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Antifreeze protein gene expression in winter flounder pre-hatch embryos: Implications for cryopreservation
Cryobiology 57 (2008) 84–90
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Antifreeze protein gene expression in winter flounder pre-hatch embryos: Implications for cryopreservation q Heather M. Young, Garth L. Fletcher * Ocean Sciences Centre and Department of Biology, Memorial University of Newfoundland, St. John’s, NL, Canada A1C 5S7
a r t i c l e
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Article history: Received 8 January 2008 Accepted 23 May 2008 Available online 2 June 2008 Keywords: Skin type antifreeze proteins (AFP) Gene expression Cryopreservation Winter flounder RT-PCR
a b s t r a c t Cryopreservation of fish embryos has proven to be an elusive goal. Two reasons for this lack of success are their high chilling sensitivity and the formation of ice crystals while in the frozen state or during the thawing process. Antifreeze proteins (AFP) that protect marine teleost fishes from freezing in subzero waters have been shown to be capable of inhibiting ice recrystallization and protecting cell membranes from cold induced damage. Therefore they have the potential to improve the success of embryo cryopreservation. A recent study demonstrated that vitrified winter flounder embryos continued to show developmental changes following thaw [V. Robles, E. Cabrita, G.L. Fletcher, M.A. Shears, M.J. King, M.P. Herráez, Vitrification assays with embryos from a cold tolerant sub-arctic fish species, Theriogenology 64 (2005) 1633–1646]. Since winter flounder produce AFP it was hypothesized that these proteins, if present in the embryos, could have contributed to this progressive step towards success. Winter flounder produce three species of type 1 AFP: a small liver type, a large ‘‘hyperactive” liver type and a skin type. This study was conducted to determine which, if any, of these AFP genes was being expressed in pre-hatch winter flounder embryos. There was no evidence of AFP activity in freshly fertilized embryos. However, low levels of AFP activity were found in embryos at 4, 8, and 11 days post-fertilization. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the AFP mRNA isolated from the embryos revealed the expression of seven different skin type AFP genes that translated into four distinct AFP. Neither of the liver type AFP genes was expressed in the embryos. Ó 2008 Elsevier Inc. All rights reserved.
Introduction Successful cryopreservation of fish embryos has been an elusive goal ever since the technique was successfully applied to mammalian species in the 1970s [25,28]. The reasons put forth for this lack of success are complex [12]. However it is believed that major problems lie with their high chilling sensitivity [43] and difficulties with cryoprotectant infiltration into all embryonic compartments due to barriers such as the chorion and the yolk syncytial layer [11,44]. Ongoing research continues to examine potential solutions to these difficulties [13,14,40]. Over the past 15 or so years there has been considerable interest in the potential use of antifreeze proteins to help protect cells, tissues, and organs from hypothermic damage and freezing injury during cryopreservation [18,17,39]. Although research to date has produced mixed results, there is no doubt that AFP can inhibit ice recrystallization [19]. In addition, there is clear evidence that AFP can protect cold sensitive cell membranes [33].
q This research was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). * Corresponding author. Fax: +1 709 737 3220. E-mail addresses: garthfl[email protected], fl[email protected] (G.L. Fletcher).
0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.05.005
Winter flounder (Pseudopleuronectes americanus) inhabit shallow inshore waters off the northeast coast of North America where ocean temperatures can decline to 1.8 °C during winter [6,34]. The flounder survive in these ice-laden waters by synthesizing antifreeze proteins (AFP) that bind to nascent ice crystals and prevent their further growth. This results in a non-colligative freezing point depression while leaving the melting point unchanged [9]. The cold hardiness of the winter flounder, along with the fact that they produce antifreeze proteins, prompted Robles et al. [30] to subject several embryonic stages to vitrification. Although none of the embryos hatched following thaw, a number of them survived for several days and showed movements and signs of continued pigmentation pattern development. The authors speculated that these promising results could be attributable, at least in part, to the presence of trace amounts of AFP in the embryos. However, nothing is known about the presence or absence of AFP at the embryonic stages of winter flounder development. Winter flounder produce type I AFP, characterized by being alanine rich and having amphipathic a-helices [3,7]. There are three species of homologous alanine-rich type I AFP encoded by three separate gene families present in adult winter flounder: the classic liver type (3–4 kDa) that are produced by the liver and secreted into the circulatory system, the skin type (3–4 kDa) that are pre-
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dominantly expressed in external epithelia to function intracellularly, and a recently discovered large hyperactive AFP (type 1hyp) (17 kDa) that is also produced in the liver and secreted into the plasma [10,22,23,35,42]. The present study was carried out to determine whether any or all of these type I species of AFP are produced in pre-hatch embryos of winter flounder by examining them for antifreeze protein activity and for the presence of AFP mRNA transcripts using reverse transcript-polymerase chain reaction (RT-PCR) techniques. Materials and methods Collection of samples Winter flounder were obtained by divers from Conception Bay (Newfoundland, Canada) and brought to the Ocean Sciences Centre at Logy Bay where they were kept at the seasonally cycling water temperatures and photoperiod experienced by the east coast of Newfoundland [6]. During the spawning period (April, 4 °C) eggs were gently stripped from ovulating females and fertilized with milt according to procedures outlined by Murray et al. [26]. The fertilized embryos were transferred to Petri dishes containing seawater and incubated at 4–6 °C. Samples of embryos were collected immediately after fertilization and at 4, 8, 11, and 15 days post-fertilization. Larval samples were collected and euthanized in MS222 as they hatched at 16–18 days. Liver and skin samples were collected from euthanized (MS222 overdose) adult flounder during March and September. These samples served as positive tissue controls for the expression of skin, liver and hyperactive type I AFP genes. All embryos, larvae and tissue samples were frozen in liquid nitrogen as soon as they were collected and stored in a 70 °C freezer until analysis. Antifreeze protein activity (thermal hysteresis) Winter flounder embryos were sampled (0.5 ml > 500 embryos) immediately after fertilization and at 4, 8, and 11 days post-fertilization were frozen in liquid nitrogen and stored at 70 °C. Samples were later homogenized in a 1:1 weight to volume concentration of 0.1 M NH4HCO3 containing Complete Protease Inhibitor Cocktail TabletsTM (Roche, Laval, Quebec), according to the manufacturer’s instructions. Samples were then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant of each sample was lyophilized overnight and resuspended in 100 ll of 0.1 M NH4HCO3 buffer to yield a fivefold concentrated extract. Antifreeze activity, measured as thermal hysteresis (TH), was determined using a Clifton Nanolitre Osmometer (Clifton Technical Physics, Hartford, NY), following the procedures outlined in