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The Role of Sperm Ion Channels in Reproduction
C H A P T E R
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The Role of Sperm Ion Channels in Reproduction P.V. Lishko, M.R. Miller, S.A. Mansell University of California, Berkeley, CA, United States
SPERM CELL: MORPHOLOGY AND ORGANIZATION Sperm cells are terminally differentiated and are thought to be transcriptionally and translationally silent, meaning that spermatozoa are largely unable to synthesize new mRNA or translate it into new polypeptides.2,3 They are divided into two easily recognized parts: the head and the tail (flagellum). The sperm head comprises a condensed nucleus, a redundant nuclear envelope, and an acrosomal vesicle. The shape and size of the sperm head differs dramatically among species: from the spatula-like head in primates and ruminants to the hook-like pointed head that usually defines rodent sperm. Motility originates from the flagellum and is powered by adenosine triphosphate (ATP) hydrolysis within the sperm tail. The flagellum has a specialized cytoskeleton called an axoneme surrounded by specialized structural components and is subdivided into three functional parts: a mitochondria-containing midpiece, a principal piece, and the endpiece (Fig. 9.1). The flagellum is in essence a motile cilium4,5 with a flagellar plasma membrane tightly attached to all underlying structures along the sperm body. This arrangement provides spermatozoa with a ridged structure to which membrane proteins, including ion channels, can be tethered to the fibrous sheath6 to ensure their strict compartmentalization.7 Sperm basal motility depends on three crucial factors: the presence of ATP, a low concentration of intracellular calcium [Ca2+]i,, and normal to alkaline intracellular pH. Intraflagellar ATP is generated during glycolysis and oxidative phosphorylation whereas the intraflagellar concentration of protons and calcium is controlled by ion channels and transporters. Because of its high motility, powered by glycolysis and oxidative phosphorylation, the sperm flagellum quickly acidifies. The removal of protons from the sperm
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FIGURE 9.1 Mammalian spermatozoon. Schematic representation of spermatozoon with cellular compartments labeled and distribution of species-specific ion channels found within each section. CatSper, cation channels of sperm; sNHE, sperm-specific Na+/H+; TMEM16, Transmembrane protein 16. Reproduced from Miller MR, Mansell SA, Meyers SA, Lishko PV. Cell Calcium. 2015 Jul;58(1):105-13, Elsevier.
flagellum is an essential because the axonemal motility is inhibited by acidic pH. To expel protons and support motility, spermatozoa possess specialized proton channels and/or transporters. With the help of bioinformatic approaches,8 the whole-cell sperm patch clamp method,9,10 and several genetic models,8–35 sperm ion channels have been comprehensively characterized, among which calcium, potassium, proton, and nonselective and various ligand-gated channels have been identified.21,27,30,36
CATSPER CHANNEL: A PRINCIPAL CALCIUM CHANNEL OF SPERM In spermatozoa, swimming behavior is controlled by rises in flagellar [Ca2+]i that changes the basal flagellar beat pattern through Ca2+-sensing proteins called calaxins.37,38 Calcium-bound calaxins inhibit the activity of the dynein motors within the axoneme of the cell, resulting in asymmetrical, whip-like bending of the flagellum, commonly referred to as hyperactivation.39 This high-amplitude asymmetric flagellar bending is essential for sperm fertility because it enables them to overcome the protective vestments of the oocyte. The propagation of a Ca2+-induced wave produced from the opening of Ca2+ channels along the flagella is an important step in sperm maturation that triggers hyperactivated motility.40–42 Therefore the identification of the molecule comprising Ca2+ permeation pathways in mammalian spermatozoa represented a significant milestone in understanding the basic molecular principles of sperm activation.8
Discovery of the CatSper Channel and CatSper Complex Organization The founding member of the family of cation channels of sperm (CatSper) was cloned in 2001 as a result of the bioinformatics search for the DNA sequences with similarities to other calcium channels.8 Named
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Catsper1, this gene showed selective expression only in developing spermatozoa,29 and its product was likely to be a pore-forming α subunit of an ion channel related to the two-pore channels and distantly related to transient receptor potential (TRP) channels. CatSper1 contained six transmembrane (TM) segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore resembled that of voltage-gated calcium channels. Male mice deficient in Catsper1 exhibited infertility, but no other phenotypical abnormalities were noted. Female Catsper1-deficient mice were healthy and fertile.8 Later studies revealed that CatSper1 functions as a calcium channel that controls calcium entry to mediate hyperactivated motility, a key flagellar function.14 Three other homologous genes were later identified based on their similarity to Catsper1.11,18,23,43 Similar to CatSper1 they also encode pore-forming subunits that contain six TM segments. In addition, all four are required for fertility, which indicates that CatSper1, CatSper2, CatSper3, and CatSper4 form a functional calcium channel complex that is likely assembled as a tetramer.28 The availability of sperm electrophysiology opened the door to comprehensive characterization of sperm channels.9 CatSper activity was eventually directly recorded and CatSper was established as the principal Ca2+ channel of mouse sperm that is activated by intracellular alkalinization.9 In addition, three other CatSper auxiliary subunits, β, γ, and δ, have been shown to colocalize and associate with the CatSper complex, suggesting that CatSper is a heteromeric ion channel complex of at least seven different subunits.15,22,32 Loss of any single member of the complex is detrimental to male fertility because of their interdependent protein expression in spermatozoa, although CatSperβ- and CatSperγ-null mice have not been generated.8,21,28,30,43,44 CatSper localization is restricted to the principal piece of the sperm tail, and recent work by Chung et al. suggests that CatSper is localized to linearly arranged Ca2+ signaling domains along the length of the flagellum.44 Loss of CatSper expression results in aberrant regulation of these Ca2+ signaling domains, indicating that CatSper not only acts as a mechanism of Ca2+ entry into the cell but also as an organizing, possibly anchoring, unit of various intracellular signaling.
