Sperm ion channels and transporters in male fertility and infertility

Huafeng Wang; Luke L McGoldrick; Jean-Ju Chung

doi:10.1038/s41585-020-00390-9

. Author manuscript; available in PMC: 2022 Jan 1.

Published in final edited form as: Nat Rev Urol. 2020 Nov 19;18(1):46–66. doi: 10.1038/s41585-020-00390-9

Sperm ion channels and transporters in male fertility and infertility

Huafeng Wang ¹, Luke L McGoldrick ¹, Jean-Ju Chung ^1,²

PMCID: PMC7852504 NIHMSID: NIHMS1664850 PMID: 33214707

Abstract

Mammalian sperm cells must respond to cues originating from along the female reproductive tract and from the layers of the egg in order to complete their fertilization journey. Dynamic regulation of ion signalling is, therefore, essential for sperm cells to adapt to their constantly changing environment. Over the past 15 years, direct electrophysiological recordings together with genetically modified mouse models and human genetics have confirmed the importance of ion channels, including the principal Ca²⁺-selective plasma membrane ion channel CatSper, for sperm activity. Sperm ion channels and membrane receptors are attractive targets for both the development of contraceptives and infertility treatment drugs. Furthermore, in this era of assisted reproductive technologies, understanding the signalling processes implicated in defective sperm function, particularly those arising from genetic abnormalities, is of the utmost importance not only for the development of infertility treatments but also to assess the overall health of a patient and his children. Future studies to improve reproductive health care and overall health care as a function of the ability to reproduce should include identification and analyses of gene variants that underlie human infertility and research into fertility-related molecules.

Regulation of ion balance is essential for sperm motility and fertility. In particular, ions pass through channels at least 1,000 times faster than through transporters¹. Thus, ion channels enable sperm to rapidly respond to guidance cues in the female reproductive tract. Calcium (Ca²⁺) influx through Ca²⁺-permeable ion channels can affect cell signalling by altering local electrostatic fields and protein conformations². The speed and effectiveness of Ca²⁺ signalling is a consequence of a more than ~10,000-fold gradient maintained across the cell plasma membrane; the intracellular Ca²⁺ concentration (10–100 nM free) is, therefore, low when compared with the extracellular concentration (~1–2 mM)^2,3.

Ca²⁺ signalling is a conserved mechanism to modulate cell motility by increasing flagellar asymmetry. Ca²⁺ influx is required for altering flagellar waveform in Chlamydomonas⁴, the steering and turning of sperm from sea urchins^5,6 and fish⁷, and sperm hyperactivation in mammals⁸. The axoneme of motile cilia and flagella have Ca²⁺-binding sites that regulate flagellar curvature by modulating the motor protein dynein ATPase^9-12. Although various molecular mechanisms can chelate, compartmentalize or extrude Ca²⁺ upon its entry into the cytosol², specialized Ca²⁺-selective ion channels are the only Ca²⁺ entry sites in the sperm flagella^13,14.

In mammals, flagellar Ca²⁺ entry is facilitated by the cation channel of sperm (CatSper), the sperm-specific Ca²⁺ channel complex^13,15-18 (FIG. 1). CatSper-dependent Ca²⁺ entry induces hyperactivated motility during sperm capacitation^16,17. Hyperactivated motility is the swimming pattern observed in most sperm retrieved from the oviductal ampulla at the time of fertilization and is characterized by a deep and asymmetrical flagellar bend^8,19,20. Sperm hyperactivation helps to free sperm cells from the oviductal epithelium^21,22, to facilitate their upstream progression²³, and to penetrate the zona pellucida to fuse with the egg¹⁵. Specific signals, such as progesterone and other secretion factors, which are present within the oviduct around ovulation, stimulate hyperactivation of bovine and human sperm^24,25. Studies have suggested that human CatSper functions as a polymodal sensor that translates physical and chemical cues in the reproductive tract into a Ca²⁺ response^23,26. Thus, elucidating the molecular regulatory mechanisms by which CatSper is regulated is fundamentally important to understanding sperm hyperactivation and mammalian fertilization.

Within the past two decades, the development of genetically modified mouse models and the application of direct electrophysiological recordings have improved our understanding of the molecular basis of mammalian sperm sensory signalling²⁷. Species-specific variations in sperm signalling such as differences in overall receptor expression, molecular composition, and/or regulatory mode of primary ion channels have also been revealed²⁸. Furthermore, application of state-of-the-art techniques such as super-resolution imaging^29-32, imaging flow cytometry^33-35 and cryo-electron tomography^36-38 to sperm cells has begun to more directly inform the molecular and structural bases of sperm motility. Thus, the signal transduction pathways that lead to the mechanical transitions in the axoneme to regulate sperm motility have, at least partially, been revealed.

In this Review, we explore the literature regarding ion channel signalling in the context of regulating mammalian sperm motility and male fertility, focusing on sperm ion channels and membrane transporters for which genetic and/or electrophysiological evidence is available to support their roles in male fertility in mice and humans. We summarize important findings, current controversies and challenges, and provide insights into molecular mechanisms, future perspectives and clinical developments in the field.

Sperm intracellular alkalinization

Mammalian sperm remain quiescent during maturation and storage in the acidic luminal environment of the epididymis (pH 6.6–6.8)^39-41. Variations in pH along the female reproductive tract is one of the physiological cues that stimulates sperm capacitation, including hyperactivated motility and the acrosome reaction⁴². In humans, the pH of luminal fluid in the female reproductive tract increases gradually from the vagina (pH ~4.4) towards the cervix (pH 6.5–7.5); the pH of cervical mucus is in the range 5.4–8.5 and the pH of the uterus is >7 (REFS^43,44). The luminal pH is normally highest in the fallopian tubes (73–7.7 in humans and ~7.9 in pigs and rabbits)⁴³. Thus, spermatozoa encounter a drastic extracellular pH change during their journey through the female reproductive tract. Upon ejaculation, human spermatozoa are mixed with seminal plasma (pH 7.2–8.4), which alkalinizes the acidic vaginal environment^45,46. The optimal pH for sperm motility is in the range 7.0–8.5 in bulls⁴⁷ and humans⁴⁸, which is consistent with the pH of the oviductal fluid. Intracellular alkalinization of sperm cells during capacitation can be caused by HCO₃⁻ uptake from the fluid in the female reproductive tract through HCO₃⁻ transporters and extrusion of H⁺ through proton carriers (FIG. 1 a).

Proton carriers

Carrier-mediated mechanisms constitute the major route for proton transport across the plasma membrane in sperm³⁹. Membrane transporters such as sodium–hydrogen exchangers (NHEs) and the voltage-gated proton-selective ion channel HV1 are the best-studied examples.

Sodium–hydrogen exchangers.

NHEs, encoded by the Slc9 gene family, transport Na⁺ into cells and H⁺ out of cells⁴⁹, thereby regulating intracellular pH (FIG. 1a). NHE1 (REF.⁵⁰), NHE5 (REF.⁵¹) and NHE8 (REF.⁵²) are expressed in multiple tissue types in mammals, including the testis. However, two NHEs — sNHE (encoded by Slc9c1)^53,54 and NHA1 (encoded by Slc9b1)⁵⁴ — are expressed specifically in sperm flagellum. Gene knockout studies in mice have demonstrated the importance of pH regulation by NHEs in sperm physiology (TABLE 1). In these studies, loss of sNHE⁵³ or NHA1 and NHA2 (encoded by Slc9b2)⁵⁵ together caused male infertility and knockout of NHE8 (encoded by Slc9a8) in male germ cells resulted in defects in acrosome formation and male infertility⁵⁶. However, the extent of the contribution made by each NHE in controlling intracellular pH in mouse sperm is not clear. For example, impaired sperm motility in sNHE-deficient sperm was only partially rescued by artificial alkalinization but was completely rescued by administration of cyclic AMP (cAMP) analogues⁵³. The intracellular cAMP levels and protein expression of soluble adenylyl cyclase (sAC) were attenuated in the absence of sNHE, NHA1 and NHA2 (REFS^55,57), suggesting a functional redundancy in NHEs for sAC expression and/or their relationship with cAMP signalling pathways to facilitate sperm motility regulation. In sea urchins, sNHE can control intracellular pH homeostasis more rapidly than typical transporters by responding to hyperpolarization and cAMP⁵⁸. As no other NHE has been reported in the sea urchin genome, sNHE might act as a solo voltage-dependent NHE. Whether other, as-yet-uncharacterized, NHEs are involved in pH regulation in mammalian sperm is unknown.

Table 1 ∣.

Genetic studies of sperm ion channels and membrane transporters implicated in male fertility

