The Role of Sperm Membrane Potential and Ion Channels in Regulating Sperm Function

The family of two-pore domain K+ channels (K2P) is composed of 15 different subunits (K2P1–7, K2P9–10, K2P12–13 and K2P15–18) [113]. They are responsible for a voltage-independent K+-selective leak that is regulated by numerous chemical and physiological stimuli such as pH, mechanical stretch, temperature, membrane phospholipid composition, second messengers or activation of G protein-coupled receptors (GPCRs) [113,131]. These channels play a central role in Em regulation in a wide range of cell types [131]. K2P subunits have a structure unique among K+ channels, with 4 TM, and the functional channel is formed by two 2P dimers, leading, in a similar way to other K+ channels, to a tetramer-like structure [113]. The K2P family is divided in six subfamilies and, among them, the presence of TREK1 (K2P2), TASK2 (K2P5) and TRAAK (K2P4) has been demonstrated in sperm from nonhuman primates [88]. By using an agonist and an antagonist of TRAAK, Chow et al., found that K2P channels participate in the regulation of several kinetic parameters of sperm movement and accelerates AR [88]. Using RT-PCR, Western blot and immunofluorescence, K2P5 has also been detected in human sperm with a major localization around the neck region [89] (Table 2).

As occurs with Kv and Kir, electrophysiological studies have failed to identify the presence of a K2P current in sperm cells. However, there is increasing evidence suggesting the participation of K+ channels other than SLO in the regulation of sperm function. Brown et al. (2016) described an infertile patient whose sperm showed deficient K+ currents but intact genes encoding SLO1 and SLO3 [132] and in pig sperm, the Kv and K2P inhibitor quinine increases progesterone-induced AR more than the specific SLO inhibitor paxilline [133]. The wide expression of different K+ channels highlights the need for more studies to elucidate the mechanisms underlying their regulation and their specific role in human sperm fertilizing capacity [104,132,133].

Sperm intracellular alkalinization is an essential requisite for acquisition of fertilization ability and sperm cells possess different ion channels and transporters that finely regulate intracellular pH. Between them, the voltage-gated proton channel Hv1 plays an essential role in the regulation of pH in human sperm [24,47,117].

Regulation of sperm pHi is also fundamental for capacitation. Babcock et al. (1983) suggested that the mechanism for proton efflux from bovine sperm was via a voltage-gated proton channel, based on the fact that the sperm cytosol becomes alkaline upon membrane depolarization [134]. Voltage-gated proton channels (Hv1) play an important role in the regulation of pH in human spermatozoa. A full-length Hv1 and a truncated isoform, lacking 68 amino acids at the N-terminal and named Hv1sper, have been detected in human sperm, although the functional differences between them are unclear [24,47,69,117]. Hv1 is an unusual member of the VGC superfamily [135]. Encoded by the HVCN1 gene, it was identified in 2006 [136,137] and, until now, its activity has only been demonstrated in human sperm [24,117,138]. In contrast to the high complexity of the rest of the family, which are large proteins with 24 TM segments, Hv1 is formed by two identical dimers, each of them with 2 TM domains, and comprise a voltage sensor -structurally homologous to the voltage-gated sensor domain of the other members of the VGC family, a pore and a gate. The dimers can function autonomously [135,138], but a cooperation between them is required to achieve a correct functionality [135,139]. The opening of these channels can be mediated by depolarization, extracellular alkalinization [139], PKA phosphorylation [76], the endocannabinoid anandamide and by the removal of extracellular zinc [76,117]. When they are in an open state, they mediate a specific outward proton current through the membrane, controlling the intracellular alkalinization of human sperm [117].

Hv1 channels are localized in the principal piece of the human sperm flagellum and their proximity to cation channels such as CatSper [24,140], make them ideally positioned to activate pH-dependent proteins of the axoneme and thus to control sperm motility [117]. However, the specific Hv1 isoform present in humans has not been found in other mammalian species, where the precise mechanisms of regulation of intracellular pH are still unclear.

