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Dual effect of spermine on acrosomal exocytosis in capacitated bovine spermatozoa
BB
ELSEVIER
Biochimica et Biophysica Acta 1266 (1995) 196-200
Biochi~ic~a et BiophysicaA~ta
Dual effect of spermine on acrosomal exocytosis in capacitated bovine spermatozoa Sara Rubinstein, Yehudit Lax, Yael Shalev, Haim Breitbart * Department of Life Sciences, Bar-llan University, Ramat-Gan 52900, Israel
Received 18 August 1994; revised 13 December 1994; accepted 9 January 1995
Abstract The influence of spermine on the acrosomal exocytosis of capacitated bovine spermatozoa was studied. Dual effect of spermine was observed, depending on its concentration. It was shown that 10 /~M spermine stimulated acrosomal exocytosis and prostaglandin F2~ production, whereas higher concentrations of spermine inhibited these processes. Acrosomal exocytosis induced by spermine was inhibited by staurosporine, a specific protein kinase C (PKC) inhibitor, indicating that PKC may be involved in this stimulation. Also, acrosomal exocytosis induced by the PKC activator phorbol 12-myristate-13-acetate was inhibited by 10 mM spermine. Therefore, these data indicate that spermine is involved in signal transduction events leading to exocytosis. We suggest that the concentration-dependent reversal of the stimulatory action of spermine could be explained by the existence of two binding sites for spermine: high affinity sites involved in inducing acrosomal exocytosis by low spermine concentration and low affinity sites mediating inhibition of acrosomal exocytosis by high concentration of spermine.
1. Introduction
Polyamines are essential cellular components which are involved in a large number of physiological processes, especially cell division, various enzymatic reactions, signal transduction and membrane fusion [1-4]. It is well established that polyamines can be taken up from the circulation [5,6] but the characterization of polyamine uptake has only begun recently. Polyamines were first discovered in the seminal plasma, where they are present in millimolar concentrations [7], much higher than in other body fluids. In spite of this unusual concentration, the role of polyamines in sperm cells is still unknown. Polyamine binding sites were characterized in ram spermatozoa. Scatchard analysis revealed the presence of two types of binding sites of different affinity [8]. A very high rate of binding and dissociation of spermine was found, as compared with other cell types, and this binding did not require
Abbreviations: PKC, protein kinase C; PMA, phorbol 12-myristate13-acetate; dbcAMP, dibutyryl cyclic AMP; BAEE, bezoylarginine ethyl ester; PGF2~, prostaglandin F2~,; STP, staurosporin; PKA, protein kinase A; EGF, epidermal growth factor; PLC, phospholipase C. * Corresponding author. Fax: +972 3-6356041. 0167-4889/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4889(95 )00007-0
metabolic energy [8]. The binding was reversible and inversely depended on the ionic strength of the medium. Heparin, which is a polyanion found in the female genital tract, facilitated the release of cell-associated spermine [8]. The aim of this study was to investigate the involvement of spermine in the acrosome reaction of capacitated bovine spermatozoa. The acrosome reaction is a tightly regulated process which occurs in sperm cells only after capacitation [9]. The acrosome reaction is a prerequisite for successful fertilization in mammals, defined as a Ca2+-de pendent exocytotic event in sperm, in which membrane fusion takes place between the outer acrosomal membrane and the overlying plasma membrane, thereby allowing release of the acrosomal contents [10,11]. Before the acrosome reaction, the mammalian sperm in the female reproductive tract undergoes a capacitation process [12,13] in which several modifications of sperm membranes occur [9,14]. The acrosome reaction is induced by signal transduction events, involving G-proteins, phospholipases A 2 and C, protein kinase C and changes in intracellular Ca 2+ concentration [14,15]. In this study we investigated the effect of various concentrations of polyamines on acrosomal exocytosis and found that low concentration was stimulatory while high concentration was inhibitory.
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
197
firmed by observing thin sections of spermatozoa in the transmission electron microscope.
2. Materials and methods
2.1. Experimental procedures Radioimmunoassay of PGF2, Sperm preparation
PGF 2~ was measured in capacitated cells after induction with spermine (1-102 /xM) or melittin (20 /zg/ml) in the presence of Ca 2÷ (2 mM) for 20 min. Incubation was terminated by centrifugation at 780 X g for 10 min at 4° C and the supernatants were extracted with ethyl acetate acidified with HC1. After evaporation under vacuum, the dried residues were stored at - 2 0 ° C and redissolved in 0.5 ml steroid buffer (phosphate buffer saline (PBS) + 0.1% gelatin) prior to the radioimmunoassay (RIA), and assayed directly by RIA as described by Shemesh et al. [32].
