Interactions between sperm and sea urchin egg jelly

DEVELOPMENTAL

BIOLOGY

98,

1-14

(1983)

Interactions

between Sperm and Sea Urchin Egg Jelly

R. CHRISTEN,* Department

of Biochemistry, *Station Received

July

R. W. SCHACKMANN,’ University Zoologique,

AND B. M. SHAPIRO

of Washington SJ-70, Seattle, Washington 06230 ViUeFranche sur Mer, France

2, 1982; accepted

in revised form December

98195, and

20, 1982

The addition of egg jelly to sea urchin sperm induces multiple changes in morphology and behavior. When jelly is added to sperm diluted in seawater, the acrosome reaction is triggered, the mitochondrion rounds up, the internal pH is transiently alkalinized and then reacidified, and respiration becomes uncoupled and rapidly decreases. Sperm also become unable to fertilize eggs within a few minutes after jelly addition. In order to explore in more detail the effect of egg jelly on sperm, we have studied the response to jelly in the presence of inhibitors of the acrosome reaction. When jelly is added to sperm under conditions which are inhibitory for the acrosome reaction, an alkalinization takes place without the subsequent reacidification, the mitochondria remain coupled, and respiration and intracellular ATP levels remain high. Sperm viability is prolonged by some of these conditions, but not others. The addition of jelly to sperm in the absence of calcium elicits an internal alkalinization but no other rapid change in sperm physiology. The capacity of egg jelly to alter sperm physiology even when the overall acrosome reaction is inhibited indicates that some of the physiological changes either are early events in the triggering pathway that happen before the inhibitory step or are unrelated to the acrosomal reaction itself. The reacidification of the internal pH, the uncoupling and decrease of the respiration, and the decrease of the ATP levels might be linked together by the large influx of calcium that occurs after the acrosome reaction. INTRODUCTION

man, 1975; Ohtake, 1976; Kopf and Garbers, 1979; Tubb et al, 1978; Kopf et a& 1979; Hansbrough and Garbers, 1981; Suzuki et al, 1981; Garbers et a& 1982). Although the physiological significance of this activation is not clear, it has been suggested that this peptide is required to activate sperm to swim through the egg coat material, which might be acidic. A mixture of high-molecular-weight components triggers the acrosome reaction, which includes the exocytosis of an apical vesicle (the acrosomal vesicle) and elongation of an actin-containing acrosomal process (Tilney et aZ., 1973, 1978; Jessen et aL, 1973). This mixture includes a polysaccharide of sulfated fucose and a sialoprotein and is relatively species specific in its interaction with sperm (Haino and Dan, 1961; Dan et ak, 1972; Hotta et aZ., 1973; Lorenzi and Hedrick, 1973; Summers and Hylander, 1975; SeGall and Lennarz, 1979, 1981). When separated from the sialoprotein, the fucose sulfate polysaccharide alone is sufficient to trigger the acrosome reaction (SeGall and Lennarz, 1979; 1981; Garbers et al, 1980; Kopf and Garbers, 1980). Since the acrosome reaction is required for fertilization, egg jelly is essential. The relationship between the triggering of the acrosome reaction and the movements of ions has been extensively analyzed since the work of Dan (1954). The acrosome reaction is accompanied by a depolarization of the plasma membrane potential, an increase in the

In order to fertilize eggs, spermatozoa proceed through a series of activation processes. In sea urchins, the sequence of events that prepares the sperm to fertilize homologous eggs includes activation of respiration and motility upon dilution into seawater (Gray, 1928; Mohri and Horiuchi, 1961; Mohri and Yasumasu, 1973; Timourian and Watchmaker, 1970; Ohtake, 1976; Nishioka and Cross, 1978), interactions with substances released from the surface of the eggs (discussed below), binding to the external envelopes that cover the unfertilized eggs (Hylander and Summers, 1975; Summers and Hylander, 1976; Vacquier and Moy, 1977; Aketa et al, 1978; Glabe and Lennarz, 1979; Kinsey et al, 1980), digestion of the vitelline layer by enzymes exposed during the acrosomal reaction (Colwin and Colwin, 1960; Schuel et aL, 1976; Hoshi et al, 1979; Green and Summers, 1980; Hoshi and Moriya, 1980), and fusion between the plasma membranes of the two gametes. Recent attention has been devoted to the action of the egg upon the sperm, and two different components of the jelly coat surrounding the egg have been identified, which have different actions on sperm behavior and morphology. Speract, a small polypeptide that has no reported effect on sperm in normal seawater, activates sperm respiration and motility at acidic pH (Garbers and Hard’ To whom correspondence should be addressed. 1

0012-1606/83 $3.00 Copyright All rights

0 1983 by Academic Press, Inc. of reproduction in any form reserved.

2

DEVELOPMENTAL

BIOLOGY

internal pH, an influx of Ca’+, an efflux of K+, and uptake of Na+ (Christen et ab, 1980, 1981; Schackmann and Shapiro, 1981; Schackmann et al., 1981). The acrosome reaction is blocked by verapamil, TEA, removal of Ca2+ or Na+, low extracellular pH (PH,J,~ or high extracellular K+ (Decker et aZ., 1976; Talbot et ah, 1976; Collins and Epel, 19’77; Schackmann et aZ., 1978; Kopf and Garbers, 1980). In addition, elevated CAMP concentrations have been demonstrated when the acrosome reaction is triggered (Kopf and Garbers, 1980; Garbers, 1981). Besides triggering the acrosome reaction, egg jelly also brings about other changes in sperm physiology and behavior, such as altered mitochondrial and flagellar position, aggregation of sperm into large clusters (Loeb, 1914; Isaka et al, 1970; Collins, 1976), and rapid sperm death (Kinsey et aL, 1979; Vacquier, 1979b). Some of these changes are striking in other phyla, for example in ascidians, where the mitochondrion slides along the tail to be expelled from the sperm (Lambert and Epel, 1979; Lambert and Lambert, 1981). In this paper we describe the effects of egg jelly on several physiological parameters of sea urchin sperm. We have examined the sperm internal pH (pHi), respiration, membrane potential, mitochondrial shape, viability, and ATP concentration. By using various inhibitors of the acrosome reaction, we have investigated the interrelationships of these several physiological changes to each other and to the induction of the acrosome reaction. MATERIALS

AND

METHODS

Gametes

from the sea urchin Strongylocentrotus purby intracoelomic injection of 0.5 M KCl. Sperm were collected as “dry” as possible (2-6 X lOlo sperm/ml) either by pipetting sperm directly from the gonopores with a Pasteur pipet or by wrapping the sea urchin body with absorbent paper. Sperm were subsequently stored on ice. Viability assay. Sperm viability was assessed by their ability to elevate fertilization membranes of eggs. Dry sperm were diluted lOOO-fold into plastic tubes containing the various media and incubated at lo-12°C. Packed eggs were diluted 200-fold into Millipore-filtered seawater (MFSW) and stirred with a propeller to ensure a homogeneous suspension. The assay for sperm viability

puratus were obtained

s Abbreviations used: EtzNH, diethylamine; g-AA, 9 aminoacridine; DMO, dimethyloxazoladine-2,4-dione; pHi, intracellular pH0, extracellular pH, Ar, accumulation ratio; ASW, artificial seawater; ChSW, choline substituted for Na+ in artificial seawater; TPP, tetraphenylphosphonium; TEA, tetraethylammonium; MFSW, Millipore-filtered seawater; 5% FME, percentage of eggs which elevated fertilization membranes; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; SITS, 4 acetamido 4’-isothiocyanostilbene-2,8-disulfonic acid.

