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September 15, 1988
ABSENCE OF ACTIVE PROTEIN KINASE C IN RAM SPERMATOZOA E. R. S. Roldan and R. A. P. Harrison Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, U.K.
Received August 2, 1988 2-1-
-dependent exocytotic event known as the acrosome reaction. As protein kinase C (PKc) has been implicated in exocytosis in some other cell systems, we have searched for PKc in ram spermatozoa. We have found that: (a) no changes in protein phosphorylation pattern could be induced in the intact cells by phorbol dibutyrate (PDBu), a compound which binds to and stimulate~ PKc; (b) no changes in protein phosphorylation pattern could be detected during the course of the CaZ+/ionophore-induced acrosome reaction (when >95% of the ceils underwent exocytosis); (c) there was no effect of PDBu on the exocytoti¢ response to various Ca 2+ and ionophore levels; (d) no specific PDBu binding could be detected in the cells (this binding is considered to be indicative of the presence of active PKc). We conclude that potentially active PKc is not present in ram spermatozoa. © 1988 Academic Press, Inc. At fertilization, mammalian spermatozoa undergo a Ca
At fertilization, the mammalian spermatozoon exocytoses its acrosomal contents in an event known as the acrosome reaction, apparently in response to egg-associated factors (1-3). The molecular processes responsible are as yet largely unknown, save that lipid changes are involved (4,5) and that there is an essential requirement for Ca 2+ (6). Recently, specific alterations in the distribution of intramembranous particles have been observed to take place over the acrosomal region following Ca 2+ influx; these precede membrane fusion, and it has been suggested that modifications in cytoskeletal elements are involved (7). In several cell systems, involvement of protein kinase C (PKc) has been implicated in exocytosis (8-10). PKc is a Ca 2+- and phospholipid- dependent enzyme that is activated by diacylglycerol (DAG); this latter metabolite is produced from receptor-mediated breakdown of polyphosphoinositides (I 1) and induces PKc to phosphorylate specific proteins whence various cellular processes are triggered or modulated (8). In addition to DAG, certain phorbol esters may also stimulate PKc; in consequence, these compounds, which are less readily metabolized than DAG, have been used as a probe for the enzyme (8-12). 2+
•
We have recently demonstrated that during the Ca honophore-induced acrosome reaction in ram and other mammalian spermatozoa, there is a rapid breakdown of the polyphosphoinositides following Ca 2+ influx (13); this breakdown results in release of DAG and seems to be linked with subsequent exocytosis (13-14). It has been reported that diacylglycerol and phorbol esters enhance the onset of the early stages of the acrosome reaction in mouse spermatozoa (15). We have therefore searched for the presence of PKc in ram spermatozoa and investigated its potential role in the 2+.
Ca honophore-induced acrosome reaction, using phorbol dibutyrate (PDBu) as a probe. We present here the results of this study.
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0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS MATERIALS AND METHODS
Materials. Semen was obtained from Clun Forest rams in the Institute's colony, by means of an artificial vagina. Plasmacytoma cell lines X63Ag8.653 (mouse) and Y3Ag1.2.3 (rat) were obtained as continuously growing suspensions (spinner cultures) in DMEM medium containing 2.5% (v/v) foetal calf serum; they were supplied by courtesy of Dr G.W. Butcher of the Institute's Monoclonal Antibody Centre. . 32 [20(n)-3H]phorbol 12,13-dibutyrate (23 Curies/mmol) and carrier-free [ PIP: (10 mCi/ml on day 0) were purchased from Amersham International, Little Chalfont, Bucks, U.I~. Unlabelled phorbol 12,13-dibutyrate, bovine serum albumin (BSA; "Fraction V"), and poly(vinyl) alcohol (PVA; Type II, cold-water-soluble, average M r I0,000) were from Sigma Chemical Co., Poole, Dorset, U.K.; polyvinylpyrrolidone (PVP; average M r 44,000) and Hepes were from BDH, Poole, Dorset, U.K.; the PVP was dialysed thoroughly against water and lyophilized before use. Ionophore A23187 was a gift from Eli Lilly, Indianapolis, IN, USA. The standard saline incubation medium used throughout consisted of 142 mM-NaCI, 2,5 mM-KOH, 10 mM-glucose and 20 mM-Hepes, adjusted to 7.55 at 20°C with NaOH (7); the sucrose washing medium contained 222 mM-suerose in place of the NaCI. Both media also contained 1 mg PVA/ml, 1 mg PVP/ml and 0.1 mM-dithiothreitol (freshly added), and had an osmolality of 305 mOsm/kg. Preparation of cells. Samples of ram semen (0.2 ml) were diluted with 0.8 ml saline medium, layered over 7.5 ml cushions of sucrose washing medium, and the cells washed through by centrifugation for 5 min at 400 gmax followed by 10 min at 1000 gmax (16). After removal of most of the supernatant, the loose pellet was resuspended in the remaining wash medium (about 1.0 