Hypothermic storage of sheep embryos with antifreeze proteins: Development in vitro and in vivo

ELSEVIER

HYPOTHERMIC

STORAGE OF SHEEP EMBRYOS WITH ANTIFREEZE PROTEINS DEVELOPMENT IN VITRO AND M VJVO

A.Baguisi,l A. Ara~,~ T.F.Crosby,l

J.F.Roche2 andM.P.Boland’

lFacuh.y of Agriculture, University College Dublin, Belfield, Dublin 4, Ireland ‘Faculty of Veterinary Medicine, University College Dublin, Dublin 4, Ireland 3Department of Mechanical Engineering, University of California, Berkeley, CA, USA Received for publication: Accepted:

February May 16,

17, 1997 1997

ABSTRACT Antifreeze proteins (AFPs) non-colhgatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. Jn Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4’C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viabilityrates werenot different between controls (18/21); BSA (9/15); WFat 1 mg/ml(l4/15); WF at 10 mg/ml(l3/15) or OP at I mg/nd (15/21), but were decreased (PC 0.05) in embryos stored in OP at 1 0 mg/ml (I l/20). Pooled data showed higher (PC 0.05) viability rates for WF (27/30) than for OP (26/41) or BSA (9/15). There was no ef%t of protein source on hatching rates, but mean hatched diameters of embryos were lower (p< 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0’ or 4OC in 4 mg/n-d BSA or 1 mgml WF. Embryos stored in WF at 4’C (WF/4’C) had comparable hatching rates (S/12) to that of controls (lo/IO), but embryos in the other treatments (WF O°C, 5/11; BSA 4’C, 6/11 and BSA O°C, 300) had significantly lower hatching rates (P2°C/min; T2, WF >2°C/min and T3, WF
1997 by Elsevier Science

InC

Key words: ovine, embryos, hypothermia, antifreeze proteins

Acknowledgments We thank A/P Proteins for providing the antifreeze proteins and G. Ramsbottom, P. Duffy and M. Wade for technical assistance. A. Baguisi was supported by BioResearch Ireland. Theriogenology 48:1017-1024, 1997 0 1997 by Elsevier Science Inc.

0093-691X/97/$17.00 PII s0093-691x(97)00328-2

1018

Theriogenology

INTRODUCTION Some degree of hypothermia can generally be tolerated by mammalian cells, but the suppression of their biochemical and physiological mechanisms as an effect of cooling, if prolonged, is normally incompatible with survival. A primary criterion for cell survival is the regulation of the normal state of ion equilibration across membranes. During sustained hypothermic exposure, a shift in ion concentration across the membrane occurs, leading to increased cellular perturbations, which, if not reversed, will ultimately lead to cell damage or death Different animal species have evolved their own adaptive mechanisms for survival in response to fluctuating cold temperatures in their environment. Polar and subpolar fish (2,3,7,8,23,26), overwintering insects and arachnids (2,3,9,10,11,29) can tolerate subzero freezing temperatures by producing thermal hystheresis proteins (THPs) that depress the temperature where freezing and supercooling of their body fluids occur. These proteins function by non-colligatively lowering the freezing point of water without changing its normal melting temperature. The proteins were isolated and grouped as either antifreeze proteins or polypeptides (AFPs) of Type I (Winter Flounder) with MW of 3.3 to 4.5 kDa, Type 2 (Sea Raven) with MW of 15 kDa and Type 3 (Ocean Pout) with MW of 5 to 6.7 kDa (2,13,19) or antifreeze glycoproteins (AFGPs) with MW of 2.6 to 34 kDa (7,s). The thermal hystberesis property is e&ted by binding to the growing plane of ice crystals, thereby altering ice growth and structure. Another fundamental property of THPs is the ability to protect cells and membranes from hypothermic damage (23,24). Evidence also suggests that the antifreexe protein from Winter Flounder exhibits the ability to block ion channels, specifically suppressing calcium and potassium fluxes across cell membranes (25). Although embryos can be preserved by hypothermia, its adoption as an alternative means of preservation is dependent on the duration of storage compatible with maintaining high viability on transfer. Current cryopreservation techniques cause a significant reduction in cell viability, resulting in variable and often lower pregnancy rates than those obtained after fresh embryo transfer. Some early studies on embryo preservation involved storage at temperatures of 5O to 10°C (4,11,12,27). More recently, lower temperatures (O’to 4OC) have been employed (6,15,16,17,20), with high survival rates in vitro and in vivo when storage was limited to 24 h, but storage for longer than 48 h significantly decreased the survival rates. The objective of this study was to determine if the polypeptide group of THPs (AFP Types 1 and 3) could maintain viability of ovine embryos during extended hypothermic storage at 0’ to 4OC in vitro as assessed by in vitro culture or transfer to synchronized recipients.

