DEVELOPMENTAL
BIOLOGY
71, 49-59 (1979)
The Effect of the Acrosome Reaction on the Respiratory Fertilizing Capacity of Echinoid Sperm’ WILLIAM Department
H. KINSEY,~
GARY K. SEGALL,”
AND WILLIAM
of Physiological Chemistry, The Johns Hopkins Unioersity 725 N. Wolfe Street, Baltimore, Maryland 21205 Received November
21, 1978; accepted in revised form January
Activity and
J. LENNARZ” School of Medick
24, 1979
We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 set with little or no loss in their capacity to fertilize eggs; (2) by 60 set there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration. and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility. INTRODUCTION
time was that, when fertilizin was added to sperm, it occupied egg recognition sites (antifertilizin) on the sperm surface, thus rendering them incapable of interacting with the fertilizin bound to the egg plasma membrane. With the observation that jelly coat can induce the acrosome reaction (Dan, 1956), in some cases species specifically (SeGall and Lennarz, 1978; SeGall and Lennarz, 1979), an additional possibility arose, namely, that sperm which undergo the acrosome reaction may only retain full fertilizing capacity for a very short time (Tyler and Tyler, 1966). Under these circumstances, prior induction of the acrosome reaction could result in an inhibition of fertilization merely because the titer of sperm capable of fertilizing eggs is greatly reduced, irrespective of whether or not egg recognition sites on the surface of the sperm were occupied. Until recently, little attempt has been made to distinguish between the above possibilities. Takahashi and Sugiyama (1973, 1977) have studied the relationship be-
Studies reported in the preceding paper (SeGall and Lennarz, 1979) have established that jelly coat consists of two macromolecules: a highly sulfated polymer of fucose which is active in inducing the acrosome reaction in sperm, and a sialoprotein of unknown function. Early studies by Lillie (1913) and Tyler (1941) suggested that jelly coat, then called fertilizin, functioned as a sperm receptor involved in the species-specific adhesive interaction between sperm and egg. This hypothesis was, in part, based on the observation that treatment of sperm with fertilizin had a speciesspecific inhibitory effect on their ability to fertilize eggs. The interpretation at that ’ This work was supported by grants from the National Institutes of Health (HD-08357) and The Rockefeller Foundation to W.J.L. ’ W.H.K. is a Special Postdoctoral Fellow supported by The Rockefeller Foundation. .‘G.K.S. is a predoctoral fellow supported by a training grant from the National Institutes of Health (GM-00184). 4 To whom reprint requests should be addressed. 49
0012.1606/79/070049-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in anv form reservfdd.
50
DEVELOPMENTAL BIOLOGY
tween the acrosome reaction and fertilization, and concluded that the fertile life of sperm that have undergone the acrosome reaction can be as long as 4 min. Numerous studies on the effects of jelly coat material on sperm motility and respiration have not clarified this issue. Some investigators have shown that motility and respiration are stimulated by jelly components (Gray, 1928; Vasseur, 1949; Hathaway, 1963; Ohtake, 1976, 1977), whereas others have demonstrated that jelly coat inhibits these processes (Hayashi, 1946; Rothschild, 1952; Spikes, 1949). In no case have these effects been correlated with the acrosome reaction. In this study, we have examined the effects of jelly coat on sperm with respect to (1) induction of the acrosome reaction, (2) fertilizing capacity, and (3) respiration. The results indicate that the acrosome reaction is followed by a dramatic decrease in fertilizing capacity within 60 sec. Similar results are obtained when the ionophore A23187 is used to induce the acrosome reaction. Shortly after the decline in fertilizing capacity, sperm exhibit a marked and progressive decrease in oxygen consumption and motility. Both the extent of the acrosome reaction and the inhibitory effect on respiration are dependent on the concentration of jelly coat and are species specific. The results suggest that the relatively short fertile lifetime of sperm which have undergone the acrosome reaction must be taken into consideration when studying spermegg interactions and the effects of various agents on fertilization.
VOLUME 71, 1979
Systems, Inc.). The concentration of sperm is expressed as mg sperm protein/ml as determined by the Lowry et al. (1951) assay, using bovine serum albumin (Sigma Chemical Co.) as a standard. Jelly coat was prepared by two methods. Jelly coat (Type A) or “egg water” was prepared by suspending gravity-settled eggs in 2 vol of artificial seawater buffered with 10 mM Tris-HCl, pH 8. The suspension was allowed to stand for 2 to 3 hr at 20°C with occasional agitation. Eggs were removed by gentle centrifugation with a hand centrifuge and the supernate was further centrifuged at 12,000g for 20 min to remove insoluble material. The concentration of jelly coat was expressed as nmoles fucose/ml as determined by the method of Dische and Shettles (1951) using L-fucose (Pfanstiehl Laboratories, Waukegan, Ill.) as a standard. Typical preparations contained 140 pg protein/pmole fucose. Jelly coat (Type B) was prepared by acidifying a 10% (v/v) suspension of eggs to pH 5.5 for 4 to 5 min. The eggs were removed by centrifugation and the supernate was adjusted to pH 8.0 with Tris-HCl buffer. Insoluble material was removed by centrifugation at 12,000g for 20 min and the supernate was dialyzed extensively against Hz0 and lyophilized. Prior to use, the material was dissolved in artificial seawater and dialyzed against artificial seawater buffered with 10 mil4 Tris-HCl at pH 8.0. The occurrence of the acrosome reaction was monitored by electron microscopy, as previously described (Decker et al., 1976). Sperm were fixed by addition of an equal MATERIALS AND METHODS volume of 6% -glutaraldehyde in artificial Strongylocentrotus purpuratus were seawater. The cells were then dried down purchased from Pacific Bio-Marine, Venice, on Formvar-coated grids, washed with waCalif. Arbacia punctulata were obtained ter, and dried again. Over 100 cells were from Florida Marine Biological Supply CO., counted for each determination and sperm Panama City, Fla. Gametes were obtained in which the acrosomal process was visible by injections of 0.5 M KC1 into the coelomic were scored as “reacted.” The ability of sperm to fertilize eggs was cavity (S. purpuratus) or by electric shock (A. punctulata) and were suspended in ar- monitored by a slight modification of the assay published earlier (Schmell et al., tificial seawater (Instant Ocean, Aquarium
KINSEY, SEGALL, AND LENNARZ
Physiological
1977). In this assay, the percentage of eggs fertilized is linearly dependent on the number of sperm added. S. purpuratus sperm (100 pg protein) were added to a preincubation tube containing 1 ml of artificial seawater buffered with 10 mM Tris-HCl, pH 8.0, and either (1) no additions (control), (2) jelly coat (Type B) from S. purpuratus at a final concentration of 80 nmoles fucase/ml, or (3) jelly coat (Type B) from A. punctulata at 80 nmoles fucose/ml. This preincubation was carried out for various periods of time. One 100~~1 aliquot of the cell suspension was fixed for electron microscopy, while a second was simultaneously transferred to a tube containing 3 ml of artificial seawater and mixed well to disrupt aggregates and dilute the cells. Observations by phase-contrast microscopy indicated that this procedure disrupts any aggregates that might be formed by the isoagglutinin activity of jelly coat and very few “rosettes” (Collins, 1976) are formed under these dilute conditions. An aliquot (100 ~1) of this dilute suspension was quickly added to 500 ~1 of a 1% (v/v) suspension of acid-dejellied eggs in buffered artificial seawater. After 5 min the reaction was stopped with a few drops of 6% glutaraldehyde and the percentage of eggs that were fertilized was determined as previously described (Schmell et al., 1977). The effect of the ionophore A23187 on the acrosome reaction and fertilization was determined in A. punctulata since its effects have been well studied in this species (Decker et al., 1976). Sperm (200 pg sperm protein) were added to 1 ml of artificial seawater containing 57 @kf A23187 and 20 mM Tris-HCl, pH 8.8. This slightly elevated pH is necessary to obtain ionophore induction of the acrosome reaction. The cells were incubated for various lengths of time. Then one 200+1 aliquot of the cell suspension was fixed for electron microscopy, and a second aliquot was simultaneously added to 4 ml of artificial seawater and mixed to dilute the cells. An aliquot (20
Effects
of the Acrosome
Reaction
51
~1) of this suspension was then added to 0.5 ml of a 1% suspension of dejellied eggs and fertilization was assayed as described above. Sperm oxygen consumption was measured with an oxygen-sensitive electrode fitted to a 2.5-ml chamber (Gilson Medical Electronics, Middleton, Wis.). The temperature was maintained at 21°C and the chamber contents were stirred at a constant rate by a magnetic stirring bar. The lower limit of the electrode sensitivity was calibrated by sodium dithionite and arbitrarily represented as 0 patom oxygen/ml. A value of 0.22 patom oxygen/ml (McLellan, 1965) was used for the solubility of oxygen in seawater at 21’C. To determine the effect of various agents on sperm respiration, lo-20 ~1 sperm (l.O1.8 mg protein) was added to the chamber containing 2.0 ml of artificial seawater buffered with 10 mM Tris-HCl, pH 8.0. The cells were allowed to respire for 2 min to establish the initial rate of oxygen consumption before the test substance (contained in 0.5 ml of Tris-HCl-buffered artificial seawater) was added to the chamber. Controls to which 0.5 ml of Tris-HCl buffered artificial seawater was added respired at a constant rate for 5 to 10 min. Oxygen consumption was almost completely inhibited by antimycin A (20 pm/ ml). In several cases, the pH of the chamber contents was measured at the end of an experiment; the pH did not change more than 0.1 pH unit. RESULTS
Effect of the Acrosome Reaction Fertilizing Capacity
on Sperm
The effect of prior induction of the acrosome reaction on the fertilizing capacity of S. purpuratus sperm was investigated using a bioassay. The method employed is a sensitive measure of the number of sperm that are capable of fertilizing eggs, since it is carried out under conditions in which
52
DEVELOPMENTAL
BIOLOGY
sperm are limiting (Schmell et al., 1977). S. purpuratus sperm were first prereacted by exposure to jelly coat (Type B) for various lengths of time, and then aliquots were simultaneously fixed in glutaraldehyde or diluted and added to eggs to measure their ability to fertilize eggs. The results shown in Fig. 1A indicate that the acrosome reaction is complete in over 80% of the sperm after a 30-set exposure to jelly coat. As seen in Fig. lB, sperm exposed to jelly coat for 30 set fertilize eggs at a level similar to control sperm, which are not exposed to jelly coat and presumably undergo the acrosomal reaction only when they contact the egg. However, prior exposure of sperm 100
A
r
5 80 XA F \ 2 60 k! E g 40 :: 5 a
X’
l-f+
x-x
20 1
o\” ,oo[
to jelly for 60 set results in a marked decrease in fertility. Exposure times longer than 1 min result in a further decrease in fertilizing ability, with the maximum effect occurring after 5 min. From these results it is clear that during the first 30 set the reacted sperm have a fertilizing capacity equal to the control sperm. However, within the next 30 set, during which time an additional 5% of the sperm react, the ability to fertilize eggs drops by approximately 70%. This indicates that the reacted 100 v
A
5
; 80z: $ 60r" 0 40E? 5 20 ; "\ / X s m !
