Cytosolic and Acrosomal pH Regulation in Mammalian Sperm.

As in most cells, intracellular pH regulation is fundamental for sperm physiology. Key sperm functions like swimming, maturation, and a unique exocytotic process, the acrosome reaction, necessary for gamete fusion, are deeply influenced by pH. Sperm pH regulation, both intracellularly and within organelles such as the acrosome, requires a coordinated interplay of various transporters and channels, ensuring that this cell is primed for fertilization. Consistent with the pivotal importance of pH regulation in mammalian sperm physiology, several of its unique transporters are dependent on cytosolic pH. Examples include the Ca2+ channel CatSper and the K+ channel Slo3. The absence of these channels leads to male infertility. This review outlines the main transport elements involved in pH regulation, including cytosolic and acrosomal pH, that participate in these complex functions. We present a glimpse of how these transporters are regulated and how distinct sets of them are orchestrated to allow sperm to fertilize the egg. Much research is needed to begin to envision the complete set of players and the choreography of how cytosolic and organellar pH are regulated in each sperm function.

The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm.

Sperm capacitation, crucial for fertilization, occurs in the female reproductive tract and can be replicated in vitro using a medium rich in bicarbonate, calcium, and albumin. These components trigger the cAMP-PKA signaling cascade, proposed to promote hyperpolarization of the mouse sperm plasma membrane through activation of SLO3 K+ channel. Hyperpolarization is a hallmark of capacitation: proper membrane hyperpolarization renders higher in vitro fertilizing ability, while Slo3 KO mice are infertile. However, the precise regulation of SLO3 opening remains elusive. Our study challenges the involvement of PKA in this event and reveals the role of Na+/H+ exchangers. During capacitation, calcium increase through CatSper channels activates NHE1, while cAMP directly stimulates the sperm-specific NHE, collectively promoting the alkalinization threshold needed for SLO3 opening. Hyperpolarization then feeds back Na+/H+ activity. Our work is supported by pharmacology, and a plethora of KO mouse models, and proposes a novel pathway leading to hyperpolarization.

Riesgos a la Salud Mental de las Personas Cuidadoras durante la Pandemia por COVID-19 en México / Mental Health Risks of Caregivers during the COVID-19 Pandemic in Mexico

Resumen Diversos estudios han reportado que el cuidado informal de adultos mayores, personas con alguna enfermedad o discapacidad, e incluso el cuidado de menores de edad, tiene un impacto en la salud física y mental de las personas cuidadoras (PC). El objetivo del presente estudio fue identificar los riesgos a la salud mental de las PC, teniendo como referencia el concepto de "carga de cuidado" que alude a las demandas físicas, emocionales, sociales y/o económicas de las actividades de cuidado, y la tensión que estas producen. Los participantes respondieron a un tamizaje entre abril y diciembre de 2020, vía una plataforma electrónica, mismo que tuvo por objeto identificar y atender problemas de salud mental que pudieran presentarse o agravarse por la pandemia y las medidas de confinamiento adoptadas para mitigarla. Más de 51,000 personas reportaron ser PC de menores, adultos mayores y/o enfermos crónicos, lo que permitió identificar variaciones en la carga de cuidado considerando el impacto de distintos perfiles de cuidadores (según el tipo y número de personas dependientes). Las condiciones de salud mental evaluadas fueron estrés agudo, ansiedad generalizada, ansiedad por la salud y depresión. Los resultados confirman que las PC que cuidan más de un tipo de persona dependiente tienen mayores probabilidades de riesgo a desarrollar alguna de las condiciones de salud mental. Así mismo, quienes cuidaban alguna persona con enfermedad crónica fueron los más vulnerables, mientras que tener un menor de edad al cuidado actuó en alguna medida como un factor protector.

