BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vo1.173, No. 3,1990
Pages 1369-1374
December 31, 1990
THE EFFECT OF ANTIFREEZE GLYCOPEPTIDES ON MEMBRANE POTENTIAL CHANGES AT HYPOTHERMIC TEMPERATURES Boris Rubinsky1,*, Amir Arav 2, Mauro Mattioli 2 and Arthur L. Devries 3 1Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94720 2Instituto di Fisiologia Veterinaria Universita di Bologna, 40126, Italy 3Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, IL 61801 Received
November
20,
1990
Summary: The research on antifreeze glycopeptides (AFGPs) from Antarctic and Arctic fishes has focused primarily on their interaction with ice crystals. This study reports results of experiments in which pig oocytes, known to be sensitive to hypothermic temperatures, were exposed to 4°C for various periods of time, in solutions of different molecular weight AFGPs from Antarctic nototheniid fishes. The membrane potential was measured across the oolemma following hypothermic exposure. The results show that a physiological combination of the different molecular weight AFGPs protects the structural integrity of the oolemma and inhibits ion leakage across the oolemma at hypothermic temperatures. The results also show that the hypothermic protection is nonlinearly dependent on concentration and that separately, the different molecular weight glycopeptides do not stop ion leakage even at very high concentration. The protection of membranes at hypothermic temperatures is a new property of AFGPs which was not known prior to our work. ~ 1990 Academic Press, Inc. Antarctic fishes live in waters where the annual temperatures fluctuate between -1.4°C to -2.15°C, (1). The fishes have adapted to survival under these conditions by producing special "antifreeze" glycopeptides (AFGPs) and peptides (AFPs) (1, 2, 3, 4, 5, 6). In the last two decades the research on the properties of the AFGPs has focused on the interaction between these compounds and ice crystals, in particular their ability to inhibit ice crystal growth, (2, 3, 7, 8). Studies show that the AFGPs inhibit ice growth on the primary prism planes of ice crystals, {10T0}, presumably by adsorbtion on these planes (2, 4, 8). Consequently, in solutions of AFGPs, ice crystals grow predominantly on the basal plane, parallel to the c-axis, to which the AFGPs do not adsorb, and take the form of very small needle like ice crystals. AFGPs isolated from the blood of Antarctic nototheniid fishes, consist of a series of eight different glycopeptides. The eight different AFGPs are referred to as AFGP 1 to AFGP 8 according to their relative migration on gel electrophoresis and range in molecular mass from 34,000 Da to 2,600 Da, respectively. Antifreeze glycopeptides 1 to 5 (the larger ones) have strong antifreeze activities and molecular weights greater than 10,500 Da. AFGPs 7 and 8 have molecular weights of 3,500 *Correspondence should be sent to this author. 0006-291X/90 $1.50 1369
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Da and 2,600 Da respectively and have a weaker antifreeze activity, while AFGP 6 with a molecular weight of 7,900 Da appears only in trace quantities. The chemical composition of these compounds has been described in several publications, i.e. (2, 3, 4). While the function of the higher molecular weight AFGPs 1 to 5 seems to be strongly related to the modification of the ice crystal structure, the biological function of the low molecular weight AFGPs remains unclear (9). They are less efficient in depressing the freezing point than the larger glycopeptides yet they seem to be present in the serum at much higher concentrations. Recently, we have found that AFGPs from Antarctic nototheniid fishes facilitate the survival of a variety of cells at cryogenic temperatures after rapid cooling and vitrification (10). The beneficial effect of the AFGPs seems to be related with the preservation of membrane integrity at cryogenic temperatures. The study reported in this paper has been undertaken in an attempt to better understand the mechanism by which the AFGPs protect cells. In particular, we wanted to find out whether the AFGPs have protective properties not directly related to freezing. To this end we choose to work with pig oocytes, which are known to be temperature sensitive and which cannot survive exposure to hypothermic temperatures as high as 10°C, (11, 12). Therefore, an experiment can be designed with pig oocytes in which the effect of the AFGPs on the oocytes can be studied at temperatures higher than the phase transition temperature, thereby having the certainty that if a protective effect of the AFGPs is found it is not directly related to the ability of the compound to modify ice crystal morphology or inhibit ice crystal formation. In this paper we report experiments in which pig oocytes were exposed to hypothermic temperatures, for various periods of time and in solutions with different concentrations and compositions of the AFGPs. The effect of the hypothermic exposure was evaluated through measurements of the membrane potentials across the oolemma. The membrane potential is a very sensitive criteria for the functional integrity of membranes, and a direct measure of ion leakage through the membrane during the exposure to hypothermic temperatures. Materials and Methods
