Properties of a Novel pH-dependent Ca(2+) Permeation Pathway Present in Male Germ Cells with Possible Roles in Spermatogenesis and Mature Sperm Function

Rises of intracellular Ca(2+) ([Ca(2+)](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 [Ca(2+)](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 Ca(2+) channels modulated by pH(i) in the origin of these Ca(2+) 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 [Ca(2+)](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 NH(4)Cl increased pH(i) by ∼1.3 U from a resting pH(i) ∼6.65. A steady pH(i) plateau was maintained during NH(4)Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca(2+)](i) drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca(2+) influx or intracellular Ca(2+) release demonstrated that the majority of the Ca(2+) rise results from plasma membrane Ca(2+) influx, although a small component likely to result from intracellular Ca(2+) release was occasionally observed. Ca(2+) transients potentiated with repeated NH(4)Cl applications, gradually obliterating the initial [Ca(2+)](i) drop. The pH-sensitive Ca(2+) permeation pathway allows the passage of other divalents (Sr(2+), Ba(2+), and Mn(2+)) and is blocked by inorganic Ca(2+) channel blockers (Ni(2+) and Cd(2+)), but not by the organic blocker nifedipine. The magnitude of these Ca(2+) transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca(2+) 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 Ca(2+) channel also present in spermatogenic cells. We postulate that the Ca(2+) permeation pathway regulated by pH(i), if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca(2+) influx required for initiating the acrosome reaction and perhaps other important sperm functions.