Abstract Several studies have reported that informal care of older adults, chronically ill, and children's, has an impact on physical and mental health of caregivers. The goal of this study was to identify mental health risks of caregivers, considering the concept of caregiver burden that points to the physical, emotional, social and/or economic demands of care activities, and the tension they produce. Participants answered a mental health screening (via an electronic platform) that intended to identify and attend mental health problems that could arise or be aggravated during the pandemic and confinement measures adopted to mitigate it. More than 51,000 people reported being caregivers of children, older people and/or chronically ill, which allows to identify variations in caregiver burden considering type and number of dependent people. The mental health conditions evaluated were acute stress, generalized anxiety, health anxiety, and depression. Results confirmed that caregivers who take care of more than one type of dependent are more likely to be at risk of developing any of the mental health conditions measured. Likewise, those who care for a chronically ill, were the most vulnerable and, to take care of children was at some extent a protective factor.

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.

Role of calcium oscillations in sperm physiology.

Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells often use cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations as a means to decodify external and internal information. [Ca2+]i oscillations faster than those usually found in other cells and correlated with flagellar beat were the first to be described in sperm in 1993 by Susan Suarez, in the boar. More than 20 years passed before similar [Ca2+]i oscillations were documented in human sperm, simultaneously examining their flagellar beat in three dimensions by Corkidi et al. 2017. On the other hand, 10 years after the discovery of the fast boar [Ca2+]i oscillations, slower ones triggered by compounds from the egg external envelope were found to regulate cell motility and chemotaxis in sperm from marine organisms. Today it is known that sperm display fast and slow spontaneous and agonist triggered [Ca2+]i oscillations. In mammalian sperm these Ca2+ transients may act like a multifaceted tool that regulates fundamental functions such as motility and acrosome reaction. This review covers the main sperm species and experimental conditions where [Ca2+]i oscillations have been described and discusses what is known about the transporters involved, their regulation and the physiological purpose of these oscillations. There is a lot to be learned regarding the origin, regulation and physiological relevance of these Ca2+ oscillations.

Epac activation induces an extracellular Ca2+ -independent Ca2+ wave that triggers acrosome reaction in human spermatozoa.

BACKGROUND: The signaling pathways of the intracellular second messengers cAMP and Ca2+ play a crucial role in numerous physiological processes in human spermatozoa. One such process is the acrosome reaction (AR), which is necessary for spermatozoa to traverse the egg envelope and to expose a fusogenic membrane allowing the egg-sperm fusion. Progesterone and zona pellucida elicit an intracellular Ca2+ increase that is needed for the AR in the mammalian spermatozoa. This increase is mediated by an initial Ca2+ influx but also by a release from intracellular Ca2+ stores. It is known that intracellular Ca2+ stores play a central role in the regulation of [Ca2+ ]i and in the generation of complex Ca2+ signals such as oscillations and waves. In the human spermatozoa, it has been proposed that the cAMP analog and specific agonist of Epac 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (2'-O-Me-cAMP) elicits an intracellular Ca2+ release involved in the AR. OBJECTIVE: To identify the molecular entities involved in the Ca2+ mobilization triggered by 2'-O-Me-cAMP in human spermatozoa. MATERIALS AND METHODS: In capacitated human spermatozoa, we monitored Ca2+ dynamics and the occurrence of the AR in real time using Fluo 3-AM and FM4-64 in a Ca2+ -free medium. RESULTS: Epac activation by 2'-O-Me-cAMP induced a Ca2+ wave that started in the midpiece and propagated to the acrosome region. This Ca2+ response was sensitive to rotenone, CGP, xestospongin, NED-19, and thapsigargin, suggesting the participation of different ion transporters (mitochondrial complex I and Na+ /Ca2+ exchanger, inositol 3-phosphate receptors, two-pore channels and internal store Ca2+ -ATPases). DISCUSSION: Our results suggest that Epac activation promotes a dynamic crosstalk between three different intracellular Ca2+ stores: the mitochondria, the redundant nuclear envelope, and the acrosome. CONCLUSION: The Ca2+ wave triggered by Epac activation is necessary to induce the AR and to enhance the flagellar beat.