During capacitation, human sperm acquire hyperactivated motility, necessary to reach the oocyte and undergo AR. Most mammalian sperm, including humans’, undergo two phases during motility acquisition: activation and hyperactivation [10,141,142]. The activation phase initiates upon ejaculation, whereas hyperactivation occurs when capacitation begins [10,128]. Active motility occurs after release from the seminal coagulum, as a result of membrane depolarization. Sperm cells acquire a low amplitude, symmetrical flagellar beat that generates progressive motility. Spermatozoa can then migrate through the female genital tract where they are exposed to an increase in the extracellular [HCO3−] and [Na+] together with a decrease in [K+] [14]. This novel milieu stimulates a higher motility acquisition characterized by a high amplitude, asymmetrical beating pattern of the sperm tail, known as hyperactivation [142,143]. Although no direct evidences exist in humans, it is thought that this asymmetrical, whip-like bending of the flagellum, commonly referred to as hyperactivation, is essential for mammalian sperm to overcome the protective vestments of the oocyte [15,22,144]. Studies in mammalian species other than human have shown that spermatozoa that reach the oviductal isthmus bind to epithelial cells forming a reservoir [15,145,146] and hyperactivation permits their release from the isthmus and their progression towards the oviductal ampulla for fertilization of the egg. Calcium is required to initiate and maintain hyperactivated motility [10,147]. In hyperactive spermatozoa, swimming behavior is controlled by the propagation of a Ca2+-induced wave that changes the flagellar beat pattern. The increase in intracellular [Ca2+] can be induced by a calcium influx through calcium channels and/or by Ca2+ release from intracellular reservoirs [48,147,148,149].

Many authors suggest that an initial influx of the cation activates a Ca2+-induced Ca2+ release (CICR) from sperm intracellular stores, generating intracellular Ca2+ oscillations, which would lead to not only hyperactivation but also AR [10,148]. Mitochondria might also play an important role in Ca2+ buffering and signaling in sperm cells, shaping the kinetics of Ca2+ signals and acting as a sink, in order to buffer cytosolic Ca2+ level [11,48,146,149,150]. An increase in [Ca2+]i is required not only for initiating but also for maintaining hyperactivated motility [10,146,147,151], which can be mediated by the activation of calcium channels [11,152]. (Table 3). The voltage-dependent CatSper channel has been proposed to be the main calcium influx regulator [14], but several studies have shown that CatSper-null mice are able to acquire hyperactivated motility when calcium is released from internal stores [147,149,153,154], suggesting that other voltage-gated calcium channels (VGCCs) may be also involved in the sperm motility hyperactivation (Table 3).

The acrosome reaction consists of the fusion of the outer acrosomal membrane with the sperm plasma membrane in order to release acrosomal enzymes necessary to penetrate the zona pellucida of the egg. Changes in ion permeability are needed to obtain sperm hyperpolarization, which is essential to induce AR [40,181], pointing out the importance of Em regulation for this process to occur. Reinforcing this idea, it has been described that non-capacitated spermatozoa with depolarized membrane potential are unable to undergo AR [181].

The acrosome is a calcium reservoir, and an IP3-mediated calcium release is necessary to complete acrosome exocytosis [42,149]. As several ion channel inhibitors can inhibit AR [182], an initial influx via Ca2+ channels may activate the opening of calcium reservoirs in the sperm neck, then cause CICR-induced acrosomal calcium release, which facilitates Ca2+-regulated membrane fusion through Soluble NSF Attachment Receptors (SNAREs) located in the acrosomal region [42,147,183,184,185]. The fusion between the outer acrosomal membrane and the overlying plasma membrane, at multiple points, would result in vesiculation and the loss of the fused outer acrosomal membrane/plasma membrane with the subsequent exocytosis of acrosomal granules [13].