MATERIALS AND METHODS Superovulation and Embryo Recovery Estrus was synchronized in crossbred ewes (n=lO5) using fluorogestone acetate (30 mg) intravaginal pesraries for 14 d (Chronogest, Intervet, Boxmeer, Holland). Superovulation was initiated 48 h prior-to pessary removal, using p-FSH (Folltrophin V-FSH equivalent to 200 mg NIH-FSH, Vetrapharm, Canada) given twice daily for 3 d by intramuscular injection at a dose of 33 mg per injection. Forty hours following pessary withdrawal, laparoscopic insemination was carried out, and the ewes were joined with fertile rams. Six days post insemination the ewes were slaughtered and embryos recovered by reverse flushing of the uterine horns with PBS (Gibco, Life Technologies Ltd., Paisley, Scotland) supplemented with 3% heat-treated ewe serum (HTES). Following recovery, the embryos were assessed and grouped according to morphological criteria on a scale of 1 to 5 as previously described (5). Grade 1 to 3 embryos were washed in handling media of PBS supplemented with 15% HTES at room temperature (22’C). The embryos were then allocated at random to different treatment groups within 2 h of collection.

Theriogenology

1019

Expenment 1 Morula- and blastocyst-stage embryos (Grades 1 and 2) were assigned to 1 of 6 treatment groups. In Treatment 1, embryos were cultured in vitro immediately following recovery (Tl; culture controls, n=2 1). Embryos were loaded into 1.5~ml microcentrifuge tubes containing 200 pl PBS solution of bovine serum albumen (BSA, Fraction V, Sigma) or fish antifreeze proteins (A/F Proteins) from Winter Flounder (WF; Type 1 AFP) or from Ocean Pout (OP; Type 3 AFP). The embryos were assigned to the following treatments: T2 (n=15) BSA at 4 mg/ml; T3 (n=21) OP at 1 mg/ml; T4 (n=20) OP at 10 mg/ml; T5 (n=15) WF at 1 mg/ml; and T6 (n=15) WF at 10 mg/ml. The embryos in the treated groups were cooled from ambient temperature (22’C) to 4’C by loading the tubes on trays floating on temperature-equilibrated distilled water and storing in a refrigerator for 4 d. Following hypothermic storage, embryos were recovered from the tubes and washed twice in PBS supplemented with 15% BTES. The embryos were then cultured III vttro in CR-l media, and embryo viability, hatching rate and hatched diameters of embryos were assessed every 24 h for 72 h. Experiment 2 Grade 1 to 3 morula- or blastocyst-stage embryos were allocated to 1 of 5 treatment groups. Treatment 1 (Tl) embryos were cultured immediately following recovery (Tl, culture controls, n=lO). Embryos assigned to Treatments 2 to 5 (T2 to T5) were exposed to hypothermic conditions in solmions containing 4 nu&nl BSA (T2 BSA, n=lO) or 1 mgAnl Winter Flounder (T3 WFl, n=ll), then stored at O°C in a mixture of crushed wet ice and water, and placed in a refrigerator; while embryos in Treatments 4 and 5 were placed in medium containing BSA (T4 BSA, n=ll) or WFl (T5, n=12) and were stored at 4OC, as described in Experiment 1. The embryos were loaded into 1.5~ml microcentrifuge tubes containing 200 ul of the protein solution and exposed to hypothermic temperatures for 4 d. Following hypc&ermic storage, the embryos were washed twice in PBS supplemented with 15% HTES and cultured in vitro for 72 h as in Experiment 1. Viability, hatching and hatched diameters were assessed every 24 h until the end of the culture period Experiment 3 Grade 1 to 3 morula- or blastocyst-stage embryos were randomly assigned to 1 of 4 treatments. Treatment I (Tl) consisted of fresh embryos transferred within 2 h of recovery (n=21); T2 embryos were stored at 4OC in BSA (4mg/ml) and cooled rapidly (n=27); T3 embryos were stored at 4OC in Winter Flounder (1 mg/ml) and cooled rapidly (n=23); and T4 embryos were stored as in Treatment 3 but were cooled slowly (n=27). Rapid cooling was achieved by cooling from 22OC to 4OC at > 2°C/min, while slow cooling involved a rate of
Theriogenology glutamine, pyruvate (Sigma; tissue culture grade) and 10% fetal calf serum (FCS; Gibco). Embryos were assessed morphologically for viability @lastulation and expansion) every 24 h for 72 h. Hatching rates and hatched diameters were recorded at the end of the culture period, Statistical Analysis Chi-square analysis was performed on the data from the 3 experiments. variance was performed on the hatched diameters in Experiments 1 and 2.

