THE JOURNAL OF EXPERIMENTALZOOLOGY 264:231-235 (1992)
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The Ionophore-InducedAcrosome Reaction Differs Structurally From the Spontaneous Acrosome Reaction P.F. WATSON, J.M. PLUMMER, AND P.S. J O N E S Department of Veterinary Basic Sciences, Royal Veterinary College, London NW1 OTU, United Kingdom
ABSTRACT
The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-inducedvesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation. 01992 Wiley-Liss, Inc.
ing better than 70% motility were considered acceptable. For experiments 1 and 2, semen from three rams was pooled. The standard diluent was a glucose-saline medium buffered with HEPESNaOH and containing 3 mM CaC12 (Watson et al., '91). For the ionophore-inducedacrosome reaction, calcium ionophore A23187 (free acid; Sigma Chemical Co. Ltd., Poole, Dorset, UK) was dissolved in dimethyl sulphoxide (DMSO)and added to a diluted suspension of spermatozoa (lo8spermatozoa/ml)at a final concentration of 1 pm A23187 and 1% DMSO (Shams-Borhan and Harrison, '81). The sample was then incubated at 39°C. Control tubes had DMSO added but no ionophore, and this treatment was shown to be without effect (Watson et al., 1991). The spontaneous acrosome reaction was investigated by incubation of the semen for 4 h at either 20 or 39°C; the semen was either undiluted or diluted to a concentration of 2 x lo8 spermatozoa/ml. At fixed periods after the commencement of incubation, samples were removed for fixation for transmission electron microscopy (Plummer and Watson, '88). Electron micrographs of thin sections were taken at a magnification of 15,000 x ,and 300 sperm head sections displaying both the acrosomal ridge and a portion of the equatorial segment were MATERIALS AND METHODS selected randomly from each treatment. SpermaSemen was collected by artificial vagina from tozoa were classified into four categories: 1)intact, Finnish Landrace crossbred rams. Immediately after transfer to the laboratory (15 min) a sample Received October 9,1991; revision accepted J u n e 26,1992. of semen was examined, and only ejaculates show-
An acrosome reaction has been initiated in the spermatozoa of several species by means of the divalent cation ionophore, A23187 (Talbot et al., '76; Green, '78; Shams-Borhan and Harrison, '81; Lee et al., '87; Varner et al. '87; Didion et al., '89; Ruknudin, '89; de Jonge et al., '89; Lax et al., '90; Zhang et al., '90). The ionophore achieves this end by facilitating the passage of calcium ions across the plasma membrane (Pressman, '761, resulting in a higher than threshold level of calcium between the outer acrosomal membrane and the overlying plasma membrane. Calcium ions are a prerequisite for the multiple fusion between these membranes which characterises the acrosome reaction (Yanagimachi and Usui, '74). Several authors have published electron micrographs of the ionophoreinduced acrosome reaction and, apart from the difference in timing of the event, have reported that this reaction resembles the spontaneous acrosome reaction (Talbot et al., '76; Green, '78; Jamil and White, '81; Flechon et al., '86). We have undertaken a study of both the spontaneous and the ionophore-induced acrosome reactions in ram spermatozoa, and in the course of this study we have observed some distinct differences which are reported here.
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2) cells displaying a classical acrosome reaction of multiple fusion between the outer acrosomal membrane and the overlying plasma membrane with the formation of hybrid vesicles, 3) cells displaying rupture of the plasma membrane with acrosomal deterioration, and 4)spermatozoa with a missing acrosome. For experiment 3, a total of 40 profiles was examined from each ram: 10 profiles each from 10 and 20 min ionophore treatments, and 10 each from the 2 and 4 h incubation treatments. The numbers of vesicles in a fixed length of membrane (and hence, by inference, the vesicle size) were estimated as follows: a 5 mm grid square on clear acetate was placed over each acrosome-reacted sperm profile just anterior to the equatorial segment; the number of vesicles within a span of 5 grid squares was counted on each face of the spermatozoon, providing a total count for 10 grid squares per sperm covering a total distance on the sperm head of 3.33 pm. The mean counts for sampling times within each treatment were compared by uncorrelated Student's t-test. The combined results from the two sampling times in each treatment were analysed by a hierarchal analysis of variance.