X
/
X
1
l
B
IOO-
p&# B
r
it Kx-x,
s
VOLUME 71, 1979
20
x-+--x--//-x PREINCUBATION TIME (MIN.1 FIG. 1. (A) Time course of the acrosome reaction in S. purpuratus sperm treated with jelly coat. Aliquots of sperm were fixed for electron microscopy after pretreatment with Tris-HCl-buffered artificial seawater as a control (W), A. punctulata jelly coat (Type B) (O-----O), or S. purpurutus jelly coat (Type B) (X-X) for the indicated length of tune. The percentage of acrosome reaction was determined as described in Materials and Methods. (B) Effect of jelly coat on the fertilizing capacity of S. purpuratus sperm. Sperm were pretreated as described in (A).
ZSO-u - xHX F i?i 60? \ I= a 402 8 20, I 12 PREINCUBATION (MIN.)
X
\, I
X
3 TIME
FIG. 2. (A) Time course of the acrosome reaction in A. punctuZuta sperm treated with A23187. Aliquots of sperm were futed for electron microscopy after pretreatment with A23187 (57 1LM) (X--X) or an equivalent amount of DMSO (solvent) (U) for the indicated length of time. (B) Effect of the ionophore A23187 on the fertilizing capacity of A. puntt&to sperm. Sperm were pretreated as described in (A).
KINSEY, SEGALL, AND LENNARZ
Physiological
Effects
of the Acrosome
Reaction
53
sperm (85% of the population) are no longer as effective in fertilization as the control, unreacted sperm. The low level of fertilization produced by sperm preincubated for as long as 10 min is probably effected by the remaining lo-15% of the sperm that remain unreacted. S. purpuratus sperm exposed to an equal concentration of jelly from A. punctulata did not undergo the acrosome reaction (Fig. 1A) and remained similar to control sperm in their capacity to fertilize S. purpuratus eggs (Fig. 1B). This species-specific effect of jelly coat material on both the acrosome reaction and the fertilizing capacity indicates that the effect on fertilization is probably the result of the occurrence of the FIG. 3. Effect of jelly coat on sperm respiration. s’. acrosome reaction, rather than some nonpurpuratus sperm were treated at zero time (arrow) specific, physical interaction of jelly with with (a) S. purpuratus jelly coat (Type B) (40 nmoles the sperm. To determine if other agents fucose/ml), or (b) Tris-HCl-buffered artificial seawawhich induce the acrosome reaction have a ter as a control, and allowed to respire for 15 min. similar effect on fertility, experiments were Aliquots (1~1) of cells were removed from the chamber carried out using the ionophore A23187 to just prior to zero time and at various times after the addition of jelly coat or seawater and fixed for electron prereact the sperm. Since A23187 induces microscopy. The percentage of acrosome reaction was the acrosome reaction more slowly than determined and is displayed in the inset: sperm plus jelly coat, we are unable to make a precise jelly coat (M), sperm plus Tris-HCI-buffered ) estimate of the fertile lifetime of sperm artificial seawater (X-X reacted by this method. The results in Fig. 2 show that the ionophore has little effect similar to that used in the previous experion either the acrosome reaction (Fig. 2A) ments. As seen in Fig. 3, a progressive deor the sperm fertilizing capacity (Fig. 2B) crease in the rate of On consumption beduring the first 60 sec. However, as the came apparent about 1.5 to 2 min after acrosome reaction begins to occur (2 min), addition of the jelly. The rate of respiration a decrease in fertilizing capacity becomes continued to decrease during the next 8 to apparent. 10 min, finally stabilizing at a greatly reduced level. During the course of this exEffect of Jelly Coat Treatment on Sperm periment, l-p1 samples were removed from Respiration the chamber and fixed for electron microsTo determine whether the jelly-induced copy. During the first 30 set, over 70% of effect on fertility could be explained by an the cells extruded acrosomal processes (ineffect on sperm viability, we examined the set, Fig. 3), yet there was no detectable effect of jelly coat material on the respiraeffect on the rate of oxygen consumption tion of S. purpuratus sperm. Sperm were during this time period. However, between added to the chamber of an oxygen elec- 1.5 and 2 min, the onset of a marked and trode apparatus and allowed to establish a continual decrease in the respiration rate constant respiration rate (Fig. 3). Once the occurs. The OZ consumption by sperm is respiration rate was constant, jelly coat almost totally inhibited by antimycin A, (Type B) was added at a concentration indicating that it represents mitochondrial
54
DEVELOPMENTAL BIOLOGY
respiration (Chance, 1957). Thus, the above findings suggest that one of the consequences of the acrosomal reaction is a cessation of mitochondrial respiration. At various times during this experiment, aliquots of sperm were removed and motility was examined by phase-contrast microscopy. An estimate of the percentage of sperm that were actively moving indicates that a general decline in the number of motile sperm occurred only after the addition of jelly. Only 5-10% of the sperm were motile 10 min after the addition of jelly. This small number of motile sperm probably represents unreacted cells and could account for the low but steady rate of oxygen consumption observed during the subsequent 10 to 15 min. In control experiments (seawater added instead of jelly), no appreciable change in the number of motile sperm was observed during the experiment, although the rate of swimming seemed to decrease after 8 to 10 min. We also found that when sperm were treated with the ionophore A23187 under conditions sufficient to induce the acrosome reaction, a similar decrease in respiration rate and motility occurred (data not shown). Induction of the acrosome reaction and the subsequent inhibition in respiration were dependent on the concentration of ionophore; concentrations of ionophore insufficient to induce the acrosome reaction had no effect on respiration. Effect of Jelly Coat Concentration on the Acrosome Reaction and on Respiration To further examine the relationship between the acrosome reaction and the respiration effect, we tested the dependence of these two events on the concentration of jelly coat. The degree of inhibition of respiration (Fig. 4A) and the extent of the acrosome reaction (Fig. 4B) increased with increasing concentrations of jelly coat (Type A). Little further change in either the respiration effect or the percentage of reacted sperm occurred at concentrations
VOLUME 71, 1979
1
5
I
I
I
I
10 15 20 25 nmFUCOSE/mL
FIG. 4. (A) Effect of the concentration of jelly coat (Type A) on sperm respiration. S. purpuratus sperm were treated (arrow) with jelly coat (Type A) at the indicated concentrations: (a) 0.0, (b) 0.8, (c) 2.0, (d) 4.0, (e) 8.0, (f) 16.0, or (g) 24.0 nmoles fucose/ml. (B) Samples were fixed for electron microscopy at the end of each run (12 to 15 min) and the percentage of sperm that underwent the acrosome reaction was determined. The rate of oxygen consumption was calculated at the 9.5- to 10.5-min interval for each run. Both parameters are expressed as a function of jelly coat concentration: acrosome reaction (M); respiration rate (X-X).
