- Email: [email protected]
The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures
Theriogenology 68 (2007) 284–289 www.theriojournal.com
Technical note
The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures V. Robles a,1,*, V. Barbosa a,1, M.P. Herra´ez b, S. Martı´nez-Pa´ramo b, M.L. Cancela a a
CCMAR, Center for Marine Sciences, University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal b Department of Cellular Biology, Faculty of Biology, University of Leo´n, 24071 Leo´n, Spain Received 14 March 2007; received in revised form 4 April 2007; accepted 1 May 2007
Abstract To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere–yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 8C (1 h) and 10 8C (15 min) when embryos reached 5-somite stage. Embryos were subjected to 10 8C chilling in a 3 M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 8C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes. # 2007 Elsevier Inc. All rights reserved. Keywords: Seabream embryos; Microinjection; AFP I; Chilling resistance; Cryopreservation
1. Introduction Successful cryopreservation of teleost embryos would have important benefits in aquaculture, conservation programs for endangered species and preservation of valuable genetic lines. However, at present it has not been achieved in any species. It is generally
* Corresponding author at: University of Algarve, FCMA, Campus de Gambelas, 8005-139 Faro, Portugal. E-mail address: [email protected] (V. Robles). 1 These authors have equally contributed to the work. 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.003
accepted that the main reason for that failure is the absence of a proper concentration of cryoprotectants within all embryonic compartments. Achievement of this optimal concentration encounters two main problems: (1) how to overcome the physical barriers within the embryo in order to reach an adequate and homogeneous concentration of the CPA throughout the embryo, and (2) how to avoid the toxic effects of the cryoprotective agent or agents at that concentration. The technique of microinjection could be a good solution for the first problem. Janik et al. [1] have used this technique for the introduction of cryoprotective agents into zebrafish embryos overcoming the permeability
V. Robles et al. / Theriogenology 68 (2007) 284–289
barriers, in particular the yolk syncytial layer, which minimized the entry and exit of most cryoprotectants [2]. This technique has been also used for the introduction of traditional CPAs (DMSO, ethylene glycol, methanol and sucrose) in seabream embryos [3]. Beira˜o et al. demonstrated that embryo viability and larval development until first feeding were not affected by the microinjection procedure in tail bud stage embryos. However, the microinjection of those cryoprotectants at the tested concentrations did not improve significantly the survival rates after cooling. For the second problem that we have described, the use of CPA with low toxicity, like sugars or some proteins, could be a solution. It is known that the traditional CPAs usually employed in the extenders cannot properly penetrate the blastoderm and the yolk [4]. On the other hand, if we introduced them artificially in those compartments, at the level in which they are required to produce enough protection during the cryopreservation process, toxic effects could be observed. We can however compensate the low CPA concentration within those compartments by the introduction of a non-toxic CPA by microinjection, and thus AFPs could be a good candidate for this purpose. The introduction of antifreeze proteins (AFPs) into embryos can be highly beneficial for cryopreservation purposes, since they inhibit the growth of ice crystals as well as recrystallization during thawing. These AFPs are naturally expressed in sub-arctic fish species whose embryos have a better tolerance to freezing [5]. In this work we have microinjected AFP I, an antifreeze protein obtained from the sub-arctic species winterflounder, into embryos of the marine teleost seabream and then analyzed its distribution within the embryo and its effect on embryo hatching and on chilling resistance. 2. Materials and methods 2.1. Embryo microinjection Embryos were supplied by IPIMAR (Portugal) and were collected at 1–2 cell stage. For embryo microinjection an electric IM-30 microinjector (Narishige, Spain) linked to a stereoscopic microscope was used, with an air entrance pressure of 3.5 bar. Needles for microinjection were made in a puller (Narishige PB-7) using glass capillaries with an internal filament (1 mm external diameter, 0.6 mm internal diameter) (GD-1 Narishige, Spain). For embryo holding, agar disks were made in 35 mm of diameter Petri dishes using 20 ml of agarose at 1.5%
285
