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Mammalian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation, and release of the eggs, i.e., ovulation. The steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation of Abhd2 caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries of Abhd2 knockout (KO) females resemble polycystic ovary morphology (PCOM) with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowed Abhd2 KO females to ovulate a significantly increased number of mature and fertile eggs in comparison with their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.

Therian female fertility is defined by a successful and strictly periodic ovarian cycle, which is under the control of gonadotropins and steroid hormones, particularly progesterone and estrogen. The latter two are produced by the ovaries that are engaged in controlled follicular growth, maturation and release of the eggs, i.e. ovulation. It is well known that steroid hormones regulate ovarian cycles via genomic signaling, by altering gene transcription and protein synthesis. However, despite this well-studied mechanism, steroid hormones can also signal via direct, non-genomic action, by binding to their membrane receptors. Here we show, that the recently discovered sperm membrane progesterone receptor α/β hydrolase domain-containing protein 2 (ABHD2) is highly expressed in mammalian ovaries where the protein plays a novel regulatory role in follicle maturation and the sexual cycle of females. Ablation ofAbhd2caused a dysregulation of the estrous cycle rhythm with females showing shortened luteal stages while remaining in the estrus stage for a longer time. Interestingly, the ovaries ofAbhd2knockout (KO) females resemble polycystic ovary morphology with a high number of atretic antral follicles that could be rescued with injection of gonadotropins. Such a procedure also allowedAbhd2KO females to ovulate a significantly increased number of mature and fertile eggs in comparison to their wild-type littermates. These results suggest a novel regulatory role of ABHD2 as an important factor in non-genomic steroid regulation of the female reproductive cycle.

Varying pH of luminal fluid along the female reproductive tract is a physiological cue that modulates sperm motility. CatSper is a sperm-specific, pH-sensitive calcium channel essential for hyperactivated motility and male fertility. Multi-subunit CatSper channel complexes organize linear Ca2+ signaling nanodomains along the sperm tail. Here, we identify EF-hand calcium-binding domain-containing protein 9 (EFCAB9) as a bifunctional, cytoplasmic machine modulating the channel activity and the domain organization of CatSper. Knockout mice studies demonstrate that EFCAB9, in complex with the CatSper subunit, CATSPERζ, is essential for pH-dependent and Ca2+-sensitive activation of the CatSper channel. In the absence of EFCAB9, sperm motility and fertility is compromised, and the linear arrangement of the Ca2+ signaling domains is disrupted. EFCAB9 interacts directly with CATSPERζ in a Ca2+-dependent manner and dissociates at elevated pH. These observations suggest that EFCAB9 is a long-sought, intracellular, pH-dependent Ca2+ sensor that triggers changes in sperm motility.

Vigorous sperm flagellar motility is essential for fertilization, and so the quantitative measurement of motility is a useful tool to assess the intrinsic fertility potential of sperm cells and explore how various factors can alter sperm's ability to reach the egg and penetrate its protective layers. Human sperm beat their flagella many times each second, and so recording and accurately quantifying this movement requires a high-speed camera. The aim of this protocol is to provide a detailed description of the tools required for quantitative beat frequency measurement of tethered human sperm at the single-cell level and to describe methods for investigating the effects of intracellular or extracellular factors on flagellar motion. This assay complements bulk measurements of sperm parameters using commercially-available systems for computer-assisted sperm analysis (CASA).

Steroids regulate cell proliferation, tissue development, and cell signaling via two pathways: a nuclear receptor mechanism and genome-independent signaling. Sperm activation, egg maturation, and steroid-induced anesthesia are executed via the latter pathway, the key components of which remain unknown. Here, we present characterization of the human sperm progesterone receptor that is conveyed by the orphan enzyme α/β hydrolase domain-containing protein 2 (ABHD2). We show that ABHD2 is highly expressed in spermatozoa, binds progesterone, and acts as a progesterone-dependent lipid hydrolase by depleting the endocannabinoid 2-arachidonoylglycerol (2AG) from plasma membrane. The 2AG inhibits the sperm calcium channel (CatSper), and its removal leads to calcium influx via CatSper and ensures sperm activation. This study reveals that progesterone-activated endocannabinoid depletion by ABHD2 is a general mechanism by which progesterone exerts its genome-independent action and primes sperm for fertilization.