VOLUME

98, 1983

was done by mixing a small aliquot (lo-20 ~1) of the sperm suspension with 3-4 ml of the egg suspension in a small plastic petri dish. After 10 min the reaction was stopped by addition of glutaraldehyde and the percentage of eggs which elevated fertilization membranes (% FME) was estimated. The sperm/egg ratio in the petri dish was adjusted for each experiment so that about 90% FME was achieved at the beginning of the experiment; under those conditions any decrease in sperm viability was immediately detected (Vacquier, 1979b; Kinsey, et al., 1979). Washing of spemz. For experiments using a Na+ and Ca2+-free medium, sperm were first washed (loo-fold dilution) by centrifugation (lOOOg, 10 min) and resuspended in the same medium. Determination of the internal pH (pHi). Dry sperm were diluted loo-fold into media containing radiolabeled [‘4C]diethylamine (Et2NH) from ICN (55.5 mCi/ mmole) at a final concentration of 2-10 FM. Separation of the sperm from the reaction medium (200-~1 samples) was by centrifugation in an Eppendorf microfuge through a layer of silicone oil; extraction of the pellet was as previously described (Christen and Sardet, 1980; Schackmann et al, 1981). Extracellular and total pellet Hz0 spaces were determined with tritiated water and [14C]inulin as previously described (Schackmann et al., 1981). The internal pH is pHi = pHo - log[Ar], where Ar, the accumulation ratio, is the ratio of the internal to external amine concentrations. This is an approximation which holds when the pHi and external pH are substantially lower than the pK of the amines used (Schuldiner et aZ., 1972; Boron, 1978). EtzNH was used because it reaches its equilibrium faster than any other amine tested (Christen et ak, 1982). In order to chelate the external calcium after jelly addition, an EGTA buffer was added. The buffer contained 100 mM EGTA, 150 mlM Tris, 190 mM NaCl, pH 8.7 (50 mM MgC12 was also present in some experiments). Both elevated pH and buffer concentrations were necessary to prevent a substantial decrease in the seawater pH by the divalent cation-EGTA interaction. The experimental procedure followed in the experiment described in Fig. 4 was as follows: 50 ~1 dry sperm were diluted loo-fold in ASW (containing 1 mM EGTA, pHo = 8.0). [14C]EtzNH was then added and sperm were sampled (150 ~1) for amine accumulation; 9 min after the addition of amine, the sperm suspension was divided into three different tubes (1.5 ml), and either jelly (150 ~1) or ASW was added, followed 45 set later by 300 ~1 ASW or EGTA buffer. The final pH in each tube was 8.0. Subsequent measurements of the pHi were done by taking 200-~1 samples. The radioactivity in each supernatant and pellet was used to calculate the accumulation ratio (Ar). In a separate experiment a solution containing 150 mM

CHRISTEN,

SCHACKMANN,

AND

SHAPIRO

Hepes, 100 mM EGTA, 190 mM NaCl, pH 9, was used as the chelating solution and a solution containing 150 mM Hepes, 400 mM NaCl at pH 8 was used as a control instead of ASW. No substantial differences were observed from the data presented using ASW as a control as described above and in Fig. 4. Membrane potentiaL Sperm were diluted lOO- to 500fold in the presence of rH]TPP (12-120 nM, 0.05-0.5 &i/ml) or S4CN- (72 PM, 0.8 &X/ml) to measure membrane potentials; measurements of uptake were done after centrifugation through silicone oil (Christen et aL, 1979,198O) and the potential was calculated as described previously (Schackmann et aZ., 1981) using the total intracellular water space. Respiration rates. Respiration rates were determined by continuous recording with a Clark-type oxygen electrode fitted to a 5-ml chamber and refrigerated to 10°C. Dry sperm (lo-25 ~1) were diluted and stirred in a final volume of 5 ml. Respiratory rates were unchanged until about 90% of the oxygen was depleted from the medium and measurements were made only up to that point. For extended experiments, the medium was reoxygenated by bubbling air for a short period of time. A 100% value in the figures is for the medium in equilibrium with air at 10°C. Spewn motility. Qualitative estimates of motility were obtained by microscopic observation of a thick droplet of sperm by dark-field microscopy. Only sperm in liquid suspension were examined to avoid artifacts due to interactions with the surface of the glass. ATP concentration, Dry sperm were diluted lOOO-fold into each medium and assayed for ATP by the luciferinluciferase technique. Purified luciferin-luciferase mixture, extraction medium, and buffer were obtained from Calbiochem or Analytical Luminescence Laboratory (San Diego). Five microliters of the sperm suspension was lysed with 200 ~1 of boiling extraction medium; then 200 ~1 Hepes buffer and 50 ~1 of luciferin-luciferase were added and the samples were counted in a scintillation counter. By maintaining the size of the sample at l/lOOth of the volume of the assay, interference from other ions present in the ASW was negligible. Standards for ATP concentrations were run with ASW. Sperm preincubated with oligomycin gave negligible background luminescence. The internal ATP concentration was calculated by assuming that the internal volume equals 50% of the volume of dry sperm; this volume represents an upper limit to the exchangeable internal water volume as measured with tritiated water and [‘4C]sucrose (Schackmann et aZ., 1981). Since most of the sperm volume is occupied by the nucleus and the mitochondrion, it is very difficult to estimate the absolute volume in which the cytoplasmic ATP might be distributed. Reasonable ATP concentrations were found

Jelly Effect on Sea Urchin Spemn

3

with these assumptions, and these numbers were used only for comparison within a single batch of sperm. Solutions. Normal artificial seawater (ASW) had the following composition: NaCl, 360 mM; KCl, 10 mM; CaClz, 10 mM; MgCl,, 50 mM; Hepes, 30 mM; pH 8.0; 20 mM Tris was also included in some experiments. Variations in this composition were obtained at constant osmotic strength by replacing one ion by another. Increase or decrease of any cation except Na+ was by exchange for Na+. For example, a solution with 200 mMKf contained only 170 mM Naf; all other cation concentrations remained constant. Removal of Na+ was done by substitution with choline chloride. Three times recrystallized choline (Sigma) was used, and fresh solutions were prepared regularly, since choline might decompose with time. All experiments were performed at lo-12°C; the pH was adjusted with HCl or NaOH (Trizma base in the case of the Na+-free solutions) at this temperature. We chose to use sodium as the complementary ion in these studies, because over the range 100 to 400 mM no variations in sperm physiology or behavior were detected (see Christen et al, 1980, 1981, 1982, and manuscript in preparation). Most chemicals used were the best available grade from Sigma or Calbiochem. Rhodamine-123 and acridine orange were from Eastman Kodak. FCCP (0.5 mM) and oligomycin (1 mM) in DMSO were used as stock solutions. Final concentrations of TEA of 10 mM and verapamil of 40 pg/ml were used unless indicated differently in the text. Because of the rapid effects of egg jelly on sperm, it is not possible to wash out the calcium ions from the external medium; for this reason we used EGTA to chelate most of the Ca’+. Ca2+ (10 mM) and Mg2+ (50 mM) are the major divalent cations in the seawater, and their dissociation constants with EGTA are 10.9 and 5.4, respectively. With 20 mM EGTA present in the medium, we calculated that the free concentrations of these ions were about 0.1 PM for calcium and 40 mM for magnesium. Triggering of the acrosome reaction. Egg jelly was prepared as previously described (Schackmann et al, 1978; Schackmann and Shapiro 1981). With sperm diluted into ASW, and unless mentioned otherwise in the text, the addition of 50 ~1 of the jelly solution to 1 ml of sperm suspension (final 4 pg fucose eq/ml) resulted in 95-100% of fully extended acrosomal processes as scored by phase microscopy. Triggering of the acrosome reaction by a low concentration of Na+ in the presence of Ca2+ (Fig. 5) resulted in about 50% fully extended acrosomal processes; the rest of the sperm appeared to have undergone partial reactions, such as the disappearance of the acrosomal granule without a visible acrosomal process. For phase contrast microscopy, sperm were fixed with 1% glutaraldehyde in seawater and photographed with a C. Zeiss (Oberkochen) microscope equipped with an

4

DEVELOPMENTAL

BIOLOGY

Olympus OM2 using Kodak 2415 emulsion film. For fluorescence, live sperm were photographed with a Leitz epifluorescence microscope using an acridine orange filter (G). Color slides were taken with a 400 ASA Ektachrome pushed to 800 (l&sec exposure). Prints were obtained via an intermediate negative. Black and white prints were obtained directly with a Tri X emulsion (400 ASA) using 15-set exposures for the fluorescence. RESULTS

(I) Changes in the Internal

pH

We have estimated the internal pH (pHJ by the accumulation of radioactive amines after centrifugation of the cells through silicone oil (Christen et aL, 19’79, 1980; Schackmann et al, 1981). A decrease in pHi is indicated by an increase in the accumulation ratio (Ar) of the amine. After dilution of sperm into seawater, triggering of the acrosome reaction by egg jelly induced changes in the sperm internal pH. The immediate result of the addition of jelly was an internal alkalinization (Christen et aL, 1980; Schackmann et al., 1981). However, this alkalinization was followed after 10 to 15 min by a substantial reacidification (Fig. lA, curve 1). We analyzed the biphasic alteration in pHi that followed the acrosome reaction by using various inhibitors of the acrosome reaction, and by changing the ionic composition of the external medium. Verapamil, a drug which blocks calcium movements in other systems (Kohlhardt et al, 1972), and TEA, an inhibitor of potassium channels (Hille, 1967), inhibited the triggering of the acrosome reaction by jelly (Schackmann et a& 1978). When sperm were incubated with either of these inhibitors prior to jelly addition, internal alkalinization still occurred even though no acrosome reaction was triggered; however, the reacidification was strongly inhibited (Fig. 1, curves 2 and 3). Removal of calcium or sodium from the dilution medium are also conditions that inhibit the acrosome reaction (Dan, 1954; Schackmann et al, 1978). Figure 1B shows that in the absence of calcium an internal alkalinization still occurred after jelly addition, but that the subsequent reacidification was totally inhibited. In the presence of calcium but with sodium replaced by choline, the sperm internal pH is much more acidic (Nishioka and Cross, 1978; Lee et al, 1980; Christen et al, 1981, 1982). Addition of jelly also induced an internal alkalinization but no reacidification took place (data not shown). Since an alkalinization was obtained in the absence of either Ca2+ or Na+, it might reflect the efflux of protons in response to either ion. To test this, sperm were washed in media lacking both Ca2+ and Na+, then resuspended in the same solution, and jelly was added. Under those conditions an internal alkalinization still