The Role of CatSper in Male Fertility The critical role of the CatSper channel in human fertility was confirmed when patients with mutations within the CatSper1 or CatSper2 genes were also shown to be infertile.11,17,45–49 Human CatSper current was finally recorded in 2010 with the adaptation of the patch-clamp technique for human ejaculated sperm.20,36 Interestingly, comparison of the CatSper currents originated from human and mouse sperm revealed significant differences in CatSper regulation. Although both currents were pH dependent, as human CatSper relies on intracellular alkalinity the same way as murine CatSper does,the voltage–current relationship of human
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CatSper was different: V1/2 human = +85 mV, whereas V1/2 mouse = +11 mV under similar conditions. This indicated that a smaller fraction of human CatSper channels would be open under normal physiological conditions in comparison to murine CatSper.9,50 This suggested that to open, human CatSper requires not only a combination of intracellular alkalinization and membrane voltage but also an additional activator. This activator was later revealed to be the steroid hormone progesterone,50,51 which is produced and released by the cumulus cells surrounding the oocyte. Progesterone has long been known as a stimulator of an immediate increase in intracellular Ca2+ within human sperm cells52,53 that coincides with onset of hyperactivated motility and initiation of the acrosome reaction.52–59 Indeed, extracellular application of progesterone resulted in fast activation of the CatSper channel, and taking into account that sperm cells are transcriptionally silent, it was suggested that the nuclear progesterone receptor was not involved in CatSper activation.50,51 Human CatSper is acutely sensitive to progesterone, with an EC50 of 7 nM.50 Progesterone activates the CatSper by shifting its V1/2 toward more physiological membrane potential.50 Further evidence for CatSper as the sole source of progesterone-induced Ca2+ influx into sperm flagellum came from studies on a CatSper-deficient infertile patient with a homozygous microdeletion in the Catsper2 gene.11,48 These CatSper-less spermatozoa did not produce any progesterone-activated current and even lacked basal CatSper activity.48 Interestingly, rodent CatSper is insensitive to progesterone,50 which indicates the evolutionary diversity of these channels. CatSper activation by progesterone happens in the absence of all intracellular soluble secondary messengers, such as Ca2+, ATP, and guanosine triphosphate, suggesting that the progesterone effect observed acts through a receptor directly associated with the CatSper channel and not through G-proteins or protein kinases. Thus sperm cells represent an example of obscure nongenomic progesterone signaling that also takes place in other tissues.60 Steroid hormones control fundamental organism functions via two pathways: by modulating gene expression in the nucleus and by signaling at the plasma membrane (Fig. 9.2).60–62 The membrane or nongenomic pathway plays a vital role in human sperm cell activation, modulation of pain perception by dorsal root ganglion (DRG) neurons, and oocyte maturation, but the molecular determinants of this pathway are poorly understood. It is possible that DRG neurons and spermatozoa are activated similarly by progesterone and share common molecular features of this cascade. Interestingly, pain thresholds in women tend to be higher during the follicular phase of the menstrual cycle, when levels of estradiol and progesterone are increased. Transcriptionally and translationally silent spermatozoa lack conventional genomic progesterone signaling; therefore they represent an ideal
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Slow: Genomic Steroid Hormone Signaling
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Fast: Membrane Steroid Hormone Signaling
[Ca2+]
Steroid hormone Gene expression nuclear receptor
Ca2+ (calcium) lipid messenger membrane receptor
FIGURE 9.2 Models of genomic and membrane steroid hormone actions. According to the classical model, steroid hormones bind to a genomic (nuclear) receptor that resides in the cytoplasm. The receptor/hormone complex then migrates to the nucleus and initiates and/or alters gene expression. The timescale of such an event is slow and may take several days. According to the nongenomic (membrane) model, steroids bind to their corresponding, and in many cases yet-to-be-identified, membrane receptors, which often are associated with various ion channels or G-protein-coupled receptors. These receptors then trigger ion channel opening or potentially initiate a lipid signaling cascade, resulting in an immediate cellular response (within seconds to minutes).
model to identify the nongenomic progesterone receptor. Although it is still possible that progesterone may bind to CatSper channel directly and hence activate the channel, it may also initiate a lipid cascade through its nongenomic progesterone receptor. The latter would produce a bioactive signaling lipid, thereby altering CatSper activity. This bioactive lipid could be a true CatSper modulator, which also can alter the activity of other ion channels. Although the molecular identity of sperm progesterone receptor is still unknown, the binding site for a cell impermeant analog of this steroid is accessible from the extracellular space.50 Furthermore, evidence for nongenomic progesterone signaling in cells lacking CatSper channel expression13 suggests that the initiation of membrane progesterone signaling is through a yet unidentified protein separate from the CatSper complex.
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Together, voltage, intracellular alkalinization, and progesterone work collectively to regulate Ca2+ influx into human spermatozoa, although other regulatory mechanisms may also exist. Further modulation of CatSper activity has been observed through direct recording of CatSper in the presence of prostaglandins.21,50 Environmental toxins including those known to disrupt the endocrine system have also been shown to induce intracellular Ca2+ elevation via the CatSper mechanism.63,64 The regulation of [Ca2+]i is critical for proper sperm function; thus compounds that directly modulate CatSper or affect [Ca2+]i pose a genuine threat to sperm fertilization potential. Alternatively, exclusive CatSper expression in sperm cells makes this channel an excellent target for novel contraceptives for both women and men.
Evolutionary Diversity of CatSper Channels The CatSper channel is evolutionarily conserved in the genome of species from mammals to invertebrates such as sea urchins and sea squirts. However, the CatSper genes are lost in teleosts, amphibians, and birds.13 So far, the only species with an electrophysiologically confirmed CatSper current were human and mouse. Characterization of Ca2+ influx within sperm of different species has been attempted using optical methods including fluorescent Ca2+ dyes and motility studies. For instance, bovine65 and equine66 sperm are both sensitive to intracellular alkalization, resulting in an influx of Ca2+ into the cell. However, the presence of a functional CatSper channel does not guarantee that it is regulated similarly in different species. Murine CatSper is insensitive to the classical activators of human CatSper, progesterone and prostaglandins, which may indicate that the spermatozoa of different species evolved their CatSpers to adjust to specific activators of the corresponding female reproductive tract. Additional work is needed to confirm the molecular arrangement of the sperm Ca2+ channel complexes within these species and characterize the role of these channels in sperm function.
VOLTAGE-GATED PROTON CHANNEL OF SPERM Intracellular pH is a key regulator of many sperm physiological processes, including initiation of motility, capacitation, hyperactivation, chemotaxis, and acrosome reaction. Even the basal sperm motility is pH sensitive because dynein’s ability to hydrolyze ATP and provide axonemal bending greatly increases with the rise of intracellular pH (pHi). The motile sperm flagellum constantly generates intracellular protons via glycolysis, ATP hydrolysis, and proton/calcium exchange.21 The faster a flagellum moves, the more acidic it becomes. In 1983 Babcock et al.67,68
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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.67,68 Later studies focused on the role of sperm-specific Na+/H+ (sNHE) and Cl − /HCO3 − exchangers as potential mechanisms for intracellular alkalization in rodent spermatozoa.69–71 An intriguing model was recently proposed72 suggesting that sNHE may function as a hyperpolarization activated proton extrusion mechanism because it possesses a putative voltage sensor.69,73–75 In 2010 direct electrophysiological recordings of human sperm revealed a large voltage-activated H+ current20 that was sensitive to Zn2+.20 It was shown that this sperm proton channel closely resembles the voltage-gated channel Hv1.76,77 Interestingly, Hv1 was highly expressed in the flagellum, indicating a potential role in the regulation of CatSper.20 Although Hv1 is ubiquitously expressed and plays an important role in several physiological processes such as the innate immune system, apoptosis, and cancer metastasis, the presence of Hv1 in human spermatozoa was intriguing. Hv1 is not a true ion channel but rather a hybrid between a transporter and an ion channel without the pore that provides rapid movement of protons across a lipid bilayer via a voltage-gated mechanism.78–80
Hv1: Principles of Proton Transport The first electrophysiological recording of voltage-dependent proton current in snail neurons was performed by Roger Thomas and Robert Meech in 1982.82 Later studies, primarily done by the Thomas DeCoursey group,83–85 found similar currents in other cell types.86 It was not until 2006 when two publications—one from the David Clapham group76 and another from the Yasushi Okamura group77—reported the molecular identity of this channel to be voltage-gated proton channel Hv1. Molecular and electrophysiological studies of Hv1 have indicated four distinct features: (1) the channel is activated by membrane depolarization and intracellular acidification, (2) is inhibited by zinc, (3) is H+ selective, and (4) it lacks pore domain. Hv1 forms four TM spanning segments homologous in structure to voltage-sensor of voltage-gated cation channels.76,77 Unlike the traditional voltage-gated ion channel, Hv1 does not possess a classical selectivity pore region. Several models for the proton conductance pathway have been proposed.78,79,87 Among them the “water wire” model79 suggests the movement of protons via the Grotthus mechanism from the intracellular water vestibules to the extracellular vestibule formed by the TM portions of the channel. Two vestibules are connected by a narrow bottleneck79 where proton selectivity occurs via a highly conserved and unique aspartate moiety (Asp112 in human Hv188). Another model78 suggests that the Hv1 closed state favors electrostatic interactions between hydrophobic residues of TM segments
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and pulls them together to form a hydrophobic plug. This hydrophobic layer is formed at the center of the voltage-sensing domain and prevents proton permeation. The latter occurs once the plug is removed by a voltage-dependent rearrangement of TMs and is followed by insertion of the protonatable residues in the center of the channel.78 The recent crystal structure of the Hv1 in the resting state sheds80 light on the possible mechanism of H+ permeation, suggesting that a combination of two hydrophobic plugs and a protonatable aspartate is required for proton movement. Hv1 forms a functional dimer in the plasma membrane through a coil–coil interaction of the C-termini89–91; however, each Hv1 subunit can function independently as a channel.