and capillaries placed on it during cooling to make parallel grooves of 1 mm of diameter. The embryos were carefully pressed into the grooves before being microinjected. Sparus aurata embryos were microinjected with 20 nl of a solution of AFP I at 10 mg/ml in embryo media (EM), an ionic buffer solution described by Westerfield [6], at stage 2–8 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. To evaluate embryo survival after AFP I microinjection, a total of 91 embryos at 2–8 cells and 104 embryos at blastula were microinjected. Three replicates were done with approximately 30–35 embryos each. Hatching rates were determined as the number of larvae per total number of microinjected embryos. To evaluate the effect of AFP I when embryos were subjected to low temperatures, a total of 847 embryos were microinjected, 435 in 2–8 cells and 412 in blastula. Six to eight replicates were done with approximately 30–36 embryos each. 2.2. AFP distribution by confocal microscope FITC-labelled protein distribution was analyzed by confocal microscopy. For protein labelling, AFP I was dissolved in reaction buffer (500 mM carbonate, pH 9.5; 1.7 g Na2CO3; 2.8 g NaHCO3, pH 9.5) at the concentration of 10 mg/ml. Fluorescein isothiocyanate (FITC) (100 mg/ml in DMSO) was added to protein at 20 mg FITC/mg protein then wrapped in foil, and incubated at RT for 1 h. It was then dialyzed against 2 l of storage buffer (1.42 g Trzma 8.0, 8.77 g NaCl, three to four drops of pHix (at 5 mg/ml in 95% EtOH) pH 8.2–1 l dH2O) at 4 8C overnight with gentle stirring to remove unreacted FITC (1000 MWCO dialysis tubing). Dialyzed protein was freeze-dried. Before microinjection freeze-dried protein was dissolved in EM and filtered. After microinjection and before fixation, embryos were incubated at room temperature (22 8C). Microinjected embryos were fixed 5 min after microinjection (in 2–8 cells and blastula) and when they reach 17-somite stage, using 2% paraformaldehyde in Sorensen buffer (8 ml solution A plus 42 ml solution B; solution A: KH2PO4 9.066 g/l distilled water, solution B: Na2HPO4 23.866 g/l distilled water) at 4 8C. After 4 h, three washes were performed in Sorensen buffer. Fixed embryos were kept refrigerated until they were analyzed by confocal microscopy. For fluorescence imaging a Radiance 2000 confocal microscope (Bio-Rad) was used. For visualization of
286
V. Robles et al. / Theriogenology 68 (2007) 284–289
FITC fluorescence emitted by the labelled proteins, the green fluorescence/transmission method was used. Images of optical sections in each embryo were recorded.
detected by the Student–Newman–Keuls multiple range test (SNK), significance being set at p < 0.05.
2.3. Chilling resistance study
The microinjection of embryos from S. aurata with 20 nl of the non-labelled AFP I (10 mg/ml) did not decrease hatching rates, regardless of the developmental stage in which embryos were microinjected (2–8 cells or blastula) (Fig. 2), hatching rates were above 90% in all cases. These results are not in agreement with observations made for turbot embryos which display a high sensitivity to the technique of microinjection [7]. Therefore, our results demonstrate not only that the protein had no toxic effect on seabream embryos, but also that the performance of the microinjection, including the selected volume of injection, did not affect hatching. This technique can be successfully used in this species, in agreement with results obtained by Beira˜o et al. [3] that used the technique of microinjection to introduce traditional cryoprotectants in seabream embryos. Furthermore, using confocal microscopy we have demonstrated that AFP-FITC is located in the cellular compartment. Proteins microinjected at early cleavage states are likely to be distributed by cytoplasmic bridges through blastomeres [8] but also proteins microinjected into the yolk in blastula are rapidly incorporated into the cells (Fig. 3). The presence
Embryos were incubated after microinjection with the non-labelled AFP in sterilized sea water at room temperature (RT) until they reached approximately 17somite stage when they were subjected to chilling. Two different chilling tests were done: 0 8C for 1 h and 10 8C for 15 min. In the last case, embryos were subjected to chilling in the presence of 3 M DMSO to avoid the freezing of the external solution. In both tests two different groups were used as controls: intact embryos and punctured-only embryos. The experimental design is explained in Fig. 1. After the chilling, embryos were carefully washed and incubated into fresh sterilized sea water until hatch. Hatching rates were established as explained above. 2.4. Statistical analyses The percentile data was normalized though arcsine transformation and the results were expressed as means S.D. and analyzed by one way ANOVA. Significant differences between hatching rates were
3. Results and discussion
Fig. 1. Diagram representing the experimental design.