above 16 nmoles fucose/ml. As shown in Fig. 5, similar results were obtained when jelly coat (Type B) was used, although much higher concentrations were necessary. The apparent decrease in the biological activity of jelly coat upon dialysis is probably due to the loss of bound Ca2+ since, as discussed in the preceding paper (SeGall and Lennarz, 1979), full activity can be restored by the addition of CaCh. Jelly coat partially purified by gel filtration (see SeGall and Lennarz, 1979) was also effective (data not shown). These experiments clearly show that both events, i.e., the acrosome reaction and respiration loss, are
KINSEY, SEGALL, AND LENNARZ
Physiological
similarly dependent on the concentration of jelly coat and that “egg water” has the same effect as the more purified forms of jelly coat. Together with the previous ex-
Effects
of the Acrosome
55
Reaction
periments, this suggests that the decrease in respiration rate represents the death of the population of sperm which have undergone the acrosome reaction.
Species Specificity of Jelly Coat in Induction of the Acrosome Reaction and Respiratory Inhibition
1
I
100
200
1
J
1
300 400 500 600 nmFUCOSE/ml FIG. 5. (A) Effect of the concentration of jelly coat (Type B) concentration on sperm respiration. S. purpuratus sperm were treated (arrow) with jelly coat (Type B) at the indicated concentrations: (a) 0.0, (b) 7.2, (c) 60, (d) 300, or (e) 600 nmoles fucose/ml. (B) Samples were fixed for electron microscopy at the end of each run and the percentage of sperm that underwent the acrosome reaction was determined. TABLE INDUCTION
Incubation
OF THE ACROSOME
REACTION
time
1
BY EGGS CONTAINING
ATTACHED
JELLY
COATS
‘% Acrosome reaction S. purpuratus Artificial seawater
15 set 30 set 1 min 2 min 5 min 10 min
SolubiIized components of the jelly coat of S. purpuratus and A. punctulata (SeGall and Lennarz, 1978, 1979) induce the acrosome reaction in a species-specific manner. However, Summers and Hylander (19751, using other species of echinoids, demonstrated nonspecific induction of the acrosome reaction by eggs containing their surrounding “native” jelly. To confirm that the species specificity observed in the solubilized jelly coat of S. purpuratus and A. punctulata is retained in the native jelly coat surrounding eggs, we tested the ability of eggs with intact jelly coat to induce the acrosome reaction. It is clear from the results presented in Table 1 that the native jelly of A. punctulata and S. purpuratu,s eggs can induce the acrosome reaction only in the homologous sperm. Thus the speciesspecific properties of solubilized jelly coat (Types A and B) are also present in the jelly surrounding freshly spawned eggs and are not an artifact of preparation. It is apparent from these findings, coupled with those reported in the preceding paper
2 4 9
sperm plus __. -~-.S. _ purpuratus A. zmnctulatn _ em eggs 72 75 1 76 75 2 86 0 86
A. punctulata Artificial seawater
sperm plus A. nunctulatn ems
0
50
1 2
57 60
” Sperm from S. purpuratus (560 pg sperm protein) or a. punctulata (500 pg sperm protein) were added to Y! ml of a 50% (v/v) suspension of freshly collected eggs containing intact jelly coats. After various periods of time the cells were fixed for electron microscopy and the occurrence of the acrosome reaction was determined.
56
DEVELOPMENTAL BIOLOGY
(SeGall and Lennarz, 1979) and by Summers and Hylander (1975), that echinoid sperm show species variation in their ability to be reacted by heterologous jelly coat; some are species specific, whereas others are not. If the jelly-induced inhibition of sperm respiration is actually a consequence of the acrosome reaction, then the respiration effect should also be species specific. The results in Fig. 6 show that this is, in fact, the case. A. punctulata jelly has no inhibitory effect on the respiration of S. purpuratus sperm over a wide concentration range in which S. purpuratus jelly is effective (Fig. 6A). As expected, S. purpuratus jelly
1
0
I
100
4
5 TIME (Mitt?
I
I
I
I
,
’
15
I
300 500 700 nm FUCOSE/mt
FIG. 6. (A) Effect of jelly coat from S. purpuratus and A. pun&data on the respiration of S. purpuratus sperm. S. purpuratus sperm were treated (arrow) with jelly coat (Type B) from S. purpuratus or A. punctuZata eggs. (a) Control; (b) S. purpuratus jelly coat, 82 nmoles fucose/ml; (c) S. purpuratus jelly coat, 508 nmoles fucose/ml; (d) A. punctulata jelly coat, 227 nmoles fucose/ml; (e) A. punctulata jelly coat, 700 nmoles fucose/ml. (B) Samples were fixed for electron microscopy after each run and the occurrence of the acrosome reaction was determined. S. purpuratus jelly coat (M); A. punctulata jelly coat (A-A).
VOLUME 71, 1979
induced the acrosome reaction in this species, whereas A. punctulata jelly did not (Fig. 6B). Similarly, S. purpuratus jelly had no effect on the respiration rate or the acrosome reaction of A. punctulata sperm at concentrations as high as 508 nmoles fucose/ml. DISCUSSION
The results presented here show that the following sequence of events occurs when S. purpuratus sperm are treated with homologous jelly coat: (1) The majority of the sperm undergo the acrosome reaction within 30 sec. Sperm which have undergone the acrosome reaction retain their ability to fertilize eggs during this time, since a sperm population containing 82% prereacted sperm can fertilize eggs as effectively as a control containing 1% reacted sperm. (2) By 60 set there is a dramatic decrease in fertilizing capacity, which stabilizes after 4 to 5 min at a greatly reduced level. (3) By 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration of the sperm is detectable. This decrease continues for 8 to 10 min, after which time the rate of respiration stabilizes at a greatly reduced rate. At this time only 5-10s of the sperm are actively swimming. The abrupt decline in fertilizing capacity and respiration observed in sperm treated with jelly coat can be explained in at least two ways. Jelly coat could cause the entire sperm population to be less effective in fertilization and to respire at a lower rate. Alternatively, the sperm which have reacted may become incapable of effecting fertilization and respire at a very low rate, while only the few unreacted sperm remain fully active and capable of fertilizing eggs. We favor the second interpretation for the following reasons. First, microscopic examination reveals that only a small proportion of the sperm population remains motile after treatment with a quantity of jelly sufficient to cause 90% of the sperm to undergo the acrosome reaction. Second, the magnitude of the jelly-induced depression
KINSEY, SEGALL, AND LENNAFU
Physiological
of respiration correlates roughly with the extent of the acrosome reaction and both events show a similar dependence on the concentration of jelly. Third, the effect of jelly on inducing the acrosome reaction, and on decreasing both the fertilization capability and the rate of respiration, is species specific, thus ruling out any nonspecific toxic effects. Our estimate of the fertile life of sperm following the acrosome reaction (30 set) is considerably shorter than that reported by Takahashi and Sugiyama (1973) for Pseudocentrodus depressus. This could be because our fertilization assays were done under sperm-limiting conditions that provide a more sensitive estimate of the number of viable sperm. The observation that jelly coat triggers a decrease in respiration rate is consistent with results obtained by several workers (Hayashi, 1946; Spikes, 1949; Rothchild, 1952) but not others (Gray, 1928; Vasseur, 1949; Hathaway, 1963; Ohtake, 1976, 1977). Ohtake, for example, has recently isolated a sperm activating substance from “egg water” (jelly coat, Type A). He found that when the pH of a sperm suspension was reduced from the normal value of 8.2 to 6.8, sperm respiration was suppressed. Addition of the sperm activating substance to the acidified suspension stimulated respiration back to the control level but not above it. However, the physiological significance of these studies involving exposure of sperm to seawater at pH 6.8 is not clear. Earlier studies reported up to threefold stimulation of respiration by egg water (Hathaway, 1963). The major difference between these early studies and our work lies in the concentration of sperm upon which the respiration measurements were made. Typical values were 1:lO diluted “semen” (Hathaway, 1963) and 1:6 diluted “dry” sperm (Vasseur, 1949). We estimate these concentrations to be about lo-15 mg sperm protein/ml, whereas we used about 0.6 mg sperm protein/ml. At the very high sperm concentrations used by those investigators, it is unlikely that a significant fraction of
Effects
of the Acrosome
Reaction
5:
