The Role of Calcium in the Acrosome Reaction: An Analysis Using lonophore A23187 P. TALBOT, R. G. SUMMERS,1*2B. L. HYLANDER? E. M. KEOUGH AND L. E. FRANKLIN Department of Biology, University of Houston, Houston, Texas 77004 and Department of Zoology, Uniuersity of Maine, Orono, Maine 04473
ABSTRACT The role of Ca+2in the acrosome reaction of echinoid and mammalian sperm was investigated using the Ca'2 transporting ionophore A23187. The ionophore induced morphologically normal acrosome reactions in both types of sperm (as assessed by electron microscopic observation of echinoid sperm and phase contrast microscopic observation of mammalian sperm). In echinoids, these reactions were immediate. In the guinea pig and hamster, ionophore significantly decreased the capacitation interval; early reactions were accompanied by activation of motility. Ionophore induced reactions were affected by sperm, ionophore and Ca' 2 concentrations. Since both ionophore induced and natural reactions require extracellular Ca'2, it is suggested that an influx of Ca+2represents the initial step of the acrosome reaction. Under natural conditions, the permeability change which results in Ca+einflux may be induced in echinoid sperm by egg jelly and may occur in mammalian sperm during capacitation. Ionophore A23187 should prove an experimentally useful drug for further study of the acrosome reaction since its effect on cells is understood, it induces synchronous reactions in a high percentage of sperm, and it conveniently reduces the capacitation interval in mammalian sperm.
The acrosome reaction of invertebrate and mammalian sperm comprises a series of morphological events which begin with membrane fusion or vesiculation and result in the exposure of the acrosomal vesicle contents by exocytosis (reviews by Colwin and Colwin, '67; Austin, '75). The absolute calcium dependency of the acrosome reaction has been well established in marine invertebrates (Dan, '54, '56, '67) and mammals (Barros, '74; Yanagimachi and Usui, '74). In the echinoderms, the presence of extracellular Ca'2 is required for the natural stimulus (egg jelly) to elicit the reaction, whereas in the mollusc, Mytilus, elevation of extracellular Cat alone will initiate reactions. Dan et al. ('72) have suggested that egg jelly might act by altering the permeability of the sperm plasma membrane to ions, especially Cat2, which are required for initiation of the reaction. In the case of the guinea pig, it has been suggested that the Cat2 binding J. EXP. ZOOL., 198: 383-392.
or transporting properties of the sperm plasma membrane are altered during capacitation (an undefined physiological change which precedes the reaction) and that these changes enable sperm to undergo acrosome reactions in the presence of extracellular Ca'2 (Yanagimachi and Usui, '74). The divalent cation ionophore A23187 binds to and transports Ca+2 across lipid barriers, including cell membranes (Reed and Lardy, '721, which makes it a useful probe for elucidating the role of Ca+2in certain cellular processes. Several exocytotic responses can be induced by A23187 (e.g., the cortical reaction of several marine invertebrate and vertebrate eggs: Chambers et al., '74; Lallier, '74; Steinhardt and Epel, '74; Steinhardt et al., '74; Schuetz, '75). 2 Present address: Department of Anatomical Sciences, State University of New York, Buffalo, New York 14216.
