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Microinjection of α-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes
Toxicon Vol. 28, No. 11, pp . 1368-1371 . 1990. Printed in Great Britain .
0041-0101/90 $3 .00 + .011 Pergamon Press plc
MICROINJECTION OF a-TOXIN FROM CLOSTRIDIUM NO VYI TYPE A PROMOTES MEIOTIC MATURATION IN XENOPUS LAEVIS OOCYTES PETER BETTE, FRANK MAULER, CHRISTIANE MOHR
and
ERNST HABERMANN*
Rudolf-Buchheim-Institut lur Pharmakologie, Justus-Liebig University, D-6300 Giessen, F .R.G . (Acceptedfor publication 19 June 1990)
Mom and E . HABERMANN . Microinjection of a-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes. Toxicon 28, 1368-1371, 1990.-Microinjection of purified a-toxin into Xenopus laevis oocytes induced meiotic maturation provided insulin was present in the medium. Induction of maturation as indicated by breakdown of the germinal vesicle depended on the amount of intracellular a-toxin (with a detection limit of 2 ng/oocyte) and on the concentration of insulin. The hormone concentrations used were inactive when given alone and so was atoxin (1 pg/ml) applied from the outside. The results demonstrate an intracellular target for a-toxin whereas an extracellular target is apparently lacking in oocytes. P. BETTE, F . MAULER, C .
Clostridium novyi type A is involved in gas gangrene infections in man and animals (AIKAT and DIELE, 1960). Its a-toxin is the only factor of the culture filtrate that is both lethal and induces edema in vivo (Izumi et al., 1983). The toxin does not possess any known enzymatic activity . It acts through formation of interendothelial gaps which lead to longlasting edema in vivo. In vitro cytopathic effects manifest themselves on numerous cell types by rounding and retraction (BETTE et al., 1989). Nothing is known about the cellular target and the molecular mode of action of this unusually potent and long acting toxin. It is even debated whether its primary target is located within the cell or on the membranal surface. We have addressed this question by intracellular vs epicellular application of the toxin to oocytes. Stage VI oocytes of Xenopus laevis (DUMONT, 1972) are very large cells (about 1 .3 mm in diameter, corresponding to a volume of 1 pl), which allow injection of volumes up to 0.1 pl. They are arrested at diakinesis of the first meiotic prophase . Upon exposure to progesterone or insulin the oocytes continue meiotic cell division with breakdown of their germinal vesicle (i.e . nucleus membrane). The process is reflected by a white spot in the animal hemisphere (for review see MALLER, 1985). a-Toxin was purified to homogeneity in SDS gel electrophoresis (BETTE et al., 1989) . Its LD50 in mice was about 200 ng/kg, and it caused cytopathic effects in cultured endothelial cells with an ED50 of 20 ng/ml. Xenopus laevis females were obtained from Institut für *Author to whom correspondence should be addressed . 1368
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Entwicklungsbiologie, Hamburg. Stage VI oocytes were selected after collagenase incubation of the ovarian tissue as described (RICHARDSON and PoTTER, 1983) and kept overnight in Barth's medium (88 mM NaCl, 1 mM KC1, 2.4 mM NaHC03, 0.82 mM MgSO,, 0.33 mM Ca(N03)3, 0.41 mM CaC12, 10 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES), pH 7.8) at 16-19°C. One hour after microinjection of 50/nliter of native or heat-inactivated (10 min at 95°C) a-toxin, oocytes were placed in Barth's medium at exactly 19°C containing insulin (Sigma ; 24.5 units/mg) at the indicated concentrations, and 0.1% bovine serum albumin . In another set of experiments progesterone (Sigma) was added for 1 hr to the medium of the injected and preincubated oocytes. At different times oocytes were scored for the appearance of a white spot in the pigmented animal hemisphere, indicating the breakdown of germinal vesicle . The number of oocytes with this phenomenon was recorded and expressed in percent. The effect of toxin was indistinguishable from the normal process of maturation by light microscopy. While injection of 20 ng of the native a-toxin into oocytes followed by incubation with 0.35,uM insulin for 18 hr led to the morphological change in 74% of the cells (Fig. 1A), heat inactivated a-toxin was not effective in this test (Fig. 1B). Maturation due to a-toxin and insulin was always 100% within 20 to 24 hr, and 50% maturation was recorded at 14 f 2 hr (three separate experiments with cell preparations from three different toads) . Injection of 20 ng of cc-toxin per oocyte without insulin did not initiate meiotic division
FIG. 1 . INDUCTION OF OOCYTE MATURATION VIA MICROINIECnON OF a-TOXIN OF Cl. novyi TYPE A . Fully grown Xenopus laevis oocytes were microinjected with 20 ng of native (A) or heat-inactivated (B) a-toxin . After 1 hr preincubated oocytes were placed in a medium containing 0 .35 ,UM insulin for 18 hr at 19°C . The white spot at the animal pole indicative of maturation is only visible in oocytes injected with native toxin . The bar scale is I mm .
