Alberto Darszon

T-type Ca2+ channels and α1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction

There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage‐dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the α 1A, α 1B, α 1C, α 1D and α 1E genes in mouse testis and in purified spermatogenic cell populations with RT‐PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only α 1E, and to a minor extent α 1A, are expressed in spermatogenic cells. In agreement with these findings, only T‐type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch‐clamp recordings of pachytene spermatocytes. The results suggest that low‐threshold Ca2+ channels are the dihydropyridine‐sensitive channels involved in the sperm 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.

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca2+ has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca2+ in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1–7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca2+ channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca2+‐dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co‐immunolocalized with caveolin‐1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca2+ flux. Furthermore, by using fluorescein‐coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin‐ and Trp‐independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.

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.

Calcium Channels and Ca2+ Fluctuations in Sperm Physiology

Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca(2+) is probably the key messenger in this information exchange. It is therefore not surprising that different Ca(2+)-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca(2+), membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.

The Intraacrosomal Calcium Pool Plays a Direct Role in Acrosomal Exocytosis

The acrosome reaction is a unique type of regulated exocytosis. The single secretory granule of the sperm fuses at multiple points with the overlying plasma membrane. In the past few years we have characterized several aspects of this process using streptolysin O-permeabilized human spermatozoa. Here we show that Rab3A triggers acrosomal exocytosis in the virtual absence of calcium in the cytosolic compartment. Interestingly, exocytosis is blocked when calcium is depleted from intracellular stores. By using a membrane-permeant fluorescent calcium probe, we observed that the acrosome actually behaves as a calcium store. Depleting calcium from this compartment by using a light-sensitive chelator prevents secretion promoted by Rab3A. UV inactivation of the chelator restores exocytosis. Rab3A-triggered exocytosis is blocked by calcium pump and inositol 1,4,5-trisphosphate (IP3)-sensitive calcium channel inhibitors. Calcium measurements inside and outside the acrosome showed that Rab3A promotes a calcium efflux from the granule. Interestingly, release of calcium through IP3-sensitive calcium channels was necessary even when exocytosis was initiated by increasing free calcium in the extraacrosomal compartment in both permeabilized and intact spermatozoa. Our results show that a calcium efflux from the acrosome through IP3-sensitive channels is necessary downstream Rab3A activation during the membrane fusion process leading to acrosomal exocytosis.

δ-Endotoxins induce cation channels in Spodoptera frugiperda brush border membranes in suspension and in planar lipid bilayers

Membrane potential measurements using a fluorescent dye indicated that two specific toxins active against Spodoptera frugiperda larvae (CryIC and CryID) cause immediate permeability changes in midgut epithelial brush border membrane vesicles (BBMV). The initial response and the sustained permeability change are cationic, notvery K+ selective, and occur at in vivo lethal doses (nM). The toxin response has a different ion selectivity and is more sensitive to Ba2+ than the intrinsic cation permeability of BBMV. Experiments incorporating BBMV into planar lipid bilayers (PLB) demonstrated that these vesicles contain cation channels (31, 47 and 76 pS). A 2–40 fold conductance increase was induced by nM concentrations of toxin in PLB containing BBMV. Cationic single channel transitions of 50, 106, 360 and 752 pS were resolved. Thus, Bacillus thuringiensis δ‐endotoxins induce an increase in cation membrane permeability involving ion channels in BBMV‐containing functional receptors.

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.

Transient receptor potential (TRPC) channels in human sperm: expression, cellular localization and involvement in the regulation of flagellar motility.

Capacitative Ca2+ entry is a process whereby the activation of Ca2+ influx through the plasma membrane is triggered by depletion of intracellular Ca2+ stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca2+ channels. Recent evidence indicates that capacitative Ca2+ entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription‐polymerase chain reaction (RT‐PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer‐assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.

Functional reassembly of membrane proteins in planar lipid bilayers.

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transduction.