the sperm had undergone the acrosome reaction since the ratio of egg jelly to sperm would have been much lower than that used in our studies. In fact, Hathaway (1963) reported that under his conditions no reacted sperm were detected. We feel that this point may explain some of the differences reported in the literature. At very high sperm concentrations, jelly coat may indeed stimulate respiration (by some unknown mechanism) without inducing the acrosome reaction. However, our results show conclusively that, under conditions which will support the acrosome reaction, jelly coat induces a decrease in respiration rate which is dependent on the concentration of jelly coat used and is roughly proportional to the percentage of sperm that have undergone the acrosome reaction. Why sperm that have undergone the acrosome reaction should so quickly lose t,heir respiration capacity is puzzling. The acrosomal vesicle is morphologically remote from the mitochondrion situated at the posterior of the sperm head, and there are no obvious interconnections between the two organelles. Moreover, while induction of the acrosome reaction causes a cessation of mitochondrial respiration, the converse is not true. Sperm that have been pretreated with antimycin A to block respiration are fully capable of undergoing the acrosome reaction (data not shown). Shackmann et al. (1978) have reported a similar insensitivity of the acrosome reaction to a number of other respiratory inhibitors and to uncouplers. Several studies (Decker et al., 1976; Collins and Epel, 1977; Tilney et al., 1977; Shackmann et al., 1978) have demonstrated that occurrence of the acrosome reaction requires an influx of Ca’+ ions. In addition, Shackmann et al. have shown that an efflux of protons occurs, and they suggest that countermovements of Na’ (in) and K’ (out) also are involved. Such ion movements during the acrosome reaction might ultimately cause inhibition of mitochondrial respiration. However, the mechanism that would
58
DEVELOPMENTAL BIOLOGY
cause such a change is unknown. One possibility is that jelly coat, in the presence of Ca2+ ions, may cause a general increase in the permeability of the plasma membrane and the mitochondria membrane, with a concomitant loss of oxidizable substrates. This seems unlikely, however, since when the mitochondrial substrates pyruvate, malate, or /?-hydroxybutyrate were added to inhibited sperm, respiration was not restored (data not shown). In any case, since not only jelly coat, but also the Ca2+ ionophore A23187, induces the acrosome reaction and the consequent respiratory inhibition, it seems clear that at least the initial step, the acrosome reaction, involves ion permeability changes. How the fucose sulfate polymer component of egg jelly brings about these changes remains a most challenging problem. As noted above, early workers implicated “fertilizin” or jelly coat material as a sperm receptor functioning in gamete recognition (see Tyler and Tyler (1966) for review). This theory is largely based on the observation that fertilizin can inhibit fertilization species specifically. Most of these studies were done by pretreating the sperm with jelly coat for as long as 15 to 20 min and then testing the ability of the sperm to fertilize eggs (Tyler and Tyler, 1966; Metz, 1961). Our results clearly show that such treatments have a very pronounced, species-specific inhibitory effect on sperm respiration. This respiration effect could account for the observed inhibitory effect of fertilizin on the fertilizing capacity of sperm. However, our work does not rule out the possibility that fertilizin also has a receptor-like function. For example, we observed that while treated sperm retain full fertilizing capacity for at least 30 see, this ability is greatly reduced by 60 sec. The fact that this decline precedes the first detectable decrease in oxygen consumption may indicate that fertilizin could have other effects on the fertilizing capacity of sperm. It is clear (Summers and Hylander, 1978;
VOLUME 71, 1979
SeGall and Lennarz, 1979) that in some echinoid species induction of the acrosome reaction, which is a prerequisite to fertilization, is not species specific. In these species, at least, jelly coat cannot be the component that dictates species specificity in fertilization. Other studies (Schmell et al., 1977; Tsuzuki et al., 1977; Glabe and Vacquier, 1977) have implicated receptors on the egg cell surface per se, rather than in the jelly coat, as the components that control species specificity. Clearly, depending on the particular echinoid used, either jelly coat or a cell surface receptor, or both, could define specificity. The results of the current study afford an approach to distinguishing between these possibilities, since it is now evident that sperm can be prereacted and utilized in fertilization studies, provided that the time interval between the acrosome reaction andfertilization is short. The authors wish to thank Mrs. Betty Earles for her technical assistance and Ms. Ann Fuhr for typing the manuscript. REFERENCES CHANCE, B. (1957). Techniques for the assay of the respiratory enzymes. Methods Enzymol. 4,273-328. COLLINS, F. (1976). A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion. Develop. 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. 106,211-222. DAN, J. C. (1956). The acrosome reaction. ht. Reu. Cytol. 5,365-393. DECKER, G. L., JOSEPH, D. B., and LENNARZ, W. J. (1976). A study of factors involved in the induction of the acrosomal reaction in sperm of the sea urchin Arbacia punctulata. Develop. Biol. 53, 115-125. DISCHE, Z., and SHETTLES, L. B. (1951). A new spectrophotometric test for the detection of methylpentose. J. Biol. Chem. 192,579-582. GLABE, C. G., and VACQUIER, V. D. (1977). Species specific agglutination of eggs by bindin isolated from sea urchin sperm. Nature 267.836-838. GRAY, J. (1928). The effect of egg secretions on the activity of spermatozoa. J. Exp. Biol. 5,262-265. HAGSTROM, B. E. (1956). Studies of the fertilization of jelly-free sea urchin eggs. Exp. Cell Res. 10, 24-28. HATHAWAY, R. R. (1963). Activation of respiration in sea urchin spermatozoa by egg water. Biol. Bull.
KINSEY, SEGALL, AND LENNARZ
Physio Ilogical Effects of the Acrosome Reaction
125,486-498. HAYASHI, T. (1946). Dilution medium and survival of the spermatozoa of Arbacia punctulata. II. Effect of the medium on respiration. Biol. Bull. 90, 177187. LILLIE, F. R. (1913). Studies on fertilization V. The behavior of the spermatozoa of Nereis and Arbacia with special reference to egg-extractives. J. Exp. Zool. 14,515-574. LOWRY, 0. H., ROSEBROLJGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MCLELLAN, H. J. (1965). Elements of Physical Oceanography. Pergamon Press, New York. METZ, C. B. (1961). Use of inhibiting agents in studies on fertilization mechanisms. Znt. Rev. Cytol. l&219253. OHTAKE, H. (1976). Respiratory behavior of sea-urchin spermatozoa I. Effect of pH and egg water on the respiratory rate. J. Enp. 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. ROTHSCHILD, L. (1952). The behavior of spermatozoa in the neighborhood of eggs. Znt. Reu. Cytol. 1,257263. SHACKMANN, R. W., EDDY, E. M., and SHAPIRO, B. M. (1978). The acrosome reaction of Strongylocentrotus purpuratus sperm: Ion requirements and movements Develop. Biol. 65,483-495. SCHMELL, E., EARLES, B. J., BREAUX, C., and LENNARZ, W. J. (1977). Identification of a sperm receptor on the surface of the eggs of the sea urchin
Arbacia punctulata. J. Cell Biol. 72, 35-46. SEGALL, G. K., and LENNARZ, W. J. (1978). The role of jelly coat in the induction of the acrosomal reaction in sperm. J. Cell Biol. 79, 170a.
59
SEGALL, G. K., and LENNARZ, W. J. (1979). Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Develop. Biol. 71,33-48. SPIKES, J. D. (1949). Metabolism of sea-urchin sperm. Amer. Natur. 83, 285-301. SUGIYAMA, M., and KATO, K. (1977). Effects of trypsin on the fertilizing capacity of sea urchin spermatozoa treated with egg-water. Develop. Growth Different. 19,23-29. SUMMERS, R. G., and HYLANDER, B. L. (1975). Species specificity of the acrosome reaction and primary gamete binding in echinoids. Exp. Cell Rex 96, 6368. TAKAHASHI, Y. M., and SUGIYAMA, M. (1973). Relation between the acrosome reaction and fertilization in the sea urchin. I. Fertilization in Ca-free sea water Develop. with egg-water-treated spermatozoa. Growth Different. 15, 261-267. 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 acrosomal reaction of echinoderm sperm. J. Cell Biol. 77, 536-550. TYLER, A. (1941). The role of fertihzin in the fertilization of eggs of the sea urchin and other animals. Biol. Bull. 81, 190-204. TYLER, A., and TYLER, B. S. (1966). Physiology of fertilization and early development. In “Physiology of Echinodermata” (R. A. Boolootian, ed.), pp. 683741. John Wiley and Sons, New York. TSUZUKI, H., YOSHIDA, M., ONITAKE, K., and AKETA, K. (1977). Purification of the sperm-binding factor from the egg of the sea urchin Hemicentrotus pul-
cherrimus. Biochem. Biophys. Res. Commun. 76, 502-511. VASSEUR, E. (1949). Effect of sea-urchin egg jelly-coat solution and calcium ions on the oxygen uptake of sea-urchin sperm. Ark. Kemi. 1,393-399.
