Research Article
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Published Online: 20 January 2015

Short-Term Chilled Storage of Zebrafish (Danio rerio) Embryos in Cryoprotectant As an Alternative to Cryopreservation

Publication: Zebrafish
Volume 12, Issue Number 1

Abstract

As zebrafish embryos have never been cryopreserved, we developed a protocol to store zebrafish embryos (50% epiboly—5.3 hour post fertilization) for up to 18 h at 0°C. Initial experiments to optimize the cryoprotectant (CPA) solution demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH+0.1 M sucrose (56±5%) compared with other combinations of methanol (0.2–0.5 M) and sucrose (0.05–0.1 M). This combination of CPAs that protects against chilling injury was further tested to assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH+0.1 sucrose compared with non-chilled controls, however the expression of both sox2 and sox3 proteins was unaffected. Significant decreases in sox2 protein expression were, however, observed in embryos that had been chilled without CPAs and these embryos also had lower hatching rates than those chilled with the optimal CPA solution. We, therefore, conclude that the CPA combination of 1 M MeOH+0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish embryos for up to 18 h without compromising transcriptional response.

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References

1.
Zhang T: Cryopresrvation of gametes and embryos of aquatic species. In: Life in the Frozen State. Fuller BJ, Lane N, and Benson EE (eds), pp. 415–435. CRC Press, London, 2004.
2.
Maddock BG. A technique to prolong the incubation period of brown trout ova. Prog Fish Cult 1974;36:219–222.
3.
Zhang T, Rawson DM. Studies on chilling sensitivity of zebrafish (Brachydanio rerio) embryos. Cryobiology 1995;32:239–246.
4.
Dinnyés A, Urbányi B, Baranyai B, Magyary I. Chilling sensitivity of carp (Cyprinus carpio) embryos at different developmental stages in the presence or absence of cryoprotectants: work in progress. Theriogenology 1998;50:1–13.
5.
Liu K-C, Chou T-C, Lin H-D. Cryosurvival of goldfish embryo after subzero freezing. Aquat Living Resour 1993;6:63–66.
6.
Ahammad MM, Bhattacharyya D, Jana BB. Hatching of common carp (Cyprinus carpio L.) embryos stored at 4 and −2°C in different concentrations of methanol and sucrose. Theriogenology 2003;60:1409–1422.
7.
Desai K, Spikings E, Zhang T. Effect of chilling on sox2, sox3 and sox19a gene expression in zebrafish (Danio rerio) embryos. Cryobiology 2011;63:96–103.
8.
Driever W. Axis formation in zebrafish. Curr Opin Genet Dev 1995;5:610–618.
9.
Helde KA, Wilson ET, Cretekos CJ, Grunwald DJ. Contribution of early cells to the fate map of the zebrafish gastrula. Science 1994;265:517–520.
10.
Strahle U, Blader P. Early neurogenesis in the zebrafish embryo. FASEB J 1994;8:692–698.
11.
Stainier DY, Lee RK, Fishman MC. Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development 1993;119:31–40.
12.
Driever W, Stemple D, Schier A, Solnica-Krezel L. Zebrafish: genetic tools for studying vertebrate development. Trends Genet 1994;10:152–159.
13.
Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, et al. The syntenic relationship of the zebrafish and human genomes. Genome Res 2000;10:1351–1358.
14.
Lefebvre V, Dumitriu B, Penzo-Méndez A, Han Y, Pallavi B. Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol 2007;39:2195–2214.
15.
Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 2003;17:126–140.
16.
Yuan H, Corbi N, Basilico C, Dailey L. Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev 1995;9:2635–2645.
17.
Bylund M, Andersson E, Novitch BG, Muhr J. Vertebrate neurogenesis is counteracted by Sox1–3 activity. Nat Neurosci 2003;6:1162–1168.
18.
Graham V, Khudyakov J, Ellis P, Pevny L. SOX2 functions to maintain neural progenitor identity. Neuron 2003;39:749–765.
19.
Dee CT, Hirst CS, Shih Y-H, Tripathi VB, Patient RK, Scotting PJ. Sox3 regulates both neural fate and differentiation in the zebrafish ectoderm. Dev Biol 2008;320:289–301.
20.
Vriz S, Joly C, Boulekbache H, Condamine H. Zygotic expression of the zebrafish Sox-19, an HMG box-containing gene, suggests an involvement in central nervous system development. Mol Brain Res 1996;40:221–228.
21.
Pevny L, Placzek M. SOX genes and neural progenitor identity. Curr Opin Neurobiol 2005;15:7–13.
22.
Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR, et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 2001;1:277–290.
23.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of the zebrafish. Am J Anat 1995;203:253–310.
24.
Lin C, Spikings E, Zhang T, Rawson DM. Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres. Cryobiology 2009;59:42–47.
25.
Lahnsteiner F. Factors affecting chilled storage of zebrafish (Danio rerio) embryos. Theriogenology 2009;72:333–340.
26.
Lin C, Spikings E, Zhang T, Rawson D. Housekeeping genes for cryopreservation studies on zebrafish embryos and blastomeres. Theriogenology 2009;71:1147–1155.
27.