ml). ' ' for 10 mln at The myeloma cells (approximately 8 x 106) were harvested by centrlfugatlon 500 ~ after which they were resuspended again gently in 1 ml of the supernatant. They were then ~x' " medmm . . . washe~through 7 ' 5 ml sucrose washmg by. centrffugatlon for 10 mm at 1000 z' - ' l ~ a . X ' resuspe n ded in remaining wash medium (about 0.5 ml), and then diluted to 4.5 ml with saline incubation medium. Cell concentrations in the washed suspensions were estimated using a haemocytometer. Labelling of spermatozoa. Washed spermatozoa (about 1.0 x 10a/ml) were incubated in 5 ml of saline medium for different periods of time at 37°C, in the presence of 250-500/iCi [32P]pi/ml; because the isotope was supplied in dilute HCI solution (pH 2-3), the incubation medium was made up from stock concentrates such that the osmolality and final pH would be as stated (see above) after allowing for isotope addition. h~duction of the acrosome reaction. The acrosome reaction was induced by treatment of spermatozoa with Ca 2+ and the divalent cation ionophore A23187 in saline medium at 37°C (17); the occurrence of the reaction was monitored by phase-contrast microscopy of glutaraldehyde-fixed samples. Analysis ofphosphoproteinsfi'om labelled spermatozoa.
At the required times, 0.5 ml aliquants of the sperm suspensions were centrifuged at 10,000 g~x for 1 min (Beckman Microfuge) and the supernatant fluid was removed. The packed spermatozoa were then resuspended as rapidly and thoroughly as possible in 0.1 ml extraction medium (2% w/v SDS, 28% w/v sucrose, 12.4 mM-N,N,N',N'-tetramethylethylenediamine and 185 mM-tris, adjusted to pH 6.8-7.0 with HCI), and immediately incubated at 100°C for 5 min. Finally, the suspension was centrifuged on the Microfuge for 4 min. Samples of these supernatants, containing equal amounts of incorporated isotope (as measured by liquid scintillation counting of TCA-precipitable material), were reduced with 2-mercaptoethanol and subjected to one-dimensional SDS-gel electrophoresis on 5-17% (w/v) gradient separating gels (18). Afterwards the gels were dried down onto filter paper supports and autoradiographed using Fuji RX film and an intensifying screen at -70°C.
Phorbol dibutyrate binding assay. (adapted from ref. 19) Suitable aliquants of the cell suspensions were incubated with [~H]PDBu in saline medium for 30 min at room temperature in glass tubes. Parallel incubations contained in addition unlabelled PDBu. The final volume of each sample was 1 ml, [3H]PDBu was added at 20 pmol/ml and, when present, unlabelled PDBu at 500 ng (991 pmol)/ml. Cells were added first, labelled ester next, and immediately afterwards unlabelled ester where required; the suspensions were then mixed thoroughly by gentle vortexing. Cells were resuspended in the same way at intervals during the 30 mm incubation period. At the end of the incubation, the suspensions were filtered under gentle vacuum through glass fibre discs (GF/C from Whatman International, Maidstone, Kent, U.K.); tubes were rinsed out with 1 ml of diluting medium, and the discs were then washed with a further 4 x 1 ml of diluting medium. Finally, the discs were subjected to liquid scintillation counting. Preliminary tests demonstrated that all ceils were retained by the filters. 902
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RESULTS Ram spermatozoa treated with Ca 2+ (3 mM) and the ionophore A23187 (1 aM) started to exocytose (display acrosome reactions) after a lag of about 5 - 10 rain. The reaction was complete in most of the sperm population after 60 min of incubation (Fig. 1). Spermatozoa incubated for up to 60 min in the presence of 0.01 - 10 a M PDBu and 3 mM Ca 2+ but in the absence of ionophore did not undergo an acrosome reaction (data not shown). Moreover, PDBu (0.01 - 1 aM) was not able to modify the time-course of the acrosome reaction as induced by A23187 (0.1 or 1.0 aM) and Ca2+ (3.0 mM) (Fig. 1). Lower or higher concentrations of PDBu (0.001 or 10/~M), and lower concentrations of A23187 (0.01 aM) and Ca 2+ (0 or 0.3 mM) were also used in an attempt to detect any interaction between reagents; again, PDBu was unable to modify the time-course of the acrosome reaction (data not shown). Extensive labelling of sperm proteins took place during 45 min incubation with 250/JCi [32p]Pi/ml (Fig. 2). The pattern of protein phosphorylation did not change when spermatozoa were subsequently incubated for 2, 5, 10 or 20 min with 0.01 - 1.0 aM A23187 and 3 mM Ca 2+. PDBu alone (0.1 aM) did not modify the pattern of protein phosphorylation when spermatozoa were incubated in its presence for 5 - 20 min, nor was it able to change the pattern seen when spermatozoa were incubated with 0.01 - 1.0 a M A23187 and 3 mM Ca 2+. (Only the key treatments demonstrating these results are shown in Fig. 2). Specific PDBu binding, in which unlabelled compound competed with labelled compound, was readily detectable in two myeloma cell lines, tested at cell concentrations of about 2 x 106/ml.
