Rediscovering sperm ion channels with the patch-clamp technique

Department of Physiology, University of California San Francisco UCSF Mail Code 2140, Genentech Hall Room N272F 600 16th Street, San Francisco, CA 94158, USA.
Molecular Human Reproduction (Impact Factor: 3.75). 06/2011; 17(8):478-99. DOI: 10.1093/molehr/gar044
Source: PubMed


Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca(2+) in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca(2+) and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (H(v)1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible.

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Available from: Polina Lishko
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    • "The molecular basis of hyperactivation is also incompletely understood. Reports in mice suggest that opening of H C and Ca 2C ion channels present in the sperm plasma membrane is sufficient to allow H C efflux from and Ca 2C influx to the sperm cytosol, along their respective concentration gradients (Kirichok & Lishko 2011). The primary source of Ca 2C for the spermatozoon is thus extracellular. "
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    ABSTRACT: Induction of hyperactivated motility is considered essential for triggering the release of oviduct-bound mammalian spermatozoa in preparation for fertilization. In this study, oviduct-bound stallion spermatozoa were exposed for 2 h to: i) pre-ovulatory and ii) post-ovulatory oviductal fluid; iii) 100% and iv) 10% follicular fluid (FF); v) cumulus cells, vi) mature equine oocytes, vii) capacitating and viii) non-capacitating medium. None of these triggered sperm release or hyperactivated motility. Interestingly, native FF was detrimental to sperm viability, an effect that was negated by heat inactivation, charcoal treatment and 30 kDa filtration alone or in combination. Moreover, sperm suspensions exposed to treated FF at pH 7.9 but not pH 7.4 showed Ca(2+)-dependent hypermotility. Fluo-4 AM staining of sperm showed elevated cytoplasmic Ca(2+) in hyperactivated stallion spermatozoa exposed to treated FF at pH 7.9 compared to a modest response in defined capacitating conditions at pH 7.9 and no response in treated FF at pH 7.4. Moreover, 1 h incubation in alkaline, treated FF induced protein tyrosine phosphorylation in 20% of spermatozoa. None of the conditions tested induced widespread release of sperm pre-bound to oviduct epithelium. However, the hyperactivating conditions did induce release of 70-120 spermatozoa per oviduct explant, of which 48% showed protein tyrosine phosphorylation and all were acrosome-intact, but capable of acrosomal exocytosis in response to calcium ionophore. We conclude that, in the presence of elevated pH and extracellular Ca(2+), a heat-resistant, hydrophilic, <30 kDa component of FF can trigger protein tyrosine phosphorylation, elevated cytoplasmic Ca(2+) and hyperactivated motility in stallion sperm, but infrequent release of sperm pre-bound to oviduct epithelium. © 2015 Society for Reproduction and Fertility.
    Full-text · Article · Sep 2015 · Reproduction
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    • "On the other hand, Darszon et al. (1999) hypothesized that the motility of spermatozoa is activated by changes in intracellular ion concentration, with intracellular Ca 2+ playing a prominent role (Kirichok & Lishko, 2011). The intracellular Ca 2+ levels are related to sperm motility and the capability of spermatozoa for fertilization (Kirichok & Lishko, 2011; Rahman et al., 2014a). According to the findings of our present study, NaF-mediated decreases in sperm motility and motion kinematics were associated with reduced [Ca 2+ ] i (Fig. 1B). "
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    ABSTRACT: Sodium fluoride (NaF), an environmental pollutant, has been tested for its impact on fertility in several species of laboratory animals. A literature demonstrated that NaF adversely affects sperm motility, morphology, capacitation, and the acrosome reaction. However, the molecular mechanisms underlying these alterations have not yet been elucidated. Therefore, present study was designed to evaluate the regulatory pathways involved in the effect of NaF on sperm function and fertilization. In this in vitro study, mouse spermatozoa were incubated with a range of concentrations (2.5, 5, and 10 mM) of NaF for 90 min in media that support in vitro fertilization. Our results showed that NaF was associated with reduced intracellular ATP generation, motility, and motion kinematics. Likewise, short-term exposure of spermatozoa to NaF significantly reduced the intracellular calcium concentration, protein kinase-A activity, and tyrosine phosphorylation of sperm proteins, which were associated with a significant decrease in the rate of capacitation and the acrosome reaction. Finally, NaF significantly reduced the fertilization and blastocyst formation during early embryonic development. On the basis of these results, we propose that NaF reduces sperm motility, capacitation, and the acrosome reaction leading to poor fertilization and suppressed embryonic development.
    Full-text · Article · Apr 2015 · Andrology
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    • "It is speculated that positively charged residues of CatSper α subunits are directly related to voltage sensitivity, and as other pore-forming CatSper α subunits except CatSper1 have a lower number of such residues, the overall voltage sensitivity of the channel is weak (Navarro et al., 2008; Ren et al., 2001). Furthermore , the V1/2 (the voltage at which half of channels are active) of human CatSper is more positive (+85 mV) than mouse (+11 mV) at the same intracellular pH (7.5) (Kirichok et al., 2006; Lishko et al., 2011). The CatSper channel is regulated by changes in intrascellular pH, such that the ICatSper is increased with an increase in intracellular pH. "
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    ABSTRACT: A number of physiological events, such as sperm hyperactivation, chemotaxis towards the egg, capacitation and acrosome reaction, are triggered by activation of sperm ion channels in response to a diverse range of chemical cues. Cation channel of sperm (CatSper), a sperm-specific ion channel, is unique in orchestrating the events for fertilization, and seems to be exclusively evolved for sperm function and male fertility. CatSper acts as a polymodal, chemosensory calcium channel and plays a vital role in the regulation of sperm hyperactivation. CatSper knockout models and application of patch clamp recordings have shown that it is indispensable for male fertility, and mutations and deletions in CatSper gene(s) may lead to infertility. In fact, mutations in CatSper1 and 2 have been identified in infertile individuals; however, CatSper3 and 4 have not been explored. Restricted localization and expression of CatSper in sperm offer an added advantage to developing gamete-based safe non-hormonal contraceptives. This review concisely covers identification, structure, function, and mechanism of action of CatSper channels. The functional importance of this complex ion channel in sperm motility and male fertility is highlighted for further research on male fertility, infertility, and contraception.
    Full-text · Article · Oct 2014 · Reproductive biomedicine online
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