Gene	Tissue expression	Protein localization in sperm	Phenotypes of mutation	Species	Refs
*Ion channels*
Kcnu1	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: impaired In vivo fertility: infertile	Knockout mouse	^70,84
Lrrc52	Testis	ND	Spermatogenesis: ND Hyperactivated motility: ND In vitro fertilization: impaired In vivo fertility: subfertile	Knockout mouse	⁸⁶
Catsper1	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: impaired In vivo fertility: infertile	Knockout mouse	^15,16
CATSPER1	Testis	Principal piece	Spermatogenesis: impaired Motility^a: reduced In vitro fertilization: ND In vivo fertility: infertile	Human^b	^100b
Catsper2	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: ND In vivo fertility: infertile	Knockout mouse	¹⁷
CATSPER2	Testis	Principal piece	Spermatogenesis: impaired Motility^a: reduced In vitro fertilization: ND In vivo fertility: infertile	Human^c,d	^{102c; 103c; 104d;111c;216c}
Catsper3	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: ND In vivo fertility: infertile	Knockout mouse	^18,95
Catsper4	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: ND In vivo fertility: infertile	Knockout mouse	^18,95
CATSPERE	Testis	Principal piece^e	Spermatogenesis: normal Motility^a: normal In vitro fertilization: impaired In vivo fertility: infertile	Human^f	^29e;106f
Catsperz	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: impaired In vivo fertility: subfertile	Knockout mouse	²⁹
Cacna1e	Ubiquitous	Head	Spermatogenesis: ND Hyperactivated motility: normal In vitro fertilization: impaired In vivo fertility: mildly subfertile	Knockout mouse	^160,161
CFTR	Multiple	Head/midpiece	Spermatogenesis: impaired Hyperactivated motility: ND In vitro fertilization: impaired In vivo fertility: impaired	Human, knockout mouse	^{178,180-183,188,251}
Pkdrej	Testis	Head^e	Spermatogenesis: normal Hyperactivated motility: normal but slow In vitro fertilization: ND In vivo fertility: fertile	Knockout mouse	^{171,172e; 252}
Trpv4	Ubiquitous	Head and tail	Spermatogenesis: normal Hyperactivated motility: normal but delayed In vitro fertilization: ND In vivo fertility: fertile	Knockout mouse	^{147,151,152,253}
Membrane transporters
Atp1a4	Testis	Principal piece	Spermatogenesis: normal Hyperactivated motility: reduced In vitro fertilization: impaired In vivo fertility: infertile	Knockout mouse	⁸²
Slc9a8	Ubiquitous	Head (acrosome)	Spermatogenesis: impaired Motility^a: reduced In vitro fertilization: ND In vivo fertility: infertile	Knockout mouse	⁵⁶
Slc9b1	Testis	Principal piece	Spermatogenesis: normal Motility^a: reduced In vitro fertilization: ND In vivo fertility: subfertile^g	Knockout mouse	⁵⁵
Slc9b2	Ubiquitous	Principal piece	Spermatogenesis: normal Motility^a: reduced In vitro fertilization: ND In vivo fertility: subfertile^g	Knockout mouse	⁵⁵
Slc9c1	Testis	Principal piece	Spermatogenesis: normal Motility^a: reduced In vitro fertilization: impaired In vivo fertility: infertile	Knockout mouse	⁵³
P2rx2	Ubiquitous	Midpiece	Spermatogenesis: normal Hyperactivated motility: normal In vitro fertilization: normal In vivo fertility: mildly subfertile	Knockout mouse	¹⁵⁴
Cnnm4	Ubiquitous	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: impaired In vivo fertility: subfertile	Knockout mouse	¹⁹⁵
Atp2b4	Ubiquitous	Principal piece	Spermatogenesis: normal Hyperactivated motility: impaired In vitro fertilization: normal In vivo fertility: infertile	Knockout mouse	^191-193
SLC26A3	Multiple	Head/midpiece	Spermatogenesis: ND Hyperactivated motility: ND In vitro fertilization: ND In vivo fertility: subfertile	Human, knockout mouse	^182-185,254

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ND, Not determined.

Motility was tested in the standard bath solution without capacitation components.

Two separate insertion mutations in exon 1 (c.539–540insT and c.948–949insATGGC).

Deaf infertility syndrome with a deletion encompassing CATSPEP2 and STRC together.

Copy number variation in the region of 43894500 to 43950000 in 15q 15.3 encompassing a heterozygous deletion of CATSPEP2.

Protein localization information only.

Homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG).

Slc9bl/Slc9b2-double-knockout mice are completely infertile.

Proton channels.

The HV1 proton channel has been suggested to control human sperm intracellular pH⁵⁹. HV1 is homologous with the voltage sensor domain (VSD) of voltage-gated channels and functions as a dimeric channel complex, but does not contain a separate pore domain^60,61. Molecular dynamic simulations of HV1 homology models suggest that HV1 probably contains an internal water wire within the central crevice of the VSD for selective proton transfer⁶². Full-length HV1 and N-terminal cleaved HV1_Sper have been detected in human sperm⁶³. Both channels exhibit voltage-dependent activation, which requires a pH difference across the membrane (ΔpH), and are inhibited by zinc^59,63. The voltage dependence of heterologously expressed HV1_Sper is also affected by simultaneous changes in intracellular and extracellular pH. Electrophysiological recordings of human sperm have not been able to distinguish HV1_Sper from HV1 current⁶³; thus, uncovering the molecular mechanisms by which HV1 and HV1_Sper regulate the pH of human sperm requires further studies. Such studies would rely on human genetic evidence because HV1 is absent in mouse sperm⁵⁹. Interestingly, a 2018 study found that the HV1 channels are arranged in bilateral lines along one side of the flagellar membrane³², which suggests that the combined effect of H⁺ efflux through many HV1 channels alters the local intraflagellar pH and, in doing so, can regulate pH-sensitive molecules.

Intracellular alkalinization has been predicted to precede membrane potential hyperpolarization and Ca²⁺ signalling in mouse and human spermatozoa after ejaculation^13,64-67. Thus, understanding the molecular mechanism of sperm pH regulation and sensing is crucial. HV1 and sNHE are both confined to the principal piece of the flagella^53,59. Interestingly, the ion channels CatSper and KSper (sperm-specific K⁺ channel), which are both pH-sensitive, are also found in the principal piece (FIG. 1a), suggesting compartmentalized pH regulation and sensing machineries.

Membrane potential hyperpolarization

Electrochemical gradients across cellular plasma membranes are generally maintained by Na⁺/K⁺ ATPase⁶⁸. Resting membrane potential is primarily set by K⁺ channels and is typically about −70 mV in somatic cells, including nerve fibres, in the absence of excitation⁶⁹. By comparison, it is less polarized in mammalian spermatozoa (for example, about −40 mV in mouse and human sperm)^64,65,70. During capacitation, the sperm membrane hyperpolarizes — K⁺ efflux through activated KSper is primarily responsible for this change in membrane potential⁶⁴ (FIG. 1a). In turn, hyperpolarization regulates various membrane proteins, including the voltage-gated proton channel HV1, ion exchangers and Ca²⁺ channels. Abnormal depolarization of membrane potential might be associated with human male subfertility^71-74; thus, understanding the molecular interactions and regulatory mechanisms of Na⁺/K⁺ exchange and KSper is critical to understanding sperm physiology. Genetic evidence regarding which mutations in genes encoding Na⁺/K⁺ ATPases and KSper are associated with fertility defects in men remains to be collected.

Na⁺/K⁺ transporters

Na⁺/K⁺ ATPase transporters contribute to the regulation of membrane potential owing to unequal exchange of cytoplasmic Na⁺ for extracellular K⁺ (REF.⁶⁸). The transporter consists of a group of isozymes that contain α-subunits and β-subunits. The catalytic α-subunit of Na⁺/K⁺ ATPase facilitates ion permeation of the plasma membrane⁶⁸. The α1 subunit is expressed in a variety of tissue and cell types in humans and rats, including sperm^75,76; however, the α4 subunit is specific to male germ cells⁷⁶. When rat or human α4 subunit is expressed in transgenic mice, it is particularly abundantly expressed in the sperm flagellum, which is consistent with its expression in wild-type rat and human sperm^77,78. Treatment of rat sperm with a low concentration of ouabain (which selectively inhibits α4) increased intracellular Na⁺, depolarized the membrane potential, increased intracellular calcium and decreased intracellular pH⁷⁹. Because α4 does not directly transport H⁺, it has been proposed that α4 provides electrochemical energy that facilitates pH regulation by NHEs^77,80. In particular, sNHE has a putative voltage sensor⁵³ and so could potentially be regulated by an α4-established membrane potential. Measuring intracellular pH under various transmembrane sodium gradients will help to test this possibility.

The potential contribution of the α1 subunit to sperm function remains to be clarified, as α1 subunit knockout is embryonically lethal in mice⁸¹. By contrast, disruption of Atp1α4, which encodes the α4 subunit, in mice causes complete sterility⁸² (TABLE 1), demonstrating that α4-mediated ion transport is critical for sperm function. Furthermore, Na⁺/K⁺ ATPase α4-deficient sperm from the knockout mice are severely bent at the junction of the midpiece and the principal piece, exhibit increased intracellular Na⁺ levels and have depolarized membrane potentials, consistent with the phenotypes observed in mouse sperm with altered osmoregulation⁸³. Contrastingly, transgenic mouse sperm that express functional rat Atp1α4 exhibit increased membrane hyperpolarization and also demonstrate increased total sperm motility and hyperactivated motility⁷⁷; these effects starkly contrast those observed in the Atp1α4-disrupted mice that are completely sterile⁸², demonstrating the importance of functional ATP1A4 in sperm motility and fertility. Expression of human ATP1A4 in transgenic mice results in similar motility phenotypes but not in a significant change in sperm membrane potential⁷⁸. The expression of rat or human α4 does not affect the acrosome reaction in transgenic mouse sperm^77,78. These studies demonstrate that α4 activity primarily maintains sperm intracellular Na⁺ levels and contributes to setting membrane potential. As a result, α4 affects several vital parameters, such as intracellular pH and Ca²⁺, which are essential for sperm motility and hyperactivation.

The KSper channel

KSper was first recorded in mouse sperm in 2007 (REF⁶⁴). Upon intracellular alkalinization, KSper is activated and further hyperpolarizes the spermatozoan cellular membrane (FIG. 1a). Genetic disruption of Kcnu1 demonstrated that SLO3, the pore-forming α-subunit, mediates KSper in mouse sperm^70,84 (TABLE 1). Despite their normal morphology and motility, mouse spermatozoa lacking SLO3 are infertile owing to impaired hyperpolarization during capacitation. However, a residual K⁺ current has been observed in Kcnu1-knockout sperm at very positive potentials⁸⁴, which suggests that another voltage-gated K⁺ current might exist in mouse sperm. However, a subsequent study clarified that all voltage-gated outward current is abolished in sperm of Kcnu1/Catsper1-double-knockout mice¹⁴, demonstrating that the residual K⁺ current observed in Kcnu1-knockout sperm is caused by K⁺ efflux through CatSper and provides evidence that KSper and CatSper are the sole mediators of voltage-dependent K⁺ and Ca²⁺ currents, respectively, in uncapacitated mouse epididymal sperm in response to alkalinization. Heterologous expression of leucine-rich repeat-containing protein 52 (LRRC52), an auxiliary subunit of SLO3, modulates SLO3 gating by shifting its voltage and pH dependence to more negative values, close to those of native KSper currents^85,86. Consistently, upon deletion of Lrrc52, the activity of KSper at rest is attenuated and its activation requires increasingly positive voltages and higher pH, resulting in a more depolarized membrane potential⁸⁶. Abrogation of LRRC52 results in male subfertility and reduced litter sizes⁸⁶ (TABLE 1), supporting the association of this abnormal depolarization of membrane potential with male subfertility in humans^71-74.

