BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 180, No. 2, 1991
Pages 566-571
October 31, 1991
HYPOTHERMIC PROPERTY
PROTECTION
- A FUNDAMENTAL
OF "ANTIFREEZE"
PROTEINS
B. Rubinskyt.*, A. Arav~, and G.L. Fletcher§ t Department of Mechanical Engineering, University of California, Berkeley, CA 94720 :~ Instituto di Fisiologia Veterinaria, Universita di Bologna, 40126 Italy § Marine Sciences Research Laboratory, Memorial University of Newfoundland St. John's, Newfoundland, Canada A1C 5S7 Received August 23, 1991
Summary: For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4°C for 24 h in the presence of type I alanine rich tx helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that coldsensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins. © 1991 Academic Press, Inc.
Scholander and his colleagues (1) were the first to show that some fish off the coast of Labrador can survive in sea-water at temperatures as low as -1.9°C, in the presence of ice crystals. They termed the responsible solute "antifreeze" but were unable to determine its chemical nature. DeVries and Wohlschlag (2) isolated the antifreeze from the blood of Antarctic nototheniid fish and demonstrated that it was a proteinaceous macromolecule. These antifreezes were characterized as a series of relatively high molecular weight (2.5 kDa to 35 kDa) glycoproteins made up primarily of a tripeptide repeat (Ala-Ala-Thr)n with a disaccharide attached to the hydroxyl oxygen of the threonine residue (3) and are referred to as "antifreeze glycoproteins" (AFGP). Essentially identical AFGP have been isolated from the blood of northern cods (4). Some species of fish produce compounds with "antifreeze" properties similar to those of the AFGP, but which do not contain any prosthetic groups. These compounds are referred to as
* To whom correspondence should be addressed.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
566
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
"antifreeze polypeptides" (AFP). While the AFGP isolated from different fish species are similar in their chemical structure there exist a variety of AFP which have been classified broadly similar in their chemical structure there exist a variety of AFP which have been Classified broadly into three major groups. AFP of types I, II, and III (5,6,7). All three types affect ice crystal growth in a manner similar to that of the AFGP. AFP of type I were first identified by Duman and DeVries (8) in the northern winter flounder (Pseudopleuronectes americanus). These AFP consist of a family of seven independently active compounds which are alanine rich (60 tool %) ranging in molecular weight from 3.3 kDa to 4.5 kDa. They are ampbiphilic ct - helices in which the majority of the hydrophilic amino acid side chains project along the length of one side of the helix while the opposite side is predominantly hydrophobic (9). The type II AFP is larger than type I (14 kDa) has a [~ structure and five disulfide bridges. This AFP was first isolated from the sea raven (Hemitripterus ameri-
canus) (10). Similar AFP have been isolated from Atlantic herring, and smelt (11) and insects (5). Hew and his colleagues (12) first isolated an AFP of type III from the Newfoundland ocean pout (Macrozoarces americanus) belonging to the family Zoaridae. These AFP range in molecular weight from 5 kDa to 6.7 kDa, are not alanine rich, contain no cysteine residues and have no distinguishing secondary structural features. Similar AFP have been isolated from Antarctic zoarcids (13). While past research on the properties of "antifreeze" proteins has focused on their ability to modify ice crystal structure and on the mechanism by which this is achieved (5,14,15,16) we have found that AFGP can protect mammalian oocytes at cryogenic temperatures (-130°C) (17) and at hypothermic temperatures (4°C) (18). Porcine oocytes are normally completely destroyed by cryogenic temperatures (17,19,20). However when they were rapidly cooled to -130°C in a vitrifying solution containing AFGP, approximately 80% of them retained intact oolemmas and 25% could undergo in vitro maturation (17). In a follow-up experiment exposing porcine oocytes to 4°C for 24h in the presence of AFGP it was observed that 80% of them retained an intact oolemma and about 70% a normal membrane potential. In the absence of AFGP the integrity of the oolemma is completely destroyed (18). The present study was conducted to answer two general questions based on the foregoing results. Are the AFGP unique in their ability to protect cell membranes from hypothermic damage, or is this protection a general property of all known antifreeze protein classes? Does the membrane protection afforded by the antifreeze proteins improve the viability of cold-sensitive mammalian cells? To answer these questions experiments were performed with immature bovine oocytes exposed to hypothermic conditions.
