Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature

Renard, Jean‐Paul; Bui-Xuan-Nguyen,; Garnier, V.

doi:10.1530/jrf.0.0710573

Cited by 82 publications

(17 citation statements)

References 30 publications

(32 reference statements)

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“…Miyamoto & Ishibashi (1983) reported that mouse embryos survive freezing by direct transfer to subzero temperatures without seeding. The viability of directly frozen embryos depends on the degree of dehydration which can be controlled by the inclusion of sucrose in the freezing medium (Nguyen et al, 1983;Renard et al, 1984).…”

Section: Introductionmentioning

confidence: 99%

Osmotic and cryoprotective effects of glycerol--sucrose solutions on Day-3 mouse embryos

Széll¹,

Shelton²

1987

Reproduction

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Summary. The relative volume of Day-3 mouse embryos changed as a linear function of the reciprocal of osmolarity of non-permeating solutes after 10 min exposure to sucrose and glycerol\p=m-\sucrose solutions at 20\s=deg\C.The slope of the linear regression line was less in glycerol\p=m-\sucrose than in sucrose solutions because glycerol permeation caused re-expansion.Before freezing by direct transfer to \m=-\ 180\s=deg\Cthe embryos were placed into gl ycerol \x=req-\ sucrose in 1-step (1-step equilibration) or first into glycerol and then into gl ycerol \x=req-\ sucrose (2-step equilibration). Using 2-step equilibration the post-thaw survival rate was substantially higher at 3\m=.\0and 4\m=.\0 m-glycerol levels and less dependent on changes in the sucrose concentration within the range of 0\m=.\125 to 1\m=.\0 M than with 1-step equilibration. Under optimal conditions 90\p=n-\95%of rapidly frozen embryos developed to blastocysts in vitro and 30% into live young in vivo.It is suggested that the cryoprotective role of glycerol is due to its ability to reduce osmotic pressure differences between the extra and intracellular spaces during rapid freezing of embryos.

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Section: Introductionmentioning

confidence: 99%

Osmotic and cryoprotective effects of glycerol--sucrose solutions on Day-3 mouse embryos

Széll¹,

Shelton²

1987

Reproduction

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“…Rabbit embryos have been successfully cryopreserved by conventional freezing (Bank and Maurer, 1974;Renard et al, 1984;Kojima et al, 1987;Vicente and Garcia-Ximenez, 1994) or by vitri®cation (Smorag et al, 1989;Kasai et al, 1992;Gajda and Smorag, 1993;Vicente and Garcia-Ximenez, 1994;Smorag and Gajda, 1998). Dimethylsulfoxide (DMSO), propylene glycol (PG), glycerol, or ethylene glycol (EG), alone or as mixtures, have been used as cryoprotective additives (CPAs) for freezing or for vitri®cation of rabbit embryos at the 2-cell and morula-stages.…”

Section: Introductionmentioning

confidence: 99%

“…Of 176 morulae vitri®ed in EG alone or in EG DMSO, 40% developed into blastocysts (Vicente and Garcia-Ximenez, 1994); however, of 235 morulae vitri®ed in a mixture of EG Ficoll sucrose (EFS), 89% developed into blastocysts (Kasai et al, 1992). With 2-cell-stage rabbit embryos, in vitro survival has ranged from 58% developing to blastocysts for 101 embryos vitri®ed in EFS (Smorag and Gajda, 1998), to 89% developing into morulae for 54 embryos frozen in PG (Renard et al, 1984). In contrast, these former investigators also reported that only 18% of rabbit zygotes vitri®ed in the same EFS solution developed into blastocysts.…”

Section: Introductionmentioning

confidence: 99%

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

Hochi

Hirabayashi²,

Hirao³

et al. 2001

Molecular Reproduction Devel

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The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

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“…It could even induce vitrification of the cytoplasm upon cooling (Pruli&e et al 1987;Pruli&e and Douzou 1989). Therefore, a range of studies involving both in vivo and in vitro investigations was undertaken to examine the action of 1,2-propanediol, an efficient cryoprotectant for early mammal embryos (Vincent et al 1990;Renard et al 1984;Glenister et al 1990), on actin, which is a major cytoplasmic protein.…”

Section: Introductionmentioning

confidence: 99%

The effect of organic cryosolvents on actin structure: studies by small angle X-ray scattering

Pajot‐Augy

Axelos

1992

Eur Biophys J

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Small-angle X-ray scattering was used to probe the structure of actin in the presence of cryosolvents: 1,2-propanediol, glycerol, or a mixture of both solvents. In media devoid of polymerizing salts, a radius of gyration of 23 A is measured, as expected from the literature. In the presence of 1,2-propanediol alone, the scattering pattern begins to exhibit the characteristic slope of elongated objects with a non-negligible thickness, such as actin filaments polymerized in 40 mM KCl and 1 mM MgCl2. However, only short fragments (radius of gyration 40 A) are generated. We infer that in a medium of low ionic strength containing 15% 1,2-propanediol, actin assumes a structure closer to that of filamentous actin. 1,2-propanediol apparently induces nucleation of oligomers, as with polymerizing salts, but no propagation occurs. Glycerol and/or propanediol induce no alteration in the structure of individual salt-polymerized actin filaments. Aggregation occurs with propanediol, even in the presence of glycerol. Glycerol alone has no such effect. No shortening is detected within the scale covered, with either solvent, although 1,2-propanediol is known to shorten actin filaments. We suggest that in the absence of salts, 1,2-propanediol induces a conformational change in monomeric actin that is necessary for nucleation. This could correlate with a conformational change of actin promoters within microfilaments observed in the presence of 1,2-propanediol by other authors using different techniques.

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Two-step freezing of two-cell rabbit embryos after partial dehydration at room temperature

Abstract: Summary. The

Cited by 82 publications

References 30 publications

Osmotic and cryoprotective effects of glycerol--sucrose solutions on Day-3 mouse embryos

Osmotic and cryoprotective effects of glycerol--sucrose solutions on Day-3 mouse embryos

Effects of cryopreservation of pronuclear‐stage rabbit zygotes on the morphological survival, blastocyst formation, and full‐term development after DNA microinjection

The effect of organic cryosolvents on actin structure: studies by small angle X-ray scattering

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