Unlike other human biological fluids, semen contains multiple types of amyloid fibrils in the absence of disease. These fibrils enhance HIV infection by promoting viral fusion to cellular targets, but their natural function remained unknown. The similarities shared between HIV fusion to host cell and sperm fusion to oocyte led us to examine whether these fibrils promote fertilization. Surprisingly, the fibrils inhibited fertilization by immobilizing sperm. Interestingly, however, this immobilization facilitated uptake and clearance of sperm by macrophages, which are known to infiltrate the female reproductive tract (FRT) following semen exposure. In the presence of semen fibrils, damaged and apoptotic sperm were more rapidly phagocytosed than healthy ones, suggesting that deposition of semen fibrils in the lower FRT facilitates clearance of poor-quality sperm. Our findings suggest that amyloid fibrils in semen may play a role in reproduction by participating in sperm selection and facilitating the rapid removal of sperm antigens. DOI: http://dx.doi.org/10.7554/eLife.24888.001, eLife digest Seminal plasma, the fluid portion of semen, helps to transport sperm cells to the egg during sexual reproduction. Seminal plasma contains numerous proteins that help the sperm to survive and, in recent years, researchers discovered that it also harbours protein deposits known as amyloid fibrils. Such protein deposits are generally associated with neurodegenerative diseases such as Alzheimer's and Parkinson’s disease, where a build-up of fibrils can damage the nervous system. Semen amyloids, however, are present in the absence of disease, but can boost infection by HIV and other sexually transmitted viruses, by shuttling virus particles to their target cells. Despite these damaging effects, some researchers had suggested that amyloids in semen could be beneficial for humans, though it was unclear what these benefits might be. Roan et al. now set out to assess how semen amyloids affect human sperm activity. The results show that semen amyloids bind to damaged sperm cells and immobilize them, which are then quickly cleared away by immune cells. This could ensure that only the fittest sperm cells reach the egg. These findings suggest that amyloids can potentially serve beneficial roles for reproduction. A next step will be to investigate how semen amyloids trap unwanted sperm and how immune cells know when to remove it. More research is needed to investigate if problems in these processes could lead to infertility in men. DOI: http://dx.doi.org/10.7554/eLife.24888.002

The calcium channel of sperm (CatSper) is essential for sperm hyperactivated motility and fertility. The steroid hormone progesterone activates CatSper of human sperm via binding to the serine hydrolase ABHD2. However, steroid specificity of ABHD2 has not been evaluated. Here, we explored whether steroid hormones to which human spermatozoa are exposed in the male and female genital tract influence CatSper activation via modulation of ABHD2. The results show that testosterone, estrogen, and hydrocortisone did not alter basal CatSper currents, whereas the neurosteroid pregnenolone sulfate exerted similar effects as progesterone, likely binding to the same site. However, physiological concentrations of testosterone and hydrocortisone inhibited CatSper activation by progesterone. Additionally, testosterone antagonized the effect of pregnenolone sulfate. We have also explored whether steroid-like molecules, such as the plant triterpenoids pristimerin and lupeol, affect sperm fertility. Interestingly, both compounds competed with progesterone and pregnenolone sulfate and significantly reduced CatSper activation by either steroid. Furthermore, pristimerin and lupeol considerably diminished hyperactivation of capacitated spermatozoa. These results indicate that (i) pregnenolone sulfate together with progesterone are the main steroids that activate CatSper and (ii) pristimerin and lupeol can act as contraceptive compounds by averting sperm hyperactivation, thus preventing fertilization.

Author(s): Bjorkgren, I; Lishko, PV | Abstract: Since their discovery in late 1970, transient receptor potential (TRP) channels have been implicated in a variety of cellular and physiological functions (Minke, 2010). The superfamily of TRP channels consists of nearly 30 members that are organized into seven major subgroups based on their specic function and sequence similarities (Owsianik et al., 2006; Ramsey et al., 2006). With the exception of TRPN channels that are only found in invertebrates and sh, mammalian genomes contain representatives of all six subfamilies: (1) TRPV (vanilloid); (2) TRPC (canonical); (3) TRPM (melastatin); (4) TRPA (ankyrin); (5) TRPML (mucolipin); and (6) TRPP (polycystin). TRP channels play crucial regulatory roles in many physiological processes, including those associated with reproductive tissues. As calcium-permeable cation channels that respond to a variety of signals (Clapham et al., 2003; Wu et al., 2010), TRP channels exert their role as sensory detectors in both male and female gametes, and play regulatory functions in germ cell development and maturation. Recent evidence obtained from Caenorhabditis elegans studies point to the importance of these proteins during fertilization where certain sperm TRP channels could migrate from a spermatozoon into an egg to ensure successful fertilization and embryo development. In this chapter we discuss how TRP channels can regulate both female and male fertility in different species and their specic roles.

The mir-34/449 family consists of six homologous miRNAs at three genomic loci. Redundancy of miR-34/449 miRNAs and their dominant expression in multiciliated epithelia suggest a functional significance in ciliogenesis. Here we report that mice deficient for all miR-34/449 miRNAs exhibited postnatal mortality, infertility and strong respiratory dysfunction caused by defective mucociliary clearance. In both mouse and Xenopus, miR-34/449-deficient multiciliated cells (MCCs) exhibited a significant decrease in cilia length and number, due to defective basal body maturation and apical docking. The effect of miR-34/449 on ciliogenesis was mediated, at least in part, by post-transcriptional repression of Cp110, a centriolar protein suppressing cilia assembly. Consistent with this, cp110 knockdown in miR-34/449-deficient MCCs restored ciliogenesis by rescuing basal body maturation and docking. Altogether, our findings elucidate conserved cellular and molecular mechanisms through which miR-34/449 regulate motile ciliogenesis.