V. Robles et al. / Theriogenology 68 (2007) 284–289
287
Fig. 2. Percentage of embryos that hatch after being microinjected with AFP I. No statistical differences were found among the groups.
of this natural cryoprotectant into the cellular compartment is of great importance, since blastoderm possesses almost 80% of the enzymes necessary for proper development [1]. The fact that this protein showed no toxic effects to the embryos is also a key factor, since most of the traditional CPAs produce toxic effects, and those enzymes present in the blastoderm are often the target of CPA toxicity [9–11]. AFP I significantly improved chilling resistance at 0 8C, particularly in 2-cell microinjected embryos, which displaced nearly 100% of hatching rates (Fig. 4). This fact is in agreement with the confocal microscopy observations which demonstrated that the protein is present in the cells (Fig. 3). On the other hand, the effect of the protein seems to be limited to a specific range of temperatures, being extremely efficient when embryos
were subjected to 0 8C for 1 h but much less when embryos where subjected to 10 8C for 15 min, when ice crystal formation within the embryos can take place (Fig. 5). It is well known that antifreeze proteins lower the freezing temperature to below the thermodynamic freezing point in plasma and extracellular fluids in living fish and avoid ice crystal formation, but also have other protective properties at low temperatures. For example, they can protect cell membranes from cold induced damage [12,13] and inhibit ice recrystallization [14]. This study demonstrates that AFP I can protect embryos from chilling and we hypothesize that this effect may be due to a stabilization of the cellular membranes during chilling rather than an inhibition of ice crystal formation. The reduction in temperature during the cooling process causes physical changes in
Fig. 3. Confocal microscope photographs of AFPI-FITC labelled microinjected embryos. (A) 17-Somite embryo microinjected in 2 cells stage (approximately 16 h after microinjection); (B) 17-somite embryo microinjected in high blastula stage (approximately 14 h after microinjection); (C) high blastula embryo 5 min after microinjection. (A0 –C0 ) are bright field images superimposed on the confocal fluorescence images.
288
V. Robles et al. / Theriogenology 68 (2007) 284–289
Fig. 4. Percentage of control, punctured and AFP I microinjected embryos that hatch after being subjected to 0 8C for 1 h at 5-somite stage. Letters show the statistical differences between three different experimental groups ( p < 0.05). Bars sharing the same letter or letters are not statistically different. Embryos microinjected in 2 cells showed a significantly higher survival after the treatment than control and only punctured embryos, however, those microinjected in blastula did not reported a significantly improvement in survival.
Fig. 5. Percentage of control, punctured and AFP I microinjected embryos that hatch after being subjected to 10 8C for 15 min at 5-somite stage. No statistical differences were found among the groups.
cellular membranes such as separation or phase transitions of the lipid components, which result in alteration of membrane function [15]. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane. Tomczak et al. [16] proposed that AFP may produce these effects by partially inserting into the membrane during chilling. The AFP protection at this level would explain its positive effect when embryos were subjected to 0 8C rather than when subjected to 10 8C. At temperatures under 0 8C, ice crystals can form within the embryo in those compartments with insufficient CPA concentration, and therefore the stabilization of plasma membranes could be insufficient for embryo survival. It is possible that optimizing AFP concentration within the embryo may achieve both protective effects, by inhibition ice crystal formation and membrane stabilization, since it is known that the protective effect of
AFPs is dependent on the concentration and type of AFP used [12,13]. This study has demonstrated that the AFP type I, at the concentration used (10 mg/ml), has no toxic effects on S. aurata embryos and can be successfully incorporated into the blastomeres thus inducing in embryos a protective effect against chilling. Accordingly, the incorporation of this antifreeze protein in cryopreservation protocols should be considered in order to protect the embryos during the range of temperatures at which occur the main deleterious effects, caused by low temperatures-induced stress rather than by ice crystal formation. Acknowledgments This work was partially financed by CCMAR. V. Robles was the recipient of a postdoctoral fellowship (SFRH/BPD/17980/2004) from The Portuguese
V. Robles et al. / Theriogenology 68 (2007) 284–289
Science and Technology Foundation (FCT). Authors would like to thank IPIMAR (Portugal) for the supply of seabream embryos and Dr. Elsa Cabrita for her comments on the manuscript.