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TmBOT, SUMMERS, HYLANDER, KEOUGH AND FRANKLIN
The Ca+2 dependency and exocytotic nature of the acrosome reaction prompted investigations which demonstrated that A23187 induces the acrosome reaction of sand dollar and guinea pig sperm (Summers et al., '76). In the present study, ionophore A23187 was used to explore further the role of Ca+2in the acrosome reaction of echinoid and mammalian sperm. MATERIALS AND METHODS
Echinoid sperm The effect of ionophore A23187 on the acrosome reaction of echinoid sperm was assessed by treating sperm with ionophore and determining the percentage of reactions under various experimental conditions. The ability of ionophore to elicit reactions was compared to that of a second artificial stimulus, ammoniated sea water (ASW) which is known to induce normal reactions in echinoderm sperm (Dan, '54; Colwin and Colwin, '56). Ionophore A23187 (M. W. 523) was dissolved lmg/ml in dimethylsulfoxide (DMSO) and then diluted in sea water to concentrations from 0.001-50 pM. All ionophore preparations and experiments were conducted in darkness to prevent inactivation by light. Sperm were obtained from Echinuruchnius parma (sand dollar) and Strongylocentrotus droebachiensis (sea urchin) by injection with 0.53 M KCI and were pooled from at least five animals for each experiment. Replicate experiments were conducted with sperm from different males. Sperm maturity and motility were assessed by phase contrast microscopy; sperm concentrations were determined by hemocytometer counts. Acrosome reactions were scored by electron microscopic examination of sperm fixed in 1% glutaraldehyde and negatively stained (Dan, '52; Sanger and Sanger, '75). Each score was based upon examination of at least 100 sperm. Ionophore treated E. p u m a sperm were also embedded, sectioned and examined with the electron microscope to assess the morphological nor-
mality of the acrosome reaction (Summers and Hylander, '74). Four series of experiments were performed to determine the effect of ionophore and/or extracellular Ca+2 on the acrosome reaction: (1) Dosage response. To evaluate the relationship between ionophore concentration and the number of sperm which undergo the acrosome reaction, sperm (lO7/ml final concentration) were suspended in varying concentrations of ionophore (0.001-50pM), fixed after three minutes, and scored for percentage of acrosome reactions, Control sperm were treated with sea water (SW), SW 2.0% DMSO, ammoniated SW (ASW, pH 9.2, 4 mM NH,,OH) and ASW + 2.0% DMSO. (2) Kinetics. The percentage of acrosome reactions induced by ionophore and ASW were compared as a function of time. Sperm (106/ml final concentration) were suspended in either ASW or ionosphore (0.02pM- E. parma; 0.01pM- S. droebachimis). Controls were prepared as above. Sperm samples were removed at intervals from 5 seconds to 10 minutes, fixed, and scored for reactions. Regression analysis was performed according to Sokal and Rohlf ('69). (3) lon dependency. The effects of extracellular Ca+2 and M g + 2 on both ASW and ionophore induced reactions were examined. Complete or ion deficient (- Ca+2, - Mg+2,or - Ca+2and Mg+2)MBL artificial SW (Cavanaugh, '56) was used to prepare ASW and 0.02 pM ionophore. The Ca+ and Ca+zjMg+2deficient SW contained 2 mM EGTA. Sperm from E. p a m (107/mlfinal concentration) and S . droebachiensis (lW/ml final concentration) were incubated in these media for three minutes, fixed, and scored for reactions. (4) Calcium concentration. The relationship between the concentration of extracellular Ca+2 and the percentage of ionophore or ASW induced reactions was investigated. Sperm of S. droebachiensis (1061ml final concentration) were suspended in MBL SW containing either 0.005
+
385
CALCIUM AND THE ACROSOME REACTION
pM ionophore or ASW and varying amounts