Human Sperm Hv1 At the time of ejaculation, spermatozoa are combined with the seminal plasma, which contains high concentrations of zinc (∼2 mM)36 and as such directly inhibits Hv1 function by binding to two histidine residues that stabilize the channel in the closed state.92 As sperm progress through the female reproductive tract, divalent zinc is chelated by proteins in the oviductal fluid, resulting in gradual activation of the Hv1 channel.36,93 To fertilize an oocyte, sperm cells have to undergo a process known as capacitation, which comprises several physiological changes including intracellular alkalization. In humans this could be triggered via proton extrusionthrough Hv120, especially because Hv1 is activated by capacitation.20 In contrast to human sperm, mouse spermatozoa do not possess Hv1, and, not surprisingly, Hv1−/− mice are fertile. Natural mutations in Hv1 appear to be extremely rare, with only one case of a single substitution mutation being reported in humans,94 which was assessed only in airway epithelial cells. The role of this channel in human fertility is still unclear and will remain so until an Hv1-deficient patient can be identified. In species that lack sperm Hv1, it is still unclear how intracellular pH is regulated in these cells, although sNHE remainsas possible mechanism.
OTHER ION CHANNELS OF SPERM Sperm membrane potential is important for fertility because both Hv1 and CatSper channels are voltage dependent (Fig. 9.3). As in other cells, sperm membrane potential is defined by the gradients of K+, Na+, and Cl−, with potassium channels playing a crucial role in its regulation. Epididymal murine spermatozoa keep their membrane potential slightly depolarized at approximately −40 mV; however, they tend to hyperpolarize up to −60 mV upon in vitro capacitation.95 This effect is attributed to
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FIGURE 9.3 Flagellar ion channels of sperm. Murine potassium channel (KSper) is composed of the Slo3 channel and associated subunit LRRC52 (gamma) as well as possible beta subunits. The identity of human KSper is still under debate but seems to share characteristics of the Slo family of ion channels. Cation channels of sperm (CatSper) in mouse and human share the same molecular composition consisting of at least seven different subunits to form calcium-permeable channel. Proton conductance via the Hv1 channel (HSper) is only detected in human sperm cells. Both CatSper and Hv1 require membrane depolarization to function. This could be achieved by inhibition of KSper. In addition, CatSper is activated by intracellular alkalinization that could be provided by the Hv1. By moving protons out of the flagellum, Hv1 further hyperpolarizes the cell and provides negative feedback inhibiting both Hv1 and CatSper. This model misses the fourth member, yet-to-be-identified “depolarizing channel of sperm” or “DSper.” This hypothetical DSper could be activated by either membrane hyperpolarization as proposed for sperm-specific Na+/H+ (sNHE)87 or other mechanisms. DSper would provide positive net charge influx resulting in depolarization, consequent activation of Hv1/CatSper, and sperm hyperactivation. Reproduced from Miller MR, Mansell SA, Meyers SA, Lishko PV. Cell Calcium. 2015 Jul;58(1):105-13, Elsevier.
specific potassium permeability, or KSper, and two members of the Slo family of potassium channels have been recently proposed to play a role in this process.27,31,96–103 Slo3 (Kcnu1), a pH-sensitive, calcium-independent, and weakly voltage-sensitive channel, has been identified as the principal potassium channel in murine sperm.31,98,99,101–105 Although Slo3 was also expected to form the potassium channel of human sperm, recent recording from ejaculated human spermatozoa indicates that in contrast to murine sperm, human sperm potassium current (KSper) is less pH
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dependent and sensitive to [Ca2+]i.24 Furthermore, it could also be inhibited by progesterone.12,24 From these biophysical properties as well as the pharmacological profile, human KSper resembled the calcium-activated big conductance potassium channel Slo1 (KCNMA1).24 Recently other models were proposed, suggesting that human capacitated spermatozoa possess a different type of Slo channel25 or even a modified version of Slo3 that is calcium-sensitive and weakly pH-dependent,12 an unusual set of properties for this type of ion channel. Slo channels exist as tetrameric complexes of alpha subunits (Slo1 or Slo3) and auxiliary gamma subunits (LRCC26 or LRCC52); additional beta subunits can change their biophysical and pharmacological properties.100,106,107 Uncovering the precise molecular identity of the human potassium channel remains an essential task for understanding the regulation of potassium homeostasis in human spermatozoa. Navarro et al. recently reported the presence of an ATP-gated P2X2 ion channel (also known as purinergic receptor P2X, ligand gated ion channel, 2) that is cation nonselective and originates from the midpiece of murine sperm cells.108 P2rx2-deficient male mice are fertile and have normal sperm morphology and other sperm parameters; however, their sperm lack ATP-evoked currents and fertility of P2rx2−/− males declines with frequent mating.108 Calcium-activated chloride channels (anoctamins) have been recently found in human sperm,109 but not in mouse,34 as well as aquaporins, which are water channels that are required for sperm osmoadaptation.110 Several members of the TRP ion channel family111–113 were thought to function in mammalian sperm cells.16,114 These include TRPM8, TRPV1, TRPA1, and others. However, mice deficient in TRPV1–4, TRPA1, and TRPM8 have no obvious defects in sperm morphology or male fertility.111,112 Moreover, CatSper promiscuity toward high concentrations of exogenous activators, such as menthols, may account for the mistaken identity of TRPM8 in human spermatozoa.115 However, it is possible that other species that do not have CatSper activity rely on different flagellar channels; therefore the role of TRP channels in sperm is yet to be determined.
CONCLUSIONS On their route to the egg, mammalian spermatozoa encounter multiple obstacles: viscous mucus, the narrow lumen of the uterotubal junction, the complex maze formed by the epithelial folds of the fallopian tubes, and finally the protective layers of the egg. To overcome these barriers, the sperm cell must sense the cues released by the egg and adapt its swimming behavior. Sperm can achieve this by increasing the amplitude and driving force of their tail bending, changing their direction of movement,
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and releasing special enzymes to dissolve the egg’s protective vestments. Such sperm responses depend upon activity of the sperm ion channels that open in response to environmental cues within the female reproductive tract. This in turn changes conductance of the sperm plasma membrane and sperm behavior. Influx of calcium through the flagellar calcium channel CatSper results in the flagellar bending and hyperactivation of motility. In turn, CatSper is activated by intracellular alkalinization, provided by perhaps the sNHE or Hv1 channel (in human sperm) and membrane depolarization, which results from KSper inhibition by progesterone. The same compartmental flagellar localization of sperm ion channels provides fine-tuned regulation of sperm motility. It is possible that many cases of idiopathic male infertility can be attributed to malfunctioning of sperm ion channels and their regulators.