The cation channel of sperm, CatSper, is a sperm-specific voltage- and pH-dependent calcium channel localized in the principal piece of sperm flagellum [151,162]. CatSper belongs to the family of voltage-gated Ca2+ channels and is the main channel responsible for Ca2+ influx in mammalian sperm [14,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,186,187,188,189,190]. CatSper channels comprise four homologous pore-forming α subunits named CATSPER1, CATSPER2, CATSPER3 and CATSPER4 [15,24,25,151,155,162,166]. Unlike other members of the VGCC family, each CatSper subunit is formed by a single repeat of six TM segments. However, functional activity requires the aggrupation of four monomers of CATSPER1–4 to form the channel pore, resulting in a heterotetramer with a primary structure similar to that of other members of the VGCC family [24,76,156,159]. The channel activity also requires the presence of a large number of auxiliary subunits: the transmembrane proteins CATSPERβ, CATSPERγ CATSPERδ and CATSPERε [156,167,169,171] interact with the adjacent voltage-sensing domain of CATSPER4, 1, 3 and 2, respectively, stabilizing the complex [159] while the cytoplasmic proteins CATSPERζ and EF-hand Ca2+-binding domain-containing protein 9 (EFCAB9) form a complex associated with the cytoplasmic mouth of the channel pore, regulating its opening and closing [158,159,171,172]. The CATSPERζ-EFCAB9 complex acts as a dual Ca2+ and pH sensor [25,172]. Thus, at low pHi, CATSPERζ-EFCAB9 stabilizes the channel which then remains closed at physiological sperm Em [172,187]. The increase in pH induces Ca2+ binding to EFCAB9 causing the dissociation of the CATSPERζ-EFCAB9 complex and permitting the activation of the CatSper channel [76,172]. Very recently, new subunits of this highly complex channel, which has been named CatSpermasome, have been characterized.

Lin et al. (2021) and Huang et al. (2023) described three new components in mouse sperm: the transmembrane protein 249 (TMEM249), now named CATSPERθ, essential for the CatSper channel assembly during sperm tail formation; the transmembrane protein 262 (TMEM262), which has been named CATSPERŋ, and the rodent- and testis-specific solute carrier organic anion transporter SCLO6C1 [159,190]. These proteins interact with other auxiliary transmembrane proteins of CatSper (CATSPERŋ with CATSPERβ and SCLO6C1 with CATSPERε) and contribute to the assembly and stability of the CatSper complex [159]. Yang et al. (2022) and Hwang et al. (2022) discovered an additional subunit, the testis-specific protein C2 calcium-dependent domain-containing protein 6 (C2CD6), which has been named CATSPERτ [173,174]. This cytoplasmic protein interacts with CATSPER1–4 subunits and with EFCAB9 and play an essential role in the localization of the CatSpermosome by regulating its targeting to the sperm flagella in developing spermatids [173,174]. All these data show that the specificity and precise identity of the subunits in different mammalian species and the complete structure of the CatSpermosome is still not completely resolved [158,159,173,174]. This highly complex channel is organized in four linear columns within the sperm flagella membrane in both humans and mice, generating a unique longitudinal signaling nanodomain in each flagellar quadrant [158,168,171,172,173]. This spatial arrangement, which is still far from being completely unraveled, is absolutely necessary for Ca2+ signaling and sperm fertilization ability.

Activation of CatSper requires membrane depolarization and intracellular alkalinization [152,187]. CatSper can also be indirectly sensitized to depolarization, through Hv1 activation and potentiation of intracellular alkalinization [79,188]. The voltage at which half of the CatSper channels are activated in humans is +85 mV versus +11 mV of mouse CatSper at the same pHi (pHi = 7.5) reflecting the profound differences that exist between CatSper channels of different species, in spite of being highly conserved throughout evolution [24,171,187]. In any case, potassium-induced membrane hyperpolarization has been proposed to induce a reduction on CatSper opening [84], suggesting a reciprocal regulation of potassium induced membrane potential alterations and CatSper in order to prevent a premature AR.

CatSper is also sensitive to different activators which vary between species [15,24,76] and insensitive to GPCRs [189,191]. Progesterone at low concentrations and prostaglandins can change the voltage sensitivity of the channel and activate CatSper in human sperm [115,189,192]. In fact, progesterone-induced Ca2+ oscillations in human sperm are generated in the flagellum principal piece by Em-sensitive activity of CatSper [152].