VOLUME

98, 1983

took place (the Ar decreased from 17.2 to 15.2) with no subsequent reacidification. The acrosome reaction is also inhibited in media of increased K+ concentration (Schackmann et al+, 1978). Although inhibitionbf the acrosome reaction occurs with a potassium concentration as low as 20 mM, at this concentration the sperm internal pH changes with time (Christen et aZ., 1982) so that the effects of jelly addition are difficult to analyze. In order to obtain a constant pHi, we incubated sperm with 150 mM potassium. Figure 1C shows that under these conditions an internal alkalinization took place after jelly addition (the Ar decreased from 10 to 8) but no subsequent reacidification occurred. A control of the internal pH by Cll/HCO, exchange has been demonstrated in other cell types (Russel and Boron, 1976; Thomas, 1977; Boron, 1978; Roos and Boron, 1981), and such a mechanism could be responsible for the alkalinization triggered by jelly. To test this hypothesis, we used SITS, an inhibitor of this Cl-/HCO, exchange mechanism (Knauf and Rothstein, 1971). Incubation with SITS (1 mM) for 5 min prior to jelly addition did not inhibit the acrosome reaction, nor did it alter the pH change that occurred upon addition of jelly (data not shown). The internal alkalinization which occurred after the triggering of the acrosome reaction could be due both to an alkalinization of the cytoplasm and to the exocytosis of an acidic acrosomal vesicle. The fluorescent amine 9-aminoacridine was used to demonstrate that acrosomal vesicles have an acidic pH (Meizel and Deamer, 1978). Acridine orange is an amine which accumulates according to pH gradients, and exhibits changes in its fluorescence spectrum when accumulated in very acidic compartments (Robbins and Marcus, 1963; Robbins et ab, 1961). Sperm incubated with acridine orange have a red fluorescence in the acrosomal region, an indication that the acrosome might be very acidic (Fig. 2A). Addition of a high concentration of NH&l, or of monensin (which catalyzes Na+/H+ exchange), resulted in total loss of the fluorescence from the vesicle, as well as an overall decrease in the green fluorescence of the sperm body (data not shown). When sperm were incubated in a Ca2+-free solution with acridine orange, and then jelly was added, the acrosomal vesicle remained fluorescent, showing that it had not partially fused with the plasma membrane and that it had retained its ability to maintain a pH gradient. When jelly was added in the presence of TEA a similar result was obtained (Fig. 3), whereas in the absence of the inhibitor the acrosomal vesicle fluorescence disappeared (Fig. 3B). This approach, as shown in Fig. 2B or C, with guinea pig and mouse sperm displaying the characteristic red color over the acrosomal region, indicates that acridine

CHRISTEN,

SCHACKMANN,

AND

Jelly

SHAPIRO

Effect

on Sea

Urchin

5

Sperm

B 20

40

60

60

20

100

min.

40

60

min.

6 a'6

;

lb

is

20

is

min. FIG. 1. Changes in the internal pH of sperm after jelly addition. Dry sperm were diluted into seawaters of the indicated composition. Radioactive EtzNH was added and the reaction mixture was divided into different tubes for incubation as follows: In A sperm were diluted in ASW pH 8.0 (A) and jelly was added after 15 min (arrow 1, V); 99% of the sperm had undergone the acrosome reaction when scored 2 min after jelly addition. To another tube 40 pg/ml verapamil was added at 20 min and jelly was added 10 min later (arrow 2, 0). To a third sample 10 mJ4 TEA was added at 40 min and jelly was added 10 min later (arrow 3, n). No sperm underwent the acrosome reaction with ASW (with 1 mM EGTA) and the amine accumulation was measured either verapamil or TEA. In B, dry sperm were diluted in Ca ‘+-free (A); addition of jelly (arrow) at 15 min induced an alkalinization, as shown by the decrease in Ar (0). In C dry sperm were diluted in ASW containing 150 m&f K+ (A). Addition of jelly (arrow) at 10 min also induced an internal alkalinization (0). No sperm reacted in either B or C.

orange might be useful for detecting the acrosome reaction of mammalian sperm. This staining of the acrosome is especially useful with mouse sperm, in which the acrosomal vesicle is not easily detected by direct light microscopy. The reacidification that follows the triggering of the acrosome reaction induced by addition of jelly might be related to the large calcium uptake that ensues (Schackmann et al, 19’78) if it reflected a Ca2+/Hf exchange

from the mitochondria (Vercesi et aL, 19’78; Kauffman and Lardy, 1980; Lee et al, 1980). To test this hypothesis, we triggered the acrosome reaction by jelly in seawater and after its completion (45 set after jelly addition) we added EGTA to complex most of the calcium present in the external medium (Fig. 4). The stock solution of EGTA was well buffered so that the final pH of the dilution medium after chelation of Ca2+ was the same as before EGTA addition (see Materials and Methods).

6

DEVELOPMENTAL

BIOLOGY

Figure 4 shows that in the presence of calcium after jelly addition, the pHi increased for a short time and then internal reacidification took place. However, when Ca2+ was chelated after jelly addition, not only did the subsequent reacidification not occur, but the alkalinization was more pronounced. This experiment shows that the reacidification requires calcium ions and that it is superimposed upon the alkalinization, so that in seawater the amplitude of the alkalinization is decreased.

(2) Changes in Mewzbrane Potential Changes in sperm membrane potential after addition of jelly were estimated by using lipophilic anions and cations. These molecules partition through membranes according to the membrane potential and allow one to calculate the potential according to the Nernst equation (Grinius et al+, 1970; Lichtshtein et al, 1979; Rottenberg, 1979). Both a lipophilic cation, TPP+, as well as an anion, SCN-, were used to estimate the membrane potential of sperm. Lipophilic cations accumulate in large quantities within the mitochondrion, which has a very negative membrane potential, as illustrated by the distribution of the fluorescent cation, rhodamine(Fig. 2D). On the other hand an anion will be almost completely excluded from the mitochondrion, its distribution providing a more accurate estimate of the plasma membrane potential (see Discussion). When jelly was added to sperm, it triggered a large depolarization of the membrane potential (Schackmann et aZ., 1981) as measured with both the anionic and cationic probes, but when jelly was added to sperm in the presence of verapamil, TEA, Ca2+-free ASW, or at pH 6.9, only small depolarizations were found (Table 1). A small decrease in TPP+ uptake occurred in Na+-Ca2+-free ASW upon addition of jelly (Table 1).

(3) Sperm Respiration Figure 5e shows that triggering the acrosome reaction with jelly resulted in a rapid decrease in sperm respiration when compared to sperm diluted in ASW without jelly (Fig. 5d) (see also Kinsey et al, 1979; Hino et aL, 1980; Fijiwara et al, 1980). The acrosome reaction

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is also triggered by low concentrations of Na+ but only in the presence of Ca2’ (Schackmann and Shapiro, 1981). Figure 5a shows that when sperm were diluted into a medium lacking Na+ and Ca2+, respiration was almost totally inhibited, due to the acidic internal pH found under these conditions. Addition of Na+ to the external medium increased the internal pH (Lee et al, 1982; Christen et al, 1982) and triggered sustained respiration, as shown in Fig. 5b, and the acrosome reaction was not triggered (Schackmann et al, 1981). If, however, calcium was present in the external medium at the time of Na+ addition (Fig. 5c), the acrosome reaction was triggered and respiration increased transiently and then ceased. Since sperm respiration and motility are highly dependent upon the pHi, and since the pHi becomes acidic soon after induction of the acrosome reaction (Fig. lA), the decreased pHi might have led to the decreased respiration that followed the acrosome reaction. However, by adding FCCP (0.75 PM) or NH&l (20 mM) or both we were unable to restore respiration after triggering of the acrosome reaction by jelly or low Na+ (data not shown). As shown in Table 2, when the acrosome reaction was inhibited by the addition of verapamil or TEA, the addition of jelly did not induce a large decrease in respiration. Elevated concentrations of K+ in the dilution medium inhibited the acrosome reaction, but these conditions also delayed the increase in respiration which follows the dilution of sperm (Christen et cd, 1981,1982). In the presence of jelly, respiration was delayed even longer when sperm were in either 45 mM or 105 m&l K+ but the rate increased with time instead of decreasing, as was the case in ASW with jelly (Table 2). When jelly was added to sperm in the absence of Ca2+ or after chelation of external calcium by EGTA, there was again no decrease in respiration (Figs. 6e and 7b). Thus, whenever jelly was added to sperm under conditions that are inhibitory for the acrosome reaction, there was no large deleterious effect on respiration. Addition of verapamil, TEA, or K+ after triggering of the acrosome reaction did not prevent the decrease in sperm respiration (data not shown). The addition of oligomycin to sperm diluted in ASW resulted in a 90-95% decrease of respiration and the