Native human KSper (hKSper), unlike mouse KSper, is sensitive to both intracellular alkalinization^65,87 and calcium^65,88. It exhibits Ca²⁺ sensitivity in the same manner as another SLO family member SLO1 (also known as BK channel and encoded by Kcnma1), and pH sensitivity in the same manner as SLO3. These unusual characteristics — whereby hKSper displays hybrid characteristics of the mouse counterpart, SLO3, which is pH-sensitive, and SLO1, which is Ca²⁺-sensitive — have resulted in controversy regarding the exact molecular composition of hKSper. However, studies have shown that heterologous expression of human SLO3 in Xenopus and 293T cells does give rise to currents that exhibit the properties of native hKSper in human sperm^65,89,90 — that is, activation by Ca²⁺ and alkalinization, inhibition by progesterone, and a single-channel conductance of 70 pS (REF.⁶⁵). Moreover, SLO3 and LRRC52 were identified in human sperm by mass spectrometry⁶⁵, further supporting the view that SLO3 and LRRC52 comprise hKSper. A 2017 study showed that a single-nucleotide polymorphism of human SLO3 (C382R) can endow the channel with enhanced pH and Ca²⁺ sensitivities⁸⁹, suggesting that species-specific SLO3 variants could have acquired different Ca²⁺ sensitivity. KSper sensitivity to intracellular pH and Ca²⁺ might define its role in mouse and human sperm: in mouse sperm, the KSper-dependent, capacitation-associated membrane hyperpolarization is an upstream signalling event that increases the force driving Ca²⁺ influx through CatSper⁹¹; in humans, KSper probably functions downstream of CatSper because activation of hKSper requires an increase in cytosolic Ca²⁺ (REF⁹²); KSper-induced hyperpolarization would further affect CatSper. An improved understanding of how sperm membrane potential is regulated during capacitation will provide insights into species-specific fertilization processes.

Ca²⁺ influx and signal transduction

The intracellular Ca²⁺ increase in sperm, which is required for hyperactivated motility and the acrosome reaction, originates primarily from influx of extracellular Ca²⁺; in these specialized cells, the organelles that are typically used for intracellular Ca²⁺ storage and release, such as the endoplasmic reticulum, are less developed than in somatic cells. Mouse spermatogenic cells possess T-type Ca_v channels⁹³, but electrophysiological recordings have demonstrated that Ca_v currents gradually decrease during spermiogenesis and become undetectable in epididymal sperm⁹⁴, suggesting that Ca_v currents are only required during spermatogenesis and not in mature sperm cells. So far, CatSper is the only Ca²⁺ channel in which genetic mutations have been reported to cause male infertility. Thus, the flagellar-specific Ca²⁺ channel CatSper is a predominant pathway by which Ca²⁺ enters mammalian sperm.

Molecular organization of CatSper

CatSper was first identified in 2001 as a gene (Catsper1) encoding a putative sperm Ca²⁺ channel with a single repeat of six transmembrane domains in human and mouse testis¹⁵. The topology of CATSPER1 is unlike that of conventional voltage-gated Ca²⁺ channels, which are typically composed of four repeats of six transmembrane domains. Subsequently, Catsper2 (REF¹⁷), Catsper3 and Catsper4 (REFS^18,95) were identified. CatSper currents are absent in mouse sperm in which one subunit (Catsper1, Catsper2, Catsper3 or Catsper4) has been knocked out¹⁸, indicating that the CatSper pore is a heterotetramer composed of CATSPER1–4 and that one of each is required for channel function. In addition, CatSper channels comprise at least six additional subunits that do not form the pore (FIG. 1b): four transmembrane proteins that are predicted to contain large extracellular domains (ECDs), denoted CATSPERβ⁹⁶, CATSPERγ⁹⁷, CATSPERδ⁹⁸ and CATSPERε²⁹; and two small cytoplasmic proteins, CATSPERζ²⁹ and EF-hand calcium-binding domain-containing protein 9 (EFCAB9)³¹. CatSper is the most complex ion channel known^31,98. Knocking out any one of the four genes that encode CATSPER1–4 in mice renders males infertile^15,18,95,99 (TABLE 1) and CATSPER loss-of-function mutations have also been identified in infertile men^100-104. Sperm cells in which Catsper1, Catsper2, Catsper3 or Catsper4 has been knocked out fail to develop hyperactivated motility^16-18,99, which is consistent with the inability of Catsper1-null sperm to fertilize oocytes with an intact zona pellucida¹⁵. Direct whole-cell patch clamp studies of spermatozoa have demonstrated that CatSper is Ca²⁺-selective channel activated by intracellular alkalinization^13,67.

Deletion of Catsperd, which encodes one of the nonpore-forming transmembrane subunits, CATSPERδ, also abrogates CatSper current and hyperactivated motility, resulting in infertility in male mice⁹⁸ (TABLE 1). This phenotype arises from the loss of not only CATSPERδ but also each transmembrane domain-containing CATSPER protein in sperm cells of Catsperd-null males, and is similar to the all-or-nothing expression pattern of the CatSper pore-forming subunits^29-31,98. By contrast, knocking out Catsperz and/or Efcab9 does not completely eliminate the formation of functional CatSper channels and correspondingly results in male subfertility^29,31 (TABLE 1). Consistently, reduced CATSPERζ protein expression has been reported in men with asthenozoospermia¹⁰⁵, suggesting that these two non-transmembrane subunits, CATSPERζ and EFCAB9, function as true auxiliary subunits that could modulate CatSper expression levels and/or channel kinetics. Interestingly, Catsperz orthologues have been found only in mammals, implying that its regulatory function might be specific to mammalian CatSper channels²⁹. Direct analyses of the functions of the other non-pore-forming transmembrane subunits, CATSPERβ, CATSPERγ and CATSPERε await their investigation using genetic disruption in mice and/or the discovery of human genetic abnormalities related to these proteins.

Spatial organization of CatSper

Flagellar Ca²⁺ signalling nanodomains.

Super-resolution microscopy has been used to demonstrate that the macromolecular CatSper channel forms four linear (also called quadrilateral) Ca²⁺ signalling nanodomains along the sperm tails in both mice and humans^29-31 (FIGS 1,2) and organizes a network of intracellular signalling molecules such as calmodulin-dependent protein kinase II (CaMKII) and calcineurin³⁰. Compartmentalized domains enable specific and fast-triggering downstream events and are common cellular adaptations for effective Ca²⁺ signalling in many biological systems². A 2019 study in mice demonstrated that each of the four CatSper nanodomains is further resolved into two row structures³¹ (FIG. 2b). CATSPERζ and/or EFCAB9 deficiency disrupts not only the linearity of the CatSper nanodomains but also the two-row organization, renders the proximal sperm flagella rigid and alters sperm motility^29,31. As a result, the presence and integrity of these nanodomains serves as an indicator of fertilizing capability. These studies also indicate that CATSPERζ and EFCAB9 regulate the compartmentalization of Ca²⁺ signalling in mammalian sperm, and might, therefore, modulate the mechanism by which CatSper facilitates Ca²⁺ influx.

Fig. 2 ∣ — a ∣ 3D stochastic optical reconstruction microscopy (STORM) images. Distributions of CATSPER1 (mouse, upper) and CATSPERε (human, lower) suggest conservation of the CatSper nanodomains in mammalian sperm. b ∣ 3D 4Pi single-molecule switching nanoscopy (SMSN) images of CATSPER1 in mouse sperm. Note that each nanodomain is further resolved into two row structures. The colour encodes the relative distance from the focal plane along the z axis (color scale bar in x–y projection). Adapted from (REF.²⁹), CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/).

CatSper assembly and trafficking to the flagellar membrane.

Despite the absolute requirement of the CatSper channel for male fertility and the importance of CatSper nanodomain formation for sperm Ca²⁺ signalling, knowledge of how CatSper is assembled and organized into nanodomains is limited. This lack is largely due to an inability to heterologously reconstitute CatSper under laboratory conditions. Although the timing of translation of CatSper subunits is not known, mouse CATSPERδ has been suggested to assist the assembly of the CatSper channel in the endoplasmic reticulum or the Golgi, as CATSPER1 is degraded in its absence⁹⁸. CATSPERδ and CATSPERε might dimerize, as they have a region of high homology at their C terminus²⁹. In 2018, a 6-bp in-frame deletion within CATSPERE in an infertile man was reported; the sperm cells of this patient did not respond to progesterone to elicit CatSper-mediated Ca²⁺ increase and in vitro fertilization (IVF) failed^106,107. This alteration occurred within the region of CATSPERε that exhibits high homology with CATSPERδ, suggesting that CATSPERε might also have a role in the assembly of the large CatSper complex together with CATSPERδ. Studies in mice have suggested that only a correctly assembled CatSper channel, probably with all of its transmembrane subunits in place, is trafficked to the flagellar membrane⁹⁸.

Questions remain regarding how CatSper traffics to the flagellar membrane and whether intraflagellar transport (IFT) machinery and adaptor proteins exist that specifically interact with CatSper or other sperm ion channels and transporters is unknown. Studies using genetically altered mouse models have shown that IFT is essential for mammalian spermiogenesis but that epididymal spermatozoa are devoid of IFT proteins¹⁰⁸. For example, IFT25 and IFT27, which are dispensable for ciliogenesis in somatic cells, are required for flagellar formation as they participate in assembling and transporting structural components of the fibrous sheath that is unique to spermatozoa^109,110. Identification of the CatSper interactome from spermatids, not mature sperm, and application of super-resolution microscopy could provide valuable information on the trafficking of flagellar membrane proteins and quadrilateral compartmentalization (FIGS 1,2) during spermiogenesis.

Human versus mouse CatSper.

Interestingly, two studies in infertile patients who lacked CatSper current demonstrated that CatSper assembly in humans is strikingly different from CatSper assembly in mice^103,104. Infertile men who had lost one genomic copy of CATSPER2 (owing to a copy number variation)¹⁰⁴ or both copies owing to contiguous deletion of a genomic region encompassing CATSPER2 and STRC (15q15.1–15q15.3, deletion of which leads to the rare deafness–infertility syndrome)^103,111,112 had otherwise normal semen parameters despite substantially reduced or absent expression of CATSPER2, respectively, in their spermatozoa. The protein expression levels of the other three CatSper pore-forming subunits in these unique human CatSper-deficient sperm cells were only marginally changed, or were not changed at all^103,104, in contrast to the changes observed in mouse CatSper-knockout sperm. Moreover, the uniquely quadrilateral sub-flagellar compartmentalization was observed for CATSPER3 and CATSPER4 in the principal piece of human CATSPER2-deficient spermatozoa¹⁰³. These results suggest that human CATSPER1, CATSPER3 and CATSPER4 proteins can still traffic to the flagella, but they cannot form functional channels without CATSPER2. Thus, the all-or-nothing expression pattern of the CatSper complex apparently does not exist in human sperm cells. Formation of trimeric complexes lacking CATSPER2 and their proper trafficking to the flagella is unlikely to happen because a tetramer of an ion channel pore is often assembled as a dimer of dimers. These findings illustrate the species-specific differences in the assembly and trafficking patterns of CatSper.