Evans et al. [5]. In this method a sub-microlitre volume of aqueous solution is introduced into an oil filled well in a metal sample holder that is situated on a cooling block. The sample is flash frozen and then warmed slowly until a single small ice crystal remains. When the ice crystal is viewed under a compound microscope it can be observed to shrink or grow as the temperature is lowered or raised. The freezing temperature of the sample is established when the crystal enlarges and the melting temperature is the point at which the crystal shrinks. In the absence of antifreeze proteins the melting and freezing temperatures are the same and the ice crystal remains spherical. However, when antifreeze proteins are present the freezing temperature is lowered while the melting temperature remains unchange. This difference between the melting and freezing temperature, termed thermal hysteresis (TH), is used as a measure of antifreeze protein activity [18]. Antifreeze protein activities (TH) were converted to antifreeze protein concentrations using a standard curve [4]. In addition to effecting a thermal hysteresis, type I antifreeze proteins alter the growth habit of the ice crystal by binding to their prism faces thus inhibiting their growth
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[35]. In the presence of low levels of AFP the ice crystals take on a hexagonal shape [16]. Sample measurements were made in triplicate and averaged. Ice crystal images were captured using the MultiImageTM light cabinet and AlphaImagerTM 1220 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). Extraction of RNA RNA was extracted using the TrizolÒ method according to the manufacturer’s directions (InvitrogenTM, Burlington, ON). Samples were resuspended in a minimum volume of 10 mM TE at pH 7.6, quantified spectrophotometrically (GeneQuant pro, Amersham Pharmacia Biotech) and stored at 70 °C. cDNA synthesis RNA samples were DNase treated according to the manufacturer’s directions (InvitrogenTM, Burlington, ON) and the cDNAs synthesized using SuperscriptÒ II RT as per the manufacturers’ instructions (InvitrogenTM, Burlington, ON), then stored at 70 °C. Antifreeze protein gene primers and PCR PCR amplifications were carried out using the following forward (F) and reverse (R) primers: Skin AFP-F 50 -AAT CAC TGA CAT CAA CAT G-30 Skin AFP-R 50 -CTG CTG AAT AAA CCT GAG AAG CT -30 Liver AFP-F 50 -AAT CAC TGA AGC CAG ACC C-30 Liver AFP-R 50 -TGG GGC GGC TGC GGC AGG GG -30 Hyper AFP-F 50 -GTA GTG AAC CAG TGC TCC CTA A-30 Hyper AFP-R 50 -TAT CTC ATT GCT CAG ATG TTC-30 The skin type AFP primers were designed using the 11-3 genomic sequence (GenBank Accession No. M63478) to yield a 219 bp amplicon. Liver type AFP primers were designed using the 2A-7b genomic sequence (GenBank Accession No. M62415) to yield a 142 bp amplicon. Hyperactive (Hyper) AFP primers were designed from a liver cDNA sequence (P.L. Davies and L. Graham, Queen’s University, Kingston Ontario, Canada (unpublished data), to yield a 742 bp amplicon. All PCRs were carried out using a Taq kit containing Q bufferTM (Qiagen Inc., Chatsworth, CA). Touchdown PCR parameters for the skin and liver type AFP PCR analyses were as follows: 90 °C for 45 s denaturation, 67 °C (1 °C for each additional cycle) for 1 min as an annealing temperature, and 72 °C for 1 min as an extension temperature, all of which were repeated 13 times. This was followed by 30 cycles of a denaturing temperature of 95 °C for 45 s, an annealing temperature of 55 °C for 1 min, and an extension temperature of 72 °C for 1 min. PCR parameters for the hyperactive AFP were as follows: denaturing temperature of 94 °C for 45 s, an annealing temperature of 59 °C for 1 min, an extension temperature of 72 °C for 1 min, for a total of 35 cycles. All PCR products obtained from the hyperactive AFP primers were reamplified using 2 ll of reaction product. PCR conditions were identical to those used initially. Gel electrophoresis Five microlitres of 11 sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in water) was added to a sample of each PCRs mixture and 12.5 ll of the resulting solution was added to the wells of a 2% agarose (InvitrogenTM, Burlington, ON) gel and electrophoresed in 1 TBE buffer (45 mM Tris–borate, 1 mM EDTA, pH 8) at 90 V until the sample buffer had migrated approximately 5 cm. The gels were stained with a 0.5 lg/ml solution of ethidium bromide (SigmaÒ, Oakville, ON) for approximately 30 min, followed by a rinse in distilled water for approximately
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primer amplification of the cDNA of 8-day-old embryos into DH5a cells using TOPO TA CloningÒ Kit (with pCRII-TOPOÒ), as per the manufacturer’s directions (InvitrogenTM, Burlington, ON). Colonies were randomly selected based on the LacZ gene disruption and were replated. Plasmids were purified from replated colonies using Qiaprep Spin Mini Prep KitTM, as per the manufacturer’s instructions (Qiagen Inc., Chatsworth, CA) and seven of them (labeled 1F, 2F, 4F, 5F, 6F, 7F, and 8F) were sent for sequencing in both directions. Sequencing
Fig. 1. Example of the ice crystals that formed in extracts of 4-, 8-, and 11-day-old post-fertilization embryos of winter flounder. Magnification 320.
15 min. Gels were exposed to UV light and photographed using the MultiImageTM light cabinet and AlphaImagerTM 1220 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). PCR products were isolated from the agarose gels and purified using a QIAquickÒ gel extraction kit (Qiagen Inc., Chatsworth, CA) and diluted with Dnase/Rnase free water (InvitrogenTM, Burlington, ON) to a final concentration of 10–20 ng/ll and sequenced, and the identity of the PCR amplicons was confirmed.
All sequencing was carried out by Mobix laboratory (McMaster University, Hamilton, Ontario, Canada). The ABI Big Dye terminator cycle sequencing chemistry was used to perform DNA sequencing. Conditions for the standard sequencing reaction were: annealing temperature 50 °C, extension 60 °C, 2.5 mM magnesium chloride, and a primer concentration of 0.2 mM. After the completion of the sequencing reaction, excess primer and unincorporated dNTPs were removed using DyeEx columns (Qiagen Inc., Chatsworth, CA) and the DNA precipitated using sodium acetate and ethanol. The purified reaction was resuspended in HiDi formamide (ABI) and the sample placed in the 3100 DNA automated sequencer. Results were provided as alphabetic sequences as well as chromatograms. Results Antifreeze protein activity in pre-hatch embryos
Cloning Since there appeared to be more than one PCR product present in the pre-hatch embryos (Fig. 2) more detailed sequence information was obtained by cloning the PCR amplicons from the skin type
There was no evidence of antifreeze protein activity in embryos sampled immediately after fertilization. In contrast, extracts of 48- and 11-day-old embryos had low levels of antifreeze activity with thermal hysteresis (TH) values ranging from 0.025 °C in 4-
Fig. 2. RT-PCR analysis of type I AFP gene expression in winter flounder liver, skin, hatched larvae, and pre-hatch embryos. PCR analysis was conducted using primers specific for the three AFP species; liver (upper panel), hyperactive (centre panel) and skin (lower panel). The hyperactive AFP central panel shows an upper component illustrating the results of the first round of PCR amplification (1st PCR), and a lower component where the samples were re-amplified (2nd PCR). Liver 1 and skin 1 samples were collected in September. Liver 2 and skin 2 samples were collected in March. Larvae 1 and 2 are duplicate samples of the same batch of newly hatched embryos. Pre-hatch embryos were analysed at 4, 8, 11, and 15 days post-fertilization. Negative controls are water. Positive controls are plasmids containing 11-3 or type I hyp AFP constructs.