Key physiological functions of organisms, such as sensory transduction, regulation of heart rate, smooth muscle contraction, bile secretion, endocrine regulation, immune responses, and various pathophysiological conditions, including neuropathic pain, diabetes, kidney failure, and cancer, are

© 2015 American Chemical Society. The basal fungus Allomyces macrogynus (A. Macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

Ion channels control the sperm ability to fertilize the egg by regulating sperm maturation in the female reproductive tract and by triggering key sperm physiological responses required for successful fertilization such as hyperactivated motility, chemotaxis, and the acrosome reaction. CatSper, a pH-regulated, calcium-selective ion channel, and KSper (Slo3) are core regulators of sperm tail calcium entry and sperm hyperactivated motility. Many other channels had been proposed as regulating sperm activity without direct measurements. With the development of the sperm patch-clamp technique, CatSper and KSper have been confirmed as the primary spermatozoan ion channels. In addition, the voltage-gated proton channel Hv1 has been identified in human sperm tail, and the P2X2 ion channel has been identified in the midpiece of mouse sperm. Mutations and deletions in sperm-specific ion channels affect male fertility in both mice and humans without affecting other physiological functions. The uniqueness of sperm ion channels makes them ideal pharmaceutical targets for contraception. In this review we discuss how ion channels regulate sperm physiology.

Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

Steroid hormone progesterone released by cumulus cells surrounding the egg is a potent stimulator of human spermatozoa. It attracts spermatozoa towards the egg and helps them penetrate the egg's protective vestments. Progesterone induces Ca(2+) influx into spermatozoa and triggers multiple Ca(2+)-dependent physiological responses essential for successful fertilization, such as sperm hyperactivation, acrosome reaction and chemotaxis towards the egg. As an ovarian hormone, progesterone acts by regulating gene expression through a well-characterized progesterone nuclear receptor. However, the effect of progesterone upon transcriptionally silent spermatozoa remains unexplained and is believed to be mediated by a specialized, non-genomic membrane progesterone receptor. The identity of this non-genomic progesterone receptor and the mechanism by which it causes Ca(2+) entry remain fundamental unresolved questions in human reproduction. Here we elucidate the mechanism of the non-genomic action of progesterone on human spermatozoa by identifying the Ca(2+) channel activated by progesterone. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of progesterone dramatically potentiate CatSper, a pH-dependent Ca(2+) channel of the sperm flagellum. We demonstrate that human CatSper is synergistically activated by elevation of intracellular pH and extracellular progesterone. Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Because our experimental conditions did not support second messenger signalling, CatSper or a directly associated protein serves as the elusive non-genomic progesterone receptor of sperm. Given that the CatSper-associated progesterone receptor is sperm specific and structurally different from the genomic progesterone receptor, it represents a promising target for the development of a new class of non-hormonal contraceptives.

Elevations of sperm intracellular pH and Ca2+ regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilise the egg. In human spermatozoa, the flagellar voltage-gated proton channel Hv1 is the main H+ extrusion pathway that controls sperm intracellular pH, and the pH-dependent flagellar Ca2+ channel CatSper is the main pathway for Ca2+ entry as measured by the whole-cell patch clamp technique. Hv1 and CatSper channels are co-localized within the principal piece of the sperm flagellum. Hv1 is dedicated to proton extrusion from flagellum and is activated by membrane depolarisation, an alkaline extracellular environment, the endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. The CatSper channel is strongly potentiated by intracellular alkalinisation. Since Hv1 and CatSper channels are located in the same subcellular domain, proton extrusion via Hv1 channels should induce intraflagellar alkalinisation and activate CatSper ion channels. Therefore the combined action of Hv1 and CatSper channels in human spermatozoa can induce elevation of both intracellular pH and Ca2+ required for sperm activation in the female reproductive tract. Here, we discuss how Hv1 and CatSper channels regulate human sperm physiology and the differences in control of sperm intracellular pH and Ca2+ between species.

SummaryHuman spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. This process is known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown because of the inability to measure membrane conductance in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, endocannabinoid anandamide, and removal of extracellular zinc, a potent Hv1 blocker. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. The importance of Hv1 for sperm activation makes it an attractive target for controlling male fertility.PaperFlick

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.

© 2014 Elsevier Ltd. Motility and fertilization potential of mammalian sperm are regulated by ion homeostasis which in turn is under tight control of ion channels and transporters. Sperm intracellular pH, membrane voltage and calcium concentration ([Ca2+]i) are all important for sperm activity within the female reproductive tract. While all mammalian sperm are united in their goal to find and fertilize an egg, the molecular mechanisms they utilize for this purpose are diverse and differ between species especially on the level of ion channels. Recent direct recording from sperm cells of different species indicate the differences between rodent, non-human primate, ruminant, and human sperm on the basic levels of their ion channel regulation. In this review we summarize the current knowledge about ion channel diversity of the animal kingdom and concentrate our attention on flagellar ion channels of mammalian sperm.