Effects of Semen Processing on Sperm Function: Differences between Swim-Up and Density Gradient Centrifugation.

PURPOSE: Andrology research has evolved notoriously in the latest years, particularly since male factor contribution to couple infertility has been undoubtedly demonstrated. However, sperm function investigations results are sometimes contradictory, probably as a result of the use of different sperm processing techniques. In this work, we underwent a systematic functional comparison of human sperm samples simultaneously processed by swim-up and density gradient centrifugation, which are the preferred sperm processing methods used in basic and clinical laboratories. MATERIALS AND METHODS: To compare functional characteristics of sperm isolated by swim-up and density gradient centrifugation followed by incubation at different times under capacitating conditions. RESULTS: Semen samples processed in parallel by these two procedures resulted in sperm preparations with significant differences in redox state, spontaneous intracellular calcium oscillations, hyperactivation, protein tyrosine phosphorylation, and acrosome reaction responsivity to calcium ionophore. Such differences showed time-dependent specific patterns for spontaneous intracellular calcium oscillations, hyperactivation and protein tyrosine phosphorylation. Sperm retrieved by density gradient centrifugation showed more hyperactivation and tyrosine phosphorylation than swim-up sperm, suggesting a higher degree of capacitation. CONCLUSIONS: Our results account for functional differences observed in spermatozoa processed with these two methods and therefore may contribute to a better interpretation of outcomes obtained in different laboratories as well as to improve experimental designs aimed to study sperm physiology and fertility potential.

[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.

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.

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.

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).

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.

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.

Sperm physiology varies according to ultradian and infradian rhythms.

The spermatozoon must be physiologically prepared to fertilize the egg, process called capacitation. Human sperm samples are heterogeneous in their ability to capacitate themselves, which leads to variability between samples from the same or different donors, and even along the seasons. Here we studied sperm variation in the capacitation state according to the ability of capacitated spermatozoa to acrosome react upon stimulation (% ARi) and to be recruited by chemotaxis (% Chex). Both indirect indicators of sperm capacitation increased along the incubation time with fluctuations. Those capacitated sperm recruited by chemotaxis showed an ultradian rhythm with a cycle every 2 h, which might be influenced by unknown intrinsic sperm factors. Two infradian rhythms of 12 months for the % ARi and of 6 months for % Chex were observed, which are associated with the joint action of temperature and photoperiod. Thus, to avoid false negative results, human sperm samples are recommended to be incubated for a long period (e.g. 18 h) preferably in spring time. This innovative point of view would lead to better comprehend human reproductive biology and to think experimental designs in the light of sperm cyclicity or to improve sperm aptitude for clinical purposes.

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.

Quantitative Intracellular pH Determinations in Single Live Mammalian Spermatozoa Using the Ratiometric Dye SNARF-5F.

Intracellular pH (pH i ) plays a crucial role in mammalian sperm physiology. However, it is a challenging task to acquire quantitative single sperm pH i images due to their small size and beating flagella. In this study, we established a robust pH i imaging system using the dual-emission ratiometric pH indicator, SNARF-5F. Simultaneous good signal/noise ratio fluorescence signals were obtained exciting with a green high-power LED (532 nm) and acquiring with an EM-CCD camera through an image splitter with two band-pass filters (550-600 nm, channel 1; 630-650 nm, channel 2). After in vivo calibration, we established an imaging system that allows determination of absolute pH i values in spermatozoa, minimizing cell movement artifacts. Using this system, we determined that bicarbonate increases non-capacitated human pH i with slower kinetics than in mouse spermatozoa. This difference suggests that distinct ionic transporters might be involved in the bicarbonate influx into human and mouse spermatozoa. Alternatively, pH i regulation downstream bicarbonate influx into spermatozoa could be different between the two species.

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.

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.

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.