Na⁺-requiring mechanisms modulate capacitation and acrosomal exocytosis in mouse spermatozoa

Mouse spermatozoa require extracellular Na⁺ for both capacitation and acrosomal exocytosis, but the minimum concentrations differ widely: > 1 ≤ 25 mmol Na⁺ l⁻¹ will support capacitation, but > 125 mmol Na⁺ l⁻¹ is needed for acrosomal exocytosis in capacitated cells. Our conclusions are based on evidence obtained from sperm cells preincubated in isoosmotic media with differing concentrations of Na⁺ and then analysed for occurrence of the acrosome reaction, capacitation-related changes in chlortetracycline (CTC) fluorescence and in vitro fertilization. The modified Tyrode's medium used as the control medium in these experiments contained 150 mmol Na⁺ l⁻¹ and supported full sperm function. At least some of the Na⁺ needs to be internalized to promote the functional changes, as evidenced by the ability of the monovalent cation ionophore monensin to accelerate capacitation and trigger acrosomal exocytosis in control medium. However, in low Na⁺ (25 mmol l⁻¹) medium, monensin could only modulate the transition to the capacitated state, assessed with CTC, indicating that higher concentrations of extracellular Na⁺ are required for initiation of acrosomal exocytosis. We suggest that changes in the composition of the female reproductive tract fluids serve to control expression of sperm functional potential. Before ovulation in the mouse, sufficient Na⁺ and Ca²⁺ are present to promote capacitation. However, the Na⁺ concentration is marginal for support of acrosomal exocytosis and the relatively high K⁺ reinforces an inhibition of exocytosis. At ovulation, the release of follicular fluid would increase the Na⁺ and decrease the K⁺ concentrations, thereby permitting full expression of fertilizing potential. Possible mechanisms that might be involved in the Na⁺-related responses, including a Na⁺–Ca²⁺ exchanger, a Na⁺–K⁺ ATPase and a Na⁺–H⁺ exchanger, were also investigated. If a Na⁺–Ca²⁺ exchanger has a role to play, it is not during capacitation per se. Incubation of sperm cells in high Na⁺, low Ca²⁺ (90 μmol CaCl₂l⁻¹) medium that supports capacitation, followed by introduction of monensin, which would have promoted an influx of Na⁺ and could have, in turn, activated a Na⁺ out, Ca²⁺ in response, did not accelerate transition to the capacitated state (B pattern of CTC fluorescence). In contrast, it is possible that a Na⁺–K⁺ ATPase may play a role during capacitation. Incubation of suspensions in control medium plus ouabain, which would inhibit the ATPase, significantly accelerated the transition from the uncapacitated to the capacitated state, although it did not trigger acrosomal exocytosis. Thus, a decline in activity of this enzyme may be an important part of capacitation. Finally, we have obtained evidence that a Na⁺–H⁺ exchange mechanism may be involved in initiation of acrosomal exocytosis. Incubation of suspensions in low Na⁺ medium for a time sufficient to promote capacitation, followed by introduction of 25 mmol NH₄Cl l⁻¹ for 10 min to raise intracellular pH, significantly stimulated the acrosome reaction even though the Na⁺ concentration was not increased to provide permissive Na⁺ conditions for exocytosis. We suggest that during fertilization an influx of Na⁺ into the fertilizing spermatozoon causes a rise in intracellular pH that in turn opens Ca²⁺ channels, thereby permitting the influx of Ca²⁺ needed to trigger acrosomal exocytosis.