The complex between the short splice variant of the ninth member of the RGS protein family and the long splice variant of type 5 G protein beta subunit (RGS9-Gbeta5L) plays a critical role in regulating the duration of the light response in vertebrate photoreceptors by activating the GTPase activity of the photoreceptor-specific G protein, transducin. RGS9-Gbeta5L is tightly associated with the membranes of photoreceptor outer segments; however, the nature of this association remains unknown. Here we demonstrate that rod outer segment membranes contain a limited number of sites for high affinity RGS9-Gbeta5L binding, which are highly sensitive to proteolysis. In membranes isolated from bovine rod outer segments, all of these sites are occupied by the endogenous RGS9-Gbeta5L, which prevents the binding of exogenous recombinant RGS9-Gbeta5L to these sites. However, treating membranes with urea or high pH buffers causes either removal or denaturation of the endogenous RGS9-Gbeta5L, allowing for high affinity binding of recombinant RGS9-Gbeta5L to these sites. This binding results in a striking approximately 70-fold increase in the RGS9-Gbeta5L ability to activate transducin GTPase. The DEP (disheveled/EGL-10/pleckstrin) domain of RGS9 plays a crucial role in the RGS9-Gbeta5L membrane attachment, as evident from the analysis of membrane-binding properties of deletion mutants lacking either N- or C-terminal parts of the RGS9 molecule. Our data indicate that specific association of RGS9-Gbeta5L with photoreceptor disc membranes serves not only as a means of targeting it to an appropriate subcellular compartment but also serves as an important determinant of its catalytic activity.

We have found that the platelet activating factor antagonist (BN52021) is an effective blocker of the glycine (Gly) receptor-mediated responses in the hippocampal pyramidal neurons of rat. Using the whole-cell voltage clamp and concentration clamp recording techniques, we investigated the mechanism underlying the inhibitory action of this terpenoid on the glycine-induced chloride current. BN52021 selectively and reversibly inhibits glycine current in a non-competitive and voltage-dependent fashion. The antagonistic effect of this substance is more pronounced at positive membrane potentials. At holding potential −70 mV and in the presence of 200 μM glycine IC 50 value for the blocking action of BN52021 was 270±10 nM. Repetitive applications of BN52021 reveal the use-dependence of its blocking action. When co-applied with strychnine (STR), a competitive glycine receptor antagonist, BN52021 does not alter the IC 50 value for strychnine. The inhibitory effect of BN52021 on gamma-aminobutyric acid (GABA) current is at least 25 times less potent than the effect on glycine current. This substance fails to affect AMPA and NMDA responses. It may be concluded that BN52021 inhibits glycine-gated Cl − channels by interacting with the pore region and does not compete for the strychnine-binding centre.

Effects of hyperforin, an acylphloroglucinol derivative isolated from antidepressive medicinal herb Hypericum perforatum (St. John's Wort), on voltage- and ligand-gated ionic conductances were investigated. Whole-cell patch clamp and concentration clamp techniques on acutely isolated hippocampal pyramidal neurons and on cerebellar Purkinje neurons of rat were used. At concentrations between 3 to 100 μM hyperforin induced a dose and time dependent inward current which completely stabilized within a few seconds. Although 1 μM hyperforin inhibited virtually all investigated conductances (GABA ≥ I Ca(N) > I Na ≥ I Ca(P) ≥ AMPA ≥ I K(A) > NMDA > I K(DR) ), its effects on several of them could not be reversed by repeated washings. Dose response studies revealed that although AMPA induced current is inhibited by hyperforin in a competitive manner, these responses are not completely blocked by very high concentration of the agent. On the contrary, however, NMDA receptor-activated ionic conductance could be completely and uncompetitively inhibited by the agent. Taken together these observation not only reconfirm that hyperforin is a major neuroactive component of hypericum extracts but also demonstrate that this structurally unique and naturally abundant molecule is a potent modulation of mechanism involved in the control of neuronal ionic conductances. Various observed effects of hyperforin do not, however, seem to be mediated by one single molecular mechanism of action of the agent.

KA-672.HCl (KA-672) is a new substance demonstrating anti-dementia properties. It shows modulatory effects on several neurotransmitter systems known to be affected in patients with Alzheimer's disease. In this study the action of KA-672 on the NMDA receptors was examined by applying patch clamp techniques to acutely isolated hippocampal neurons. KA-672 antagonizes NMDA responses in a voltage-dependent manner. At a holding potential of -90 mV the IC50 value for the blocking action of KA-672 was 20+/-7 microM. This action of KA-672 is independent on the concentration either of agonist or coagonist of NMDA receptor. Ketamine, which interacts with the PCP center, does not occlude the action of KA-672. Evidently, KA-672.HCl is a weak NMDA receptor-operated channel blocker. This property may account for its pharmacological profile.

Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K+ efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca2+] to produce hyperactivation and allowing sperm to fertilize an oocyte. DOI: http://dx.doi.org/10.7554/eLife.01009.001, eLife digest The sperm cells that are released into the female reproductive tract when a mammal ejaculates, are not capable of fertilizing an egg right away, so they must go through a process called maturation. The early stages of this process involve interactions with the seminal fluid that increase the motility of the sperm cells, and the latter stages involve interactions with the walls of the reproductive tract and vaginal secretions to ensure that the sperm cells move toward the egg. Many of these interactions involve positive ions entering and leaving the sperm cells via ion channels. The properties of the ion channels that allow protons and calcium ions to move into and out of human sperm cells are well understood, but little is known about the channels that control the movement of the potassium (K) ions. At first it was assumed that the molecular structure of these channels was similar to that of the Slo3 potassium channel in mouse sperm, but crucial differences between human and mouse sperm cells have been reported in recent years. Now Mannowetz et al. have shown that the potassium channel in human sperm is opened by increased levels of calcium ions inside the sperm cells. Moreover, the pH inside the sperm cells had no influence on this process. Furthermore, the channel was blocked by three toxins that have no effect on the Slo3 potassium channels in mice, but are known to block a type of potassium channel known as Slo1. Mannowetz et al. then used a technique called Western blotting to confirm the presence of Slo1 potassium channels in the tails of human sperm cells. Mannowetz et al. also showed that the Slo1 potassium channel can be blocked by the female hormone progesterone. This is important because blocking the potassium channels causes the calcium ion channels in the cells to open fully, and the resulting influx of calcium ions triggers a process called sperm hyperactivation that makes it possible for the sperm cell to fertilize the egg. By clearly showing the fundamental differences between human sperm cells and mouse sperm cells, this work stresses the need to exercise caution in using mice as a model of male fertility in humans. DOI: http://dx.doi.org/10.7554/eLife.01009.002

The female steroid hormone progesterone regulates ovulation and supports pregnancy, but also controls human sperm function within the female reproductive tract. Progesterone causes elevation of sperm intracellular Ca 2+ leading to sperm hyperactivation, acrosome reaction, and perhaps chemotaxis toward the egg. Although it has been suggested that progesterone-dependent Ca 2+ influx into human spermatozoa is primarily mediated by cationic channel of sperm (CatSper), the principal flagellar Ca 2+ channel of sperm, conclusive loss-of-function genetic evidence for activation of CatSper by progesterone has yet to be provided. Moreover, it is not clear whether the responsiveness of CatSper to progesterone is an innate property of human spermatozoa or is acquired as the result of exposure to the seminal plasma. Here, by recording ionic currents from spermatozoa of an infertile CatSper-deficient patient, we demonstrate that CatSper is indeed the principal Ca 2+ channel of human spermatozoa, and that it is strongly potentiated by progesterone. In addition, by recording CatSper currents from human epididymal and testicular spermatozoa, we show that CatSper sensitivity to progesterone arises early in sperm development and increases gradually to a peak when spermatozoa are ejaculated. These results unambiguously establish an important role of CatSper channel in human sperm nongenomic progesterone signaling and demonstrate that the molecular mechanism responsible for activation of CatSper by progesterone arises early in sperm development concurrently with the CatSper channel itself.

Sperm intracellular pH and calcium concentration ([Ca(2+)]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca(2+)] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels.

The sperm connecting piece is a complex structure that, from a mechanical perspective, appears to play a role in stabilizing the proximal part of the sperm tail. We report the three-dimensional structure of the intact bovine sperm connecting piece, revealing an intricate, asymmetrical architecture with the segmented columns held together by filamentous linkages. The columns fuse, at the proximal end, with each other into structures that form the centriolar vault, and at the distal end, with the outer dense fibers (ODFs). The grouping of the fibers into these structures is consistent with bending only in the plane of the head. Structures reminiscent of the proximal centriole were observed in the vault, while the association of a novel bar structure with ODFs 3 and 8 organizes the distal centriolar vault. It has been proposed that the elastic compliance of the connecting piece provides the underlying mechanism behind initiation of the sperm beat cycle and bend propagation. According to the basal sliding theory of sperm movement, distortion of the connecting piece may store energy that initiates a new beat. The intersegment linkers could serve as mechanosensitive elements that regulate alternation of the sperm tail's bending direction in the beat cycle in addition to providing structural stabilization for the connecting piece segmented structures. On the other hand, our video recordings of the bull sperm movement show little bending of the head with respect to the tail, so it appears that there may be normally little strain within the connecting piece.

Two isoforms of the GTPase-activating protein, regulator of G protein signaling 9 (RGS9), control such fundamental functions as vision and behavior. RGS9–1 regulates phototransduction in rods and cones, and RGS9–2 regulates dopamine and opioid signaling in the basal ganglia. To determine their functional differences in the same intact cell, we replaced RGS9–1 with RGS9–2 in mouse rods. Surprisingly, RGS9–2 not only supported normal photoresponse recovery under moderate light conditions but also outperformed RGS9–1 in bright light. This versatility of RGS9–2 results from its ability to inactivate the G protein, transducin, regardless of its effector interactions, whereas RGS9–1 prefers the G protein-effector complex. Such versatility makes RGS9–2 an isoform advantageous for timely signal inactivation across a wide range of stimulus strengths and may explain its predominant representation throughout the nervous system.

SummaryTRPV1 plays a key role in nociception, as it is activated by heat, low pH, and ligands such as capsaicin, leading to a burning pain sensation. We describe the structure of the cytosolic ankyrin repeat domain (ARD) of TRPV1 and identify a multiligand-binding site important in regulating channel sensitivity within the TRPV1-ARD. The structure reveals a binding site that accommodates triphosphate nucleotides such as ATP, and biochemical studies demonstrate that calmodulin binds the same site. Electrophysiology experiments show that either ATP or PIP2 prevent desensitization to repeated applications of capsaicin, i.e., tachyphylaxis, while calmodulin plays an opposing role and is necessary for tachyphylaxis. Mutations in the TRPV1-ARD binding site eliminate tachyphylaxis. We present a model for the calcium-dependent regulation of TRPV1 via competitive interactions of ATP and calmodulin at the TRPV1-ARD-binding site and discuss its relationship to the C-terminal region previously implicated in interactions with PIP2 and calmodulin.