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40. White DR, Aitken RJ. Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility. Gamete Res 1989;22(2):163–77. 41. Suarez SS, Varosi SM, Dai X. Intracellular calcium increases with hyperactivation in intact, moving hamster sperm and oscillates with the flagellar beat cycle. Proc Natl Acad Sci USA 1993;90(10):4660–4. 42. Ho HC, Granish KA, Suarez SS. Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP. Dev Biol 2002;250(1):208–17. 43. Carlson AE, Quill TA, Westenbroek RE, Schuh SM, Hille B, Babcock DF. Identical phenotypes of CatSper1 and CatSper2 null sperm. J Biol Chem 2005;280(37):32238–44. 44. Chung JJ, Shim SH, Everley RA, Gygi SP, Zhuang X, Clapham DE. Structurally distinct Ca(2+) signaling domains of sperm flagella orchestrate tyrosine phosphorylation and motility. Cell 2014;157(4):808–22. 45. Zhang Y, Malekpour M, Al-Madani N, et al. Sensorineural deafness and male infertility: a contiguous gene deletion syndrome. J Med Genet 2007;44(4):233–40. 46. Avenarius MR, Hildebrand MS, Zhang Y, et al. Human male infertility caused by mutations in the CATSPER1 channel protein. Am J Hum Genet 2009;84(4):505–10. 47. Hildebrand MS, Avenarius MR, Smith RJH. CATSPER-related male infertility. 2009. 48. Smith JF, Syritsyna O, Fellous M, et al. Disruption of the principal, progesterone-activated sperm Ca2+ channel in a CatSper2-deficient infertile patient. Proc Natl Acad Sci USA 2013;110(17):6823–8. 49. Jaiswal D, Singh V, Dwivedi US, Trivedi S, Singh K. Chromosome microarray analysis: a case report of infertile brothers with CATSPER gene deletion. Gene 2014;542(2): 263–5. 50. Lishko PV, Botchkina IL, Kirichok Y. Progesterone activates the principal Ca2+ channel of human sperm. Nature 2011;471:387–91. 51. Strünker T, Goodwin N, Brenker C, et al. The CatSper channel mediates progesteroneinduced Ca2+ influx in human sperm. Nature 2011;471:382–6. 52. Blackmore PF, Beebe SJ, Danforth DR, Alexander N. Progesterone and 17 alphahydroxyprogesterone. Novel stimulators of calcium influx in human sperm. J Biol Chem 1990;265(3):1376–80. 53. Baldi E, Casano R, Falsetti C, Krausz C, Maggi M, Forti G. Intracellular calcium accumulation and responsiveness to progesterone in capacitating human spermatozoa. J Androl 1991;12(5):323–30. 54. Osman RA, Andria ML, Jones AD, Meizel S. Steroid induced exocytosis: the human sperm acrosome reaction. Biochem Biophys Res Commun 1989;160(2):828–33. 55. Ralt D, Goldenberg M, Fetterolf P, et al. Sperm attraction to a follicular factor(s) correlates with human egg fertilizability. Proc Natl Acad Sci USA 1991;88(7):2840–4. 56. Uhler ML, Leung A, Chan SY, Wang C. Direct effects of progesterone and antiprogesterone on human sperm hyperactivated motility and acrosome reaction. Fertil Steril 1992;58(6):1191–8. 57. Roldan ER, Murase T, Shi QX. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science 1994;266(5190):1578–81. 58. Schaefer M, Hofmann T, Schultz G, Gudermann T. A new prostaglandin E receptor mediates calcium influx and acrosome reaction in human spermatozoa. Proc Natl Acad Sci USA 1998;95(6):3008–13. 59. Shimizu Y, Yorimitsu A, Maruyama Y, Kubota T, Aso T, Bronson RA. Prostaglandins induce calcium influx in human spermatozoa. Mol Hum Reprod 1998;4(6):555–61. 60. Luconi M, Francavilla F, Porazzi I, Macerola B, Forti G, Baldi E. Human spermatozoa as a model for studying membrane receptors mediating rapid nongenomic effects of progesterone and estrogens. Steroids 2004;69(8–9):553–9. 61. Revelli A, Massobrio M, Tesarik J. Nongenomic actions of steroid hormones in reproductive tissues. Endocr Rev 1998;19(1):3–17.
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62. Wendler A, Albrecht C, Wehling M. Nongenomic actions of aldosterone and progesterone revisited. Steroids 2012;77(10):1002–6. 63. Schiffer C, Muller A, Egeberg DL, et al. Direct action of endocrine disrupting chemicals on human sperm. EMBO Rep 2014;15(7):758–65. 64. Tavares RS, Mansell S, Barratt CL, Wilson SM, Publicover SJ, Ramalho-Santos J. p,p′DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations. Hum Reprod 2013;28(12):3167–77. 65. Marquez B, Suarez SS. Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx. Biol Reprod 2007;76(4):660–5. 66. Loux SC, Crawford KR, Ing NH, et al. CatSper and the relationship of hyperactivated motility to intracellular calcium and pH kinetics in equine sperm. Biol Reprod 2013;89(5):123. 67. Babcock DF. Examination of the intracellular ionic environment and of ionophore action by null point measurements employing the fluorescein chromophore. J Biol Chem 1983;258(10):6380–9. 68. Babcock DF, Rufo Jr GA, Lardy HA. Potassium-dependent increases in cytosolic pH stimulate metabolism and motility of mammalian sperm. Proc Natl Acad Sci USA 1983;80(5):1327–31. 69. Garcia MA, Meizel S. Regulation of intracellular pH in capacitated human spermatozoa by a Na+/H+ exchanger. Mol Reprod Dev 1999;52(2):189–95. 70. Woo AL, James PF, Lingrel JB. Roles of the Na,K-ATPase alpha4 isoform and the Na+/ H+ exchanger in sperm motility. Mol Reprod Dev 2002;62(3):348–56. 71. Zeng Y, Oberdorf JA, Florman HM. pH regulation in mouse sperm: identification of Na(+)-, Cl(−)-, and HCO3(−)-dependent and arylaminobenzoate-dependent regulatory mechanisms and characterization of their roles in sperm capacitation. Dev Biol 1996;173(2):510–20. 72. Chavez JC, Ferreira Gregorio J, Butler A, et al. SLO3 K+ Channels control calcium entry through CATSPER channels in sperm. J Biol Chem 2014;289(46). 73. Donowitz M, Ming Tse C, Fuster D. SLC9/NHE gene family, a plasma membrane and organellar family of Na(+)/H(+) exchangers. Mol Aspects Med 2013;34(2–3):236–51. 74. Wang D, Hu J, Bobulescu IA, et al. A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC). Proc Natl Acad Sci USA 2007;104(22):9325–30. 75. Wang D, King SM, Quill TA, Doolittle LK, Garbers DL. A new sperm-specific Na+/H+ exchanger required for sperm motility and fertility. Nat Cell Biol 2003;5(12):1117–22. 76. Ramsey IS, Moran MM, Chong JA, Clapham DE. A voltage-gated proton-selective channel lacking the pore domain. Nature 2006;440(7088):1213–6. 77. Sasaki M, Takagi M, Okamura Y. A voltage sensor-domain protein is a voltage-gated proton channel. Science 2006;312(5773):589–92. 78. Chamberlin A, Qiu F, Rebolledo S, Wang Y, Noskov SY, Larsson HP. Hydrophobic plug functions as a gate in voltage-gated proton channels. Proc Natl Acad Sci USA 2014;111(2):E273–82. 79. Ramsey IS, Mokrab Y, Carvacho I, Sands ZA, Sansom MS, Clapham DE. An aqueous H+ permeation pathway in the voltage-gated proton channel Hv1. Nat Struct Mol Biol 2010;17(7):869–75. 80. Takeshita K, Sakata S, Yamashita E, et al. X-ray crystal structure of voltage-gated proton channel. Nat Struct Mol Biol 2014;21(4):352–7. 81. Fogel M, Hastings JW. Bioluminescence: mechanism and mode of control of scintillon activity. Proc Natl Acad Sci USA 1972;69(3):690–3. 82. Thomas RC, Meech RW. Hydrogen ion currents and intracellular pH in depolarized voltage-clamped snail neurones. Nature 1982;299(5886):826–8. 83. DeCoursey TE. Hydrogen ion currents in rat alveolar epithelial cells. Biophys J 1991;60(5):1243–53.
