Capacitation-associated alkalization in human sperm is differentially controlled at the subcellular level.

Capacitation in mammalian sperm involves the accurate balance of intracellular pH (pHi), but the mechanisms controlling this process are not fully understood, particularly regarding the spatiotemporal regulation of the proteins involved in pHi modulation. Here, we employed an image-based flow cytometry technique combined with pharmacological approaches to study pHi dynamics at the subcellular level during capacitation. We found that, upon capacitation induction, sperm cells undergo intracellular alkalization in the head and principal piece regions. The observed localized pHi increases require the initial uptake of HCO3-, which is mediated by several proteins acting consistently with their subcellular localization. Hv1 proton channel (also known as HVCN1) and cAMP-activated protein kinase (protein kinase A, PKA) antagonists impair alkalization mainly in the principal piece. Na+/HCO3- cotransporter (NBC) and cystic fibrosis transmembrane regulator (CFTR) antagonists impair alkalization only mildly, predominantly in the head. Motility measurements indicate that inhibition of alkalization in the principal piece prevents the development of hyperactivated motility. Altogether, our findings shed light on the complex control mechanisms of pHi and underscore their importance during human sperm capacitation.This article has an associated First Person interview with the first author of the paper.

Hyperpolarization induces cytosolic alkalization of mouse sperm flagellum probably through sperm Na+/H+ exchanger.

In brief: Hyperpolarization of the membrane potential is a crucial step for mammalian sperm maturation. This work demonstrates that this membrane potential change likely activates a sperm-specific sodium/proton exchanger to induce alkalization in mouse sperm flagellum. Abstract: The sperm-specific sodium/proton exchanger (sNHE) is an indispensable protein for male fertility in mammals. Nevertheless, it is still unknown how mammalian sNHE is regulated. Evidence obtained from sea urchin sNHE indicates that hyperpolarization of plasma membrane potential (Vm), which is a hallmark of mammalian capacitation, positively regulates the sNHE. Therefore, we explored the activity of sNHE in mouse and human sperm by fluorescence imaging of intracellular pH (pHi) with a ratiometric dye, SNARF-5F. A valinomycin-induced Vm hyperpolarization elevated sperm flagellar pHi of WT mouse but not in sNHE-KO mouse. Moreover, this pHi increase was inhibited in a high K+ (40 mM) medium. These results support the idea that mouse sNHE is activated by Vm hyperpolarization. Interestingly, we observed different types of kinetics derived from valinomycin-induced alkalization, including some (30%) without any pHi changes. Our quantitative pHi determinations revealed that unresponsive cells had a high resting pHi (>7.5), suggesting that the activity of mouse sNHE is regulated by the resting pHi. On the other hand, valinomycin did not increase the pHi of human sperm in the head or the flagellum, regardless of their resting pHi values. Our findings suggest that the regulatory mechanisms of mammalian sNHEs are probably distinct depending on the species.

Differences and Similarities: The Richness of Comparative Sperm Physiology.

Species preservation depends on the success of fertilization. Sperm are uniquely equipped to fulfill this task, and, although several mechanisms are conserved among species, striking functional differences have evolved to contend with particular sperm-egg environmental characteristics. This review highlights similarities and differences in sperm strategies, with examples within internal and external fertilizers, pointing out unresolved issues.

[Ca2+]i oscillations in human sperm are triggered in the flagellum by membrane potential-sensitive activity of CatSper.

STUDY QUESTION: How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated? SUMMARY ANSWER: P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane potential (Vm)-sensitive Ca2+-influx through CatSper channels. WHAT IS KNOWN ALREADY: A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required. STUDY DESIGN, SIZE, DURATION: This was a laboratory study that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1000 individual cells over a period of 2 years. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp. MAIN RESULTS AND THE ROLE OF CHANCE: P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1-2 s). K+-ionophore valinomycin (1 µM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly 'reset' upon manipulation of [K+]o. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n = 257; P = 5 × 10-55 compared to control) and significantly reduced both the amplitude and frequency of persisting oscillations (P = 0.0001). Upon valinomycin washout, oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+], the inhibitory effect of valinomycin was reduced and was dependent on EK (P = 10-25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P < 0.01). The CatSper inhibitor RU1968 (4.8 and 11 µM) caused immediate and reversible arrest of activity in 36% and 96% of oscillating cells, respectively (P < 10-10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in-vitro study and caution must be taken when extrapolating these results to in-vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the regulation and generation (or failure) of these oscillations. STUDY FUNDING/COMPETING INTEREST(S): E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest.