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and 8-day embryos to 0.032 °C in the 11 day embryos. In addition, the extracts of these 4- to 11-day embryos exhibited hexagonal ice crystal shaping typical of low AFP concentrations (Fig. 1) [16]. Since the extracts were concentrated fivefold, the TH within the embryos ranged from approximately 0.005 °C to 0.006 °C. Antifreeze protein gene expression There was no evidence for liver type AFP gene expression at any of the pre-hatch embryo stages or in the hatched larvae or skin samples. The only tissue that showed evidence for expression was the adult liver tissue where the expected 142 bp band was observed in samples collected from adult winter flounder during the months of March and September (Fig. 2). Despite repeated PCR attempts, the hyperactive-type AFP did not appear to be expressed in any of the pre- and post-hatched embryos, nor was there expression in the adult skin or liver samples following the first round of PCR amplification (Fig. 2). However when the reaction mixtures from the first round of PCR amplifica-
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tion were reamplified, a 742 bp band was evident for the liver sample collected from an adult winter flounder sampled in March (Fig. 2). In contrast there was no evidence for expression of the hyperactive AFP in the September liver sample or the skin, hatched larvae, and pre-hatch embryo samples. Skin type AFP were expressed at all of the pre-hatch embryo stages of development as evidenced by the 219 bp band (Fig. 2). In addition similar evidence for skin type AFP expression was observed for the larvae, skin, and liver samples (Fig. 2). There appeared to be an additional band of approximately 230 bp in the pre-hatch embryos that was not apparent for any of the other samples (Fig. 2). However repeated electrophoretic analyses failed to result in a clear separation of the 219 and 230 bp bands. Nucleotide sequencing Nucleotide sequencing of the seven clones (1F, 2F, 4F, 5F, 6F, 7F, and 8F) derived from a single PCR product revealed that seven different skin type AFP genes were being expressed in the pre-hatch
Fig. 3. Comparison of nucleotide sequences expressed by pre-hatch embryos (clones 1F–8F), with sequences of known skin type antifreeze proteins 11-3, sAFP1, sAFP3, and sAFP5. All sequence data were aligned with AFP 11-3 using Vector NTI (Invitrogen, Burlington, Ontario, Canada). The 11-3 genomic sequence was obtained from GenBank (Accession No. M63478), while sAFP1, sAFP3, and sAFP5 sequences were obtained from Ref. [16]. The protein coding region is highlighted in bold. Underlined nucleotides indicate where differences exist between the various sequences. Dashes represent sequence gaps while dots indicate where sequence data are lacking.
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Fig. 3 (continued)
embryos. A detailed analysis of the sequences (Fig. 3) revealed that clones 2F, 6F, and 7F were identical to that of sAFP3, [15], while clone 5F was identical in sequence to sAFP5 [15]. The nucleotide sequence of clone 4F differed from that of skin type AFP 11-3 by a single base substitution at position 1148 which is within the 3’ UTR (Fig. 3) The sequences of clones 8F and 1F were very similar to that of skin type sAFP1 [15], with clone 8F differing by a single base within the 30 UTR at position 167, while clone 1F differed by three bases at positions 125, 149, and 167 (Fig. 3) The base substitution at position 125, which is within the protein reading frame, did not result in an amino acid change (Fig 4). When the nucleotide sequences were translated into amino acids it was evident that only four different skin type AFP were being produced (Fig. 4). Two of the four proteins (sAFP1 and sAFP3) that were expressed in these embryos have been isolated and purified from adult winter flounder skin, indicating that they are likely present at all life stages [10]. Discussion The results of this study clearly indicate that winter flounder begin to express skin type AFP genes, synthesize AFP, and accumu-
late low but measurable levels of the protein very early during embryonic development. This result lends credence to the hypothesis put forward by Robles et al. [30] that the brief continuation of pigmentation development and active movements observed in winter flounder embryos that had been vitrified was in part due to the presence of AFP. As pointed out in the introduction, there are two general hypotheses as to how AFP could help protect cryopreserved embryos: inhibition of ice recrystallization, and protection of cell membranes from cold induced damage. The discovery that only skin type AFP are expressed in pre-hatch embryos suggests that they exert their cryoprotective effect intracellularly. None of these hypotheses are meant to be mutually exclusive. Ice recrystallization occurs when larger ice crystals grow at the expense of smaller ones creating considerable physical damage within the frozen tissue. This phenomenon is believed to be a major cause for cellular damage associated with cryogenic procedures [2,13,14,29]. AFP have been shown to be effective in reducing this cellular and tissue damage by binding to the ice and inhibiting or blocking the recrystallization process [2,21,24,27]. Walker et al. [38] examined the ability of type I AFP to inhibit ice recrystallization (IR) using an IR assay [37], and found that AFP concentrations
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Fig. 4. Amino acid sequences of winter flounder skin type AFP expressed in pre-hatch embryos. The amino acid sequences were deduced from cDNA clones 1F, 2F, 4F, 5F, 6F, 7F, 8F and aligned with skin type AFP 11-3 (GenBank Accession No. M63478), sAFP1, sAFP3, and sAFP5 [15]. Residue differences between the proteins are located at positions 6, 23, 32, and 38. Asterisks represent positions where the amino acid is identical to that of the 11-3 sequence.
