Carmen Beltrán

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.

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.

TRPM8, a Versatile Channel in Human Sperm

Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.

Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications

During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

A high-conductance voltage-dependent multistate Ca2' channel found in sea urchin and mouse spermatozoa

Ion fluxes through poorly understood channel‐mediated mechanisms participate in the interaction between spermatozoa and egg. Previously, we reported the characterization in planar bilayers of a high conductance Ca2+‐selective, voltage‐dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes [14]. Here we show that this ion channel can be directly transferred to planar lipid bilayers upon sperm addition, from sea urchin [S. purpuratus and L. pictus) and from mouse. We found that spermatozoa from these species posses a conspicuous Ca2+‐selective, high conductance, multi‐state, voltage‐dependent channel, which displays similar voltage dependence and equal PBa2+/PK + ~4 in the three species. The presence of this Ca2+ channel in such diverse species suggests it plays a relevant role in sperm physiology. The high sensitivity of planar bilayers to detect single ion channels can now be used to study ion channel regulation and gamete interaction.

4 Ion Channels: Key Elements in Gamete Signaling

Publisher Summary This chapter discusses the participation of ion channels in the information exchange between gametes themselves and with the environment. Since the best known sperm ion transport systems have been described in the sea urchin, a marine invertebrate, and in mouse, bull, and pig in mammals, these species will be referred to more extensively. The success of fertilization depends on gamete information processing from the environment. There are long- and short-range signals, emitted by the egg, that influence sperm function and lead to proper gamete interaction, and finally to fertilization. Although the factors that mediate the sperm-egg dialogue have been studied for close to a century, the detailed molecular mechanisms involved in these events remain elusive. However, there is growing evidence that ion channels are deeply involved in gamete signaling. For instance, in echinoderm, fish, and mammalian spermatozoa, the acrosome reaction (AR), a necessary process for fertilization in many species, is inhibited by ion channel blockers.

Identification of voltage-dependent Ca2+ channels in sea urchin sperm

Functional evidence indicates that voltage‐dependent Ca2+ (Cav) channels participate in sea urchin sperm motility and the acrosome reaction (AR), however, their molecular identity remains unknown. We have identified transcripts for two Ca2+ channel α1 subunits in sea urchin testis similar in sequence to Cav1.2 and Cav2.3. Antibodies against rat Cav1.2 and Cav2.3 channels differentially label proteins in the flagella and acrosome of mature sea urchin sperm. The Cav channel antagonists nifedipine and nimodipine, which inhibit the AR, diminish the intracellular Ca2+ elevation induced by a K+‐induced depolarization in valinomycin‐treated sperm. These findings reveal that Cav1.2 and Cav2.3 channels could participate in motility and/or the AR in sea urchin sperm.

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.

Human spermatozoa possess a calcium‐dependent chloride channel that may participate in the acrosomal reaction

•  Ion channels participate in crucial sperm functions such as motility, capacitation and the acrosome reaction. •  Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology. •  We implemented a modified perforated patch‐clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa to investigate their ion channels. •  This work presents the first evidence for the presence of calcium‐dependent chloride channels (CaCCs) in human spermatozoa; they could be constituted by TMEM16. •  The CaCCs play an important role in the physiology of human spermatozoa and participate in the acrosome reaction.