Intracellular Ca2+-Mg2+-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa

Abstract

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca2+-Mg2+-ATPase (Ca2+ pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca2+, since incubation of cells in the absence of added Ca2+ or in the presence of the calcium channel blocker, La3+, completely inhibited thapsigargin-induced acrosomal exocytosis. ATP- Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca2+ concentration ([Ca2+](i)), and this increase was inhibited when extracellular Ca2+ was chelated by EGTA, indicating that this rise in Ca2+ is derived from the external medium. This rise of [Ca2+](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP3-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP3-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca2+ elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca2+ store. We suggest here that the cytosolic Ca2+ is actively transported into the acrosome by an ATP- dependent, thapsigargin-sensitive Ca2+ pump and that the accumulated Ca2+ is released from the acrosome via an IP3-gated calcium channel. The ability of thapsigargin to increase [Ca2+](i) could be due to depletion of Ca2+ in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca2+](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca2+](i) during capacitation and the AR.