BIOLOGY
71, 49-59 (1979)
The Effect of the Acrosome Reaction on the Respiratory Fertilizing Capacity of Echinoid Sperm’ WILLIAM Department
H. KINSEY,~
GARY K. SEGALL,”
AND WILLIAM
of Physiological Chemistry, The Johns Hopkins Unioersity 725 N. Wolfe Street, Baltimore, Maryland 21205 Received November
21, 1978; accepted in revised form January
Activity and
J. LENNARZ” School of Medick
24, 1979
We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 set with little or no loss in their capacity to fertilize eggs; (2) by 60 set there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration. and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility. INTRODUCTION
time was that, when fertilizin was added to sperm, it occupied egg recognition sites (antifertilizin) on the sperm surface, thus rendering them incapable of interacting with the fertilizin bound to the egg plasma membrane. With the observation that jelly coat can induce the acrosome reaction (Dan, 1956), in some cases species specifically (SeGall and Lennarz, 1978; SeGall and Lennarz, 1979), an additional possibility arose, namely, that sperm which undergo the acrosome reaction may only retain full fertilizing capacity for a very short time (Tyler and Tyler, 1966). Under these circumstances, prior induction of the acrosome reaction could result in an inhibition of fertilization merely because the titer of sperm capable of fertilizing eggs is greatly reduced, irrespective of whether or not egg recognition sites on the surface of the sperm were occupied. Until recently, little attempt has been made to distinguish between the above possibilities. Takahashi and Sugiyama (1973, 1977) have studied the relationship be-
Studies reported in the preceding paper (SeGall and Lennarz, 1979) have established that jelly coat consists of two macromolecules: a highly sulfated polymer of fucose which is active in inducing the acrosome reaction in sperm, and a sialoprotein of unknown function. Early studies by Lillie (1913) and Tyler (1941) suggested that jelly coat, then called fertilizin, functioned as a sperm receptor involved in the species-specific adhesive interaction between sperm and egg. This hypothesis was, in part, based on the observation that treatment of sperm with fertilizin had a speciesspecific inhibitory effect on their ability to fertilize eggs. The interpretation at that ’ This work was supported by grants from the National Institutes of Health (HD-08357) and The Rockefeller Foundation to W.J.L. ’ W.H.K. is a Special Postdoctoral Fellow supported by The Rockefeller Foundation. .‘G.K.S. is a predoctoral fellow supported by a training grant from the National Institutes of Health (GM-00184). 4 To whom reprint requests should be addressed. 49
0012.1606/79/070049-11$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in anv form reservfdd.
50
DEVELOPMENTAL BIOLOGY
tween the acrosome reaction and fertilization, and concluded that the fertile life of sperm that have undergone the acrosome reaction can be as long as 4 min. Numerous studies on the effects of jelly coat material on sperm motility and respiration have not clarified this issue. Some investigators have shown that motility and respiration are stimulated by jelly components (Gray, 1928; Vasseur, 1949; Hathaway, 1963; Ohtake, 1976, 1977), whereas others have demonstrated that jelly coat inhibits these processes (Hayashi, 1946; Rothschild, 1952; Spikes, 1949). In no case have these effects been correlated with the acrosome reaction. In this study, we have examined the effects of jelly coat on sperm with respect to (1) induction of the acrosome reaction, (2) fertilizing capacity, and (3) respiration. The results indicate that the acrosome reaction is followed by a dramatic decrease in fertilizing capacity within 60 sec. Similar results are obtained when the ionophore A23187 is used to induce the acrosome reaction. Shortly after the decline in fertilizing capacity, sperm exhibit a marked and progressive decrease in oxygen consumption and motility. Both the extent of the acrosome reaction and the inhibitory effect on respiration are dependent on the concentration of jelly coat and are species specific. The results suggest that the relatively short fertile lifetime of sperm which have undergone the acrosome reaction must be taken into consideration when studying spermegg interactions and the effects of various agents on fertilization.
VOLUME 71, 1979
Systems, Inc.). The concentration of sperm is expressed as mg sperm protein/ml as determined by the Lowry et al. (1951) assay, using bovine serum albumin (Sigma Chemical Co.) as a standard. Jelly coat was prepared by two methods. Jelly coat (Type A) or “egg water” was prepared by suspending gravity-settled eggs in 2 vol of artificial seawater buffered with 10 mM Tris-HCl, pH 8. The suspension was allowed to stand for 2 to 3 hr at 20°C with occasional agitation. Eggs were removed by gentle centrifugation with a hand centrifuge and the supernate was further centrifuged at 12,000g for 20 min to remove insoluble material. The concentration of jelly coat was expressed as nmoles fucose/ml as determined by the method of Dische and Shettles (1951) using L-fucose (Pfanstiehl Laboratories, Waukegan, Ill.) as a standard. Typical preparations contained 140 pg protein/pmole fucose. Jelly coat (Type B) was prepared by acidifying a 10% (v/v) suspension of eggs to pH 5.5 for 4 to 5 min. The eggs were removed by centrifugation and the supernate was adjusted to pH 8.0 with Tris-HCl buffer. Insoluble material was removed by centrifugation at 12,000g for 20 min and the supernate was dialyzed extensively against Hz0 and lyophilized. Prior to use, the material was dissolved in artificial seawater and dialyzed against artificial seawater buffered with 10 mil4 Tris-HCl at pH 8.0. The occurrence of the acrosome reaction was monitored by electron microscopy, as previously described (Decker et al., 1976). Sperm were fixed by addition of an equal MATERIALS AND METHODS volume of 6% -glutaraldehyde in artificial Strongylocentrotus purpuratus were seawater. The cells were then dried down purchased from Pacific Bio-Marine, Venice, on Formvar-coated grids, washed with waCalif. Arbacia punctulata were obtained ter, and dried again. Over 100 cells were from Florida Marine Biological Supply CO., counted for each determination and sperm Panama City, Fla. Gametes were obtained in which the acrosomal process was visible by injections of 0.5 M KC1 into the coelomic were scored as “reacted.” The ability of sperm to fertilize eggs was cavity (S. purpuratus) or by electric shock (A. punctulata) and were suspended in ar- monitored by a slight modification of the assay published earlier (Schmell et al., tificial seawater (Instant Ocean, Aquarium
KINSEY, SEGALL, AND LENNARZ
Physiological
1977). In this assay, the percentage of eggs fertilized is linearly dependent on the number of sperm added. S. purpuratus sperm (100 pg protein) were added to a preincubation tube containing 1 ml of artificial seawater buffered with 10 mM Tris-HCl, pH 8.0, and either (1) no additions (control), (2) jelly coat (Type B) from S. purpuratus at a final concentration of 80 nmoles fucase/ml, or (3) jelly coat (Type B) from A. punctulata at 80 nmoles fucose/ml. This preincubation was carried out for various periods of time. One 100~~1 aliquot of the cell suspension was fixed for electron microscopy, while a second was simultaneously transferred to a tube containing 3 ml of artificial seawater and mixed well to disrupt aggregates and dilute the cells. Observations by phase-contrast microscopy indicated that this procedure disrupts any aggregates that might be formed by the isoagglutinin activity of jelly coat and very few “rosettes” (Collins, 1976) are formed under these dilute conditions. An aliquot (100 ~1) of this dilute suspension was quickly added to 500 ~1 of a 1% (v/v) suspension of acid-dejellied eggs in buffered artificial seawater. After 5 min the reaction was stopped with a few drops of 6% glutaraldehyde and the percentage of eggs that were fertilized was determined as previously described (Schmell et al., 1977). The effect of the ionophore A23187 on the acrosome reaction and fertilization was determined in A. punctulata since its effects have been well studied in this species (Decker et al., 1976). Sperm (200 pg sperm protein) were added to 1 ml of artificial seawater containing 57 @kf A23187 and 20 mM Tris-HCl, pH 8.8. This slightly elevated pH is necessary to obtain ionophore induction of the acrosome reaction. The cells were incubated for various lengths of time. Then one 200+1 aliquot of the cell suspension was fixed for electron microscopy, and a second aliquot was simultaneously added to 4 ml of artificial seawater and mixed to dilute the cells. An aliquot (20
Effects
of the Acrosome
Reaction
51
~1) of this suspension was then added to 0.5 ml of a 1% suspension of dejellied eggs and fertilization was assayed as described above. Sperm oxygen consumption was measured with an oxygen-sensitive electrode fitted to a 2.5-ml chamber (Gilson Medical Electronics, Middleton, Wis.). The temperature was maintained at 21°C and the chamber contents were stirred at a constant rate by a magnetic stirring bar. The lower limit of the electrode sensitivity was calibrated by sodium dithionite and arbitrarily represented as 0 patom oxygen/ml. A value of 0.22 patom oxygen/ml (McLellan, 1965) was used for the solubility of oxygen in seawater at 21’C. To determine the effect of various agents on sperm respiration, lo-20 ~1 sperm (l.O1.8 mg protein) was added to the chamber containing 2.0 ml of artificial seawater buffered with 10 mM Tris-HCl, pH 8.0. The cells were allowed to respire for 2 min to establish the initial rate of oxygen consumption before the test substance (contained in 0.5 ml of Tris-HCl-buffered artificial seawater) was added to the chamber. Controls to which 0.5 ml of Tris-HCl buffered artificial seawater was added respired at a constant rate for 5 to 10 min. Oxygen consumption was almost completely inhibited by antimycin A (20 pm/ ml). In several cases, the pH of the chamber contents was measured at the end of an experiment; the pH did not change more than 0.1 pH unit. RESULTS
Effect of the Acrosome Reaction Fertilizing Capacity
on Sperm
The effect of prior induction of the acrosome reaction on the fertilizing capacity of S. purpuratus sperm was investigated using a bioassay. The method employed is a sensitive measure of the number of sperm that are capable of fertilizing eggs, since it is carried out under conditions in which
52
DEVELOPMENTAL
BIOLOGY
sperm are limiting (Schmell et al., 1977). S. purpuratus sperm were first prereacted by exposure to jelly coat (Type B) for various lengths of time, and then aliquots were simultaneously fixed in glutaraldehyde or diluted and added to eggs to measure their ability to fertilize eggs. The results shown in Fig. 1A indicate that the acrosome reaction is complete in over 80% of the sperm after a 30-set exposure to jelly coat. As seen in Fig. lB, sperm exposed to jelly coat for 30 set fertilize eggs at a level similar to control sperm, which are not exposed to jelly coat and presumably undergo the acrosomal reaction only when they contact the egg. However, prior exposure of sperm 100
A
r
5 80 XA F \ 2 60 k! E g 40 :: 5 a
X’
l-f+
x-x
20 1
o\” ,oo[
to jelly for 60 set results in a marked decrease in fertility. Exposure times longer than 1 min result in a further decrease in fertilizing ability, with the maximum effect occurring after 5 min. From these results it is clear that during the first 30 set the reacted sperm have a fertilizing capacity equal to the control sperm. However, within the next 30 set, during which time an additional 5% of the sperm react, the ability to fertilize eggs drops by approximately 70%. This indicates that the reacted 100 v
A
5
; 80z: $ 60r" 0 40E? 5 20 ; "\ / X s m !