The AFGPs used in this work were obtained from Antarctic fishes belonging to the family Nototheniidae (Dissostichus Mawsoni). A physiological composition of AFGPs was used in most of the experiments of one part weight AFGPs 1 to 5 to three part weights AFGPs 7, 8. We will refer to this composition as AFGPs 1-8. Experiments were also performed separately with solutions containing AFGPs I to 5, (AFGPs 1-5), and AFGPs 7 and 8 (AFGPs 7, 8). The basic solution used in this work was a standard buffer solution, PBS, (Dulbecco's phosphate buffered saline supplemented with 0.4 w/v BSA (Bovine Serum Albumin), 0.34 mM pyruvate, 5.5 mM glucose and 70 I.tm/ml kanamycin). The different compositions used in the experiments are listed here: a) PBS; b) PBS with 0.1 mg/ml AFGPs 1-8; c) PBS with 1.0 mg/ml AFGPs 1-8; d) PBS with 40.0 mg/ml AFGPs 1-8; e) PBS with 40.0 mg/ml AFGPs 1-5; f) PBS with 40.0 mg/ml AFGPs 7, 8. Immature pig oocytes surrounded by the cumulus were obtained from selected follicles of cyclic sows 20 minutes after slaughter, at 20°C, and isolated according to the procedure by Mattioli et. al. (13). The oocytes were then introduced in vials containing the different compositions and concentrations of AFGPs listed above. The vials were exposed to a constant temperature of 4°C, in a constant temperature chamber, for 4 hours and 24 hours. After removing the oocytes from the 4°C environment the integrity of the oolemma was determined by measuring the membrane potential of the oocytes at room temperature, 22°C, and comparing that measurement to the membrane potential measured in fresh oocytes from the same batch. The membrane potential was measured using a procedure by Mattioli et al. (14). According to that procedure intra1370
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cellular voltage measurements were made using single microelectrodes made from borosilicate glass tubes (Hilgenberg, FDR). To record the membrane potential the tip of the microelectrode was maneuvered to the surface of the cell using a micromanipulator controlled through 400x magnification with a Leitz Fluovert microscope equipped with Nomarski contrast. When the tip just dimpled the surface of the cell the final penetration was achieved by briefly causing an electrical oscillation induced by turning the capacity compensation of the amplifier. The electrical potential values, which remained constant for at least 1-2 minutes, were recorded. Results and Discussion To establish a criteria for the normal membrane potential across the oolemma in pig oocytes exposed to hypothermic conditions, preliminary experiments were performed for each batch of oocytes used in the experiments. In the preliminary experiments the membrane potentim was measured at 22°C, in fresh oocytes in a PBS solution and in fresh oocytes in a PBS solution with 40 mg/ml AFGPs 1-8. The mean value of the membrane potential, and the standard deviation, were calculated for each batch. The mean of all means for all batches was -31 mV, and the mean standard deviation was 4.5 mV. These values are within the normal range of membrane potentials for immature pig oocytes, (14). (The results also show that the AFGPs do not effect the membrane potential in fresh oocytes.) After hypothermic exposure the membrane potential in each of the exposed oocytes was measured at 22°C and compared to the the mean membrane potential and the standard deviation of the membrane potential in the fresh oocytes from the same batch. Figures 1 and 2 summarize the results obtained from measuring the membrane potential across the oolemma in oocytes exposed to hypothermic conditions, for 4 hours and 24 hours, respectively, in the different solutions. The figures give the percentage of oocytes which after exposure to hypothermic conditions had a membrane potential within two standard deviations from the mean membrane potential in the fresh oocytes from the same batch (membrane potential was measured in groups of 5 oocytes and the number, n, represents the number of groups studied). It should be emphasized that the resting potential is a very sensitive measure of the functional integrity of the oolemma. A drop in voltage difference across the oolemma is a direct measure of the ion leakage through the oolemma during hypothermia. Figure 1 shows that after 4 hours of exposure to 4°C in a PBS solution, the membrane potential of 20.4% of the exposed oocytes was within two standard deviations from the mean value of the membrane potential in fresh oocytes from the same batch. The voltage difference across the oolemma dropped below two standard deviations from the mean in fresh oocytes, in 79.6% of the hypothermically exposed oocytes (P < 0.05). This suggests that there was significant ion leakage across the oolemma in the majority of the oocytes exposed to hypothermic conditions in the PBS solution. In contrast, in the presence of 40 mg/ml AFGPs 1-8, 83.3% of the oocytes retained a membrane potential across the oolemma which was within two standard deviations from the mean in the fresh oocytes. Obviously the ion leakage across the oolemma was significantly reduced by the presence of AFGPs 1-8, which appear to protect the oolemma and inhibit ion leakage at hypothermic temperatures. Results from experiments in which the concentration of AFGPs 1-8 was varied, show that the protection afforded by AFGPs 1-8 is consistently high at concentrations ranging from 40 mg/ml to 1 mg/ml. Then, at the lower concentration of 0.1 mg/ml AFGPs 1-8, the ion leakage increases significantly. 1371
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Figure 1. Percentage of oocytes with a membrane potential within two standard deviations from the mean in fresh oocytes, (oocytes not included are statistically different (P < 0.05) from control), after 4 hours of exposure to 4°C, in different solutions. Values are mean + one standard deviation. Each experimental group consists of 5 oocytes and n represents the number of groups.