Frozen bovine sperm ceils were thawed at 37°C in medium containing NaC1 (150 mM) and histidine (10 mM) (pH 7.4). The cells were wa:~hed by three centrifugations at 780 × g for 10 min, with NKM medium containing NaCI (110 mM), KC1 (5 mM) and MOPS (10 mM) (pH 7.4). and resuspended in NKM to final concentration 1-3. 109 cell/ml.
Sperm capacitation Washed sperm (108 celh;/ml) were capacitated for 4 h in 39° C in modified Tyrode's (mTALP medium) containing NaC1 (100 raM), KC1 (3.1 mM), MgC1z (1.5 mM), K H 2 P O 4 (0.29 M), NaHCO 3 (25 mM), sodium pyruvate (0.1 mM), sodium lactate (21.6 mM), penicillin (10 IU/ml), BSA (50 /~g/ml) and Hepes (20 mM) (pH 7.4), supplemented with heparin (20 /xg/ml).
2.2. Materials Labeled PGF2~ [[5,6,8,9,11,12,14,15], 3H] (SA 164 C i / m m o l - 1) was purchased from Amersham, Buckinghamshire. All other materials were purchased from Sigma.
Determination of acrosomal exocytosis
3. Results
Acrosomal exocytosis was induced in capacitated cells by incubating the cells for additional 20 min in the presence of 2 mM CaC12 and one of the following inducers: A23187 (10/zM), PMA (1 ng/ml), dbcAMP (0.1 mM) or spermine (1-104 /xM) in 39° C. At the end of the incubation, the cells were pelleted by centrifugation (12930 X g, 5 min) and the extent of the acrosomal exocytosis was determined by measuring the activity of the released acrosin in the supematant as described previously in bovine sperm [16,17]. Briefly, the supematant was adjusted to pH 3.0 with HC1, and acrosin activity was determined by the esterolytic assay using benzoylarginine ethyl ester (BAEE) as substrate and recording the increase in absorbance at 259 nm with time. The molar absorption coefficient was 1150. The occurrence of the acrosome reaction was con-
3.1. Influence of spermine concentration on acrosomal exocytosis Capacitated bovine spermatozoa were exposed to increased concentrations of spermine (0.1-104 /zM) and acrosin release was measured after 20 min of incubation. It was found (Fig. 1) that spermine in the micromolar range induced acrosin release, while at the millimolar range this effect was reduced, leading to a bell shaped dose-response curve with a maximum at 10/zM. This observation probably results from an additional action of spermine, masking its stimulatory effect on acrosomal exocytosis. The effect of spermine at the millimolar range counteracts its enhancing effect on acrosomal exocytosis at the micromolar
Table 1 Effect of different inducers and inhibitors on acrosomal exocytosis Inhibitors
None Spermine (10 mM) Neomycin (10 mM) STP (10 nM)
Inducer:~ Spermine (10/zM)
PMA (1.6/xM)
Acrosin release
Inhibition %
Acrosin release
233 + 51 56 + 18 n.d. * 19 + 7
76 92
171 + 46 89 + 71 101 + 24 14 5:6
A23187 (10/zM)
dbc-AMP (0.1 mM)
Inhibition %
Acrosin release
Inhibition %
Acrosin release
Inhibition %
48 42 82
282 ± 65 131 5:- 28 139 5:37 110 ± 42
54 51 61
130 + 35 130 + 38 n.d. 140 5:25
0 0
Capacitated bovine spermatozoa were incubated in the presence of 2 mM CaC12 and one of the following inducers: spermine, PMA, A23187 and dbcAMP. The inhibitors (spermine, neomycin or STP) were added 10 min prior to the inducers. Acrosine release was measured (see Sction 2) after 20 min and expressed as nmol BAEE/108 cells X min. Spontaneous acrosin release was subtracted from all samples. The assay for acrosin activity itself was not affected by spermine (10 mM), neomycin (10 raM) or by STP (10 nm). Data represent the mean _ S.D obtained from three experiments each done in duplicate. * n.d., not done.
198
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
range. This maximal effect of spermine on acrosin release was about 75% of the maximal acrosin release obtained by the Ca 2÷ ionophore A23187 while in the presence of 10 mM spermine, only 16% of the maximal acrosin release was obtained. The stimulatory effect of micromolar spermine on acrosin release was seen whether calcium was present or absent from the incubation medium.
