Membrane hyperpolarization during human sperm capacitation.

Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca(2+) concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K(+) concentration (60 mM), indicating the participation of K(+) channels. In order to identify, which of the potential K(+) channels were involved in this hyperpolarization, we used different K(+) channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K(+) channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K(+) currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 µM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.

K+ and Cl- channels and transporters in sperm function.

To succeed in fertilization, spermatozoa must decode environmental cues which require a set of ion channels. Recent findings have revealed that K(+) and Cl(-) channels participate in some of the main sperm functions. This work reviews the evidence indicating the involvement of K(+) and Cl(-) channels in motility, maturation, and the acrosome reaction, and the advancement in identifying their molecular identity and modes of regulation. Improving our insight on how these channels operate will strengthen our ability to surmount some infertility problems, improve animal breeding, preserve biodiversity, and develop selective and secure male contraceptives.

Potassium channels in C. elegans.

Ion channels are the "transistors" (electronic switches) of the brain that generate and propagate electrical signals in the aqueous environment of the brain and nervous system. Potassium channels are particularly important because, not only do they shape dynamic electrical signaling, they also set the resting potentials of almost all animal cells. Without them, animal life as we know it would not exist, much less higher brain function. Until the completion of the C. elegans genome sequencing project the size and diversity of the potassium channel extended gene family was not fully appreciated. Sequence data eventually revealed a total of approximately 70 genes encoding potassium channels out of the more than 19,000 genes in the genome. This seemed to be an unexpectedly high number of genes encoding potassium channels for an animal with a small nervous system of only 302 neurons. However, it became clear that potassium channels are expressed in all cell types, not only neurons, and that many cells express a complex palette of multiple potassium channels. All types of potassium channels found in C. elegans are conserved in mammals. Clearly, C. elegans is "simple" only in having a limited number of cells dedicated to each organ system; it is certainly not simple with respect to its biochemistry and cell physiology.

Dissection of K+ currents in Caenorhabditis elegans muscle cells by genetics and RNA interference.

GFP-promoter experiments have previously shown that at least nine genes encoding potassium channel subunits are expressed in Caenorhabditis elegans muscle. By using genetic, RNA interference, and physiological techniques we revealed the molecular identity of the major components of the outward K+ currents in body wall muscle cells in culture. We found that under physiological conditions, outward current is dominated by the products of only two genes, Shaker (Kv1) and Shal (Kv4), both expressing voltage-dependent potassium channels. Other channels may be held in reserve to respond to particular circumstances. Because GFP-promoter experiments indicated that slo-2 expression is prominent, we created a deletion mutant to identify the SLO-2 current in vivo. In both whole-cell and single-channel modes, in vivo SLO-2 channels were active only when intracellular Ca2+ and Cl- were raised above normal physiological conditions, as occurs during hypoxia. Under such conditions, SLO-2 is the largest outward current, contributing up to 87% of the total current. Other channels are present in muscle, but our results suggest that they are unlikely to contribute a large outward component under physiological conditions. However, they, too, may contribute currents conditional on other factors. Hence, the picture that emerges is of a complex membrane with a small number of household conductances functioning under normal circumstances, but with additional conductances that are activated during unusual circumstances.

Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction.

The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca(2+)](i) mediated by Ca(2+) channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca(2+) channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca(2+) currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC(50) values of approximately 10 and approximately 12 microM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca(2+)](i) transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC(50) of approximately 10 microM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca(2+) channel. They are also consistent with the involvement of T-channels in the AR.

Molecular and functional characterization of a family of rat brain T-type calcium channels.

Voltage-gated calcium channels represent a heterogenous family of calcium-selective channels that can be distinguished by their molecular, electrophysiological, and pharmacological characteristics. We report here the molecular cloning and functional expression of three members of the low voltage-activated calcium channel family from rat brain (alpha(1G), alpha(1H), and alpha(1I)). Northern blot and reverse transcriptase-polymerase chain reaction analyses show alpha(1G), alpha(1H), and alpha(1I) to be expressed throughout the newborn and juvenile rat brain. In contrast, while alpha(1G) and alpha(1H) mRNA are expressed in all regions in adult rat brain, alpha(1I) mRNA expression is restricted to the striatum. Expression of alpha(1G), alpha(1H), and alpha(1I) subunits in HEK293 cells resulted in calcium currents with typical T-type channel characteristics: low voltage activation, negative steady-state inactivation, strongly voltage-dependent activation and inactivation, and slow deactivation. In addition, the direct electrophysiological comparison of alpha(1G), alpha(1H), and alpha(1I) under identical recording conditions also identified unique characteristics including activation and inactivation kinetics and permeability to divalent cations. Simulation of alpha(1G), alpha(1H), and alpha(1I) T-type channels in a thalamic neuron model cell produced unique firing patterns (burst versus tonic) typical of different brain nuclei and suggests that the three channel types make distinct contributions to neuronal physiology.