The effect of AFGPs 1-8 becomes more obvious after 24 hours at 4°C. Figure 2 shows that for concentrations between 40 mg/ml AFGPs 1-8 and 1 mg/ml AFGPs 1-8, between 59% to 65% of the oocytes had a membrane potential within two standard deviations from the membrane potential in fresh oocytes from the same batch, i.e. retained a normal electrical potential across the oolemma. In contrast, none of the oocytes preserved in PBS or PBS with 0.1 mg/ml AFGPs 1-8 had any measurable membrane potential after 24 hours at 4°C. It should be emphasized that the membrane potential in all the oocytes preserved in PBS or PBS with 0.1 mg/ml AFGPs 1-8, was, after a 24 hour exposure to 4°C, either exactly zero or very close to zero, (P < 0.0005).
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0.1mglml 1.0 mg/ml 40rnglml 40mglml 40mglml AFGPI-8 AFGPI-8
A F G P I - 8 AFGPI-5 AFGP7,8
Figure 2. Percentage of oocytes with a membrane potential within two standard deviations from the mean in fresh oocytes (oocytes not included were statistically different (P < 0.05) from control) after 24 hours exposure to 4°C in different solutions. In cases with a percentage of 0%, the value is given in the figure (in these cases oocytes not included were statistically different (P < 0.0005) from control). Values are mean + one standard deviation. Each experimental group consists of 5 oocytes and n represents the number of groups. 1372
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It is obvious from Figures 1 and 2 that in the absence of AFGPs 1-8 the potential difference across the oolemma drops rapidly with exposure to hypothermic temperatures which implies membrane damage and ion leakage. However, the membrane potential in the presence of AFGPs 1-8 at concentrations higher than 1 mg/ml, is retained even after 24 hours at 4°C. Therefore, it is possible to conclude from this part of the study that AFGPs 1-8 interact with the oolemma at hypothermic temperatures, protect the oolemma from damage caused by hypothermic exposure and inhibit ion leakage. This is a property of AFGPs 1-8 that was not known prior to our work, and may become very important in attempts to preserve biological materials at hypothermic conditions. Another intriguing property of the antifreeze glycoproteins emerges from results presented in Figures 1 and 2. The Figures show that while the combination of AFGPs 1-8 at a concentration of 40 mg/ml protects the oolemma and inhibits ion leakage, AFGPs 1-5 and AFGPs 7,8 separately, do not protect the oolemma or stop the ion leakage, even at concentrations as high as 40 mg/ml. (It should be emphasized that the combination AFGPs 1-8 was obtained by actually mixing 1 part weight AFGP 1-5 with three parts weight AFGP 7,8.) After 4 hours exposure to 4°C, only 49% of the oocytes in 40 mg/ml AFGPs 1-5, and only 36.5% of the oocytes in 40 mg/ml AFGP 7, 8 retained a membrane potential which was within two standard deviations from the mean in fresh oocytes. In comparison, in a solution with 40 mg/ml AFGPs 1-8, 84% of the oocytes retained a membrane potential within two standard deviations from the mean in fresh oocytes. After 24 hours exposure to a temperature of 4°C all the oocytes exposed in solutions with 40 mg/ml AFGPs 1-5, and 40 mg/ml AFGPs 7, 8 showed practically zero potential difference across the oolemma, similarly to unprotected oocytes (P < 0.0005). In comparison, 59% of the oocytes protected by 40 mg/ml AFGPs 1-8 had a membrane potential within two standard deviations from that in fresh oocytes. It is obvious that the whole physiological combination of AFGPs 1 to 8 is needed for protection and that AFGPs 1 to 5 and AFGPs 7, 8 separately do not protect