Reactive oxygen species are involved in the signaling of equine sperm chemotaxis.

Sperm chemotaxis may facilitate the finding of the oocyte. Only capacitated spermatozoa can orient their movement by chemotaxis, which as well as capacitation, is regulated in part by the cAMP-PKA pathway. Reactive oxygen species (ROS) are produced during sperm capacitation which is closely related to chemotaxis. Then, the ROS participation in the chemotactic signaling can be expected. Here we studied the role of ROS in the chemotaxis signaling of equine spermatozoa which produce high quantities of ROS because of their energy metabolism. The level of capacitated and chemotactic spermatozoa was increased with 0.1 and 0.2 mM hydrogen peroxide (H2O2), which was involved in the chemotactic signaling. By combining a concentration gradient of H2O2 with inhibitors/chelators of some of the signaling pathway elements, we showed that the activation of NOX (membrane NADPH oxidase) increases the intracellular ROS which activate the chemotaxis AMPc-PKA pathway. Our results provide evidence about the participation of ROS in the chemotactic signaling mediated by progesterone (P).

Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm.

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

CFTR/ENaC-dependent regulation of membrane potential during human sperm capacitation is initiated by bicarbonate uptake through NBC.

To fertilize an egg, sperm must reside in the female reproductive tract to undergo several maturational changes that are collectively referred to as capacitation. From a molecular point of view, the HCO3--dependent activation of the atypical soluble adenylyl cyclase (ADCY10) is one of the first events that occurs during capacitation and leads to the subsequent cAMP-dependent activation of protein kinase A (PKA). Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. We previously reported that PKA activation is necessary for CFTR (cystic fibrosis transmembrane conductance regulator channel) activity and for the modulation of membrane potential (Em). However, the main HCO3- transporters involved in the initial transport and the PKA-dependent Em changes are not well known nor characterized. Here, we analyzed how the activity of CFTR regulates Em during capacitation and examined its relationship with an electrogenic Na+/HCO3- cotransporter (NBC) and epithelial Na+ channels (ENaCs). We observed that inhibition of both CFTR and NBC decreased HCO3- influx, resulting in lower PKA activity, and that events downstream of the cAMP activation of PKA are essential for the regulation of Em. Addition of a permeable cAMP analog partially rescued the inhibitory effects caused by these inhibitors. HCO3- also produced a rapid membrane hyperpolarization mediated by ENaC channels, which contribute to the regulation of Em during capacitation. Altogether, we demonstrate for the first time, that NBC cotransporters and ENaC channels are essential in the CFTR-dependent activation of the cAMP/PKA signaling pathway and Em regulation during human sperm capacitation.

Analyzing the functional divergence of Slo1 and Slo3 channel subfamilies.

Slo1 and Slo3 encode close paralogues of the Slo potassium (K+) channels family. Despite their evolutionary relatedness, Slo1 and Slo3 channels show marked functional differences and evolutionary dynamics. Whereas Slo1 is a highly conserved and widely expressed channel, Slo3 is a rapidly evolving channel restricted to sperm. However, the molecular mechanisms behind the structural-functional differences of Slo1 and Slo3 channels are unknown. In this study, we explored the functional divergence of Slo1 and Slo3 subfamilies in vertebrates and examined the structure-function relationships of our predictions using experimental data. We found that ∼25% of sites between Slo1 and Slo3 underwent altered functional constraints, affecting some residues with important roles in Slo1 channel gating. Because functional divergence was principally generated by accelerated evolution of Slo3 after gene duplication, we explored selective forces behind Slo3 diversification. We observed that Slo3 subjected was principally subjected to relaxation of purifying selection, but we also identified several sites evolving under positive selection in the cytosolic domain of this channel . Concerning Slo1, this channel presented strong purifying selection. Whether residues evolving under different selection in Slo1 and Slo3 are responsible for functional differences observed between these channels, as well as among Slo3 orthologs, remains to be established.