[8] [9]
References [10] [1] Janik M, Kleinhans FW, Hagedorn M. Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio). Cryobiology 2000;41:25–34. [2] Hagedorn M, Hsu EW, Pilatus U, Wildt DE, Rall WF, Blackband SJ. Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system. Proc Natl Acad Sci USA 1996;93:7454–9. [3] Beira˜o J, Robles V, Herra´ez MP, Sarasquete C, Dinis MT, Cabrita E. Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos. Aquaculture 2006;261:897–903. [4] Hagedorn M, Kleinhans FW. Problems and prospects in cryopreservation of fish embryos. In: Tiersch TR, Mazik PM, editors. Cryopreservation in aquatic species. Baton Rouge, LA, USA: The World Aquaculture Society; 2000. p. 161–78. [5] Robles V, Cabrita E, Fletcher GL, Shears MA, King MJ, Herra´ez MP. Vitrification assays with embryos from a cold tolerant subarctic fish species. Theriogenology 2005;64:1633–46. [6] Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio). Eugene, University of Oregon Press; 1993. [7] Robles V, Cabrita E, Anel L, Herra´ez MP. Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus
[11]
[12]
[13]
[14]
[15]
[16]
289
maximus) embryos: toxicity and protein distribution. Aquaculture 2006;261:1299–306. Kimmel CB, Law RD. Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer. Dev Biol 1985;108:86–93. Adam MM, Rana KJ, McAndrew BJ. Effect of cryoprotectants on activity of selected enzymes in fish embryos. Cryobiology 1995;32:92–104. Arakawa T, Carpenter JF, Kita YA, Crowe JH. The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 1990;27:401–15. Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT. Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms. Cryobiology 1990;27:247–68. Rubinsky B, Arav A, DeVries AL. Cryopreservation of oocytes using directional cooling and antifreeze glycoproteins. Cryo Lett 1991;12:93–106. Rubinsky B, Arav A, Fletcher GL. Hypothermic protection: a fundamental property of antifreeze proteins. Biochem Biophys Res Commun 1991;180:566–71. Knight CA, Duman JG. Inhibition of recrystallization of ice by insect thermal hysteresis proteins: a possible cryoprotective role. Cryobiology 1986;23:256–62. Pugh PA, Ankersmit AEL, McGowan LT, Tervit HR. Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival. Theriogenology 1998;50: 495–506. Tomczak MM, Hincha DK, Estrada SD, Wolkers WF, Crowe LM, Feeney RE, et al. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein. Biophys J 2002;82:874–81.