as low as 2 lg/ml were highly efficacious. Type I AFP have also been shown to effectively enhance the survival of cryopreserved red blood cells during thaw at concentrations of 5–150 lg/ml [2]. The levels of AFP measured in the embryos of the present study were approximately 0.16–0.22 mg/ml, as estimated from the thermal hysteresis values (0.025–0.032 °C) obtained for the homogenates using the AFP activity curves generated by Evans and Fletcher [4]. Since the homogenates were concentrated fivefold, the AFP concentration in the embryos was approximately 30– 40 lg/ml, well within the range required to inhibit ice recrystallization [2,37]. A number of studies have demonstrated that AFP can protect cold sensitive mammalian cells, tissues, and organs from hypothermic non-freezing damage [1,20,32,33,36]. However, no comparable studies have been carried out on fish tissues or cells, thus it is not known whether AFP could play a similar protective role. Further details on antifreeze and cold adaptation in fish can be found in Fletcher et al. [7]. Recently Robles et al. [31] have found evidence that AFP can reduce the chilling sensitivity of seabream embryos (a warm water species). These studies demonstrated that the hatching rates of embryos from this species microinjected with the small liver type I AFP and exposed to 0 °C for an hour approached 100% whereas the mean value for controls with no AFP was less than 50%. Confocal microscopy indicated that microinjected FITC labeled AFP rapidly became associated within the cellular compartment of the embryo prompting the suggestion that the AFP had improved the cold resistance of the embryos by protecting cell membranes. Winter flounder skin type AFP lack a secretory signal sequence suggesting that they remain and function intracellularly [10]. Since the pre-hatch embryos of the present study only express skin type AFP genes, it is possible that the AFP remained within the cells and served to prevent the formation or the recrystallization of intracellular ice during the process of freezing or thawing the vitrified winter flounder embryos studied by Robles et al. [30]. One interesting discovery made in the Robles et al. [30] study was that the only embryos showing continued development following thaw were the 11-day-old group that were cryopreserved using dimethyl sulfoxide and thawed relatively slowly at 0 °C. Slow thawing would be expected to increase the probability of recrystallization. In contrast there was no evidence of continued embryonic development in any of the vitrified embryos thawed rapidly at 25 °C; conditions that minimize recrystallization. Although it could be argued that AFP prevented recrystallization in the slowly thawed embryos it is dif-
ficult to reconcile why the embryos that were rapidly thawed did not fare as well. Winter flounder are a cold water species whose embryos normally develop in the range of 0 °C–12 °C. In addition the upper lethal temperature has been reported as 15 °C [41]. Therefore, exposure of the cryopreserved embryos to 25 °C may have contributed to their mortality. Acknowledgments We thank Drs Peter Davies and Laurie Graham (Department of Biochemistry, Queen’s University) for the hyperactive AFP cDNA sequence and primers, and Madonna King (Ocean Sciences Centre) for her expert advice and assistance. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). References [1] G. Amir, L. Horowitz, B. Rubinsky, S.Y. Yousif, J. Lavee, Smolinsky, Subzero nonfreezing cryopreservation of rat hearts using antifreeze protein I and antifreeze protein III, Cryobiology 48 (2004) 273–282. [2] J.F. Carpenter, T.N. Hansen, Antifreeze protein modulates cell survival during cryopreservation: Mediation through influence on ice crystal growth, Proc. Nat. Acad. Sci. USA 89 (1992) 8953–8957. [3] P.L. Davies, C.L. Hew, Biochemistry of fish antifreeze proteins, FASEB J. 4 (1990) 2460–2468. [4] R.P. Evans, G.L. Fletcher, Isolation and characterization of type I antifreeze proteins from Atlantic snailfish (Liparis atlanticus) and dusky snailfish (Liparis gibbus), Biochim. Biophys. Acta 1547 (2001) 235–247. [5] R.P. Evans, R.S. Hobbs, S.V. Goddard, G.L. Fletcher, The importance of dissolved salts to the in vivo efficacy of antifreeze proteins, Comp. Biochem. Physiol. A148 (2007) 556–561. [6] G.L. Fletcher, Circannual cycles of blood plasma freezing points and Na+ and Cl concentrations in Newfoundland winter flounder (Pseudopleuronectes americanus): correlation with water temperature and photoperiod, Can. J. Zool. 55 (1977) 789–795. [7] G.L. Fletcher, S.V. Goddard, P.L. Davies, Z. Gong, K.V. Ewart, C.L. Hew, New insights into fish antifreeze proteins: physiological significance and molecular regulation, in: H.O. Portner, R. Playle (Eds.), Cold Ocean Physiology, Cambridge University Press, Cambridge, 1998, pp. 239–265. [8] G.L. Fletcher, S.V. Goddard, Y. Wu, Antifreeze proteins and their genes: From basic research to business opportunities, Chemtech 29 (1999) 17–28. [9] G.L. Fletcher, C.L. Hew, P.L. Davies, Antifreeze proteins of teleost fishes, Annu. Rev. Physiol. 63 (2001) 359–390. [10] Z. Gong, K.V. Ewart, Z. Hu, G.L. Fletcher, C.L. Hew, Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretary signal and prosequences, J. Biol. Chem. 245 (1996) 2901–2913. [11] M. Hagedorn, F.W. Kleinhans, D. Artemov, U. Pilatus, Characterization of a major permeability barrier in the zebrafish embryo, Biol. Reprod. 59 (1998) 1240–1250. [12] M. Hagedorn, F.W. Kleinhans, Cryopreservation in aquatic species, in: T.R. Tiersch, P.M. Mazik (Eds.), Problems and Prospects in Cryopreservation of Fish
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[29] V. Robles, E. Cabrita, M. Real, R. Alvarez, Herraez, Vitrification of turbot embryos: preliminary assays, Cryobiology 47 (2003) 30–39. [30] V. Robles, E. Cabrita, G.L. Fletcher, M.A. Shears, M.J. King, M.P. Herráez, Vitrification assays with embryos from a cold tolerant sub-arctic fish species, Theriogenology 64 (2005) 1633–1646. [31] V. Robles, V. Barbosa, M.P. Herraez, S. Martinez-Paramo, M.L. Cancela, The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures, Theriogenology 68 (2007) 284–289. [32] B. Rubinsky, A. Arav, M. Mattioli, A.L. DeVries, The Effect of antifreeze glycoproteins on membrane potential changes at hypothermic temperatures, Biochem. Biophys. Res. Commun. 173 (1990) 1369–1374. [33] B. Rubinsky, A. Arav, G.L. Fletcher, Hypothermic protection: a Fundamental property of ‘‘antifreeze” protein, Biochem. Biophys. Res. Commun. 180 (1991) 566–571. [34] W.B. Scott, M.G. Scott, Atlantic Fishes of Canada, University of Toronto Press, 1988. [35] A.J. Scotter, C.B. Marshall, L.A. Graham, J.A. Gilbert, C.P. Garnham, P.L. Davies, The basis for hyperactivity of antifreeze proteins, Cryobiology 52 (2006) 229– 239. [36] F. Tablin, A.E. Oliver, N.J. Walker, A.E. Crowe, J.H. Crowe, Membrane phase transition of intact human platelets: correlation with cold-induced activation, J. Cell. Physiol. 168 (1996) 305–313. [37] M.M. Tomczak, C.B. Marshall, J.A. Gilbert, P.L. Davies, A facile method for determining ice recrystallization inhibition by antifreeze proteins, Biochem. Biophys. Res. Commun. 311 (2003) 1041–1046. [38] V.K. Walker, J.R. Palmer, G. Voordouw, Freeze–thaw tolerance and clues to the winter survival of a soil community, Appl. Environ. Microbiol. 72 (2006) 1784– 1792. [39] J.H. Wang, A comprehensive evaluation of the effects and mechanisms of antifreeze proteins during low temperature preservation, Cryobiology 41 (2000) 1–9. [40] R.Y. Wang, T. Zhang, Q. Bao, D.M. Rawson, Study on fish embryo responses to the treatment of cryoprotective chemicals using impedance spectroscopy, Eur. Biophys. J. 35 (2006) 224–230. [41] G.C. Williams, Viable embryogenesis of the winter flounder Pseudopleuronectes americanus from 1.8 °C to 15 °C, Mar. Biol. 33 (1975) 71–74. [42] D.S. Yang, M. Sax, A. Chakrabartty, C.L. Hew, Crystal structure of an antifreeze polypeptide and its mechanistic implications, Nature 333 (1988) 232–237. [43] T. Zhang, D.M. Rawson, Studies on chilling sensitivity of zebrafish (Brachydanio rerio) embryos, Crybiology 38 (1995) 239–246. [44] T.T. Zhang, D.M. Rawson, Feasibility studies on vitrification of intact zebrafish (Brachydanio rerio) embryos, Cryobiology 33 (1996) 13–21.