1013
so 8
~
40
2 20
3.2. Dual effect of spermine on acrosomal exocytosis 0
The influence of spermine both as an acrosomal exocytosis inducer (10/xM) and inhibitor (10 mM) compared to other known acrosomal exocytosis inducers and inhibitors, is shown in Table 1. Spermine has a dual effect on acrosomal exocytosis: a stimulatory influence in low concentration and an additional inhibitory effect on acrosomal exocytosis invoked by different inducers. The potency of the inducers was: A23187 > spermine > PMA > dbcAMP (namely, spermine was the second best inducer after the ionophore which is known to give the maximal effect as an inducer of acrosomal exocytosis). Spermine at 10 mM caused about 50% inhibition of acrosin release induced by A23187 or PMA but did not inhibit acrosin release induced by dbcAMP. Similarly, neomycin, another known polyamine, had almost the same inhibitory effect as spermine. As expected, staurosporine (STP), a specific PKC inhibitor, inhibited (82%) the acrosomal exocytosis induced by PMA, a PKC activator, and also inhibited the acrosin release induced by A23187, but to a lesser extent (61%). STP interestingly inhibited (92%) the acrosomal exocytosis induced by spermine (10 /zM), but did not
350
0
1
10
100
Spermine (BM) Fig. 2. Influence of spermine concentration on PGF2, , production. Different concentrations of spermine (1-102 p~M) were added to capacitated bovine spermatozoa. After 20 min of incubation the produced PGF2,, was measured as described in Section 2. Data represent the mean+S.D. obtained from three experiments each done in duplicate.
inhibit acrosomal exocytosis induced by dbcAMP, an activator of protein kinase A (PKA). These data indicate the specificity of STP to PKC and imply that spermine (10 p~M) can induce acrosome exocytosis via PKC activity.
3.3. Influence of spermine concentration on PGF2, production Capacitated bovine spermatozoa were exposed to increased concentrations of spermine (1-102/xM) and PGF 2 production was measured after 20 min of incubation. A bell-shaped curve was obtained where the maximal effect was at 10/zM (Fig. 2), similar to the effect of spermine on acrosin release (Fig. 1). Incubation of capacitated sperms with melittin (20 ;zg/ml), a known PLA 2 activator, resuited in production of 160 pg/108 cells PGE2~.
~)0 x
250
"~ .~
2.0
..~
4. Discussion
< m
,~
,00
T
50
0.1
I
r
1
T
l0
102
l
II I,. 103
104
Spermine(pM) Fig. 1. Effect of sperrnine concentration on acrosomal exocytosis. Different concentrations of spermine (0.1-104 p,M) were added to capacitated bull spermatozoa. After 20 min of incubation, acrosin release was measured and expressed as nmole BAEE/108 cells×min (see Section 2). The extent of spontaneous acrosine release without any supplement was subtracted from each experiment. The maximal acrosin release was determined with Ca2+/A23187 and was 360+79 nmol BAEE/108 cells× min. This value was considered 100% acrosin release and under these conditions, maximum 55% of the cells were acrosome reacted as was examined in thin sections under electron microscope analysis. Data represent the mean_+ S.D. obtained from three experiments each done in duplicate.
We have presented in this study a dual effect of spermine on acrosomal exocytosis depending on its concentration. Spermine in the micromolar range induced the acrosomal exocytosis, whereas millimolar concentration inhibited acrosomal exocytosis. There is a concentration-dependent reversal of the stimulatory action of spermine which may be explained by the existence of a second low affinity binding site for spermine which mediates an inhibition of the acrosomal exocytosis rather than stimulation which is mediated by a high affinity binding site. Indeed, we have found high and low-affinity binding sites in ram spermatozoa [8]. Similar results were found in bovine spermatozoa (unpublished data). Therefore, the most straightforward explanation appears to be that, at high concentration, spermine has an additional (low affinity) site of action which counteracts its enhancing effect on acrosomal exocytosis, mediated by a binding site of higher affinity. The effect of spermine on acrosomal exocytosis was dose-dependent and
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
revealed a bell-shaped curve. Likewise, PMA and EGF have similar effects on bovine sperm acrosomal exocytosis [16,17]. The down-regulation at relatively high concentration of an inducer or ligand is a well known phenomenon in signal transduction. It was suggested that signal transduction events are involved in the cascade leading to acrosomal exocytosis [14,15]. We propose that the induction of acrosomal exocytosis by micromolar spermine is derived from the involvement of spermine in the signal transduction pathway leading to exocytosis. In a recent paper, we have shown thai: PKC was involved in bovine sperm acrosomal exocytosis [16]. In this study, we have found that the acrosomal exocytosis induced by spermine (10 /xM) was inhibited by STP (Table 1), a specific inhibitor of PKC. This finding indicates