A more thorough understanding of the organization of CatSper into Ca²⁺ signalling nanodomains requires further investigation. For example, probing the absence or presence and the sub-flagellar localizations of other CatSper auxiliary subunits in these human CATSPER2-deficient sperm will provide more insight into their roles in channel assembly and trafficking and domain organization. Additional unidentified channel subunits that specifically function in trafficking might also exist. This possibility was implied by a recent CatSper proteome study of mouse sperm cells, which showed several candidate CatSper-associated proteins, including one with a conserved domain involved in membrane trafficking³¹. The likely existence of additional unknown CatSper subunits highlights the molecular and regulatory complexity of this important channel.

Modulation of CatSper activity

Molecular mechanisms of alkaline activation and Ca²⁺ sensitivity of CatSper.

Ca²⁺ entry through CatSper requires channel activation by intracellular alkalinization^13,31,66,67. The pH sensitivity of CatSper was initially attributed to a conserved histidine-rich region in the N terminus of CatSper1 among mammals^13,15, but, subsequently, the molecular basis by which CatSper can sense pH and Ca²⁺ has been elucidated. The mechanism relies upon EFCAB9, a testis-specific protein that has co-evolved with other core members of the CatSper complex³¹. EFCAB9 was identified in a comparative proteomics screen for proteins differentially expressed in Catsper1-null sperm when compared with wild-type sperm³¹. EFCAB9 is the only CatSper subunit that contains known Ca²⁺- and calmodulin-binding motifs. Three EF-hand motifs were identified in EFCAB9 and it was shown to bind CATSPERζ in a pH-dependent and Ca²⁺-dependent manner³¹. An increased Ca²⁺ concentration facilitated EFCAB9–CATSPERζ complex formation, whereas alkalinization impeded the interaction³¹. That EFCAB9 and CATSPERζ work as one functional unit is supported by mouse models in which either Efcab9 or Catsperz or both has been knocked out. These three mouse models exhibit identical phenotypes and demonstrate that the expression of these proteins is interdependent: if Efcab9 has been knocked out, CATSPERζ is absent, and vice versa³¹. Although EFCAB9-deficient sperm exhibit reduced CatSper expression, when intracellular pH is low, wild-type and Efcab9-null sperm show a similarly dense CatSper current (I_CatSper)³¹, indicating that EFCAB9–CATSPERζ normally limits CatSper-mediated Ca²⁺ entry. When intracellular pH rises, CatSper is activated in a Ca²⁺-dependent manner, but in the absence of EFCAB9, the channel is less sensitive to intracellular Ca²⁺ changes and less responsive to alkalinization³¹. Thus, a working model was generated: before capacitation, the pH-sensing and Ca²⁺-binding EFCAB9–CATSPERζ complex stabilizes the closed CatSper pore; upon alkalinization and Ca²⁺ entry through CatSper, EFCAB9–CATSPERζ undergoes structural rearrangements releasing its inhibition of the channel. Further opening of the channel is followed by Ca²⁺ entry, which would be bound by EFCAB9, stabilizing its prolonged open state³¹.

Steroid hormones in human CatSper regulation.

Both mouse and human CatSper channels are activated by intracellular alkalinization; however, activation of CatSper by physiological ligands has only been reported in humans^66,67 and rhesus macaques¹¹³, suggesting that CatSper is regulated by species-specific mechanisms. For example, progesterone, a steroid hormone secreted by cumulus cells, can robustly evoke human^66,67,102, but not mouse⁶⁷, CatSper currents. Similarly, prostaglandin E₁ (PGE₁), which is abundant in the seminal plasma and also secreted by cumulus cells, was found to activate human, but not mouse, CatSper⁶⁷. α/β hydrolase domain-containing protein 2 (ABHD2) has been identified as the non-genomic progesterone receptor in human sperm¹¹⁴. ABHD2 abolishes endocannabinoid 2-arachidonoylglycerol (2-AG) inhibition of CatSper as a progesterone-dependent lipid hydrolase, enabling CatSper activation¹¹⁴. Other steroid hormones, such as pregnenolone sulfate and testosterone, also modulate CatSper-mediated Ca²⁺ influx into human sperm; they probably bind to the same sites as progesterone¹¹⁵. Whether testosterone and other steroids function as human CatSper agonists or antagonists remains controversial^115-117, and the mechanisms by which these ligands bind to and modulate CatSper activity remain largely unknown.

Additional ligands and chemicals have been reported to activate human CatSper directly or indirectly^115,118-120. These include structurally diverse endocrine-disrupting chemicals (EDCs) such as p,p′-dichlorodiphenyldichloroethylene, 4-methylbenzylidene camphor and triclosan^119-121. Thus, the presence of any of these EDCs in the female reproductive tract could interfere with human sperm function by modulating CatSper activity.

The non-pore-forming CatSper transmembrane subunits have been suggested to bind factors that alter CatSper gating as they have large ECDs^29,96-98 (FIG. 1b). They could potentially bind ligands and function as sensory transducers in the polymodal Ca²⁺ signalling exhibited by human sperm. Disrupting one of the genes, CatSperd, in mice results in an identical phenotype to that seen in mice with knockout of the pore-forming CatSper subunits at the organismal and cellular levels⁹⁸, illustrating that CATSPERδ is essential to form a functional CatSper. In the future, biochemical and structural characterization of isolated ECDs from the non-pore-forming CatSper transmembrane subunits, in addition to characterization of the cytoplasmic CatSper subunits themselves, is a sensible approach to improving our understanding of the molecular mechanisms of CatSper.

Signalling pathways in capacitation

Sperm capacitation involves a cascade of signalling pathways (FIG. 1a). Studies have indicated that different pathways directly or indirectly regulate CatSper, the central signalling hub in network with multiple signalling proteins.

Signalling crosstalk and CatSper

During capacitation, the rise in intracellular pH activates not only CatSper but also KSper. Activation of KSper hyperpolarizes the membrane to further drive Ca²⁺ influx through CatSper in mouse sperm^64,70,84. HCO₃⁻, Ca²⁺ and bovine serum albumin (BSA) have long been recognized as indispensable for sperm capacitation and fertilization in vitro¹²², engaging signalling pathways that regulate CatSper-mediated calcium signalling.

HCO₃⁻ uptake and cAMP-dependent PKA activation.

At the molecular level, capacitation is initiated when spermatozoa are exposed to a high concentration of HCO₃⁻ in the luminal fluid of the female reproductive tract, which also has a higher Ca²⁺ concentration than the luminal fluid of the epididymis³⁹. HCO₃⁻ enters the sperm through HCO₃⁻ transporters⁴² and activates a unique sAC¹²³, resulting in increased cAMP levels. The cAMP-dependent PKA pathway is important in the regulation of sperm motility: sAC-deficient male mice are infertile¹²⁴, as are mice deficient in the catalytic subunit of PKA, Cα2 (REF.¹²⁵). HCO₃⁻ also stimulates Ca²⁺ entry into sperm by raising intracellular pH (FIG. 1a). However, the role of cAMP in the regulation of Ca²⁺ influx is less clear. Membrane-permeable analogues of cyclic nucleotides stimulate Ca²⁺ entry in mouse^{15,96,126,127} and human sperm²⁶. However, a series of studies have also shown that an increase in intracellular cAMP (stimulated by HCO₃⁻, 3-isobutyl-1-methylxanthine, uncaging of cAMP, or adenosine) fails to stimulate Ca²⁺ influx in mouse¹²⁸ and human sperm⁶⁶. Furthermore, membrane-permeable analogues of cyclic nucleotides, but not physiological concentrations of cAMP or cGMP, activate human CatSper when applied extracellularly, but not intracellularly^26,129. However, these data are inconsistent with the findings of studies in mouse sperm, in which cAMP directly applied intracellularly through a pipette activated CatSper¹³⁰, but cyclic nucleotide analogues applied extracellularly to the bath solution had no effect¹³. Thus, cAMP and PKA regulation of CatSper-mediated Ca²⁺ influx is probably species-specific and needs to be further clarified.

Calcium influx induced by loss of cholesterol.

Together with high levels of HCO₃⁻ and Ca²⁺ concentration, serum albumin is a key component in mammalian sperm capacitation in vivo as well as in vitro^131,132. Although BSA is known to induce Ca²⁺ influx in sperm in vitro, the molecular mechanism by which this occurs has not been fully elucidated. However, BSA-induced Ca²⁺ influx is obliterated in Catsper1-null spermatozoa¹³³. As cholesterol release from the sperm plasma membrane by BSA is associated with activation of cAMP–PKA pathways during sperm capacitation in both mice¹³⁴ and humans¹³⁵, lipid signalling by cholesterol efflux might participate in regulating CatSper-mediated Ca²⁺ signalling via the cAMP–PKA pathway. In this respect, caveolin 1 — a protein associated with cholesterol-rich lipid rafts — has been found in the CatSper Ca²⁺ signalling nanodomains in mouse sperm. The role of the CatSper channel as a domain organizer is notable, as caveolin 1 localization is dysregulated in the absence of the CatSper channel but CatSper nanodomains remain intact in the absence of caveolin 1 (REF.³⁰).

Downstream signalling that leads to motility regulation at the axoneme.