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80-
C 0
20
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FIG. 2. GERMINAL VESICLE BREAKDOWN IN OOCYTFS INJECTED WITH 20 ng a-TOXIN DEPENDS ON TIME AND ON THE CONCENTRATION OF INSULIN . Fully grown Xenopus laevis oocytes were microinjected with 20 ng of a-toxin . After l hr, oocytes were placed in a medium containing 350 nM (Q ; n = 20(1 .), 19(2 .), 22(3)) or 35 nM (O ; n = 22) or 3.5 nM (p ; n = 20) insulin . The progress was followed for at least 24 hr and given in percent (ordinate). Control oocytes were incubated with 350 nM insulin without previous injection . ;n = 23) or after injection of 20 ng bovine serum albumin (/ ; n - 22) or 0.2 ng toxin (A ; n = 23). Injection of 20 ng at-toxin alone did not initiate meiosis (0 ; n = 27). At the indicated times oocytes were scored for germinal vesicle breakdown, and the percentage of reacting oocytes was calculated . The experiment with a-toxin and the highest insulin concentration was repeated twice with different cell preparations (1,2,3) but essentially produced the same results.
(Fig. 2). Ten-fold higher concentrations of insulin alone were required for maturation of oocytes (not shown) . Kinetics of germinal vesicle breakdown in oocytes injected with 20 ng of a-toxin depended on the concentration of insulin in the Barth's medium (Fig. 2). The detection limit of a-toxin-induced maturation in the presence of 0.35 uM insulin was below 2 ng/oocyte (data not shown) . This amount initiated meiotic division in 90% of injected oocytes within 26 hr. Ten-fold lower (i.e. 0.2 ng) quantities of a-toxin per oocyte or 20 ng bovine serum albumin per oocyte did not induce maturation (Fig. 2). a-Toxin did not significantly accelarate maturation induced by 5 pM progesterone or promote it in the presence of 0.5 pM progesterone (not shown) . Whereas a-toxin in concentrations between 2 and 20 ng/ml in the surrounding medium gives rise to cytopathic alterations in a great variety of cells (BErIE et al., 1989) it was inactive when applied (1 hg/ml) from the outside in the presence or absence of 0.35 pM insulin. Germinal vesicle breakdown was the only visible event resulting from toxin injection. The data presented strongly support the assumption that a-toxin can act intracellularly, at least in oocytes. It remains to be shown whether cells cultivated from homeiothermic animals retract upon injection of a-toxin. It is still disputed how insulin acts on oocytes . The hormone initiates meiotic division synergistically with protein kinase C, with inositol1,4,5-trisphosphate (STrrH and MALLER, 1987), with the sarc gene product pp60"arc (SPIVACK and MALLER, 1985), and with progesterone (HiRm et al ., 1983 ; LE GOASCOGNE et al ., 1987).