FIG. 2. Accumulation of fluorescent compounds by sperm. Sperm were diluted into ASW containing 1 rg/ml acridine orange (A-C) or 1 @g/ml rhodamine 123 (D) incubated for 15 to 30 min and photographed with an epifluorescence microscope. (A) Fluorescent image of sea urchin sperm incubated with acridine orange, showing that the fluorescence is concentrated over the sperm body and excluded from the mitochondrial region. The red fluorescence over the acrosomal region suggests this area to be very acidic. (B) Fluorescent picture of guinea pig sperm incubated with 1 pg/ml acridine orange in Gibco BMOC-3 medium with 12 mM Hepes at pH 7.4. The acrosomal region is a red crescent. Sperm which have lost their acrosomal vesicle (as observed with phase contrast) have also lost their red fluorescence. (C) Mouse sperm under the same conditions as in B. (D) Composite image of fluorescence and phase contrast showing the morphology of the sea urchin sperm and the fact that rhodamine-123, a lipophilic cation, is localized in the mitochondria. Sperm photographed were adhering to the slide and were therefore immotile. No significant difference in the fluorescence staining pattern was observed between those in solution and those attached to the slide.

CI-IRISTEN,

SCHACKMANN,

AND

SHAPIRO

full respiratory rate was recovered after addition of FCCP (Fig. 6b). These results indicate that before jelly addition mitochondria are tightly coupled. Addition of oligomycin to sperm after the acrosome reaction was induced with jelly resulted in only a partial inhibition of respiration (Fig. 6d) showing that the mitochondria had become uncoupled. Since sperm respiration was decreased after jelly addition, the respiration rate in the presence of oligomycin should be compared to the one

Jelly

Effect

on Sea

Urchin

7

Sperm

7.2 7.3

.I P

7.5 7.6

7.6

10

.

.

,

20

30

.

. 40

min. FIG. 4. Influence of Ca*+ on the sperm reacidification after triggering of the acrosomal reaction. Dry sperm were diluted into ASW at pH0 8.0 (with 1 mM EGTA), [‘“C]Et2NH was added, and the mixture was divided into three tubes. To tube 1 (V) jelly was added (large arrow), and 45 set later EGTA was added to complex the extracellular Ca2+ (final EGTA = 16.4 mM). To tube 2 (A) jelly was added and 45 set later ASW was added to the suspension as a control for the EGTA addition. To tube 3 seawater was added equivalent in volume to the jelly and EGTA additions above (0). Each tube was sampled for amine accumulation and the pHi was calculated as described under Materials and Methods. In tubes 1 and 2 sperm were 85-90s acrosome reacted. No sperm reacted in tube 3. Special care was taken so that EGTA addition did not change the pH of the solutions (see Materials and Methods).

FIG. 3. Influence of jelly on acridine orange fluorescence. Sea urchin sperm were incubated 30 min in ASW with 1 pgg/ml acridine orange and treated as follows: (A) Sperm are photographed prior to jelly addition; acrosomal vesicles are bright dots at the apical tip of the head. (B) Sperm were photographed 5 min after jelly addition; the vesicles have exocytosed and are no longer visible. (C) Sperm were incubated for 5 min with 10 mM TEA, jelly was added, and after an additional 5-min incubation, photographs were taken.

obtained at the same time after jelly addition in the absence of oligomyein (Fig. 6a). Addition of FCCP to sperm in the presence of jelly and oligomycin (Fig. 6d, thick arrow) increased respiration to the same level obtained in the absence of oligomycin (compare with Fig. 6a). If jelly was added in the presence of an inhibitor (TEA, or after Ca2+ removal: Figs. 6c and e), uncoupling of respiration was not observed. In both cases, the inhibition of respiration by oligomycin was overcome by FCCP (Figs. 6c and e, thick arrows). The massive calcium uptake observed after induction of the acrosome reaction might be involved in uncoupling the mitochondria as found with isolated mitochondria (Vercesi et al., 1978). In order to probe for the role of calcium uptake in respiration, EGTA was added to complex the external Ca2+ either before or after the acrosome reaction was induced by jelly (Fig. 7). When EGTA was added before jelly, the acrosome reaction was inhibited and the mitochondria remained tightly coupled (Fig. 7b). If EGTA was added after triggering (Fig. 7c), respiration could still be inhibited by oligomycin, although the inhibition was not as substantial as that seen when the acrosome reaction had not been induced. For example, the ratio (respiratory rate 10 min

8

DEVELOPMENTAL

JELLY-INDUCED

CHANGES

TABLE IN rH]TPP

1 AND S4CN-

ACCUMULATION

TPP+ -Jelly Conditions ASW, pH 8.0 ASW + 40 rg/ml verapamil ASW + 5 mM TEA Na+, Caa+-free ASW, pH 8.0 Ca’+-free ASW ASW, pH 6.9

BIOLOGY

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98, 1983

with EGTA 45 set after jelly addition, a smaller protective effect was observed (Table 3, experiment 2).

SCN+Jelly

-Jelly

+Jelly

[Arl

[Ar (&)I

[Arl

[Ar (A+)1

128 136 135 611 224 196

38 111 100 542 164 145

0.25 0.15 0.30

0.80 (+31) 0.14 (-2) 0.34 (+3)

(+30) (+5) (+7) (+3) (+8) (+6)

Note. Dry sperm were diluted into the different media containing either [‘HFPP’ or S”CN-, as detailed under Materials and Methods. For TPP+ measurements, two different hatches of sperm (10’ and 5.5 X 10s sperm/ml) were incubated for 4.5 and 6 hr, respectively, before egg jelly (50 PI/ml, 4 pg fucose eq/ml reaction mixture) or distilled water (50 pi/ml) was added. These long incubations are required for an apparent equilibrium to be reached (Schackmann et d, 1981). Efflux of TPP was measured at 10 min after jelly addition. Verapamil and TEA were added 5 min before addition of egg jelly and did not significantly alter uptake levels in control cells. Under these conditions 74% of the sperm underwent the acrosome reaction when exposed to jelly in the absence of inhibitors; less than 6% of the cells reacted under inhibitory conditions. For S”CNuptake sperm were incubated for 2 hr before jelly was added. The jelly-induced changes were complete within 10 min for both TPP+ and SCN-. The data are given as accumulation ratios, as defined under Materials and Methods. The change in membrane potential (AG, mV) upon jelly addition is indicated within parentheses. Accumulation ratios and membrane potentials were calculated as described under Materials and Methods.

(5) ATP Concentration Addition of jelly to sperm diluted in ASW induced an immediate decrease in the ATP concentration, a result which was probably related to the mitochondrial uncoupling described above. Ten minutes after jelly addition the ATP decreased from 5 to 2 mM when jelly was added in ASW. In the presence of TEA, or in the absence of calcium, a small decrease in ATP concentration (4.5 to 3.5 mM or 5.5 to 4.5 mM, respectively) was still observed, probably because of the effect of increased pHi upon the rate of the internal ATPase of sperm (Christen et al., 1982). When the ATP concentrations were measured under the same conditions where viability was measured (Table 3) ATP levels remained high under all inhibitory conditions although viability was highly variable; for example, 10 min after jelly addition, ATP concentrations measured in sperm were 2 mM after dilution in ASW pHo 8.0,3.5 mM after dilution in ASW at pHo 7.0, and 3.5 mM with 50 mM K+ at pHo 7.0. At the same time, the percentages of fertilization obtained with those sperm solutions were 0% for sperm diluted in ASW pHo 8.0, 5% for sperm diluted in ASW at pH 7.0, and 70% for sperm in 50 mM at pH 7.0 (control sperm in ASW without jelly was 75%). Thus, there was no direct correlation between ATP levels and sperm viability after jelly addition.

after oligomycin addition)/(respiratory rate without oligomycin) was 0.04 in the absence of jelly. In the presence of jelly, it was 0.04 when jelly was added after chelation of the calcium by EGTA, 0.16 when EGTA was added after jelly, and 0.67 when no EGTA was added (see Fig. 7).