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Antifreeze protein gene expression in winter flounder pre-hatch embryos: Implications for cryopreservation q Heather M. Young, Garth L. Fletcher * Ocean Sciences Centre and Department of Biology, Memorial University of Newfoundland, St. John’s, NL, Canada A1C 5S7
a r t i c l e
i n f o
Article history: Received 8 January 2008 Accepted 23 May 2008 Available online 2 June 2008 Keywords: Skin type antifreeze proteins (AFP) Gene expression Cryopreservation Winter flounder RT-PCR
a b s t r a c t Cryopreservation of fish embryos has proven to be an elusive goal. Two reasons for this lack of success are their high chilling sensitivity and the formation of ice crystals while in the frozen state or during the thawing process. Antifreeze proteins (AFP) that protect marine teleost fishes from freezing in subzero waters have been shown to be capable of inhibiting ice recrystallization and protecting cell membranes from cold induced damage. Therefore they have the potential to improve the success of embryo cryopreservation. A recent study demonstrated that vitrified winter flounder embryos continued to show developmental changes following thaw [V. Robles, E. Cabrita, G.L. Fletcher, M.A. Shears, M.J. King, M.P. Herráez, Vitrification assays with embryos from a cold tolerant sub-arctic fish species, Theriogenology 64 (2005) 1633–1646]. Since winter flounder produce AFP it was hypothesized that these proteins, if present in the embryos, could have contributed to this progressive step towards success. Winter flounder produce three species of type 1 AFP: a small liver type, a large ‘‘hyperactive” liver type and a skin type. This study was conducted to determine which, if any, of these AFP genes was being expressed in pre-hatch winter flounder embryos. There was no evidence of AFP activity in freshly fertilized embryos. However, low levels of AFP activity were found in embryos at 4, 8, and 11 days post-fertilization. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of the AFP mRNA isolated from the embryos revealed the expression of seven different skin type AFP genes that translated into four distinct AFP. Neither of the liver type AFP genes was expressed in the embryos. Ó 2008 Elsevier Inc. All rights reserved.
Introduction Successful cryopreservation of fish embryos has been an elusive goal ever since the technique was successfully applied to mammalian species in the 1970s [25,28]. The reasons put forth for this lack of success are complex [12]. However it is believed that major problems lie with their high chilling sensitivity [43] and difficulties with cryoprotectant infiltration into all embryonic compartments due to barriers such as the chorion and the yolk syncytial layer [11,44]. Ongoing research continues to examine potential solutions to these difficulties [13,14,40]. Over the past 15 or so years there has been considerable interest in the potential use of antifreeze proteins to help protect cells, tissues, and organs from hypothermic damage and freezing injury during cryopreservation [18,17,39]. Although research to date has produced mixed results, there is no doubt that AFP can inhibit ice recrystallization [19]. In addition, there is clear evidence that AFP can protect cold sensitive cell membranes [33].
q This research was funded by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC). * Corresponding author. Fax: +1 709 737 3220. E-mail addresses: garthfl[email protected], fl[email protected] (G.L. Fletcher).