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84. DeCoursey TE, Cherny VV. Potential, pH, and arachidonate gate hydrogen ion currents in human neutrophils. Biophys J 1993;65(4):1590–8. 85. Cherny VV, Markin VS, DeCoursey TE. The voltage-activated hydrogen ion conductance in rat alveolar epithelial cells is determined by the pH gradient. J Gen Physiol 1995;105(6):861–96. 86. DeCoursey TE. Voltage-gated proton channels: molecular biology, physiology, and pathophysiology of the H(V) family. Physiol Rev 2013;93(2):599–652. 87. Hong L, Pathak MM, Kim IH, Ta D, Tombola F. Voltage-sensing domain of voltagegated proton channel Hv1 shares mechanism of block with pore domains. Neuron 2013;77(2):274–87. 88. Musset B, Smith SM, Rajan S, Morgan D, Cherny VV, Decoursey TE. Aspartate 112 is the selectivity filter of the human voltage-gated proton channel. Nature 2011;480(7376):273–7. 89. Koch HP, Kurokawa T, Okochi Y, Sasaki M, Okamura Y, Larsson HP. Multimeric nature of voltage-gated proton channels. Proc Natl Acad Sci USA 2008;105(26):9111–6. 90. Lee SY, Letts JA, Mackinnon R. Dimeric subunit stoichiometry of the human voltagedependent proton channel Hv1. Proc Natl Acad Sci USA 2008;105(22):7692–5. 91. Tombola F, Ulbrich MH, Isacoff EY. The voltage-gated proton channel Hv1 has two pores, each controlled by one voltage sensor. Neuron 2008;58(4):546–56. 92. Cherny VV, DeCoursey TE. pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations. J Gen Physiol 1999;114(6):819–38. 93. Lu J, Stewart AJ, Sadler PJ, Pinheiro TJ, Blindauer CA. Albumin as a zinc carrier: properties of its high-affinity zinc-binding site. Biochem Soc Trans 2008;36(Pt 6):1317–21. 94. Iovannisci D, Illek B, Fischer H. Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation, M91T. J Gen Physiol 2010;136(1):35–46. 95. Munoz-Garay C, De la Vega-Beltran JL, Delgado R, Labarca P, Felix R, Darszon A. Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation. Dev Biol 2001;234(1):261–74. 96. Navarro B, Kirichok Y, Clapham DE. KSper, a pH-sensitive K+ current that controls sperm membrane potential. Proc Natl Acad Sci USA 2007;104(18):7688–92. 97. Nishigaki T, Jose O, Gonzalez-Cota AL, Romero F, Trevino CL, Darszon A. Intracellular pH in sperm physiology. Biochem Biophys Res Commun 2014;450(3). 98. Santi CM, Martinez-Lopez P, de la Vega-Beltran JL, et al. The SLO3 sperm-specific potassium channel plays a vital role in male fertility. FEBS Lett 2010;584(5):1041–6. 99. Tang QY, Zhang Z, Xia XM, Lingle CJ. Block of mouse Slo1 and Slo3 K+ channels by CTX, IbTX, TEA, 4-AP and quinidine. Channels (Austin, Tex) 2010;4(1):22–41. 100. Yang C, Zeng XH, Zhou Y, Xia XM, Lingle CJ. LRRC52 (leucine-rich-repeat-containing protein 52), a testis-specific auxiliary subunit of the alkalization-activated Slo3 channel. Proc Natl Acad Sci USA 2011;108(48):19419–24. 101. Yang CT, Zeng XH, Xia XM, Lingle CJ. Interactions between beta subunits of the KCNMB family and Slo3: beta4 selectively modulates Slo3 expression and function. PLoS One 2009;4(7):e6135. 102. Zeng XH, Yang C, Kim ST, Lingle CJ, Xia XM. Deletion of the Slo3 gene abolishes alkalization-activated K+ current in mouse spermatozoa. Proc Natl Acad Sci USA 2011;108(14):5879–84. 103. Zhang X, Zeng X, Lingle CJ. Slo3 K+ channels: voltage and pH dependence of macroscopic currents. J Gen Physiol 2006;128(3):317–36. 104. Leonetti MD, Yuan P, Hsiung Y, Mackinnon R. Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel. Proc Natl Acad Sci USA 2012;109(47):19274–9. 105. Tang QY, Zhang Z, Xia J, Ren D, Logothetis DE. Phosphatidylinositol 4,5-bisphosphate activates Slo3 currents and its hydrolysis underlies the epidermal growth factorinduced current inhibition. J Biol Chem 2010;285(25):19259–66.
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106. Behrens R, Nolting A, Reimann F, Schwarz M, Waldschutz R, Pongs O. hKCNMB3 and hKCNMB4, cloning and characterization of two members of the large-conductance calcium-activated potassium channel beta subunit family. FEBS Lett 2000;474(1):99–106. 107. Yan J, Aldrich RW. LRRC26 auxiliary protein allows BK channel activation at resting voltage without calcium. Nature 2010;466(7305):513–6. 108. Navarro B, Miki K, Clapham DE. ATP-activated P2X2 current in mouse spermatozoa. Proc Natl Acad Sci USA 2011;103(34). 109. Orta G, Ferreira G, Jose O, Trevino CL, Beltran C, Darszon A. Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction. J Physiol 2012;590(Pt 11):2659–75. 110. Chen Q, Peng H, Lei L, et al. Aquaporin3 is a sperm water channel essential for postcopulatory sperm osmoadaptation and migration. Cell Res 2011;21(6):922–33. 111. Clapham DE, Montell C, Schultz G, Julius D, International Union of Pharmacology. International Union of Pharmacology. XLIII. Compendium of voltage-gated ion channels: transient receptor potential channels. Pharmacol Rev 2003;55(4):591–6. 112. Julius D. TRP channels and pain. Annu Rev Cell Dev Biol 2013;29:355–84. 113. Montell C, Birnbaumer L, Flockerzi V, et al. A unified nomenclature for the superfamily of TRP cation channels. Mol Cell 2002;9(2):229–31. 114. Jungnickel MK, Marrero H, Birnbaumer L, Lemos JR, Florman HM. Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3. Nat Cell Biol 2001;3(5):499–502. 115. Brenker C, Goodwin N, Weyand I, et al. The CatSper channel: a polymodal chemosensor in human sperm. EMBO J 2012;31(7):1654–65.
