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Ca²⁺-related changes in the mouse sperm capacitation state: a possible role for Ca²⁺-ATPase

Summary. Mammalian spermatozoa require extracellular Ca²⁺, some of which must be internalized, to undergo complete capacitation. At a critical threshold, a rise in intracellular Ca²⁺ will trigger acrosomal exocytosis. We used chlortetracycline (CTC) fluorescence patterns to assess changes in the capacitation state of mouse spermatozoa after incubation under various conditions that would affect their intracellular Ca²⁺ concentrations. Under standard conditions with 1·80 mmol CaCl₂ l^–1 known to support capacitation within 120 min and subsequent fertilization in vitro, a rise in the number of capacitated, acrosome-intact cells (B pattern) was observed over the first 60 min, followed by a decline. A detectable increase in capacitated, acrosome-reacted cells (AR pattern) coincided with the maximum of B pattern cells and a continued rise was observed over the following 60 min. With incubation in 3·60 mmol Ca²⁺ l^–1, the rise in AR cells began at 30 min, suggesting that this treatment accelerates capacitation. Introduction of ionophore A23187 [GenBank] at 15 min to cells in standard Ca²⁺ produced a similar but even more rapid response, with a maximum in B pattern cells and a noticeable rise in AR cells within 10 min. Thus ionophore-treated cells proceed through capacitation, but do so very quickly. However, ionophore in the presence of 90 µmol Ca²⁺ l^–1 could promote transition from the uncapacitated F pattern to the capacitated B pattern, but could not trigger acrosomal exocytosis, indicating that the latter requires high extracellular Ca²⁺. After preincubation in Ca²⁺-deficient medium, most cells exhibited the uncapacitated F pattern and the introduction of millimolar Ca²⁺ altered this distribution only slowly, over a period of 50 min. In contrast, preincubation in 90 µmol Ca²⁺ l^–1 resulted in a minority of F pattern cells and, within 10 min of millimolar Ca²⁺ introduction, a significant increase in AR cells was observed. These results suggest that changes in intracellular Ca²⁺ are important both for transition from the uncapacitated to the capacitated state, and for triggering of acrosomal exocytosis; furthermore, changes in CTC fluorescence patterns reflect alterations in Ca²⁺ within the cells. Both quercetin and ethacrynic acid, compounds that inhibit Ca²⁺ -ATPase in somatic cells, caused a significant increase in the proportion of B pattern capacitated cells, suggesting that intrinsic Ca²⁺ -ATPase activity may play a role during capacitation. The limited effect on acrosomal exocytosis per se suggests that another mechanism, possibly involving calcium channels, may control this latter event.

Keywords: mouse; ionophore; Ca²⁺-ATPase; quercetin; ethacrynic acid; decapacitation factors; spermatozoa

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