Brain and testis: more alike than previously thought?

Several strands of evidence indicate the presence of marked similarities between human brain and testis. Understanding these similarities and their implications has become a topic of interest among the scientific community. Indeed, an association of intelligence with some semen quality parameters has been reported and a relation between dysfunctions of the human brain and testis has also been evident. Numerous common molecular features are evident when these tissues are compared, which is reflected in the huge number of common proteins. At the functional level, human neurons and sperm share a number of characteristics, including the importance of the exocytotic process and the presence of similar receptors and signalling pathways. The common proteins are mainly involved in exocytosis, tissue development and neuron/brain-associated biological processes. With this analysis, we conclude that human brain and testis share several biochemical characteristics which, in addition to their involvement in the speciation process, could, at least in part, be responsible for the expression of a huge number of common proteins. Nonetheless, this is an underexplored topic, and the connection between these tissues needs to be clarified, which could help to understand the dysfunctions affecting brain and testis, as well as to develop improved therapeutic strategies.

Human sperm ion channel (dys)function: implications for fertilization.

BACKGROUND: Intensive research on sperm ion channels has identified members of several ion channel families in both mouse and human sperm. Gene knock-out studies have unequivocally demonstrated the importance of the calcium and potassium conductances in sperm for fertility. In both species, the calcium current is carried by the highly complex cation channel of sperm (CatSper). In mouse sperm, the potassium current has been conclusively shown to be carried by a channel consisting of the pore forming subunit SLO3 and auxiliary subunit leucine-rich repeat-containing 52 (LRRC52). However, in human sperm it is controversial whether the pore forming subunit of the channel is composed of SLO3 and/or SLO1. Deciphering the role of the proton-specific Hv1 channel is more challenging as it is only expressed in human sperm. However, definitive evidence for a role in, and importance for, human fertility can only be determined through studies using clinical samples. OBJECTIVE AND RATIONALE: This review aims to provide insight into the role of sperm ion channels in human fertilization as evidenced from recent studies of sperm from infertile men. We also summarize the key discoveries from mouse ion channel knock-out models and contrast the properties of mouse and human CatSper and potassium currents. We detail the evidence for, and consequences of, defective ion channels in human sperm and discuss hypotheses to explain how defects arise and why affected sperm have impaired fertilization potential. SEARCH METHODS: Relevant studies were identified using PubMed and were limited to ion channels that have been characterized in mouse and human sperm. Additional notable examples from other species are included as appropriate. OUTCOMES: There are now well-documented fundamental differences between the properties of CatSper and potassium channel currents in mouse and human sperm. However, in both species, sperm lacking either channel cannot fertilize in vivo and CatSper-null sperm also fail to fertilize at IVF. Sperm-lacking potassium currents are capable of fertilizing at IVF, albeit at a much lower rate. However, additional complex and heterogeneous ion channel dysfunction has been reported in sperm from infertile men, the causes of which are unknown. Similarly, the nature of the functional impairment of affected patient sperm remains elusive. There are no reports of studies of Hv1 in human sperm from infertile men. WIDER IMPLICATIONS: Recent studies using sperm from infertile men have given new insight and critical evidence supporting the supposition that calcium and potassium conductances are essential for human fertility. However, it should be highlighted that many fundamental questions remain regarding the nature of molecular and functional defects in sperm with dysfunctional ion channels. The development and application of advanced technologies remains a necessity to progress basic and clinical research in this area, with the aim of providing effective screening methodologies to identify and develop treatments for affected men in order to help prevent failed ART cycles. Conversely, development of drugs that block calcium and/or potassium conductances in sperm is a plausible strategy for producing sperm-specific contraceptives.

Homozygous in-frame deletion in CATSPERE in a man producing spermatozoa with loss of CatSper function and compromised fertilizing capacity.

STUDY QUESTION: Does a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene? SUMMARY ANSWER: Patient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE. WHAT IS KNOWN ALREADY: CatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex. STUDY DESIGN, SIZE, DURATION: This was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ. PARTICIPANTS/MATERIALS, SETTING, METHODS: The original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant. MAIN RESULTS AND THE ROLE OF CHANCE: Patient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious. LIMITATIONS, REASONS FOR CAUTION: The nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Population genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

A novel cross-species inhibitor to study the function of CatSper Ca2+ channels in sperm.

BACKGROUND AND PURPOSE: Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH: We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS: RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS: We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.

Single-cell analysis of [Ca2+]i signalling in sub-fertile men: characteristics and relation to fertilization outcome.