Technical note
The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures V. Robles a,1,*, V. Barbosa a,1, M.P. Herra´ez b, S. Martı´nez-Pa´ramo b, M.L. Cancela a a
CCMAR, Center for Marine Sciences, University of Algarve, Campus de Gambelas, Faro 8005-139, Portugal b Department of Cellular Biology, Faculty of Biology, University of Leo´n, 24071 Leo´n, Spain Received 14 March 2007; received in revised form 4 April 2007; accepted 1 May 2007
Abstract To date, all attempts at fish embryo cryopreservation have failed. One of the main reasons for this to occur is the high chilling sensitivity reported in fish embryos thus emphasizing the need for further testing of different methods and alternative cryoprotective agents (CPAs) in order to improve our chances to succeed in this purpose. In this work we have used the antifreeze protein type I (AFP I) as a natural CPA. This protein is naturally expressed in sub-arctic fish species, and inhibits the growth of ice crystals as well as recrystallization during thawing. Embryos from Sparus aurata were microinjected with AFP I at different developmental stages, 2 cells and blastula, into the blastomere–yolk interface and into the yolk sac, respectively. Control, punctured and microinjected embryos were subjected to chilling at two different temperatures, 0 8C (1 h) and 10 8C (15 min) when embryos reached 5-somite stage. Embryos were subjected to 10 8C chilling in a 3 M DMSO extender to avoid ice crystal formation in the external solution. Survival after chilling was established as the percentage of embryos that hatch. To study the AFP I distribution in the microinjected embryos, a confocal microscopy study was done. Results demonstrate that AFP I can significantly improve chilling resistance at 0 8C, particularly in 2-cell microinjected embryos, displaying nearly 100% hatching rates. This fact is in agreement with the confocal microscopy observations which confirmed the presence of the AFP protein in embryonic cells. These results support the hypothesis that AFP protect cellular structures by stabilizing cellular membranes. # 2007 Elsevier Inc. All rights reserved. Keywords: Seabream embryos; Microinjection; AFP I; Chilling resistance; Cryopreservation
1. Introduction Successful cryopreservation of teleost embryos would have important benefits in aquaculture, conservation programs for endangered species and preservation of valuable genetic lines. However, at present it has not been achieved in any species. It is generally
* Corresponding author at: University of Algarve, FCMA, Campus de Gambelas, 8005-139 Faro, Portugal. E-mail address: [email protected] (V. Robles). 1 These authors have equally contributed to the work. 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.05.003
accepted that the main reason for that failure is the absence of a proper concentration of cryoprotectants within all embryonic compartments. Achievement of this optimal concentration encounters two main problems: (1) how to overcome the physical barriers within the embryo in order to reach an adequate and homogeneous concentration of the CPA throughout the embryo, and (2) how to avoid the toxic effects of the cryoprotective agent or agents at that concentration. The technique of microinjection could be a good solution for the first problem. Janik et al. [1] have used this technique for the introduction of cryoprotective agents into zebrafish embryos overcoming the permeability
V. Robles et al. / Theriogenology 68 (2007) 284–289
barriers, in particular the yolk syncytial layer, which minimized the entry and exit of most cryoprotectants [2]. This technique has been also used for the introduction of traditional CPAs (DMSO, ethylene glycol, methanol and sucrose) in seabream embryos [3]. Beira˜o et al. demonstrated that embryo viability and larval development until first feeding were not affected by the microinjection procedure in tail bud stage embryos. However, the microinjection of those cryoprotectants at the tested concentrations did not improve significantly the survival rates after cooling. For the second problem that we have described, the use of CPA with low toxicity, like sugars or some proteins, could be a solution. It is known that the traditional CPAs usually employed in the extenders cannot properly penetrate the blastoderm and the yolk [4]. On the other hand, if we introduced them artificially in those compartments, at the level in which they are required to produce enough protection during the cryopreservation process, toxic effects could be observed. We can however compensate the low CPA concentration within those compartments by the introduction of a non-toxic CPA by microinjection, and thus AFPs could be a good candidate for this purpose. The introduction of antifreeze proteins (AFPs) into embryos can be highly beneficial for cryopreservation purposes, since they inhibit the growth of ice crystals as well as recrystallization during thawing. These AFPs are naturally expressed in sub-arctic fish species whose embryos have a better tolerance to freezing [5]. In this work we have microinjected AFP I, an antifreeze protein obtained from the sub-arctic species winterflounder, into embryos of the marine teleost seabream and then analyzed its distribution within the embryo and its effect on embryo hatching and on chilling resistance. 2. Materials and methods 2.1. Embryo microinjection Embryos were supplied by IPIMAR (Portugal) and were collected at 1–2 cell stage. For embryo microinjection an electric IM-30 microinjector (Narishige, Spain) linked to a stereoscopic microscope was used, with an air entrance pressure of 3.5 bar. Needles for microinjection were made in a puller (Narishige PB-7) using glass capillaries with an internal filament (1 mm external diameter, 0.6 mm internal diameter) (GD-1 Narishige, Spain). For embryo holding, agar disks were made in 35 mm of diameter Petri dishes using 20 ml of agarose at 1.5%
