Influence of recipient cytoplasm cell stage on transcription in bovine nucleus transfer embryos.

Abstract

Nucleus transfer for the production of multiple embryos derived from a donor embryo relies upon the reprogramming of the donor nucleus so that it behaves similar to a zygotic nucleus. One indication of nucleus reprogramming is the RNA synthetic activity. In normal bovine embryogenesis, the embryo relies upon maternally derived RNA transcripts up to the 8-cell stage, at which time it begins to transcribe its own RNA. In this experiment, RNA synthesis was detected in nucleus transfer embryos (NTE) and control embryos by pulsing with 3H-uridine, fixation, and autoradiography on semithin sections. NTE were produced using either a MII phase (nonactivated) cytoplasts at 32 hr of maturation or S-phase (activated) cytoplasts activated with calcium ionophore A23187 and cycloheximide treatment approximately 8 hr prior to fusion with a blastomere from an in-vitro-produced morula stage embryo at 32 hr of maturation. Control in-vitro-produced embryos were 3H-uridine-labelled and fixed at the 2-, 4-, early 8-, and late 8-cell stages. NTE were similarly prepared at 1, 3, and 20 hr postfusion and at the 2-, 4-, and 8-cell stages. In the control embryos, RNA synthesis was absent in the 2-, 4-, and early 8-cell stages, whereas in all late 8-cell stages, it was present. In NTE from nonactivated (MII phase) cytoplasts, there was a sharp decline in RNA synthesis at 1 hr and 3 hr after fusion and a total absence by 20 hr after fusion. In contrast, NTE from activated (S phase) cytoplasts exhibited continued high levels of RNA synthesis at 1 hr and moderate levels at 3 hr after fusion, although it had ceased by 20 hr after fusion. In all NTE (activated and nonactivated cytoplasts), there was no RNA synthesis seen at the 2-cell stage. However, at the 4-cell stage, weak RNA synthesis was seen in all NTE from activated cytoplasts, whereas none was observed in those from MII nonactivated cytoplasts. At the 8-cell stage, nearly all NTE from S-phase cytoplasts showed weak to moderate levels of RNA synthesis. We conclude that the nucleus reprogramming differs between NTE reconstructed from activated and nonactivated cytoplast with the former undergoing a slower cessation of RNA synthesis after fusion and earlier resumption of RNA synthesis, occurring as early as the 4-cell stage.