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Although similar events in maturation are induced by insulin and progesterone the early sequelae appear to be distinct (MORGAN et al., 1986 ; STITH and MALLER, 1987 ; DESHPANDE and KUNG, 1987; KORN et al ., 1987) . Since a-toxin like protein kinase C (STITH and MALLER, 1987) acts synergistically with insulin but not with progesterone, microinjection of a-toxin into oocytes may help to elucidate the mechanism of insulin-induced maturation . Conversely the action on cytoskeletal components can be studied in the oocyte system (MERRIAM et al., 1983 ; CHRISTENSEN et al., 1984) . Since cell retraction due to atoxin (BETTE et al., 1989) suggests a primary interaction between the toxin and the cytoskeleton, the toxin's mode of action may be sought for in oocytes. Acknowledgements-Work was supported by the Deutsche Forschungsgemeinschaft (SFB 249) and the Fonds der Chemischen Industrie. REFERENCES AIKAT, B. K . and DIBIE, J . H . (1960) The local and general effects of cultures and culture filtrates of Clostridium oedematiens, C1. septicum, C1. sporogenes and C1. histolyticwn . J. Pathol. Bacteriol. 79, 227-241 . Berm, P., FREVERT, J ., MAULER, F ., SuTroRP, N . and HABERMANN, E . (1989) Pharmacological and biochemical studies of cytotoxicity of C1. novyi type A a-toxin . Infect . Immun . 57, 2507-2513 . CHRISTENSEN, K ., SAuTERm, R. and MERRIAM, R. W., (1984) Role of soluble myosin in cortical contractions of Xenopus eggs. Nature 310, 150-151 . DESHPANDE, A. K ., and KUNG, H . (1987) Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mot. Cell. BioL 7, 1285-1288. DumoNT, J . N . (1972) Oogenesis in Xenopus laevis (Daudin). I. Stage of oocyte development in laboratory maintained animals. J. Morphol. 136,153-179. HIRm, S ., LE GoAscoGNE, C . and BAULIEU, E.-E . (1983) Induction of germinal vesicle breakdown in Xenopus laevis oocytes : Response of denuded oocytes to progesterone and insulin . Dev. Mot. 100, 214-221 . Izumi, N ., KoNDo, H ., OmsEn, l . and SAKAGUCHI, G . (1983) Purification and characterization of a-toxin of Clostridium oedematiens type A . Jpn . J. Med. Sci . Biol. 36, 135-145 . KORN, L . J ., SIEREL, C . W ., MCCORMICK, F. and ROTH, R . A . (1987) Ras p2l as a potential mediator of insulin action in Xenopus oocytes . Science 236, 840-843 . LE GoAScoGNE, C., SANANm, N ., GouEzou, M . and BAULIEU, E .-E . (1987) Alkaline phosphatase activity in the membrane of Xenopus laevis oocytes : effects of steroids, insulin, and inhibitors during meiosis reinitiation . Dev. Biol. 119, 511-519 . MALLER, J . L . (1985) Regulation of amphibian oocyte maturation . Cell Diff. 16, 211-221 . MERRIAM, R . W ., SAuTEREER, R . A . and CHRISTENSEN, K . (1983) A subcortical, pigment containing structure in Xenopus eggs with contractile properties . Dev . Biol. 95, 439-446 . MORGAN, D. 0 ., Ho, L ., KORN, L . J . and RoTH, R . A . (1986) Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase. Proc. Natn . Acad. Sci . USA 83, 328-332 . RICHARDSON, C . and POTTER, R . L . (1983) Changes in nucleotide-binding proteins during oocyte maturation in Xenopus laevis. Dev . Biol. 99, 240-247 . SPIVACK. J . G . and MALLER, J . L . (1985) Phosphorylation and proteinsysnthetic events in Xenopus laevis oocytes microinjected with pp60-1 . Mol. Cell. Biol. S, 3629-3633 . STrnt, B . J. and MALLER, J. L . (1987) Induction of meiotic maturation in Xenopus oocytes by 12-0tetradecanoylphorbol 13-acetate . Exp. Cell. Res. 169, 514-523 .