RESULTS The timing of the ionophore-induced acrosome reaction is shown in Table 1.At time zero, few sperm showed evidence of an acrosome reaction (defined as multiple fusions between the plasma membrane and the underlying outer acrosomal membrane in the region of the main part of the acrosome). By 15 min, a proportion of spermatozoa displayed the acrosome reaction, and by 30 min greater than 90% were showing an acrosome reaction. In this experiment, very few sperm showed evidence of deterioration of the acrosome. The timing of the spontaneous acrosome reaction induced by dilution and incubation at 39°C is shown TABLE 1. Timing of the ionophore-induced acrosome reaction i n ram spermatozoa' 0
Normal intact Acrosome reacted Damaged or missing acrosome'
95 4
1
Time ( m i d 15 30
60
37 61 2
0 93 7
1 93 6
'Numbers show the percentage of spermatozoa, observed by electron microscopy, showing evidence of the acrosome reaction as shown in Figure 1. 2Damaged cells had lost plasma membranes or showed degeneration of the acrosome not involving fusion of the plasma and outer acrosomal membranes. A missing acrosome was identified in a small percentage of cells which had also lost the plasma membrane over the acrosome.
in Table 2. A greater proportion of the spermatozoa were damaged at the commencement of the experiment compared with the previous experiment, but the proportion did not change markedly with incubation. Greater than 75%of the cells were intact at time zero, and in those samples held at room temperature no change took place over the 4 h incubation. However, when incubated at 39"C,the sample diluted before incubation, but not that remaining undiluted, showed a considerable development of acrosomal fusion characteristic of an acrosomal reaction. On closer examination of the pattern of acrosome reactions seen in the two treatments incubated at 39"C, diluted, and ionophore-induced, the mean number of vesicles in 3.33 pm span of membrane profile in each treatment group was found to differ (Table 3). No significant difference was observed in numbers of vesicles between 10 and 20 min ionophore treatments, nor between 2 and 4 h incubation treatments although in two rams (A and B) fewer vesicles were seen at 4 h than at 2 h. The results from the two times were pooled for each treatment, and the pooled data were analysed by hierarchal analysis of variance. Differencesbetween rams were not significant, but differences between treatments within individual were very highly significant (P< 0.001). There were fewer vesicles in a fixed span of membrane in the ionophore treatment than in the spontaneous acrosome reaction. Typical sperm profiles from the two treatments are shown in Figure 1.The ionophore-inducedacrosome reaction revealed a larger range of sizes of vesicles which were more irregularly shaped compared with those formed in the spontaneous acrosomereaction. Especially in the ionophore-induced acrosome reactions, vesiculation of the equatorial segment was frequently noted. The actual vesicle sizes were frequently too small to measure accurately. However, the vesicle counts for individual 5 mm grid squares were the data most closely reflecting vesicle size. The apparent greater heterogeneity of vesicle size in ionophore-treated spermatozoa was examined formally by a two-tailed variance ratio test of these counts for each treatment. The variance ratio for 600 data items in each treatment was 1.665L.287 = 1.29 (P< 0.05).
DISCUSSION The calcium ionophore, A23187, induced an acrosome reaction as previously reported by others (Shams-Borhanand Harrison, '81; Jamil and White, '81; Flechon et al., '86; Watson et al., '91). In this quantitative electron microscopy study, it was clear
IONOPHORE VS. SPONTANEOUS ACROSOME REACTIONS
233
TABLE 2. Eflect of temperature and ditution on the spontaneous acrosome reaction in ram spermatozoa'
Time (h) Treatment2 Intact Acrosome reacted Damaged or missing acrosome3
0 Undil
0 Dil
20 4 Undil
76 1 23
75 3 22
70 5 24
Temperature ("C) 20 39 4 4 Dil Undil 70 4 26
39 4 Dil
74 5 21
-
4 85 11
'Numbers give the percentage of spermatozoa, observed by electron microscopy, showing evidence of the acrosome reaction as shown in Figue 1. 'Undil = undiluted semen; dil = diluted to 2 x 10' spermatozodmil. 3Seefootnote to Table 1.