(4) Sperm Viability Sperm viability, the ability of sperm to raise the fertilization membrane in sea urchin eggs, was reduced after triggering of the acrosomal reaction (Hagstrom, 1959; Kinsey et al, 1979; Vacquier, 1979b Talbot et al, 1976). Some conditions inhibitory for the acrosomal reaction had a partial protective effect on sperm viability (Table 3). Elevated K+ and H+ were able to increase sperm viability, but at concentrations higher than those required to inhibit the acrosome reaction, and they acted synergistically to protect sperm. Verapamil did not increase sperm viability after jelly addition but was also toxic to sperm in the absence of jelly. One of the most potent conditions that preserved sperm viability in the presence of jelly was removal of the external Ca” before jelly addition. When the external Ca2+ was chelated

d ‘b FIG. 5. Effect of triggering the acrosomal reaction on sperm respiration, 10 ~1 dry sperm were diluted in 5 ml of the following media: (a) Na+, Cal+-free ASW (with 1 mM EGTA); (b) as in (a) but 20 mM Na+ was added at the arrow; (c) as in (a) hut 20 mMNa+ and 12 mM Ca*+ was added at the arrow; (d) ASW, (e) ASW with jelly. Respiration was followed as described under Materials and Methods.

CHRISTEN,

SCHACKMANN,

AND

TABLE 2 IN RESPIRATORY RATES AFTER JELLY ADDITION TO SPERM IN DIFFERENT MEDIA

CHANGES

Conditions Medium

-

Respiratory rate (pmole Oa/min)

Jelly

10 min

25 min

-

17.5 9.5 16.9 15.5

12.8 0.7 8.1 9.3

ASW ASW ASW ASW

+ verapamil + TEA

+ + +

ASW, ASW,

45 mM 45 mM

+

10.6 2.5

13.8 11.4

ASW, ASW,

105 mh4 K+ 105 mM K’

+

1.1 0.6

5.4 4.5

K+ K+

-

Note. 25 ~1 dry sperm were diluted into 5 ml of the indicated medium and egg jelly was added 5 min later. Respiration was measured continuously as described under Materials and Methods. The respiratory rates presented were those obtained 10 and 25 min after sperm dilution (i.e., 5 and 20 min after jelly addition). (6) Mitochondrial

Jelly Efect

SHAPIRO

Movem~ents

Movements or changes in the shape of sperm mitochondria after encountering egg jelly have been dem-

9

onstrated in starfish (Schroeder and Christen, 1982) and ascidians (Lambert and Epel, 1979). In the sea urchin these changes were far less dramatic, but still occurred (Fig. 8). Before jelly addition the sperm mitochondrion was an integral part of the sperm head and could hardly be distinguished by phase microscopy (Fig. 8a); after jelly addition it became a round sphere detached from the nucleus and appeared darker by phase contrast (Fig. 8~). When jelly was added in the presence of TEA, 30 mM K’, verapamil, or low pHO, the mitochondria did not round up, but rather moved to the side of the nucleus (Fig. 8d). This sliding phenomenon showed a large heterogeneity in its occurrence from one sperm to another as well as from one batch of sperm to another. In the absence of calcium, jelly addition triggered no visible mitochondrial movement (Fig. 8b). When jelly was added to sperm in seawater lacking inhibitors, the small percentage of sperm that did not undergo the acrosomal reaction had mitochondria that were displaced, showing the sliding behavior typical of sperm that had encountered jelly in the presence of inhibitors (i.e., as in Fig. 8d). DISCUSSION

Since the discovery of a Ca2+ requirement for induction of the acrosome reaction by egg jelly (Dan, 1954), it has become clear that triggering of the reaction is affected by changes in the composition of the seawater. An analysis of the ionic fluxes after interaction with a

FIG. 6. Inhibition of sperm respiration by oligomycin and the influence of jelly addition. Dry sperm (25 ~1) were diluted into 5 ml of the following media: (a) ASW with jelly; (b) ASW; (c) ASW with 10 mM TEA (preincubation 5 min) followed by jelly; (d) ASW with jelly; (e) Ca’+-free ASW (1 mMEGTA) with jelly. Thin arrows indicate the time of oligomycin (1 pM final) addition and thick arrows designate addition of 0.75 & FCCP. Respiration was measured as described under Materials and Methods.

on Sea Urchin Sperm

b

C

d

FIG. 7. Dependence of mitochondrial uncoupling of the external Ca2+ after triggering of the acrosomal reaction. Dry sperm (10 ~1) were diluted into 5 ml of different media: (a) ASW; (b) ASW + 20 mM EGTA + jelly; (c) ASW + jelly for 30 set, then EGTA was added at a final concentration of 20 mM, (d) ASW + jelly. Oligomycin was added as indicated by the arrows. Respiration in the absence of oligomycin is shown by dashed lines; respiration with oligomycin is shown by the solid lines.

10

DEVELOPMENTAL

EFFECTS

OF K+ AND p&

TABLE 3 ON SPERM VIABILITY

OF THE ACROSOME

Conditions

f

Expt 1 Klo pH 8.0 7.5 6.5 K, pH 8.0 7.5 KS0 pH 8.0 Expt 2 (pH 8.0) ASW ASW ASW + verapamil ASW t TEA Caa+-free Car+ removal after jelly addition

jelly

BIOLOGY

AND INHIBITION

REACTION

Sperm viability (W FME)

% Acrosome reaction

+ + + + + +

0 36 73 36 50 92

96 50 0 4 0 0

+ + t t

99 5 0 21 85

0 95 0 0 0

t

36

95

Note. Sperm were diluted into the indicated medium; then jelly was added. The ability of sperm to fertilize eggs was measured as follows: 10 min after jelly addition to the various sperm suspensions a small aliquot of each sperm suspension was mixed with a suspension of eggs in seawater and the number of fertilization membranes was scored (% FME). A small aliquot from each tube was also taken at 10 min to measure the percentage of acrosome reactions as described under Materials and Methods. All conditions gave 90-95% fertilization when sperm were mixed with eggs immediately after jelly addition.

egg surface material unveiled a sequence of steps in the acrosome reaction triggering pathway (Schackmann and Shapiro, 1981). In this paper, we dissect further the sequence of events that follows the interaction with egg jelly. Since the jelly we use is a mixture of different components (SeGall and Lennarz, 1979,198l) these multiple effects could reflect interaction with several components, all of which are part of the cascade of events that take place when sperm meet eggs. In the absence of calcium, jelly can be added to sperm, then removed, and subsequently readded in the presence of Ca2+ to induce the acrosome reaction. Of the multiple inhibitory conditions for the acrosome reaction so far investigated, Ca2+ omission was the only one that could be reversed by such a sequential addition (Schackmann and Shapiro, 1981). We show that even in the absence of Ca2+, addition of jelly induces an internal alkalinization (Fig. lb). Thus, calcium does not seem necessary for all interactions between sperm and jelly, and the pHi change might be one of the initial events occurring after such interaction. We investigated several mechanisms which might explain the increased pHi following jelly addition. The internal alkalinization that occurs with induction of the acrosome reaction cannot be entirely explained by exocytosis of the acrosomal vesicle, since the pHi increase

VOLUME

98, 1983

occurs even in the presence of inhibitors, conditions under which the acrosomal vesicle does not exocytose and still accumulates fluorescent amines (Fig. 3). Since removal of Na+ and Ca2+ does not prevent the alkaline shift, this shift is probably not due to a Na+:H+ or Ca2+:H+ exchange. However, in high potassium or in the absence of sodium, the internal pH is much more acidic than in ASW, and it is possible that the alkalinization seen after addition of jelly might be a different process than the one which occurs during the normal acrosome reaction. The possibility of a Cl-:HCO; exchange also exists; SITS, an inhibitor of the chloride/ bicarbonate exchange in the red blood cell, does not inhibit the acrosome reaction or the alkalinization when added shortly before jelly. However, when sperm were preincubated with SITS (1 hr, 1 mlM) induction of the acrosome reaction by jelly (but not by nigericin) was inhibited (data not shown). Since SITS is a broad-range labeling reagent for membrane proteins (Maddy, 1964), it is yet unclear whether the inhibition is due to a specific inhibition of the chloride/bicarbonate exchange or to a nonspecific effect. Another possibility was that the pHi might in part be a result of the passive permeability of protons through the membrane, in response to the membrane potential. The membrane potential of sea urchin sperm is depolarized after the acrosome reaction, and if protons equilibrated passively in response to this depolarization, the pHi would shift to a more basic value. Any alteration of the membrane potential after addition of jelly in the presence of acrosome reaction inhibitors might thereby affect the pHi. To examine this point, we measured the membrane potential alterations with lipophilic cations and anions. Lipophilic cations accumulate in negatively charged compartments, and thus not only distribute within the cytoplasm, but concentrate in the mitochondrion, as shown by Fig. 2d. Since the mitochondrion occupies a substantial fraction of the internal volume of the sperm (about 30%), the accumulation of lipophilic cations is a complex function of both the mitochondrial and plasma membrane potentials. In order to examine the contribution of the plasma membrane potential, we used the lipophilic anion SCN-, which is partially excluded from the cytoplasm and totally excluded from the mitochondria; its accumulation ratio will thus reflect only changes of the plasma membrane potential. Addition of jelly to trigger the acrosome reaction induces a large depolarization of the plasma membrane (see Schackmann et al, 1981). In the presence of TEA, verapamil, Ca2+-free ASW, or at low pH, a decrease in the accumulation of TPP+ suggests that jelly induces a slight depolarization of the sperm. These changes, if they reflect depolarization of the plasma membrane potential, are almost large enough

