ATP increases head volume in capacitated human sperm via a purinergic channel.

Scanning ion-conductance microscopy allowed us to document an external Ca2+ dependent ATP driven volume increase (ATPVI) in capacitated human sperm heads. We examined the involvement of purinergic receptors (PRs) P2X2R and P2X4R in ATPVI using their co-agonists progesterone and Ivermectin (Iver), and Cu2+, which co-activates P2X2Rs and inhibits P2X4Rs. Iver enhanced ATPVI and Cu2+ and 5BDBD inhibited it, indicating P2X4Rs contributed to this response. Moreover, Cu2+ and 5BDBD inhibited the ATP-induced acrosome reaction (AR) which was enhanced by Iver. ATP increased the concentration of intracellular Ca2+ ([Ca2+]i) in >45% of individual sperm, most of which underwent AR monitored using FM4-64. Our findings suggest that human sperm P2X4R activation by ATP increases [Ca2+]i mainly due to Ca2+ influx which leads to a sperm head volume increase, likely involving acrosomal swelling, and resulting in AR.

Carbonic anhydrases and their functional differences in human and mouse sperm physiology.

Fertilization is a key reproductive event in which sperm and egg fuse to generate a new individual. Proper regulation of certain parameters (such as intracellular pH) is crucial for this process. Carbonic anhydrases (CAs) are among the molecular entities that control intracellular pH dynamics in most cells. Unfortunately, little is known about the function of CAs in mammalian sperm physiology. For this reason, we re-explored the expression of CAI, II, IV and XIII in human and mouse sperm. We also measured the level of CA activity, determined by mass spectrometry, and found that it is similar in non-capacitated and capacitated mouse sperm. Importantly, we found that CAII activity accounts for half of the total CA activity in capacitated mouse sperm. Using the general CA inhibitor ethoxyzolamide, we studied how CAs participate in fundamental sperm physiological processes such as motility and acrosome reaction in both species. We found that capacitated human sperm depend strongly on CA activity to support normal motility, while capacitated mouse sperm do not. Finally, we found that CA inhibition increases the acrosome reaction in capacitated human sperm, but not in capacitated mouse sperm.

The anti-inflammatory drug celecoxib inhibits T-type Ca2+ currents in spermatogenic cells yet it elicits the acrosome reaction in mature sperm.

Celecoxib (Cx), an anti-inflammatory drug designed to inhibit COX2, can affect some ion channels. T-type (CaV3) channels have been implicated in sperm physiology. Here we report and characterize the Cx induced inhibition of T-type channels in mouse spermatogenic cells. Unexpectedly, Cx can also induce the acrosome reaction (AR), an intracellular Ca(2+) ([Ca(2+)]i) increase and a sperm depolarization. This [Ca(2+)]i increase possibly results from the ability Cx has to alkalinize intracellular pH (pHi), which is known to activate the sperm specific Ca(2+) channel CatSper. As the Cx induced [Ca(2+)]i increase is sensitive to mibefradil, a CatSper blocker, this channel may mediate the Cx-induced Ca(2+) entry leading to the AR. Our observations demonstrate that Cx can compromise fertilization.

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.

Inwardly rectifying K(+) channels in spermatogenic cells: functional expression and implication in sperm capacitation.

To fertilize, mammalian sperm must complete a maturational process called capacitation. It is thought that the membrane potential of sperm hyperpolarizes during capacitation, possibly due to the opening of K(+) channels, but electrophysiological evidence is lacking. In this report, using patch-clamp recordings obtained from isolated mouse spermatogenic cells we document the presence of a novel K(+)-selective inwardly rectifying current. Macroscopic current activated at membrane potentials below the equilibrium potential for K(+) and its magnitude was dependent on the external K(+) concentration. The channels selected K(+) over other monovalent cations. Current was virtually absent when external K(+) was replaced with Na(+) or N-methyl-D-glucamine. Addition of Cs(+) or Ba(2+) (IC(50) of approximately 15 microM) to the external solution effectively blocked K(+) current. Dialyzing the cells with a Mg(2+)-free solution did not affect channel activity. Cytosolic acidification reversibly inhibited the current. We verified that the resting membrane potential of mouse sperm changed from -52 +/- 6 to -66 +/- 9 mV during capacitation in vitro. Notably, application of 0.3-1 mM Ba(2+) during capacitation prevented this hyperpolarization and decreased the subsequent exocytotic response to zona pellucida. A mechanism is proposed whereby opening of inwardly rectifying K(+) channels may produce hyperpolarization under physiological conditions and contribute to the cellular changes that give rise to the capacitated state in mature sperm.

Dual regulation of the T-type Ca(2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells.

This study provides evidence for a novel mechanism of voltage-gated Ca(2+) channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch-clamp experiments demonstrated that serum albumin induced an increase in Ca(2+) T current density in a concentration-dependent manner, and significant shifts in the voltage dependence of both steady-state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, beta-estradiol significantly inhibited Ca(2+) channel activity in a concentration-dependent and essentially voltage-independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

Anion channel blockers differentially affect T-type Ca(2+) currents of mouse spermatogenic cells, alpha1E currents expressed in Xenopus oocytes and the sperm acrosome reaction.

The direct electrophysiological characterization of sperm Ca(2+) channels has been precluded by their small size and flat shape. An alternative to study these channels is to use spermatogenic cells, the progenitors of sperm, which are larger and easier to patch-clamp. In mouse and rat, the only voltage-dependent Ca(2+) currents displayed by these cells are of the T type. Because compounds that block these currents inhibit the zona pellucida-induced Ca(2+) uptake and the sperm acrosome reaction (AR) at similar concentrations, it is likely that they are fundamental for this process. Recent single channel recordings in mouse sperm demonstrated the presence of a Cl(-) channel. This channel and the zona pellucida (ZP)-induced AR were inhibited by niflumic acid (NA), an anion channel blocker [Espinosa et al. (1998): FEBS Lett 426:47-51]. Because NA and other anion channel blockers modulate cationic channels as well, it became important to determine whether they affect the T-type Ca(2+) currents of spermatogenic cells. These currents were blocked in a voltage-dependent manner by NA, 1, 9-dideoxyforskolin (DDF), and 5-nitro-2-(3-phenylpropylamine)benzoic acid (NPPB). The IC(50) values at -20 mV were 43 microM for NA, 28 microM for DDF, and 15 microM for NPPB. Moreover, DDF partially inhibited the ZP-induced AR (40% at 1 microM) and NPPB displayed an IC(50) value of 6 microM for this reaction. These results suggest that NA and DDF do not inhibit the ZP-induced AR by blocking T-type Ca(2+) currents, while NPPB may do so. Interestingly 200 microM NA was basically unable to inhibit alpha1E Ca(2+) channels expressed in Xenopus oocytes, questioning that this alpha subunit codes for the T-type Ca(2+) channels present in spermatogenic cells. Evidence for the presence of alpha1C, alpha1G, and alpha1H in mouse pachytene spematocytes and in round and condensing spermatids is presented.

Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.