Complex CatSper-dependent and independent [Ca2+]i signalling in human spermatozoa induced by follicular fluid.
ABSTRACT: 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?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.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.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.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.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 ?M 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.N/A.This was an in vitro study. Caution must be taken when extrapolating these results in vivo.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.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.
Project description:Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the CatSper Ca2+-channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca2+ influx through CatSper, thus mimicking the effect of progesterone on Ca2+ signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca2+ influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca2+ signaling in human sperm cells can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo.
Project description:CatSper channel has been considered the principal sperm Ca2+ channel responsible for the cytosolic Ca2+ elevation required for various sperm functions necessary for fertilization [1-4]. However, the mechanism underlying the activation of CatSper channel by various physiological ligands remain incompletely understood. We have recently demonstrated the expression of C-C chemokine receptor 6 (CCR6) in sperm and Ca2+ influx upon binding of human ?-defensin 1 (DEFB1) to CCR6, which is important for sperm motility . In the present study, we have demonstrated that CCR6 receptor and CatSper channel are both required for the Ca2+ entry/current induced by physiological ligands DEFB1, chemokine (C-C motif) ligand 20 (CCL20) and progesterone in human sperm. CCR6 is co-localized and interacts with CatSper in human sperm. Ca2+ influx mediated by CCR6 and CatSper is required for essential sperm functions, including motility, hyperactivation and acrosome reaction, which are impaired in infertile sperm showing reduced levels of CCR6 and CatSper. The present finding suggests a critical role of CCR6 receptor in mediating ligand-induced, CatSper-dependent Ca2+ influx required for various sperm functions and thus male fertility.
Project description:Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1-2 s before responses in the head and neck. Pre-treatment with 5 ?M 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] 'amplified' progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 ?M) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50-100 ?M 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50-100 ?M 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.
Project description:The Catsper channel is a sperm-specific, Ca2+-permeable, pH-dependent, and low voltage-dependent channel that is essential for the hyperactivity of sperm flagellum, chemotaxis towards the egg, capacitation and acrosome reaction. All of these physiological events require calcium entry into sperm cells. Remarkably, Catsper genes are exclusively expressed in the testis during spermatogenesis, and are sensitive to ion channel-induced pH change, such as NHEs, Ca2+ATPase, K+ channel, Hv1 channel and HCO3- transporters. Furthermore, the Catsper channel is regulated by some physiological stimulants, such as progesterone, cyclic nucleotides (e.g., cAMP, cGMP), zona pellucida (ZP) glycoproteins and bovine serum albumin (BSA). All of these factors normally stimulate Ca2+ entry into sperm through the Catsper channel. In addition, the Catsper channel may be a potential target for male infertility treatment or contraception. This review will focus on the structure, functions, regulation mechanisms and medicinal targets of the Catsper channel.
Project description:Aim: Evidence suggests that bisphenol A diglycidyl ether (BADGE), bisphenol A (BPA), and BPA analogs can interfere with human male fertility. However, the effect directly on human sperm function is not known. The CatSper Ca2+ channel in human sperm controls important sperm functions and is necessary for normal male fertility. Environmental chemicals have been shown to activate CatSper and thereby affect Ca2+ signaling in human sperm. BPA has previously been investigated for effects on Ca2+ signaling human sperm, whereas the effects of other BPA analogs are currently unknown. The aim of this study is thus to characterize the effect of BADGE, BPA, and the eight analogs BPG, BPAF, BPC, BPB, BPBP, BPE, BPF, BPS on Ca2+ signaling, and CatSper in human sperm. Methods: Direct effects of the bisphenols on Ca2+ signaling in human sperm cells were evaluated using a Ca2+ fluorimetric assay measuring changes in intracellular Ca2+. Effects via CatSper were assessed using the specific CatSper inhibitor RU1968. Effects on human sperm function was assessed using an image cytometry-based acrosome reaction assay and the modified Kremer's sperm-mucus penetration assay. Results: At 10 ?M the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP induced Ca2+ signals in human sperm cells, whereas BPE, BPF, BPS, and BPA had no effect. The efficacy of the chemicals at 10 ?M is BPG > BPAF > BPC > BADGE > BPB > BPBP. Dose-response relations of BPG, BPAF, BPC, BADGE, BPB, and BPBP yielded EC50-values in the nM-?M range. The induced Ca2+ signals were almost completely abolished using the CatSper inhibitor RU1968, indicating an effect of the bisphenols on CatSper. All bisphenols, except BPBP, were found to dose-dependently inhibit progesterone-induced Ca2+ signals, with BPG and BPAF displaying inhibition even in low ?M doses. BPG and BPAF were shown to affect human sperm function in a progesterone-like manner. Conclusion: Our results show that the bisphenols BPG, BPAF, BPC, BADGE, BPB, and BPBP can affect Ca2+ signaling in human sperm cells through activation of CatSper. This could potentially disrupt human sperm function by interfering with normal CatSper-signaling and thus be a contributing factor in human infertility, either alone or in mixtures with other chemicals.