the oolemma or stop the ion leakage, even at very high concentrations. This result is extremely surprising because studies on the effects of AFGPs on depressing the phase transition temperature of water and modifying the pattern of ice crystal formation show that AFGPs 1-5 depress the phase transition temperature almost as effectively as the whole combination of AFGPs 1 to 8 (2, 4). On the other hand, AFGPs 1 to 5 separately do not protect the oolemma and do not block ion leakage, and neither do AFGPs 7, 8 separately. In summary, the study performed here demonstrates by means of a model ideally suited for this purpose, the pig oocyte, through measurements of the membrane potential across the oolemma, that AFGPs 1-8 interact with cell membranes, protect the membranes from damaging hypothermic conditions and inhibit ion leakage. AFGPs 1-5 and AFGPs 7, 8 separately do not afford the same protection. It is possible to speculate that since hypothermic damage to the oolemma could be associated with different mechanisms such as destabilization of lipid structures and ion leakage the protection must occur through an interaction between the Antarctic fish glycopeptides and the oolemma It is possible that the glycopeptides may offer protection by binding to available sites on the oolemma. These sites could be the hydrophi llic part of membrane proteins. Regardless of the accuracy of these speculations it is obvious mat much work 1373
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
remains to be done on this topic, on the mechanism of membrane protection, the inhibition of ion leakage and the intriguing findings concerning the lack of effective protection with AFGPs 1-5 and AFGPs 7-8 separately. Obviously our finding opens a new area of research on the properties of the AFGPs in relation to protection of cell membranes and ion leakage, on the significance of the different AFGPs in the adaption of fishes to the Antarctic environment and on the potential application of these properties to hypothermic and cryogenic preservation of ceils and organs.
ACKNOWLEDGMENTS: We would like to express our gratitude to Professor E. Seren, Drs. G. Galeati and M.L. Bacci for their help with these experiments. One of the authors, B.R. wishes to express his gratitude to the Institute of Veterinary Physiology at the University of Bologna, Italy for hosting him while this work was performed at the Institute. Partial support from the NSF through grant NSF CTS-8914832 is acknowledged. The authors A.A. and M.M. were supported by CNR-RA15A: Project Agrobiotechnologies in animal production No. 1.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
DeVries, A.L., Wohlschlag, D.E., (1965) Science, 163, 1074-1075. DeVries, A.L., (1984) Trans. R. Soc. Lond. B304, 575-588. DeVries, A.L., (1988) Comp. Biochem. Physiol. 90B (3), 611-621. Aranthanarayanan, V.S., (1989) Life Chemistry Reports, 7, 1-32. Lin, Y., Duman, J.G., DeVries, A.L., (1971) Biochem. Biophys. Res. Commun., 46, 89-92. Hew, L.C., Slaughter, D., Fletcher, G.L., Joshi, S.B., (1981) Can. J. Zool., 59, 2186-2192. Raymond, J.A. and DeVries, A.L., (1977) Proc. Natl. Acad. Sci. USA 74, 2589-2593. Raymond, J.A., Wilson, P.W. and DeVries, A.L., (1989) Proc. Natl. Acad. Sci., 86(3), 881-885. Schrag, J.D.and DeVries, A.L., (1983) Comp. Biochem. Physiol., 74A, 381-385. Rubinsky, B., Arav, A., and DeVries, A.L., Cryobiology, (in review). Fahning; M.L., de Garcia, M., (1989) Cryobiology, 26, 563. Newmann, H., (1985) Theriogenology, 23, 213. Mattioli, M. Galeati, G. Bacci, M.L., Seren, E., (1988) Gamete Research, 21, 223-232. Mattioli, M., Barboni, B., Bacci, M.L., and Seren, E., (1990) Biology of Rep., 43, 318-323.