that virtually the entire population of spermatozoa responded to A23187. However, we and others (Talbot et al., '76; Green, '78; Shams-Borhan and Harrison, '81; Watson et al., '91) have reported that the timescale is very different from that of a spontaneous event. The latter was clearly dependent both on temperature and on dilution. The temperature dependence was more closely studied and it was also demonstrated that at the temperature used in the present study individual variation was minimal (Watson et al., '91). The effect of dilution was suprising, and may be due t o the dilution of a calcium chelating protein in the seminal plasma (San Agustin et al., '87) and/or to an effect upon calcium permeability induced by dilution (Robertson and Watson, '86). It is not due to the concentration of calcium in the diluent, since some spermatozoa underwent an acrosome reaction in the absence of added calcium (unpublished observation). The region just anterior to the equatorial segment was chosen for vesicle measurement because it has previously been shown that this region is the site of the initiation of the acrosome reaction (Flechon et al., '86; Watson and Plummer, '86), and
could be assumed to display the most complete stage of the acrosome reaction anywhere in the acrosomal membrane. Although the appearance of the acrosome reaction induced by the ionophore A23187 is similar to that occurring spontaneously, it is not identical. The observation presented here that the number of vesicles in a given span of membrane is fewer in an ionophore-induced acrosome reaction provides clear evidence of the difference. The counting of the number of vesicles in a fixed span permitted the inference to be drawn that the average size of the vesicles formed in the ionophoreinduced reaction was larger. Indeed, visual inspection of the micrographs suggested that under this treatment, the vesicles were heterogeneous in size, in contrast to those formed in the spontaneous event where they appeared smaller and more homogeneous in size. The mean calculated diameter ( + SEM) of the vesicles was 60.13 i- 1.61and 84.47 & 1.01 nm for the spontaneous and the ionophoreinduced events, respectively. A variance ratio test of the data confirmed that this difference was significant. It is probable that this observed difference per-
TABLE 3 . Mean numbers of vesicles i n the acrosome reaction' Time Ram A Ram B Ram C Mean
10 min
Ionophore-induced 20 min
Mean
38.7 t 1.11 44.8 rt 2.32 35.1 2 1.10 39.5 & 1.17
39.9 t 1.30 39.2 t 2.03 38.8 t 1.74 39.3 rt 0.96
39.3 2 0.84 42.0 rt 1.63 37.0 2 1.09 39.4 t 0.75
2h 60.0 t 63.6 2 48.1 & 57.2 k
-
Spontaneous 4h 1.53 1.59 1.82 1.54
54.0 t 1.03 54.6 rt 1.71 52.0 t 2.00 53.5 k 0.94
Mean 57.0 t 1.13 59.1 1: 1.53 50.1 t 1.40 55.4 f 0.92 -
Summary of hierarchal analysis of variance ofpooled data from different times within treatment and ram Source of variation Between rams Treatments within ram Cells within treatment
Degrees of freedom
Mean square
2 3 114
532.06 2548.42 34.45
-
Variance ratio 0.21 73.97***
'Figures are mean numbers of vesicle counted in a 3.33 +m section of sperm plasma membrane just anterior to the equatorial segment. Means of 10 sperm head sections. ***P< 0.001.
234
P.F. WATSON ET AL.
tains to the state of capacitation of the membranes. Whereas by the action of ionophore, calcium is permitted access in high concentration to the entire cytoplasmicspace between the reactive membranes, it is only able to gain access slowly and progressively during the incubation preceding the spontaneous reaction as the membranes become more permeable to calcium. Capacitation also involves subtle modifications of the membranes in preparation for membrane fusion, perhaps including isothermic phase separations of the lipids (Friend et al., '77; Flechon, '85) which favour fusion. In the ionophore-inducedacrosome reaction, the formation of larger, more irregular vesicles suggests a more disordered progression of membrane fusion. Indeed, Klausner et al. ('79) suggested that A23187 actually disrupts membrane structure by disordering protein-lipid interactions. The equatorial segment vesiculation seen in the presence of ionophore also suggests a more pronounced fusion event than that seen spontaneously; Shams-Borhan and Harrison ('81) also noticed this phenomenon. However, perhaps not too much should be made of this since veisculation of the equatorial segment has been noted before in fertilizing spermatozoa (Yanagimachi and Noda, '70; Vigil, '89). In contrast, in the spontaneous event, a gradual influx of calcium, perhaps restricted to discrete regions and coinciding with the alterations of membrane organization facilitating fusion, results in an orderly progression of fusion events. The small, more evenly sized, regularly shaped vesicles suggest that in this case the points of fusion are more frequent and regularly spaced along the membranes. While there is no presumption that the spontaneous acrosome reaction is necessarily normal, it would be a very difficult proposition to prove. It is unlikely t o result in spermatozoa that could penetrate the egg, most likely due not t o the artificial environment or abnormality of the event, but to its occurring far from the zona surface (Crozet and Dumont, '84). Nevertheless, in the spontaneous event, the temperature was comparable to that of the female tract, the timing of the event compared favourably with the suggested capacitation time for ram spermatozoa of 1.5 h (Mattner, '631, and there was no known cellular toxin present. In addition, motility was maintained until after the acrosome reactions had occurred (Watson et al., '91). In contrast, the ionophore-induced acrosome reaction occurred rapidly and extensively, and motility declined rapFig. 1. Electron micrographs of acrosome reactions in ram idly, in a manner quite different from that which spermatozoa (a)occurring spontaneously, or (b) induced by is presumed to happen in vivo. ionophore A23187. Bar = 1 pm.