Project description: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.
Project description:BACKGROUND AND PURPOSE:Asthenozoospermia is a leading cause of male infertility, but development of pharmacological agents to improve sperm motility is hindered by the lack of effective screening platforms and knowledge of suitable molecular targets. We have demonstrated that a high-throughput screening (HTS) strategy and established in vitro tests can identify and characterise compounds that improve sperm motility. Here, we applied HTS to identify new compounds from a novel small molecule library that increase intracellular calcium ([Ca2+ ]i ), promote human sperm cell motility, and systematically determine the mechanism of action. EXPERIMENTAL APPROACH:A validated HTS fluorometric [Ca2+ ]i assay was used to screen an in-house library of compounds. Trequinsin hydrochloride (a PDE3 inhibitor) was selected for detailed molecular (plate reader assays, electrophysiology, and cyclic nucleotide measurement) and functional (motility and acrosome reaction) testing in sperm from healthy volunteer donors and, where possible, patients. KEY RESULTS:Fluorometric assays identified trequinsin as an efficacious agonist of [Ca2+ ]i , although less potent than progesterone. Functionally, trequinsin significantly increased cell hyperactivation and penetration into viscous medium in all donor sperm samples and cell hyperactivation in 22/25 (88%) patient sperm samples. Trequinsin-induced [Ca2+ ]i responses were cross-desensitised consistently by PGE1 but not progesterone. Whole-cell patch clamp electrophysiology confirmed that trequinsin activated CatSper and partly inhibited potassium channel activity. Trequinsin also increased intracellular cGMP. CONCLUSION AND IMPLICATIONS:Trequinsin exhibits a novel pharmacological profile in human sperm and may be a suitable lead compound for the development of new agents to improve patient sperm function and fertilisation potential.
Project description: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.
Project description: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?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.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.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.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).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).This is an in vitro study and caution must be taken when extrapolating these results in vivo.This study reveals important details of impaired [Ca2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies.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.
Project description:Aim:Exposure of boar sperm cells to Bisphenol A diglycidyl ether (BADGE) has been shown to lead to reproductive failure in sows, however, the mode of action is unknown. As we have recently shown that BADGE can interfere with Ca2 + signaling in human sperm cells through an action on CatSper, and as CatSper has been shown to be expressed in boar sperm cells, we hypothesized that a similar mechanism in the boar sperm cells could be responsible for the reproductive failure. Methods:Direct effects of BADGE and the endogenous ligand of human CatSper, progesterone, on Ca2+ signaling in human and boar sperm cells were evaluated side-by-side using a Ca2+ fluorimetric assay measuring changes in intracellular Ca2+. Effects of BADGE on Ca2+ signaling in boar sperm were furthermore assessed by flow cytometry by an independent laboratory. Results:The exact same solutions of BADGE and progesterone induced transient biphasic Ca2+ signals in human sperm cells, but failed to do so in both non-capacitated and capacitated boar sperm cells. BADGE also failed to induce transient biphasic Ca2+ signals in boar sperm cells in the flow cytometric assay. Conclusion:BADGE and progesterone failed to induce Ca2+ signals in boar sperm cells. This indicates that the signaling mechanisms leading to activation of CatSper differs between human and boar sperm cells, and suggests that the mode of action by which exposure of boar sperm cells to BADGE can lead to reproductive failure in sows does not involve effects on Ca2+ signaling.