STUDY QUESTION Are significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success? SUMMARY ANSWER Sperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF. WHAT IS KNOWN ALREADY In human spermatozoa, Ca2+ influx induced by progesterone is mediated by CatSper, a sperm-specific Ca2+ channel. A suboptimal Ca2+ influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca2+]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception. STUDY DESIGN, SIZE, DURATION Spermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS Samples were primarily screened using the Ca2+ influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca2+ response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca2+ response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis. MAIN RESULTS AND THE ROLE OF CHANCE A total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca2+ response. The mean (±SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca2+ response. Three men were initially identified with a defective Ca2+ influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF. LIMITATIONS, REASONS FOR CAUTION A key limitation relates to working with a specific functional parameter (Ca2+ influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼22% of IVF patients) could not be screened. As such the incidence of significant Ca2+ abnormalities induced by progesterone may be higher than the ∼1% observed here. Additionally, we used a strict definition of a defective Ca2+ influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca2+ influx. WIDER IMPLICATIONS OF THE FINDINGS These data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception. STUDY FUNDING/COMPETING INTEREST(S) Initial funding was from NHS Tayside, Infertility Research Trust, TENOVUS, Chief Scientist Office NRS Fellowship, the Wellcome Trust, University of Abertay. The majority of the data were obtained using funding from a MRC project grant (# 4190). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER Not applicable.

Flagella potassium channel of sperm (KSper) is required for successful fertilization. In mice the dominant K+ conductance is via KCNU1 or Slo3, a pH regulated K+ channel. In humans KSper is activated by Ca2+, not pH, and is inhibited by scorpion toxin. Based on these unique properties, it was suggest that Slo1 (KCNMA1) underlies the molecular identity of human KSper. Recent conflicting work indicated that human KSper maybe a Ca2+ sensitive Slo3 variant. Due to the lack of a Ca2+ sensing domain in the structure of Slo3, it seems unlikely that it would be activated by Ca2+. Therefore to address this conundrum, we characterized a KSper defective patient and present here the molecular composition of this channel complex. Infertile patients attending Ninewells Hospital, Dundee, were screened for KSper current (IK+) by using sperm patch-clamp method. One out of 13 patients showed a complete loss of IK+ and was insensitive to changes in intracellular Ca2+. MiSeq genome analysis showed no mutations in KCNMA1 but a single nucleotide polymorphism (SNP) at position 768 (W768R) in KCNU1 was present. Electrophysiological assessment of heterologously expressed mutated KCNU1 in CHO cells cloned from human spermatozoa showed no loss IK+. Further analysis of patient genome showed additional mutation in auxiliary subunits which were not present in fertile donors. Co-expression of mutated subunits in CHO cells showed a complete loss of IK+ identical to patient. Interestingly, we were unable to show Ca2+ activated IK+ in CHO cells with wild type KCNU1. This suggests human KSper may be more complex than initially thought and requires an additional Ca2+ sensitive module for channel activation.

Photoreceptor cells have a remarkable capacity to adapt the sensitivity and speed of their responses to ever changing conditions of ambient illumination. Recent studies have revealed that a major contributor to this adaptation is the phenomenon of light-driven translocation of key signaling proteins into and out of the photoreceptor outer segment, the cellular compartment where phototransduction takes place. So far, only two such proteins, transducin and arrestin, have been established to be involved in this mechanism. To investigate the extent of this phenomenon we examined additional photoreceptor proteins that might undergo light-driven translocation, focusing on three Ca(2+)-binding proteins, recoverin and guanylate cyclase activating proteins 1 (GCAP1) and GCAP2. The changes in the subcellular distribution of each protein were assessed quantitatively using a recently developed technique combining serial tangential sectioning of mouse retinas with Western blot analysis of the proteins in the individual sections. Our major finding is that light causes a significant reduction of recoverin in rod outer segments, accompanied by its redistribution toward rod synaptic terminals. In both cases the majority of recoverin was found in rod inner segments, with approximately 12% present in the outer segments in the dark and less than 2% remaining in that compartment in the light. We suggest that recoverin translocation is adaptive because it may reduce the inhibitory constraint that recoverin imposes on rhodopsin kinase, an enzyme responsible for quenching the photo-excited rhodopsin during the photoresponse. To the contrary, no translocation of rhodopsin kinase itself or either GCAP was identified.

DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for solubleN-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.