IONOPHORE VS. SPONTANEOUS ACROSOME REACTIONS
235
reaction in mammalian spermatozoa. Biochim. Biophys. Acta These observations represent a caution to the use 1043:12-18. of A23187 as a simple means of inducing acrosome M.A., G.S. "rucco, K.B. Bechtol, N. Wummer, G.S. Kopf, reactions. Although ionophore-treatedram sperma- Lee, L. Blasco, and B.T. Storey (1987) Capacitation and acrosome tozoa were found to be capable of fusing with zonareactions in human spermatozoa monitored by a chlortetrafree sheep eggs, Shams-Borhan and Harrison ('81) cycline fluorescence assay. Fertil. Steril. 48:649-658. were unable t o achieve in vitro fertilization. Thus, Mattner, P.E. (1963) Capacitation of ram spermatozoa and penetration of the ovine egg. Nature 199:772-773. there is insufficient evidence t o sustain the arguPlummer, J.M., and P.F. Watson (1988) The quantitative ultrament that the ionophore-induced reaction is, in structural assessment of head membrane damage in boar sperevery respect, normal. Indeed, if the morphology matozoa subjected to varying degrees of cold shock. Anim. of the events is under examination it is essential Reprod. Sci. 16:265-275. to use a method of inducing the acrosome reaction Pressman, B.C. (1976) Biological applications of ionophores. Annu. Rev. Biochem. 45500-530. which results in events which resemble the true Robertson, L., and P.F. Watson (1986) Calcium transport in acrosome reaction as closely as possible.
ACKNOWLEDGMENTS This work was supported by grants from The Wellcome Trust and from the University of London Special Research Fund.
LITERATURE CITED Crozet, N., and M. Dumont (1984) The site of the acrosome reaction during in vivo penetration of the sheep oocyte. Gamete R ~ s10:97-105. . De Jonge, C.J., S.R. Mack, and L.J. Zaneveld (1989) Synchronous assay for human sperm capacitation and the acrosome reaction. J. Androl. 10:232-239. Didion, B.A., J.R. Dobrinsky, J.R. Giles, and C.N. Graves (1989) Staining procedure t o detect viability and the true acrosome reaction in spermatozoa of various species. Gamete Res., 22:51-57. Flechon, J.-E. (1985)Sperm surface changes during the acrosome reaction as observed by freeze-fracture. Am. J . Anat. 174:239-248. Flechon, J.-E., R.A.P. Harrison, B. Flechon, and J. Escaig (1986) Membrane fusion events in the Ca'+iionophore-induced acrosome reaction of ram spermatozoa. J. Cell Sci. 81:43-63. Friend, D.S., L. Orci, A. Perrelet, and R. Yanagimachi (1977) Membrane particle changes attending the acrosome reaction in guinea-pig spermatozoa. J. Cell Biol. 74.561-577. Green, D.P.L. (1978) The induction of the acrosome reaction in guinea-pig spermatozoa by the divalent cation ionophore A23187. J. Cell Sci. 32:137-151. Jamil, K., and I.G. White (1981) Induction of acrosome reaction in spermatozoa with ionophore A23187 and calcium. Arch. Androl. 7:283-292. Klausner, R.D., M.C. Fishman, and M.J. Karnovsky (1979) Ionophore A23187 disrupts membrane structure by modifying protein-lipid interactions. Nature 281 :280-281. Lax, Y., S. Grossman, S. Rubenstein, N. Magid, and H. Breitbart (1990) Role of lipoxygenase in the mechanism of acrosome
diluted or cooled ram semen. J. Reprod. Fert. 77:177-185. Ruknudin, A. (1989) Cytochemical study of intracellular calcium in hamster spermatozoa during the acrosome reaction. Gamete Res. 22:375-384. San Agustin, J.T., P. Hughes, and H.A. Lardy (1987) Properties and function of caltrin, the calcium-transport inhibitor of bull seminal plasma. FASEB J . 1 :60-66. Shams-Borhan, G., and R.A.P. Harrison (1981)Production, characterization, and use of ionophore-induced,calcium-dependent acrosomereaction in ram spermatozoa.Gamete Res.4:407-432. Talbot, P., R.G. Summers, B.L. Hylander, E.M. Keough, and L.E. Franklin (1976)The role of calcium in the acrosome reaction: An analysis using ionophore A23187. J. Exp. Zool. 198:383-392. Varner, D.D., C.R. Ward, B.T. Storey, and R.M. Kenney (1987) Induction and characterization of acrosome reaction in equine spermatozoa. Am. J. Vet. Res. 48:1383-1389. Vigil, P. (1989) Gamete membrane fusion in hamster spermatozoa with reacted equatorial segments. Gamete Res. 23: 203-213. Watson, P.F., and J.M. Plummer (1986) Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa. J . Exp. Zool. 238:113-118. Watson, P.F., P.S. Jones, and J.M. Plummer (1991) A quantitative comparison of the spontaneous and ionophore-induced acrosome reaction in ejaculated ram spermatozoa: the effects of temperature, time and individual. Anim. Reprod. Sci. 24 :93-108. Yanagimachi, Y., and Y.D. Noda (1970) Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res. 31:465-485. Yanagimachi, Y., and N. Usui (1974) Calcium dependence of the acrosome reaction and activation of guinea-pig spermatozoa. Exp. Cell Res. 89:161-174. Zhang, J., M.S. Boyle, C.A. Smith, and H.D.M. Moore (1990) Acrosome reaction of stallion spermatozoa evaluated with monoclonal antibody and zona-free hamster eggs. Mol. Reprod. Dev. 27:152-158.