9
The Role of Sperm Ion Channels in Reproduction P.V. Lishko, M.R. Miller, S.A. Mansell University of California, Berkeley, CA, United States
SPERM CELL: MORPHOLOGY AND ORGANIZATION Sperm cells are terminally differentiated and are thought to be transcriptionally and translationally silent, meaning that spermatozoa are largely unable to synthesize new mRNA or translate it into new polypeptides.2,3 They are divided into two easily recognized parts: the head and the tail (flagellum). The sperm head comprises a condensed nucleus, a redundant nuclear envelope, and an acrosomal vesicle. The shape and size of the sperm head differs dramatically among species: from the spatula-like head in primates and ruminants to the hook-like pointed head that usually defines rodent sperm. Motility originates from the flagellum and is powered by adenosine triphosphate (ATP) hydrolysis within the sperm tail. The flagellum has a specialized cytoskeleton called an axoneme surrounded by specialized structural components and is subdivided into three functional parts: a mitochondria-containing midpiece, a principal piece, and the endpiece (Fig. 9.1). The flagellum is in essence a motile cilium4,5 with a flagellar plasma membrane tightly attached to all underlying structures along the sperm body. This arrangement provides spermatozoa with a ridged structure to which membrane proteins, including ion channels, can be tethered to the fibrous sheath6 to ensure their strict compartmentalization.7 Sperm basal motility depends on three crucial factors: the presence of ATP, a low concentration of intracellular calcium [Ca2+]i,, and normal to alkaline intracellular pH. Intraflagellar ATP is generated during glycolysis and oxidative phosphorylation whereas the intraflagellar concentration of protons and calcium is controlled by ion channels and transporters. Because of its high motility, powered by glycolysis and oxidative phosphorylation, the sperm flagellum quickly acidifies. The removal of protons from the sperm
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FIGURE 9.1 Mammalian spermatozoon. Schematic representation of spermatozoon with cellular compartments labeled and distribution of species-specific ion channels found within each section. CatSper, cation channels of sperm; sNHE, sperm-specific Na+/H+; TMEM16, Transmembrane protein 16. Reproduced from Miller MR, Mansell SA, Meyers SA, Lishko PV. Cell Calcium. 2015 Jul;58(1):105-13, Elsevier.
flagellum is an essential because the axonemal motility is inhibited by acidic pH. To expel protons and support motility, spermatozoa possess specialized proton channels and/or transporters. With the help of bioinformatic approaches,8 the whole-cell sperm patch clamp method,9,10 and several genetic models,8–35 sperm ion channels have been comprehensively characterized, among which calcium, potassium, proton, and nonselective and various ligand-gated channels have been identified.21,27,30,36
CATSPER CHANNEL: A PRINCIPAL CALCIUM CHANNEL OF SPERM In spermatozoa, swimming behavior is controlled by rises in flagellar [Ca2+]i that changes the basal flagellar beat pattern through Ca2+-sensing proteins called calaxins.37,38 Calcium-bound calaxins inhibit the activity of the dynein motors within the axoneme of the cell, resulting in asymmetrical, whip-like bending of the flagellum, commonly referred to as hyperactivation.39 This high-amplitude asymmetric flagellar bending is essential for sperm fertility because it enables them to overcome the protective vestments of the oocyte. The propagation of a Ca2+-induced wave produced from the opening of Ca2+ channels along the flagella is an important step in sperm maturation that triggers hyperactivated motility.40–42 Therefore the identification of the molecule comprising Ca2+ permeation pathways in mammalian spermatozoa represented a significant milestone in understanding the basic molecular principles of sperm activation.8
Discovery of the CatSper Channel and CatSper Complex Organization The founding member of the family of cation channels of sperm (CatSper) was cloned in 2001 as a result of the bioinformatics search for the DNA sequences with similarities to other calcium channels.8 Named
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Catsper1, this gene showed selective expression only in developing spermatozoa,29 and its product was likely to be a pore-forming α subunit of an ion channel related to the two-pore channels and distantly related to transient receptor potential (TRP) channels. CatSper1 contained six transmembrane (TM) segments, making it more similar to the voltage-gated potassium channels, but the ion selectivity pore resembled that of voltage-gated calcium channels. Male mice deficient in Catsper1 exhibited infertility, but no other phenotypical abnormalities were noted. Female Catsper1-deficient mice were healthy and fertile.8 Later studies revealed that CatSper1 functions as a calcium channel that controls calcium entry to mediate hyperactivated motility, a key flagellar function.14 Three other homologous genes were later identified based on their similarity to Catsper1.11,18,23,43 Similar to CatSper1 they also encode pore-forming subunits that contain six TM segments. In addition, all four are required for fertility, which indicates that CatSper1, CatSper2, CatSper3, and CatSper4 form a functional calcium channel complex that is likely assembled as a tetramer.28 The availability of sperm electrophysiology opened the door to comprehensive characterization of sperm channels.9 CatSper activity was eventually directly recorded and CatSper was established as the principal Ca2+ channel of mouse sperm that is activated by intracellular alkalinization.9 In addition, three other CatSper auxiliary subunits, β, γ, and δ, have been shown to colocalize and associate with the CatSper complex, suggesting that CatSper is a heteromeric ion channel complex of at least seven different subunits.15,22,32 Loss of any single member of the complex is detrimental to male fertility because of their interdependent protein expression in spermatozoa, although CatSperβ- and CatSperγ-null mice have not been generated.8,21,28,30,43,44 CatSper localization is restricted to the principal piece of the sperm tail, and recent work by Chung et al. suggests that CatSper is localized to linearly arranged Ca2+ signaling domains along the length of the flagellum.44 Loss of CatSper expression results in aberrant regulation of these Ca2+ signaling domains, indicating that CatSper not only acts as a mechanism of Ca2+ entry into the cell but also as an organizing, possibly anchoring, unit of various intracellular signaling.