Mammalian spermatozoa are unable to fertilize an oocyte immediately after their deposit in the female genital tract. To gain competence to fertilize, they must undergo a gradual maturation process termed capacitation that is accompanied by: 1) sperm intracellular alkalinization, evoked by proton extrusion through proton channels; 2) elevation of the intracellular [Ca2+], evoked by calcium influx through calcium ion channels, and 3) membrane hyperpolarization that is evoked by K+ efflux. Whereas the proton and calcium channels of human sperm are identified as Hv1 and CatSper, the identity of the principal potassium channel of human sperm (hKSper) was unknown. Potassium channels are indispensable for normal sperm physiology as they regulate cell membrane potential and cell motility. Recently, a pH-sensitive sperm K+ channel, encoded by the Slo3 gene was shown to be regulated by intracellular alkalinization and essential for male fertility in mice. It has been assumed, but never proven, that the K+ channel of human sperm has a similar molecular identity. Here we present full electrophysiological and pharmacological characterization of hKSper in both mature and developing human spermatozoa, and report that the molecular identity of hKSper is different from that of mouse KSper. In human sperm, the KSper current is conveyed by the big conductance, calcium-activated potassium channel (Slo1) which differs from mouse Slo3 in both its regulation and functional relationship with other sperm ion channels.

Mammalian spermatozoa gain their competence to fertilize an oocyte as they travel through the female genital tract. This process - termed capacitation - goes along with an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K+ efflux. However, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper and show that this chanel is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also demonstrate that human KSper is inhibited by charybdotoxin, iberiotoxin and paxilline, while mouse KSper is insensitive to these compounds. These properties suggest that the Slo1 ion channel is the molecular identity for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone, in turn, may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca2+] to produce hyperactivation and allowing sperm to fertilize an oocyte.

Elevation of intracellular Ca2+ regulate sperm motility, chemotaxis, capacitation and the acrosome reaction, and play a vital role in the ability of the sperm cell to reach and fertilize the egg. In mammalian spermatozoa, the flagellar pH-dependent Ca2+ channel CatSper is the main pathway for calcium entry as measured by the whole-cell patch clamp technique. Mouse CatSper channel is activated by alkaline intracellular pH, but human CatSper requires also a ligand. By applying the patch-clamp technique to mature human spermatozoa, we found that nanomolar concentrations of female hormone progesterone activate CatSper and this action explains the mechanism of non-genomic action of progesterone on human sperm cells. Progesterone is released by cumulus cells surrounding the oocyte and serves as a chemoattractant for human spermatozoa.Interestingly, human CatSper can be further potentiated by prostaglandins, but apparently through a binding site other than that of progesterone. Behavior of CatSper channel gradually changes during spermiogenesis with CatSper channel being fully operational only in mature spermatozoa. Physiological regulation of CatSper channel by different components of seminal plasma as well as hormones of female reproductive tract will be discussed.

Brown adipose tissue (BAT) is a specialized mammalian organ that coverts body fat reserves into heat. This conversion is mediated by BAT mitochondria and is important for maintaining body temperature and reducing fat depositions. Burning fat in BAT mitochondria does not result in ATP synthesis due to unusually high conductance of the inner mitochondrial membrane (IMM). This BAT-specific conductance dissipates the mitochondrial electrochemical gradient used in other tissues for ATP production and converts the energy of fatty acid oxidation into heat. The molecule responsible for the high conductance of the IMM and uncoupling of oxidative phosphorylation in BAT mitochondria is uncoupling protein 1 (UCP1). UCP1 is a BAT-specific protein that belongs to the superfamily of mitochondrial solute carriers (SLC25). UCP1-mediated uncoupling is activated by fatty acids and inhibited by purine nucleotides. In spite of the fact that UCP1 was identified decades ago, the mechanism of UCP1 operation is unknown due to lack of direct methods to study its activity. Here, we resolve the problem by directly recording UCP1 currents across the IMM of BAT mitochondria using the patch-clamp technique. To identify UCP1 current, we compare currents recorded from the wild-type and UCP1 (-/-) mitochondria. The detailed electrophysiological analysis demonstrated that UCP1 translocates fatty acid anion in symport with H+, while the fatty acid hydrophobic tail prevents dissociation of fatty acid from UCP1, causing UCP1 to operate as H+ carrier. This work establishes the mechanism of fatty acid dependent mitochondrial uncoupling which is implicated in metabolic and age-related diseases.

Uncoupling proteins (UCP1- UCP5) are six-transmembrane-domain transport proteins of the inner mitochondrial membrane (IMM). They increase electrical conductance of the IMM, thus dissipating the electrochemical proton gradient across this membrane and uncoupling mitochondrial respiration and ATP synthesis. By controlling mitochondrial membrane potential, UCPs can affect many aspects of mitochondrial function and have been implicated in regulation of body's energy efficiency, reducing fat depositions, thermogenesis, diabetes, and protecting the cell against oxidative damage and ageing. The founding member of the family, UCP1, is specifically expressed in brown adipose tissue (BAT) and is responsible for adaptive thermogenesis mediated by this tissue. Due to its unusually high level of expression, upon activation UCP1 completely uncouples BAT mitochondria and converts the energy of the substrate oxidation into heat. Since UCP1 can dissipate large amounts of energy, it has attracted attention as a potential target to treat obesity. In spite of their physiological and therapeutic significance, the mechanism of operation of uncoupling proteins including their ionic selectivity has long remained unknown due to the lack of direct methods to study their activity in their native membrane environment. Here, by applying the patch-clamp technique to the whole inner membrane of BAT mitochondria and for the first time directly measuring transmembrane currents produced by UCP1, we show that UCP1 is a ligand-gated hydroxide (OH-) ion channel activated by fatty acids. UCP1 is the only hydroxide ion channel reported to date. Thus, BAT thermogenesis involves the outward transport of protons by the electron transport chain along with the outward transport of OH- by UCP1, thereby amounting to cycling of water across the IMM and not to futile cycling of protons as was largely considered before.