of Ca+2(1/3-3times normal concentration = 9.27 mM). In controls, sperm were treated with SW containing varying Ca+2 concentrations but wrhout ionophore or ammonia. Mammalian sperm The effect of ionophore A23187 on the acrosome reaction of guinea pig and golden hamster sperm was assessed by incubating sperm in varying concentrations of ionophore and monitoring the percentage of reactions as a function of time. Sperm from the cauda epididymis and vas deferens of mature guinea pigs were collected in calcium free minimum capacitation medium (CF-MCM: Barros, '741, washed by centrifugation and resuspended in CF-MCM to a concentration of 108 sperm/ml. Minimum capacitation medium (MCM, pH 7.4-7.61, which supports occurrence of acrosome reactions and activated motility was used for incubation. Ionophore stock solutions were prepared i n DMSO and handled in darkness. Incubation tubes contained 450 pl MCM or CFMCM, 50 pl sperm suspension, and 5 pl of ionophore stock solution (2.4 or 7.4 pM). Final concentrations were 0.024 pM or 0.074 pM ionophore, 1%DMSO and lo7 spermlml. Control tubes contained either MCM or 1% DMsO/MCM in place of ionophore, In preliminary experiments, ionophore concentrations of 0.008 pM or less exhibited no effect and 0.22 pM or greater were lethal to guinea pig sperm. Incubations were performed under air in sealed Falcon test tubes (no. 2095) wrapped in aluminum foil and held horizontally at 37°C. The relationship between sperm and ionophore concentration was further investigated by incubating varying concentrations of guinea pig sperm (serial dilutions: 4 x lo7 - 2.5 x 106/ml) in 0.074 pM ionophore , Hamster sperm were collected and washed in normal saline. Since a defined medium which will consistently support the reaction and activation of hamster
sperm is not available, sperm were incubated in heat pretreated (60°C for 60 minutes) human serum. On the basis of preliminary experiments, final concentrations of ionophore were 0.5, 2.0, or 10 pM; incubation tubes were prepared and handled as above. For both species, incubations were initiated by the addition of sperm to the tubes. At various intervals up to four hours sperm samples were assessed for: (1)the percentage of motile sperm (100counted/sample) and (2) the percentage of motile sperm which had undergone acrosome reactions (50 counted/sample). Motile sperm which had lost the acrosomal cap were identified as reacted (compare figs. 10 and 11).Evaluations were made at 100 x (guinea pig) or 400 x (hamster) with a phase contrast microscope. Coverslips were not used with guinea pig sperm as such treatment may induce the reaction (Barros et al., '73). The motility of the majority of the sperm was qualitatively assessed as activated or nonactivated (Yanagimachi, '70; Yanagimachi and Usui, '74). Photomicrographs were made using a Wild-M-20 phase contrast microscope. RESULTS
Echinoid sperm Dosage-response data are presented in table 1. Ionophore A23187 induces acrosome reactions in both E. parma and S. droebachiensis sperm in a concentration dependent manner. Treatment of E. parma sperm with 0.01-10 pM ionophore or S. droebachiensis sperm with 0.01-0.02 pM ionophore elicits percentages of reactions not significantly different (> 0.05) from those obtained with ASW. Ionophore concentrations above and below these ranges induce significantly higher and lower percentages of reaction, respectively (< 0.05). Ultrastructural observations on negatively stained and sectioned spermatozoa indicate that the reactions are morphologically normal (figs. 6-91. The presence of DMSO has no effect on the occurrence of reactions (table 11, and sperm motility was normal in all cases.
386
TALBOT, SUMMERS, HYLANDER, KEOUCH AND FFUNKLIN TABLE 1
Dosage-response of echinoid sperm to ionophore A23187 % acrosome reaction
Ionophore concentration
6.W
0 (SW) 0 (SW+DMSO) 0 (ASW) 0 (ASW+DMSO) 0.001 0.0025
0.005 0.010 0.015 0.020 10.0 20.0 50.0 1
E. aarma (X 3 replicates 5 SEM)
S. droebachiensis (X 2 replicates t SEMI
3.0 t O 2.3 k 0.4 48.0 + O 48.0 + 0 6.3 20.4 12.0 + o 27.3 k0.4 41.0 k 0.6 46.3 I r t 0 50.0 1 0 58.0 Ik 0.6 60.0 kO.6 66.0 f 0.6
1.0 + 1.0 1.5 kO.41 62.0 + O 62.0 0 5.5 rt0.5 12.5 k0.5 26.0 k 1 54.5 1 k 0.5 65.0 1 0 74.5 12 0.5 85.0 k O 85.0 1 84.5 k 0.5
+
+ +
+
Not significantly different (p> 0.05) from ASW controls.