One-factor analysis of

RESULTS Experiment 1 The viability rates of embryos after 4 d of hypothermic storage and subsequent culture for 3 d were decreased in embryos stored in 10 mg/ml OP (PC 0.05) compared with the other treatments (Table 1); however, the results were not different between controls, BSA, WF or OP at a concentration of 1 mg/ml. When the data were pooled by protein type, the viability rates of embryos were higher (,P < 0.01) following storage in WF (QO%, 27/30) than in OP (63%, 26/41) or BSA (60%, Q/15) There was no effect of protein source on the hatching rates in vitro (P> 0.05). Diameters of hatched embryos at the end of culture were comparable for controls and embryos stored in 1 mg/ml WF; however, they were smaller when embryos were stored in 10 mg/ml WF or in 1 and 10 mg/ml OP (PC 0.05). Diameters of hatched embryos stored in BSA were smaller (PC 0.05) than in all other treatments,

Table 1. Viability rates in vitro (72 hours) of ovine embryos following storage at 4OC for 4 days in PBS supplemented with bovine serum albumen (BSA) at 4 n@nl or in Winter Flounder Crype 1) or Ocean Pout (‘Type 3) antifreeze proteins at 1 and 10 mg/ml Treatment

Viability rate

group

(%)

Tl-Control TZBSA T3-OPl T4-OPlO TS-WFl T6-WFlO

18/21 (86)a Q/15 (60)ab 15/21 (7 1 ab 1 l/20 (55) L 14/15 (93)a 13/15 (87)a

Hatching rate (%) 15/21(7 l)a 7/15(47)a 12/21 (57)a Q/20 (45)a 11/15(73)a Q/15 (60)a

Diameter (pm)

453+7oa 260&10d 316*74bc 337*33c 415*125ab 353k137C

a,b$ Values within cohmms without common superscripts are diflbrent (P
Experiment 2 The viability (Table 2) of control embryos was not ditMent from that of embryos following hypothermic storage for 4 d in BSA irrespective of the temperature of storage, or of embryos stored in WF at 4’C; however, storage at O°C in WF decreased viability (PC 0.05). Hatching rates and hatched diameters of embryos were similar for control embryos and those stored in WF at 4OC; but these

1021

Theriogenology

parameters were significantly decreased (PC 0.01 and P< 0.025, respectively) following storage in BSA at 4OC or O°C, or in WF at O’C. When the data for tempemture (4’ or O°C) or protein type (BSA vs WF) were pooled, there were no diflbrences (P> 0.05) in the viability or hatching rates of the embryos.

Table 2. Viability and hatching rates of ovine embryos following hypothermic storage for 4 days at O°C or 4OC in PBS containing either bovine serum albumen (BSA) at 4 mgAnl or Winter Flounder (WF) at 1 mgkrd and subsequent culture in vitro for 72 hours Treatment

Viability rate

Hatching rate

Diameter (pm)

IO/10 (1oo)a 6/10 (60)ab 8/l 1 (73)ab 6/l 1 (54)b 9112 (75)ab

lO/lO (lOO)d 3/10 (3o)e 6/11 (54)e 5/l 1 (45)e 8/12 (67)de

432*46a 263*1Zc 275*20= 345*62b 383k35ab

group Tl-controls T2-BSA O°C T3-BSA 4OC T4-WF O°C TS-WF 4OC

a-e Values within columns with no common superscript are significantly different: az PcO.025; due P
Experiment 3 A total of 30 of the 39 recipient ewes was pregnant following surgical transfer of fresh or hypothermic-stored embryos at Days 28 to 30 post transfer (Table 3). There were no ditI+nces in pregnancy rates, in the number of viable fetuses, or in the percentage of viable fetuses recovered from pregnant ewes (P> 0.05) between any of the treatments. These data indicate that it is possible to store sheep embryos at 4OC for 4 d and transfer them into recipients previously synchronized with the donors and obtain reasonable pregnancy rates at 28 d post transfer.