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The Ionophore-InducedAcrosome Reaction Differs Structurally From the Spontaneous Acrosome Reaction P.F. WATSON, J.M. PLUMMER, AND P.S. J O N E S Department of Veterinary Basic Sciences, Royal Veterinary College, London NW1 OTU, United Kingdom
ABSTRACT
The ultrastructure of the spontaneous acrosome reaction in ram spermatozoa has been compared with that induced by the ionophore, A23187. The spontaneous event was dependent on incubation for 4 h, on the temperature, and on dilution. Apart from the more rapid occurrence of the ionophore-induced event, the mean diameter and distribution of vesicle size was also different. The ionophore-inducedvesicles were larger, more irregular, and heterogeneous in size compared with those occurring in the spontaneous acrosome reaction (average diameter 84 nm vs. 60 nm in the spontaneous acrosome reaction). These observations are interpreted in relation to capacitation. 01992 Wiley-Liss, Inc.
ing better than 70% motility were considered acceptable. For experiments 1 and 2, semen from three rams was pooled. The standard diluent was a glucose-saline medium buffered with HEPESNaOH and containing 3 mM CaC12 (Watson et al., '91). For the ionophore-inducedacrosome reaction, calcium ionophore A23187 (free acid; Sigma Chemical Co. Ltd., Poole, Dorset, UK) was dissolved in dimethyl sulphoxide (DMSO)and added to a diluted suspension of spermatozoa (lo8spermatozoa/ml)at a final concentration of 1 pm A23187 and 1% DMSO (Shams-Borhan and Harrison, '81). The sample was then incubated at 39°C. Control tubes had DMSO added but no ionophore, and this treatment was shown to be without effect (Watson et al., 1991). The spontaneous acrosome reaction was investigated by incubation of the semen for 4 h at either 20 or 39°C; the semen was either undiluted or diluted to a concentration of 2 x lo8 spermatozoa/ml. At fixed periods after the commencement of incubation, samples were removed for fixation for transmission electron microscopy (Plummer and Watson, '88). Electron micrographs of thin sections were taken at a magnification of 15,000 x ,and 300 sperm head sections displaying both the acrosomal ridge and a portion of the equatorial segment were MATERIALS AND METHODS selected randomly from each treatment. SpermaSemen was collected by artificial vagina from tozoa were classified into four categories: 1)intact, Finnish Landrace crossbred rams. Immediately after transfer to the laboratory (15 min) a sample Received October 9,1991; revision accepted J u n e 26,1992. of semen was examined, and only ejaculates show-
An acrosome reaction has been initiated in the spermatozoa of several species by means of the divalent cation ionophore, A23187 (Talbot et al., '76; Green, '78; Shams-Borhan and Harrison, '81; Lee et al., '87; Varner et al. '87; Didion et al., '89; Ruknudin, '89; de Jonge et al., '89; Lax et al., '90; Zhang et al., '90). The ionophore achieves this end by facilitating the passage of calcium ions across the plasma membrane (Pressman, '761, resulting in a higher than threshold level of calcium between the outer acrosomal membrane and the overlying plasma membrane. Calcium ions are a prerequisite for the multiple fusion between these membranes which characterises the acrosome reaction (Yanagimachi and Usui, '74). Several authors have published electron micrographs of the ionophoreinduced acrosome reaction and, apart from the difference in timing of the event, have reported that this reaction resembles the spontaneous acrosome reaction (Talbot et al., '76; Green, '78; Jamil and White, '81; Flechon et al., '86). We have undertaken a study of both the spontaneous and the ionophore-induced acrosome reactions in ram spermatozoa, and in the course of this study we have observed some distinct differences which are reported here.