CHRISTEN,

SCHACKMANN,

AND

SHAPIRO

Jelly Efect

on Sea Urchin Sperm

11

to account for the changes in the accumulation of EbNH under the inhibitory conditions. We have attempted to confirm that these depolarizations do not represent changes in the mitochondrial membrane potential by using SCN- uptake, but the changes in the accumulation of this probe under conditions in which the reaction is prevented are too small to be considered significant. Thus, this mechanism of generating a change in the internal pH remains plausible, but with no substantial support. The primary alkalinization induced by jelly is followed after 10 min by a marked reacidification (Christen et al., 1981) but only when the acrosome reaction is triggered. The depolarization of the plasma membrane might be involved in the process. Indeed following jelly addition, this strong reacidification occurs only when the membrane is depolarized (Table 1) and in the absence of jelly, depolarization of the plasma membrane with high concentrations of external potassium also leads to an internal acidification (Christen et al, 1982). Since the membrane is depolarized and yet the pHi decreases, protons cannot be in equilibrium with the membrane potential. However, a depolarization of the membrane potential might inactivate the proton efflux mechanism, so that protons produced by metabolism would accumulate and acidify the cytoplasm (see also below). The encounter of sperm with substances released by the egg triggers changes other than the acrosome reaction, including a decrease in sperm respiration (Figs. 5 and 6). Conditions which are inhibitory for the triggering of the acrosome reaction by jelly prevent this decrease in respiration (Table 2). However, since some inhibitors of the acrosome reaction also affect the respiratory rate in the absence of jelly (Table 2, see also Christen et al., 1982), the analysis is somewhat difficult. The use of oligomycin to block the mitochondrial ATPase clarified the situation, since it indicated that the mitochondria are tightly coupled before the addition of jelly and that they become uncoupled after triggering of the acrosome reaction (Fig. 6). When the acrosome reaction was prevented by any of several of the inhibitory conditions, respiration remained coupled despite jelly addition (Fig. 6). The uncoupling of mitochondria and, as proposed by Lee et al. (1982), the internal reacidification that follow the acrosomal reaction might be related to the large calcium uptake which occurs after FIG. 8. Phase contrast observations of the effect of jelly addition on sperm morphology. Sperm were incubated for 5 min in the following media: (a) ASW without jelly; (b) C a*+-free ASW (1 mMEGTA) + jelly; (c) ASW + jelly; (d) ASW + 10 mMTEA (5-min preincubation) + jelly. Arrows point to the location of the mitochondria. Before addition of jelly or in the absence of calcium, the mitochondrion is

tightly packed at the rear of the head, so that it cannot be clearly distinguished by phase contrast (compare to Fig. 2). After jelly addition in ASW, mitochondria round up and are clearly visible as a dark sphere (c); in the presence of TEA (and verapamil or low pHo as well), they slide to the side of the nucleus and become clearly visible(d).

12

DEVELOPMENTAL BIOLOGY

the acrosome reaction. This hypothesis is supported by the following observations: (1) The influx of Ca2+ is inhibited by cyanide azide and FCCP (Schackmann et aZ., 1978; Schackmann and Shapiro, 1981); (2) calcium is accumulated primarily within the mitochondria (Cantino, 1981); (3) when the acrosome reaction is inhibited by TEA, verapamil, or elevated K+, the calcium influx and the reacidification do not occur and the respiration remains coupled; (4) if calcium is chelated after the acrosome reaction is completed, respiration is inhibited by oligomycin (Fig. 7). In addition it has been shown that isolated mitochondria take up calcium in exchange for protons, in a process linked to the respiratory chain (Vercesi et al., 1978). Under these conditions respiration occurs independently of ATP synthesis so that mitochondria are uncoupled; moreover calcium uptake takes priority over ATP phosphorylation, so that no ATP is produced while calcium is accumulating. These considerations suggest to us that the Ca2+ uptake, cytoplasmic mitochondrial uncoupling, and dereacidification, creased ATP levels after the acrosome reaction are linked processes. The progressive decrease in respiration might also be due to a calcium poisoning of mitochondrial function, as previously described (Vercesi et al., 1978). Since we do not know the affinity of sperm mitochondria for calcium (as compared to EGTA), we cannot tell whether the partial uncoupling and poisoning that follow the addition of EGTA after jelly (Fig. 7~) are due to a residual calcium uptake or to some other mechanism. An analysis of calcium influx under the same conditions would help clarify this point. The mechanism by which sperm rapidly lose fertility (and are biologically dead) after jelly addition is not known (Kinsey et aL, 1979, Vacquier, 1979b). The reasons for sperm “death” might be different after the triggering of the reaction and in the presence of an inhibitor, since when the complete acrosome reaction is triggered, death could be secondary to the decrease in ATP levels, the internal pH change, the depolarization of the plasma membrane potential, or the release and inactivation of components exposed during exocytosis of the acrosomal vesicle. Calcium is involved in the inactivation phenomenon, for removal of calcium is one of the best conditions for protecting sperm, although a combination of low pH and high potassium is also very effective (Table 3). According to the hypothesis of Tilney et ah (1978), an internal alkalinization should lead to the polymerization of actin; it is thus possible that jelly addition in the presence of inhibitors of the acrosome reaction induces the polymerization of actin located behind the intact acrosomal vesicle, leading to a semireacted sperm which would not be able to carry out normal fertilization. In sea urchin sperm, it is difficult to analyze the state of actin polymerization because of the small quantity of actin present in the

VOLUME 98, 1983

acrosome region, This question might be better answered with starfish sperm which have a much larger stock of unpolymerized actin and in which a specific fluorescent dye (NBD-phallacidin) can be used to probe for actin polymerization (Schroeder and Christen, 1982). The data in this paper expand our understanding of the multiple physiological alterations that attend the interaction between sperm and egg jelly. When sperm are diluted in the absence of sodium at acidic pHo, or with an elevated concentration of potassium, they have a more acidic internal pH and they do not swim or respire (Nishioka and Cross, 1978; Lee et d., 1980,1982; Christen et CLZ., 1980,1981,1982). This acidic internal pH is also inhibitory for the acrosome reaction. When the acrosome reaction is triggered by jelly, sperm enter a state characterized by a depolarization of the plasma membrane potential, an increased intracellular pH, and a loss of intracellular K+, all of which occur as a result of transient events (within seconds) (Shackmann et al., 1981; Schackmann and Shapiro, 1981). Inhibitory conditions, such as TEA, verapamil, or calcium removal have no effect by themselves on the pHi, moreover they do not greatly reduce the initial internal alkalinization triggered by jelly; they inhibit the acrosome reaction probably by affecting other necessary ionic movements. Their most prominent action is an inhibition of the membrane depolarization (Table l), suggesting that this depolarization might be one of the crucial steps leading to the acrosome reaction. This hypothesis is also supported by the observation that NH&l does not trigger the acrosome reaction when sperm are diluted in seawater, but NH&l does trigger the acrosome reaction when sperm are diluted in a medium containing a high concentration of potassium (Schackmann, manuscript in preparation). Thus both a membrane depolarization and an internal alkalinization appear to be necessary for the acrosome reaction. Lasting changes which follow the acrosome reaction include Ca2+ uptake into the mitochondrion, intracellular reacidification, mitochondrial uncoupling, and ATP depletion. We suggest that these are linked processes. Inhibition of respiration and the characteristic mitochondrial rounding up phenomenon (Fig. 8) may also be associated with the other changes in mitochondrial function. In conclusion, the encounter with the substances that cover the surface of the egg induces a complex response in the sea urchin sperm, and also drastically alters its metabolism. Following this interaction, sperm must fertilize eggs quickly, before their physiology becomes too deranged (Hagstrom, 1959a,b; see also Vacquier, 1979a; Vacquier et a& 1979; Dale and Monroy, 1981). This work was supported by National Institutes Grant GM 23910 and National Science Foundation to B. M. Shapiro, and by the Centre National de tifique, and DGRST to R. Christen. The authors

of Health Research Grant PCM ‘77204’72 la Recherche Scienwish to thank Dr.