that spermine (10 /~M) activates PKC directly or indirectly in the process leading to acrosomal exocytosis. We have recently found that PKC is involved in PLA 2 activation in bovine sperm [18]. It was also demonstrated that spermine (/xM) stimulates the biosynthesis of prostaglandins (Fig. 2) in bovine spermatozoa, probably via activation of PLA 2. Therefore, it can be speculated that spermine can induce acrosomal exocytosis via activation of PLA z which causes release of arachidonic acid, a precursor for leukotriens and prostaglandins, products important to acrosomal exocytosis [1820]. It was reported that spermine [21] and neomycin [22] stimulated histamine exocytosis from mast cells through activation of G-proteins. In the same way, spermine might affect sperm cells which contain G-proteins [23], and influence acrosomal exocytosis [24]. Furthermore, we found that calcium or micromolar spermine in absence of calcium enhanced specific fusion between acrosomal and plasma membranes isolated from capacitated bovine sperm [25]. We also found that 1 mM spermine inhibits the calcium-induced membrane fusion in this cell free system (unpublished data). We mentioned in the results section that p~M spermine can stimulate acrosomal exocytosis even in absence of calcium during incubation. Thus, spermine as a polycation in physiological condition, can substitute calcium in the induction of acrosomal exocytosis or in the fusion between isolated acrosomal and plasma membranes. This significant correlation between membrane fusion in intact cells and in the cell free system of isolated membranes, further supports our notion concerning the importance of micromolar spermine in inducing acrosomal exocytosis. Contrary to the micromolar range, the millimolar concentration of spermine inhibited acrosomal exocytosis induced by A23187 and PIVIA but not dbcAMP, a result which may imply specificity of spermine activity. Similarly, neomycin, another polyamine, inhibited acrosomal exocytosis. The fact that spermine inhibited acrosomal exocytosis induced by PMA, a known PKC activator, may be explained by the possibility that spermine in the mM range directly inhibited PKC, as known from other cell types [26-28], or it inhibited other events downstream to
199
PKC in the cascade leading to acrosomal exocytosis. Since 10 mM spermine did not affect acrosomal exocytosis induced by dbcAMP which activates protein kinase A (PKA), we suggest that spermine does not affect the PKA activity. It is also known that spermine inhibits phospholipase C (PLC) in other cell types [29-31], suggesting that spermine may inhibit acrosomal exocytosis by inhibiting PLC activity in spermatozoa. In summary, we suggest that polyamines can regulate acrosomal exocytosis in two opposite directions which may determine the precise timing of this process, in order to achieve fertilization. During ejaculation, spermatozoa are exposed to a high (millimolar) concentration of spermine, which binds rapidly to the cells. At this concentration, spermine acts as a major decapacitating factor which inhibits premature acrosome reaction. As sperm cells migrate through the female genital tract, they leave the seminal plasma behind and are now exposed to a heparincontaining medium that might rapidly reduce the level of cell-bound spermine as was shown previously [8]. Under such conditions, the remaining spermine concentrations (probably at the micromolar range) can activate processes which trigger and enhance acrosomal exocytosis, an essential step towards successful fertilization.
Acknowledgements This work was supported by a grant from the Ihel Foundation to H.B. We thank Avrille Goldreich for language editing and typing this manuscript.
References [1] Pegg, E.A, and McCann, P.P. (1982) Am. J. Physiol. 243, C212C221. [2] Tabor, C.W. and Tabor, H. (1984) Ann Rev. Biochem. 53, 749-790. [3] Seiler, N. and Heby, D. (1988) Arch. Biochem. Biophys. Hung. 23, 1-35. [4] Schuber, F. (1989) Bioehem J. 260, 1-10. [5] Seiler, N. and Dezeure, F. (1990) Int. J. Biochem. 20, 211-218. [6] Khan, A.H., Quemener, V. and Moulinoux, J.-Ph. (1991) Cell. Biol. Int. Rep. 15, 9-15. [7] Mann, T. (1964) Biochemistry of semen and of the male reproductive tract p 339. Wiley, New York. [8] Rubinstein, S. and Breitbart, H. (1991) Biochem. J. 278: 25-28. [9] Yanagimachi, R. (1988) in The Physiology of Reproduction (E. Knobil, and J. Neill, Eds.), pp. 135-185. Raven Press, New York. [10] Yanagimachi, R. (1981) in Fertilization and Embryonic Development In Vitro, (L. Mastroianni, and J.D. Biggers, Eds.), pp. 82-182. Plenum Press, New York. [11] Russell, L., Peterson, R. and Freund, M. (1979) J. Exp. Zool. 208, 41-56. 112] Chang, M.C. (1951) Nature 168, 697-699. [13] Austin, C.R. (1952) Nature 170, 326-328. [14] Saling, P.M. (1989) in Oxford Reviews of Reproductive Biology. (S.R. Milligan, Ed.), pp. 339-388. Oxford University Press, Oxford. [15] Kopf, S.G. (1990) in Fertilization in Mammals. (B.D. Bavister, J. Cummins, and E.R.S. Roldan, Eds.), pp. 253-266, Serono Symposia, USA.