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.

Properties of a novel pH-dependent Ca2+ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function.

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2',7')-bis(carboxymethyl)- (5, 6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30-60 s) application of 25 mM NH4Cl increased pHi by approximately 1.3 U from a resting pHi approximately 6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions.

A dihydropyridine-sensitive T-type Ca2+ current is the main Ca2+ current carrier in mouse primary spermatocytes.

Ca2+ entry through Ca2+ channels is likely to play an important role in the differentiation of male germ cells as well as in fertilization by mature sperm. Here we present a detailed analysis of Ca2+ currents expressed in acutely dissociated mouse primary spermatocytes. Patch-clamp recordings demonstrated that the only voltage-gated Ca2+ channels present belong to the family of T-type Ca2+ currents. Accordingly, Ni2+ (200 microM) and amiloride (500 microM) reduced current amplitude by 75 and 62%, respectively. To our knowledge, this is the first report of a system where T-type Ca2+ channels are expressed in isolation. Unexpectedly, 5 and 10 microM nifedipine also reduced peak currents by 38 and 53%, respectively significant inhibition of the Ca2+ current occurred at concentrations as low as 2 microM. Because mature sperm cells are unable to synthesize new proteins, these Ca2+ channels are also likely to be present in these cells, where they may contribute to the Ca2+ influx required to trigger the acrosome reaction. This notion is supported by the fact that concentrations of Ni2+ and nifedipine, which block these Ca2+ currents, also inhibit the acrosome reaction. Because these channels represent the primary pathway for voltage-gated Ca2+ entry in mouse spermatocytes, they may also participate in regulating meiotic cell division and sperm differentiation.

T-type Ca2+ channels and alpha1E 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 alpha 1A, alpha 1B, alpha 1C, alpha 1D and alpha 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 alpha 1E, and to a minor extent alpha 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. These results suggest that low-threshold Ca2+ channels are the dihydropyridine-sensitive channels involved in the sperm acrosome reaction.

A significant fraction of calcium transients in intact guinea pig ventricular myocytes is mediated by Na(+)-Ca2+ exchange.

Ca2+ mobilization elicited by simulation with brief pulses of high K+ were monitored with confocal laser scanned microscopy in intact, guinea pig cardiac myocytes loaded with the calcium indicator fluo-3. Single wavelength ratioing of fluorescence images obtained after prolonged integration times revealed non-uniformities of intracellular Ca2+ changes across the cell, suggesting the presence of significant spatial Ca2+ gradients. Treatment with 20 microM ryanodine, an inhibitor of Ca2+ release from the SR, and 10 microM verapamil, a calcium channel blocker, reduced by 42% and 76% respectively the changes in [Ca2+]i elicited by membrane depolarization. The overall spatial distribution of [Ca2+]i changes appeared unchanged. Ca2+ transients recorded in the presence of verapamil and ryanodine (about 20% of the size of control responses), diminished in the presence of 50 microM 2-4 Dichlorbenzamil (DCB) or 5 mM nickel, two relatively specific inhibitors of the Na+/Ca2+ exchange mechanism. Conversely, when the reversal potential of the Na+/Ca2+ exchange was shifted to negative potentials by lowering [NA+]o or by increasing [Na+]i by treatment with 20 microM monensin, the amplitude of these Ca2+ transients increased. Ca2+ transients elicited by membrane depolarization and largely mediated by reverse operation of Na(+)-Ca2+ exchange could be recorded in the presence of ryanodine, verapamil and monensin. These finding suggest that in intact guinea pig cardiac cells, Ca2+ influx through the Na+/Ca2+ exchange mechanism activated by a membrane depolarization in the physiological range can be sufficient to play a significant role in excitation-contraction coupling.