The Role of CatSper in Male Fertility The critical role of the CatSper channel in human fertility was confirmed when patients with mutations within the CatSper1 or CatSper2 genes were also shown to be infertile.11,17,45–49 Human CatSper current was finally recorded in 2010 with the adaptation of the patch-clamp technique for human ejaculated sperm.20,36 Interestingly, comparison of the CatSper currents originated from human and mouse sperm revealed significant differences in CatSper regulation. Although both currents were pH dependent, as human CatSper relies on intracellular alkalinity the same way as murine CatSper does,the voltage–current relationship of human
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CatSper was different: V1/2 human = +85 mV, whereas V1/2 mouse = +11 mV under similar conditions. This indicated that a smaller fraction of human CatSper channels would be open under normal physiological conditions in comparison to murine CatSper.9,50 This suggested that to open, human CatSper requires not only a combination of intracellular alkalinization and membrane voltage but also an additional activator. This activator was later revealed to be the steroid hormone progesterone,50,51 which is produced and released by the cumulus cells surrounding the oocyte. Progesterone has long been known as a stimulator of an immediate increase in intracellular Ca2+ within human sperm cells52,53 that coincides with onset of hyperactivated motility and initiation of the acrosome reaction.52–59 Indeed, extracellular application of progesterone resulted in fast activation of the CatSper channel, and taking into account that sperm cells are transcriptionally silent, it was suggested that the nuclear progesterone receptor was not involved in CatSper activation.50,51 Human CatSper is acutely sensitive to progesterone, with an EC50 of 7 nM.50 Progesterone activates the CatSper by shifting its V1/2 toward more physiological membrane potential.50 Further evidence for CatSper as the sole source of progesterone-induced Ca2+ influx into sperm flagellum came from studies on a CatSper-deficient infertile patient with a homozygous microdeletion in the Catsper2 gene.11,48 These CatSper-less spermatozoa did not produce any progesterone-activated current and even lacked basal CatSper activity.48 Interestingly, rodent CatSper is insensitive to progesterone,50 which indicates the evolutionary diversity of these channels. CatSper activation by progesterone happens in the absence of all intracellular soluble secondary messengers, such as Ca2+, ATP, and guanosine triphosphate, suggesting that the progesterone effect observed acts through a receptor directly associated with the CatSper channel and not through G-proteins or protein kinases. Thus sperm cells represent an example of obscure nongenomic progesterone signaling that also takes place in other tissues.60 Steroid hormones control fundamental organism functions via two pathways: by modulating gene expression in the nucleus and by signaling at the plasma membrane (Fig. 9.2).60–62 The membrane or nongenomic pathway plays a vital role in human sperm cell activation, modulation of pain perception by dorsal root ganglion (DRG) neurons, and oocyte maturation, but the molecular determinants of this pathway are poorly understood. It is possible that DRG neurons and spermatozoa are activated similarly by progesterone and share common molecular features of this cascade. Interestingly, pain thresholds in women tend to be higher during the follicular phase of the menstrual cycle, when levels of estradiol and progesterone are increased. Transcriptionally and translationally silent spermatozoa lack conventional genomic progesterone signaling; therefore they represent an ideal
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Fast: Membrane Steroid Hormone Signaling
[Ca2+]
Steroid hormone Gene expression nuclear receptor
Ca2+ (calcium) lipid messenger membrane receptor
FIGURE 9.2 Models of genomic and membrane steroid hormone actions. According to the classical model, steroid hormones bind to a genomic (nuclear) receptor that resides in the cytoplasm. The receptor/hormone complex then migrates to the nucleus and initiates and/or alters gene expression. The timescale of such an event is slow and may take several days. According to the nongenomic (membrane) model, steroids bind to their corresponding, and in many cases yet-to-be-identified, membrane receptors, which often are associated with various ion channels or G-protein-coupled receptors. These receptors then trigger ion channel opening or potentially initiate a lipid signaling cascade, resulting in an immediate cellular response (within seconds to minutes).
model to identify the nongenomic progesterone receptor. Although it is still possible that progesterone may bind to CatSper channel directly and hence activate the channel, it may also initiate a lipid cascade through its nongenomic progesterone receptor. The latter would produce a bioactive signaling lipid, thereby altering CatSper activity. This bioactive lipid could be a true CatSper modulator, which also can alter the activity of other ion channels. Although the molecular identity of sperm progesterone receptor is still unknown, the binding site for a cell impermeant analog of this steroid is accessible from the extracellular space.50 Furthermore, evidence for nongenomic progesterone signaling in cells lacking CatSper channel expression13 suggests that the initiation of membrane progesterone signaling is through a yet unidentified protein separate from the CatSper complex.
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Together, voltage, intracellular alkalinization, and progesterone work collectively to regulate Ca2+ influx into human spermatozoa, although other regulatory mechanisms may also exist. Further modulation of CatSper activity has been observed through direct recording of CatSper in the presence of prostaglandins.21,50 Environmental toxins including those known to disrupt the endocrine system have also been shown to induce intracellular Ca2+ elevation via the CatSper mechanism.63,64 The regulation of [Ca2+]i is critical for proper sperm function; thus compounds that directly modulate CatSper or affect [Ca2+]i pose a genuine threat to sperm fertilization potential. Alternatively, exclusive CatSper expression in sperm cells makes this channel an excellent target for novel contraceptives for both women and men.
Evolutionary Diversity of CatSper Channels The CatSper channel is evolutionarily conserved in the genome of species from mammals to invertebrates such as sea urchins and sea squirts. However, the CatSper genes are lost in teleosts, amphibians, and birds.13 So far, the only species with an electrophysiologically confirmed CatSper current were human and mouse. Characterization of Ca2+ influx within sperm of different species has been attempted using optical methods including fluorescent Ca2+ dyes and motility studies. For instance, bovine65 and equine66 sperm are both sensitive to intracellular alkalization, resulting in an influx of Ca2+ into the cell. However, the presence of a functional CatSper channel does not guarantee that it is regulated similarly in different species. Murine CatSper is insensitive to the classical activators of human CatSper, progesterone and prostaglandins, which may indicate that the spermatozoa of different species evolved their CatSpers to adjust to specific activators of the corresponding female reproductive tract. Additional work is needed to confirm the molecular arrangement of the sperm Ca2+ channel complexes within these species and characterize the role of these channels in sperm function.
VOLTAGE-GATED PROTON CHANNEL OF SPERM Intracellular pH is a key regulator of many sperm physiological processes, including initiation of motility, capacitation, hyperactivation, chemotaxis, and acrosome reaction. Even the basal sperm motility is pH sensitive because dynein’s ability to hydrolyze ATP and provide axonemal bending greatly increases with the rise of intracellular pH (pHi). The motile sperm flagellum constantly generates intracellular protons via glycolysis, ATP hydrolysis, and proton/calcium exchange.21 The faster a flagellum moves, the more acidic it becomes. In 1983 Babcock et al.67,68
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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.67,68 Later studies focused on the role of sperm-specific Na+/H+ (sNHE) and Cl − /HCO3 − exchangers as potential mechanisms for intracellular alkalization in rodent spermatozoa.69–71 An intriguing model was recently proposed72 suggesting that sNHE may function as a hyperpolarization activated proton extrusion mechanism because it possesses a putative voltage sensor.69,73–75 In 2010 direct electrophysiological recordings of human sperm revealed a large voltage-activated H+ current20 that was sensitive to Zn2+.20 It was shown that this sperm proton channel closely resembles the voltage-gated channel Hv1.76,77 Interestingly, Hv1 was highly expressed in the flagellum, indicating a potential role in the regulation of CatSper.20 Although Hv1 is ubiquitously expressed and plays an important role in several physiological processes such as the innate immune system, apoptosis, and cancer metastasis, the presence of Hv1 in human spermatozoa was intriguing. Hv1 is not a true ion channel but rather a hybrid between a transporter and an ion channel without the pore that provides rapid movement of protons across a lipid bilayer via a voltage-gated mechanism.78–80
Hv1: Principles of Proton Transport The first electrophysiological recording of voltage-dependent proton current in snail neurons was performed by Roger Thomas and Robert Meech in 1982.82 Later studies, primarily done by the Thomas DeCoursey group,83–85 found similar currents in other cell types.86 It was not until 2006 when two publications—one from the David Clapham group76 and another from the Yasushi Okamura group77—reported the molecular identity of this channel to be voltage-gated proton channel Hv1. Molecular and electrophysiological studies of Hv1 have indicated four distinct features: (1) the channel is activated by membrane depolarization and intracellular acidification, (2) is inhibited by zinc, (3) is H+ selective, and (4) it lacks pore domain. Hv1 forms four TM spanning segments homologous in structure to voltage-sensor of voltage-gated cation channels.76,77 Unlike the traditional voltage-gated ion channel, Hv1 does not possess a classical selectivity pore region. Several models for the proton conductance pathway have been proposed.78,79,87 Among them the “water wire” model79 suggests the movement of protons via the Grotthus mechanism from the intracellular water vestibules to the extracellular vestibule formed by the TM portions of the channel. Two vestibules are connected by a narrow bottleneck79 where proton selectivity occurs via a highly conserved and unique aspartate moiety (Asp112 in human Hv188). Another model78 suggests that the Hv1 closed state favors electrostatic interactions between hydrophobic residues of TM segments
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and pulls them together to form a hydrophobic plug. This hydrophobic layer is formed at the center of the voltage-sensing domain and prevents proton permeation. The latter occurs once the plug is removed by a voltage-dependent rearrangement of TMs and is followed by insertion of the protonatable residues in the center of the channel.78 The recent crystal structure of the Hv1 in the resting state sheds80 light on the possible mechanism of H+ permeation, suggesting that a combination of two hydrophobic plugs and a protonatable aspartate is required for proton movement. Hv1 forms a functional dimer in the plasma membrane through a coil–coil interaction of the C-termini89–91; however, each Hv1 subunit can function independently as a channel.
