Felipe Rafael Reyna Espinosa

Mouse sperm membrane potential: changes induced by Ca2+

Mouse sperm resting membrane potential (E r) (−42±8.8 mV), determined with a potential sensitive dye, depended on extracellular K+ and, in the absence of extracellular Ca2+ ([Ca2+]e), on external Na+ ([Na+]e). Ca2+ addition (>5 μM) to sperm in Ca‐free media induced a transient hyperpolarization (Ca‐ith) which strongly depended on [Na+]e and less on external Cl− ([Cl−]e). Cd2+ and Mn2+ (μM) mimicked the Ca2+ effect, but not Ba2+. The Ca‐ith was partially inhibited by ouabain (74%, IC 50 = 5.8 μM) and niflumic acid (38%, IC 50 = 240 μM), indicating the participation of the Na‐K ATPase and Cl− channels. In Ca‐free low‐Na+ media, Ca2+ addition caused a depolarization sensitive to: nimodipine (25 μM), trifluoperazine (12.5 μM) and Mg2+ (1.2 mM), suggesting the participation of Ca2+ channels. Since some inhibitors of the sperm Ca‐ith block the acrosome reaction (AR), both processes may share transport systems.

Dual regulation of the T-type Ca2+ current by serum albumin and β-estradiol in mammalian spermatogenic cells.

This study provides evidence for a novel mechanism of voltage‐gated Ca2+ 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 Ca2+ 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, β‐estradiol significantly inhibited Ca2+ channel activity in a concentration‐dependent and essentially voltage‐independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

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 (∼3 μm 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 μM). 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.

Anion channel blockers differentially affect t‐type Ca2+ currents of mouse spermatogenic cells, α1E 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.

7 – Regulation of Sperm Ion Currents

Publisher Summary This chapter focuses on the regulation of sperm ion currents—that is, the participation of the sperm ion channels in the information exchange between gametes and between gametes and their environment. In all species whose sperm cells possess an acrosome, successful fertilization requires the acrosome reaction. This reaction allows spermatozoa to penetrate through the outer vestments of the egg and to recognize and fuse with the egg plasma membrane. Induction of this fundamental process involves short-range interactions of spermatozoa with components from the eggs outer layers, and also with other components of the female reproductive tract for internal fertilizers. Spermatozoa experience important alterations in their ionic milieu that influence their functional state. This chapter ends with concluding remark that cell signaling is fundamental in determining the behavior of organisms. The propagation of life in many species depends on the dialogue between gametes, ion channels being elementary tools of cell communication. Currently, there is background information about some of the ion channels present in spermatozoa. Future study will determine the molecular mechanisms that regulate these channels in the cell. Combining molecular biological strategies and electrophysiology in spermatogenic cells, and the transfer of ion channels directly from spermatozoa to planar bilayers, opens new avenues to explore the participation of channels in spermatogenesis, and their regulation in mature spermatozoa cells.