However,
no such binding was observed in three separate sperm preparations, tested at cell concentrations of 107/ml (Fig. 3). In other experiments (data not shown), no specific binding was detected in sperm suspensions compared at cell concentrations of 107/ml and 10S/ml, nor was specific binding discerned at any time point when 106 cells/ml were incubated with PDBu for periods between 5 and 75 min.
100
"
80-
0 t~
60"
o 0
40"
20"
0 10
20
30
40
50
60
Time (min)
Fig. 1o Effect of PDBu on the time-course of the Ca2÷lA23187-induced acrosome reaction of ram spermatozoa. Spermatozoa were exposed to 3 mM Ca2+ and combinations of A23187 and PDBu in
saline medium at 37°C. At various times after the initiation of treatment subsamples were analysed for occurrence of acrosome reactions. Open symbols, 1 aM A23187; dark symbols, 0.1,uMA23187. ( 0 , 0 ) control, noPDBu; ( n , B ) 0 . 0 1 a M P D B u ; (A A)0.1BMPDBu; (V, • ) 1 tiM PDBu.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
M~ x 10 ~
_..94 -"68 "~ 43 30 20 Caa+ (mE):
-
q
-
l
-
PDBu ( ~ ) :
-
-
0.1
Time:
0
4
A23187
(pM):
3
~
9
0.01 0.1 0.1
0.1
5 ~in---,-~
3
l
-
-
0.1
•
0.01 0.1 0.1
0.1
20 min....~
Fig. 2. Effect of Ca2+/A23187 and PDBu on the pattern of protein phosphorylation of ram spermatozoa. Spermatozoa were labelled for 45 min in a saline medium containing 250 pCi [S2P]P,/ml, and then exposed to combinations of Ca2+ (3 mM), A23187 (0.01 - 1 aM) and PDBu (0.1 ,ui~I) for 0, 5 or 20 min. After the different treatments, spermatozoa were centrifuged and extracted (see Materials and Methods section). After reduction, samples of the extracts were subjected to one-dimensional SDS-gel electrophoresis on 5-17% (w/v) gradient separating gels and autoradiographed.
DISCUSSION
The results of the present study indicate that no major protein phosphorylation events are involved in the ram sperm acrosome reaction down-stream of Ca 2+ entry, and that ram spermatozoa do not contain active PKc.
8" 7-
• []
Blank Sperm A
[]
M1
[] M2
x
~
j']"
• []
Blank Sperm B
[]
Sparm C
[] M1 [] M2
,4 4 . d = 3m
~2-
0
Label only + cold PDBu
Label only
Exp. 1
+ cold PDBu Exp. 2
Fig. 3. Absence of specific bindings sites for PDBu in ram spermatozoa. Sperm samples from 3 different males (A, B and C) and two plasmacytoma cell lines [X63Ag8.653 of mouse origin (M1) and Y3Agl.2.3 of rat origin (M2)], were incubated with [ H]PDBu for 30 min at room temperature; parallel incubations also contained unlabelled PDBu. The suspensions were then filtered through glass fibre discs, and the discs were subjected to liquid scintillation counting. In Exp. 1, Sperm A contained 1 x 107cells, M1 0.23 x 10 cells, and M2 0.29 x 107 cells; in 7 ? 7 Exp. 2, Sperm B and C contained 1 x 10 cells, MI 0.20 x l0 livecells, and M2 0.21 x 10 live cells. In addition, increased vacuum was used to wash the filters in Exp. 2, and this reduced the blank values considerably.