A cytoplasmic Slo3 isoform is expressed in somatic tissues.

Slo3 is a pH-sensitive and weakly voltage-sensitive potassium channel that is essential for male fertility in mouse and whose expression is regarded as sperm-specific. These properties have proposed Slo3 as a candidate target for male contraceptive drugs. Nonetheless, the tissue distribution of Slo3 expression has not been rigorously studied yet. Applying computational and RT-PCR approaches, we identified expression of two short Slo3 isoforms in somatic mouse tissues such as brain, kidney and eye. These isoforms, which seem to result of transcription starting sites between exons 20 and 21, have an identical open reading frame, both encoding the terminal 381 amino acids of the cytosolic Slo3 domain. We corroborated the expression of these isoforms in mouse brain and testis by Western-blot. The complete isoform encoding the Slo3 ion channel was uniquely detected in testis, both at transcript and protein level. Although the functional role of the cytosolic Slo3 isoforms remains to be established, we propose that they may have a functional effect by modulating Slo channels trafficking and/or activity. This study confirms that expression of full-length Slo3 is sperm-specific but warns against developing contraceptive drugs targeting the C-terminal tail of Slo3 channels.

Acrosomal alkalization triggers Ca2+ release and acrosome reaction in mammalian spermatozoa.

The sperm acrosome reaction (AR), an essential event for mammalian fertilization, involves Ca2+ permeability changes leading to exocytosis of the acrosomal vesicle. The acrosome, an intracellular Ca2+ store whose luminal pH is acidic, contains hydrolytic enzymes. It is known that acrosomal pH (pHacr ) increases during capacitation and this correlates with spontaneous AR. Some AR inducers increase intracellular Ca2+ concentration ([Ca2+ ]i ) through Ca2+ release from internal stores, mainly the acrosome. Catsper, a sperm specific Ca2+ channel, has been suggested to participate in the AR. Curiously, Mibefradil and NNC55-0396, two CatSper blockers, themselves elevate [Ca2+ ]i by unknown mechanisms. Here we show that these compounds, as other weak bases, can elevate pHacr , trigger Ca2+ release from the acrosome, and induce the AR in both mouse and human sperm. To our surprise, µM concentrations of NNC55-0396 induced AR even in nominally Ca2+ free media. Our findings suggest that alkalization of the acrosome is critical step for Ca2+ release from the acrosome that leads to the acrosome reaction.

Calcium channels in the development, maturation, and function of spermatozoa.

A proper dialogue between spermatozoa and the egg is essential for conception of a new individual in sexually reproducing animals. Ca(2+) is crucial in orchestrating this unique event leading to a new life. No wonder that nature has devised different Ca(2+)-permeable channels and located them at distinct sites in spermatozoa so that they can help fertilize the egg. New tools to study sperm ionic currents, and image intracellular Ca(2+) with better spatial and temporal resolution even in swimming spermatozoa, are revealing how sperm ion channels participate in fertilization. This review critically examines the involvement of Ca(2+) channels in multiple signaling processes needed for spermatozoa to mature, travel towards the egg, and fertilize it. Remarkably, these tiny specialized cells can express exclusive channels like CatSper for Ca(2+) and SLO3 for K(+), which are attractive targets for contraception and for the discovery of novel signaling complexes. Learning more about fertilization is a matter of capital importance; societies face growing pressure to counteract rising male infertility rates, provide safe male gamete-based contraceptives, and preserve biodiversity through improved captive breeding and assisted conception initiatives.

Positive Selection in the Evolution of Mammalian CRISPs.