STUDY QUESTION: What are the characteristics of progesterone-induced (CatSper-mediated) single cell [Ca2+]i signals in spermatozoa from sub-fertile men and how do they relate to fertilizing ability? SUMMARY ANSWER: Single cell analysis of progesterone-induced (CatSper-mediated) [Ca2+]i showed that reduced progesterone-sensitivity is a common feature of sperm from sub-fertile patients and is correlated with fertilization rate. WHAT IS KNOWN ALREADY: Stimulation with progesterone is a widely used method for assessing [Ca2+]i mobilization by activation of CatSper in human spermatozoa. Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability. STUDY DESIGN, SIZE, DURATION: This was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA). MAIN RESULTS AND THE ROLE OF CHANCE: For analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization; 62 samples), IVF-FF (failed fertilization; eight samples) and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response (progesterone-induced increase in fluorescence normalized to resting level) was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study and caution must be taken when extrapolating these results in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by a MRC project grant (MR/M012492/1; MR/K013343/1). Additional funding was provided by Chief Scientist Office/NHS research Scotland.

On-Demand Electrical Switching of Antibody-Antigen Binding on Surfaces.

The development of stimuli-responsive interfaces between synthetic materials and biological systems is providing the unprecedented ability to modulate biomolecular interactions for a diverse range of biotechnological and biomedical applications. Antibody-antigen binding interactions are at the heart of many biosensing platforms, but no attempts have been made yet to control antibody-antigen binding in an on-demand fashion. Herein, a molecular surface was designed and developed that utilizes an electric potential to drive a conformational change in surface bound peptide moiety, to give on-demand control over antigen-antibody interactions on sensor chips. The molecularly engineered surfaces allow for propagation of conformational changes from the molecular switching unit to a distal progesterone antigen, resulting in promotion (ON state) or inhibition (OFF state) of progesterone antibody binding. The approach presented here can be generally applicable to other antigen-antibody systems and meets the technological needs for in situ long-term assessment of biological processes and disease monitoring on-demand.

Complex CatSper-dependent and independent [Ca2+]i signalling in human spermatozoa induced by follicular fluid.

STUDY QUESTION: Does progesterone in human follicular fluid (hFF) activate CatSper and do other components of hFF modulate this effect and/or contribute separately to hFF-induced Ca2+ signaling? SUMMARY ANSWER: hFF potently stimulates CatSper and increases [Ca2+]i, primarily due to high concentrations of progesterone, however, other components of hFF also contribute to [Ca2+]i signaling, including modulation of CatSper channel activity and inhibition of [Ca2+]i oscillations. WHAT IS KNOWN ALREADY: CatSper, the principal Ca2+ channel in spermatozoa, is progesterone-sensitive and essential for fertility. Both hFF and progesterone, which is present in hFF, influence sperm function and increase their [Ca2+]i. STUDY DESIGN, SIZE, DURATION: This basic medical research study used semen samples from >40 donors and hFF from >50 patients who were undergoing surgical oocyte retrieval for IVF/ICSI. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service REC1. Activities of CatSper and KSper were assessed by patch clamp electrophysiology. Sperm [Ca2+]i responses were examined in sperm populations and single cells. Computer-assisted sperm analysis (CASA) parameters and penetration into viscous media were used to assess functional effects. MAIN RESULTS AND THE ROLE OF CHANCE: hFF and progesterone significantly potentiated CatSper currents. Under quasi-physiological conditions, hFF (up to 50%) failed to alter membrane K+ conductance or current reversal potential. hFF and progesterone (at an equivalent concentration) stimulated similar biphasic [Ca2+]i signals both in sperm populations and single cells. At a high hFF concentration (10%), the sustained (plateau) component of the [Ca2+]i signal was consistently greater than that induced by progesterone alone. In single cell recordings, 1% hFF-induced [Ca2+]i oscillations similarly to progesterone but with 10% hFF generation of [Ca2+]i oscillations was suppressed. After treatment to 'strip' lipid-derived mediators, hFF failed to significantly stimulate CatSper currents but induced small [Ca2+]i responses that were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+]i responses were observed when sperm pretreated with 3 µM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped hFF. hFF stimulated viscous media penetration and was more effective than the equivalent does of progesterone. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study. Caution must be taken when extrapolating these results in vivo. WIDER IMPLICATIONS OF THE FINDINGS: This study directly demonstrates that hFF activates CatSper and establishes that the biologically important effects of hFF reflect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [Ca2+]i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution. STUDY FUNDING/COMPETING INTEREST(S): Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P., C.L.R.B.) and University of Abertay (sabbatical for S.G.B.). Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest.

Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.

STUDY QUESTION: Are significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success? SUMMARY ANSWER: Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF. WHAT IS KNOWN ALREADY: Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects. STUDY DESIGN, SIZE AND DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities. MAIN RESULTS AND THE ROLE OF CHANCE: Patch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function. LIMITATIONS, REASONS FOR CAUTION: For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects. WIDER IMPLICATIONS OF THE FINDINGS: These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized. STUDY FUNDING/COMPETING INTERESTS: The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

p,p'-DDE activates CatSper and compromises human sperm function at environmentally relevant concentrations.

STUDY QUESTION: Is the environmental endocrine disruptor p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) able to induce non-genomic changes in human sperm and consequently affect functional sperm parameters? SUMMARY ANSWER: p,p'-DDE promoted Ca(2+) flux into human sperm by activating CatSper channels even at doses found in human reproductive fluids, ultimately compromising sperm parameters important for fertilization. WHAT IS KNOWN ALREADY: p,p'-DDE may promote non-genomic actions and interact directly with pre-existing signaling pathways, as already observed in other cell types. However, although often found in both male and female reproductive fluids, its effects on human spermatozoa function are not known. STUDY DESIGN, SIZE, DURATION: Normozoospermic sperm samples from healthy individuals were included in this study. Samples were exposed to several p,p'-DDE concentrations for 3 days at 37°C and 5% CO2 in vitro to mimic the putative continuous exposure to this toxicant in the female reproductive tract in vivo. Shorter p,p'-DDE incubation periods were also performed in order to monitor sperm rapid Ca(2+) responses. All experiments were repeated on a minimum of five sperm samples from different individuals. PARTICIPANTS/MATERIALS, SETTING, METHODS: All healthy individuals were recruited at the Biosciences School, University of Birmingham, the Medical Research Institute, University of Dundee and in the Human Reproduction Service at University Hospitals of Coimbra. Intracellular Ca(2+) concentration ([Ca(2+)]i) was monitored by imaging single spermatozoa loaded with Oregon Green BAPTA-1AM and further whole-cell patch-clamp recordings were performed to validate our results. Sperm viability and acrosomal integrity were assessed using the LIVE/DEAD sperm vitality kit and the acrosomal content marker PSA-FITC, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: p,p'-DDE rapidly increased [Ca(2+)]i (P < 0.05) even at extremely low doses (1 pM and 1 nM), with magnitudes of response up to 200%, without affecting sperm viability, except after 3 days of continuous exposure to the highest concentration tested (P < 0.05). Furthermore, experiments performed in a low Ca(2+) medium demonstrated that extracellular Ca(2+) influx was responsible for this Ca(2+) increase (P < 0.01). Mibefradil and NNC 55-0396, both inhibitors of the sperm-specific CatSper channel, reversed the p,p'-DDE-induced [Ca(2+)]i rise, suggesting the participation of CatSper in this process (P < 0.05). In fact, whole-cell patch-clamp recordings confirmed CatSper as a target of p,p'-DDE action by monitoring an increase in CatSper currents of >100% (P < 0.01). Finally, acrosomal integrity was adversely affected after 2 days of exposure to p,p'-DDE concentrations, suggesting that [Ca(2+)]i rise may cause premature acrosome reaction (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study, and caution must be taken when extrapolating the results. WIDER IMPLICATIONS OF THE FINDINGS: A novel non-genomic p,p'-DDE mechanism specific to sperm is shown in this study. p,p'-DDE was able to induce [Ca(2+)]i rise in human sperm through the opening of CatSper consequently compromising male fertility. The promiscuous nature of CatSper activation may predispose human sperm to the action of some persistent endocrine disruptors. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by both the Portuguese National Science Foundation (FCT; PEst-C/SAU/LA0001/2011) and the UK Wellcome Trust (Grant #86470). SM was supported by the Infertility Research Trust. RST is a recipient of a PhD fellowship from FCT (SFRH/BD/46002/2008). None of the authors has any conflict of interest to declare.

The clinical significance of calcium-signalling pathways mediating human sperm hyperactivation.