Table 3. Pregnancy rates (Days 28 to 32) following surgical transfer of ovine embryos either fresh or following storage at 4OC for 4 d prior to transfer Treatment group Fresh (control) BSA > 2OC/m WF > 2OCfm WF < 1°C/m

Pregnancy rate (%) 618 (75) 10/l 1 (91) 6/l 0 (60) 8/10 (80)

No. viable fetuses (%) 11/21 (52) 14127 (52) lo/23 (44) 15/27 (56)

No.(%) viable ftises from pregnant ewes 1 l/16 (68) 14125 (56) IO/14 (71) 15/21 (71)

DISCUSSION This study demonstrates that in vivo viability of ovine embryos is not compromised following storage at hypothermic temperatures (4OC) for 4 d in BSA or in antifreeze proteins compared with that of

1022

Theriogenology

embryos transferred immediately following recovery. However, in vitro development was compromised using a high dose of TVpe 3 antifreeze protein. In many of the early studies with hypothermic preservation of embryos of different animal species, the storage temperatures used (5’ to 10°C) were relatively high (1,4,12,27). Harper and Rowson (12) reported that no ovine embryos stored at 7OC for 3 or more days survived after transfer. More recently, lower temperatures (O”, to 4OC) have been employed, but development in vitro and in vivo was considerably reduced when embryos were stored for longer than 48 h (6,15,16,17,20). Lindner et al. (17) reported higher in vitro survival rates with bovine embryos stored for 1 d than for 2, 3 or 5 d at 4’C, and no recipient that received embryos stored for 3 or 5 d became pregnant. Leibo and Winninger (15) reported a pregnancy rate of 58% when bovine embryos were stored at O°C for 12 to 24 h. The results of our current study suggest that it is possible to achieve relatively high survival rates in vitro and in vivo when embryos are cooled in a solution containing either antifreeze proteins or BSA and stored at 4’C rather than O°C for 4 d with subsequent development comparable to that of fresh embryos, Furthermore, the results suggest that cooling rates higher than 2°C/min do not compromise in vivo survival compared with embryos cooled at less than l°C/min or of embryos transferred immediately following recovery. The high hatching rates and hatched diameters attained by embryos in Experiments 1 and 2 provide an objective indication that embryo quality and thus potential development is not compromised following hypothermic storage. Furthermore, the normal hatched diameters observed from treated embryos point to minimal loss of cells or cell function where extended hypothermic exposure over long periods could potentially cause extensive and irreversible damage. Although there is a significant loss of quality in vitro (lower hatch diameters) in embryos stored in BSA compared with those stored in antifreeze proteins, tbe loss in quality did not have a detrimental effect on the actual number of viable fetuses following transfer. This suggests that the purified AFP or the more complex BSA both confer hypothermic tolerance on ovine embryos. We hypothesize that AFP and BSA may possibly interact with cell membrane function and structure, since these proteins have large molecular structures and therefore are not permeable. Functionally, AFPs and BSA may significantly decrease membrane permeability relating to ion charmel gating to match the depressed physiological status of the embryos, and they may maintain the microstructural milieu of the cell membrane. It is also possible that at a certain hypothermic temperature, intracelhrlar ion concentrations are regulated to match the effects of temperatuknduced decreases in metabolic rates and thus slow down the cell cycle and allow for extended storage. By binding to the membranes, these proteins may also maintain the structural organization of the membrane bi-layer and minimize cold-induced perturbations affecting the function of membrane bound StNChreS. Unlike storage at cryogenic temperatures, metabolism is not totally arrested in embryos stored in an unfrozen state. As a consequence, the period of time during which embryos can be stored at hypothermic temperatures although longer than at 37OC is limited due to decreased embryo viability. ‘Ibis short storage period (5 48 h) is a major factor limiting the use of hypothermic storage as an alternative preservation technique. For it to be of potential practical value, embryos need be rendered cold-tolerant for periods longer than 48 h and with subsequent acceptable viability rates compared to that of fresh embryos. The findings of most of the earlier hypothermic studies using various mammalian embryos have been discouraging when embryos were cooled below lS°C for more than 48 h with proteins normally used to supplement handling media. In our present study, the results of Experiment 1 suggest that BSA is inferior to AFPs and that WF is superior to OP, based on pooled viability rates. Hatched embryo diameters also point to a deleterious effect of high AFP concentrations on embryo quality. This indicates that the AFPs protective activity is type- and concentrationdepend. In a similar study, Ramsbottom et al. (21) reported the effectiveness of WF (AFP Type 1) at a lower concentration (1 vs 10 mg/ml) for hypcthermic storage of ovine embryos, whereas in bovine embryos, OP (AFP Type 3) seems to provide better prctecticn than WF (‘Type 1) and at higher concentrations (unpublished data). This implies that the mechanism of

