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 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

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen–thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate poly-acrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both uncapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of ³²P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6–22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M _r of about 95 000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M _r 116 000, 105 000, 95 000, 86 000, and 76 000, and possibly a sixth at 54 000. The 95 000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.

Adenosine and its analogues, known to stimulate adenylate cyclase activity in somatic cells via A₂ receptors, can accelerate capacitation in mouse spermatozoa and thereby enhance fertilizing ability in vitro. Indirect evidence has suggested that adenosine can modulate mouse sperm adenylate cyclase, implicating this enzyme and cAMP in the observed functional responses. In the present study we provide evidence that [³H]5′-N-ethylcarboxamidoadenosine (NECA), an adenosine analogue with specificity for stimulatory A₂ adenosine receptors, can bind to mouse spermatozoa. This binding can be displaced by both unlabelled NECA and 2-chloroadenosine, another A₂ receptor agonist, but not by cyclopentyladenosine, an inhibitory A₁ receptor agonist, suggesting that the NECA binding is specific for A₂ receptors. The presence of S-(p-nitrobenzyl)-6-thioinosine, an adenosine transport inhibitor, did not affect binding, indicating an external site for interaction with sperm cells. Saturable specific binding of [³H]NECA to mouse spermatozoa incubated at 37°C was observed, with a B _max of 5.17 pmol mg⁻¹ protein and a K _d value of 930 nmol l⁻¹. Binding data were consistent with the presence of a single major class of receptor. In addition to demonstrable binding of [³H]NECA, both NECA and 2-chloroadenosine significantly stimulated adenylate cyclase activity in a concentration-dependent manner, with NECA being effective at a lower concentration. Furthermore, the hydrolysis-resistant GTP analogue Gpp(NH)p, alone and in the presence of either NECA or 2-chloroadenosine, also significantly stimulated enzyme activity. In somatic cells, expression of responses to adenosine usually requires GTP and G proteins. These results are consistent, therefore, with the hypothesis that adenosine-induced enhancement of sperm functional ability is due to stimulation of adenylate cyclase, via A₂ adenosine receptors, and hence increasing availability of intracellular cAMP. They also suggest that G proteins play a role in the receptor-mediated response.

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.

Mouse sperm capacitation in vitro, leading to hyperactivated motility, acrosomal exocytosis and rapid fertilization, takes approximately 120 min in a medium containing sufficient Ca²⁺ During that period, spermatozoa incubated in ⁴⁵Ca²⁺ exhibited a biphasic pattern of Ca²⁺ uptake, with the first and lower peak occurring from 10 to 50 min and the second and higher peak from 60 to 90 min. When the exogenously supplied glucose was reduced from 5.56 mmol l⁻¹ to 5.56 μmol l⁻¹, the latter supporting capacitation but not fertilization, only the first peak of ⁴⁵Ca₂₊ uptake was observed. Increasing the glucose to a millimolar concentration produced a second peak of uptake. We therefore propose that the first phase of ⁴⁵Ca²⁺ uptake is associated with capacitation and the second phase with acrosomal exocytosis, which are both necessary prerequisites for fertilization. In micromolar glucose the rate of ⁴⁵Ca²⁺ uptake during the first 30 min was 47% higher than in millimolar glucose, suggesting that the former conditions might promote a precocious rise in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and hence accelerate capacitation. This hypothesis was confirmed by demonstrating both significantly accelerated transition from the uncapacitated F pattern of chlortetracycline (CTC) fluorescence to the capacitated B and AR patterns and significantly higher fertility in vitro in suspensions preincubated for 30 min in micromolar glucose, compared with those maintained continuously in millimolar glucose. These results suggest that an ATP-dependent mechanism, for example a Ca²⁺-ATPase, may be involved in maintaining a low [Ca²⁺]_i. In micromolar glucose, available ATP would be limited and hence the ATPase activity would decline, allowing [Ca²⁺]_i to rise. The possibility that such an ATPase might be calmodulin-sensitive was investigated by incubating sperm suspensions in the presence of trifluoperazine (TFP), a calmodulin antagonist. TFP significantly accelerated both the initial uptake of ⁴⁵Ca₂₊ and the transition from uncapacitated to capacitated CTC patterns, suggesting a role for a calmodulin-sensitive Ca²⁺-ATPase during capacitation. Finally, inclusion of the mitochondrial inhibitor azide had little effect on ⁴⁵Ca²⁺ uptake, indicating that most of the observed uptake in this study was occurring in the sperm head.

Mammalian spermatozoa require extracellular Ca²⁺, some of which must be internalized, to undergo capacitation and acrosomal exocytosis. The mechanisms controlling the intracellular Ca²⁺ concentration are unclear, but current evidence suggests that a Ca²⁺-ATPase may be involved. Using treatments that potentially modulate enzyme activity, we investigated this possibility in human spermatozoa; the capacitation state and acrosomal integrity were monitored by chlortetracycline fluorescence. Incubation of cells in the presence of quercetin, a Ca²⁺-ATPase inhibitor, significantly accelerated the transition from the uncapacitated F pattern of chlortetracycline fluorescence to the capacitated, acrosome-intact B pattern within 1 h. This was followed by an increase in the number of cells displaying the capacitated, acrosome-reacted AR pattern. Since most Ca²⁺-ATPases in somatic cells are sensitive to calmodulin, we also investigated the effect of the calmodulin antagonist W-7 on chlortetracycline patterns. At 1–125 μmol l⁻¹, W-7 significantly stimulated capacitation and acrosomal exocytosis. Furthermore, W-7 at 1 μmol l⁻¹ proved to be more effective than W-5, a less potent antagonist, suggesting that the observed responses in human spermatozoa did reflect a calmodulin-sensitive mechanism. When the glucose concentration in the culture medium was varied (from 0 to 5.56 mmol l−1) to alter the availability of ATP for enzyme activity, it was found that a reduced concentration of glucose promoted capacitation more rapidly than did the standard concentration of 5.56 mmol glucose l⁻¹. However, maximal changes, particularly in promoting the shift from the B to the AR pattern of chlortetracycline fluorescence, required millimolar concentrations of glucose during the last few minutes before assessment. Finally, the addition of partially purified mouse sperm decapacitation factor (proposed to activate a Ca²⁺-ATPase and thus maintain a low intracellular Ca²⁺ concentration) to capacitated human sperm suspensions caused a significant reversal in the capacitation state of cells (from the B to the F pattern). The F pattern of chlortetracycline fluorescence predominates in conditions favouring low concentrations of intracellular Ca²⁺. From these results, we suggest that a Ca²⁺-ATPase may play an important role during human sperm capacitation. A time-dependent decrease in endogenous enzyme activity would allow the intracellular concentration of Ca²⁺ to rise to a critical value necessary for initiation acrosomal exocytosis and subsequent successful fertilization.

