High pH-induced acrosome reaction and Ca uptake in sea urchin sperm suspended in Na-free seawater

The egg jelly-induced acrosome reaction of sea urchin sperm requires the presence of Ca²⁺ and Na⁺ in seawater at its normal pH 8. Sperm suspended in seawater at pH 9 undergo the acrosome reaction in the absence of jelly. We have attempted to understand the role of external Na⁺ in this reaction. Sperm were suspended in Na⁺-free seawater and the percentage of acrosome reaction and the amount of Ca²⁺ uptake were determined as a function of external pH. High pH (9.0) in Na⁺-free medium without jelly triggered a high percentage (above 65%) of sperm acrosome reactions and a two to fourfold increase in Ca²⁺ uptake. Both the percentage of acrosome reactions and the amount of Ca²⁺ uptake were similar to those induced by either jelly or pH 9 in Na⁺-containing seawater. On the other hand, the absence of Na⁺ in seawater inhibits jelly from inducing Ca²⁺ uptake and acrosome reactions at pH 8.0 and even at pH 8.5. These results indicate that the Na⁺ requirement for the acrosome reaction induced by jelly is lost when triggering is by high pH. In contrast, Ca²⁺ was strictly required since sperm did not react in Ca²⁺-free seawater at pH 9. We also found that like the jelly-induced acrosome reaction the high-pH-induced acrosome reaction and Ca²⁺ uptake in complete and Na⁺-free seawater were inhibited by D600. This finding suggests that the same transport system for Ca²⁺ uptake associated with the acrosome reaction operates at both triggering conditions, i.e., jelly or pH 9. Although D600 is not now considered a specific blocker, its effect has suggested the involvement of Ca²⁺ channels in the acrosome reaction. This proposal is supported by our results with nisoldipine, a highly specific inhibitor of calcium channels. The drug inhibited both the sperm acrosome reaction and Ca²⁺ uptake induced by jelly or pH 9 in complete seawater.     

The ascidian sperm reaction: Ca2+ uptake in relation to H+ efflux

During the ascidian sperm reaction the single large cylindrical mitochondrion which lies next to the nucleus in the head swells, becomes spherical, and migrates along the tail to be lost when it reaches the end. This sequence is initiated by eggs, egg water, high pH, low Na⁺, or the ionophore X537A. Accompanying the sperm reaction induced by low Na⁺ are H⁺ efflux and Ca²⁺ influx in a ratio of near 100:1 as determined by ⁴⁵Ca²⁺ and atomic absorption analysis. Simultaneous pH and Ca²⁺ electrode measurements suggest that the movement of H⁺ begins 10–13 sec before the movement of Ca²⁺. Ca²⁺ uptake can be inhibited by verapamil without affecting H⁺ efflux or the sperm reaction. Acid release and Ca²⁺ uptake are proportional to the initial pH of the medium when the reaction is triggered by high pH. Acid release initiated by low Na⁺ is proportional to Ca²⁺ concentrations above 2 mM. H⁺ and Ca²⁺ movements differ in magnitude, kinetics, and inhibition by verapamil, thus suggesting that H⁺ is probably not exchanged for Ca²⁺. Instead we propose that loss of H⁺ triggers the uptake of Ca²⁺, which initiates the sperm reaction.     

Mitochondrial movement during the ascidian sperm reaction

The ascidian sperm reaction, Which involves swelling, migration, and loss of the single large mitochondrion, can be triggered in vitro by raising the seawater pH to 9.3 or lowering Na⁺ to 20 mM, but only if the sperm are allowed to attach to a suitable Substate. Mitochondrial translocation does not usually occur in the absence of sperm attachment. Extracellular Ca²⁺ is necessary for triggering the reaction with low Na⁺ but not high pH; however, the intrecellular Ca²⁺ blocker, TMB-8, inhibits high pH-induced mitochondrial movement in the absence of extracellular Ca²⁺. After swelling, the mitochondrion fluoresces in the presence of chlortetracycline, suggesting that Ca²⁺ becomes membranebound after activation. Elevated cAMP and theophylline both inhibit mitochondrial move ment but not sperm motility. The antiactin drug cytochalasin B(10μM) and the calmodulinblocking drugs TFP (1 μM) and W-13 (10 μM) block mitochondrial movement, suggesting roles for actin and calmodulin in mitochondrial movement. A model is proposed relating intracellular alkalinization, Ca²⁺ influx, actin, myosin, and calmodulin in mitochondrial translocation.     