0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.05.005
Winter flounder (Pseudopleuronectes americanus) inhabit shallow inshore waters off the northeast coast of North America where ocean temperatures can decline to 1.8 °C during winter [6,34]. The flounder survive in these ice-laden waters by synthesizing antifreeze proteins (AFP) that bind to nascent ice crystals and prevent their further growth. This results in a non-colligative freezing point depression while leaving the melting point unchanged [9]. The cold hardiness of the winter flounder, along with the fact that they produce antifreeze proteins, prompted Robles et al. [30] to subject several embryonic stages to vitrification. Although none of the embryos hatched following thaw, a number of them survived for several days and showed movements and signs of continued pigmentation pattern development. The authors speculated that these promising results could be attributable, at least in part, to the presence of trace amounts of AFP in the embryos. However, nothing is known about the presence or absence of AFP at the embryonic stages of winter flounder development. Winter flounder produce type I AFP, characterized by being alanine rich and having amphipathic a-helices [3,7]. There are three species of homologous alanine-rich type I AFP encoded by three separate gene families present in adult winter flounder: the classic liver type (3–4 kDa) that are produced by the liver and secreted into the circulatory system, the skin type (3–4 kDa) that are pre-
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dominantly expressed in external epithelia to function intracellularly, and a recently discovered large hyperactive AFP (type 1hyp) (17 kDa) that is also produced in the liver and secreted into the plasma [10,22,23,35,42]. The present study was carried out to determine whether any or all of these type I species of AFP are produced in pre-hatch embryos of winter flounder by examining them for antifreeze protein activity and for the presence of AFP mRNA transcripts using reverse transcript-polymerase chain reaction (RT-PCR) techniques. Materials and methods Collection of samples Winter flounder were obtained by divers from Conception Bay (Newfoundland, Canada) and brought to the Ocean Sciences Centre at Logy Bay where they were kept at the seasonally cycling water temperatures and photoperiod experienced by the east coast of Newfoundland [6]. During the spawning period (April, 4 °C) eggs were gently stripped from ovulating females and fertilized with milt according to procedures outlined by Murray et al. [26]. The fertilized embryos were transferred to Petri dishes containing seawater and incubated at 4–6 °C. Samples of embryos were collected immediately after fertilization and at 4, 8, 11, and 15 days post-fertilization. Larval samples were collected and euthanized in MS222 as they hatched at 16–18 days. Liver and skin samples were collected from euthanized (MS222 overdose) adult flounder during March and September. These samples served as positive tissue controls for the expression of skin, liver and hyperactive type I AFP genes. All embryos, larvae and tissue samples were frozen in liquid nitrogen as soon as they were collected and stored in a 70 °C freezer until analysis. Antifreeze protein activity (thermal hysteresis) Winter flounder embryos were sampled (0.5 ml > 500 embryos) immediately after fertilization and at 4, 8, and 11 days post-fertilization were frozen in liquid nitrogen and stored at 70 °C. Samples were later homogenized in a 1:1 weight to volume concentration of 0.1 M NH4HCO3 containing Complete Protease Inhibitor Cocktail TabletsTM (Roche, Laval, Quebec), according to the manufacturer’s instructions. Samples were then centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant of each sample was lyophilized overnight and resuspended in 100 ll of 0.1 M NH4HCO3 buffer to yield a fivefold concentrated extract. Antifreeze activity, measured as thermal hysteresis (TH), was determined using a Clifton Nanolitre Osmometer (Clifton Technical Physics, Hartford, NY), following the procedures outlined in Evans et al. [5]. In this method a sub-microlitre volume of aqueous solution is introduced into an oil filled well in a metal sample holder that is situated on a cooling block. The sample is flash frozen and then warmed slowly until a single small ice crystal remains. When the ice crystal is viewed under a compound microscope it can be observed to shrink or grow as the temperature is lowered or raised. The freezing temperature of the sample is established when the crystal enlarges and the melting temperature is the point at which the crystal shrinks. In the absence of antifreeze proteins the melting and freezing temperatures are the same and the ice crystal remains spherical. However, when antifreeze proteins are present the freezing temperature is lowered while the melting temperature remains unchange. This difference between the melting and freezing temperature, termed thermal hysteresis (TH), is used as a measure of antifreeze protein activity [18]. Antifreeze protein activities (TH) were converted to antifreeze protein concentrations using a standard curve [4]. In addition to effecting a thermal hysteresis, type I antifreeze proteins alter the growth habit of the ice crystal by binding to their prism faces thus inhibiting their growth
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[35]. In the presence of low levels of AFP the ice crystals take on a hexagonal shape [16]. Sample measurements were made in triplicate and averaged. Ice crystal images were captured using the MultiImageTM light cabinet and AlphaImagerTM 1220 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). Extraction of RNA RNA was extracted using the TrizolÒ method according to the manufacturer’s directions (InvitrogenTM, Burlington, ON). Samples were resuspended in a minimum volume of 10 mM TE at pH 7.6, quantified spectrophotometrically (GeneQuant pro, Amersham Pharmacia Biotech) and stored at 70 °C. cDNA synthesis RNA samples were DNase treated according to the manufacturer’s directions (InvitrogenTM, Burlington, ON) and the cDNAs synthesized using SuperscriptÒ II RT as per the manufacturers’ instructions (InvitrogenTM, Burlington, ON), then stored at 70 °C. Antifreeze protein gene primers and PCR PCR amplifications were carried out using the following forward (F) and reverse (R) primers: Skin AFP-F 50 -AAT CAC TGA CAT CAA CAT G-30 Skin AFP-R 50 -CTG CTG AAT AAA CCT GAG AAG CT -30 Liver AFP-F 50 -AAT CAC TGA AGC CAG ACC C-30 Liver AFP-R 50 -TGG GGC GGC TGC GGC AGG GG -30 Hyper AFP-F 50 -GTA GTG AAC CAG TGC TCC CTA A-30 Hyper AFP-R 50 -TAT CTC ATT GCT CAG ATG TTC-30 The skin type AFP primers were designed using the 11-3 genomic sequence (GenBank Accession No. M63478) to yield a 219 bp amplicon. Liver type AFP primers were designed using the 2A-7b genomic sequence (GenBank Accession No. M62415) to yield a 142 bp amplicon. Hyperactive (Hyper) AFP primers were designed from a liver cDNA sequence (P.L. Davies and L. Graham, Queen’s University, Kingston Ontario, Canada (unpublished data), to yield a 742 bp amplicon. All PCRs were carried out using a Taq kit containing Q bufferTM (Qiagen Inc., Chatsworth, CA). Touchdown PCR parameters for the skin and liver type AFP PCR analyses were as follows: 90 °C for 45 s denaturation, 67 °C (1 °C for each additional cycle) for 1 min as an annealing temperature, and 72 °C for 1 min as an extension temperature, all of which were repeated 13 times. This was followed by 30 cycles of a denaturing temperature of 95 °C for 45 s, an annealing temperature of 55 °C for 1 min, and an extension temperature of 72 °C for 1 min. PCR parameters for the hyperactive AFP were as follows: denaturing temperature of 94 °C for 45 s, an annealing temperature of 59 °C for 1 min, an extension temperature of 72 °C for 1 min, for a total of 35 cycles. All PCR products obtained from the hyperactive AFP primers were reamplified using 2 ll of reaction product. PCR conditions were identical to those used initially. Gel electrophoresis Five microlitres of 11 sample buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in water) was added to a sample of each PCRs mixture and 12.5 ll of the resulting solution was added to the wells of a 2% agarose (InvitrogenTM, Burlington, ON) gel and electrophoresed in 1 TBE buffer (45 mM Tris–borate, 1 mM EDTA, pH 8) at 90 V until the sample buffer had migrated approximately 5 cm. The gels were stained with a 0.5 lg/ml solution of ethidium bromide (SigmaÒ, Oakville, ON) for approximately 30 min, followed by a rinse in distilled water for approximately
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primer amplification of the cDNA of 8-day-old embryos into DH5a cells using TOPO TA CloningÒ Kit (with pCRII-TOPOÒ), as per the manufacturer’s directions (InvitrogenTM, Burlington, ON). Colonies were randomly selected based on the LacZ gene disruption and were replated. Plasmids were purified from replated colonies using Qiaprep Spin Mini Prep KitTM, as per the manufacturer’s instructions (Qiagen Inc., Chatsworth, CA) and seven of them (labeled 1F, 2F, 4F, 5F, 6F, 7F, and 8F) were sent for sequencing in both directions. Sequencing
Fig. 1. Example of the ice crystals that formed in extracts of 4-, 8-, and 11-day-old post-fertilization embryos of winter flounder. Magnification 320.