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2) cells displaying a classical acrosome reaction of multiple fusion between the outer acrosomal membrane and the overlying plasma membrane with the formation of hybrid vesicles, 3) cells displaying rupture of the plasma membrane with acrosomal deterioration, and 4)spermatozoa with a missing acrosome. For experiment 3, a total of 40 profiles was examined from each ram: 10 profiles each from 10 and 20 min ionophore treatments, and 10 each from the 2 and 4 h incubation treatments. The numbers of vesicles in a fixed length of membrane (and hence, by inference, the vesicle size) were estimated as follows: a 5 mm grid square on clear acetate was placed over each acrosome-reacted sperm profile just anterior to the equatorial segment; the number of vesicles within a span of 5 grid squares was counted on each face of the spermatozoon, providing a total count for 10 grid squares per sperm covering a total distance on the sperm head of 3.33 pm. The mean counts for sampling times within each treatment were compared by uncorrelated Student's t-test. The combined results from the two sampling times in each treatment were analysed by a hierarchal analysis of variance.
RESULTS The timing of the ionophore-induced acrosome reaction is shown in Table 1.At time zero, few sperm showed evidence of an acrosome reaction (defined as multiple fusions between the plasma membrane and the underlying outer acrosomal membrane in the region of the main part of the acrosome). By 15 min, a proportion of spermatozoa displayed the acrosome reaction, and by 30 min greater than 90% were showing an acrosome reaction. In this experiment, very few sperm showed evidence of deterioration of the acrosome. The timing of the spontaneous acrosome reaction induced by dilution and incubation at 39°C is shown TABLE 1. Timing of the ionophore-induced acrosome reaction i n ram spermatozoa' 0
Normal intact Acrosome reacted Damaged or missing acrosome'
95 4
1
Time ( m i d 15 30
60
37 61 2
0 93 7
1 93 6
'Numbers show the percentage of spermatozoa, observed by electron microscopy, showing evidence of the acrosome reaction as shown in Figure 1. 2Damaged cells had lost plasma membranes or showed degeneration of the acrosome not involving fusion of the plasma and outer acrosomal membranes. A missing acrosome was identified in a small percentage of cells which had also lost the plasma membrane over the acrosome.
in Table 2. A greater proportion of the spermatozoa were damaged at the commencement of the experiment compared with the previous experiment, but the proportion did not change markedly with incubation. Greater than 75%of the cells were intact at time zero, and in those samples held at room temperature no change took place over the 4 h incubation. However, when incubated at 39"C,the sample diluted before incubation, but not that remaining undiluted, showed a considerable development of acrosomal fusion characteristic of an acrosomal reaction. On closer examination of the pattern of acrosome reactions seen in the two treatments incubated at 39"C, diluted, and ionophore-induced, the mean number of vesicles in 3.33 pm span of membrane profile in each treatment group was found to differ (Table 3). No significant difference was observed in numbers of vesicles between 10 and 20 min ionophore treatments, nor between 2 and 4 h incubation treatments although in two rams (A and B) fewer vesicles were seen at 4 h than at 2 h. The results from the two times were pooled for each treatment, and the pooled data were analysed by hierarchal analysis of variance. Differencesbetween rams were not significant, but differences between treatments within individual were very highly significant (P< 0.001). There were fewer vesicles in a fixed span of membrane in the ionophore treatment than in the spontaneous acrosome reaction. Typical sperm profiles from the two treatments are shown in Figure 1.The ionophore-inducedacrosome reaction revealed a larger range of sizes of vesicles which were more irregularly shaped compared with those formed in the spontaneous acrosomereaction. Especially in the ionophore-induced acrosome reactions, vesiculation of the equatorial segment was frequently noted. The actual vesicle sizes were frequently too small to measure accurately. However, the vesicle counts for individual 5 mm grid squares were the data most closely reflecting vesicle size. The apparent greater heterogeneity of vesicle size in ionophore-treated spermatozoa was examined formally by a two-tailed variance ratio test of these counts for each treatment. The variance ratio for 600 data items in each treatment was 1.665L.287 = 1.29 (P< 0.05).