285
and capillaries placed on it during cooling to make parallel grooves of 1 mm of diameter. The embryos were carefully pressed into the grooves before being microinjected. Sparus aurata embryos were microinjected with 20 nl of a solution of AFP I at 10 mg/ml in embryo media (EM), an ionic buffer solution described by Westerfield [6], at stage 2–8 cells and blastula, into the blastomere-yolk interface and into the yolk sac, respectively. To evaluate embryo survival after AFP I microinjection, a total of 91 embryos at 2–8 cells and 104 embryos at blastula were microinjected. Three replicates were done with approximately 30–35 embryos each. Hatching rates were determined as the number of larvae per total number of microinjected embryos. To evaluate the effect of AFP I when embryos were subjected to low temperatures, a total of 847 embryos were microinjected, 435 in 2–8 cells and 412 in blastula. Six to eight replicates were done with approximately 30–36 embryos each. 2.2. AFP distribution by confocal microscope FITC-labelled protein distribution was analyzed by confocal microscopy. For protein labelling, AFP I was dissolved in reaction buffer (500 mM carbonate, pH 9.5; 1.7 g Na2CO3; 2.8 g NaHCO3, pH 9.5) at the concentration of 10 mg/ml. Fluorescein isothiocyanate (FITC) (100 mg/ml in DMSO) was added to protein at 20 mg FITC/mg protein then wrapped in foil, and incubated at RT for 1 h. It was then dialyzed against 2 l of storage buffer (1.42 g Trzma 8.0, 8.77 g NaCl, three to four drops of pHix (at 5 mg/ml in 95% EtOH) pH 8.2–1 l dH2O) at 4 8C overnight with gentle stirring to remove unreacted FITC (1000 MWCO dialysis tubing). Dialyzed protein was freeze-dried. Before microinjection freeze-dried protein was dissolved in EM and filtered. After microinjection and before fixation, embryos were incubated at room temperature (22 8C). Microinjected embryos were fixed 5 min after microinjection (in 2–8 cells and blastula) and when they reach 17-somite stage, using 2% paraformaldehyde in Sorensen buffer (8 ml solution A plus 42 ml solution B; solution A: KH2PO4 9.066 g/l distilled water, solution B: Na2HPO4 23.866 g/l distilled water) at 4 8C. After 4 h, three washes were performed in Sorensen buffer. Fixed embryos were kept refrigerated until they were analyzed by confocal microscopy. For fluorescence imaging a Radiance 2000 confocal microscope (Bio-Rad) was used. For visualization of
286
V. Robles et al. / Theriogenology 68 (2007) 284–289
FITC fluorescence emitted by the labelled proteins, the green fluorescence/transmission method was used. Images of optical sections in each embryo were recorded.
detected by the Student–Newman–Keuls multiple range test (SNK), significance being set at p < 0.05.
2.3. Chilling resistance study
The microinjection of embryos from S. aurata with 20 nl of the non-labelled AFP I (10 mg/ml) did not decrease hatching rates, regardless of the developmental stage in which embryos were microinjected (2–8 cells or blastula) (Fig. 2), hatching rates were above 90% in all cases. These results are not in agreement with observations made for turbot embryos which display a high sensitivity to the technique of microinjection [7]. Therefore, our results demonstrate not only that the protein had no toxic effect on seabream embryos, but also that the performance of the microinjection, including the selected volume of injection, did not affect hatching. This technique can be successfully used in this species, in agreement with results obtained by Beira˜o et al. [3] that used the technique of microinjection to introduce traditional cryoprotectants in seabream embryos. Furthermore, using confocal microscopy we have demonstrated that AFP-FITC is located in the cellular compartment. Proteins microinjected at early cleavage states are likely to be distributed by cytoplasmic bridges through blastomeres [8] but also proteins microinjected into the yolk in blastula are rapidly incorporated into the cells (Fig. 3). The presence
Embryos were incubated after microinjection with the non-labelled AFP in sterilized sea water at room temperature (RT) until they reached approximately 17somite stage when they were subjected to chilling. Two different chilling tests were done: 0 8C for 1 h and 10 8C for 15 min. In the last case, embryos were subjected to chilling in the presence of 3 M DMSO to avoid the freezing of the external solution. In both tests two different groups were used as controls: intact embryos and punctured-only embryos. The experimental design is explained in Fig. 1. After the chilling, embryos were carefully washed and incubated into fresh sterilized sea water until hatch. Hatching rates were established as explained above. 2.4. Statistical analyses The percentile data was normalized though arcsine transformation and the results were expressed as means S.D. and analyzed by one way ANOVA. Significant differences between hatching rates were
3. Results and discussion
Fig. 1. Diagram representing the experimental design.
