title: Expression of the T85A mutant of zebrafish aquaporin 3b improves post-thaw survival of cryopreserved early mammalian embryos.
abstract: While vitrification has become the method of choice for preservation of human oocytes and embryos, cryopreservation of complex tissues and of large yolk-containing cells, remains largely unsuccessful. One critical step in such instances is appropriate permeation while avoiding potentially toxic concentrations of cryoprotectants. Permeation of water and small non-charged solutes, such as those used as cryoprotectants, occurs largely through membrane channel proteins termed aquaporins (AQPs). Substitution of a Thr by an Ala residue in the pore-forming motif of the zebrafish (Dario rerio) Aqp3b paralog resulted in a mutant (DrAqp3b-T85A) that when expressed in Xenopus or porcine oocytes increased their permeability to ethylene glycol at pH 7.5 and 8.5. The main objective of this study was to test whether ectopic expression of DrAqp3b-T85A also conferred higher resistance to cryoinjury. For this, DrAqp3b-T85A + eGFP (reporter) cRNA, or eGFP cRNA alone, was microinjected into in vivo fertilized 1-cell mouse zygotes. Following culture to the 2-cell stage, appropriate membrane expression of DrAqp3b-T85A was confirmed by immunofluorescence microscopy using a primary specific antibody directed against the C-terminus of DrAqp3b. Microinjected 2-cell embryos were then cryopreserved using a fast-freezing rate and low concentration (1.5 M) of ethylene glycol in order to highlight any benefits from DrAqp3b-T85A expression. Notably, post-thaw survival rates were higher (P<0.05) for T85A-eGFP-injected than for -uninjected or eGFP-injected embryos (73±7.3 vs. 28±7.3 or 14±6.7, respectively). We propose that ectopic expression of mutant AQPs may provide an avenue to improve cryopreservation results of large cells and tissues in which current vitrification protocols yield low survival.
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Sylvia J
authors.initials: SJ
authors.lastname: Bedford-Guaus
authors.affiliation: Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Institut de Ciències del Mar,Consejo Superior de Investigaciones Científicas (CSIC),Barcelona,Spain.
authors.forename: François
authors.initials: F
authors.lastname: Chauvigné
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Eva
authors.initials: E
authors.lastname: Mejía-Ramírez
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Mercè
authors.initials: M
authors.lastname: Martí
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Antoni
authors.initials: A
authors.lastname: Ventura-Rubio
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Raya
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Joan
authors.initials: J
authors.lastname: Cerdà
authors.affiliation: Centre de Medicina Regenerativa de Barcelona (CMRB),Barcelona,Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Veiga
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D030801:Animals, Genetically Modified; D051400:Aquaporin 3 / Q000235:genetics* / Q000378:metabolism; D001757:Blastomeres; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008297:Male; D008815:Mice, Inbred Strains; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D034421:Sus scrofa; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D015053:Zygote / Q000502:physiology*
chemical_list: D003451:Cryoprotective Agents; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D051400:Aquaporin 3; D019855:Ethylene Glycol
keywords: Aquaporins; Cryopreservation; Embryo; Ethylene glycol; Mouse