Calcium-dependent binding of mouse epididymal spermatozoa to the zona pellucida.

The minimal requirements and characteristics of epididymal sperm binding to the zona pellucida of the mouse egg were investigated using a new stop-fix centrifugation technique. This assay provided a precise physical definition of the association between the spermatozoon and the zona and permitted quantitation of the binding reaction at short time intervals. The results demonstrated that Ca²⁺ is an essential physiological component required for binding to occur. Sperm preincubated for 60 min in a simplified medium lacking Ca²⁺ did not acquire the ability to bind to eggs. In contrast, if sperm preincubation occurred in this medium supplemented with 1.7 mM Ca²⁺, binding was identical to that observed following sperm preincubation in the complete culture medium which supports both capacitation and fertilization in vitro. The Ca²⁺-dependent binding reaction was rapid, reversed by EGTA, specific for Ca²⁺, and did not require the transport of Ca²⁺ into the cell. Sperm bound to the zona surface following preincubation with Ca²⁺ were capable of fertilization in vitro when the eggs were subsequently transferred to the culture medium. It is proposed that this binding reaction represents a part of capacitation and not the acrosome reaction.     

Importance of sodium ion to the progesterone-initiated acrosome reaction in human sperm

Progesterone (P) has previously been shown to rapidly increase free intracellular calcium concentration ([Ca²⁻]_i), and subsequently to initiate the acrosome reaction (AR) in capacitated human sperm. The present study used cytochemical analysis of the AR, and spectrofluorometric determination of sperm [Ca²⁻]_i and intracellular pH (pH_i) in Na⁺-containing and Na⁺-deficient bicarbonate/CO₂-buffered media to investigate the role of Na⁺ in these P-initiated changes. We found that P failed to initiate the AR in Na⁺-deficient medium, and that the initial rise in [Ca²⁺]_i following P (1 μg/ml) stimulation was similar for both media; however, the [Ca²⁺]_i in the Na⁺-deficient medium regressed more rapidly and plateaued at a significantly lower [Ca²⁺]_i. Moreover, the differences in plateau [Ca²⁺]_i were directly related to the percentage of acrosome reactions, suggesting that the plateau phase is not due to [Ca²⁺]_i, but rather to the release of intracellular fura-2 into the medium during the AR. These [Ca²⁺]_i and AR results are in contrast to those reported previously by others for human sperm and suggest that a Na⁺-dependent mechanism is important in the P-initiated human sperm AR. Such a Na⁺ requirement may reflect the involvement of this ion in pH_i regulation, as capacitated sperm that were incubated in a Na⁺-deficient medium for ≥ 30 min displayed a significantly lower pH_i. © 1996 Wiley-Liss, Inc.     