Cysteine-RIch Secretory Proteins (CRISPs) constitute a versatile family, with functions in reptilian venom and mammalian reproduction. Mammals generally express three CRISPs, four in mice, and all are highly expressed in male reproductive tissues, either testis or accessory organs. Because reproductive proteins often evolve adaptively in response to post-copulatory sexual selection, we hypothesized that mammalian CRISPs, with important roles in male reproduction, could have undergone positive selection promoting their divergence. We explored the molecular adaptation of mammalian CRISPs applying phylogenetic methods. Our analyses revealed the evidence of positive selection in all mammalian CRISPs. The intensity of positive selection was heterogeneous among CRISP members, being stronger in CRISP3 than in CRISP1 and CRISP2, and also across functional domains, having stronger impact on Pathogenesis-Related 1 (PR-1) in CRISP2 and on Ion Channel Regulator (ICR) in CRISP1 and CRISP3. In addition, we discovered a new CRISP in some rodent species, suggesting that the acquisition of new CRISP components could contribute to male reproductive success or to acquire new physiological roles. Signatures of positive selection were not focused on any particular mammalian group, suggesting that adaptive evolution is a recurrent pattern in mammalian CRISPs. Our findings support a model of CRISP family diversification driven by episodes of duplication and posterior neofunctionalization, and provide potential adaptive changes responsible for interspecific differences in CRISPs activity.

pH-dependent Ca+2 oscillations prevent untimely acrosome reaction in human sperm.

During transit through the female reproductive tract, sperm encounter metabolites and environmental conditions that modulate various processes leading to fertilization. Intracellular Ca2+ dynamics regulate the acrosome reaction (AR), which involves exocytosis of the acrosomal granule, a prerequisite for successful fertilization. We explored the ability of progesterone, prostanglandin-E1, and GABA to induce Ca2+ mobilization and AR in single human spermatozoa capacitated under external pH (pHe) conditions found in different regions of the female reproductive tract (pHe 6.5, 7.4 and 8.0). The highest percentage of AR induction, regardless of the inducer, occurred when sperm were capacitated at pHe 7.4. Interestingly, at pHe 6.5 a high percentage of cells exhibit Ca2+ oscillations, which prevent AR. These oscillations involve extracellular and intracellular Ca2+ channels. Pharmacological inhibition of Ca2+ oscillations restores the ability of spermatozoa to undergo the AR when exposed to progesterone, even if capacitated at pHe 6.5.

Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation.