The kinetics of ionophore and ASW responses of E. parmu are compared in figure 1. The concentration of 0.02 pM was selected for these experiments because it induces approximately 50% reaction at three minutes (as does ASW) allowing maximum rate sensitivity to both treatments on either side of the 3-minute time point. Reaction kinetics were similar using S. droebachiensis sperm with 0.01 pM ionophore and ASW (not shown). During the first minute of treatment, the rates of acrosome reaction with A23187 and ASW do not differ significantly as indicated by regression analysis (E. parma: F = 1.14, df = 1,11, p > 0.05; S. droebachiensis: F = 1.13, df = 1,11, p>O.O5). Figure 1 also suggests that the rate of response of E. parma sperm to the ionophore from five seconds to one minute differs sharply from the rate during one to four minutes. Regression analysis confinns that the response is biphasic (F = 8.47, df = 1, 11, p < 0.05). The response of S. droebachiensis to 0.01 pM ionophore (not shown) is also biphasic when the time intervals five seconds to one minute and one to ten minutes are compared (F = 8.47, df = 1,11, p < 0.05). It is evident that the ionophore has induced the acrosome reaction in a population of sperm which is not responsive to ASW. However,
such a population is not distinguishable ultrastructurally . In order for either the ionophore or ASW to induce the acrosome reaction, exogenous Ca+2 must be present (table 2). M g ' 2 is not necessary nor may it substitute for Cat%in these species. Sperm motility was normal in all cases. Not only is exogenous Ca+2essential for induction of the reaction, but the percentage of acrosome reactions induced by 0.005 pM ionophore (fig. 2) is related stoichiometrically ke., directly proportional) to Ca+2concentration. As Ca+2concentration is increased, the percentage of acrosome reactions approaches 100%. ASW induced acrosome reactions are also limited by Ca+2concentrations below that of normal SW; however, in contrast to the ionophore effect, elevating the Ca'2 above the physiological level does not increase the percentage of acrosome reactions (60%)induced by ASW (fig. 2). In controls, elevation of Ca+2concentration alone did not induce reactions. Sperm motility was normal in all controls; however, in the sample containing 0.005 pM ionophore and 3 x Ca+2(= 27.8p M ) sperm motility had ceased by three minutes and some membrane damage was observed ultrastructurally. Membrane damage and loss of
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CALCIUM AND THE ACROSOME REACTION
motility also accompanied the use of ASW derwent early acrosome reactions and activation of motility. The results of a repreat high Ca+2concentrations. sentative experiment (4 performed) are Mammalian sperm shown in figure 3. Sperm incubated in Guinea pig sperm incubated in the pres- MCM alone (fig. 3D) reached a maximum ence of 0.074 or 0.024 pM ionophore un- percentage of acrosome reactions and showed activated motility by three hours. Incubation in 1%DMSO (fig. 3C) did not affect either event. In contrast, sperm reached the maximum response of controls within 15 minutes in 0.074 pM ionophore (fig. 3A) and within two hours in 0.024 pM ionophore (fig. 3B). It was determined that ionophore mediated reactions and activation are dependent on the presence of extracellular Ca+2.Neither acrosome reactions nor activation occurred during incubation of sperm in 0.074 or 0.024 pM
:I 70
0
1
2
3
4
5
TIME
6
7
8
9
1
0
(min)
Fig. 1 Kinetics of the acrosomereaction in E. parma as induced with 0.02 pM A23187 ( *-* ) and ammoniated sea water ( m 4 ) .Abscissa: Time in minutes. Ordinate: Percentage acrosome reactions. Points for two replicates are shown. TABLE 2
Percentage of acrosome reactions induced by ionophore A231 87 in MBL seawater of different ionic composition Complete ions -Ca*Z
- Ca'2 -&+2
-&+2
E. P a m
sw ASW SW + 0.02pM
A23187 S. droebachiensis
sw
ASW SW 0.02pM A23187
+
* Includes 2 mM EGTA.
5 55
1 2
4 55
1 3
55
61
54
41
1 61
0
1
1
1
59
0
60
0'
60
1'
0
2'
I 1
I
I 2
I
I 3
Co CONCENTRATION Fig. 2 Effect of Ca+*concentration on 0.005 pM, ionophore ( -0 ) and ammoniated seawater (A-A) induced acrosome reactions in S. droebachiensis. Abscissa: Ca' 2 concentration x 9.27 mM. Ordinate: Percentage of acrosome reactions. Points for two or three replicates are shown. Mean percentages of reactions at all concentrationsof Ca+2without ionophore or ASW were < 2%.