V. Robles et al. / Theriogenology 68 (2007) 284–289
287
Fig. 2. Percentage of embryos that hatch after being microinjected with AFP I. No statistical differences were found among the groups.
of this natural cryoprotectant into the cellular compartment is of great importance, since blastoderm possesses almost 80% of the enzymes necessary for proper development [1]. The fact that this protein showed no toxic effects to the embryos is also a key factor, since most of the traditional CPAs produce toxic effects, and those enzymes present in the blastoderm are often the target of CPA toxicity [9–11]. AFP I significantly improved chilling resistance at 0 8C, particularly in 2-cell microinjected embryos, which displaced nearly 100% of hatching rates (Fig. 4). This fact is in agreement with the confocal microscopy observations which demonstrated that the protein is present in the cells (Fig. 3). On the other hand, the effect of the protein seems to be limited to a specific range of temperatures, being extremely efficient when embryos
were subjected to 0 8C for 1 h but much less when embryos where subjected to 10 8C for 15 min, when ice crystal formation within the embryos can take place (Fig. 5). It is well known that antifreeze proteins lower the freezing temperature to below the thermodynamic freezing point in plasma and extracellular fluids in living fish and avoid ice crystal formation, but also have other protective properties at low temperatures. For example, they can protect cell membranes from cold induced damage [12,13] and inhibit ice recrystallization [14]. This study demonstrates that AFP I can protect embryos from chilling and we hypothesize that this effect may be due to a stabilization of the cellular membranes during chilling rather than an inhibition of ice crystal formation. The reduction in temperature during the cooling process causes physical changes in
Fig. 3. Confocal microscope photographs of AFPI-FITC labelled microinjected embryos. (A) 17-Somite embryo microinjected in 2 cells stage (approximately 16 h after microinjection); (B) 17-somite embryo microinjected in high blastula stage (approximately 14 h after microinjection); (C) high blastula embryo 5 min after microinjection. (A0 –C0 ) are bright field images superimposed on the confocal fluorescence images.
288
V. Robles et al. / Theriogenology 68 (2007) 284–289
Fig. 4. Percentage of control, punctured and AFP I microinjected embryos that hatch after being subjected to 0 8C for 1 h at 5-somite stage. Letters show the statistical differences between three different experimental groups ( p < 0.05). Bars sharing the same letter or letters are not statistically different. Embryos microinjected in 2 cells showed a significantly higher survival after the treatment than control and only punctured embryos, however, those microinjected in blastula did not reported a significantly improvement in survival.
Fig. 5. Percentage of control, punctured and AFP I microinjected embryos that hatch after being subjected to 10 8C for 15 min at 5-somite stage. No statistical differences were found among the groups.
