Jean-Paul Renard

Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol

Eighty-five human embryos fertilized in vitro were frozen and thawed with 1,2 propanediol as a cryoprotective agent. The effects of viability of in vitro culture duration, stage, and morphologic appearance of embryos were examined after thawing. Survival was higher for 2-day-old embryos than for 3-day-old embryos (56% versus 18%) and for 2-, 4-, and 8-cell embryos than for intermediate-cleavage-stage embryos (67% versus 22%). Among 19 regular-cell-size embryos at the 2-, 4-, and 8-cell stage, 15 (79%) kept 50% or more of their initial number of blastomeres after thawing and 9 were intact. The average viability of all 2-day-old frozen-thawed embryos can be estimated at 19%.

Effects of cryoprotectants on actin filaments during the cryopreservation of one-cell rabbit embryos.

A dynamic equilibrium between globular and filamentous actin plays a crucial role in cell structure and motility. Many factors such as pH, ionic strength, temperature, and divalent cations, are known to influence this equilibrium. Some organic solvents, such as those used for the cryopreservation of cells, may also alter the dynamic equilibrium of this system. Fluorescence staining with NBD-phallacidin permits polymerized actin to be visualized in embryos and provides evidence that propanediol depolymerizes actin, whereas dimethyl sulfoxide does not. This depolymerizing effect is reversible after propanediol removal. Biochemical techniques were used to study the influence of these solvents on rabbit skeletal actin. Results obtained by sedimentation, fluorescence, DNase inhibition, electron microscopy, and viscometry analysis demonstrate that propanediol has a dual effect on actin polymers in vitro: it decreases the proportion of filamentous actin and the remaining filaments appear shorter and aggregate to form bundles. In contrast, dimethyl sulfoxide does not alter dramatically the actin polymer integrity. Propanediol is shown to exert a good cryoprotective action on rabbit embryos, while dimethyl sulfoxide does not. We suggest that the depolymerization of actin filaments by propanediol prior to cooling may facilitate the cryopreservation of one-cell rabbit embryos.

Dynamics of chromatin changes in live one-cell mouse embryos: A continuous follow-up by fluorescence microscopy

Conditions of minimal dye concentration and minimal irradiation which allow the continuous observation of pronuclei in live unicellular mouse eggs by fluorescence microscopy have been found with the use of Hoechst 33342 as fluorophore and a camera of high sensitivity coupled with an image processing system allowing true integration of weak fluorescent signals and further treatment and analysis. Under these conditions the developmental potential of the embryos is not affected. Using such an approach, which avoids eventual artifacts due to fixation procedure, we describe the changes in the nuclear organization and chromatin structure, from formation of pronuclei to mitosis, with particular attention to the chromatin associated with nucleoli and the timing process of chromatin condensation.

Participation of the paternal genome is not required before the eight-cell stage for full-term development of mouse embryos

Differential expression of the paternal and maternal genomes during mouse embryonic development is considered a reason for both genomes being required for development to term. Extending previous studies performed on two-cell embryos, we show here that diploid embryos reconstituted at the four-cell stage from uniparental haploid blastomeres can produce living offspring. This result shows that for normal development to occur, a paternal genome does not need to be associated with a maternal genome within the same nucleus before the eight-cell stage.

Genetic engineering in farm animals: The lessons from the genetic mouse model

Abstract The present paper takes into account the numerous observations now available from transgenic mice to examine the potential of the methods that allow the direct transfer of genes into farm animals. Tissue specific, or ubiquitous expression of foreign genes has already been obtained in mice with evidence of coordinated activation with endogeneous genes. These results show that organs such as liver or mammary gland or tissues such as exocrine pancreas or B lymphocytes involved in traits of zootechnical interest (food assimilation, milk production, immune response) can be appropriately stimulated with transgenes. Today however abnormal expression and transmission of the foreign genes, is occasionaly observed; in some cases the fertility of transgenic animals is severly lowered. These uncontrolled events together with the still low efficiency of the methods used to introduce the foreign DNA into the germ line constitute important barriers for the application of genetic engineering to farm animals.

Rheological changes in mammalian egg cytoskeleton in response to cold adaptation

The ability of mammalian eggs to support freezing is shown to be related to a reversible reorganization of their cytoskeleton induced by organic compounds used as protectants. 1,2 propanediol which is very effective for the successful freezing of early embryonic stages induces the formation of a fairly homogeneous gel within the cytoplasm in which the cortical layer of highly entangled polymers is no longer apparent. In vitro, propanediol influences specifically interactions between actin and binding proteins and induces the formation of microporous and homogeneous gels able to retain a significant fraction of water within their pores. When eggs are first submitted to a partial dehydration at room temperature they become able to support rapid freezing and thawing in the presence of propanediol probably because the remaining cellular water becomes osmotically inactive. This indicates that the rheological changes of the cytoplasm that occur in the presence of propanediol make the cell able to support the physical and chemical modifications that affect their microenvironment at low temperature.