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[16] Breitbart, H., Lax, Y., Rotem, R. and Naor, Z. (1992) Biochem. J. 281,473-476. [17] Lax, Y., Rubinstein, S. and Breitbart, H. (1994) FEBS Lett. 339, 234-238. [18] Shalev, Y., Shemesh, M., Levinshal, T. Marcus, S. and Breitbart, H. (1994) J. Reprod. Fert., in press. [19] Joyce, C., Nuzzo, N.A., Wilson, L., Zaneveld, L.J.D. (1987) J. Androl. 8, 74-82. [20] Lax, Y., Grossman, S., Rubinstein, S., Magid, N. and Breitbart, H. (1990) Biochim. Biophys. Acta 1043, 12-18. [21] Bueb, J.L., DaSilva, A., Mousli, M. and Landry, Y. (1992) Biochem. J. 282, 545-550. [22] Aridor, M. and Sagi-Eisenberg, R. (1990) J. Cell. Biol. l 11, 28852891. [23] Garty, N.B., Galiani, D., Aharonheim, A., Yee Kim, H., Phillips, D.M., DeKel, N. and Salomon, Y. (1988) J. Cell Sci. 91, 21-31. [24] Florman, H.M., Tombes, R.M., First, N., Babcock, D.F. (1989) Dev. Biol. 135, 133-149.
[25] Spungin, B., Levinshal, T., Rubinstein, S. and Breitbart, H. (1992) FEBS Lett. 311, 155-160. [26] Qi, D.-F., Schatzman, R.C., Mazzei, G.J., Turner, R.S., Raynor, R.L., Liao, S. and Kuo, J.F. (1983) Biochem. J. 213, 281-288. [27] Thams, P., Capito, K. and Hedeskov, C.J. (1986) Biochem. J. 237, 131-138. [28] Moruzzi, M., Barbiroli, B., Monti, M.G., Tadolini, B., Hakim, G. and Mezzetti, G. (1987) Biochem. J. 247, 175-180. [29] Eichberg, J., Zetusky, W.J., Bell, M.E. and Cavanagh, E. (1981) J. Neurochern. 36, 1868-1871. [30] Nahas, N. and Graft, G. (1982) Biochem. Biophys. Res. Commun. 109, 1035-1040. [31] Vergara, J., Tsien, R.Y. and Delay, M. (1985) Proc. Natl. Acad. Sci. USA 82, 6352-6356. [32] Shemesh, M., Milaguin, F., Ayalon, N. and Hansel, W. (1979) J. Reprod. Fertil. 56, 181-185.
ELSEVIER
Biochimica et Biophysica Acta 1266 (1995) 196-200
Biochi~ic~a et BiophysicaA~ta
Dual effect of spermine on acrosomal exocytosis in capacitated bovine spermatozoa Sara Rubinstein, Yehudit Lax, Yael Shalev, Haim Breitbart * Department of Life Sciences, Bar-llan University, Ramat-Gan 52900, Israel
Received 18 August 1994; revised 13 December 1994; accepted 9 January 1995
Abstract The influence of spermine on the acrosomal exocytosis of capacitated bovine spermatozoa was studied. Dual effect of spermine was observed, depending on its concentration. It was shown that 10 /~M spermine stimulated acrosomal exocytosis and prostaglandin F2~ production, whereas higher concentrations of spermine inhibited these processes. Acrosomal exocytosis induced by spermine was inhibited by staurosporine, a specific protein kinase C (PKC) inhibitor, indicating that PKC may be involved in this stimulation. Also, acrosomal exocytosis induced by the PKC activator phorbol 12-myristate-13-acetate was inhibited by 10 mM spermine. Therefore, these data indicate that spermine is involved in signal transduction events leading to exocytosis. We suggest that the concentration-dependent reversal of the stimulatory action of spermine could be explained by the existence of two binding sites for spermine: high affinity sites involved in inducing acrosomal exocytosis by low spermine concentration and low affinity sites mediating inhibition of acrosomal exocytosis by high concentration of spermine.
