RESUMEN
In this study 874 mouse embryos were allocated to six groups including a control, co-culture, and four groups that underwent partial zona dissection (PZD): at the 2-cell (PZD-2) and morula stages (PZD-M) both with and without co-culture. Rates of complete blastocyst hatching on day 5 increased in the following order: control, co-culture alone, PZD-2 alone, PZD-M alone, PZD-2 with co-culture and PZD-M with co-culture (P < 0.00001). PZD-M led to significantly higher rates of complete blastocyst hatching compared to PZD-2 (P < 0.03). This study showed also that co-culture apparently compensates for any minor damage incurred during the PZD technique at the 2-cell and morula stages, (P < 0.01 and P < 0.01) respectively. Therefore PZD and co-culture seem mutually beneficial techniques that promote early blastocyst hatching in the mouse.
Asunto(s)
Blastocisto/fisiología , Micromanipulación , Zona Pelúcida , Animales , Técnicas de Cultivo , Desarrollo Embrionario y Fetal/fisiología , Femenino , Ratones , Ratones EndogámicosRESUMEN
The development of 1-cell mouse embryos in explanted oviducts, on mouse and bovine oviduct epithelial cells and on two established cell line supports is compared. The best rates of blastocyst formation were obtained using explanted oviducts; mouse and to a lesser extent, bovine oviduct epithelial cells allow good embryonic development, associated with high viability after transfer of the blastocysts obtained in co-culture. MDBK (from bovine kidney) and Vero (from Green monkey kidney) have been tested. MDBK allows high rates of blastocyst formation (67%) and the blastocysts obtained are viable. Vero does not allow the 2-cell block to be overcome. Maintenance of cell polarity for all the feeder layers did not improve embryo development. A preliminary study on the metabolic modifications induced by the feeder layers showed no modifications at all related to a decrease in glucose, an increase in lactate and early embryonic development. On the other hand, for the free amino acids, cellular supports with high embryotrophic activity seem to mimic tubal secretions, especially with a high level of glycine. Neither a genital tract origin, nor a hormonal contribution are strictly necessary for embryo co-culture, as already demonstrated by co-culture with trophoblastic tissue. Established cell lines, which are easy to handle and control, could be useful tools in embryo biotechnology.
Asunto(s)
Blastocisto/citología , Aminoácidos/análisis , Animales , Blastocisto/metabolismo , Bovinos , Línea Celular , Células Cultivadas , Células Epiteliales , Trompas Uterinas/citología , Femenino , Viabilidad Fetal/fisiología , Glucosa/metabolismo , Glicina/biosíntesis , Inmunohistoquímica , Lactatos/metabolismo , Metionina/metabolismo , Ratones , Control de CalidadRESUMEN
In early embryos, methylation is involved in "gamete imprinting" and inactivation of artificially introduced foreign genes. We studied the biosynthesis of the universal methylation cofactor: S-Adenosyl methionine (SAM). In the mouse, SAM conversion from methionine is limited by saturation of the methionine endogenous pool. SAM is present at a practically unchanged level from the unfertilized oocyte to early morula. SAM synthesis is increased at the time of compaction. In blastocysts, although methionine uptake is increased, the conversion rate from methionine is lowered. We observed no differences between C57 Black and Swiss albino random bred strains. In few experiments with human unfertilized oocytes and spared embryos, we observed higher methionine incorporation, and higher conversion to SAM. Next, the effect of two methylation inhibitors was tested, on early mouse embryonic development, at the one-cell and the two-cell stage. We found that ethionine is very toxic, even at the lowest tested concentration of 25 microM. Homocysteine is more potent at the one-cell stage than at the 2-cell stage, and it only partially blocks blastocyst formation from the 2-cell stage even at a concentration of 500 microM. It clearly acts as a methylation inhibitor; it lowers the SAM pool and the methylation index, SAH/SAM ratio (SAH: S-Adenosyl Homocysteine). We also found that homocysteine is an unexpected competitor for methionine influx and efflux.