Materials and Methods
Immature bovine oocytes, in multilayered dense cumulus and with no alteration of the cytoplasm were obtained from selected follicles (2-6 mm) of cow and heifer ovaries brought to the laboratory in warm saline (0.9%). The follicles were aspirated with a 15 gauge needle within 80 minutes after slaughter. The oocytes were kept in a standard buffer solution, PBS, (Dulbecco's phosphate buffered saline supplemented with 0.4 v/v BSA (bovine serum albumin), 0.34 mM pyruvate, 5.5 mM glucose and 15 mM Kanamycin). Prior to the hypothermic experiments the 567
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
oocytes were introduced into Eppendort vials containing 0.~ ml of the following soluUons: a) PBS; b) PBS with 20 mg/ml AFP of type I (Winter flounder), c) PBS with 20 mg/ml AFP of type II (Sea raven), d) PBS with 20 mg/ml AFP of type llI (Ocean pout). (A concentration of 20 mg/ml was chosen because it is within the physiological range for all the "antifreeze" proteins). All three antifreeze proteins were purified from fish blood plasma using Sephadex G75 as described in Kao et al., (21). Additional control experiments were performed with oocytes in a PBS solution with 0.1 M sucrose and in a PBS solution with 20% v/v FCS (fetal calf serum). The oocytes were incubated in the various solutions at 4°C for 24h. When the oocytes were removed from the 4°C environment the integrity of their oolemma was determined using three different tests; morphological examination, fluoroscein diacetate (FDA) staining, and trypan blue (TB) exclusion. The morphological examinations were performed using a Leitz microscope with phase contrast. The FDA staining test employs the fact that fluoroscein diacetate is converted to a fluorescent compound only when it contacts hydrolases in the live cell. Oocyte viability and oolemma intactness results in intense fluorescence, while nonfluorescence indicates nonviability (20,22,23). In our experiments the oocytes were exposed to a PBS solution containing 5 t.tg/ml FDA as described by Didion et al. (20). After 3 minutes exposure to the FDA the oocytes were placed on a slide under a coverslip. The oocytes were screened in a fluorescent microscope (Leitz), to which a photoreceptor was connected. The photoreceptor can evaluate quantitatively the intracellular fluorescence. Prior to the experiments with oocytes exposed to hypothermic conditions, similar, control experiments were performed with fresh oocytes. The mean fluorescence intensity and the standard deviation was determined for fresh oocytes, and used to establish a quantitative criteria for evaluating the integrity of the oolemma. An oocyte was considered to have an intact oolemma after hypothermic exposure if the quantitative reading of the photoreceptor was within two standard deviations of the mean in fresh oocytes. Trypan blue exclusion (TB) is commonly used to determine membrane integrity (20). Exclusion of trypan blue is indicative of an intact oolemma. In the TB exclusion test an aqueous solution containing 0.1% TB was added to the oocytes in the PBS solution for 3 minutes. The oocytes were then examined with a phase contrast microscope, (Leitz) to determine if the trypan blue was excluded. Additional tests were performed to determine the ability of the oocytes to undergo in vitro maturation and in vitro fertilization. Following hypothermic exposure, the oocytes with their cumulus cells were incubated in a standard maturation medium, TCM-199 with 10% v/v FCS supplemented with 500 ng/ml hormones, LH (NiH-LH-B9) and FSH (bFsH-LER-1596-1) and granulosa cells. The granulosa cells were used at concentration of approximately 5 x 106 cells/ml and were obtained by dissection of small, fresh follicles, which were washed and recollected in the maturation medium. The oocytes were cultured in the incubation medium at 39°C in an atmosphere of 5% CO2 in air, for 24 hours. After incubation, the oocytes were fixed in an acetic acid: alcohol mixture (1:3 v/v) and stained after 24 hours with lacmoid stain to determine the percentage which had undergone in vitro maturation. Microscopic evidence of the second metaphase stage, MII, and/or extrusion of the first polar body was used to determine oocyte meiotic maturation. Incubated oocytes which were not used to study in vitro maturation