STUDY QUESTION: What is the prevalence of defects in the Ca(2+)-signalling pathways mediating hyperactivation (calcium influx and store mobilization) among donors and sub-fertile patients and are they functionally significant, i.e. related to fertilization success at IVF? SUMMARY ANSWER: This study identifies, for the first time, the prevalence of Ca(2+) store defects in sperm from research donors, IVF and ICSI patients. It highlights the biological role and importance of Ca(2+) signalling (Ca(2+) store mobilization) for fertilization at IVF. WHAT IS KNOWN ALREADY: Sperm motility and hyperactivation (HA) are important for fertility, mice with sperm incapable of HA are sterile. Recently, there has been significant progress in our knowledge of the factors controlling these events, in particular the generation and regulation of calcium signals. Both pH-regulated membrane Ca(2+) channels (CatSper) and Ca(2+) stores (potentially activating store-operated Ca(2+) channels) have been implicated in controlling HA. STUDY DESIGN, SIZE, AND DURATION: This was a prospective study examining a panel of 68 donors and 181 sub-fertile patients attending the Assisted Conception Unit, Ninewells Hospital Dundee for IVF and ICSI. Twenty-five of the donors gave a second sample (∼4 weeks later) to confirm consistency/reliability of the recorded responses. Ca(2+) signalling was manipulated using three agonists, NH4Cl (activates CatSper via pH), progesterone (direct activation of CatSper channels, potentially enhancing mobilization of stored Ca(2+) by CICR) and 4-aminopyridine (4-AP) (effect on pH equivalent to NH4Cl and mobilizes stored Ca(2+)). The broad-spectrum phosphodiesterase inhibitor 3-isobutyl-1-methyxanthine (IBMX), a potent activator of HA was also used for comparison. For patient samples, an aliquot surplus to requirements for IVF/ICSI treatment was examined, allowing direct comparison of Ca(2+) signalling and motility data with functional competence of the sperm. MATERIALS, SETTING, METHODS: The donors and sub-fertile patients were screened for HA (using CASA) and changes in intracellular Ca(2+) were assessed by loading with Fura-2 and measuring fluorescence using a plate reader (FluoStar). MAIN RESULTS AND THE ROLE OF CHANCE: The relative efficacy of the stimuli in inducing HA was 4-AP >> IBMX > progesterone. NH4Cl increased [Ca(2+)]i similarly to 4-AP and progesterone but did not induce a significant increase in HA. Failure of samples to generate HA (no significant increase in response to stimulation with 4-AP) was seen in just 2% of research donors but occurred in 10% of IVF patients (P = 0.025). All donor samples generated a significant [Ca(2+)]i increase when stimulated with 4-AP but 3.3% of IVF and 28.6% of ICSI patients failed to respond. Amplitudes of HA and [Ca(2+)]i responses to 4-AP were correlated with fertilization rate at IVF (P= 0.029; P = 0.031, respectively). Progesterone reliably induced [Ca(2+)]i responses (97% of donors, 100% of IVF patients) but was significantly less effective than 4-AP in inducing HA. Twenty seven per cent of ICSI patients failed to generate a [Ca(2+)]i response to progesterone (P= 0.035). Progesterone-induced [Ca(2+)]i responses were correlated with fertilization rate at IVF (P= 0.037) but induction of HA was not. In donor samples examined on more than one occasion consistent responses for 4-AP-induced [Ca(2+)]i (R(2) = 0.97) and HA (R(2) = 0.579) were obtained. In summary, the data indicate that defects in Ca(2+) signalling leading to poor HA do occur and that ability to undergo Ca(2+) -induced HA affects IVF fertilizing capacity. The data also confirm that release of stored Ca(2+) is the crucial component of Ca(2+) signals leading to HA and that Ca(2+) store defects may therefore underlie HA failure. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study of sperm function. While the repeatability of the [Ca(2+)]i and HA responses in samples from the same donor were confirmed, data for patients were from 1 assessment and thus the robustness of the failed responses in patients' needs to be established. The focus of this study was on using 4AP, which mobilizes stored Ca(2+) and is a potent inducer of HA. The n values for other agonists, especially calcium assessments, are smaller. WIDER IMPLICATIONS OF THE FINDINGS: Previous studies have shown a significant relationship between basal levels of HA, calcium responses to progesterone and IVF fertilization rates. Here, we have systematically investigated the ability/failure of human sperm to generate Ca(2+) signals and HA in response to targeted pharmacological challenge and, related defects in these responses to IVF success. [Ca(2+)]i signalling is fundamental for sperm motility and data from this study will lead to assessment of the nature of these defects using techniques such as single-cell imaging and patch clamping. STUDY FUNDING/COMPETING INTEREST(S): Resources from a Wellcome Trust Project Grant (#086470, Publicover and Barratt PI) primarily funded the study. The authors have no competing interests.