STUDY QUESTION: Is image-based flow cytometry a useful tool to study intracellular events in human sperm such as protein tyrosine phosphorylation or signaling processes? SUMMARY ANSWER: Image-based flow cytometry is a powerful tool to study intracellular events in a relevant number of sperm cells, which enables a robust statistical analysis providing spatial resolution in terms of the specific subcellular localization of the labeling. WHAT IS KNOWN ALREADY: Sperm capacitation is required for fertilization. During this process, spermatozoa undergo numerous physiological changes, via activation of different signaling pathways, which are not completely understood. Classical approaches for studying sperm physiology include conventional microscopy, flow cytometry and Western blotting. These techniques present disadvantages for obtaining detailed subcellular information of signaling pathways in a relevant number of cells. This work describes a new semi-automatized analysis using image-based flow cytometry which enables the study, at the subcellular and population levels, of different sperm parameters associated with signaling. The increase in protein tyrosine phosphorylation during capacitation is presented as an example. STUDY DESIGN SIZE, DURATION: Sperm cells were isolated from seminal plasma by the swim-up technique. We evaluated the intensity and distribution of protein tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h under different experimental conditions. We used an antibody against FER kinase and pharmacological inhibitors in an attempt to identify the kinases involved in protein tyrosine phosphorylation during human sperm capacitation. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples from normospermic donors were obtained by masturbation after 2-3 days of sexual abstinence. We used the innovative technique image-based flow cytometry and image analysis tools to segment individual images of spermatozoa. We evaluated and quantified the regions of sperm where protein tyrosine phosphorylation takes place at the subcellular level in a large number of cells. We also used immunocytochemistry and Western blot analysis. Independent experiments were performed with semen samples from seven different donors. MAIN RESULTS AND THE ROLE OF CHANCE: Using image analysis tools, we developed a completely novel semi-automatic strategy useful for segmenting thousands of individual cell images obtained using image-based flow cytometry. Contrary to immunofluorescence which relies on the analysis of a limited sperm population and also on the observer, image-based flow cytometry allows for unbiased quantification and simultaneous localization of post-translational changes in an extended sperm population. Interestingly, important data can be independently analyzed by looking to the frame of interest. As an example, we evaluated the capacitation-associated increase in tyrosine phosphorylation in sperm incubated in non-capacitation and capacitation-supporting media for 1 and 18 h. As previously reported, protein tyrosine phosphorylation increases in a time-depending manner, but our method revealed that this increase occurs differentially among distinct sperm segments. FER kinase is reported to be the enzyme responsible for the increase in protein tyrosine phosphorylation in mouse sperm. Our Western blot analysis revealed for the first time the presence of this enzyme in human sperm. Using our segmentation strategy, we aimed to quantify the effect of pharmacological inhibition of FER kinase and found a marked reduction of protein tyrosine phosphorylation only in the flagellum, which corresponded to the physical localization of FER in human sperm. Our method provides an alternative strategy to study signaling markers associated with capacitation, such as protein tyrosine phosphorylation, in a fast and quantitative manner. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: This is an in vitro study performed under controlled conditions. Chemical inhibitors are not completely specific for the intended target; the possibility of side effects cannot be discarded. WIDER IMPLICATIONS OF THE FINDINGS: Our results demonstrate that the use of image-based flow cytometry is a very powerful tool to study sperm physiology. A large number of cells can be easily analyzed and information at the subcellular level can be obtained. As the segmentation process works with bright-field images, it can be extended to study expression of other proteins of interest using different antibodies or it can be used in living sperm to study intracellular parameters that can be followed using fluorescent dyes sensitive to the parameter of interest (e.g. pH, Ca2+). Therefore, this a versatile method that can be exploited to study several aspects of sperm physiology. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported DGAPA (IN203116 to C. Treviño), Fronteras-CONACyT No. 71 and Eunice Kennedy Shriver National Institute of Child Health and Human Development NIH (RO1 HD38082) to P.E. Visconti and by a Lalor Foundation fellowship to M.G. Gervasi. A. Matamoros is a student of the Maestría en Ciencias Bioquímicas-UNAM program supported by CONACyT (416400) and DGAPA-UNAM. A. Moreno obtained a scholarship from Red MacroUniversidades and L. Giojalas obtained a schloarhip from CONICET and Universidad Nacional de Cordoba. The authors declare there are not conflicts of interest.

Essential Role of CFTR in PKA-Dependent Phosphorylation, Alkalinization, and Hyperpolarization During Human Sperm Capacitation.

Mammalian sperm require to spend a limited period of time in the female reproductive tract to become competent to fertilize in a process called capacitation. It is well established that HCO3- is essential for capacitation because it activates the atypical soluble adenylate cyclase ADCY10 leading to cAMP production, and promotes alkalinization of cytoplasm, and membrane hyperpolarization. However, how HCO3- is transported into the sperm is not well understood. There is evidence that CFTR activity is involved in the human sperm capacitation but how this channel is integrated in the complex signaling cascades associated with this process remains largely unknown. In the present work, we have analyzed the extent to which CFTR regulates different events in human sperm capacitation. We observed that inhibition of CFTR affects HCO3- -entrance dependent events resulting in lower PKA activity. CFTR inhibition also affected cAMP/PKA-downstream events such as the increase in tyrosine phosphorylation, hyperactivated motility, and acrosome reaction. In addition, we demonstrated for the first time, that CFTR and PKA activity are essential for the regulation of intracellular pH, and membrane potential in human sperm. Addition of permeable cAMP partially recovered all the PKA-dependent events altered in the presence of inh-172 which is consistent with a role of CFTR upstream of PKA activation. J. Cell. Physiol. 232: 1404-1414, 2017. © 2016 Wiley Periodicals, Inc.

Luteinizing hormone modulates intracellular calcium, protein tyrosine phosphorylation and motility during human sperm capacitation.