1374
Vo1.173, No. 3,1990
Pages 1369-1374
December 31, 1990
THE EFFECT OF ANTIFREEZE GLYCOPEPTIDES ON MEMBRANE POTENTIAL CHANGES AT HYPOTHERMIC TEMPERATURES Boris Rubinsky1,*, Amir Arav 2, Mauro Mattioli 2 and Arthur L. Devries 3 1Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94720 2Instituto di Fisiologia Veterinaria Universita di Bologna, 40126, Italy 3Department of Physiology and Biophysics, University of Illinois at Urbana-Champaign, IL 61801 Received
November
20,
1990
Summary: The research on antifreeze glycopeptides (AFGPs) from Antarctic and Arctic fishes has focused primarily on their interaction with ice crystals. This study reports results of experiments in which pig oocytes, known to be sensitive to hypothermic temperatures, were exposed to 4°C for various periods of time, in solutions of different molecular weight AFGPs from Antarctic nototheniid fishes. The membrane potential was measured across the oolemma following hypothermic exposure. The results show that a physiological combination of the different molecular weight AFGPs protects the structural integrity of the oolemma and inhibits ion leakage across the oolemma at hypothermic temperatures. The results also show that the hypothermic protection is nonlinearly dependent on concentration and that separately, the different molecular weight glycopeptides do not stop ion leakage even at very high concentration. The protection of membranes at hypothermic temperatures is a new property of AFGPs which was not known prior to our work. ~ 1990 Academic Press, Inc. Antarctic fishes live in waters where the annual temperatures fluctuate between -1.4°C to -2.15°C, (1). The fishes have adapted to survival under these conditions by producing special "antifreeze" glycopeptides (AFGPs) and peptides (AFPs) (1, 2, 3, 4, 5, 6). In the last two decades the research on the properties of the AFGPs has focused on the interaction between these compounds and ice crystals, in particular their ability to inhibit ice crystal growth, (2, 3, 7, 8). Studies show that the AFGPs inhibit ice growth on the primary prism planes of ice crystals, {10T0}, presumably by adsorbtion on these planes (2, 4, 8). Consequently, in solutions of AFGPs, ice crystals grow predominantly on the basal plane, parallel to the c-axis, to which the AFGPs do not adsorb, and take the form of very small needle like ice crystals. AFGPs isolated from the blood of Antarctic nototheniid fishes, consist of a series of eight different glycopeptides. The eight different AFGPs are referred to as AFGP 1 to AFGP 8 according to their relative migration on gel electrophoresis and range in molecular mass from 34,000 Da to 2,600 Da, respectively. Antifreeze glycopeptides 1 to 5 (the larger ones) have strong antifreeze activities and molecular weights greater than 10,500 Da. AFGPs 7 and 8 have molecular weights of 3,500 *Correspondence should be sent to this author. 0006-291X/90 $1.50 1369
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Da and 2,600 Da respectively and have a weaker antifreeze activity, while AFGP 6 with a molecular weight of 7,900 Da appears only in trace quantities. The chemical composition of these compounds has been described in several publications, i.e. (2, 3, 4). While the function of the higher molecular weight AFGPs 1 to 5 seems to be strongly related to the modification of the ice crystal structure, the biological function of the low molecular weight AFGPs remains unclear (9). They are less efficient in depressing the freezing point than the larger glycopeptides yet they seem to be present in the serum at much higher concentrations. Recently, we have found that AFGPs from Antarctic nototheniid fishes facilitate the survival of a variety of cells at cryogenic temperatures after rapid cooling and vitrification (10). The beneficial effect of the AFGPs seems to be related with the preservation of membrane integrity at cryogenic temperatures. The study reported in this paper has been undertaken in an attempt to better understand the mechanism by which the AFGPs protect cells. In particular, we wanted to find out whether the AFGPs have protective properties not directly related to freezing. To this end we choose to work with pig oocytes, which are known to be temperature sensitive and which cannot survive exposure to hypothermic temperatures as high as 10°C, (11, 12). Therefore, an experiment can be designed with pig oocytes in which the effect of the AFGPs on the oocytes can be studied at temperatures higher than the phase transition temperature, thereby having the certainty that if a protective effect of the AFGPs is found it is not directly related to the ability of the compound to modify ice crystal morphology or inhibit ice crystal formation. In this paper we report experiments in which pig oocytes were exposed to hypothermic temperatures, for various periods of time and in solutions with different concentrations and compositions of the AFGPs. The effect of the hypothermic exposure was evaluated through measurements of the membrane potentials across the oolemma. The membrane potential is a very sensitive criteria for the functional integrity of membranes, and a direct measure of ion leakage through the membrane during the exposure to hypothermic temperatures. Materials and Methods