0041-0101/90 $3 .00 + .011 Pergamon Press plc
MICROINJECTION OF a-TOXIN FROM CLOSTRIDIUM NO VYI TYPE A PROMOTES MEIOTIC MATURATION IN XENOPUS LAEVIS OOCYTES PETER BETTE, FRANK MAULER, CHRISTIANE MOHR
and
ERNST HABERMANN*
Rudolf-Buchheim-Institut lur Pharmakologie, Justus-Liebig University, D-6300 Giessen, F .R.G . (Acceptedfor publication 19 June 1990)
Mom and E . HABERMANN . Microinjection of a-toxin from Clostridium novyi type A promotes meiotic maturation in Xenopus laevis oocytes. Toxicon 28, 1368-1371, 1990.-Microinjection of purified a-toxin into Xenopus laevis oocytes induced meiotic maturation provided insulin was present in the medium. Induction of maturation as indicated by breakdown of the germinal vesicle depended on the amount of intracellular a-toxin (with a detection limit of 2 ng/oocyte) and on the concentration of insulin. The hormone concentrations used were inactive when given alone and so was atoxin (1 pg/ml) applied from the outside. The results demonstrate an intracellular target for a-toxin whereas an extracellular target is apparently lacking in oocytes. P. BETTE, F . MAULER, C .
Clostridium novyi type A is involved in gas gangrene infections in man and animals (AIKAT and DIELE, 1960). Its a-toxin is the only factor of the culture filtrate that is both lethal and induces edema in vivo (Izumi et al., 1983). The toxin does not possess any known enzymatic activity . It acts through formation of interendothelial gaps which lead to longlasting edema in vivo. In vitro cytopathic effects manifest themselves on numerous cell types by rounding and retraction (BETTE et al., 1989). Nothing is known about the cellular target and the molecular mode of action of this unusually potent and long acting toxin. It is even debated whether its primary target is located within the cell or on the membranal surface. We have addressed this question by intracellular vs epicellular application of the toxin to oocytes. Stage VI oocytes of Xenopus laevis (DUMONT, 1972) are very large cells (about 1 .3 mm in diameter, corresponding to a volume of 1 pl), which allow injection of volumes up to 0.1 pl. They are arrested at diakinesis of the first meiotic prophase . Upon exposure to progesterone or insulin the oocytes continue meiotic cell division with breakdown of their germinal vesicle (i.e . nucleus membrane). The process is reflected by a white spot in the animal hemisphere (for review see MALLER, 1985). a-Toxin was purified to homogeneity in SDS gel electrophoresis (BETTE et al., 1989) . Its LD50 in mice was about 200 ng/kg, and it caused cytopathic effects in cultured endothelial cells with an ED50 of 20 ng/ml. Xenopus laevis females were obtained from Institut für *Author to whom correspondence should be addressed . 1368
Short Communications
1369
Entwicklungsbiologie, Hamburg. Stage VI oocytes were selected after collagenase incubation of the ovarian tissue as described (RICHARDSON and PoTTER, 1983) and kept overnight in Barth's medium (88 mM NaCl, 1 mM KC1, 2.4 mM NaHC03, 0.82 mM MgSO,, 0.33 mM Ca(N03)3, 0.41 mM CaC12, 10 mM N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid (HEPES), pH 7.8) at 16-19°C. One hour after microinjection of 50/nliter of native or heat-inactivated (10 min at 95°C) a-toxin, oocytes were placed in Barth's medium at exactly 19°C containing insulin (Sigma ; 24.5 units/mg) at the indicated concentrations, and 0.1% bovine serum albumin . In another set of experiments progesterone (Sigma) was added for 1 hr to the medium of the injected and preincubated oocytes. At different times oocytes were scored for the appearance of a white spot in the pigmented animal hemisphere, indicating the breakdown of germinal vesicle . The number of oocytes with this phenomenon was recorded and expressed in percent. The effect of toxin was indistinguishable from the normal process of maturation by light microscopy. While injection of 20 ng of the native a-toxin into oocytes followed by incubation with 0.35,uM insulin for 18 hr led to the morphological change in 74% of the cells (Fig. 1A), heat inactivated a-toxin was not effective in this test (Fig. 1B). Maturation due to a-toxin and insulin was always 100% within 20 to 24 hr, and 50% maturation was recorded at 14 f 2 hr (three separate experiments with cell preparations from three different toads) . Injection of 20 ng of cc-toxin per oocyte without insulin did not initiate meiotic division
FIG. 1 . INDUCTION OF OOCYTE MATURATION VIA MICROINIECnON OF a-TOXIN OF Cl. novyi TYPE A . Fully grown Xenopus laevis oocytes were microinjected with 20 ng of native (A) or heat-inactivated (B) a-toxin . After 1 hr preincubated oocytes were placed in a medium containing 0 .35 ,UM insulin for 18 hr at 19°C . The white spot at the animal pole indicative of maturation is only visible in oocytes injected with native toxin . The bar scale is I mm .