In order to fertilize, spermatozoa must undergo physiological and biochemical changes during their transit along the female reproductive tract before reaching and fusing with the oocyte, process known as capacitation. Sperm modifications associated with capacitation are modulated by their interaction with molecules present in the female reproductive tract. During the woman fertile window, some reproductive hormones reach their maximum concentrations in serum, such as the luteinizing hormone (LH). Since spermatozoa preparing to fertilize may be exposed to LH, the purpose of this work was to study the effects of this hormone on intracellular Ca2+ concentrations ([Ca2+]i), protein tyrosine phosphorylation, sperm motility and acrosome reaction under capacitating conditions. The results showed that LH increases the duration and amplitude of Ca2+ oscillations. Furthermore, motility analysis indicated that LH decreases rapid progressive motility and that sperm hyperactivation as well as several kinetic parameters augment in the presence of 0.5 and 1 µg/ml of the hormone. In addition, these two hormone concentrations also consistently promoted protein tyrosine phosphorylation. However, no effects on acrosome reaction were observed. In conclusion, the evidence indicates that LH modulates several sperm function variables involved in capacitation, suggesting that may have an important and unexplored role during human fertilization.

Regulation of Spermatogenic Cell T-Type Ca(2+) Currents by Zn(2+): Implications in Male Reproductive Physiology.

Zn(2+) is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis, and prostate contain high Zn(2+) concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV 3.2-encoded T-type Ca(2+) currents (ICaT) which are positively or negatively modulated by Zn(2+) in other tissues. To explore whether ICaT could be regulated by Zn(2+) and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn(2+) concentrations. Zn(2+) decreased ICaT in a concentration-dependent manner (IC50 = 2 µM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn(2+) . Further analysis showed that Zn(2+) did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady-state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non-stationary fluctuation analysis, we found that Zn(2+) mainly reduced the number of available Ca(2+) channels without changing the single channel current amplitude. ICaT modulation by Zn(2+) could be relevant for spontaneous Ca(2+) oscillations during spermatogenesis and in pathophysiological conditions such as diabetes.

A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm.

During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca(2+)]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca(2+)]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca(2+)]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca(2+)]i originating in the sperm head promotes the initiation of AR.

Role of Ion Channels in the Sperm Acrosome Reaction.

The acrosome reaction (AR) is a unique exocytotic process where the acrosome, a single membrane-delimited specialized organelle, overlying the nucleus in the sperm head of many species, fuses with the overlying plasma membrane. This reaction, triggered by physiological inducers from the female gamete, its vicinity, or other stimuli, discharges the acrosomal content modifying the plasma membrane, incorporating the inner acrosomal membrane, and exposing it to the extracellular medium. The AR is essential for sperm-egg coat penetration, fusion with the eggs' plasma membrane, and fertilization. As in most exocytotic processes Ca(2+) is crucial for the AR, as well as intracellular pH and membrane potential changes. Thus, among the required processes needed for this reaction, ion permeability changes involving channels are pivotal. In spite of the key role ion channels play in the AR, their identity and regulation is not fully understood. Though molecular and pharmacological evidence indicates that various ionic channels participate during the AR, such as store-operated Ca(2+) channels and voltage-dependent Ca(2+) channels, whole cell patch clamp recordings have failed to detect some of them until now. Since sperm display a very high resistance and a minute cytoplasmic volume, very few channels are needed to achieve large membrane potential and concentration changes. Functional detection of few channels in the morphologically complex and tiny sperm poses technical problems, especially when their conductance is very small, as in the case of SOCs. Single channel recordings and novel fluorescence microscopy strategies will help to define the participation of ionic channels in the intertwined signaling network that orchestrates the AR.

SLO3 K+ channels control calcium entry through CATSPER channels in sperm.

Here we show how a sperm-specific potassium channel (SLO3) controls Ca(2+) entry into sperm through a sperm-specific Ca(2+) channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca(2+) entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca(2+) entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca(2+) entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na(+)/H(+) exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na(+) gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na(+)/H(+) exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.