José Luis de la Vega-Beltrán

Ion channels, phosphorylation and mammalian sperm capacitation

Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies.

Involvement of a Na+/HCO-3 cotransporter in mouse sperm capacitation.

Mammalian sperm are incapable of fertilizing eggs immediately after ejaculation; they acquire fertilization capacity after residing in the female tract for a finite period of time. The physiological changes sperm undergo in the female reproductive tract that render sperm able to fertilize constitute the phenomenon of “sperm capacitation.” We have demonstrated that capacitation is associated with an increase in the tyrosine phosphorylation of a subset of proteins and that these events are regulated by an HCO 3 − /cAMP-dependent pathway involving protein kinase A. Capacitation is also accompanied by hyperpolarization of the sperm plasma membrane. Here we present evidence that, in addition to its role in the regulation of adenylyl cyclase, HCO 3 − has a role in the regulation of plasma membrane potential in mouse sperm. Addition of HCO 3 − but not Cl− induces a hyperpolarizing current in mouse sperm plasma membranes. This HCO 3 − -dependent hyperpolarization was not observed when Na+ was replaced by the non-permeant cation choline+. Replacement of Na+ by choline+ also inhibited the capacitation-associated increase in protein tyrosine phosphorylation as well as the zona pellucida-induced acrosome reaction. The lack of an increase in protein tyrosine phosphorylation was overcome by the presence of cAMP agonists in the incubation medium. The lack of a hyperpolarizing HCO 3 − current and the inhibition of the capacitation-dependent increase in protein tyrosine phosphorylation in the absence of Na+ suggest that a Na+/HCO 3 − cotransporter is present in mouse sperm and is coupled to events regulating capacitation.

The SLO3 sperm-specific potassium channel plays a vital role in male fertility

Here we show a unique example of male infertility conferred by a gene knockout of the sperm‐specific, pH‐dependent SLO3 potassium channel. In striking contrast to wild‐type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent “hairpin” shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation‐induced hyperpolarization, and membrane hyperpolarization is crucial to the AR.

Dual regulation of the T-type Ca2+ current by serum albumin and β-estradiol in mammalian spermatogenic cells.

This study provides evidence for a novel mechanism of voltage‐gated Ca2+ channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch‐clamp experiments demonstrated that serum albumin induced an increase in Ca2+ T current density in a concentration‐dependent manner, and significant shifts in the voltage dependence of both steady‐state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, β‐estradiol significantly inhibited Ca2+ channel activity in a concentration‐dependent and essentially voltage‐independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

Compartmentalization of distinct cAMP signaling pathways in mammalian sperm.

Background: cAMP is essential for the acquisition of sperm fertilizing capacity. The presence of transmembrane adenylyl cyclases (tmACs) in sperm remains controversial. Results: tmAC activity and its activator Gs are detected in the sperm head. Conclusion: Two cAMP synthesis pathways coexist in sperm and lead to capacitation. Significance: Understanding capacitation is essential for improvement of assisted fertilization and for finding novel contraceptive targets. Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO3−-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric Gs proteins; however, the presence of Gs or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the Gs presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although Gs immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, Gs reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca2+ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with Gs and tmAC in the head and Adcy10 and PKA in the flagellum.

Cysteine-rich secretory protein 4 is an inhibitor of transient receptor potential M8 with a role in establishing sperm function

