PROPERTIES OF A NOVEL PH-DEPENDENT CA2-CELLS WITH POSSIBLE ROLES IN SPERMATOGENESIS AND MATURE SPERM FUNCTION( PERMEATION PATHWAY PRESENT IN MALE GERM)

Rises of intracellular Ca2+ ([Ca2+](i)) are key signals for cell divis ion, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, ca rried 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 re action. Evidence implicates the activity of Ca2+ channels modulated by pH(i) in the origin of these Ca2+ elevations, but their nature remain s unexplored, in part because work in individual spermatozoa are hampe red by formidable experimental difficulties. Recently, late spermatoge nic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we descri be the first study on the influence of controlled intracellular alkali nization on [Ca2+](i) on identified spermatogenic cells from mouse adu lt testes. In BCECF [ (2',7')-bis (carboxymethyl) (5,6)-carboxyfluores cein]-AM-loaded spermatogenic cells: a brief (30-60 s) application of 25 mM NH4Cl increased pH(i) by similar to 1.3 U from a resting pH(i) s imilar to 6.65. A steady pH(i) plateau was maintained during NH4Cl app lication, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an init ial [Ca2+](i) drop followed by a two- to threefold rise. Maneuvers tha t inhibit either Ca2+ influx or intracellular Ca2+ release demonstrate d that the majority of the Ca2+ rise results from plasma membrane Ca2 influx, although a small component likely to result from intracellula r 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 pas sage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorg anic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blo cker nifedipine. The magnitude of these Ca2+ transients increased as m aturation advanced, with the largest responses being recorded in testi cular sperm. By extrapolation, these findings suggest that the pH-depe ndent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it cor responds to an ion channel different from the voltage-gated T-type Ca2 + channel also present in spermatogenic cells. We postulate that the C a2+ permeation pathway regulated by pH(i), if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx re quired for initiating the acrosome reaction and perhaps other importan t sperm functions.