DISCUSSION The calcium ionophore, A23187, induced an acrosome reaction as previously reported by others (Shams-Borhanand Harrison, '81; Jamil and White, '81; Flechon et al., '86; Watson et al., '91). In this quantitative electron microscopy study, it was clear
IONOPHORE VS. SPONTANEOUS ACROSOME REACTIONS
233
TABLE 2. Eflect of temperature and ditution on the spontaneous acrosome reaction in ram spermatozoa'
Time (h) Treatment2 Intact Acrosome reacted Damaged or missing acrosome3
0 Undil
0 Dil
20 4 Undil
76 1 23
75 3 22
70 5 24
Temperature ("C) 20 39 4 4 Dil Undil 70 4 26
39 4 Dil
74 5 21
-
4 85 11
'Numbers give the percentage of spermatozoa, observed by electron microscopy, showing evidence of the acrosome reaction as shown in Figue 1. 'Undil = undiluted semen; dil = diluted to 2 x 10' spermatozodmil. 3Seefootnote to Table 1.
that virtually the entire population of spermatozoa responded to A23187. However, we and others (Talbot et al., '76; Green, '78; Shams-Borhan and Harrison, '81; Watson et al., '91) have reported that the timescale is very different from that of a spontaneous event. The latter was clearly dependent both on temperature and on dilution. The temperature dependence was more closely studied and it was also demonstrated that at the temperature used in the present study individual variation was minimal (Watson et al., '91). The effect of dilution was suprising, and may be due t o the dilution of a calcium chelating protein in the seminal plasma (San Agustin et al., '87) and/or to an effect upon calcium permeability induced by dilution (Robertson and Watson, '86). It is not due to the concentration of calcium in the diluent, since some spermatozoa underwent an acrosome reaction in the absence of added calcium (unpublished observation). The region just anterior to the equatorial segment was chosen for vesicle measurement because it has previously been shown that this region is the site of the initiation of the acrosome reaction (Flechon et al., '86; Watson and Plummer, '86), and
could be assumed to display the most complete stage of the acrosome reaction anywhere in the acrosomal membrane. Although the appearance of the acrosome reaction induced by the ionophore A23187 is similar to that occurring spontaneously, it is not identical. The observation presented here that the number of vesicles in a given span of membrane is fewer in an ionophore-induced acrosome reaction provides clear evidence of the difference. The counting of the number of vesicles in a fixed span permitted the inference to be drawn that the average size of the vesicles formed in the ionophoreinduced reaction was larger. Indeed, visual inspection of the micrographs suggested that under this treatment, the vesicles were heterogeneous in size, in contrast to those formed in the spontaneous event where they appeared smaller and more homogeneous in size. The mean calculated diameter ( + SEM) of the vesicles was 60.13 i- 1.61and 84.47 & 1.01 nm for the spontaneous and the ionophoreinduced events, respectively. A variance ratio test of the data confirmed that this difference was significant. It is probable that this observed difference per-
TABLE 3 . Mean numbers of vesicles i n the acrosome reaction' Time Ram A Ram B Ram C Mean
10 min
Ionophore-induced 20 min
Mean
38.7 t 1.11 44.8 rt 2.32 35.1 2 1.10 39.5 & 1.17
39.9 t 1.30 39.2 t 2.03 38.8 t 1.74 39.3 rt 0.96
39.3 2 0.84 42.0 rt 1.63 37.0 2 1.09 39.4 t 0.75
2h 60.0 t 63.6 2 48.1 & 57.2 k
-
Spontaneous 4h 1.53 1.59 1.82 1.54
54.0 t 1.03 54.6 rt 1.71 52.0 t 2.00 53.5 k 0.94
Mean 57.0 t 1.13 59.1 1: 1.53 50.1 t 1.40 55.4 f 0.92 -
Summary of hierarchal analysis of variance ofpooled data from different times within treatment and ram Source of variation Between rams Treatments within ram Cells within treatment
Degrees of freedom
Mean square
2 3 114
532.06 2548.42 34.45
-
Variance ratio 0.21 73.97***
'Figures are mean numbers of vesicle counted in a 3.33 +m section of sperm plasma membrane just anterior to the equatorial segment. Means of 10 sperm head sections. ***P< 0.001.