CHRISTEN, SCHACKMANN, AND SHAPIRO David Shellenbarger for assistance with the mammalian spermatozoa, and Pam Horbett for careful typing of the manuscript. REFERENCES

Jelly

Eflect

ture

(London)

257, 677-678.

GARBERS, D. L., TUBB, J. I)., and KOPF, G. S. (1980). Regulation of sea urchin sperm cyclic AMP-dependent protein kinases by an egg associated factor. BioL Reprod. 22, 526-532. GARBERS, D. L., WATKINS, H. D., HANSBROUGH, J. R., SMITH, A., and MISONO, K. S., (1982). The amino acid sequence and chemical synthesis of speract and of speract analogues. J. Biol. Chem. 257,27342737.

13

GLABE, C. G., and LENNARZ, W. J. (1978). Species specific sperm adhesion in sea urchins-A quantitative investigation of bindin-mediated egg agglutination. J. CeU BioL 83, 595-604. GRAY, J. (1928). The effect of dilution on the activity of spermatozoa. J. Exp.

AKETA, K., MIYAZAKI, S., YOSHIDA, M., and TSUZUKI, H. (1978). A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs. Biochem. Biophys. Res. Commun 80,91’7-922. BORON,W. F. (1978). Active control of intracellular pH. Resp. PhysioL 33, 59-62. CANTINO, M. E. (1981). The use of x-ray microanalysis in studies of the acrosome reaction in sea urchin sperm. In “Microprobe Analysis of Biological Systems” (T. E. Hutchinson and A. P. Somlyo, eds.). Academic Press, New York/San Francisco. CHRISTEN, R., and SARDET, C. (1980). Changes in permeability of sea urchin egg membrane to urea after fertilization or activation. J. PhysioL &on&& 305, l-11. CHRISTEN,R., SARDET, C., and LALLIER, R. (1979). Chloride permeability of sea urchin eggs. Cell BioL Int Rep. 3,121-128. CHRISTEN, R., SCHACKMANN, R. W., and SHAPIRO, B. M. (1980). Regulation of viability and the acrosome reaction of Strongylowntratus purpuratus sperm by pH and K+. J. Cell Bid 87,140a (Abstract). CHRISTEN, R., SCHACKMANN, R. W., and SHAPIRO, B. M. (1981). Coupling of sperm respiration, motility and intracellular pH (pHi) in Strongylocentratus purpuratus. J. Cell BioL 91, 174a. CHRISTEN,R., SCHACKMANN,R. W., and SHAPIRO,B. M. (1982). Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin. J. BioL Chum 257,14881-14890. COLLINS, F. (1976). A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion. Dev. BioL 49, 381-394. COLLINS, F., and EPEL, D. (1977). The role of calcium ions in the acrosome reaction of sea urchin sperm. Exp. Cell Res. 196.211-222. COLWIN, A. L., and COLWIN, L. H. (1960). Egg membrane lytic activity of sperm extracts and its significance in relation to sperm entry in Hydroides hexagonus (Annelida). J. Biophys. B&hem. CytoL 7,321328. DALE, B., and MONROY,A. (1981). How is polyspermy presented. Gamete Res. 4, 151-169. DAN, J. C. (1954). Studies on the acrosome. III. Effects of calcium deficiency. BioL Bull 107, 335-350. DAN, J. C., KAKIZAWA, Y., KUSHIDA, J., and FUJITA, K. (1972). Acrosomal triggers. Exp. Cell Res. 72, 60-68. DECKER, G. L., JOSEPH, D. B., and LENNARZ, W. J. (1976). A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arabacia punctulata. Dev. BioL 53, 115-125. EPEL, D. (1978). Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes. Curr. Zbp. Dev. BioL 12, 186246. FUJIWARA, A., HINO, A., and YASUMASU, I. (1980). Inhibition of respiration in sea urchin spermatozoa following interaction with fixed unfertilized eggs. III. Inhibition of sperm respiration by heat stable substance removed from glutaraldehyde fixed eggs. Dev. Growth D&f 22, 763-771. GARBERS, D. L. (1981). The elevation of cyclic AMP concentrations in flagellaless sea urchin sperm heads. J. Biol. Chem 256, 620-624. GARBERS, D. L., and HARDMAN, J. G. (1975). Factors released from sea urchin eggs affect cyclic nucleotide metabolism in sperm. No-

07~ Sea Urchin Sperm

BioL

5, 337-344.

GREEN, J. D., and SUMMERS, R. G. (1980). Ultrastructural demonstration of trypsin-like protease in acrosomes of sea urchin sperm. Science 209, 399-400. GRINIUS, L. L., JASAITIS, A. A., KADZIAUSKAS, Y. P., LIBERMANN, E. A., SKULACHEV, V. P., TOPALI, V. P., TSOFINA, L. N., and VLADIMIROVA, M. A. (1970). Conversion of biomembrane-produced energy into electric form. B&him. Biuphys. Acta 216, 1-12. HAGSTROM, B. E. (1959a). Further experiments on jelly free sea urchin eggs. Exp. Cell Res. 17, 256-261. HAGSTROM, B. E. (1959b). The influence of jelly coat solution on sea urchin spermatozoa. Exp. Cell Res. 16, 184-192. HAINO, K., and DAN, J. C. (1961). Some quantitative aspects of the acrosomal reaction to jelly substance in the sea urchin. Embryologia 5, 376-383. HANSBROUGH, J. R., and GARBERS, D. L. (1981). Sodium dependent activation of sea urchin spermatozoa by speract and monensin. J. BioL Chem 256, 22352241. HILLE, B. (1967). The selective inhibition of delayed potassium currents in nerve by tetraethylammonium ions. J. Gen PhysioL 50, 12871302. HINO, A., HIRUMA, T., FUJIWARA,A., and YASUMASU,I. (1980). Inhibition of respiration in sea urchin spermatozoa following interaction with fixed unfertilized eggs. Dew. Growth D$ 22. 813-820. HOSHI, M., and MORIYA, T. (1980). Arysulfatase of sea urchin sperm. 2. Arysulfatase as a lysin of sea urchins. Dev. BioL 74, 343-350. HOSHI, M., MORIYA, T., AOYAGI, T., UMEZAWA, H., MOHRI, H., and NAGAI, Y. (1979). Effects of hydrolase inhibitors on fertilization of sea urchins. I. protease inhibitors. Gamete Res. 2, 107-119. HOTTA, K., KUROKAWA, M., and ISAKA, S. (1973). A novel sialic acid and fucose containing disaccharide isolated from the jelly coat of the sea urchin eggs. J. BioL Chem. 248, 629-631. HYLANDER, B. L., and SUMMERS, R. G. (1975). Species specificity of acrosome reaction and primary gamete binding in echinoids. Exp. Cell Res. 96, 63-68.

ISAKA, S., HOWA, K., and KUROKAWA, M. (1970). Jelly coat substances of sea urchin eggs. I. Sperm isoagglutination and sialopolysaccharides in the jelly. Exp. Cell Res. 59, 37-42. JESSEN,H., BEHNKE, O., WINGSTRAND, K. G., and ROSTGAEND,J. (1973). Actin-like filaments in the acrosomal apparatus of spermatozoa of a sea urchin. Exp. Cell Res. 80, 47-54. KAUFFMAN, R. F., and LARDY, H. A. (1980). Biphasic uptake of Ca++ by rat liver mitochondria. J. BioL Chem. 255, 4228-4235. KINSEY, W. H., RUBIN, J. A., and LENNARZ, W. J. (1980). Studies on the specificity of sperm binding in echinoderm fertilization. Dev. Biol

74, 245-250.

KINSEY, W. H., SEGALL, G. K., and LENNARZ, W. J. (1979). The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm. Dev. BioL 71, 49-59. KNAUF, P. A., and ROTHSTEIN, A. (1971). Chemical modification of membranes. I. Effects of sulfhydryl and amino reactive agents on anion and cation permeability of the human red blood cell. J. Gen. PhysioL 58, 190-210. KOHLHARDT, M., BAUER, B., KRAUSE, H., and FLECKENSTEIN, A. (1972). Differentiation of the transmembrane Na and Ca channels in memmalian cardiac fibers by the use of specific inhibitors. Pjeuegers Arch

335,309-322.

