Mammalian sperm acquire fertilizing ability in the female tract during a process known as capacitation. In mouse sperm, this process is associated with increases in protein tyrosine phosphorylation, membrane potential hyperpolarization, increase in intracellular pH and Ca 2؉, and hyperactivated motility. The molecular mechanisms involved in these changes are not fully known. Present evidence suggests that in mouse sperm the capacitation-associated membrane hyperpolarization is regulated by a cAMP/protein kinase A-dependent pathway involving activation of inwardly rectifying K ؉ channels and inhibition of epithelial sodium channels (ENaCs). The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl ؊ channel that controls the activity of several transport proteins, including ENaCs. Here we explored whether CFTR is involved in the regulation of ENaC inhibition in sperm and therefore is essential for the capacitation-associated hyperpolarization. Using reverse transcription-PCR, Western blot, and immunocytochemistry, we document the presence of CFTR in mouse and human sperm. Interestingly, the addition of a CFTR inhibitor (diphenylamine-2-carboxylic acid; 250 M) inhibited the capacitation-associated hyperpolarization, prevented ENaC closure, and decreased the zona pellucida-induced acrosome reaction without affecting the increase in tyrosine phosphorylation. Incubation of sperm in Cl ؊ -free medium also eliminated the capacitationassociated hyperpolarization. On the other hand, a CFTR activator (genistein; 5-10 M) promoted hyperpolarization in mouse sperm incubated under conditions that do not support capacitation. The addition of dibutyryl cyclic AMP to noncapacitated mouse sperm elevated intracellular Cl ؊ . These results suggest that cAMPdependent Cl ؊ fluxes through CFTR are involved in the regulation of ENaC during capacitation and thus contribute to the observed hyperpolarization associated with this process.

Capacitative Ca2+ entry is a process whereby the activation of Ca 2+ in£ux through the plasma membrane is triggered by depletion of intracellular Ca 2+ stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca 2+ channels. Recent evidence indicates that capacitative Ca 2+ 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 immuno£uores-cence revealed the presence of TRPC1, 3, 4 and 6 di¡erentially 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 £agellum, TRPC channel antagonists were tested in sperm motility using a computer-assisted assay. Our results provide what is to our knowledge the ¢rst evidence that these channels may in£uence human sperm motility. ß

BackgroundThe 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 FindingsHere 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.ConclusionsThis 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.

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca 2+ in£ux through voltage-gated Ca 2+ (Ca V ) channels. Although the electrophysiological characterization of Ca 2+ currents in mature sperm has proven di⁄cult, functional studies have revealed the presence of lowthreshold (Ca V 3) channels in spermatogenic cells. However, the molecular identity of these proteins remains unde¢ned. Here, we identi¢ed by reverse transcription polymerase chain reaction the expression of Ca V 3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three Ca V 3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only Ca V 3.1 and Ca V 3.2 were detected in the head, suggesting its participation in the AR. Ca V 3.1 and Ca V 3.3 were found in the principal and the midpiece of the £agella. All Ca V 3 channels are also present in human sperm, but only to a minor extent in the head. These ¢ndings were corroborated by immunogold transmission electron microscopy. Tail localization of Ca V 3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit Ca V 3 channels did not signi¢cantly a¡ect human sperm motility. Only higher mibefradil doses that can block high-threshold (HVA) Ca V channels caused small but signi¢cant motility alterations. Antibodies to HVA channels detected Ca V 1.3 and Ca V 2.3 in human sperm £agella. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Trichomonas vaginalis (Tv) is a flagellated parasite commonly spread through sexual transmission. This protozoan initiates a severe inflammatory process, inducing nitric oxide, interleukin-6 (IL-6), IL-8, IL-10, IL-17 and IL-22 production by host immune cells. The parasites elicit these responses by releasing surface lipophosphoglycan, small extracellular vesicles (exosomes) and other factors. Tv exosomes are similar to mammalian exosomes and have been implicated in the modulation of IL-8 secretion by epithelial cells. Here, we report that exosome-like vesicles from T. vaginalis (Tv-ELVs) induced a more than 15-fold increase in IL-10 expression in RAW264.7 macrophages but only a two fold increase in IL-6 and TNF-α expression levels measured by RT-PCR. Because Tv-ELVs modulated the macrophage response, we also explored the effect of Tv-ELVs in a murine model of infection. Pretreatment with Tv-ELVs significantly increased IL-10 production as measured in vaginal washes by days 8 and 16 post-infection. Remarkably, Tv-ELVs-pretreated mice exhibited a decrease in IL-17 production and a significant decrease in vulvar inflammation. In addition, IL-6 and IL-13 were decreased during infection. Our results suggest that Tv-ELVs have an immunomodulatory role on the cytokine profile induced by the parasite and promote a decrease in the inflammatory process in mice infected with T. vaginalis.