388
TALBOT, SUMMEXS, HYLANDER, KEOUGH AND FRANKLIN
80 -
a
-
0
2
I
3
4
(hr) Fig. 3 Induction of guinea pig sperm acrosome reaction by various concentrations of ionophore A23187 in MCM. Abscissa:Time in hours. Ordinate: Percentage of motile sperm which have undergone reaction. A. 0.074 pM A23187; B. 0.024pM A23187; C. 1%DMSO; D. MCM only. Sperm concentration = lO7/ml. Inset: Percentage of motile unreacted sperm ( * - - ) and percentage of motile reacted sperm ( -0 ) during four hours incubation in MCM. Reactions were induced by ionophore (0.07pM). TIME
- - *
'
0
°
TIME ( h r ) Fig. 4 Effect of sperm concentration on ionophore (0.07 pM) induced acrosome reactions of guinea pig sperm. Abscissa: Time in hours. Ordinate: Percentage of motile sperm which have undergone reaction. ( * - * ) 2 x 106 sperm/ml + A23187; ( D 4 ) lo7 sperm/ml A23187; (A-A) 4 x lo7sperm/& A23187; ( * - - - - 0 ) 2 x 106 sperm/ml 1%DMSO; .I - - - 4 )107 sperm/ml 1% DMSO: (A - - - - A 1 4 x lo7 spermlml 1% DMSO.
+
+
+
ionophore in CF-MCM. However, sperm acrosomes did become crenulated (fig. 121, resembling early stages of the normal reaction. During in vitro incubation, guinea pig sperm suspensions exhibited a period of maximum reaction, following which both reacted and unreacted sperm died. Death of reacted sperm preceded that of unreacted sperm, and consequently the percentage of reacted sperm declined following the maximal period of reaction. This se-
+
+
quence of events is reflected in figure 3 (inset). The viability of reacted sperm, i.e., the breadth of the reaction peak, was not affected by ionophore induction of reactions at the concentrations employed. As the concentration of sperm to ionophore was increased, induction of both the reaction and activation were increasingly delayed (fig.4). For each sperm concentration, the maximum percentage of reaction surpassed that of the corresponding control. Additionally, control data establish
~
389
CALCIUM AND THE ACROSOME REACTION 100
80 W
60 Z W
k? W
40
a
20
0
2
I
TIME
3
4
(hr)
Fig. 5 Induction of hamster sperm acrosome reactions by ionophore A23187 in serum. Abscissa: Time in hours. Ordinate: Percentage of motile sperm which have undergone reactions. A. 10 pM A23187; B. 2 pM A23187; C. 1% DMSO; D. Serum only. Sperm concentration = lO7/ml.
reacting cells can be readily assessed under experimental conditions and thus the possibility of secretion as a consequence of cell death eliminated. The magnitude of the secretory response in a sperm population is easy to evaluate quantitatively since the reaction involves ex- * ocytosis of a single vesicle and in many species the reaction is directly observable with phase contrast microscopy. Unlike other systems which can be triggered by intracellular Ca+2 stores, extracellular Ca+2is required for the acrosome reaction and thus the function of Ca+2in regulating secretion can be directly examined in this system. Since ionophore A23187 transports Ca+2 across cellular membranes, it was used for examining the function of Ca+2in the sequential events of the acrosome reaction. Our results establish that ionophore induces: (1) immediate and morphologically normal acrosome reactions in echinoid sperm, (2)early acrosome reactions and activation of motility in mammalian sperm. These ionophore induced responses are dependent on the presence of extracellular Ca+2. DISCUSSION The Ca+2 dependency of natural acroThe acrosome reaction provides several some reactions has been previously estabadvantages over other systems for the lished. In echinoids it was been shown that study of exocytotic secretion. Since the egg jelly fails to elicit reactions in Ca+2dereaction occurs in motile sperm, viability of ficient media (Dan, '54; Gregg and Metz, that the percentage of guinea pig sperm which normally react in MCM alone varies as a function of sperm concentration (fig. 4) as previously reported for other mammalian species (e.g, Talbot et al., '74). The guinea pig sperm acrosome reaction is unusual in that it occurs readily in a simple defined medium. Since this is not the case for most mammalian sperm, the effect of A23187 on the acrosome reaction of hamster sperm was assessed during incubation in serum. A representative experiment (5 performed) is shown in figure 5. Ionophore induced: (1) early occurrence of acrosome reactions and (2) a higher percentage of reactions than in controls. In contrast to the situation for guinea pig sperm, ionophore induced reactions did not occur before one and a half hours in any of the experiments. The absolute ionophore concentration which produced results equivalent to those in figure 5 was variable among sera. Hamster sperm did not undergo normal reactions in the presence of ionophore when incubations were carried out in MCM, Tyrode's or Hanks media.