1. Introduction
Polyamines are essential cellular components which are involved in a large number of physiological processes, especially cell division, various enzymatic reactions, signal transduction and membrane fusion [1-4]. It is well established that polyamines can be taken up from the circulation [5,6] but the characterization of polyamine uptake has only begun recently. Polyamines were first discovered in the seminal plasma, where they are present in millimolar concentrations [7], much higher than in other body fluids. In spite of this unusual concentration, the role of polyamines in sperm cells is still unknown. Polyamine binding sites were characterized in ram spermatozoa. Scatchard analysis revealed the presence of two types of binding sites of different affinity [8]. A very high rate of binding and dissociation of spermine was found, as compared with other cell types, and this binding did not require
Abbreviations: PKC, protein kinase C; PMA, phorbol 12-myristate13-acetate; dbcAMP, dibutyryl cyclic AMP; BAEE, bezoylarginine ethyl ester; PGF2~, prostaglandin F2~,; STP, staurosporin; PKA, protein kinase A; EGF, epidermal growth factor; PLC, phospholipase C. * Corresponding author. Fax: +972 3-6356041. 0167-4889/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0167-4889(95 )00007-0
metabolic energy [8]. The binding was reversible and inversely depended on the ionic strength of the medium. Heparin, which is a polyanion found in the female genital tract, facilitated the release of cell-associated spermine [8]. The aim of this study was to investigate the involvement of spermine in the acrosome reaction of capacitated bovine spermatozoa. The acrosome reaction is a tightly regulated process which occurs in sperm cells only after capacitation [9]. The acrosome reaction is a prerequisite for successful fertilization in mammals, defined as a Ca2+-de pendent exocytotic event in sperm, in which membrane fusion takes place between the outer acrosomal membrane and the overlying plasma membrane, thereby allowing release of the acrosomal contents [10,11]. Before the acrosome reaction, the mammalian sperm in the female reproductive tract undergoes a capacitation process [12,13] in which several modifications of sperm membranes occur [9,14]. The acrosome reaction is induced by signal transduction events, involving G-proteins, phospholipases A 2 and C, protein kinase C and changes in intracellular Ca 2+ concentration [14,15]. In this study we investigated the effect of various concentrations of polyamines on acrosomal exocytosis and found that low concentration was stimulatory while high concentration was inhibitory.
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
197
firmed by observing thin sections of spermatozoa in the transmission electron microscope.
2. Materials and methods
2.1. Experimental procedures Radioimmunoassay of PGF2, Sperm preparation
PGF 2~ was measured in capacitated cells after induction with spermine (1-102 /xM) or melittin (20 /zg/ml) in the presence of Ca 2÷ (2 mM) for 20 min. Incubation was terminated by centrifugation at 780 X g for 10 min at 4° C and the supernatants were extracted with ethyl acetate acidified with HC1. After evaporation under vacuum, the dried residues were stored at - 2 0 ° C and redissolved in 0.5 ml steroid buffer (phosphate buffer saline (PBS) + 0.1% gelatin) prior to the radioimmunoassay (RIA), and assayed directly by RIA as described by Shemesh et al. [32].
Frozen bovine sperm ceils were thawed at 37°C in medium containing NaC1 (150 mM) and histidine (10 mM) (pH 7.4). The cells were wa:~hed by three centrifugations at 780 × g for 10 min, with NKM medium containing NaCI (110 mM), KC1 (5 mM) and MOPS (10 mM) (pH 7.4). and resuspended in NKM to final concentration 1-3. 109 cell/ml.
Sperm capacitation Washed sperm (108 celh;/ml) were capacitated for 4 h in 39° C in modified Tyrode's (mTALP medium) containing NaC1 (100 raM), KC1 (3.1 mM), MgC1z (1.5 mM), K H 2 P O 4 (0.29 M), NaHCO 3 (25 mM), sodium pyruvate (0.1 mM), sodium lactate (21.6 mM), penicillin (10 IU/ml), BSA (50 /~g/ml) and Hepes (20 mM) (pH 7.4), supplemented with heparin (20 /xg/ml).
2.2. Materials Labeled PGF2~ [[5,6,8,9,11,12,14,15], 3H] (SA 164 C i / m m o l - 1) was purchased from Amersham, Buckinghamshire. All other materials were purchased from Sigma.
Determination of acrosomal exocytosis
3. Results
Acrosomal exocytosis was induced in capacitated cells by incubating the cells for additional 20 min in the presence of 2 mM CaC12 and one of the following inducers: A23187 (10/zM), PMA (1 ng/ml), dbcAMP (0.1 mM) or spermine (1-104 /xM) in 39° C. At the end of the incubation, the cells were pelleted by centrifugation (12930 X g, 5 min) and the extent of the acrosomal exocytosis was determined by measuring the activity of the released acrosin in the supematant as described previously in bovine sperm [16,17]. Briefly, the supematant was adjusted to pH 3.0 with HC1, and acrosin activity was determined by the esterolytic assay using benzoylarginine ethyl ester (BAEE) as substrate and recording the increase in absorbance at 259 nm with time. The molar absorption coefficient was 1150. The occurrence of the acrosome reaction was con-
3.1. Influence of spermine concentration on acrosomal exocytosis Capacitated bovine spermatozoa were exposed to increased concentrations of spermine (0.1-104 /zM) and acrosin release was measured after 20 min of incubation. It was found (Fig. 1) that spermine in the micromolar range induced acrosin release, while at the millimolar range this effect was reduced, leading to a bell shaped dose-response curve with a maximum at 10/zM. This observation probably results from an additional action of spermine, masking its stimulatory effect on acrosomal exocytosis. The effect of spermine at the millimolar range counteracts its enhancing effect on acrosomal exocytosis at the micromolar
Table 1 Effect of different inducers and inhibitors on acrosomal exocytosis Inhibitors
None Spermine (10 mM) Neomycin (10 mM) STP (10 nM)
Inducer:~ Spermine (10/zM)
PMA (1.6/xM)
Acrosin release
Inhibition %
Acrosin release
233 + 51 56 + 18 n.d. * 19 + 7
76 92
171 + 46 89 + 71 101 + 24 14 5:6
A23187 (10/zM)
dbc-AMP (0.1 mM)
Inhibition %
Acrosin release
Inhibition %
Acrosin release
Inhibition %
48 42 82
282 ± 65 131 5:- 28 139 5:37 110 ± 42
54 51 61
130 + 35 130 + 38 n.d. 140 5:25
0 0
Capacitated bovine spermatozoa were incubated in the presence of 2 mM CaC12 and one of the following inducers: spermine, PMA, A23187 and dbcAMP. The inhibitors (spermine, neomycin or STP) were added 10 min prior to the inducers. Acrosine release was measured (see Sction 2) after 20 min and expressed as nmol BAEE/108 cells X min. Spontaneous acrosin release was subtracted from all samples. The assay for acrosin activity itself was not affected by spermine (10 mM), neomycin (10 raM) or by STP (10 nm). Data represent the mean _ S.D obtained from three experiments each done in duplicate. * n.d., not done.