1370
Short Communications
100-,
80-
C 0
20
0-
FIG. 2. GERMINAL VESICLE BREAKDOWN IN OOCYTFS INJECTED WITH 20 ng a-TOXIN DEPENDS ON TIME AND ON THE CONCENTRATION OF INSULIN . Fully grown Xenopus laevis oocytes were microinjected with 20 ng of a-toxin . After l hr, oocytes were placed in a medium containing 350 nM (Q ; n = 20(1 .), 19(2 .), 22(3)) or 35 nM (O ; n = 22) or 3.5 nM (p ; n = 20) insulin . The progress was followed for at least 24 hr and given in percent (ordinate). Control oocytes were incubated with 350 nM insulin without previous injection . ;n = 23) or after injection of 20 ng bovine serum albumin (/ ; n - 22) or 0.2 ng toxin (A ; n = 23). Injection of 20 ng at-toxin alone did not initiate meiosis (0 ; n = 27). At the indicated times oocytes were scored for germinal vesicle breakdown, and the percentage of reacting oocytes was calculated . The experiment with a-toxin and the highest insulin concentration was repeated twice with different cell preparations (1,2,3) but essentially produced the same results.
(Fig. 2). Ten-fold higher concentrations of insulin alone were required for maturation of oocytes (not shown) . Kinetics of germinal vesicle breakdown in oocytes injected with 20 ng of a-toxin depended on the concentration of insulin in the Barth's medium (Fig. 2). The detection limit of a-toxin-induced maturation in the presence of 0.35 uM insulin was below 2 ng/oocyte (data not shown) . This amount initiated meiotic division in 90% of injected oocytes within 26 hr. Ten-fold lower (i.e. 0.2 ng) quantities of a-toxin per oocyte or 20 ng bovine serum albumin per oocyte did not induce maturation (Fig. 2). a-Toxin did not significantly accelarate maturation induced by 5 pM progesterone or promote it in the presence of 0.5 pM progesterone (not shown) . Whereas a-toxin in concentrations between 2 and 20 ng/ml in the surrounding medium gives rise to cytopathic alterations in a great variety of cells (BErIE et al., 1989) it was inactive when applied (1 hg/ml) from the outside in the presence or absence of 0.35 pM insulin. Germinal vesicle breakdown was the only visible event resulting from toxin injection. The data presented strongly support the assumption that a-toxin can act intracellularly, at least in oocytes. It remains to be shown whether cells cultivated from homeiothermic animals retract upon injection of a-toxin. It is still disputed how insulin acts on oocytes . The hormone initiates meiotic division synergistically with protein kinase C, with inositol1,4,5-trisphosphate (STrrH and MALLER, 1987), with the sarc gene product pp60"arc (SPIVACK and MALLER, 1985), and with progesterone (HiRm et al ., 1983 ; LE GOASCOGNE et al ., 1987).