Pfaffl MW: Quantification strategies in real-time PCR. In: A-Z of Quantitative PCR. Bustin SA (ed), pp. 87–112, International University Line (IUL), La Jolla, CA, 2003.
28.
Luczynski M. A technique for delaying embryogenesis of vendace (Coregonus albula L.) eggs in order to synchronize mass hatching with optimal conditions for lake stocking. Aquaculture 1984;41:113–117.
29.
Hagedorn M, Kleinhans FW, Wildt DE, Rall WF. Chill sensitivity and cryoprotectant permeability of dechorionated zebrafish embryos, Brachydanio rerio. Cryobiology 1997;34:251–263.
30.
Lahnsteiner F. The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos. Theriogenology 2008;69:384–396.
31.
Zhang T, Isayeva A, Adams SL, Rawson DM. Studies on membrane permeability of zebrafish (Danio rerio) oocytes in the presence of different cryoprotectants. Cryobiology 2005;50:285–293.
32.
Zhang T, Liu XH, Rawson DM. Effects of methanol and developmental arrest on chilling injury in zebrafish (Danio rerio) embryos. Theriogenology 2003;59:1545–1556.
33.
Desai K: Studies on the Effect of Chilling on Sox Genes and Protein Expression in Zebrafish (Danio rerio) embryos. University of Bedfordshire, Luton, United Kingdom, 2012.
34.
Ahammad MM, Bhattacharyya D, Jana BB. Effect of different concentrations of cryoprotectant and extender on the hatching of Indian major carp embryos (Labeo rohita, Catla catla, and Cirrhinus mrigala) stored at low temperature. Cryobiology 1998;37:318–324.
35.
Crowe JH, Crowe LM, Carpenter JF, Aurell Wistrom C. Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem J 1987;242:1–10.
36.
Rudolph AS, Crowe JH. Membrane stabilization during freezing: the role of two natural cryoprotectants, trehalose and proline. Cryobiology 1985;22:367–377.
37.
Beattie GM, Crowe JH, Lopez AD, Cirulli V, Ricordi C, Hayek A. Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage. Diabetes 1997;46:519–523.
38.
Morris GJ, Watson PF. Cold shock injury—a comprehensive bibliography. Cryoletters 1984;5:352–372.
39.
Mazur P, Schneider U, Mahowald AP. Characteristics and kinetics of subzero chilling injury in Drosophila embryos. Cryobiology 1992;29:39–68.
40.
Behneke O, Forer A. Evidence for four classes of microtubules in individual cells. J Cell Sci 1967;2:169–192.
41.
Weber K, Pollack R, Bibring T. Antibody against tuberlin: the specific visualization of cytoplasmic microtubules in tissue culture cells. Proc Natl Acad Sci U S A1975;72:459–463.
42.
Magistrini M, Szollosi D. Effects of cold and of isopropyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur J Cell Biol 1980;22:699–707.
43.
Martino A, Pollard JW, Nakagawa A, Leibo SP. The kinetics of chilling sensitivity of bovine oocytes cooled to non-physiological temperatures. Theriogenology 1995;43:272.
44.
Smith GD, Ane Silva E, Silva C. Developmental consequences of cryopreservation of mammalian oocytes and embryos. Reprod BioMed Online 2004;9:171–178.
45.
Kamachi Y, Uchikawa M, Kondoh H. Pairing SOX off: with partners in the regulation of embryonic development. Trends Genet 2000;16:182–187.
46.
Wilson M, Koopman P. Matching SOX. Partner proteins and co-factors of the SOX family of transcriptional regulators. Curr Opin Genet Dev 2002;12:441–446.
47.
Okuda Y, Ogura E, Kondoh H, Kamachi Y. B1 SOX coordinate cell specification with patterning and morphogenesis in the early zebrafish embryo. PLoS Genet 2010;6:e1000936.
48.
Nageshan RK, Roy N, Hehl AB, Tatu U. Post-transcriptional repair of a split heat shock protein 90 gene by mRNA trans-splicing. J Biol Chem 2011;286:7116–7122.

Information & Authors

Information

Published In

cover image Zebrafish
Zebrafish
Volume 12Issue Number 1February 2015
Pages: 111 - 120
PubMed: 25545702

History

Published in print: February 2015
Published online: 20 January 2015
Published ahead of print: 29 December 2014

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Authors

Affiliations

Kunjan Desai
Department of Neuroscience and Regenerative Medicine, Georgia Regents University, Augusta, Georgia.
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
Emma Spikings
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
Tiantian Zhang
Institute of Biomedical Science and Environmental Science and Technology, University of Bedfordshire, Luton, United Kingdom.
School of Applied Sciences, Bournemouth University, Poole, United Kingdom.

Notes

Address correspondence to:Tiantian Zhang, PhDSchool of Applied SciencesBournemouth UniversityPoole BH12 5BBUnited Kingdom
E-mail: [email protected]

Author's Contributions

K.D. performed all the experiments and wrote the first draft of the article. E.S., contributed to data analysis and interpretation, codesigned the experiments, helped with the qPCR experiments, and the article preparation and revision. T.Z., participated in experimental design and article preparation. All authors approved the current version of the article.

Disclosure Statement

No competing financial interests exist.

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