This proposal addresses cryobiological mechanisms that will assist in developing effective cryopreservation procedures for the germ plasm of teleost fish. Although freezing teleost spermatozoa is commonly practiced, the successful cryopreservation of teleost eggs or embryos has not been achieved. Teleost eggs are large, yolk-filled and usually damaged by conventional freezing approaches. Systematic examination of the response of teleost embryos to low temperatures and the use of a novel cryopreservation approach (vitrification) may overcome these problems. The major cryobiological factors involved in vitrification are: 1) the use of a concentrated solution of cryoprotectants in which embryos are equilibrated and dehydrated before cooling; and 2) rapid cooling of the embryo suspension leading to the formation of a transparent, glassy solid. Vitrification provides a simple, rapid cryopreservation method and has yielded improved embryo survival in some species. The zebrafish (Brachydanio rerio) is being used as a model for our basic studies of cryobiology and vitrification of teleost embryos. Our basic research consists of two components. First, we will study the cryobiological properties of intact embryos.
Specific aims will include developing a fundamental understanding of the: l) permeability of embryos to water and cryoprotectants; and 2) the toxicity of cryoprotectants to embryos. Water and cryoprotectant permeabilities will be quantified using magnetic resonance microimaging. We will measure the relative intensities of pixels within the embryo versus the external medium using diffusion- weighted coefficient gradients and chemical-shift microimaging. From these measurements, vitrification solutions and protocols will be designed and cryopreservation trials conducted. The second component concerns studying transplantation and in vivo maturation on of immature teleost oocytes. The inherent cellular and physiological properties of intact embryos may make cryopreservation impossible; for this reason, cryopreserving previtellogenic (immature) oocytes will be considered. This approach would require the development of methods for in vivo maturation (i.e., growth, absorption of vitellogenin and ovulation) after cryopreservation. Specific studies include: l) correlating the developmental onset of vitellogenin receptors with oocyte size; 2) measuring the uptake of labeled vitellogenin in vivo; 3) determining whether host-rejection exists in zebrafish; and 4) directly testing in vivo maturation by removing immature oocytes from pigmented donor females and transplanting them into the ovaries of host albino (a recessive mutant) females. When crossed with an albino male, the presence of pigmented offspring would indicate the successful in vivo maturation of the donated oocytes. Since the follicle of the teleost oocyte is critical for the absorption of the oocyte, these cells will be transplanted together in an oocyte/follicle complex. Vitrification of teleost embryos will permit storing a diverse gene pool, maintaining valuable transgenic lines and hybrids, preserving genetic diversity and assisting in preventing extinctions of fish species in natural aquatic ecosystems.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
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Reproductive Biology Study Section (REB)
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Smithsonian Institution
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Hagedorn, M; Peterson, A; Mazur, P et al. (2004) High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option. Cryobiology 49:181-9
Hagedorn, M; Lance, S L; Fonseca, D M et al. (2002) Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation. Biol Reprod 67:961-6
Janik, M; Kleinhans, F W; Hagedorn, M (2000) Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio). Cryobiology 41:25-34