The AFGPs used in this work were obtained from Antarctic fishes belonging to the family Nototheniidae (Dissostichus Mawsoni). A physiological composition of AFGPs was used in most of the experiments of one part weight AFGPs 1 to 5 to three part weights AFGPs 7, 8. We will refer to this composition as AFGPs 1-8. Experiments were also performed separately with solutions containing AFGPs I to 5, (AFGPs 1-5), and AFGPs 7 and 8 (AFGPs 7, 8). The basic solution used in this work was a standard buffer solution, PBS, (Dulbecco's phosphate buffered saline supplemented with 0.4 w/v BSA (Bovine Serum Albumin), 0.34 mM pyruvate, 5.5 mM glucose and 70 I.tm/ml kanamycin). The different compositions used in the experiments are listed here: a) PBS; b) PBS with 0.1 mg/ml AFGPs 1-8; c) PBS with 1.0 mg/ml AFGPs 1-8; d) PBS with 40.0 mg/ml AFGPs 1-8; e) PBS with 40.0 mg/ml AFGPs 1-5; f) PBS with 40.0 mg/ml AFGPs 7, 8. Immature pig oocytes surrounded by the cumulus were obtained from selected follicles of cyclic sows 20 minutes after slaughter, at 20°C, and isolated according to the procedure by Mattioli et. al. (13). The oocytes were then introduced in vials containing the different compositions and concentrations of AFGPs listed above. The vials were exposed to a constant temperature of 4°C, in a constant temperature chamber, for 4 hours and 24 hours. After removing the oocytes from the 4°C environment the integrity of the oolemma was determined by measuring the membrane potential of the oocytes at room temperature, 22°C, and comparing that measurement to the membrane potential measured in fresh oocytes from the same batch. The membrane potential was measured using a procedure by Mattioli et al. (14). According to that procedure intra1370
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cellular voltage measurements were made using single microelectrodes made from borosilicate glass tubes (Hilgenberg, FDR). To record the membrane potential the tip of the microelectrode was maneuvered to the surface of the cell using a micromanipulator controlled through 400x magnification with a Leitz Fluovert microscope equipped with Nomarski contrast. When the tip just dimpled the surface of the cell the final penetration was achieved by briefly causing an electrical oscillation induced by turning the capacity compensation of the amplifier. The electrical potential values, which remained constant for at least 1-2 minutes, were recorded. Results and Discussion To establish a criteria for the normal membrane potential across the oolemma in pig oocytes exposed to hypothermic conditions, preliminary experiments were performed for each batch of oocytes used in the experiments. In the preliminary experiments the membrane potentim was measured at 22°C, in fresh oocytes in a PBS solution and in fresh oocytes in a PBS solution with 40 mg/ml AFGPs 1-8. The mean value of the membrane potential, and the standard deviation, were calculated for each batch. The mean of all means for all batches was -31 mV, and the mean standard deviation was 4.5 mV. These values are within the normal range of membrane potentials for immature pig oocytes, (14). (The results also show that the AFGPs do not effect the membrane potential in fresh oocytes.) After hypothermic exposure the membrane potential in each of the exposed oocytes was measured at 22°C and compared to the the mean membrane potential and the standard deviation of the membrane potential in the fresh oocytes from the same batch. Figures 1 and 2 summarize the results obtained from measuring the membrane potential across the oolemma in oocytes exposed to hypothermic conditions, for 4 hours and 24 hours, respectively, in the different solutions. The figures give the percentage of oocytes which after exposure to hypothermic conditions had a membrane potential within two standard deviations from the mean membrane potential in the fresh oocytes from the same batch (membrane potential was measured in groups of 5 oocytes and the number, n, represents the number of groups studied). It should be emphasized that the resting potential is a very