Theriogenology

1023

different types of AFPs is dependent and functionally related to the species of embryo and structural milieu of the membranes. Rubinsky et al.(25), in an earlier study, also reported evidence of a dose response in the ability of AFP Type 1 to effectively suppress calcium and potassium currents in porcine granulosa cells, and further suggested an AFP-membrane protein interaction in the inhibition and suppression of the ion channels. In Experiment 2, viability and hatching rates in vitro were not &rent when data were pooled according to temperature or protein type, but they point to a signiticant temperature efkt on embryo quality when embryos were stored in WF. In Experiment 3, cooling rates and the source of protein showed no differences in survival in viva, possibly indicating that the ditibrence in embryo quality in terms of hatched diameter or viability in vitro does not correlate directly with in vivo survival; but hatching rates, for which no significant diflbrence was shown in Experiments 1 and 2, were probably a better measure of developmental competence in viva. During hypothermia, the advantage of decreasing metabolic rates for preservation also becomes a Sustained exposure wilJ inevitably lead to disadvantage if the duration of exposure is prolonged. equilibrium ion concentration resulting from metabolic arrest and temperaturedependent destabihzation of membrane function (14). These perturbations if not directly lethal could lead to apoptosis as a consequence of environmental insults such as increased intracellular pH (14), solute toxicity (28) and cytosolic Ca2+ reaching toxic proportions. The above conditions ahhough not the focus of this study, were expressed in some embryos within 2 h of culture in the form of cellular lysis (membrane disruption); excessive cell expansion (loss of osmotic regulation); and cell extrusion (loss of intercellular junction complexes). Within 48 h of culture, partial or complete degeneration was observed. Viable embryos, cm the other hand, showed good blastulation and expansion within this period, To be of practical value, the future prospect for embryo preservation at hypothermic temperatures must allow cells to remain viable for periods longer than 48 h. An intervention strategy to be effective should provide the cells with the ability to maintain near normal intracellular ion concentrations (i.e., control of ion channel densities and reduced membrane permeability) even at a depressed metabolic state. A noninvasive strategy must also be deemed a prerequisite. Thus, based on these concepts of intervention AFPs and, to some degree, BSA can play a vital role in ameliorating some of the problems encountered in prolonged imposed hypothermia, since both function as active ion channel regulators and because of their large moleculai structure they are not invasive. In conclusion these data indicate that AFPs and BSA can protect ovine embryos during storage at O” or 4OC in vitro for 4 d and result in high survi~l after in vitro crdture or in vivo transfer. If the mechanism of protection against hypothermia-related perturbations could be extended for longer periods (beyond 2 d) and across a wider range of biological cell types (gametes, tissues and organs), then it would have enormous theoretical and practical implications. A specific role for AFPs has not been fully established, and it will require further elucidation to optimize their properties.

REFERENCES 1. Ananthanarayanan VS, Hew CL. Structure studies on the freexing point depressing protein of the Biochem Biophys Res Comm 1977; 74: 685-689. winter flounder w ame&nms. 2. Ananthanarayanan VS. Antifreeze proteins: structural diversity and mechanism of action. Life Chem Rep 1989; 7: l-32. 3. Anderson GB, Foote RH. Development of rabbit embryos after storage at 10°C. J krim Sci 1975; 40: 900-904.