15 min. Gels were exposed to UV light and photographed using the MultiImageTM light cabinet and AlphaImagerTM 1220 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA). PCR products were isolated from the agarose gels and purified using a QIAquickÒ gel extraction kit (Qiagen Inc., Chatsworth, CA) and diluted with Dnase/Rnase free water (InvitrogenTM, Burlington, ON) to a final concentration of 10–20 ng/ll and sequenced, and the identity of the PCR amplicons was confirmed.
All sequencing was carried out by Mobix laboratory (McMaster University, Hamilton, Ontario, Canada). The ABI Big Dye terminator cycle sequencing chemistry was used to perform DNA sequencing. Conditions for the standard sequencing reaction were: annealing temperature 50 °C, extension 60 °C, 2.5 mM magnesium chloride, and a primer concentration of 0.2 mM. After the completion of the sequencing reaction, excess primer and unincorporated dNTPs were removed using DyeEx columns (Qiagen Inc., Chatsworth, CA) and the DNA precipitated using sodium acetate and ethanol. The purified reaction was resuspended in HiDi formamide (ABI) and the sample placed in the 3100 DNA automated sequencer. Results were provided as alphabetic sequences as well as chromatograms. Results Antifreeze protein activity in pre-hatch embryos
Cloning Since there appeared to be more than one PCR product present in the pre-hatch embryos (Fig. 2) more detailed sequence information was obtained by cloning the PCR amplicons from the skin type
There was no evidence of antifreeze protein activity in embryos sampled immediately after fertilization. In contrast, extracts of 48- and 11-day-old embryos had low levels of antifreeze activity with thermal hysteresis (TH) values ranging from 0.025 °C in 4-
Fig. 2. RT-PCR analysis of type I AFP gene expression in winter flounder liver, skin, hatched larvae, and pre-hatch embryos. PCR analysis was conducted using primers specific for the three AFP species; liver (upper panel), hyperactive (centre panel) and skin (lower panel). The hyperactive AFP central panel shows an upper component illustrating the results of the first round of PCR amplification (1st PCR), and a lower component where the samples were re-amplified (2nd PCR). Liver 1 and skin 1 samples were collected in September. Liver 2 and skin 2 samples were collected in March. Larvae 1 and 2 are duplicate samples of the same batch of newly hatched embryos. Pre-hatch embryos were analysed at 4, 8, 11, and 15 days post-fertilization. Negative controls are water. Positive controls are plasmids containing 11-3 or type I hyp AFP constructs.
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and 8-day embryos to 0.032 °C in the 11 day embryos. In addition, the extracts of these 4- to 11-day embryos exhibited hexagonal ice crystal shaping typical of low AFP concentrations (Fig. 1) [16]. Since the extracts were concentrated fivefold, the TH within the embryos ranged from approximately 0.005 °C to 0.006 °C. Antifreeze protein gene expression There was no evidence for liver type AFP gene expression at any of the pre-hatch embryo stages or in the hatched larvae or skin samples. The only tissue that showed evidence for expression was the adult liver tissue where the expected 142 bp band was observed in samples collected from adult winter flounder during the months of March and September (Fig. 2). Despite repeated PCR attempts, the hyperactive-type AFP did not appear to be expressed in any of the pre- and post-hatched embryos, nor was there expression in the adult skin or liver samples following the first round of PCR amplification (Fig. 2). However when the reaction mixtures from the first round of PCR amplifica-
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tion were reamplified, a 742 bp band was evident for the liver sample collected from an adult winter flounder sampled in March (Fig. 2). In contrast there was no evidence for expression of the hyperactive AFP in the September liver sample or the skin, hatched larvae, and pre-hatch embryo samples. Skin type AFP were expressed at all of the pre-hatch embryo stages of development as evidenced by the 219 bp band (Fig. 2). In addition similar evidence for skin type AFP expression was observed for the larvae, skin, and liver samples (Fig. 2). There appeared to be an additional band of approximately 230 bp in the pre-hatch embryos that was not apparent for any of the other samples (Fig. 2). However repeated electrophoretic analyses failed to result in a clear separation of the 219 and 230 bp bands. Nucleotide sequencing Nucleotide sequencing of the seven clones (1F, 2F, 4F, 5F, 6F, 7F, and 8F) derived from a single PCR product revealed that seven different skin type AFP genes were being expressed in the pre-hatch
Fig. 3. Comparison of nucleotide sequences expressed by pre-hatch embryos (clones 1F–8F), with sequences of known skin type antifreeze proteins 11-3, sAFP1, sAFP3, and sAFP5. All sequence data were aligned with AFP 11-3 using Vector NTI (Invitrogen, Burlington, Ontario, Canada). The 11-3 genomic sequence was obtained from GenBank (Accession No. M63478), while sAFP1, sAFP3, and sAFP5 sequences were obtained from Ref. [16]. The protein coding region is highlighted in bold. Underlined nucleotides indicate where differences exist between the various sequences. Dashes represent sequence gaps while dots indicate where sequence data are lacking.
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Fig. 3 (continued)
embryos. A detailed analysis of the sequences (Fig. 3) revealed that clones 2F, 6F, and 7F were identical to that of sAFP3, [15], while clone 5F was identical in sequence to sAFP5 [15]. The nucleotide sequence of clone 4F differed from that of skin type AFP 11-3 by a single base substitution at position 1148 which is within the 3’ UTR (Fig. 3) The sequences of clones 8F and 1F were very similar to that of skin type sAFP1 [15], with clone 8F differing by a single base within the 30 UTR at position 167, while clone 1F differed by three bases at positions 125, 149, and 167 (Fig. 3) The base substitution at position 125, which is within the protein reading frame, did not result in an amino acid change (Fig 4). When the nucleotide sequences were translated into amino acids it was evident that only four different skin type AFP were being produced (Fig. 4). Two of the four proteins (sAFP1 and sAFP3) that were expressed in these embryos have been isolated and purified from adult winter flounder skin, indicating that they are likely present at all life stages [10]. Discussion The results of this study clearly indicate that winter flounder begin to express skin type AFP genes, synthesize AFP, and accumu-
late low but measurable levels of the protein very early during embryonic development. This result lends credence to the hypothesis put forward by Robles et al. [30] that the brief continuation of pigmentation development and active movements observed in winter flounder embryos that had been vitrified was in part due to the presence of AFP. As pointed out in the introduction, there are two general hypotheses as to how AFP could help protect cryopreserved embryos: inhibition of ice recrystallization, and protection of cell membranes from cold induced damage. The discovery that only skin type AFP are expressed in pre-hatch embryos suggests that they exert their cryoprotective effect intracellularly. None of these hypotheses are meant to be mutually exclusive. Ice recrystallization occurs when larger ice crystals grow at the expense of smaller ones creating considerable physical damage within the frozen tissue. This phenomenon is believed to be a major cause for cellular damage associated with cryogenic procedures [2,13,14,29]. AFP have been shown to be effective in reducing this cellular and tissue damage by binding to the ice and inhibiting or blocking the recrystallization process [2,21,24,27]. Walker et al. [38] examined the ability of type I AFP to inhibit ice recrystallization (IR) using an IR assay [37], and found that AFP concentrations
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Fig. 4. Amino acid sequences of winter flounder skin type AFP expressed in pre-hatch embryos. The amino acid sequences were deduced from cDNA clones 1F, 2F, 4F, 5F, 6F, 7F, 8F and aligned with skin type AFP 11-3 (GenBank Accession No. M63478), sAFP1, sAFP3, and sAFP5 [15]. Residue differences between the proteins are located at positions 6, 23, 32, and 38. Asterisks represent positions where the amino acid is identical to that of the 11-3 sequence.