sensitive measure of the functional integrity of the oolemma. A drop in voltage difference across the oolemma is a direct measure of the ion leakage through the oolemma during hypothermia. Figure 1 shows that after 4 hours of exposure to 4°C in a PBS solution, the membrane potential of 20.4% of the exposed oocytes was within two standard deviations from the mean value of the membrane potential in fresh oocytes from the same batch. The voltage difference across the oolemma dropped below two standard deviations from the mean in fresh oocytes, in 79.6% of the hypothermically exposed oocytes (P < 0.05). This suggests that there was significant ion leakage across the oolemma in the majority of the oocytes exposed to hypothermic conditions in the PBS solution. In contrast, in the presence of 40 mg/ml AFGPs 1-8, 83.3% of the oocytes retained a membrane potential across the oolemma which was within two standard deviations from the mean in the fresh oocytes. Obviously the ion leakage across the oolemma was significantly reduced by the presence of AFGPs 1-8, which appear to protect the oolemma and inhibit ion leakage at hypothermic temperatures. Results from experiments in which the concentration of AFGPs 1-8 was varied, show that the protection afforded by AFGPs 1-8 is consistently high at concentrations ranging from 40 mg/ml to 1 mg/ml. Then, at the lower concentration of 0.1 mg/ml AFGPs 1-8, the ion leakage increases significantly. 1371
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< t . u 100 n-,~ ~o':, LtJ O
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Figure 1. Percentage of oocytes with a membrane potential within two standard deviations from the mean in fresh oocytes, (oocytes not included are statistically different (P < 0.05) from control), after 4 hours of exposure to 4°C, in different solutions. Values are mean + one standard deviation. Each experimental group consists of 5 oocytes and n represents the number of groups.
The effect of AFGPs 1-8 becomes more obvious after 24 hours at 4°C. Figure 2 shows that for concentrations between 40 mg/ml AFGPs 1-8 and 1 mg/ml AFGPs 1-8, between 59% to 65% of the oocytes had a membrane potential within two standard deviations from the membrane potential in fresh oocytes from the same batch, i.e. retained a normal electrical potential across the oolemma. In contrast, none of the oocytes preserved in PBS or PBS with 0.1 mg/ml AFGPs 1-8 had any measurable membrane potential after 24 hours at 4°C. It should be emphasized that the membrane potential in all the oocytes preserved in PBS or PBS with 0.1 mg/ml AFGPs 1-8, was, after a 24 hour exposure to 4°C, either exactly zero or very close to zero, (P < 0.0005).
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0.1mglml 1.0 mg/ml 40rnglml 40mglml 40mglml AFGPI-8 AFGPI-8
A F G P I - 8 AFGPI-5 AFGP7,8
Figure 2. Percentage of oocytes with a membrane potential within two standard deviations from the mean in fresh oocytes (oocytes not included were statistically different (P < 0.05) from control) after 24 hours exposure to 4°C in different solutions. In cases with a percentage of 0%, the value is given in the figure (in these cases oocytes not included were statistically different (P < 0.0005) from control). Values are mean + one standard deviation. Each experimental group consists of 5 oocytes and n represents the number of groups. 1372
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It is obvious from Figures 1 and 2 that in the absence of AFGPs 1-8 the potential difference across the oolemma drops rapidly with exposure to hypothermic temperatures which implies membrane damage and ion leakage. However, the membrane potential in the presence of AFGPs 1-8 at concentrations higher than 1 mg/ml, is retained even after 24 hours at 4°C. Therefore, it is possible to conclude from this part of the study that AFGPs 1-8 interact with the oolemma at hypothermic temperatures, protect the oolemma from damage caused by hypothermic exposure and inhibit ion leakage. This is a property of AFGPs 1-8 that was not known prior to our work, and may become very important in attempts to preserve biological materials at hypothermic conditions. Another intriguing property of the antifreeze glycoproteins emerges from results presented in Figures 1 and 2. The Figures show that while the combination of AFGPs 1-8 at a concentration of 40 mg/ml protects the oolemma and inhibits ion leakage, AFGPs 1-5 and AFGPs 7,8 separately, do not protect the oolemma or stop the ion leakage, even at concentrations as high as 40 mg/ml. (It should be emphasized that the combination AFGPs 1-8 was obtained by actually mixing 1 part weight AFGP 1-5 with three parts weight AFGP 