Under capacitating conditions, cAMP-stimulated PKA activity leads to sperm motility changes within a minute^136-138. By contrast, hyperactivated motility and a capacitation-associated increase in tyrosine phosphorylation (P-Tyr) occur much later in time¹³⁹. The cAMP–PKA pathway is thought to control downstream P-Tyr because permeable cAMP analogues are able to induce P-Tyr, even in the absence of HCO₃⁻ and calcium ions¹⁴⁰ and administration of PKA inhibitors completely blocks P-Tyr development¹⁴¹. Interestingly, earlier onset and increased P-Tyr was observed in Catsper1-null and Catsperd-null spermatozoa following incubation under capacitation conditions³⁰, suggesting that PKA activity and P-Tyr are suppressed by a CatSper-mediated Ca²⁺ signalling pathway. Super-resolution imaging has revealed a striking spatial confinement of P-Tyr to the axoneme in capacitated wild-type sperm (FIG. 3a); P-Tyr spreads and fills the extra-axonemal region in the absence of CatSper³⁰. As P-Tyr requires PKA activation, the signal transduction to suppress P-Tyr in the periaxoneme has been suggested to involve active protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) to limit PKA activity and protein tyrosine phosphatase³⁰. In the same study, multiple tyrosine-phosphorylated proteins were identified from capacitated mouse sperm³⁰, including axonemal proteins and a testis-specific tyrosine kinase, FER/FERT (FIG. 3b). Subsequently, a 2016 study revealed that the capacitation-associated P-Tyr increases are mostly eliminated in sperm from kinase-inactivating mutant (D743R) Fer^DR/DR males¹⁴², demonstrating that FER/FERT is the master tyrosine kinase responsible for capacitation-associated P-Tyr. However, Fer^DR/DR males are fertile¹⁴³, although sperm from these mice do display reduced fertilizing ability in vitro¹⁴². Thus, P-Tyr is not essential for mouse fertilization in vivo but might have a functional role, such as timing motility regulation and/or determining the lifespan of sperm in the female oviduct.

Fig. 3 ∣ — a ∣ Tyrosine phosphorylation (P-Tyr) exhibits subflagellar localization. A cross-sectional view of the principal piece (left) shows CATSPER (red), fibrous sheath (blue), outer dense fibres (brown), and 9+2 axoneme (line structures in the centre). A two-colour 3D stochastic optical reconstruction microscopy (STORM) cross-sectional image of a capacitated wild-type spermatozoon (right) shows CATSPER1 (red) and P-Tyr (green). Note that P-Tyr is localized in the centre of the cross-section, corresponding to the axoneme. b ∣ P-Tyr identified by quantitative whole-sperm proteome analysis from capacitated wild-type and *Catsper1*-null mice. Each protein is represented as a dot in a volcano plot of statistical significance (y-axis) against the average protein fold-change (x-axis) of *Catsper1*-null compared with wild-type spermatozoa. Four axonemal proteins (more than twofold change and P < 0.05) are marked in colour.

A Ca²⁺ signal can be directly translated into mechanical changes in the axoneme: transient treatment with the Ca²⁺ ionophore A23187 can bypass the P-Tyr development in mouse sperm and the CatSper signalling network to induce hyperactivated motility in vitro^144,145. Nevertheless, CatSper-mediated Ca²⁺ signal transduction, originating from the linear CatSper nanodomains, is required for flagellar Ca²⁺ regulation in vivo. Disruption of the nanodomain compartmentalization^29,31, or loss of calcineurin^30,146 or EFCAB9 (REF.³¹), both of which are Ca²⁺-binding proteins from the nanodomains, leads to changes in the flagellar envelope and fertility defects in mice. Discovery of more Ca²⁺-dependent molecules associated with the CatSper nanodomains and/or the axoneme will further elucidate the downstream Ca²⁺ signalling affecting dynamic motility regulation.

Other channels in sperm

In addition to CatSper and KSper, HIV1 and sNHE, a number of other channels are involved in regulating sperm function.

DSper and TRPV4

The presence of a channel that facilitates influx of Na⁺, as is observed in neuronal excitation, has been hypothesized in sperm and named ‘DSper’²⁸. The DSper current was recorded in human sperm as a non-CatSper, non-selective cation conductance with outward rectification and pronounced temperature sensitivity¹⁴⁷. In this study, DSper was potentiated during capacitation and was not diminished by either Mg²⁺ or NNC55-0396 administration, which block and inhibit CatSper, respectively. DSper is reversibly activated by warm temperatures (22–37°C; T_1/2 34°C in uncapacitated human sperm, T_1/2 31 °C in capacitated human sperm). Pharmacological screening of human sperm has suggested that TRPV4 mediates this temperature-dependent DSper current¹⁴⁷. Capacitated rabbit and human sperm cells have been shown to move towards higher temperatures in vitro¹⁴⁸, and subsequent study showed that capacitated human sperm swim up a temperature gradient by modulating hyperactivated motility¹⁴⁹, However, in humans, sperm thermotaxis remains controversial because temperature differences within the female reproductive tract have not been reported and sperm capacitation is not linked to ovulation¹⁵⁰. Regardless, direct recording of human spermatozoa by whole-sperm patch clamp¹⁴⁷ and mouse genetics studies¹⁵¹ suggest that TRPV4 could be a molecular basis of temperature sensing at least in mouse and human sperm. However, there is a discrepancy regarding the protein distribution of TRPV4 in human sperm: data from two separate studies that used antibodies raised against the same epitope are conflicting. In one study, TRPV4 was reported in the post-acrosomal and neck regions, but not in the flagella¹⁵², whereas in the other study, TRPV4 was found in both the acrosome and the flagella¹⁴⁷. This discrepancy highlights the limitation of immunolabelling in determining protein distribution in sperm cells without stringent controls. In mouse sperm, TRPV4 was detected along the tail and in the head and the signal was absent in Trpv4-knockout sperm¹⁵¹, validating this specific distribution in mice. Mouse genetics support the involvement of TRPV4 in sperm thermotaxis¹⁵¹. Unlike wild-type sperm, Trpv4-knockout sperm failed to respond to increasing temperature and exhibited delayed hyperactivated motility, despite still being capable of fertilization¹⁵¹. Specific DSper current via TRPV4 might have been undetectable in the previous mouse sperm recordings¹³, as in the absence of external Ca²⁺, the Na+ moving through CatSper might have been masking the Na⁺ movement through DSper. Isolating the DSper current and confirming the existence of DSper-dependent thermotactic behaviour in mouse or human CatSper-deficient sperm will clarify whether and how TRPV4 contributes to the molecular basis of mammalian sperm thermotaxis.

P2X2

Extracellular ATP has been reported to raise the intracellular concentration of Ca²⁺ and to stimulate the acrosome reaction in sperm¹⁵³. Mouse spermatozoa have an ATP-gated current (I_ATP), which is the only ion current detected from the midpiece of mammalian spermatozoa by patch-clamp recordings¹⁵⁴. I_ATP is an intrinsically inwardly rectifying, cation-non-selective and divalent-permeable current, and is mediated by the homomeric P2X2 purinergic receptor. Consistent with the properties of P2X2 current in Xenopus oocytes^155,156, I_ATP is also activated by Zn²⁺ and by an acidic extracellular pH in mouse sperm¹⁵⁴. In light of these observations, P2X2 might function during sperm maturation in the epididymis where Zn²⁺ is abundant and the pH is more acidic than in the oviduct. Neither oviductal fluid nor cumulus cells from ovulated mice evoked sperm I_ATP, suggesting that the female reproductive tract is not the primary site of sperm P2X2 function in mice¹⁵⁴. The genetic disruption of P2rx2 abolishes I_ATP in mouse sperm but does not affect sperm progressive motility, hyperactivation or the acrosome reaction¹⁵⁴. However, frequent mating renders P2rx2-null male mice less fertile, suggesting that ATP-activated Ca²⁺ influx confers an advantage under high sexual demands¹⁵⁴.

Subtle sperm fertility phenotypes in the ion-channelknockout mice discussed in this Review could be manifested more prominently in natural mating settings, in which sperm competition presumably exerts a stronger force than it does in a more controlled environment. In vitro, ATP supplementation rescues the immobility observed in metabolite starvation-induced sperm from both wild-type and P2rx2-null males¹⁵⁴. Presumably, I_ATP can deliver Ca²⁺ to the mitochondria to drive ATP production. The function of I_ATP in human sperm is not clear, as ATP-gated current has not been found in human sperm²⁶. The source of extracellular ATP for P2X2 activation also remains uncertain.

Ca_v2.3

The voltage-gated calcium channel Ca_v2.3, encoded by Cacnale, mediates R-type Ca²⁺ currents in neurons¹⁵⁷. Ca_v2.3 was thought to have a function in sperm physiology based on its immunological detection in mouse sperm^158,159. However, male mice lacking Ca_v2.3 are only mildly subfertile¹⁶⁰ (TABLE 1). Non-capacitated Ca_v2.3-lacking sperm swim more linearly and exhibit a lower rising rate of Ca²⁺ transients induced by BSA in their heads than wild-type sperm¹⁶⁰. This subtle but interesting difference prompted the question as to whether Ca_v2.3 functions in the acrosome reaction, and this was investigated in a study to determine whether membrane lipids can stimulate the acrosome reaction via modulation of Ca_v2.3 during capacitation¹⁶¹. SNX-482, a Ca_v2.3-specific blocker, reduced acrosome reactions induced by cholera toxin B or GM1 in sperm incubated under capacitating conditions, and Cacnale-null sperm exhibited significant reductions in the rates of the acrosome reaction and successful IVF¹⁶¹. Intriguingly, Ca²⁺ transients in the sperm head were associated with lipid modulation of the activity and localization of Ca_v2.3 (REF.¹⁶¹). The calcium transients occurred only at the apical acrosome of wild-type sperm but in the equatorial segment of Cacna1e-null sperm with faster kinetics. These Ca_v2.3 data were not recorded using the current gold-standard method of patch clamping mouse sperm, implying that the resulting readout could be indirect^14,162. Careful modification of the recording conditions required for recording from mouse sperm incubated under capacitated conditions and/or acrosome-reacted sperm might enable further biophysical characterization of the contribution of Ca_v2.3 to the acrosome reaction. Using CATSPER-null sperm for Ca_v2.3 patch clamp measurements will also help visualize the current mediated by Ca_v2.3 by eliminating the major contribution of Ca²⁺ influx mediated by CatSper.

PKD1, PKDREJ and PKD2

PKD1 and PKD2 encode polycystin 1 (PC1) and polycystin 2 (PC2 or TRPP2), respectively. PC1 is a putative transmembrane receptor, whereas PC2 can independently form a TRP-like ion channel¹⁶³. Together PC1 and PC2 can form a heterotetrameric receptor channel complex and co-localize in renal cilia^164-166. Mutations in either PKD1 or PKD2 can cause autosomal dominant polycystic kidney disease (ADPKD), which results in the formation and expansion of collecting tubule-derived renal cysts^164,167. Men with ADPKD also exhibit an increased rate of infertility and necrospermia^168-170. Additionally, immotile sperm that lack the two central axoneme microtubules (9+0) have been identified in infertile men with PKD¹⁷⁰.