A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentrotus purpuratus

Relationships among several of the ion movements associated with the acrosome reaction of S. purpuratus were investigated. Egg jelly initiates ⁴⁵Ca²⁺ and ²²Na⁺ uptake, and K⁺ and H⁺ efflux. H⁺ efflux and ²²Na⁺ uptake occur with approximately equivalent stoichiometries as rapidly as the appearance of acrosomal rods, perhaps reflecting a linked process. Most K⁺ loss, as measured either by ⁴²K⁺ efflux or K⁺-ion-selective electrodes, occurs after the acrosome reaction is complete. Since an elevation of seawater K⁺ (from 10 to 15 mM) or the addition of 0.5 mM tetraethylammonium (TEA), an inhibitor of K⁺ channels, inhibits the acrosome reaction half-maximally, K⁺ movements or alterations of K⁺-dependent membrane potentials may regulate the triggering by jelly. Most, but not all, of the ⁴⁵Ca²⁺ influx is inhibited with a mixture of 10 μM FCCP, 1 mM CN^?, and 2 μg/ml oligomycin, suggesting that the mitochondria store most of the Ca²⁺. The extracellular Na⁺ concentration affects Ca²⁺ fluxes: sperm placed into 5 mM Na⁺ seawater have enhanced ⁴⁵Ca²⁺ uptake, but do not undergo the acrosome reaction, unless 30 mM Na⁺ is also added. Low Na⁺ concentrations lead to spontaneous triggering, by allowing for both Ca²⁺ influx and Na⁺-dependent H⁺ efflux. At least one early Ca²⁺ requirement precedes the Na⁺ and H⁺ movements, as inferred from attempts at reversing the inhibitors of jelly induction of the acrosome reaction. When sperm are incubated with jelly in the absence of Ca²⁺, then washed and incubated with jelly in the presence of Ca²⁺, the acrosome reaction is triggered only upon the second incubation. However, when sperm are mixed with jelly in the presence of the other inhibitors (verapamil, TEA, 5 mM Na⁺ seawater, low pH, or elevated K⁺), they are altered so that even upon subsequent washing, jelly-mediated triggering is no longer possible. This suggests the existence of an intermediate state in the reaction pathway, that follows an event for which Ca²⁺ is required, but that precedes the Na⁺ and H⁺ movements, which are inhibited by all inhibitors of the acrosome reaction. These data are used to develop a partial sequence of ionic changes associated with the triggering mechanism.     

Seminal plasma features of Prochilodus lineatus and Brycon orbignyanus throughout two consecutives spawning seasons

This study aims to describe seminal plasma characteristics, detect changes during and between two consecutive spawning seasons (SS), and compare plasma features between two important South American fish species. Prochilodus lineatus and Brycon orbignyanus sperm was collected over two (SS1; SS2). Each season was divided into first and second sampling periods (P1; P2). Thus, the four experimental periods were referred to as SS1P1, SS1P2, SS2P1, and SS2P2. Seminal plasma was analyzed for osmolality, pH, and Na⁺, K⁺, and Ca²⁺ concentration. Additionally, sperm concentration, motility rate, and velocities (curvilinear = VCL; straight line = VSL) were determined and correlated with plasma features. In P. lineatus, plasma osmolality was lower in SS1P2, pH was higher in SS2P2, Na⁺ was higher and K⁺ and Ca²⁺ were lower in SS2P1 compared with other experimental periods. Positive correlations were observed between motility and plasma osmolality, motility and Na⁺, and VCL and Na⁺. In B. orbignyanus, plasma osmolality was higher in SS2P1 and SS2P2 and K⁺ concentration was higher in SS1P1 compared with other experimental periods; no correlation was observed. Seminal plasma parameters change during SS; therefore, the composition of a sperm extender and artificial fertilization methods should be adapted to maximize fertilization rates.     

The ascidian sperm reaction: evidence for Cl- and HCO3- involvement in acid release

Ascidia callosa sperm are triggered to undergo initiation of the sperm reaction (mitochondrial swelling) by increasing the pH or lowering the Na⁺ concentration of the medium. The optimal [Na⁺] for acid release is 20 mM with excellent correlation between acid release and initiation of morphological changes. Increasing the [K⁺] to around 20 mM inhibits acid release when applied up to 1 min after triggering the sperm but with less inhibition at 2 and 4 min, suggesting that K⁺ inhibits initiation of acid release rather than acid release itself. Acid release and the sperm reaction can also be triggered by Cl^?-free (NO^?₃ or glutamate substituted) seawater (SW). Cl^? efflux accompanies H⁺ efflux with twice as many Cl^? being released as H⁺. Both H⁺ and Cl^? release in Cl^?-free SW are dependent upon CO₂ being present in HCO^?₃-free medium, suggesting that H⁺ efflux is in part Cl^? and HCO^?₃-mediated. However, the chloride channel blocking agent SITS has no effect on H⁺ release and augments Cl^? release. Acid release results in a substantial increase in internal pH as determined by partitioning of 9-amino acridine. We envision acid release from ascidian sperm as involving two systems, the Na⁺-dependent acidification system of unreacted sperm and the Cl^?- and HCO^?₃-mediated H⁺ release at activation. The mechanism controlling acid release would then involve inactivation of the internal acidification process and activation of the chloride-bicarbonate-mediated alkalinization process.     