7,8.) After 4 hours exposure to 4°C, only 49% of the oocytes in 40 mg/ml AFGPs 1-5, and only 36.5% of the oocytes in 40 mg/ml AFGP 7, 8 retained a membrane potential which was within two standard deviations from the mean in fresh oocytes. In comparison, in a solution with 40 mg/ml AFGPs 1-8, 84% of the oocytes retained a membrane potential within two standard deviations from the mean in fresh oocytes. After 24 hours exposure to a temperature of 4°C all the oocytes exposed in solutions with 40 mg/ml AFGPs 1-5, and 40 mg/ml AFGPs 7, 8 showed practically zero potential difference across the oolemma, similarly to unprotected oocytes (P < 0.0005). In comparison, 59% of the oocytes protected by 40 mg/ml AFGPs 1-8 had a membrane potential within two standard deviations from that in fresh oocytes. It is obvious that the whole physiological combination of AFGPs 1 to 8 is needed for protection and that AFGPs 1 to 5 and AFGPs 7, 8 separately do not protect the oolemma or stop the ion leakage, even at very high concentrations. This result is extremely surprising because studies on the effects of AFGPs on depressing the phase transition temperature of water and modifying the pattern of ice crystal formation show that AFGPs 1-5 depress the phase transition temperature almost as effectively as the whole combination of AFGPs 1 to 8 (2, 4). On the other hand, AFGPs 1 to 5 separately do not protect the oolemma and do not block ion leakage, and neither do AFGPs 7, 8 separately. In summary, the study performed here demonstrates by means of a model ideally suited for this purpose, the pig oocyte, through measurements of the membrane potential across the oolemma, that AFGPs 1-8 interact with cell membranes, protect the membranes from damaging hypothermic conditions and inhibit ion leakage. AFGPs 1-5 and AFGPs 7, 8 separately do not afford the same protection. It is possible to speculate that since hypothermic damage to the oolemma could be associated with different mechanisms such as destabilization of lipid structures and ion leakage the protection must occur through an interaction between the Antarctic fish glycopeptides and the oolemma It is possible that the glycopeptides may offer protection by binding to available sites on the oolemma. These sites could be the hydrophi llic part of membrane proteins. Regardless of the accuracy of these speculations it is obvious mat much work 1373
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remains to be done on this topic, on the mechanism of membrane protection, the inhibition of ion leakage and the intriguing findings concerning the lack of effective protection with AFGPs 1-5 and AFGPs 7-8 separately. Obviously our finding opens a new area of research on the properties of the AFGPs in relation to protection of cell membranes and ion leakage, on the significance of the different AFGPs in the adaption of fishes to the Antarctic environment and on the potential application of these properties to hypothermic and cryogenic preservation of ceils and organs.
ACKNOWLEDGMENTS: We would like to express our gratitude to Professor E. Seren, Drs. G. Galeati and M.L. Bacci for their help with these experiments. One of the authors, B.R. wishes to express his gratitude to the Institute of Veterinary Physiology at the University of Bologna, Italy for hosting him while this work was performed at the Institute. Partial support from the NSF through grant NSF CTS-8914832 is acknowledged. The authors A.A. and M.M. were supported by CNR-RA15A: Project Agrobiotechnologies in animal production No. 1.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
DeVries, A.L., Wohlschlag, D.E., (1965) Science, 163, 1074-1075. DeVries, A.L., (1984) Trans. R. Soc. Lond. B304, 575-588. DeVries, A.L., (1988) Comp. Biochem. Physiol. 90B (3), 611-621. Aranthanarayanan, V.S., (1989) Life Chemistry Reports, 7, 1-32. Lin, Y., Duman, J.G., DeVries, A.L., (1971) Biochem. Biophys. Res. Commun., 46, 89-92. Hew, L.C., Slaughter, D., Fletcher, G.L., Joshi, S.B., (1981) Can. J. Zool., 59, 2186-2192. Raymond, J.A. and DeVries, A.L., (1977) Proc. Natl. Acad. Sci. USA 74, 2589-2593. Raymond, J.A., Wilson, P.W. and DeVries, A.L., (1989) Proc. Natl. Acad. Sci., 86(3), 881-885. Schrag, J.D.and DeVries, A.L., (1983) Comp. Biochem. Physiol., 74A, 381-385. Rubinsky, B., Arav, A., and DeVries, A.L., Cryobiology, (in review). Fahning; M.L., de Garcia, M., (1989) Cryobiology, 26, 563. Newmann, H., (1985) Theriogenology, 23, 213. Mattioli, M. Galeati, G. Bacci, M.L., Seren, E., (1988) Gamete Research, 21, 223-232. Mattioli, M., Barboni, B., Bacci, M.L., and Seren, E., (1990) Biology of Rep., 43, 318-323.
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