were fertilized, in vitro. Immediately prior to insemination the oocytes were transferred for 5 minutes to a PBS solution containing 0.1% hyaluronidase (Sigma) to remove part of the cumulus cells. Then the oocytes were placed in an insemination medium, (Brackett and Oliphant) B-O, (24), supplemented with 1.9 mg/ml caffeine. Semen frozen in straws was used for in vitro fertilization. After thawing the straws in 35°C water the semen was washed by centrifugation in B-O medium. Capacitation was achieved by incubating the sperm for 15 min. in B-O medium supplemented with heparin (100 mg/ml) as described by Fukui et al., (25). Capacitated bull sperm at a final concentration of 106 cells/ml were then coincubated with in vitro matured oocytes. After 14-16 hours of culture the oocytes were fixed and stained using the technique described earlier for studies on maturation. Oocyte morphology (cytoplasm, oolemma) and fertilization were evaluated
Results and Discussion
Approximately 1000 oocytes were individually studied in the experiments reported here. The results are summarized in Figs. 1 and 2. Figure 1 shows the percentage of oocytes with an intact oolemma after hypothermic exposure as determined by three different tests, morphological examination, fluorescein diacetate (FDA) staining and trypan blue (TB) exclusion. It is evident 568
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 80
[~ r n=-20
Morph. .
.
[]]] TB .
.
•
FDA
~
.
~=__
2 E
60
/
II1111 llilil!
20
I,,~ 0
,
CONTROL AFP(WF)
CON]tK~
[]
AFRWF)
62 52 4~ ~ \ ~
[]
~P
Vol. 180, No. 2, 1991
Pages 566-571
October 31, 1991
HYPOTHERMIC PROPERTY
PROTECTION
- A FUNDAMENTAL
OF "ANTIFREEZE"
PROTEINS
B. Rubinskyt.*, A. Arav~, and G.L. Fletcher§ t Department of Mechanical Engineering, University of California, Berkeley, CA 94720 :~ Instituto di Fisiologia Veterinaria, Universita di Bologna, 40126 Italy § Marine Sciences Research Laboratory, Memorial University of Newfoundland St. John's, Newfoundland, Canada A1C 5S7 Received August 23, 1991
Summary: For the last two decades fish antifreeze proteins have been considered to function exclusively in conferring freeze-resistance to fish by binding to ice crystals and thereby depressing blood plasma freezing points non-colligatively. We report here the discovery of a second fundamental property of antifreeze proteins, the ability to protect cells and their membranes from hypothermic damage. Experiments were carried out exposing immature bovine oocytes to 4°C for 24 h in the presence of type I alanine rich tx helical antifreeze polypeptides (AFP) from winter flounder, type II cysteine-rich AFP from sea raven or type III AFP from ocean pout. The presence of AFP in the incubation medium resulted in an approximate four fold increase in the number of oocytes retaining an intact oolemma and a three fold increase in the number of oocytes able to undergo in vitro maturation. None of the control oocytes could be fertilized, whereas, of those incubated in AFP, the percentage which developed normally following fertilization was comparable to that observed for fresh oocytes. These results indicate that coldsensitive mammalian cells can be rendered cold-tolerant through the addition of "antifreeze" proteins. © 1991 Academic Press, Inc.
Scholander and his colleagues (1) were the first to show that some fish off the coast of Labrador can survive in sea-water at temperatures as low as -1.9°C, in the presence of ice crystals. They termed the responsible solute "antifreeze" but were unable to determine its chemical nature. DeVries and Wohlschlag (2) isolated the antifreeze from the blood of Antarctic nototheniid fish and demonstrated that it was a proteinaceous macromolecule. These antifreezes were characterized as a series of relatively high molecular weight (2.5 kDa to 35 kDa) glycoproteins made up primarily of a tripeptide repeat (Ala-Ala-Thr)n with a disaccharide attached to the hydroxyl oxygen of the threonine residue (3) and are referred to as "antifreeze glycoproteins" (AFGP). Essentially identical AFGP have been isolated from the blood of northern cods (4). Some species of fish produce compounds with "antifreeze" properties similar to those of the AFGP, but which do not contain any prosthetic groups. These compounds are referred to as
* To whom correspondence should be addressed.