198
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
range. This maximal effect of spermine on acrosin release was about 75% of the maximal acrosin release obtained by the Ca 2÷ ionophore A23187 while in the presence of 10 mM spermine, only 16% of the maximal acrosin release was obtained. The stimulatory effect of micromolar spermine on acrosin release was seen whether calcium was present or absent from the incubation medium.
1013
so 8
~
40
2 20
3.2. Dual effect of spermine on acrosomal exocytosis 0
The influence of spermine both as an acrosomal exocytosis inducer (10/xM) and inhibitor (10 mM) compared to other known acrosomal exocytosis inducers and inhibitors, is shown in Table 1. Spermine has a dual effect on acrosomal exocytosis: a stimulatory influence in low concentration and an additional inhibitory effect on acrosomal exocytosis invoked by different inducers. The potency of the inducers was: A23187 > spermine > PMA > dbcAMP (namely, spermine was the second best inducer after the ionophore which is known to give the maximal effect as an inducer of acrosomal exocytosis). Spermine at 10 mM caused about 50% inhibition of acrosin release induced by A23187 or PMA but did not inhibit acrosin release induced by dbcAMP. Similarly, neomycin, another known polyamine, had almost the same inhibitory effect as spermine. As expected, staurosporine (STP), a specific PKC inhibitor, inhibited (82%) the acrosomal exocytosis induced by PMA, a PKC activator, and also inhibited the acrosin release induced by A23187, but to a lesser extent (61%). STP interestingly inhibited (92%) the acrosomal exocytosis induced by spermine (10 /zM), but did not
350
0
1
10
100
Spermine (BM) Fig. 2. Influence of spermine concentration on PGF2, , production. Different concentrations of spermine (1-102 p~M) were added to capacitated bovine spermatozoa. After 20 min of incubation the produced PGF2,, was measured as described in Section 2. Data represent the mean+S.D. obtained from three experiments each done in duplicate.
inhibit acrosomal exocytosis induced by dbcAMP, an activator of protein kinase A (PKA). These data indicate the specificity of STP to PKC and imply that spermine (10 p~M) can induce acrosome exocytosis via PKC activity.
3.3. Influence of spermine concentration on PGF2, production Capacitated bovine spermatozoa were exposed to increased concentrations of spermine (1-102/xM) and PGF 2 production was measured after 20 min of incubation. A bell-shaped curve was obtained where the maximal effect was at 10/zM (Fig. 2), similar to the effect of spermine on acrosin release (Fig. 1). Incubation of capacitated sperms with melittin (20 ;zg/ml), a known PLA 2 activator, resuited in production of 160 pg/108 cells PGE2~.
~)0 x
250
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2.0
..~
4. Discussion
< m
,~
,00
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50
0.1
I
r
1
T
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102
l
II I,. 103
104
Spermine(pM) Fig. 1. Effect of sperrnine concentration on acrosomal exocytosis. Different concentrations of spermine (0.1-104 p,M) were added to capacitated bull spermatozoa. After 20 min of incubation, acrosin release was measured and expressed as nmole BAEE/108 cells×min (see Section 2). The extent of spontaneous acrosine release without any supplement was subtracted from each experiment. The maximal acrosin release was determined with Ca2+/A23187 and was 360+79 nmol BAEE/108 cells× min. This value was considered 100% acrosin release and under these conditions, maximum 55% of the cells were acrosome reacted as was examined in thin sections under electron microscope analysis. Data represent the mean_+ S.D. obtained from three experiments each done in duplicate.