The crucial role of CatSper is corroborated by the important alterations produced in sperm function as a consequence of mutations in any of the subunits that forms this complex channel (Table 3). In mice, congenital ablation of CatSper1, CatSper2, CatSper3 or CatSper4 genes inhibits CatSper currents and sperm hyperactivation and causes infertility, although spermatogenesis and initial motility are unaffected [150,151,155,161,162,168]. Sperm from these null mice are unable to fertilize oocytes surrounded by ZP demonstrating that CatSper are necessary to penetrate the zona pellucida [76]. Similarly, CatSperd−/− male mice, lacking the gene that encodes CATSPER∂, are infertile and their sperm do not express Catsper channels [156]. On the contrary, CatSperz−/− and Efcab9−/− mice lines or the double CatSperz/Efcab9 KO mice show severe subfertility but a functional, although disorganized, channel [158,171,172]. Sperm from either single or double-null males show apparently normal morphology and progressive motility but were unable to hyperactivate [171,172]. The loss of EFCAB9-CATSPERζ largely eliminates the pH-dependent activation of CatSper and impairs its regulation by intracellular Ca2+, providing further evidence for the involvement of this dual complex in the modulation of Ca2+ influx mechanisms of CatSper channels [158,172]. The strong alterations produced in the architecture of the channel complex by the loss of CATSPERζ-EFCAB9 suggest an additional structural role for these two subunits [158,171,172]. With respect to the new subunit CATSPERτ, C2cd6−/− mice show a disorganized but functional CatSper channel, with a highly reduced conductance [173,174]. C2cd6−/− mice have an apparently normal spermatogenesis, and spermatozoa with normal morphology and progressive motility.

However, sperm cells from these mice are unable to acquire hyperactivated motility and to fertilize eggs, and are therefore infertile [173,174].

Studies in human patients have also been able to detect several deletions and/or mutations in the genes encoding different CATSPER subunits and their association with infertility [24,157,163,164,165,170] (Table 3).

Although Ca2+ influx is proposed to be mainly dependent on CatSper in spermatozoa, it may also be mediated by classic voltage-gated calcium channels, Cav, which are also present in these cells of different species [10,48,193,194]. VGCCs belong to the of VGC superfamily and consist of a pore-containing α subunit, named α1, and one or more auxiliary subunits. The α subunit shows the typical structure of VGC members and is formed by a four repeat of 6 TM segments in a single protein [25,66]. There are ten different α1 subunits and, depending on which of them is present, Cav have different opening characteristics and can be classified into two functional groups: high-voltage activated (HVA) and low-voltage activated (LVA) channels [10,66,194]. HVA channels can be grouped in L-Type (Cav1.1–Cav1.4), P/Q-Type (Cav2.1), N-Type (Cav2.2) and R-Type (Cav2.3) whereas LVA channels comprise T-Type channels (Cav3.1–Cav3.3) [48,66,193,194]. With respect to auxiliary subunits, four α2∂ (α2∂1–α2∂4), four β (β1–β4) and eight different γ subunits are currently known. The number and identity of the associated auxiliary subunits depend on the type of Cav channel [66,195].

VGCCs are expressed in sperm from a wide number of species [25,48,69,175,176,177,178,179,180,193,194]. Among them, Cav1.2, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2 and Cav3.3 transcripts have been identified in mature human spermatozoa [160,177] and the presence of the auxiliary subunits α2∂, β1, β3 and β4 has also been reported [180] (Table 3).

Patch clamp studies have not revealed Cav1 or Cav2 currents in spermatogenic cells [10]. In contrast, Cav3 currents have been well documented in spermatogenic cells by patch clamp experiments [10,176], probably because weak depolarizations are needed to open Cav3 channels in comparison to Cav1 and Cav2 isoforms, that need high depolarizations. Electrophysiological recordings have demonstrated a gradual decrease in Cav3 currents during spermiogenesis, which become undetectable in epididymal sperm [24]. The blockade of L-type (Cav1) or T-type (Cav3) channels caused an inhibition of ZP3-induced Ca2+ influx, leading to AR [180], suggesting that these channels may be present in an electrophysiologically inactive state and be activated during sperm differentiation and maturation [34]. In fact, Cav3.3 colocalizes with CatSper1 in the principal piece of the human flagellum and this association inhibits the T-type Ca2+ current, measured in CATSPER1 and Cav3.3 co-transfected mammalian HEK cells [160]. In addition, several authors have reported that capacitation-induced hyperpolarization and its downstream physiological changes are needed to remove Cav from an inactive state, which generates a voltage-dependent Ca2+ influx, proving that Cav channels may play a role in modulating AR [34,194].