cellular membranes such as separation or phase transitions of the lipid components, which result in alteration of membrane function [15]. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane. Tomczak et al. [16] proposed that AFP may produce these effects by partially inserting into the membrane during chilling. The AFP protection at this level would explain its positive effect when embryos were subjected to 0 8C rather than when subjected to 10 8C. At temperatures under 0 8C, ice crystals can form within the embryo in those compartments with insufficient CPA concentration, and therefore the stabilization of plasma membranes could be insufficient for embryo survival. It is possible that optimizing AFP concentration within the embryo may achieve both protective effects, by inhibition ice crystal formation and membrane stabilization, since it is known that the protective effect of
AFPs is dependent on the concentration and type of AFP used [12,13]. This study has demonstrated that the AFP type I, at the concentration used (10 mg/ml), has no toxic effects on S. aurata embryos and can be successfully incorporated into the blastomeres thus inducing in embryos a protective effect against chilling. Accordingly, the incorporation of this antifreeze protein in cryopreservation protocols should be considered in order to protect the embryos during the range of temperatures at which occur the main deleterious effects, caused by low temperatures-induced stress rather than by ice crystal formation. Acknowledgments This work was partially financed by CCMAR. V. Robles was the recipient of a postdoctoral fellowship (SFRH/BPD/17980/2004) from The Portuguese
V. Robles et al. / Theriogenology 68 (2007) 284–289
Science and Technology Foundation (FCT). Authors would like to thank IPIMAR (Portugal) for the supply of seabream embryos and Dr. Elsa Cabrita for her comments on the manuscript.
[8] [9]
References [10] [1] Janik M, Kleinhans FW, Hagedorn M. Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio). Cryobiology 2000;41:25–34. [2] Hagedorn M, Hsu EW, Pilatus U, Wildt DE, Rall WF, Blackband SJ. Magnetic resonance microscopy and spectroscopy reveal kinetics of cryoprotectant permeation in a multicompartmental biological system. Proc Natl Acad Sci USA 1996;93:7454–9. [3] Beira˜o J, Robles V, Herra´ez MP, Sarasquete C, Dinis MT, Cabrita E. Cryoprotectant microinjection toxicity and chilling sensitivity in gilthead seabream (Sparus aurata) embryos. Aquaculture 2006;261:897–903. [4] Hagedorn M, Kleinhans FW. Problems and prospects in cryopreservation of fish embryos. In: Tiersch TR, Mazik PM, editors. Cryopreservation in aquatic species. Baton Rouge, LA, USA: The World Aquaculture Society; 2000. p. 161–78. [5] Robles V, Cabrita E, Fletcher GL, Shears MA, King MJ, Herra´ez MP. Vitrification assays with embryos from a cold tolerant subarctic fish species. Theriogenology 2005;64:1633–46. [6] Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio). Eugene, University of Oregon Press; 1993. [7] Robles V, Cabrita E, Anel L, Herra´ez MP. Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus
[11]
[12]
[13]
[14]
[15]
[16]
289
maximus) embryos: toxicity and protein distribution. Aquaculture 2006;261:1299–306. Kimmel CB, Law RD. Cell lineage of zebrafish blastomeres. II. Formation of the yolk syncytial layer. Dev Biol 1985;108:86–93. Adam MM, Rana KJ, McAndrew BJ. Effect of cryoprotectants on activity of selected enzymes in fish embryos. Cryobiology 1995;32:92–104. Arakawa T, Carpenter JF, Kita YA, Crowe JH. The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 1990;27:401–15. Fahy GM, Lilley TH, Linsdell H, Douglas MS, Meryman HT. Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms. Cryobiology 1990;27:247–68. Rubinsky B, Arav A, DeVries AL. Cryopreservation of oocytes using directional cooling and antifreeze glycoproteins. Cryo Lett 1991;12:93–106. Rubinsky B, Arav A, Fletcher GL. Hypothermic protection: a fundamental property of antifreeze proteins. Biochem Biophys Res Commun 1991;180:566–71. Knight CA, Duman JG. Inhibition of recrystallization of ice by insect thermal hysteresis proteins: a possible cryoprotective role. Cryobiology 1986;23:256–62. Pugh PA, Ankersmit AEL, McGowan LT, Tervit HR. Cryopreservation of in vitro-produced bovine embryos: effects of protein type and concentration during freezing or of liposomes during culture on post-thaw survival. Theriogenology 1998;50: 495–506. Tomczak MM, Hincha DK, Estrada SD, Wolkers WF, Crowe LM, Feeney RE, et al. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein. Biophys J 2002;82:874–81.