390
TALBOT, SUMMERS, HYLANDER. KEOUGH AND FRANKLIN
'76). Similarly, guinea pig sperm become capacitated, but do not react in the absence of Ca+2(Yanagimachiand Usui, '74). The present investigation establishes that artificial induction of the echinoid sperm reaction by ASW and ionophore is also dependent on the presence of extracellular Ca+2 (also Collins, '76). Likewise, ionophore induced reactions of guinea pig sperm have an absolute requirement for extracellular Ca+2. Sperm of E. p a m a and S. droebachiensis, which do not require capacitation, undergo immediate and morphologicallynormal acrosome reactions in the presence of ionophore and extracellular Ca+2.The percentage of ionophore induced reactions varies as a function of sperm (lO8-lO9/ml, unpublished data), ionophore (table 11, and extracellular Ca+2 concentrations (fig. 2). Since the percentage of ionophore induced reactions (over the entire scale 0-100%) and the concentration of extracellular Ca+2 are stoichiometrically related, it appears that exogenous Ca+2 concentration is the most important variable which directly limits the percentage of reactions induced by ionophore. These observations strongly suggest that the initial step of the acrosome reaction is the influx of extracellular Ca+2. A change in membrane permeability which would allow such an influx may be brought about naturally by egg jelly or artificially by pH change and/or ammonia. The 2- to 4-hour incubation period preceding in vitro reaction of guinea pig and hamster sperm is required for capacitation. In the presence of ionophore, the capacitation interval is reduced and sperm are induced to undergo early acrosome reactions. The magnitude of this effect is dependent on both ionophore and sperm concentration. These results support the idea that: (1)capacitation involves a change in membrane permeability to Ca+2 and (2) Ca+2influx initiates the sequential events of the acrosome reaction. It is interesting that in contrast to guinea pig sperm (which underwent ionophore induced reactions immediately), hamster sperm did not respond to ionophore during
the first one and a half hours of incubation. Apparently capacitation of hamster sperm involves requisite events which precede those initiated by Ca+2influx. It is important to emphasize that although capacitation requirements are variable among phylogenetic groups, the mechanism for initiating the acrosome reaction in the invertebrate and mammalian species of this study appears to involve an influx of Ca+2.Although the manner in which egg jelly, increased pH, ammonia, or capacitation mediates this influx remains to be determined, it seems probable that stimuli which induce the reaction in these and perhaps other species act to increase membrane permeability to Ca+2. ACKNOWLEDGMENTS
This investigation was supported by NIH Grant HD07346 awarded to L. €3. F. and by NSF pre-doctoral Fellowship HES 74 22400 awarded to E. M. K. We thank Dr. R. Hamill of Eli Lilly Co.for his generous gift of ionophore A23187 and Drs. R. J. Talbot and J. Ringo for their suggestions regarding the manuscript. LITERATURE CITED Austin, C. R. 1975 Membrane fusion events in fertilization. J. Reprod. Fertil., 44: 155-166. Barros, C. 1974 Capacitation of mammalian spermatozoa. In: Physiology and Genetics of Reproduction. Part B. E. Coutinho and F. Fuchs, eds. Plenum Publishing Co.,New York, pp. 3-24. Barros, C., M. J3errios and E. Herrera 1973 Capacitation in uitro of guinea pig spermatozoa in a saline solution. J. Reprod. Fertil., 34: 547-550. Cavanaugh G . M. 1956 Formulae and methods V. of the Marine Biological Laboratory Chemical Room. Fifth ed. Marine Biological Laboratory, Woods Hole, 87 pp. Chambers, E. L., B. C. Pressman and B. Rose 1974 The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A. Biochem. Biophys. Res. Comm., 60: 126-132. Collins, F. 1976 A re-evaluation of the fertilizin hypothesis of sperm agglutination and the description of a novel form of sperm adhesion. Devel. Biol., 49: 381-394. Colwin, L. H., and A. L. Colwin 1956 The acrosome filament and sperm entry in Thyone briarezrs (Holothuria) and Asterins. Biol. Bull., 11 0: 243-257. 1967 Membrane fusion in relation to sperm egg association. In: Fertilization. Comparative Mor-