Is there a role for the Ca influx during fertilization of the sea urchin egg?

Both isotopic and microelectrode studies reveal a significant Ca²⁺ influx at fertilization which if freely distributed in the cytoplasm would equal 1–2 × 10⁻⁵ M. The role, if any, of this influx is disputed. We have attempted to reevaluate contradictory findings by others on this role. Our results with Strongylocentrotus purpuratus and Lytechinus pictus eggs, assessing fertilization with acrosome-reacted sperm in EGTA-buffered media (free [Ca²⁺], 4.4 × 10⁻⁸ M) indicate that exogenous Ca²⁺ is not required for fertilization and subsequent cleavage. The contradictory findings by others may have resulted from reduced fertilizability in Ca²⁺-free seawater, which can be circumvented by higher sperm concentration and by a sensitivity to temperature in Ca²⁺-free medium, which can be bypassed by carrying out fertilization at lower temperature. Also consistent with the absence of a requirement for this Ca²⁺ influx, we found that Ca²⁺ uptake can be induced in eggs by depolarizing the membrane with high [K⁺], but there is no resultant activation of egg metabolism. Under our conditions for fertilization in Ca²⁺-free media, there is no effect on the block to polyspermy but the initiation of the cortical reaction may be delayed. The data support the hypothesis that sperm induce release of Ca²⁺ from intracellular stores, perhaps by affecting an equilibrium between Ca²⁺ sequestration and Ca²⁺ release.     

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pH_i) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na⁺-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pH_i is more acidic than the external medium (pH_o = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na⁺ to sperm suspended in Na⁺-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pH_i by 0.4–0.5 pH units. That this pH_i change is the causal trigger of motility was suggested by experiments using NH₄Cl and nigericin, which increased the pH_i and resulted in activation of motility in the absence of Na⁺. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pH_i was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca²⁺ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pH_i by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca²⁺ uptake. Either mitochondrial agents or the removal of external Ca²⁺ could also block this pH_i change suggesting a similar mechanism is involved.     

The Ascidian Sperm Reaction

SYNOPSIS. Ascidian sperm are simplified by omission of the midpieceand proximal centriole with the single mitochondrion locatednext to the nucleus in the head. Small acrosome- like vesiclesappear in some species but their distribution is unclear. Spermstructure correlates well with the location of fertilization;members of the orders Phlebobranchiata and Stolidobranchiatawith external fertilization have sperm with short heads andlong tails, while sperm from the exclusively internally fertilizingAplousobranchiata have relatively longer heads and shorter tails. The mitochondrion swells when the sperm contacts the chorion,then translocates along the tail as it enters the chorion tobe discarded when the tail disappears inside. Structural changesalso occur in the anterior sperm head that have been interpretedas an acrosome reaction. Proteolytic enzymes are involved inpenetration of the chorion. The mitochondrial transformationis under control of intracellular pH and Ca²⁺ levels with thesperm releasing H⁺ and taking up Ca²⁺ during the sperm reaction.Acid release is from inactivation of a Na⁺ requiring acidificationsystem and triggering of a Cl^– releasing HCO₃ requiringacid release system. An increase in intracellular pH increasesthe permeability to Ca²⁺, resulting in increased intracellularCa²⁺, the proximal trigger to the mitochondrial reaction.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Regulation of spermatozoa motility in response to cations in Russian sturgeon Acipenser gueldenstaedtii