234
P.F. WATSON ET AL.
tains to the state of capacitation of the membranes. Whereas by the action of ionophore, calcium is permitted access in high concentration to the entire cytoplasmicspace between the reactive membranes, it is only able to gain access slowly and progressively during the incubation preceding the spontaneous reaction as the membranes become more permeable to calcium. Capacitation also involves subtle modifications of the membranes in preparation for membrane fusion, perhaps including isothermic phase separations of the lipids (Friend et al., '77; Flechon, '85) which favour fusion. In the ionophore-inducedacrosome reaction, the formation of larger, more irregular vesicles suggests a more disordered progression of membrane fusion. Indeed, Klausner et al. ('79) suggested that A23187 actually disrupts membrane structure by disordering protein-lipid interactions. The equatorial segment vesiculation seen in the presence of ionophore also suggests a more pronounced fusion event than that seen spontaneously; Shams-Borhan and Harrison ('81) also noticed this phenomenon. However, perhaps not too much should be made of this since veisculation of the equatorial segment has been noted before in fertilizing spermatozoa (Yanagimachi and Noda, '70; Vigil, '89). In contrast, in the spontaneous event, a gradual influx of calcium, perhaps restricted to discrete regions and coinciding with the alterations of membrane organization facilitating fusion, results in an orderly progression of fusion events. The small, more evenly sized, regularly shaped vesicles suggest that in this case the points of fusion are more frequent and regularly spaced along the membranes. While there is no presumption that the spontaneous acrosome reaction is necessarily normal, it would be a very difficult proposition to prove. It is unlikely t o result in spermatozoa that could penetrate the egg, most likely due not t o the artificial environment or abnormality of the event, but to its occurring far from the zona surface (Crozet and Dumont, '84). Nevertheless, in the spontaneous event, the temperature was comparable to that of the female tract, the timing of the event compared favourably with the suggested capacitation time for ram spermatozoa of 1.5 h (Mattner, '631, and there was no known cellular toxin present. In addition, motility was maintained until after the acrosome reactions had occurred (Watson et al., '91). In contrast, the ionophore-induced acrosome reaction occurred rapidly and extensively, and motility declined rapFig. 1. Electron micrographs of acrosome reactions in ram idly, in a manner quite different from that which spermatozoa (a)occurring spontaneously, or (b) induced by is presumed to happen in vivo. ionophore A23187. Bar = 1 pm.
IONOPHORE VS. SPONTANEOUS ACROSOME REACTIONS
235
reaction in mammalian spermatozoa. Biochim. Biophys. Acta These observations represent a caution to the use 1043:12-18. of A23187 as a simple means of inducing acrosome M.A., G.S. "rucco, K.B. Bechtol, N. Wummer, G.S. Kopf, reactions. Although ionophore-treatedram sperma- Lee, L. Blasco, and B.T. Storey (1987) Capacitation and acrosome tozoa were found to be capable of fusing with zonareactions in human spermatozoa monitored by a chlortetrafree sheep eggs, Shams-Borhan and Harrison ('81) cycline fluorescence assay. Fertil. Steril. 48:649-658. were unable t o achieve in vitro fertilization. Thus, Mattner, P.E. (1963) Capacitation of ram spermatozoa and penetration of the ovine egg. Nature 199:772-773. there is insufficient evidence t o sustain the arguPlummer, J.M., and P.F. Watson (1988) The quantitative ultrament that the ionophore-induced reaction is, in structural assessment of head membrane damage in boar sperevery respect, normal. Indeed, if the morphology matozoa subjected to varying degrees of cold shock. Anim. of the events is under examination it is essential Reprod. Sci. 16:265-275. to use a method of inducing the acrosome reaction Pressman, B.C. (1976) Biological applications of ionophores. Annu. Rev. Biochem. 45500-530. which results in events which resemble the true Robertson, L., and P.F. Watson (1986) Calcium transport in acrosome reaction as closely as possible.
ACKNOWLEDGMENTS This work was supported by grants from The Wellcome Trust and from the University of London Special Research Fund.
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