X
/
X
1
l
B
IOO-
p&# B
r
it Kx-x,
s
VOLUME 71, 1979
20
x-+--x--//-x PREINCUBATION TIME (MIN.1 FIG. 1. (A) Time course of the acrosome reaction in S. purpuratus sperm treated with jelly coat. Aliquots of sperm were fixed for electron microscopy after pretreatment with Tris-HCl-buffered artificial seawater as a control (W), A. punctulata jelly coat (Type B) (O-----O), or S. purpurutus jelly coat (Type B) (X-X) for the indicated length of tune. The percentage of acrosome reaction was determined as described in Materials and Methods. (B) Effect of jelly coat on the fertilizing capacity of S. purpuratus sperm. Sperm were pretreated as described in (A).
ZSO-u - xHX F i?i 60? \ I= a 402 8 20, I 12 PREINCUBATION (MIN.)
X
\, I
X
3 TIME
FIG. 2. (A) Time course of the acrosome reaction in A. punctuZuta sperm treated with A23187. Aliquots of sperm were futed for electron microscopy after pretreatment with A23187 (57 1LM) (X--X) or an equivalent amount of DMSO (solvent) (U) for the indicated length of time. (B) Effect of the ionophore A23187 on the fertilizing capacity of A. puntt&to sperm. Sperm were pretreated as described in (A).
KINSEY, SEGALL, AND LENNARZ
Physiological
Effects
of the Acrosome
Reaction
53
sperm (85% of the population) are no longer as effective in fertilization as the control, unreacted sperm. The low level of fertilization produced by sperm preincubated for as long as 10 min is probably effected by the remaining lo-15% of the sperm that remain unreacted. S. purpuratus sperm exposed to an equal concentration of jelly from A. punctulata did not undergo the acrosome reaction (Fig. 1A) and remained similar to control sperm in their capacity to fertilize S. purpuratus eggs (Fig. 1B). This species-specific effect of jelly coat material on both the acrosome reaction and the fertilizing capacity indicates that the effect on fertilization is probably the result of the occurrence of the FIG. 3. Effect of jelly coat on sperm respiration. s’. acrosome reaction, rather than some nonpurpuratus sperm were treated at zero time (arrow) specific, physical interaction of jelly with with (a) S. purpuratus jelly coat (Type B) (40 nmoles the sperm. To determine if other agents fucose/ml), or (b) Tris-HCl-buffered artificial seawawhich induce the acrosome reaction have a ter as a control, and allowed to respire for 15 min. similar effect on fertility, experiments were Aliquots (1~1) of cells were removed from the chamber carried out using the ionophore A23187 to just prior to zero time and at various times after the addition of jelly coat or seawater and fixed for electron prereact the sperm. Since A23187 induces microscopy. The percentage of acrosome reaction was the acrosome reaction more slowly than determined and is displayed in the inset: sperm plus jelly coat, we are unable to make a precise jelly coat (M), sperm plus Tris-HCI-buffered ) estimate of the fertile lifetime of sperm artificial seawater (X-X reacted by this method. The results in Fig. 2 show that the ionophore has little effect similar to that used in the previous experion either the acrosome reaction (Fig. 2A) ments. As seen in Fig. 3, a progressive deor the sperm fertilizing capacity (Fig. 2B) crease in the rate of On consumption beduring the first 60 sec. However, as the came apparent about 1.5 to 2 min after acrosome reaction begins to occur (2 min), addition of the jelly. The rate of respiration a decrease in fertilizing capacity becomes continued to decrease during the next 8 to apparent. 10 min, finally stabilizing at a greatly reduced level. During the course of this exEffect of Jelly Coat Treatment on Sperm periment, l-p1 samples were removed from Respiration the chamber and fixed for electron microsTo determine whether the jelly-induced copy. During the first 30 set, over 70% of effect on fertility could be explained by an the cells extruded acrosomal processes (ineffect on sperm viability, we examined the set, Fig. 3), yet there was no detectable effect of jelly coat material on the respiraeffect on the rate of oxygen consumption tion of S. purpuratus sperm. Sperm were during this time period. However, between added to the chamber of an oxygen elec- 1.5 and 2 min, the onset of a marked and trode apparatus and allowed to establish a continual decrease in the respiration rate constant respiration rate (Fig. 3). Once the occurs. The OZ consumption by sperm is respiration rate was constant, jelly coat almost totally inhibited by antimycin A, (Type B) was added at a concentration indicating that it represents mitochondrial
54
DEVELOPMENTAL BIOLOGY
respiration (Chance, 1957). Thus, the above findings suggest that one of the consequences of the acrosomal reaction is a cessation of mitochondrial respiration. At various times during this experiment, aliquots of sperm were removed and motility was examined by phase-contrast microscopy. An estimate of the percentage of sperm that were actively moving indicates that a general decline in the number of motile sperm occurred only after the addition of jelly. Only 5-10% of the sperm were motile 10 min after the addition of jelly. This small number of motile sperm probably represents unreacted cells and could account for the low but steady rate of oxygen consumption observed during the subsequent 10 to 15 min. In control experiments (seawater added instead of jelly), no appreciable change in the number of motile sperm was observed during the experiment, although the rate of swimming seemed to decrease after 8 to 10 min. We also found that when sperm were treated with the ionophore A23187 under conditions sufficient to induce the acrosome reaction, a similar decrease in respiration rate and motility occurred (data not shown). Induction of the acrosome reaction and the subsequent inhibition in respiration were dependent on the concentration of ionophore; concentrations of ionophore insufficient to induce the acrosome reaction had no effect on respiration. Effect of Jelly Coat Concentration on the Acrosome Reaction and on Respiration To further examine the relationship between the acrosome reaction and the respiration effect, we tested the dependence of these two events on the concentration of jelly coat. The degree of inhibition of respiration (Fig. 4A) and the extent of the acrosome reaction (Fig. 4B) increased with increasing concentrations of jelly coat (Type A). Little further change in either the respiration effect or the percentage of reacted sperm occurred at concentrations
VOLUME 71, 1979
1
5
I
I
I
I
10 15 20 25 nmFUCOSE/mL
FIG. 4. (A) Effect of the concentration of jelly coat (Type A) on sperm respiration. S. purpuratus sperm were treated (arrow) with jelly coat (Type A) at the indicated concentrations: (a) 0.0, (b) 0.8, (c) 2.0, (d) 4.0, (e) 8.0, (f) 16.0, or (g) 24.0 nmoles fucose/ml. (B) Samples were fixed for electron microscopy at the end of each run (12 to 15 min) and the percentage of sperm that underwent the acrosome reaction was determined. The rate of oxygen consumption was calculated at the 9.5- to 10.5-min interval for each run. Both parameters are expressed as a function of jelly coat concentration: acrosome reaction (M); respiration rate (X-X).