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CALCIUM AND THE ACROSOME REACTION
phology, Biochemistry and Immunology. C. B. Metz and A. Monroy, eds. Academic Press, New York, pp. 295-367. Dan, J. C. 1952 Studies on the acrosome I. Reaction to egg water and other stimuli. Biol. Bull., 103: 54-66. 1954 Studies on the acrosome 111. Effect of calcium deficiency. Biol. Bull., 107: 335-349. 1956 The acrosome reaction. Int. Rev. Cytology, 5: 365-393. 1967 Acrosome reaction and lysins. In: Fertilization. Comparative Morphology, Biochemistry and Immunology. C. B. Metz and A. Monroy, eds. Academic Press, New York, pp. 237-293. Dan, J. C., Y. Kakizawa, H. Kushida and K. Fujita 1972 Acrosomal triggers. Exp. Cell Res., 72: 60-68. Gregg, K. W., and C. B. Metz 1976 Physiological parameters of the sea urchin acrosome reaction. Biol. Reprod., 14: 405-41 1. Lallier, R. 1974 Induction of fertilization membrane elevation by ionophore A23187 in sea urchin egg Paracentrotus lividus. Exp. Cell Res., 89: 425-426. Reed, P. W., and H. A. Lardy 1972 A23187: A divalent cation ionophore. J. Biol. Chem., 247: 6970-6977. Sanger, J. W., and J. M. Sanger 1975 Polymerization of sperm actin in the presence of cytochalasin-B. J. EXP.hi. 193: , 441-447. Schuetz, A. W. 1975 Cytoplasmic activation of
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39 1
starfish oocytes by sperm and divalent ionophore A23187. J. Cell Biol., 66: 86-94. Sokal, R. R., and F. J. Rohlf 1969 Biometry: The Principles and Practice of Statistics in Biological Research. W. H. Freeman, San Francisco. Steinhardt, R. A., and D. Epel 1974 Activation of sea urchin eggs by a calcium ionophore. Proc. Nat. Acad. Sci. (USA.), 71: 1915-1919. Steinhardt, R. A., D. Epel, E. J. Carroll and R. Yanagimachi 1974 Is calcium ionophore a universal activator for unfertilized eggs? Nature, 252: 41-43. Summers, R. G., and B. L. Hylander 1974 An ultrastructural analysis of early fertilization in the sand dollar, Echinarachnius parma. Cell Tiss. Res., 150: 343-368. Summers, R. G., P. Talbot, E. M. Keough, B. L. Hylander and L. E. Franklin 1976 Ionophore A23187 induces acrosome reactions in sea urchin and guinea pig spermatozoa. J. Exp. Zool., 196: 381-386. Talbot, P., L. E. Franklin and E. N. Fussel 1974 The effect of the concentration of golden hamster spermatozoa on the acrosome reaction and penetration in oitro. J. Reprod. Fert., 36: 429-432. Yanagimachi, R. 1970 The movement of golden hamster spermatozoa before and after capacitation. J. Reprod. Fert., 23: 193-196. Yanagimachi, R., and N. Usui 1974 Calcium dependence of the acrosome reaction and activation of the guinea pig spermatozoon. Exp. Cell Res., 89: 161-174.
CALCIUM AND THE ACROSOME REACTION Talbot, Summers, Hylander, Keough and Franklin
EXPLANATION OF FIGURES
6 Negatively stained, unreacted E. p u m sperm. x 62,000.
7 Mid-sagittal section of the acrosomal region of an unreacted E. p u m a sperm. x 58,000.
8 Negatively stained, ionophore reacted E. purrnu sperm. x 51,000. 9 Section of ionophore reacted E. p u m sperm. x 51,000. 10 Unreacted guinea pig sperm with intact acrosomal cap. x 2,500. 11 Acrosome reacted guinea pig sperm which has lost the acrosomal cap. x 2,500.
12 Guinea pig sperm incubated in 0.074 pM ionophore/CF-MCM with crenulated acrosomal cap characteristic of the early stages of acrosome reaction. x 2,500.
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PLATE 1