A PKD1 homologue, Pkdrej, has also been implicated in fertility and has been studied in some detail in mice¹⁷¹. The PKD1 homologue PKDREJ (polycystic kidney disease and receptor for egg jelly) was found to be expressed specifically in the testicular tissues of humans¹⁷² and mice¹⁷³ and localizes to the plasma membrane of the mouse sperm head¹⁷³. However, no direct evidence supports a role for PKDREJ in acrosomal exocytosis. Mice that are homozygous for a disrupted Pkdrej allele (Pkdrej^tm/tm) are still fertile (TABLE 1) but less fertile than wild-type mice when compared using sequential mating trials or artificial insemination assays¹⁷¹. Pkdrej^tm/tm sperm navigate the female reproductive tract in vivo with reduced efficiency compared with wild-type sperm, as shown by fewer sperm reaching the cumulus matrix surrounding the egg over the same amount of time, and are slower to develop the ability to undergo a zona pellucida-induced acrosome reaction under capacitating conditions. However, as a comparable proportion of Pkdrej^tm/tm sperm and wild-type sperm developed hyperactivated motility over the same time course in vitro, PKDREJ has been suggested to be a chronoregulator, not a master regulator, of capacitation¹⁷¹.

CFTR and functionally related transporters

Cystic fibrosis transmembrane regulator (CFTR) is a Cl⁻-permeable and HCO₃⁻-permeable anion channel. HCO₃⁻ conductance through CFTR is low compared with that of Cl⁻^174,175. Mutations in the gene encoding CFTR are the cause of cystic fibrosis, an autosomal recessive, monogenetic disease that results in severe phenotypes including progressive lung disease. Another cystic fibrosis phenotype is male infertility, which affects 97–98% of men with cystic fibrosis owing to congenital bilateral absence of the vas deferens (CBAVD)¹⁷⁶. However, mutations in CFTR have also been found in 8.9% of otherwise healthy men with reduced sperm counts and/or with poor sperm quality; for example, sperm that exhibited reduced motility or abnormal morphology, or both¹⁷⁷.

Xu and colleagues characterized the role of CFTR in sperm using a heterozygous (Cftr^+/−) cystic fibrosis mouse model¹⁷⁸ — a heterozygous mouse was used because the homozygotes rarely survive past weaning¹⁷⁹. When compared with wild-type sperm, fewer Cftr^+/− sperm could achieve capacitation; they exhibited decreased membrane hyperpolarization and cAMP production in response to HCO₃⁻, decreased motility and reduced fertility in vitro and in vivo¹⁷⁸ In accordance with these data, CFTR inhibitor-172, a CFTR channel blocker, inhibits the acrosome reaction, HCO₃⁻-dependent increases in intracellular pH and membrane hyperpolarization, and inhibits an HCO₃⁻-dependent increase in cAMP concentration in mouse sperm^178,180. Likewise, another CFTR inhibitor, diphenylamine-2-carboxylic acid (DPC), also blocks capacitation-associated hyperpolarization (and seems to inhibit capacitation in general) as well as Cl⁻ influx¹⁸¹. By contrast, genistein, which activates CFTR, induced hyperpolarization under noncapacitating conditions in mouse sperm and resulted in Cl⁻ influx: intracellular Cl⁻ was measured using N-(ethoxycarbonylmethyl)-6-meth oxyquinolinium bromide (MQAE), a fluorescent Cl⁻ probe¹⁸¹. External Cl⁻ was required for the genistein-induced hyperpolarization¹⁸¹. This external Cl⁻ dependence of capacitation was also observed in guinea pig sperm¹⁸², suggesting a role of CFTR in sperm capacitation in guinea pig. In the same study, the Cl⁻/HCO₃⁻ exchanger known as solute carrier family 26, number 3 (SLC26A3) was suggested to work together with CFTR by pumping out Cl⁻ that had entered through CFTR¹⁸². In support of this interaction, mouse SLC26A3 and CFTR were co-immunoprecipitated, together with other members of solute carriers (SLC26A6 and SLC9A3R1)¹⁸³. This CFTR-SLC interaction model was supported by a study showing that pharmacological inhibition or blocking SLC26A3 with an antibody could inhibit the acrosome reaction and hyperactivated motility¹⁸². Furthermore inhibition of CFTR and SLC26A3 was found to inhibit db-cAMP-induced Cl⁻ influx, capacitation-associated hyperpolarization and pH change, and HCO₃⁻ was found to induce hyperpolarization¹⁸³.

Human mutations in SLC26A3 can result in congenital chloride diarrhoea (CLD), and men with CLD often also have subfertility and oligoasthenoteratozoospermia¹⁸⁴. Accordingly, Slc26A3 knockout in mice also results in CLD and subfertility¹⁸⁵. As both CFTR and SLC26A3 are expressed in the epithelial cells of the male reproductive tract as well as in the sperm cells themselves^184,186, their respective roles in sperm versus other fertility-related processes can be difficult to determine. In the future, assessing SLC26A3 protein expression and function in heterozygous Cftr^+/− mice could inform its role in CFTR regulation and sperm capacitation.

Direct whole-cell patch recordings from mouse testicular sperm demonstrated a Cl⁻ component to the membrane current that is ATP-dependent and is stimulated by cAMP, cGMP and genistein, and inhibited by CFTR inhibitor-172 and DPC¹⁸⁷. However, Cl⁻ current could still be recorded from CFTR loss-of-function (ΔF508) mouse testicular sperm, although in ΔF508 mice this current is less sensitive to cAMP and CFTR inhibitor-172 (REF.¹⁸⁷). In the same study, Cl⁻ current was also recorded from mouse wild-type epididymal sperm, but the effects of the CFTR-modulating compounds observed were not as substantial as seen in the testicular sperm. Thus, further work is required to clarify the extent to which CFTR conducts Cl⁻ current in mature sperm cells.

The precise localization of CFTR within sperm also requires clarification. Immunocytochemistry studies have shown that CFTR is localized in the equatorial segment of human^178,180, mouse¹⁷⁸ and guinea pig sperm cells¹⁸² but have also demonstrated its presence in the midpiece of human and mouse sperm^181,183. CFTR has also been found simultaneously in both the equatorial segment of mouse sperm heads and the sperm midpiece¹⁸⁸. Likewise, immunocytochemistry has demonstrated that SLC26A3 localizes within the heads of guinea pig sperm¹⁸² but was shown to localize to the midpiece of mouse sperm in a separate study¹⁸³. This discrepancy highlights the importance of genetic studies including the generation of knockout mice, for example, in sperm studies, as sperm cells have been repeatedly shown to be prone to non-specific antibody binding.

To date, the mouse and human sperm ion channels and membrane transporters that are clearly implicated in male fertility have been largely localized in the flagella (FIG. 1a; TABLE 1). Identifying and investigating channels located in other compartments of the sperm cells, such as acrosomal channels, requires further studies using current state-of-the-art techniques that provide specificity, sensitivity and high resolution in time and space, as well as evidence from genetic studies in both mice and humans.

Sperm Ca²⁺ homeostasis

To support dynamic cellular Ca²⁺ signalling and to prevent Ca²⁺ intoxication, Ca²⁺ must be efficiently cleared from the cytosol after its entry through Ca²⁺-permeable channels.

PMCA4

Plasma membrane Ca²⁺-ATPases (PMCAs) are highly conserved Ca²⁺ extrusion pumps that maintain low basal levels of intracellular Ca²⁺ (REF.¹⁸⁹). Two isoforms, PMCA1 and PMCA4 (encoded by Atp2b1 and Atp2b4, respectively), are expressed abundantly in testis and highly conserved across species¹⁹⁰. Genetic knockout of PMCA4 in mice results in a >90% reduction in total PMCA expression¹⁹¹, indicating that PMCA4 is the primary PMCA in sperm cells. PMCA4-deficient male mice produce sperm cells with normal morphology but are totally infertile owing to impaired sperm motility and defective hyperactivated motility^191-193 (TABLE 1). Surprisingly, PMCA4-deficient mouse sperm are able to bind to zona pellucida and fertilize eggs in vitro¹⁹², suggesting that infertility in vivo is probably due to inefficient sperm navigation in the female reproductive tract. Interestingly, PMCA4 is primarily localized in the sperm principal piece but, unlike CatSper, does not exhibit a distinct pattern of distribution on the flagellar surface³⁰. Ultrastructural analysis has revealed that mitochondria from PMCA4-deficient sperm are more condensed than wild-type sperm when incubated under capacitating conditions¹⁹¹; this characteristic is indicative of Ca²⁺ overload. Whether PMCA4 participates in regulating CatSper-relevant Ca²⁺ signalling and how sperm mitochondria are involved in Ca²⁺ signalling during capacitation remains to be clarified.

Mg²⁺ transporter

A Mg²⁺ transporter, CNNM4 (also known as ancient conserved domain-containing protein 4, ACDP4) is highly expressed in mature ameloblasts and intestinal epithelia¹⁹⁴. Interestingly, CNNM4-deficient male mice are almost infertile¹⁹⁵. Cnnm4-null spermatozoa exhibit rapid motility decreases and fail to develop hyperactivated motility¹⁹⁵. Mg²+ levels are significantly increased in CNNM4-deficient mouse sperm, whereas Ca²⁺ levels are not affected and removal of Mg²⁺ from the medium can rescue Cnnm4-null sperm motility¹⁹⁵, suggesting that abnormally high levels of Mg²⁺ are detrimental to sperm function. In addition, Cnnm4-null sperm exhibit excessive P-Tyr with impaired Ca²⁺ influx¹⁹⁵, similar to the phenotype of CatSper-deficient sperm³⁰. These results suggest that CNNM4 might be involved in sperm Ca²⁺ homeostasis and/or could be functionally linked to CatSper. Additional manipulation to a Cnnm2 allele, which encodes another CNNM family Mg²⁺ transporter, to make CNNM2 non-functional rendered the resulting Cnnm2^+/−/Cnnm4^−/− male mice completely infertile. The Cnnm2^+/−/Cnnm4^−/− sperm exhibited a more severe reduction in motility and a loss of BSA-induced Ca²⁺ response, compared with Cnnm4^−/− sperm, suggesting that CNNM2 works together with CNNM4 to regulate intracellular Mg²⁺ homeostasis and male fertility¹⁹⁶. Whether the Mg²⁺ efflux activities of CNNMs are associated with Ca+ homeostasis and how they can potentially regulate sperm Ca²⁺ signalling await further investigation.