Chlortetracycline (CTC) fluorescence patterns were used to assess Ca -related changes in the capacitation state of human spermatozoa incubated under conditions that would affect their intracellular Ca²⁺ levels. Initial experiments were designed to identify consistently occurring patterns and to correlate these with acrosomal status. Incubation for up to 1 h with the ionophore A23187 (10 μmol l⁻¹), known to promote capacitation and acrosomal exocytosis, allowed the identification of three different CTC staining patterns which were very similar to those described for mouse spermatozoa. For this reason, they were given the same nomenclature: 'F' – characteristic of uncapacitated, acrosome-intact cells; 'B' – characteristic of capacitated, acrosome-intact cells; and 'AR' – characteristic of capacitated, acrosome-reacted cells. The distribution of the three patterns in the ionophore-treated suspensions was very different from that in control suspensions treated with dimethylsulfoxide only, with a significantly higher proportion of cells displaying the B and AR patterns and a significantly lower number of cells displaying the F pattern in the ionophore-treated group at all times. A strong concordance was found between the acrosomal status of cells determined using both CTC and fluorescein-conjugated Pisum sativum agglutinin (PSA) staining methods on the same cells. Verification of PSA staining patterns with acrosomal status was obtained by means of transmission electron microscopy. The proportion of cells with uniform fluorescence in the acrosomal region correlated with acrosome-intact cells; those with only equatorial segment staining correlated with fully-reacted cells, and those exhibiting equatorial fluorescence and patchy fluorescence over the rest of the acrosomal region correlated with cells in intermediate stages of exocytosis. Having established and verified the morphological basis for the CTC staining patterns, we then incubated cells in medium containing standard (1.80 mmol l⁻¹) and high (3.60 mmol l⁻¹) CaCl₂. In both media the proportion of F cells decreased with time, whereas the B and AR patterns increased, but the high Ca²⁺ treatment significantly accelerated the change from F to B to AR at all time points. In contrast, when spermatozoa were incubated in a Ca -deficient medium for up to 22 h, the majority of cells displayed the uncapacitated F pattern. The introduction of millimolar Ca²⁺ during the final 15 min of incubation failed to alter the CTC patterns, thus confirming the fact that human spermatozoa require the continuous presence of extracellular Ca to undergo capacitation and the acrosome reaction. These results suggest that changes in CTC fluorescence patterns indicate Ca^{2 +}-related changes in the functional state of human spermatozoa and therefore that CTC assessment may prove useful in clinical assessment of human sperm fertilizing potential.

Cumulus-enclosed pig oocytes were matured in vitro, freed from cumulus cells, and inseminated with frozen–thawed ejaculated spermatozoa in a chemically defined protein-free medium containing 37.0 mmol NaHCO₃ l⁻¹ and 5 mmol caffeine l⁻¹. When the medium was supplemented with 1 mg polyvinylalcohol (PVA) ml⁻¹, more penetrated oocytes were observed 14 h after insemination with 7–12 × 10⁶ cells ml⁻¹ than with 4–5 × 10⁶ cells ml⁻¹ and the incidence of polyspermy reflected the sperm concentration used. Varying the NaHCO₃ concentration but maintaining the sperm concentration at 8 × 10⁶ cells ml⁻¹ resulted in significantly more oocytes being penetrated in media containing 45.83–50.25 than 37.0–41.42 mmol NaHCO₃ l⁻¹; there were no significant differences in the incidence of either male pronuclear formation or polyspermy. In medium containing 45.83 mmol NaHCO₃ l⁻¹, the inclusion of PVA at 0–5 mg ml⁻ had no effect on proportions of penetrated oocytes, male pronuclear formation or polyspermy. However, when spermatozoa from three different boars were evaluated, the penetration and male pronuclear formation rates were highly variable, unlike the incidence of polyspermy. Penetration of cumulus-free oocytes was first detected at 6 h. When spermatozoa were incubated for 6 h in the absence of oocytes, motility, but not vitality, decreased whether or not PVA was included in the medium. Chlortetracycline (CTC) fluorescence analysis of the capacitation state indicated a rapid decline in the proportion of live uncapacitated, acrosome-intact cells and a rapid rise in the proportion of live capacitated, acrosome-reacted cells during the first hour. Smaller changes in the distribution of CTC patterns occurred during the later stages, suggesting that the rapidly responding cells were non-fertilizing, owing to damage by freeze–thawing, and that the fertilizing spermatozoa were drawn from the remaining pool of cells which underwent capacitation more slowly. This is the first report indicating that capacitation of frozen–thawed ejaculated boar spermatozoa and penetration of oocytes matured in culture are possible in a chemically defined, protein-free medium.