Short Communications
137 1
Although similar events in maturation are induced by insulin and progesterone the early sequelae appear to be distinct (MORGAN et al., 1986 ; STITH and MALLER, 1987 ; DESHPANDE and KUNG, 1987; KORN et al ., 1987) . Since a-toxin like protein kinase C (STITH and MALLER, 1987) acts synergistically with insulin but not with progesterone, microinjection of a-toxin into oocytes may help to elucidate the mechanism of insulin-induced maturation . Conversely the action on cytoskeletal components can be studied in the oocyte system (MERRIAM et al., 1983 ; CHRISTENSEN et al., 1984) . Since cell retraction due to atoxin (BETTE et al., 1989) suggests a primary interaction between the toxin and the cytoskeleton, the toxin's mode of action may be sought for in oocytes. Acknowledgements-Work was supported by the Deutsche Forschungsgemeinschaft (SFB 249) and the Fonds der Chemischen Industrie. REFERENCES AIKAT, B. K . and DIBIE, J . H . (1960) The local and general effects of cultures and culture filtrates of Clostridium oedematiens, C1. septicum, C1. sporogenes and C1. histolyticwn . J. Pathol. Bacteriol. 79, 227-241 . Berm, P., FREVERT, J ., MAULER, F ., SuTroRP, N . and HABERMANN, E . (1989) Pharmacological and biochemical studies of cytotoxicity of C1. novyi type A a-toxin . Infect . Immun . 57, 2507-2513 . CHRISTENSEN, K ., SAuTERm, R. and MERRIAM, R. W., (1984) Role of soluble myosin in cortical contractions of Xenopus eggs. Nature 310, 150-151 . DESHPANDE, A. K ., and KUNG, H . (1987) Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. Mot. Cell. BioL 7, 1285-1288. DumoNT, J . N . (1972) Oogenesis in Xenopus laevis (Daudin). I. Stage of oocyte development in laboratory maintained animals. J. Morphol. 136,153-179. HIRm, S ., LE GoAscoGNE, C . and BAULIEU, E.-E . (1983) Induction of germinal vesicle breakdown in Xenopus laevis oocytes : Response of denuded oocytes to progesterone and insulin . Dev. Mot. 100, 214-221 . Izumi, N ., KoNDo, H ., OmsEn, l . and SAKAGUCHI, G . (1983) Purification and characterization of a-toxin of Clostridium oedematiens type A . Jpn . J. Med. Sci . Biol. 36, 135-145 . KORN, L . J ., SIEREL, C . W ., MCCORMICK, F. and ROTH, R . A . (1987) Ras p2l as a potential mediator of insulin action in Xenopus oocytes . Science 236, 840-843 . LE GoAScoGNE, C., SANANm, N ., GouEzou, M . and BAULIEU, E .-E . (1987) Alkaline phosphatase activity in the membrane of Xenopus laevis oocytes : effects of steroids, insulin, and inhibitors during meiosis reinitiation . Dev. Biol. 119, 511-519 . MALLER, J . L . (1985) Regulation of amphibian oocyte maturation . Cell Diff. 16, 211-221 . MERRIAM, R . W ., SAuTEREER, R . A . and CHRISTENSEN, K . (1983) A subcortical, pigment containing structure in Xenopus eggs with contractile properties . Dev . Biol. 95, 439-446 . MORGAN, D. 0 ., Ho, L ., KORN, L . J . and RoTH, R . A . (1986) Insulin action is blocked by a monoclonal antibody that inhibits the insulin receptor kinase. Proc. Natn . Acad. Sci . USA 83, 328-332 . RICHARDSON, C . and POTTER, R . L . (1983) Changes in nucleotide-binding proteins during oocyte maturation in Xenopus laevis. Dev . Biol. 99, 240-247 . SPIVACK. J . G . and MALLER, J . L . (1985) Phosphorylation and proteinsysnthetic events in Xenopus laevis oocytes microinjected with pp60-1 . Mol. Cell. Biol. S, 3629-3633 . STrnt, B . J. and MALLER, J. L . (1987) Induction of meiotic maturation in Xenopus oocytes by 12-0tetradecanoylphorbol 13-acetate . Exp. Cell. Res. 169, 514-523 .