Clinical implications

The manipulation of ion channels in order to affect fertility could be leveraged for clinical application; for example, to produce male contraceptives or fertility treatments. Additionally, male fertility can reflect overall health as the dysregulated expression of ubiquitous proteins implicated in non-reproductive diseases might also result in altered sperm function.

Novel non-hormonal male contraceptives

Most contraceptive strategies have been developed for use by women, but interest in generating a novel male contraceptive remains. One strategy is the use of hormones to inhibit endogenous testosterone production and, as a result, to block spermatogenesis¹⁹⁷. To bypass potential adverse effects associated with hormone use, non-hormonal contraceptives are more desirable.

Ion channels in sperm are attractive targets for the development of contraceptives and, conversely, for drugs that could be used to treat infertility. Like G-protein-coupled receptors, ion channels are good drug targets as they are implicated in a variety of pathophysiologies and present druggable sites at cell surfaces^198,199. In fact, ~15% of current drug targets are ion _channels^199,200_.

The sperm-specific nature of the CatSper ion channel complex means that targeting it should result in few unintended off-target effects. Some compounds have been identified that inhibit CatSper, but they are non-specific and also inhibit KSper with comparable potency and so are probably not specific enough to be used as contraceptives²⁰¹. RU1968, a ligand of steroidal sigma receptors, has been shown to suppress progesteronestimulated Ca²⁺ signalling and prostaglandinstimulated Ca²⁺ signalling in human sperm²⁰². RU1968 has also been shown to inhibit human CatSper with ~15-fold higher potency than human KSper, and not to inhibit mouse KSper at all²⁰¹, demonstrating the specificity for CatSper inhibition. Hopefully RU1968 and other CatSper inhibitors can be used as a template for the design of drugs that could be used in contraception.

Many sperm ion channels have yet to be thoroughly explored as therapeutic targets at least partially (as has been asserted for K⁺ channel pharmacology) owing to difficulties in establishing robust, high-capacity functional assays that could be used to interrogate large chemical libraries for potential drugs with activity against not only the targets themselves but also related targets to determine the specificity of screening hits¹⁹⁹. This difficulty has been somewhat overcome in sperm with the development of a high-throughput, automated screening platform to assess the effect of small molecules on human sperm motility and ability to undergo the acrosome reaction²⁰³. The drug library (ReFRAME) used in the study comprises drugs that have either been approved for use or have undergone preclinical profiling²⁰⁴; thus, a hit in this type of screen could accelerate the search for a marketable drug for use as a male contraceptive or to modify fertility.

Fertility as a proxy for overall health in men

The relationship between subfertility and overall health in men is becoming increasingly apparent^205,206. For example, as ~10% of the genome is implicated in fertility, subfertility and/or infertility, phenotypes can indicate abnormalities in other biological processes, such as those resulting in fibrosis²⁰⁵.

In addition to genetic fertility associations, subfertility and infertility are also associated with developmental, lifestyle, oncological and cardiovascular disorders²⁰⁵. Lifestyle factors associated with infertility in men include obesity²⁰⁷, tobacco abuse²⁰⁸ and stress²⁰⁸. For example, a large meta-analysis of 21 studies including 13,077 men showed a J-shaped association between the risk of abnormal sperm concentration and BMI, in which increased BMI was negatively correlated with semen sperm concentration²⁰⁷. It is well known that cancer treatment can impair male fertility²⁰⁹, and studies have now also supported a link between male infertility and the risk of developing testicular cancer^210,211, suggesting that male infertility might serve as a marker of oncological risk. Thus, assessing fertility and sperm functionality could offer unique insights into overall male health: each sperm cell is designed to function outside of the male body as a single cell and is, therefore, particularly amenable to in vitro analysis²¹². However, although sperm analysis can be a convenient and non-invasive tool, male reproductive capability is generally not evaluated until late in a man’s life, usually after failure to reproduce^206,213. Thus, it is recommended that men who exhibit health issues such as cystic fibrosis or altered fertility should consider genetic counselling to better understand other health problems to which they might be predisposed and the risk associated with passing their genes onto their children.

Specific notable mutations.

Mutations in CFTR have been identified not only in men who exhibit cystic fibrosis but also in those with CBAVD and/or sperm of reduced quality^176,177. Although cystic fibrosis is a recessive disease, mutagenesis of one CFTR copy can result in altered sperm parameters in mice¹⁷⁸ and could be associated with CBAVD in men²¹⁴. Abnormal mucociliary clearance is associated with cystic fibrosis, although this primarily results from the abnormal biophysical properties of the airway mucus and not ciliopathy²¹⁵. However, a relationship is seen between fertility and ciliopathies²¹⁶.

Mutations in the genes encoding PKD1 or PKD2, which both localize to primary cilia, can result in ADPKD, which is associated with increased rates of infertility^164,167-170. As the motility of cilia and flagella are both conferred by an axoneme, it is tempting to speculate that similar axonemal defects could result in similar phenotypes.

Because the CATSPER complex is a sperm-specific ion channel, mutations in CATSPER genes are less likely to cause more widespread health problems than alterations in other ion channels such as CFTR. For example, a CATSPERE in-frame 6-bp deletion results in normal sperm motility in humans but failure to fertilize in IVF owing to defective hyperactivation and lack of Ca²⁺ response to progesterone^106,107. In such cases, intracyto-plasmic sperm injection can be used to achieve fertilization and can result in clinical pregnancy. Although mutations in CATSPER are most likely to affect fertility, they can be associated with other health problems, such as deafness. Deafness–infertility syndrome is a very rare syndrome characterized by both deafness and male infertility and is associated with homozygous deletions of STRC (which is expressed in the inner ear) and CATSPER2 on chromosome 15q15 (REFS^111,112).

These examples illustrate the importance of diagnosis of the underlying cause of male infertility. Mutations giving rise to channelopathies that can affect male fertility can also have more wide-ranging effects on other body systems that men might wish to be aware of before beginning to consider assisted reproductive technology (ART).

Current controversies

A large body of evidence exists regarding the role of ion channels in sperm, but limitations of the experimental approaches used in the field must be taken into account and whether the findings from in vitro and/or animal studies are physiologically relevant and not species-specific must be considered before these data can be extrapolated to humans.

Interpreting data from indirect approaches

Determining protein locations in sperm cells solely based on antibody detection has generated controversies in the field and needs to be considered critically. Studies have produced inconsistent data, including the specific localizations of some ion channels and receptors, including CFTR, SLC26A3 and TRPV4, in sperm cells.

The identification of CatSper as a primary Ca²⁺ channel in sperm illustrates the importance of genetic and/or other direct evidence, such as direct electrophysiological recording from sperm cells. Before the discovery of CatSper, the N-type and R-type voltage-gated Ca²⁺ channels, Ca_v2.2 and Ca_v2.3, were thought to be the Ca²⁺ entry channels in sperm, as they had been immunologically detected in mouse sperm^159,217. T-type voltage-gated Ca²⁺ currents were also suggested to contribute to the Ca²⁺ influx in spermatozoa, as the corresponding currents were recorded in mouse testicular sperm⁹³-²¹⁸-²¹⁹. However, whole-cell patch clamping of mouse epididymal sperm cells, combined with genetic analysis, clarified that CatSper is the primary facilitator of Ca²⁺ influx in mature sperm and is specifically localized in the principal piece of the sperm tail^13,18. Furthermore, T-type currents were shown to be diminished in spermatids and were not detected in mature sperm^13,94, highlighting the importance of a direct approach. Immunological methods such as immunostaining and immunoblotting have also been used to demonstrate the expression of many neurotransmitter receptors in mammalian sperm, including receptors for norepinephrine, aspartate, serotonin, acetylcholine, GABA and glycine^220,221. However, no neurotransmitter-mediated currents were detected by whole-sperm cell electrophysiological recordings when functional expression of the corresponding receptors was tested¹⁵⁴, suggesting either a non-functional presence of these receptors or non-specific detection.

Studies that solely relied on pharmacological interrogation also illustrate the unreliable nature of indirect approaches. Sperm cells are particularly prone to non-specific antibody binding and are particularly amenable to chemical inhibitors and/or activators, probably owing to their small dimensions and relative lack of cytoplasm (that is, their low copy number of the channels or receptors and large lipid surface-to-volume ratio), which demonstrates the risk of detecting artefacts in sperm cells when genetic or other more direct evidence (for example, electrophysiology data) is lacking.

Compounding implications from in vitro studies

Various capacitation-associated changes have been described, including activation of cAMP/PKA¹⁴¹, increases in protein P-Tyr¹³⁹, a rise in intracellular pH²²² and Ca²⁺ (REFS^223-226), membrane hyperpolarization^227,228, and modulation of the lipid content of the sperm plasma membrane¹³². In particular, a substantial body of work has documented the involvement of P-Tyr in regulating sperm motility and fertility during sperm capacitation²²⁹. As a result, P-Tyr has been used as a hallmark of sperm capacitation for decades. However, mounting evidence now indicates that P-Tyr is not actually required for hyperactivated motility¹⁴⁴ or fertility¹⁴² in mice. This new interpretation has arisen because sperm capacitation is typically studied using in vitro analyses that reflect an average value for entire sperm populations at a given time. Inducing capacitation in mouse or human sperm in vitro using buffer containing HCO₃⁻ and BS A results in a heterogeneous population of sperm, in which as few as ~15% of mouse sperm are hyperactivated²³⁰; 2–14% of human sperm are acrosome-reacted²³¹. Similar to the fact that in vitro experiments do not necessarily reflect the in vivo processes, contact with the zona pellucida was believed to induce the acrosome reaction, especially after an observation that the mouse acrosome reaction can be induced by zona pellucida sperm-binding protein 3 (ZP3; one of four glycoproteins that make up the zona pellucida) in vitro²³². Subsequently, mouse spermatozoa were found to begin undergoing acrosome reaction in the isthmus region of the fallopian tube before arriving at the ampulla^233,234 and reacted spermatozoa were able to penetrate the zona in vivo²³⁵; the small number of mouse spermatozoa that arrive at the lumen of the ampulla and cumulus oophorus are all fully capacitated and acrosome-reacted in vivo²³⁶. Even so, determining the exact site of the acrosome reaction in other mammalian species awaits further technological development and the physiological functions of the acrosome reaction and P-Tyr are currently being re-evaluated.