Several ion channels are involved in the correct functioning of sperm cells and their maturation. Thus, ion channel dysfunctions, as well as membrane potential alterations, have important consequences for fertilization. Mutations in different ion channels could be responsible for Em alterations in infertile patients and several ion channel dysfunctions have been associated with subfertility/infertility and related pathologies. The study of defective ion channels would lead to both, determining the origin of some infertility cases and the comprehension of the role of ion channels. Many studies have reported that depolarized human sperm membrane potential values are associated with lower fertilization rates [41,132] and asthenozoospermia [196]. A depolarized membrane potential can also inhibit the progesterone-induced acrosome reaction [65], and accordingly, more hyperpolarized resting Em (−75 to −35 mV) has been associated with fertile men compared with sperm from infertile men (−35 to −10 mV) [132,196].

Alterations in sodium currents have also been associated with altered semen parameters. Zhang et al., demonstrated that the expression of the Na+/H+ exchanger sNHE is downregulated in asthenozoospermic men [197]. Moreover, Wang et al. (2003) showed that sNHE-null mice were completely infertile with severely reduced sperm motility [198], which could be partially rescued by raising intracellular pH [24], probing the role of sodium channels in the maintenance of sperm motility.

Several studies in spermatozoa from infertile men have also highlighted the importance of potassium conductance in the achievement of human fertility. Brown et al. (2016) showed that 10% of the IVF and ICSI patients with subfertility had abnormalities in K+ conductance, generating a depolarization of the sperm plasma membrane [132]. Depolarization of the plasma membrane has been associated with low fertility rates and several K+ channels seem to be involved in this process. For example, SLO3 mutant mice are infertile, and they suffer membrane depolarization during capacitation [92]. Mice with congenital ablation of Kir5.1 are subfertile [107]. In addition, Inanobe et al. (1999) found that Kir3.2d mutant mice are infertile and they propose a role for this channel in spermatogenesis [106]. More studies need to be carried out in human sperm to elucidate the potassium channels involved in this process.

It has been described that CATSPER1 and CATSPER2 mutations in human sperm are associated with infertility [157,165]. Semen parameters from CatSper defective men are consistent with asthenoteratozoospermia and, therefore, abnormalities in human CatSper lead to in vitro fertilization failures [199] which may also involve a participation of other voltage-gated calcium channels [160]. Studies in human patients have been able to detect several deletions and/or mutations in different CATSPER subunits, i.e., CATSPER1, CATSPER2, CATSPER3 or CATSPERE and their association with infertility [24,157,163,164,165,170].

The second most common male factor infertility cause is a ductal obstruction or dysfunction, which is produced by CFTR mutations [200]. Defective CFTR exchangers in human sperm alter Cl− and HCO3− transport compromising human sperm fertilizing capacity [15,81]. It has recently been reported that there is a functional interaction between CFTR and SLC26A3—a solute carrier of the SCL26 family—which drives sperm capacitation [201]. The role of SLC26A8 has not been characterized in human sperm, but a nonsense mutation in this protein can cause asthenozoospermia [202]. Moreover, mutations in SLC26A3 have been associated with subfertility and asthenozoospermia [203,204].

To fully understand the effect of sperm ion channel dysfunctions and their association with infertility, the role of ion channels and transporters in human sperm has to be deeply studied. With a better comprehension of the ion channel operation system, the description of new infertility etiologies will be elucidated, and idiopathic infertility could be reduced.

The control of sperm Em is a complex process that involves the activation of a great number of ion channels, transporters and protein receptors present in the sperm surface. Between them, ion channels play a central role in the maintenance of sperm physiology and the achievement of a successful fertilization. The introduction of electrophysiological techniques has permitted a great advance in our knowledge of the role of ion channels, particularly CatSper, KSper and Hv1, and their important participation in different sperm maturation processes. However, although a large, increasing number of ion channels have been detected in spermatozoa, only a few of them have been functionally characterized in electrophysiological studies, probably reflecting the complex nature of this highly polarized cell and the difficulty of performing patch clamp studies in regions other than the flagellum. Important advances have also been produced by using ion-channel-null mice models, or by the finding of mutations in specific human genes and their association with male infertility. However, in many cases, men infertility is not associated with mutations in a certain gene, but is acquired throughout life, due to age, environmental conditions or other unknown factors. It must be considered that, in the particular case of humans, studies are performed in mature spermatozoa, following ejaculation and, in many cases, after a selection of the more motile cells present in the semen sample, thus precluding an analysis of the precise role of different sperm proteins. At this point, a better understanding of the physiological role of sperm ion channel activities appears essential to discover the mechanisms underlying a successful fertilization.