above 16 nmoles fucose/ml. As shown in Fig. 5, similar results were obtained when jelly coat (Type B) was used, although much higher concentrations were necessary. The apparent decrease in the biological activity of jelly coat upon dialysis is probably due to the loss of bound Ca2+ since, as discussed in the preceding paper (SeGall and Lennarz, 1979), full activity can be restored by the addition of CaCh. Jelly coat partially purified by gel filtration (see SeGall and Lennarz, 1979) was also effective (data not shown). These experiments clearly show that both events, i.e., the acrosome reaction and respiration loss, are
KINSEY, SEGALL, AND LENNARZ
Physiological
similarly dependent on the concentration of jelly coat and that “egg water” has the same effect as the more purified forms of jelly coat. Together with the previous ex-
Effects
of the Acrosome
55
Reaction
periments, this suggests that the decrease in respiration rate represents the death of the population of sperm which have undergone the acrosome reaction.
Species Specificity of Jelly Coat in Induction of the Acrosome Reaction and Respiratory Inhibition
1
I
100
200
1
J
1
300 400 500 600 nmFUCOSE/ml FIG. 5. (A) Effect of the concentration of jelly coat (Type B) concentration on sperm respiration. S. purpuratus sperm were treated (arrow) with jelly coat (Type B) at the indicated concentrations: (a) 0.0, (b) 7.2, (c) 60, (d) 300, or (e) 600 nmoles fucose/ml. (B) Samples were fixed for electron microscopy at the end of each run and the percentage of sperm that underwent the acrosome reaction was determined. TABLE INDUCTION
Incubation
OF THE ACROSOME
REACTION
time
1
BY EGGS CONTAINING
ATTACHED
JELLY
COATS
‘% Acrosome reaction S. purpuratus Artificial seawater
15 set 30 set 1 min 2 min 5 min 10 min
SolubiIized components of the jelly coat of S. purpuratus and A. punctulata (SeGall and Lennarz, 1978, 1979) induce the acrosome reaction in a species-specific manner. However, Summers and Hylander (19751, using other species of echinoids, demonstrated nonspecific induction of the acrosome reaction by eggs containing their surrounding “native” jelly. To confirm that the species specificity observed in the solubilized jelly coat of S. purpuratus and A. punctulata is retained in the native jelly coat surrounding eggs, we tested the ability of eggs with intact jelly coat to induce the acrosome reaction. It is clear from the results presented in Table 1 that the native jelly of A. punctulata and S. purpuratu,s eggs can induce the acrosome reaction only in the homologous sperm. Thus the speciesspecific properties of solubilized jelly coat (Types A and B) are also present in the jelly surrounding freshly spawned eggs and are not an artifact of preparation. It is apparent from these findings, coupled with those reported in the preceding paper
2 4 9
sperm plus __. -~-.S. _ purpuratus A. zmnctulatn _ em eggs 72 75 1 76 75 2 86 0 86
A. punctulata Artificial seawater
sperm plus A. nunctulatn ems
0
50
1 2
57 60
” Sperm from S. purpuratus (560 pg sperm protein) or a. punctulata (500 pg sperm protein) were added to Y! ml of a 50% (v/v) suspension of freshly collected eggs containing intact jelly coats. After various periods of time the cells were fixed for electron microscopy and the occurrence of the acrosome reaction was determined.
56
DEVELOPMENTAL BIOLOGY
(SeGall and Lennarz, 1979) and by Summers and Hylander (1975), that echinoid sperm show species variation in their ability to be reacted by heterologous jelly coat; some are species specific, whereas others are not. If the jelly-induced inhibition of sperm respiration is actually a consequence of the acrosome reaction, then the respiration effect should also be species specific. The results in Fig. 6 show that this is, in fact, the case. A. punctulata jelly has no inhibitory effect on the respiration of S. purpuratus sperm over a wide concentration range in which S. purpuratus jelly is effective (Fig. 6A). As expected, S. purpuratus jelly
1
0
I
100
4
5 TIME (Mitt?
I
I
I
I
,
’
15
I
300 500 700 nm FUCOSE/mt
FIG. 6. (A) Effect of jelly coat from S. purpuratus and A. pun&data on the respiration of S. purpuratus sperm. S. purpuratus sperm were treated (arrow) with jelly coat (Type B) from S. purpuratus or A. punctuZata eggs. (a) Control; (b) S. purpuratus jelly coat, 82 nmoles fucose/ml; (c) S. purpuratus jelly coat, 508 nmoles fucose/ml; (d) A. punctulata jelly coat, 227 nmoles fucose/ml; (e) A. punctulata jelly coat, 700 nmoles fucose/ml. (B) Samples were fixed for electron microscopy after each run and the occurrence of the acrosome reaction was determined. S. purpuratus jelly coat (M); A. punctulata jelly coat (A-A).
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induced the acrosome reaction in this species, whereas A. punctulata jelly did not (Fig. 6B). Similarly, S. purpuratus jelly had no effect on the respiration rate or the acrosome reaction of A. punctulata sperm at concentrations as high as 508 nmoles fucose/ml. DISCUSSION
The results presented here show that the following sequence of events occurs when S. purpuratus sperm are treated with homologous jelly coat: (1) The majority of the sperm undergo the acrosome reaction within 30 sec. Sperm which have undergone the acrosome reaction retain their ability to fertilize eggs during this time, since a sperm population containing 82% prereacted sperm can fertilize eggs as effectively as a control containing 1% reacted sperm. (2) By 60 set there is a dramatic decrease in fertilizing capacity, which stabilizes after 4 to 5 min at a greatly reduced level. (3) By 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration of the sperm is detectable. This decrease continues for 8 to 10 min, after which time the rate of respiration stabilizes at a greatly reduced rate. At this time only 5-10s of the sperm are actively swimming. The abrupt decline in fertilizing capacity and respiration observed in sperm treated with jelly coat can be explained in at least two ways. Jelly coat could cause the entire sperm population to be less effective in fertilization and to respire at a lower rate. Alternatively, the sperm which have reacted may become incapable of effecting fertilization and respire at a very low rate, while only the few unreacted sperm remain fully active and capable of fertilizing eggs. We favor the second interpretation for the following reasons. First, microscopic examination reveals that only a small proportion of the sperm population remains motile after treatment with a quantity of jelly sufficient to cause 90% of the sperm to undergo the acrosome reaction. Second, the magnitude of the jelly-induced depression
KINSEY, SEGALL, AND LENNAFU
Physiological
of respiration correlates roughly with the extent of the acrosome reaction and both events show a similar dependence on the concentration of jelly. Third, the effect of jelly on inducing the acrosome reaction, and on decreasing both the fertilization capability and the rate of respiration, is species specific, thus ruling out any nonspecific toxic effects. Our estimate of the fertile life of sperm following the acrosome reaction (30 set) is considerably shorter than that reported by Takahashi and Sugiyama (1973) for Pseudocentrodus depressus. This could be because our fertilization assays were done under sperm-limiting conditions that provide a more sensitive estimate of the number of viable sperm. The observation that jelly coat triggers a decrease in respiration rate is consistent with results obtained by several workers (Hayashi, 1946; Spikes, 1949; Rothchild, 1952) but not others (Gray, 1928; Vasseur, 1949; Hathaway, 1963; Ohtake, 1976, 1977). Ohtake, for example, has recently isolated a sperm activating substance from “egg water” (jelly coat, Type A). He found that when the pH of a sperm suspension was reduced from the normal value of 8.2 to 6.8, sperm respiration was suppressed. Addition of the sperm activating substance to the acidified suspension stimulated respiration back to the control level but not above it. However, the physiological significance of these studies involving exposure of sperm to seawater at pH 6.8 is not clear. Earlier studies reported up to threefold stimulation of respiration by egg water (Hathaway, 1963). The major difference between these early studies and our work lies in the concentration of sperm upon which the respiration measurements were made. Typical values were 1:lO diluted “semen” (Hathaway, 1963) and 1:6 diluted “dry” sperm (Vasseur, 1949). We estimate these concentrations to be about lo-15 mg sperm protein/ml, whereas we used about 0.6 mg sperm protein/ml. At the very high sperm concentrations used by those investigators, it is unlikely that a significant fraction of
Effects
of the Acrosome
Reaction
5:
the sperm had undergone the acrosome reaction since the ratio of egg jelly to sperm would have been much lower than that used in our studies. In fact, Hathaway (1963) reported that under his conditions no reacted sperm were detected. We feel that this point may explain some of the differences reported in the literature. At very high sperm concentrations, jelly coat may indeed stimulate respiration (by some unknown mechanism) without inducing the acrosome reaction. However, our results show conclusively that, under conditions which will support the acrosome reaction, jelly coat induces a decrease in respiration rate which is dependent on the concentration of jelly coat used and is roughly proportional to the percentage of sperm that have undergone the acrosome reaction. Why sperm that have undergone the acrosome reaction should so quickly lose t,heir respiration capacity is puzzling. The acrosomal vesicle is morphologically remote from the mitochondrion situated at the posterior of the sperm head, and there are no obvious interconnections between the two organelles. Moreover, while induction of the acrosome reaction causes a cessation of mitochondrial respiration, the converse is not true. Sperm that have been pretreated with antimycin A to block respiration are fully capable of undergoing the acrosome reaction (data not shown). Shackmann et al. (1978) have reported a similar insensitivity of the acrosome reaction to a number of other respiratory inhibitors and to uncouplers. Several studies (Decker et al., 1976; Collins and Epel, 1977; Tilney et al., 1977; Shackmann et al., 1978) have demonstrated that occurrence of the acrosome reaction requires an influx of Ca’+ ions. In addition, Shackmann et al. have shown that an efflux of protons occurs, and they suggest that countermovements of Na’ (in) and K’ (out) also are involved. Such ion movements during the acrosome reaction might ultimately cause inhibition of mitochondrial respiration. However, the mechanism that would
58
DEVELOPMENTAL BIOLOGY
cause such a change is unknown. One possibility is that jelly coat, in the presence of Ca2+ ions, may cause a general increase in the permeability of the plasma membrane and the mitochondria membrane, with a concomitant loss of oxidizable substrates. This seems unlikely, however, since when the mitochondrial substrates pyruvate, malate, or /?-hydroxybutyrate were added to inhibited sperm, respiration was not restored (data not shown). In any case, since not only jelly coat, but also the Ca2+ ionophore A23187, induces the acrosome reaction and the consequent respiratory inhibition, it seems clear that at least the initial step, the acrosome reaction, involves ion permeability changes. How the fucose sulfate polymer component of egg jelly brings about these changes remains a most challenging problem. As noted above, early workers implicated “fertilizin” or jelly coat material as a sperm receptor functioning in gamete recognition (see Tyler and Tyler (1966) for review). This theory is largely based on the observation that fertilizin can inhibit fertilization species specifically. Most of these studies were done by pretreating the sperm with jelly coat for as long as 15 to 20 min and then testing the ability of the sperm to fertilize eggs (Tyler and Tyler, 1966; Metz, 1961). Our results clearly show that such treatments have a very pronounced, species-specific inhibitory effect on sperm respiration. This respiration effect could account for the observed inhibitory effect of fertilizin on the fertilizing capacity of sperm. However, our work does not rule out the possibility that fertilizin also has a receptor-like function. For example, we observed that while treated sperm retain full fertilizing capacity for at least 30 see, this ability is greatly reduced by 60 sec. The fact that this decline precedes the first detectable decrease in oxygen consumption may indicate that fertilizin could have other effects on the fertilizing capacity of sperm. It is clear (Summers and Hylander, 1978;
VOLUME 71, 1979
SeGall and Lennarz, 1979) that in some echinoid species induction of the acrosome reaction, which is a prerequisite to fertilization, is not species specific. In these species, at least, jelly coat cannot be the component that dictates species specificity in fertilization. Other studies (Schmell et al., 1977; Tsuzuki et al., 1977; Glabe and Vacquier, 1977) have implicated receptors on the egg cell surface per se, rather than in the jelly coat, as the components that control species specificity. Clearly, depending on the particular echinoid used, either jelly coat or a cell surface receptor, or both, could define specificity. The results of the current study afford an approach to distinguishing between these possibilities, since it is now evident that sperm can be prereacted and utilized in fertilization studies, provided that the time interval between the acrosome reaction andfertilization is short. The authors wish to thank Mrs. Betty Earles for her technical assistance and Ms. Ann Fuhr for typing the manuscript. REFERENCES CHANCE, B. (1957). Techniques for the assay of the respiratory enzymes. Methods Enzymol. 4,273-328. COLLINS, F. (1976). A reevaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion. Develop. 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. 106,211-222. DAN, J. C. (1956). The acrosome reaction. ht. Reu. Cytol. 5,365-393. DECKER, G. L., JOSEPH, D. B., and LENNARZ, W. J. (1976). A study of factors involved in the induction of the acrosomal reaction in sperm of the sea urchin Arbacia punctulata. Develop. Biol. 53, 115-125. DISCHE, Z., and SHETTLES, L. B. (1951). A new spectrophotometric test for the detection of methylpentose. J. Biol. Chem. 192,579-582. GLABE, C. G., and VACQUIER, V. D. (1977). Species specific agglutination of eggs by bindin isolated from sea urchin sperm. Nature 267.836-838. GRAY, J. (1928). The effect of egg secretions on the activity of spermatozoa. J. Exp. Biol. 5,262-265. HAGSTROM, B. E. (1956). Studies of the fertilization of jelly-free sea urchin eggs. Exp. Cell Res. 10, 24-28. HATHAWAY, R. R. (1963). Activation of respiration in sea urchin spermatozoa by egg water. Biol. Bull.
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125,486-498. HAYASHI, T. (1946). Dilution medium and survival of the spermatozoa of Arbacia punctulata. II. Effect of the medium on respiration. Biol. Bull. 90, 177187. LILLIE, F. R. (1913). Studies on fertilization V. The behavior of the spermatozoa of Nereis and Arbacia with special reference to egg-extractives. J. Exp. Zool. 14,515-574. LOWRY, 0. H., ROSEBROLJGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. MCLELLAN, H. J. (1965). Elements of Physical Oceanography. Pergamon Press, New York. METZ, C. B. (1961). Use of inhibiting agents in studies on fertilization mechanisms. Znt. Rev. Cytol. l&219253. OHTAKE, H. (1976). Respiratory behavior of sea-urchin spermatozoa I. Effect of pH and egg water on the respiratory rate. J. Enp. 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. ROTHSCHILD, L. (1952). The behavior of spermatozoa in the neighborhood of eggs. Znt. Reu. Cytol. 1,257263. SHACKMANN, R. W., EDDY, E. M., and SHAPIRO, B. M. (1978). The acrosome reaction of Strongylocentrotus purpuratus sperm: Ion requirements and movements Develop. Biol. 65,483-495. SCHMELL, E., EARLES, B. J., BREAUX, C., and LENNARZ, W. J. (1977). Identification of a sperm receptor on the surface of the eggs of the sea urchin
Arbacia punctulata. J. Cell Biol. 72, 35-46. SEGALL, G. K., and LENNARZ, W. J. (1978). The role of jelly coat in the induction of the acrosomal reaction in sperm. J. Cell Biol. 79, 170a.
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SEGALL, G. K., and LENNARZ, W. J. (1979). Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Develop. Biol. 71,33-48. SPIKES, J. D. (1949). Metabolism of sea-urchin sperm. Amer. Natur. 83, 285-301. SUGIYAMA, M., and KATO, K. (1977). Effects of trypsin on the fertilizing capacity of sea urchin spermatozoa treated with egg-water. Develop. Growth Different. 19,23-29. SUMMERS, R. G., and HYLANDER, B. L. (1975). Species specificity of the acrosome reaction and primary gamete binding in echinoids. Exp. Cell Rex 96, 6368. TAKAHASHI, Y. M., and SUGIYAMA, M. (1973). Relation between the acrosome reaction and fertilization in the sea urchin. I. Fertilization in Ca-free sea water Develop. with egg-water-treated spermatozoa. Growth Different. 15, 261-267. 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 acrosomal reaction of echinoderm sperm. J. Cell Biol. 77, 536-550. TYLER, A. (1941). The role of fertihzin in the fertilization of eggs of the sea urchin and other animals. Biol. Bull. 81, 190-204. TYLER, A., and TYLER, B. S. (1966). Physiology of fertilization and early development. In “Physiology of Echinodermata” (R. A. Boolootian, ed.), pp. 683741. John Wiley and Sons, New York. TSUZUKI, H., YOSHIDA, M., ONITAKE, K., and AKETA, K. (1977). Purification of the sperm-binding factor from the egg of the sea urchin Hemicentrotus pul-
cherrimus. Biochem. Biophys. Res. Commun. 76, 502-511. VASSEUR, E. (1949). Effect of sea-urchin egg jelly-coat solution and calcium ions on the oxygen uptake of sea-urchin sperm. Ark. Kemi. 1,393-399.