Currently available tools based on flow cytometry combined with sorting have been used to detect and/or separate the in vitro capacitated heterogeneous sperm populations based on various capacitation parameters: P-Tyr^35,237, acrosome reaction status^238,239 and membrane hyperpolarization^74,240. However, the dynamics of how these parameters change in each individual sperm and the extent to which these parameters reflect the fertilizing ability of each sperm has not been established. The direct linking of molecular information with motility and fertilizing capacity awaits a new approach to be developed.

Critical appreciation of species-specific regulation

Conclusions from studies using different animal models should be carefully interpreted for two reasons. First, it is becoming apparent that some controversies in the field are partly due to a lack of understanding of marked species-specific regulation. Studies that exemplify this are those that have investigated the different molecular mechanisms of CatSper and KSper activation: CatSper is universally activated by intracellular alkalinization as demonstrated by sperm cells from sea urchin²⁴¹, mouse¹³, rat²⁸, human⁶⁷, cow²⁴² and horse²⁴³; however, only human and primate CatSper currents are robustly potentiated by hormones such as progesterone^66,67,113. Similarly, human KSper is more sensitive to Ca²⁺ than to alkalinization compared with mouse KSper. These discrepancies illustrate the divergence of molecular mechanisms that regulate CatSper and KSper across different species. In the future, these phenomena should be kept in mind, as other sperm ion channels and membrane receptors might have also diverged in a species-specific manner.

Second, the mouse model is limited in its ability to reflect the sperm biology of other species such as humans and domestic animals, at least in part because epididymal, not ejaculated, mouse sperm are usually used in in vitro experiments^28,244. Ejaculated sperm cells are mixed with secretory factors from male glands and ultimately with other molecules and ions in the female reproductive tract. Conclusions drawn from in vitro experiments using mouse sperm must consider the absence of these factors. Future studies should ensure that in vitro experiments are combined with genetic modifications and/or that in vitro systems are supplemented with the appropriate secretory factors from the male and female reproductive tracts in order to better inform our understanding of species-specific regulation of ion channels and membrane receptors including CatSper.

Future of the field

In order to better understand the regulation and role of ion channels in sperm, future studies must seek more evidence from in vivo studies and genetics evidence.

Seeking more in vivo context

Sperm analysis is currently performed using functionally heterogeneous sperm populations; these populations are typically capacitated in vitro and include degenerating cells³⁰. Researchers must consider that the population mean of these analyses of the changes undergone by the sperm is often presented and cannot be used to faithfully recapitulate the time-dependent and space-dependent changes that sperm undergo in the oviduct^245,246, nor the physiological and signalling state of the small number of spermatozoa that reach the vicinity of, and fertilize, the eggs^247,248. Consistent with this notion, a greater number of sperm are required for IVF than the number of sperm present at the site of fertilization in vivo²⁴⁹. Studies that better emulate in vivo conditions and/or the development of new systems that can directly link the molecular changes of individual cells to motility and fertilizing ability are research priorities in the future. Examples of such studies include motility-correlative molecular imaging of sperm cells³⁰, probing acrosome reaction states of sperm from genetically modified mice encoding fluorescence proteins in the acrosome along the female reproductive tracts^233,236,250, and electrophysiological recordings taken from capacitated human sperm¹⁴⁷. Such studies, in which an effort is made to understand sperm function in a more physiologically relevant manner, are anticipated in the future.

Identifying gene variants in human fertility

Decades of mouse genetics studies have resulted in an extensive list of genes encoding spermatozoan ion channels and transporters implicated in male infertility (TABLE 1). However, only a few of these genes, for example CatSper-encoding genes or CFTR, have been implicated in human infertility, which is surprising considering advances in tools such as whole-exome analysis, but illustrates the practical difficulties of studying the inheritance of infertility traits in humans. Damaging mutations in CatSper might have occurred and, therefore, have been detected, with increased frequency owing to the large number of genes required to form this multi-subunit channel complex. Targeted genomic analysis of large cohorts with a specific functional characteristic (for example, infertile men with normal semen parameters but with deficits in Ca²⁺ signalling or abnormal membrane potential) and analyses of large families that exhibit Mendelian inheritance of these infertility traits might accelerate the discovery of human variants in these genes. As our understanding has grown that clinically assessing male reproductive health could provide unique insights into the general health of a patient and his children, identification of human infertility genetic variants could serve as a guideline for counselling general health, as well as candidate drug targets for screening for contraception, and could also provide insights into drug design and mechanisms of action.

In the current era of ARTs, in which natural barriers to egg fertilization are removed, defining the genetic defects underlying infertility is of the utmost importance, as genetic mutations that lead to male infertility are able to be passed to the next generation. Understanding the long-term effect of using ARTs needs to be prioritized in research.

Conclusions

Technical advances have substantially improved our understanding of the role of ion channels and membrane receptors in sperm function during fertilization. Current knowledge of the ion channels and membrane transporters found in sperm have been greatly shaped by gene knockout studies in mice and genetic evidence from humans. In particular, our understanding of the CatSper channel — its molecular and spatial organization, the regulatory mechanism by which it is regulated, and the CatSper-based signalling pathways that are required to trigger hyperactivated motility — are now fairly well understood. However, the field is not without controversies and debates, in particular regarding the way in which in vitro data can be applied in vivo, and how data collected from one species can be applied to others; these areas need to be addressed. Many features of ion channels are unique to sperm as the sperm-specific channel isoforms have unique properties that are not found in other cell types. Thus, understanding the ion channel mechanisms in sperm cells will advance our knowledge of causes of male infertility and should inspire improvements in assisted reproduction and the development of new contraceptives, and improve diagnoses of infertility.

Key points.

Mammalian sperm cells undergo intracellular alkalinization during their fertilization journey as they encounter a drastic extracellular pH change in the female reproductive tract.
During capacitation,the sperm membrane potential hyperpolarizes, primarily via KSper activation and K⁺ efflux.
Increases in intracellular Ca²⁺ are required for inducing hyperactivated motility and acrosome reaction, two key physiological events essential for fertilization.
CatSper,the multi-subunit Ca²⁺ channel, is the predominant Ca²⁺ entry pathway in sperm cells and organizes into linear Ca²⁺ signalling nanodomains along the flagella.
CatSper-mediated Ca²⁺ signalling is integrated into other sperm capacitation signalling pathways including phosphorylation cascades.
Improved understanding of spermatozoan ion channels and transporters will help elucidate the delicate and dynamic regulation of Ca²⁺ homeostasis in sperm motility and fertility.

Acknowledgements

The authors thank Jae Yeon Hwang for valuable discussion and critical reading of the draft manuscript. This work was supported by start-up funds from Yale University School of Medicine, Grantham Foundation, and NIH (R01 HD 096745) to J.-J.C.

Glossary

CatSper: Sperm-specific calcium channel.
NHEs: Sodium–hydrogen exchangers.
HV1: Proton channel.
KSper: Native sperm-specific potassium current/channel.
Na⁺/K⁺ ATPase: Sodium–potassium adenosine triphosphatase; also known as the sodium–potassium pump.
SLO3: The mediator of KSper, which is also used as the name of the protein or channel expressed heterologously.
Quadrilateral compartmentalization: The four linear Ca²⁺ signalling nanodomains.
DSper: Depolarizing channel of sperm.
TRPV4: Transient receptor potential cation channel subfamily V member 4.
P2X2: P2X purinoceptor 2.
Ca_v2.3: R type, voltage-dependent, calcium channel, α 1 E subunit.
PKD: Polycystin, transient receptor potential cation channel, autosomal dominant polycystic kidney disease protein.
PKDREJ: Polycystin family receptor for egg jelly.
CFTR: Cystic fibrosis transmembrane conductance regulator.
PMCA4: Plasma membrane calcium ATPase 4.
CNNM4: Cyclin and CBS domain divalent metal cation transport mediator 4.

Footnotes

Competing interests

The authors declare no competing interests.

Peer review information

Nature Reviews Urology thanks P. Lishko, M. Yeste, Y. Okamura, P. Visconti and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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PERMALINK

Sperm ion channels and transporters in male fertility and infertility

Huafeng Wang

Luke L McGoldrick

Jean-Ju Chung

Abstract

Fig. 1 ∣. Spermatozoan ion channels and membrane receptors.

Sperm intracellular alkalinization

Proton carriers

Sodium–hydrogen exchangers.

Table 1 ∣.

Proton channels.

Membrane potential hyperpolarization

Na+/K+ transporters

The KSper channel

Ca2+ influx and signal transduction

Molecular organization of CatSper

Spatial organization of CatSper

Flagellar Ca2+ signalling nanodomains.

Fig. 2 ∣. Quadrilinear CatSper Ca2+ signalling nanodomains in mammalian sperm.

CatSper assembly and trafficking to the flagellar membrane.

Human versus mouse CatSper.

Modulation of CatSper activity

Molecular mechanisms of alkaline activation and Ca2+ sensitivity of CatSper.

Steroid hormones in human CatSper regulation.

Signalling pathways in capacitation

Signalling crosstalk and CatSper

HCO3− uptake and cAMP-dependent PKA activation.

Calcium influx induced by loss of cholesterol.

Downstream signalling that leads to motility regulation at the axoneme.

Fig. 3 ∣. Capacitation-associated protein tyrosine phosphorylation.

Other channels in sperm

DSper and TRPV4

P2X2

Cav2.3

PKD1, PKDREJ and PKD2

CFTR and functionally related transporters

Sperm Ca2+ homeostasis

PMCA4

Mg2+ transporter

Clinical implications

Novel non-hormonal male contraceptives

Fertility as a proxy for overall health in men

Specific notable mutations.

Current controversies

Interpreting data from indirect approaches

Compounding implications from in vitro studies

Critical appreciation of species-specific regulation

Future of the field

Seeking more in vivo context

Identifying gene variants in human fertility

Conclusions

Key points.

Acknowledgements

Glossary

Footnotes

References

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Na⁺/K⁺ transporters

Ca²⁺ influx and signal transduction

Flagellar Ca²⁺ signalling nanodomains.

Fig. 2 ∣. Quadrilinear CatSper Ca²⁺ signalling nanodomains in mammalian sperm.

Molecular mechanisms of alkaline activation and Ca²⁺ sensitivity of CatSper.

HCO₃⁻ uptake and cAMP-dependent PKA activation.

Ca_v2.3

Sperm Ca²⁺ homeostasis

Mg²⁺ transporter