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
566
Vol. 180, No. 2, 1 9 9 1
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
"antifreeze polypeptides" (AFP). While the AFGP isolated from different fish species are similar in their chemical structure there exist a variety of AFP which have been classified broadly similar in their chemical structure there exist a variety of AFP which have been Classified broadly into three major groups. AFP of types I, II, and III (5,6,7). All three types affect ice crystal growth in a manner similar to that of the AFGP. AFP of type I were first identified by Duman and DeVries (8) in the northern winter flounder (Pseudopleuronectes americanus). These AFP consist of a family of seven independently active compounds which are alanine rich (60 tool %) ranging in molecular weight from 3.3 kDa to 4.5 kDa. They are ampbiphilic ct - helices in which the majority of the hydrophilic amino acid side chains project along the length of one side of the helix while the opposite side is predominantly hydrophobic (9). The type II AFP is larger than type I (14 kDa) has a [~ structure and five disulfide bridges. This AFP was first isolated from the sea raven (Hemitripterus ameri-
canus) (10). Similar AFP have been isolated from Atlantic herring, and smelt (11) and insects (5). Hew and his colleagues (12) first isolated an AFP of type III from the Newfoundland ocean pout (Macrozoarces americanus) belonging to the family Zoaridae. These AFP range in molecular weight from 5 kDa to 6.7 kDa, are not alanine rich, contain no cysteine residues and have no distinguishing secondary structural features. Similar AFP have been isolated from Antarctic zoarcids (13). While past research on the properties of "antifreeze" proteins has focused on their ability to modify ice crystal structure and on the mechanism by which this is achieved (5,14,15,16) we have found that AFGP can protect mammalian oocytes at cryogenic temperatures (-130°C) (17) and at hypothermic temperatures (4°C) (18). Porcine oocytes are normally completely destroyed by cryogenic temperatures (17,19,20). However when they were rapidly cooled to -130°C in a vitrifying solution containing AFGP, approximately 80% of them retained intact oolemmas and 25% could undergo in vitro maturation (17). In a follow-up experiment exposing porcine oocytes to 4°C for 24h in the presence of AFGP it was observed that 80% of them retained an intact oolemma and about 70% a normal membrane potential. In the absence of AFGP the integrity of the oolemma is completely destroyed (18). The present study was conducted to answer two general questions based on the foregoing results. Are the AFGP unique in their ability to protect cell membranes from hypothermic damage, or is this protection a general property of all known antifreeze protein classes? Does the membrane protection afforded by the antifreeze proteins improve the viability of cold-sensitive mammalian cells? To answer these questions experiments were performed with immature bovine oocytes exposed to hypothermic conditions.
Materials and Methods
Immature bovine oocytes, in multilayered dense cumulus and with no alteration of the cytoplasm were obtained from selected follicles (2-6 mm) of cow and heifer ovaries brought to the laboratory in warm saline (0.9%). The follicles were aspirated with a 15 gauge needle within 80 minutes after slaughter. The oocytes were kept in a standard buffer solution, PBS, (Dulbecco's phosphate buffered saline supplemented with 0.4 v/v BSA (bovine serum albumin), 0.34 mM pyruvate, 5.5 mM glucose and 15 mM Kanamycin). Prior to the hypothermic experiments the 567
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
oocytes were introduced into Eppendort vials containing 0.~ ml of the following soluUons: a) PBS; b) PBS with 20 mg/ml AFP of type I (Winter flounder), c) PBS with 20 mg/ml AFP of type II (Sea raven), d) PBS with 20 mg/ml AFP of type llI (Ocean pout). (A concentration of 20 mg/ml was chosen because it is within the physiological range for all the "antifreeze" proteins). All three antifreeze proteins were purified from fish blood plasma using Sephadex G75 as described in Kao et al., (21). Additional control experiments were performed with oocytes in a PBS solution with 0.1 M sucrose and in a PBS solution with 20% v/v FCS (fetal calf serum). The oocytes were incubated in the various solutions at 4°C for 24h. When the oocytes were removed from the 4°C environment