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Intracellular Ca²⁺-Mg²⁺-ATPase Regulates Calcium Influx and Acrosomal Exocytosis in Bull and Ram Spermatozoa¹

^a Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

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.

¹ This research was supported by the Israel Science Foundation funded by The Academy of Sciences and Humanities and by Ihel Foundation to H.B.

² Correspondence. FAX: 972 3 5344766; breith{at}mail.biu.ac.il

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