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). Upon activation by long-chain fatty acids (LCFAs), UCP1 increases the conductance of the inner mitochondrial membrane (IMM) to make BAT mitochondria generate heat rather than ATP. Despite being a member of the family of mitochondrial anion carriers (SLC25), UCP1 is believed to transport H(+) by an unusual mechanism that has long remained unresolved. Here, we achieved direct patch-clamp measurements of UCP1 currents from the IMM of BAT mitochondria. We show that UCP1 is an LCFA anion/H(+) symporter. However, the LCFA anions cannot dissociate from UCP1 due to hydrophobic interactions established by their hydrophobic tails, and UCP1 effectively operates as an H(+) carrier activated by LCFA. A similar LCFA-dependent mechanism of transmembrane H(+) transport may be employed by other SLC25 members and be responsible for mitochondrial uncoupling and regulation of metabolic efficiency in various tissues.

Mammalian sperm motility and fertility potential are regulated by ion homeostasis which is controlled by a suite of ion permeable channels and transporters working in concert. While all mammalian sperm share in their goal to navigate the female reproductive tract in search of an egg, the molecular regulation of this process is extremely diverse and differs dramatically among species. Here we report that electrophysiological recordings from sperm cells of different species illustrate marked differences between rodent, human and ruminant sperm on the basis of their ion channel make-up and regulation. Furthermore, localization patterning of ion channels along the length of sperm flagella show species specific configuration. It is from these observed differences that we can now begin to explain the disparity in motility parameters among species as well as determine the effect proper spatiotemporal regulation of ion concentrations have on proper sperm motility.

Human spermatozoa are quiescent in the male reproductive system and must undergo activation once introduced into the female reproductive tract. After ejaculation, and during their transit through the female reproductive tract, sperm motility is initiated and then hyperactivated to allow the spermatozoa to penetrate through the viscous oviductal mucus and the egg's protective vestments. These processes are known to require alkalinization of sperm cytoplasm, but the mechanism responsible for transmembrane proton extrusion has remained unknown due to the inability to measure membrane conductances in human sperm. Here, by successfully patch clamping human spermatozoa, we show that proton channel Hv1 is their dominant proton conductance. Hv1 is confined to the principal piece of the sperm flagellum, where it is expressed at an unusually high density. Robust flagellar Hv1-dependent proton conductance is activated by membrane depolarization, an alkaline extracellular environment, and removal of extracellular zinc, a potent Hv1 blocker.Surprisingly, endocannabinoid anandamide was also found to modulate Hv1 activity in the sperm cells as well as in the heterologously expression system. Hv1 allows only outward transport of protons and is therefore dedicated to inducing intracellular alkalinization and activating spermatozoa. In contrast to the large Hv1 current in human sperm cells, the amplitude of the outward current recorded under similar conditions from mouse spermatozoa was 30 times smaller; therefore, mouse sperms seem to have a different mechanism for acid extrusion.In human sperm, Hv1-induced intracellular alkalinisation should activate pH-dependent Ca2+ channel CatSper and control intracellular Ca2+ concentration. Finally, since Hv1 lies upstream in the signalling cascades leading to sperm activation, hyperactivation, and capacitation, Hv1 is an attractive target for the control of male fertility.

Mature mammalian spermatozoa are stored in quiescent state in the male reproductive tract. Upon ejaculation and during their transit through the female reproductive tract, they acquire progressive motility and undergo other important functional changes that enable them to reach and fertilize the egg. Sperm intracellular pH controls intracellular Ca2+ concentration, membrane potential and motility of the axoneme, and appears to be a key regulator of the sperm functional changes in the female reproductive tract. Unfortunately, the mechanisms controlling sperm intracellular pH remain poorly understood. Here we applied the whole-cell configuration of the patch-clamp technique to identify and characterize these mechanisms. In human sperm, when pH of the pipette and bath solutions was 6.0 and 7.4 correspondingly, we observed a robust voltage-gated proton current with a half-activation voltage +13 mV. Similar to voltage-gated proton channels found in other cell types, the sperm proton channel (sHv) was strongly up-regulated by unsaturated fatty acids and potently blocked by Zn2+ with IC50 = 340 nM. Millimolar concentrations of Ca2+ and Mg2+ slowed down sHv activation kinetic but did not significantly reduced its amplitude. The amplitude of the voltage-gated proton current observed in human sperm was one of the highest among different cell types, with average current density ∼ 50 pA/pF at +100 mV; however in mouse sperm the amplitude of the voltage-gated proton current at the same conditions was only about 5 pA/pF. Intracellular alkalinization induced by sHv can lead to activation of pH-sensitive CatSper calcium channel resulting in well-known phenomenon of voltage-gated Ca2+ entry into the sperm cell. Here we present a model of how sHv may regulate sperm motility and discuss its role in male fertility and contraception.