Human Sperm Hv1 At the time of ejaculation, spermatozoa are combined with the seminal plasma, which contains high concentrations of zinc (∼2 mM)36 and as such directly inhibits Hv1 function by binding to two histidine residues that stabilize the channel in the closed state.92 As sperm progress through the female reproductive tract, divalent zinc is chelated by proteins in the oviductal fluid, resulting in gradual activation of the Hv1 channel.36,93 To fertilize an oocyte, sperm cells have to undergo a process known as capacitation, which comprises several physiological changes including intracellular alkalization. In humans this could be triggered via proton extrusionthrough Hv120, especially because Hv1 is activated by capacitation.20 In contrast to human sperm, mouse spermatozoa do not possess Hv1, and, not surprisingly, Hv1−/− mice are fertile. Natural mutations in Hv1 appear to be extremely rare, with only one case of a single substitution mutation being reported in humans,94 which was assessed only in airway epithelial cells. The role of this channel in human fertility is still unclear and will remain so until an Hv1-deficient patient can be identified. In species that lack sperm Hv1, it is still unclear how intracellular pH is regulated in these cells, although sNHE remainsas possible mechanism.
OTHER ION CHANNELS OF SPERM Sperm membrane potential is important for fertility because both Hv1 and CatSper channels are voltage dependent (Fig. 9.3). As in other cells, sperm membrane potential is defined by the gradients of K+, Na+, and Cl−, with potassium channels playing a crucial role in its regulation. Epididymal murine spermatozoa keep their membrane potential slightly depolarized at approximately −40 mV; however, they tend to hyperpolarize up to −60 mV upon in vitro capacitation.95 This effect is attributed to
Other Ion Channels of Sperm
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FIGURE 9.3 Flagellar ion channels of sperm. Murine potassium channel (KSper) is composed of the Slo3 channel and associated subunit LRRC52 (gamma) as well as possible beta subunits. The identity of human KSper is still under debate but seems to share characteristics of the Slo family of ion channels. Cation channels of sperm (CatSper) in mouse and human share the same molecular composition consisting of at least seven different subunits to form calcium-permeable channel. Proton conductance via the Hv1 channel (HSper) is only detected in human sperm cells. Both CatSper and Hv1 require membrane depolarization to function. This could be achieved by inhibition of KSper. In addition, CatSper is activated by intracellular alkalinization that could be provided by the Hv1. By moving protons out of the flagellum, Hv1 further hyperpolarizes the cell and provides negative feedback inhibiting both Hv1 and CatSper. This model misses the fourth member, yet-to-be-identified “depolarizing channel of sperm” or “DSper.” This hypothetical DSper could be activated by either membrane hyperpolarization as proposed for sperm-specific Na+/H+ (sNHE)87 or other mechanisms. DSper would provide positive net charge influx resulting in depolarization, consequent activation of Hv1/CatSper, and sperm hyperactivation. Reproduced from Miller MR, Mansell SA, Meyers SA, Lishko PV. Cell Calcium. 2015 Jul;58(1):105-13, Elsevier.
specific potassium permeability, or KSper, and two members of the Slo family of potassium channels have been recently proposed to play a role in this process.27,31,96–103 Slo3 (Kcnu1), a pH-sensitive, calcium-independent, and weakly voltage-sensitive channel, has been identified as the principal potassium channel in murine sperm.31,98,99,101–105 Although Slo3 was also expected to form the potassium channel of human sperm, recent recording from ejaculated human spermatozoa indicates that in contrast to murine sperm, human sperm potassium current (KSper) is less pH
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dependent and sensitive to [Ca2+]i.24 Furthermore, it could also be inhibited by progesterone.12,24 From these biophysical properties as well as the pharmacological profile, human KSper resembled the calcium-activated big conductance potassium channel Slo1 (KCNMA1).24 Recently other models were proposed, suggesting that human capacitated spermatozoa possess a different type of Slo channel25 or even a modified version of Slo3 that is calcium-sensitive and weakly pH-dependent,12 an unusual set of properties for this type of ion channel. Slo channels exist as tetrameric complexes of alpha subunits (Slo1 or Slo3) and auxiliary gamma subunits (LRCC26 or LRCC52); additional beta subunits can change their biophysical and pharmacological properties.100,106,107 Uncovering the precise molecular identity of the human potassium channel remains an essential task for understanding the regulation of potassium homeostasis in human spermatozoa. Navarro et al. recently reported the presence of an ATP-gated P2X2 ion channel (also known as purinergic receptor P2X, ligand gated ion channel, 2) that is cation nonselective and originates from the midpiece of murine sperm cells.108 P2rx2-deficient male mice are fertile and have normal sperm morphology and other sperm parameters; however, their sperm lack ATP-evoked currents and fertility of P2rx2−/− males declines with frequent mating.108 Calcium-activated chloride channels (anoctamins) have been recently found in human sperm,109 but not in mouse,34 as well as aquaporins, which are water channels that are required for sperm osmoadaptation.110 Several members of the TRP ion channel family111–113 were thought to function in mammalian sperm cells.16,114 These include TRPM8, TRPV1, TRPA1, and others. However, mice deficient in TRPV1–4, TRPA1, and TRPM8 have no obvious defects in sperm morphology or male fertility.111,112 Moreover, CatSper promiscuity toward high concentrations of exogenous activators, such as menthols, may account for the mistaken identity of TRPM8 in human spermatozoa.115 However, it is possible that other species that do not have CatSper activity rely on different flagellar channels; therefore the role of TRP channels in sperm is yet to be determined.
CONCLUSIONS On their route to the egg, mammalian spermatozoa encounter multiple obstacles: viscous mucus, the narrow lumen of the uterotubal junction, the complex maze formed by the epithelial folds of the fallopian tubes, and finally the protective layers of the egg. To overcome these barriers, the sperm cell must sense the cues released by the egg and adapt its swimming behavior. Sperm can achieve this by increasing the amplitude and driving force of their tail bending, changing their direction of movement,
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and releasing special enzymes to dissolve the egg’s protective vestments. Such sperm responses depend upon activity of the sperm ion channels that open in response to environmental cues within the female reproductive tract. This in turn changes conductance of the sperm plasma membrane and sperm behavior. Influx of calcium through the flagellar calcium channel CatSper results in the flagellar bending and hyperactivation of motility. In turn, CatSper is activated by intracellular alkalinization, provided by perhaps the sNHE or Hv1 channel (in human sperm) and membrane depolarization, which results from KSper inhibition by progesterone. The same compartmental flagellar localization of sperm ion channels provides fine-tuned regulation of sperm motility. It is possible that many cases of idiopathic male infertility can be attributed to malfunctioning of sperm ion channels and their regulators.
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