KOPF, G. S., and GARBERS,D. L. (1979). A low molecular weight factor from sea urchin eggs elevates sperm cyclic nucleotide concentrations and respiration rates. J. Reprod FertiL 57, 353-361. KOPF, G. S., and GARBERS, D. L. (1980). Calcium and fucose-sulfate

14

DEVELOPMENTAL

BIOLOGY

rich polymer regulate sperm cyclic nucleotide metabolism and the acrosome reaction. Biol Reprod 22, 1118-1126. KOPF, G. S., TUBB, D. J., and GARBERS, D. L. (1979). Activation of sperm respiration by low molecular weight egg factor and by 8bromoguanosine 3’5’-monophosphate. J. BioL Cfiem 254,8554-8560. LAMBERT, C. C., and EPEL, D. (1979). Calcium mediated mitochondrial movement in ascidian sperm during fertilization. Dw. BioL 69,296304. LAMBERT, C. C., and LAMBERT, G. (1981). The ascidian sperm reaction: Cat+ uptake in relation to H+ efflux. Dev. BioL 88, 312-317. LEE, H. C., FORTE, J. G., and EPEL, D. (1982). In “Intracellular pH, Its measurement, Regulation and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 136-158. Alan R. Liss, New York. LEE, H. C., SCHULDINER, S., JOHNSON, C., and EPEL, D. (1980). Sperm motility initiation: Changes in intracellular pH, Ca” and membrane potential. J. Cell Biol 87, 39a. LICHTSHTEIN, D., KABACK, H. R., and BLUME, A. J. (1979). Use of a lipophilic cation for determination of membrane potential in neuroblastoma-glyoma hybrid cell suspensions. Proc. Nut. Acad Sci. USA 76, 650-654. LOEB, J. (1914). Cluster formation of spermatozoa caused by specific substances from eggs. J. Exp. 2001. 17, 123-240. LORENZI, M., and HEDRICK, J. L. (1973). On the macromolecular composition of the jelly coat from S. purpuratus eggs. Exp. Cell Res. 79,417-422. MADDY, A. J. (1964). A fluorescent label for the outer components of the plasma membrane. Biochim Biophys. Acta 88, 390-399. MEIZEL, S., and DEAMER, D. W. (1978). The pH of the hamster sperm acrosome. J. Histochem. Cytochem. 26, 98-105. MOHRI, H., and HORIUCHI, K. (1961). Studies on the respiration of sea urchin spermatozoa. III. Respiratory quotient. J. Exp. BioL 38,249257. MOHRI, H., and YASUMASU, I. (1963). Studies on the respiration of sea urchin spermatozoa. V. The effect of PCOz. J. Exp. BioL 40, 573586. NISHIOKA, D., and CROSS, N. (1978). The role of external sodium in sea urchin fertilization. In “Cell Reproduction” (E. R. Dirksen, D. Prescott, and C. F. Fox, eds.). Academic Press, New York. OHTAKE, H. (1976). Respiratory behavior of sea urchin spermatozoa. I. Effect of pH and egg water on the respiratory rate. J. Exp. ZOOL 198, 303-312. OHTAKE, H. (1976). Respiratory behavior of sea urchin spermatozoa. II. Sperm activating substance obtained from jelly coat of sea urchin eggs. J. Exp. ZooL 198, 313-322. ROBBINS, E., and MARCUS, P. I. (1963). Dynamics of acridine cell interactions-Interrelationships of acridine orange and cytoplasmic reddening. J. Cell BioL 18, 237-250.

orangeparticles

ROBBINS, E., MARCUS, P. I., and GONATAS, N. K. (1964). Dynamics of acridine orange-cell interaction. II. Dye-induced ultrastructural changes in multivesicular bodies (acridine orange particles). J. Cell BioL 21, 49-61. ROOS, A., and BORON, 296-434.

W. F. (1981).

Intracellular

pH. PhysioL

Rev. 61,

ROTTENBERG, H. (1979). The measurement of membrane potential and A pH in cells, organelles, and vesicles. In “Methods in Enzymology” (S. Fleischer and L. Packer, eds.), Vol. 55, pp. 547-569. Academic Press, New York. RUSSEL, J. R., and BORON, W. F. (1976). Role of chloride transport in regulation of intracellular pH. Nature (London) 264, 43-74. SCHACKMANN, R. W., CHRISTEN, R., and SHAPIRO, B. M. (1981). Membrane potential depolarization and increased intracellular pH accompany the acrosome reaction of sea urchin sperm. Pra: Nat. Acad Sci USA 78, 6066-6070. SCHACKMANN, R. W., EDDY, E. M., and SHAPIRO, B. M. (1978). The

VOLUME

98, 1983

acrosome reaction of Strongylocentratus sperm-Ion requirements and movements. Dev. BioL 65,483-495. SCHACKMANN, R. W., and SHAPIRO, B. M. (1981). A partial sequence of ionic changes associated with the acrosome reaction of Strongylocentratus purpuratus. Dew. BioL 81, 145-154. SCHROEDER, T. E., and CHRISTEN, R. (1982). Polymerization of actin without acrosomal exocytosis in starfish sperm. Visualization with NBD phallacidin. Exp. Cell Res. 140, 363-371. SCHUEL, H., LONGO, F. J., WILSON, W. L., and TROLL, W. (1976). Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface. Dev. BioL 49,178-184. SCHULDINER, S., RO’ITENBERG, H., and AVRON, M. (1972). Determination of ApH in chloroplasts. 2. Fluorescent amines as a probe for the determination of ApH in chloroplasts. Eur. J. B&hem. 25, 54-73. SEGALL, G. K., and LENNARZ, W. 3. (1979). Chemical characterization of the components of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev. BioL 71, 33-48. SEGALL, G. K., and LENNARZ, W. J. (1981). Jelly coat induction of the acrosome reaction in echinoid sperm. Dev. BioL 86, 87-93. SUMMERS, R. G., and HYLANDER, B. L. (1975). Species specificity of acrosome reaction and primary gamete binding in echinoids. Exp. Cell Res. 96, 63-68. SUMMERS, R. G., and HYLANDER, B. L. (1976). Primary gamete binding, quantitative determination of its specificity in echinoid fertilization. Exp. Cell Res. 100, 180-194. SUZUKI, W., NOMURA, K., OHTAKE, H., and ISAKA, S. (1981). Purification and the primary structure of sperm-activating peptides from the jelly coat of sea urchin eggs. B&hem. Biophys. Res. Commun 99, 1238-1244. TALBOT, P., SUMMERS, R. G., HYLANDER, B. L., KEOUGH, E. M., and FRANKLIN, L. E. (1976). The role of calcium in the acrosome reaction: An analysis using ionophore A23187. J. Exp. ZooL 198. 383-392. THOMAS, R. C. (1977). The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurons. J. Physiol 273,317-388. TILNEY, L. G., HATANO, S., ISHIKAWA, H., and MOOSEKER, M. S. (1973). The polymerization of actin: Its role in the generation of the acrosomal process on certain echinoderm sperm. J. Cell Biol 59,109126. TILNEY, L. G., KIEHART, D. P., SARDET, C., and TILNEY, M. (1978). Polymerization of actin. IV. Role of Ca++ and H+ in the assembly of actin and in membrane fusion in the aerosomal reaction of echinoderm sperm. J. Cell BioL 77,536-550. TIMOURIAN, J., and WATCHMAKER, G. (1970). Determination of spermatozoa motility. Dew. BioL 21, 62-72. TUBB, D. J., KOPF, G. S., and GARBERS, D. L. (1978). The elevation of sperm adenosine 3’5’ monophosphate concentrations by factors released from eggs requires calcium. BioL Reprod 18, 181-185. UNO, Y., and HOSHI, M. (1977). Separation of the sperm agglutinin and the acrosome reaction-inducing substance in egg jelly of starfish. Science 200, 58-59. VACQUIER, V. D. (1979a). The interactions of sea urchin ing fertilization. Amer. ZooL 19, 839-849.

gametes

dur-

VACQUIER, V. D. (1979b). The fertilizing capacity of sea urchin sperm rapidly decreases after induction of the acrosome reaction. Dew. GroLoth D@ 21, 61-69. VACQUIER, V. D., BRANDRIFF, B., and GLABE, C. (1979). The effect of soluble egg jelly on the fertilizability of acid-dejellied sea urchin eggs. Dev. Growth DQf 21, 47-60. VACQUIER, V. D., and MOY, G. W. (1977). Isolation of bindin: The protein responsible for adhesion of sperm to sea urchin eggs. Proc. Nat. Acad. Sci. USA 74, 2456-2460. VERCESI, A., REYNAFARJE, B., and LEHNINGER, A. L. (1978). Stoichiometry of H+ ejection and Ca++ uptake coupled to electron transport in heart mitochondria. J. Biol. C&m. 253, 6379-6385.