to fertilize an egg. The evaluation of the presence or absence of the acrosome is usually performed by using lectins or dyes in fixed cells. With this approach, it is neither possible to monitor the dynamic process of exocytosis and related molecular events while discriminating between live and dead cells, nor to evaluate the acrosomal status while sperm reside in the female reproductive tract. However, over the last two decades, several new methodologies have been used to assess the occurrence of AR in living cells allowing different groups to obtain information that was not possible in the past. These techniques have revolutionized the whole study of this process. This review summarizes current methods available to analyze AR in living cells as well as the important information that emerged from studies using these approaches.

CatSper channels are regulated by protein kinase A.

Mammalian sperm must undergo capacitation as a preparation for entering into hyperactivated motility, undergoing the acrosome reaction, and acquiring fertilizing ability. One of the initial capacitation events occurs when sperm encounter an elevated HCO3- concentration. This anion activates the atypical adenylyl cyclase Adcy10, increases intracellular cAMP, and stimulates protein kinase A (PKA). Moreover, an increase in intracellular Ca2+ concentration ([Ca2+] i ) is essential for sperm capacitation. Although a cross-talk between cAMP-dependent pathways and Ca2+ clearly plays an essential role in sperm capacitation, the connection between these signaling events is incompletely understood. Here, using three different approaches, we found that CatSper, the main sperm Ca2+ channel characterized to date, is up-regulated by a cAMP-dependent activation of PKA in mouse sperm. First, HCO3- and the PKA-activating permeable compound 8-Br-cAMP induced an increase in [Ca2+] i , which was blocked by the PKA peptide inhibitor PKI, and H89, another PKA inhibitor, also abrogated the 8-Br-cAMP response. Second, HCO3- increased the membrane depolarization induced upon divalent cation removal by promoting influx of monovalent cations through CatSper channels, which was inhibited by PKI, H89, and the CatSper blocker HC-056456. Third, electrophysiological patch clamp, whole-cell recordings revealed that CatSper activity is up-regulated by HCO3- and by direct cAMP injection through the patch-clamp pipette. The activation by HCO3- and cAMP was also blocked by PKI, H89, Rp-cAMPS, and HC-056456, and electrophysiological recordings in sperm from CatSper-KO mice confirmed CatSper's role in these activation modes. Our results strongly suggest that PKA-dependent phosphorylation regulates [Ca2+] i homeostasis by activating CatSper channel complexes.

Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm.

Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.