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 (pH_i) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca²⁺ channels modulated by pH_i 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 pH_i by ∼1.3 U from a resting pH_i ∼6.65. A steady pH_i 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 pH_i, 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.