The aim of this study was to investigate the response of Russian sturgeon (Acipenser gueldenstaedtii) sperm to external cations (Na⁺, K⁺, Ca²⁺, and Mg²⁺) and their susceptibility on the induction of motility and swimming behavior. An in vitro spermatozoa motility assay was used by a computer-aided Motion-Analysis system. Sperm motility was inhibited by 60 mm NaCl (∼140 mOsm/kg) and 0.7 mm KCl solutions (∼ 21.4 mOsm/kg). The Ca²⁺ and Mg²⁺ ions were not able to inhibit spermatozoa motility. By contrast, Na⁺ within a limited concentration range (between 45 and 55 mm) was able to reverse the inhibitory effect of K⁺ at the critical concentration (0.7 mm). Ca²⁺ and Mg²⁺ were also able to reverse the K⁺-mediated spermatozoa motility restriction at concentrations starting at 0.01 and 0.1 mm, respectively. These results provide evidence for the role of K⁺ in suppressing spermatozoa motility, and suggest that Ca²⁺, Mg²⁺, and possibly Na⁺ trigger motility in Russian sturgeon sperm.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog,Rana pipiens

The membrane potential of the frog egg undergoes a transient positive shift at fertilization which is a block to polyspermy. This paper addresses the question of how a sperm elicits this “fertilization potential.” Iontophoretic injection of Ca²⁺ activates Rana pipiens eggs to develop and initiates a transient, positive-going shift in the membrane potential (the activation potential) which is like the sperm-induced fertilization potential in amplitude, duration, and Cl^? dependence. Activation potentials are elicited by Ca² injection into both animal and vegetal regions of the egg, but the rate of the initial depolarization is much less when Ca²⁺ is injected into the vegetal region. Injections of K⁺, Na⁺, Cl^?, or Mg²⁺ do not result in activation potentials, but the Ca²⁺ analogs, Sr²⁺ and Ba²⁺, can substitute for Ca²⁺. Treatment of eggs with the divalent cation ionophore, A23187, also initiates a transient, positive-going depolarization. Because injection of Ca²⁺ is sufficient to elicit a response almost identical to a fertilization potential, the ion transport mechanisms necessary to produce a fertilization potential must preexist in the unfertilized eggs; the sperm contributes only the stimulus to activate these mechanisms. The results reported here suggest that the stimulus may be a rise in free Ca²⁺.     

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs

Extracellular Ca²⁺ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca²⁺. In this study, Sr²⁺, Ba²⁺, and Mg²⁺ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba²⁺ proved to be ineffective, but Mg²⁺-containing medium was able to support capacitation to a greater extent than unsupplemented Ca²⁺-deficient media; despite this, Ca²⁺ was required for fertilization. In contrast, Sr²⁺ proved capable of substituting for Ca²⁺ in all events. Furthermore, Sr²⁺-induced responses were indistinguishable from the corresponding Ca²⁺-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr²⁺ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr²⁺. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr²⁺ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr²⁺ can profoundly affect both gametes in the mouse: It substitutes completely for Ca²⁺ in sperm responses and rapidly activates eggs, possibly by displacing Ca²⁺ from intracellular stores into the cytoplasm, where the Ca²⁺ can then trigger the various events of activation.     

Requirement of extracellular calcium ions for various stages of fertilization and fertilization-related phenomena in the hamster

Using a semi-chemically defined medium, the requirement of extracellular Ca²⁺ for survival, capacitation, and acrosome reaction of spermatozoa as well as various stages of fertilization in the hamster was studied. A Ca²⁺-deficient environment is unfavorable for long-term survival of spermatozoa. Sperm capacitation may occur in Ca²⁺-deficient media, but not as efficiently as in normal media. The acrosome reaction definitely requires extracellular Ca²⁺. Other processes or phenomena that require extracellular Ca²⁺ are initiation and maintenance of hyperactivated motility of spermatozoa, penetration of acrosome-reacted spermatozoa into the zona pellucida, fusion of the spermatozoa with eggs, and the development of pronuclear eggs into two-cell embryos. Extracellular Ca²⁺ is apparently unnecessary for the attachment of spermatozoa to the zona and egg surfaces, decondensation of the sperm nucleus, and the development of sperm and egg pronuclei within the egg. These results were compared with data obtained in other species such as the sea urchin, mouse, rat and guinea pig.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.