The cysteine-rich secretory proteins (CRISPs) are a group of four proteins in the mouse that are expressed abundantly in the male reproductive tract, and to a lesser extent in other tissues. Analysis of reptile CRISPs and mouse CRISP2 has shown that CRISPs can regulate cellular homeostasis via ion channels. With the exception of the ability of CRISP2 to regulate ryanodine receptors, the in vivo targets of mammalian CRISPs function are unknown. In this study, we have characterized the ion channel regulatory activity of epididymal CRISP4 using electrophysiology, cell assays, and mouse models. Through patch-clamping of testicular sperm, the CRISP4 CRISP domain was shown to inhibit the transient receptor potential (TRP) ion channel TRPM8. These data were confirmed using a stably transfected CHO cell line. TRPM8 is a major cold receptor in the body, but is found in other tissues, including the testis and on the tail and head of mouse and human sperm. Functional assays using sperm from wild-type mice showed that TRPM8 activation significantly reduced the number of sperm undergoing the progesterone-induced acrosome reaction following capacitation, and that this response was reversed by the coaddition of CRISP4. In accordance, sperm from Crisp4 null mice had a compromised ability to undergo to the progesterone-induced acrosome reaction. Collectively, these data identify CRISP4 as an endogenous regulator of TRPM8 with a role in normal sperm function.

Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction

Background: Sperm capacitation, a process associated with phosphorylation and membrane potential changes, is required for acrosome reaction and fertilization. Results: Inducing hyperpolarization in non-capacitated sperm does not result in protein tyrosine phosphorylation but allows physiologically-induced [Ca2+]i increases and acrosome reaction. Conclusion: Sperm hyperpolarization appears to be necessary and sufficient for acrosome reaction. Significance: Advancing our understanding of capacitation, the acrosome reaction and fertilization. Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction.

TRPM8 in mouse sperm detects temperature changes and may influence the acrosome reaction.

Changes in the concentration of intracellular Ca2+ ([Ca2+]i) trigger and/or regulate principal sperm functions during fertilization, such as motility, capacitation, and the acrosome reaction (AR). Members of the large TRP channel family participate in a variety of Ca2+‐dependent cell signaling processes. The eight TRPM channel members constitute one of the seven groups belonging to this family. Here we document using RT‐PCR experiments the presence of Trpm2, 4, 7, and 8 in mouse spermatogenic cells. Trpm8 transcription is up‐regulated after day 30. The localization of TRPM8 protein in mouse sperm was confirmed by immunocytochemistry and Western blots. Patch clamp recordings in testicular mouse sperm revealed TRPM8 agonist (menthol and icilin) activated currents sensitive to TRPM8 inhibitors N‐(4‐t‐Butylphenyl)‐4‐(3‐Chloropyridin‐2‐yl)tetrahydropyrazine‐1(2H)‐carboxamide (BCTC) and capsazepine. These findings are consistent with the presence of functional TRPM8 in mouse sperm. Furthermore, menthol induced a [Ca2+]i increase and the AR in these cells, that were inhibited by capsazepine (20 µM) and BCTC (1.6 µM). Notably, the progesterone and zona pellucida‐induced AR was significantly (>40%) inhibited by BCTC and capsazepine, suggesting the possible participation of TRPM8 channels in this reaction. TRPM family members present in sperm could be involved in other important signaling events, such as thermotaxis, chemotaxis, and mechanosensory transduction. J. Cell. Physiol. 226: 1620–1631, 2011.

Scorpion toxins that block T-type Ca2+ channels in spermatogenic cells inhibit the sperm acrosome reaction

The acrosome reaction (AR) is a Ca(2+)-dependent event required for sperm to fertilize the egg. The activation of T-type voltage-gated Ca(2+) channels plays a key role in the induction of this process. This report describes the actions of two toxins from the scorpion Parabuthus granulatus named kurtoxin-like I and II (KLI and KLII, respectively) on sperm Ca(2+) channels. Both toxins decrease T-type Ca(2+) channel activity in mouse spermatogenic cells and inhibit the AR in mature sperm. Saturating concentrations of the toxins inhibited at most approximately 70% of the whole-cell Ca(2+) current, suggesting the presence of a toxin-resistant component. In addition, both toxins inhibited approximately 60% of the AR, which is consistent with the participation of T-type Ca(2+) channels in the sperm AR.

CRISP1 as a novel CatSper regulator that modulates sperm motility and orientation during fertilization

CRISP1 is expressed by cumulus cells and plays a role in fertilization by modulating sperm orientation, hyperactivation, and key Ca2+ channels in sperm.