the integrity of their oolemma was determined using three different tests; morphological examination, fluoroscein diacetate (FDA) staining, and trypan blue (TB) exclusion. The morphological examinations were performed using a Leitz microscope with phase contrast. The FDA staining test employs the fact that fluoroscein diacetate is converted to a fluorescent compound only when it contacts hydrolases in the live cell. Oocyte viability and oolemma intactness results in intense fluorescence, while nonfluorescence indicates nonviability (20,22,23). In our experiments the oocytes were exposed to a PBS solution containing 5 t.tg/ml FDA as described by Didion et al. (20). After 3 minutes exposure to the FDA the oocytes were placed on a slide under a coverslip. The oocytes were screened in a fluorescent microscope (Leitz), to which a photoreceptor was connected. The photoreceptor can evaluate quantitatively the intracellular fluorescence. Prior to the experiments with oocytes exposed to hypothermic conditions, similar, control experiments were performed with fresh oocytes. The mean fluorescence intensity and the standard deviation was determined for fresh oocytes, and used to establish a quantitative criteria for evaluating the integrity of the oolemma. An oocyte was considered to have an intact oolemma after hypothermic exposure if the quantitative reading of the photoreceptor was within two standard deviations of the mean in fresh oocytes. Trypan blue exclusion (TB) is commonly used to determine membrane integrity (20). Exclusion of trypan blue is indicative of an intact oolemma. In the TB exclusion test an aqueous solution containing 0.1% TB was added to the oocytes in the PBS solution for 3 minutes. The oocytes were then examined with a phase contrast microscope, (Leitz) to determine if the trypan blue was excluded. Additional tests were performed to determine the ability of the oocytes to undergo in vitro maturation and in vitro fertilization. Following hypothermic exposure, the oocytes with their cumulus cells were incubated in a standard maturation medium, TCM-199 with 10% v/v FCS supplemented with 500 ng/ml hormones, LH (NiH-LH-B9) and FSH (bFsH-LER-1596-1) and granulosa cells. The granulosa cells were used at concentration of approximately 5 x 106 cells/ml and were obtained by dissection of small, fresh follicles, which were washed and recollected in the maturation medium. The oocytes were cultured in the incubation medium at 39°C in an atmosphere of 5% CO2 in air, for 24 hours. After incubation, the oocytes were fixed in an acetic acid: alcohol mixture (1:3 v/v) and stained after 24 hours with lacmoid stain to determine the percentage which had undergone in vitro maturation. Microscopic evidence of the second metaphase stage, MII, and/or extrusion of the first polar body was used to determine oocyte meiotic maturation. Incubated oocytes which were not used to study in vitro maturation were fertilized, in vitro. Immediately prior to insemination the oocytes were transferred for 5 minutes to a PBS solution containing 0.1% hyaluronidase (Sigma) to remove part of the cumulus cells. Then the oocytes were placed in an insemination medium, (Brackett and Oliphant) B-O, (24), supplemented with 1.9 mg/ml caffeine. Semen frozen in straws was used for in vitro fertilization. After thawing the straws in 35°C water the semen was washed by centrifugation in B-O medium. Capacitation was achieved by incubating the sperm for 15 min. in B-O medium supplemented with heparin (100 mg/ml) as described by Fukui et al., (25). Capacitated bull sperm at a final concentration of 106 cells/ml were then coincubated with in vitro matured oocytes. After 14-16 hours of culture the oocytes were fixed and stained using the technique described earlier for studies on maturation. Oocyte morphology (cytoplasm, oolemma) and fertilization were evaluated
Results and Discussion
Approximately 1000 oocytes were individually studied in the experiments reported here. The results are summarized in Figs. 1 and 2. Figure 1 shows the percentage of oocytes with an intact oolemma after hypothermic exposure as determined by three different tests, morphological examination, fluorescein diacetate (FDA) staining and trypan blue (TB) exclusion. It is evident 568
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 80
[~ r n=-20
Morph. .
.
[]]] TB .
.
•
FDA
~
.
~=__
2 E
60
/
II1111 llilil!
20
I,,~ 0
,
CONTROL AFP(WF)
CON]tK~
[]
AFRWF)
62 52 4~ ~ \ ~
[]
~P