as low as 2 lg/ml were highly efficacious. Type I AFP have also been shown to effectively enhance the survival of cryopreserved red blood cells during thaw at concentrations of 5–150 lg/ml [2]. The levels of AFP measured in the embryos of the present study were approximately 0.16–0.22 mg/ml, as estimated from the thermal hysteresis values (0.025–0.032 °C) obtained for the homogenates using the AFP activity curves generated by Evans and Fletcher [4]. Since the homogenates were concentrated fivefold, the AFP concentration in the embryos was approximately 30– 40 lg/ml, well within the range required to inhibit ice recrystallization [2,37]. A number of studies have demonstrated that AFP can protect cold sensitive mammalian cells, tissues, and organs from hypothermic non-freezing damage [1,20,32,33,36]. However, no comparable studies have been carried out on fish tissues or cells, thus it is not known whether AFP could play a similar protective role. Further details on antifreeze and cold adaptation in fish can be found in Fletcher et al. [7]. Recently Robles et al. [31] have found evidence that AFP can reduce the chilling sensitivity of seabream embryos (a warm water species). These studies demonstrated that the hatching rates of embryos from this species microinjected with the small liver type I AFP and exposed to 0 °C for an hour approached 100% whereas the mean value for controls with no AFP was less than 50%. Confocal microscopy indicated that microinjected FITC labeled AFP rapidly became associated within the cellular compartment of the embryo prompting the suggestion that the AFP had improved the cold resistance of the embryos by protecting cell membranes. Winter flounder skin type AFP lack a secretory signal sequence suggesting that they remain and function intracellularly [10]. Since the pre-hatch embryos of the present study only express skin type AFP genes, it is possible that the AFP remained within the cells and served to prevent the formation or the recrystallization of intracellular ice during the process of freezing or thawing the vitrified winter flounder embryos studied by Robles et al. [30]. One interesting discovery made in the Robles et al. [30] study was that the only embryos showing continued development following thaw were the 11-day-old group that were cryopreserved using dimethyl sulfoxide and thawed relatively slowly at 0 °C. Slow thawing would be expected to increase the probability of recrystallization. In contrast there was no evidence of continued embryonic development in any of the vitrified embryos thawed rapidly at 25 °C; conditions that minimize recrystallization. Although it could be argued that AFP prevented recrystallization in the slowly thawed embryos it is dif-
ficult to reconcile why the embryos that were rapidly thawed did not fare as well. Winter flounder are a cold water species whose embryos normally develop in the range of 0 °C–12 °C. In addition the upper lethal temperature has been reported as 15 °C [41]. Therefore, exposure of the cryopreserved embryos to 25 °C may have contributed to their mortality. Acknowledgments We thank Drs Peter Davies and Laurie Graham (Department of Biochemistry, Queen’s University) for the hyperactive AFP cDNA sequence and primers, and Madonna King (Ocean Sciences Centre) for her expert advice and assistance. This research was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). References [1] G. Amir, L. Horowitz, B. Rubinsky, S.Y. Yousif, J. Lavee, Smolinsky, Subzero nonfreezing cryopreservation of rat hearts using antifreeze protein I and antifreeze protein III, Cryobiology 48 (2004) 273–282. [2] J.F. Carpenter, T.N. Hansen, Antifreeze protein modulates cell survival during cryopreservation: Mediation through influence on ice crystal growth, Proc. Nat. Acad. Sci. USA 89 (1992) 8953–8957. [3] P.L. Davies, C.L. Hew, Biochemistry of fish antifreeze proteins, FASEB J. 4 (1990) 2460–2468. [4] R.P. Evans, G.L. Fletcher, Isolation and characterization of type I antifreeze proteins from Atlantic snailfish (Liparis atlanticus) and dusky snailfish (Liparis gibbus), Biochim. Biophys. Acta 1547 (2001) 235–247. [5] R.P. Evans, R.S. Hobbs, S.V. Goddard, G.L. Fletcher, The importance of dissolved salts to the in vivo efficacy of antifreeze proteins, Comp. Biochem. Physiol. A148 (2007) 556–561. [6] G.L. Fletcher, Circannual cycles of blood plasma freezing points and Na+ and Cl concentrations in Newfoundland winter flounder (Pseudopleuronectes americanus): correlation with water temperature and photoperiod, Can. J. Zool. 55 (1977) 789–795. [7] G.L. Fletcher, S.V. Goddard, P.L. Davies, Z. Gong, K.V. Ewart, C.L. Hew, New insights into fish antifreeze proteins: physiological significance and molecular regulation, in: H.O. Portner, R. Playle (Eds.), Cold Ocean Physiology, Cambridge University Press, Cambridge, 1998, pp. 239–265. [8] G.L. Fletcher, S.V. Goddard, Y. Wu, Antifreeze proteins and their genes: From basic research to business opportunities, Chemtech 29 (1999) 17–28. [9] G.L. Fletcher, C.L. Hew, P.L. Davies, Antifreeze proteins of teleost fishes, Annu. Rev. Physiol. 63 (2001) 359–390. [10] Z. Gong, K.V. Ewart, Z. Hu, G.L. Fletcher, C.L. Hew, Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretary signal and prosequences, J. Biol. Chem. 245 (1996) 2901–2913. [11] M. Hagedorn, F.W. Kleinhans, D. Artemov, U. Pilatus, Characterization of a major permeability barrier in the zebrafish embryo, Biol. Reprod. 59 (1998) 1240–1250. [12] M. Hagedorn, F.W. Kleinhans, Cryopreservation in aquatic species, in: T.R. Tiersch, P.M. Mazik (Eds.), Problems and Prospects in Cryopreservation of Fish
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