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Despite previous reports to the contrary (19,21), we found that sperm phosphoproteins could be readily labelled with [32P]Pi in populations of intact, living spermatozoa incubated in a simple medium. As we show elsewhere (13), the rate of uptake of [32PIPi is dependent upon the incubation medium, and this may explain the difficulties other workers seem to have experienced. The spectrum of actively phosphorylated proteins that could be extracted from sperm suspensions was broad, and enabled ready comparison of phosphorylatlon patterns between control and experimental groups. However, we did not observe any changes in phosphorylation pattern when the acrosome reaction was induced by Ca2+/ionophore treatment, either in the absence or the presence of added PDBu. The extracts were made from sperm populations in which a very large number of cells were responding synchronously to stimulus. Thus, since the morphological changes seen during the acrosome reaction occur over the whole of the acrosomal region (which constitutes a considerable proportion of the total surface area of the cell), it seems very unlikely that key phosphorylation events involved would have gone undetected. Our results do not prove that protein phosphorylation is not involved in processes leading to Ca 2÷ influx during the physiologically induced acrosome reaction, but they do suggest strongly that events occurring down-stream of Ca 2+ entry, such as the changes in distribution of intramembranous particles (7), are not the result of changes in protein phosphorylation. Two pieces of evidence lead us to conclude that spermatozoa do not contain potentially active PKc. Firstly, we have been unable to induce any detectable changes in phosphorylation pattern by treating spermatozoa with PDBu, regardless of whether the spermatozoa were being simultaneously treated with A23187 or not. Although the levels of PDBu used were higher than those used by some workers to obtain cellular responses, they were based upon data for receptor binding constants (12,22); moreover, we were looking for an acute short-term response. Because the effect of PDBu on PKc is mediated through lowering the enzyme's requirement for Ca 2+ (23), such an effect might have been hidden in the presence of 1/.tM A23187 and 3 mM Ca 2+, due to the resultant high free levels of Ca2+; however, no effect was seen at sub-optimal, limiting levels of ionophore or cation (when only slow induction of acrosome reactions took place). Other workers have been able to provoke specific phosphorylation in exocytosing systems using such combinations of A23187 and phorbol esters (9,24,25). The second piece of evidence against PKc is the apparent absence of specific PDBu binding sites in sperm suspensions. Competitive PDBu binding is now recognized as indicative of the presence of PKc (8,12); using this method, the presence of the enzyme has been detected in many cell types (8,12,19,22) with the exception of human and mouse erythrocytes (19). Our assay was clearly able to detect specific binding in two myeloma cell lines under identical conditions, testing the myeloma cells at one-fifth the concentration of the sperm samples (the myeloma cells appeared to be of similar dimensions to sperm heads, and the two cell types were therefore approximately quantitatively comparable). Although the assay conditions used may not have resulted in optimal binding of the labelled PDBu, there is no evidence from other cell types that they would have failed to detect potential binding. At first sight, it may appear that our findings are in direct contradiction to those of Lee et al. (15) who reported that phorbol esters and a diacylglycerol enhanced the onset of the early
stages of the acrosome reaction in mouse spermatozoa. The two experimental systems are not, of course, entirely comparable: the animal species are different, and the enhancement observed only occurred if the reaction was induced by zona components (15), whereas we have employed ionophore 905
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
induction. However, since common molecular mechanisms are assumed to be involved, our failure to detect PKc activity in ram spermatozoa appears to us significant. Thus, although it is possible that there may be species differences with respect to sperm content and role of PKc, it is also possible that the effects Lee et al. (15) noted were not mediated via PKc (see ref. 10 for discussion). We consider that the absence of PKc in spermatozoa is not unlikely. This enzyme is known to be continuously synthesized and catabolized (10), whereas protein synthesis in spermatozoa (save for intramitochondrial metabolism) ceases in the testis during the maturation stage of spermiogenesis (26,27), at least 8 days before the cells appear in the ejaculate (28). Thus, unless catabolism ceases with or before synthesis, levels of the enzyme could be expected to decline drastically during the later stages of sperm development and therefore be absent in the mature spermatozoon. It is noteworthy that the only other cell type in which PKc has not been detected is the mammalian erythrocyte (19), a cell in which protein synthesis has ceased to occur. We wish to thank Dr K.D. Brown for helpful advice and discussion, and A. Tilley for his cooperation. ERSR is a recipient of Research Fellowships from Journals of Reproduction & Fertility Ltd. and The Wellcome Trust. Acknowledgements.