1024

Theriogenology

4. Averill RLW, Rowson LEA. Attempts at storage of sheep ova at low temperatures. J Agric Sci 1959; 52:-392-395. 5 Boland MP, Crosby FT, Gordon I. Morphological normality of cattle embryos following superovulation using PMSG. Theriogenology 1987; 10: 175-180. 6. Bon Durant RI-I, Anderson GB, Boland MP, Cupps PT, Hughes MA. Preliminary studies on bovine embryo survival f&wing short term storage at 4’C. l’heriogenology 1982; 17: 233-230. 7 DeVries AL, Wohlschlag DE. Freezing resistance in some Antarctic fish. Science 1969; 163: 10741075. 8. DeVries AL, Komatsu SK, Feeney RE. Chemical and physical properties of freezing point depressing glycoproteins from Antarctic fish. J Biol Chem 1970; 245: 2901-2913. 9 Duman JG. Thermal hystheresis factors in overwintering insects. J Insect Physiol 1979; 25: 805-810. 10. Duman JG. Subzero temperature tolerance in spiders. J Comp Physioll979; 131: 347-352. 11. Duman JG, Horwath K. The role of hemolymph proteins in the cold tolerance of insects. AM Rev Physiol 1983; 45: 261-270. 12. Harper MJK, Rowson LEA. Attempted storage of sheep ova at 7OC. J Reprod Fertil 1963;6: 183191. 13. Hew CL, Slaughter D, Shashikant BJ, Fletcher GF. Antifreeze polypeptides from the Newfoundland ocean pout, Macrozoarces americanus: presence of multiple and compositionally diverse composition. J Comp Physiol 1984; B.155: 81-88. 14. Hochacka PW. Defense strategies against hypoxia and hypothermia. Science 1986; 231: 234-241. 15. Leibo SP, Winninger D. Production of bovine pregnancies from embryos transported at O°C by air. lheriogenology 1986; 25: 165 abstr. 16. Lindner GM, Anderson GB, Bon Durant RI-i, Cupps PT. Survival of bovine embryos stored at 4OC. Theriogenology 1983; 20: 311-319. 17. Lindner GM, Ellis DE. Refrigeration of bovine embryos. Theriogenology 1985; 23: 202 abstr. 18. Murray AW. Creative blocks: cell cycle checkpoints and feedback controls. Nature 1992;359: 599604. 19. Ng NF, Trinh KY, Hew CL. Structure of an antifreeze polypeptide precursor from the sea raven, IJm&~&~americanus. J Biol Chem 1986; 261: 15690-15695. 20. Pashen RL. Short-term storage and survival of horse embryos after refrigeration at 4’C. J Reprod Fertil 1987; 35 (Suppl): 697-698. 21. Ramsbottom G, Arav A, Baguisi A, Rubinsky B, Roche JF, Boland MP. Hypothermia preservation of ovine embryos with antifreeze proteins. Cryobiology 1993; 30: 65 abstr. 22. Rownkrans CF Jr, First NL. Culture of bovine zygotes to the blastocyst stage: effect of amino acids and vitamins. Theriogenology 1991; 35: 266 abstr. 23. Rubinsky B, Arav A, Fletcher GL. Hypothermia protection: a tidamental property of “antifreeze” proteins. Biochem Biophys Res Comm 1991; 180 : 566-571. 24. Rubinsky B, Arav A, Mattioli M, DeVries AL. The efl%ct of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Comm 1990; 173:1369-1374. 25. Rubinsky B, Mattioli M, Arav A, Barboni B, Fletcher GL. Inhibition of Ca* and K+ currents by “antifreeze” proteins. Am J Physiol 1992; 262: R542-R545. 26. Scholander PF, Van Dam L, Kanwisher JW, Hammal HT, Gordon MS. Supercooling and osmoregulation in arctic fish. J Cell Compar Physiol 1957; 49: 5-24. 27. Sreenan J, Scanlon P and Gordon I. Storage of fertilized cattle ova in vitro. J Agric Sci 1970; 74: 593594. 28. Trump BF, Berezesky IK, Osomio-Vargas AR. Cell death in Biology and Pathology. In Bowem IO, Lockshin RA (eds.). Lundon: Chapman and Hall, 198 1; 209-242. 29. Zachariassen KE, Hammel I-IT Nucleating agents in the hemolymph of insects tolerant to freezing. Nature 1976; 262: 285-287.