We have presented in this study a dual effect of spermine on acrosomal exocytosis depending on its concentration. Spermine in the micromolar range induced the acrosomal exocytosis, whereas millimolar concentration inhibited acrosomal exocytosis. There is a concentration-dependent reversal of the stimulatory action of spermine which may be explained by the existence of a second low affinity binding site for spermine which mediates an inhibition of the acrosomal exocytosis rather than stimulation which is mediated by a high affinity binding site. Indeed, we have found high and low-affinity binding sites in ram spermatozoa [8]. Similar results were found in bovine spermatozoa (unpublished data). Therefore, the most straightforward explanation appears to be that, at high concentration, spermine has an additional (low affinity) site of action which counteracts its enhancing effect on acrosomal exocytosis, mediated by a binding site of higher affinity. The effect of spermine on acrosomal exocytosis was dose-dependent and
S. Rubinstein et al. / Biochimica et Biophysica Acta 1266 (1995) 196-200
revealed a bell-shaped curve. Likewise, PMA and EGF have similar effects on bovine sperm acrosomal exocytosis [16,17]. The down-regulation at relatively high concentration of an inducer or ligand is a well known phenomenon in signal transduction. It was suggested that signal transduction events are involved in the cascade leading to acrosomal exocytosis [14,15]. We propose that the induction of acrosomal exocytosis by micromolar spermine is derived from the involvement of spermine in the signal transduction pathway leading to exocytosis. In a recent paper, we have shown thai: PKC was involved in bovine sperm acrosomal exocytosis [16]. In this study, we have found that the acrosomal exocytosis induced by spermine (10 /xM) was inhibited by STP (Table 1), a specific inhibitor of PKC. This finding indicates that spermine (10 /~M) activates PKC directly or indirectly in the process leading to acrosomal exocytosis. We have recently found that PKC is involved in PLA 2 activation in bovine sperm [18]. It was also demonstrated that spermine (/xM) stimulates the biosynthesis of prostaglandins (Fig. 2) in bovine spermatozoa, probably via activation of PLA 2. Therefore, it can be speculated that spermine can induce acrosomal exocytosis via activation of PLA z which causes release of arachidonic acid, a precursor for leukotriens and prostaglandins, products important to acrosomal exocytosis [1820]. It was reported that spermine [21] and neomycin [22] stimulated histamine exocytosis from mast cells through activation of G-proteins. In the same way, spermine might affect sperm cells which contain G-proteins [23], and influence acrosomal exocytosis [24]. Furthermore, we found that calcium or micromolar spermine in absence of calcium enhanced specific fusion between acrosomal and plasma membranes isolated from capacitated bovine sperm [25]. We also found that 1 mM spermine inhibits the calcium-induced membrane fusion in this cell free system (unpublished data). We mentioned in the results section that p~M spermine can stimulate acrosomal exocytosis even in absence of calcium during incubation. Thus, spermine as a polycation in physiological condition, can substitute calcium in the induction of acrosomal exocytosis or in the fusion between isolated acrosomal and plasma membranes. This significant correlation between membrane fusion in intact cells and in the cell free system of isolated membranes, further supports our notion concerning the importance of micromolar spermine in inducing acrosomal exocytosis. Contrary to the micromolar range, the millimolar concentration of spermine inhibited acrosomal exocytosis induced by A23187 and PIVIA but not dbcAMP, a result which may imply specificity of spermine activity. Similarly, neomycin, another polyamine, inhibited acrosomal exocytosis. The fact that spermine inhibited acrosomal exocytosis induced by PMA, a known PKC activator, may be explained by the possibility that spermine in the mM range directly inhibited PKC, as known from other cell types [26-28], or it inhibited other events downstream to
199
PKC in the cascade leading to acrosomal exocytosis. Since 10 mM spermine did not affect acrosomal exocytosis induced by dbcAMP which activates protein kinase A (PKA), we suggest that spermine does not affect the PKA activity. It is also known that spermine inhibits phospholipase C (PLC) in other cell types [29-31], suggesting that spermine may inhibit acrosomal exocytosis by inhibiting PLC activity in spermatozoa. In summary, we suggest that polyamines can regulate acrosomal exocytosis in two opposite directions which may determine the precise timing of this process, in order to achieve fertilization. During ejaculation, spermatozoa are exposed to a high (millimolar) concentration of spermine, which binds rapidly to the cells. At this concentration, spermine acts as a major decapacitating factor which inhibits premature acrosome reaction. As sperm cells migrate through the female genital tract, they leave the seminal plasma behind and are now exposed to a heparincontaining medium that might rapidly reduce the level of cell-bound spermine as was shown previously [8]. Under such conditions, the remaining spermine concentrations (probably at the micromolar range) can activate processes which trigger and enhance acrosomal exocytosis, an essential step towards successful fertilization.
Acknowledgements This work was supported by a grant from the Ihel Foundation to H.B. We thank Avrille Goldreich for language editing and typing this manuscript.
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