REFERENCES 1. Harrison, R.A.P. (1983) In The Sperm Cell (J. Andre, Ed.), pp. 259-273, Martinus Nijhoff, The Hague. 2. Wassarman, P.M., Bleil, J.D., Florman, H.M., Greve, J.M., Roller, R.J., and Salzmann, G.S. (1986) In The Molecular and Cellular Biology of Fertilization (J.L. Hedrick, Ed.), pp. 55-77, Plenum Press, New York. 3. Yanagimachi, R. (1988) In The Physiology of Reproduction (E. Knobil and J. Neill, Eds.), pp. 135-185, Raven Press, New York. 4. Fleming, A.D., and Yanagimachi, R. (1981) Gamete Res. 4, 253-273. 5. Fleming, A.D., Kosower, N.S., and Yanagimachi, R. (1982) Gamete Res. 5, 19-33. 6. Yanagimachi, R., and Usui, N. (1974) Expl. Cell Res. 89, 161-174. 7. Flechon, J.E., Harrison, R.A.P., Flechon, B., and Escaig, J. (1986) J. Cell Sci. 81, 43-63. 8. Nishizuka, Y. (1984) Nature (London) 308, 693-698. 9. Nishizuka, Y. (1986) Science 233, 305-312. 10. Woodgett, J.R., Hunter, T., and Gould, K.L. (1987) In Cell Membranes. Methods and Reviews (E. Elson, W. Frazier and L. Glaser, Eds.), Vol. III, pp. 215-340. Plenum Press, New York. 11. Berridge, M.J. (1987) Ann. Rev. Biochem. 56, 159-193. 12. Ashendel, C.L. (1985) Biochim. Biophys. Acta 822, 219-242. 13. Roldan, E.R.S., and Harrison, R.A.P. (1988) Biochem. J. (submitted) 14. Bennet, P.J., Moatli, J.P., Mansat, A., Pdbbes, H., Cayrac, J.C., Pontonnier, F., Chap, H., and Douste-Blazy, L. (1987) Biochim. Biophys. Acta 919, 255-265. 15. Lee, M.A., Kopf, G.S., and Storey, B.T. (1987) Biol. Reprod. 36, 617-627. 16. Harrison, R.A.P., Dott, H.M., and Foster, G.C. (1982) J. Exp. Zool. 222, 81-88. 17. Shams-Borhan, G., and Harrison, R.A.P. (1981) Gamete Res. 4, 407-432. 18. Harrison, R.A.P., and Gaunt, S.J. (1988) J. Reprod. Fert. 82, 777-785. 19. Shoyab, M., and Todaro, G.J. (1980) Nature (London) 288, 451-455. 20. Babcock, D.F., First, N.L., and Lardy, H.A. (1975) J. Biol. Chem. 250, 6488-6495. 21. Noland, T.D., Abumrad, N.A., Beth, A.H., and Garbers, D.L. (1987) Biol. Reprod. 37, 171-180. 22. Blumberg, P.M., Jaken, S., Konig, B., Sharkey, N.A., Leach, K.L., Jeng, A.Y., and Yeh, E. (1984) Biochem. Pharmacol. 33, 933-940. 23. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U., and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276, 24. Burnham, D.B., Munowitz, P., and Hootman, S.R. (1986) Biochem. J. 235, 125-131. 25. Barrett, P.Q., Kojima, I., Kojima, K., Zawalich, K., Kales, C.M., Rasmussen, H. (1986) Biochem J. 238, 893-903. 26. Harrison, R.A.P. (1977) In Frontiers in Reproduction and Fertility Control (R.O. Greep and M.A. Koblinsky, Eds.), pp. 379-401. MIT Press, Cambridge, MA. 27. Erickson, R.P., Lewis, S.E., and Butley, M. (t981) J. Reprod. Immunol. 3, 195-217. 28. Courot, M. (1981) In Epididymis and Fertility: Biology and Pathology (C. Bollack and A. Clavert, Eds.), pp. 67-79. S. Karger, Basel. 906