³H‐uridine incorporation in early porcine embryos

Abstract

The present study investigated the ontogeny of 3H-uridine incorporation into RNA as a measure for RNA synthesis in preimplantation porcine embryos from the two-cell stage up to the stage of the newly hatched blastocyst. A total of 568 embryos were cultured in vitro for 3 hr in medium (KRB plus lamb serum) containing 9 microM 3H-uridine. After disruption of cell membranes, RNA was isolated on DEAE cellulose filters, and the radioactivity was taken as a measure for the rate of RNA synthesis. No RNA synthesis was detected at the two-cell stage. From the four-cell to the morula stage, 3H-uridine incorporation per embryo increased about ninefold (P less than 0.001); in blastocyst stages, the increase between developmental stages was not statistically significant. Hatched blastocysts had the highest genomic activity. On a per cell basis, 3H-uridine incorporation was not different from the four-cell stage up to the zona pellucida-intact blastocyst and amounted to 0.29-0.37 fmol 3H-uridine incorporation/cell/3 hr. In hatched blastocysts, 3H-uridine incorporation per blastomere was increased (P less than 0.01 compared with younger stages) and amounted to 0.86 fmol 3H-uridine incorporation/cell/3 hr. It is concluded that 1) the rate of uridine incorporation depends on the cell stage in zona pellucida-intact porcine embryos and 2) uridine incorporation per blastomere is significantly increased in hatched blastocysts compared with earlier stages.

Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

“…This confirms the data on low-grade transcription during the first three cell cycles in bovine embryos (Barnes & First 1991, Plante et al 1994, Hyttel et al 1996, Viuff et al 1996, Memili & First 1998, Kues et al 2008) and extends our knowledge about porcine embryos that were considered transcriptionally inactive until late in the third cell cycle, i.e. at the four-cell stage (Tomanek et al 1989, Freitag et al 1991, Jarrell et al 1991. However, during further IVC, iSCNT embryos did not show the important increase in RNA Pol II activity observed in control embryos, which went through normal EGA.…”

“…Since the efficiency of NT (Boiani et al 2005, Lagutina et al 2006) and even more of iSCNT , Zhao et al 2006, Sugimura et al 2009) embryo development crucially depends on embryo culture conditions, we tested both SOFaa and CR1aa, which have been previously reported to support bovine (Galli et al 2003) and porcine (Lagutina et al 2006) embryo development and pig-bovine iSCNT blastocyst development (Dominko et al 1999, Uhm et al 2007 respectively. No difference was observed, and pig-bovine/ bovine-pig iSCNT embryos were not able to escape the block at the 16-25-cell/four-cell stage, which corresponds to the time of bovine MET (8-16-cell stage; Kanka 2003) and porcine MET (four-cell stage; Tomanek et al 1989, Freitag et al 1991, Jarrell et al 1991. The reasons of such developmental failure in our culture conditions are not clear, but it should not be ascribed to the quality of our in vitro maturation-in vitro culture (IVC) media or to the NT technique used, since several offspring were born following bovine and porcine NT.…”

Lamins A and C are present in the nuclei of early porcine embryos, with lamin A being distributed in large intranuclear foci

“…Thus it is possible that the lamin A foci we observed are involved in sites that will be used in transcription in the four-cell embryo, as this is when embryonic genome transcription is first initiated in the pig (Tomanek et al 1989, Freitag et al 1991, Jarrell et al 1991, Schoenbeck et al 1992, Hyttel et al 2000, Viuff et al 2002. It is also possible that the lamin foci are involved in the first rounds of embryonic replication.…”

Differences in Trophectoderm Mitotic Rate and P450 17α-Hydroxylase Expression between Late Preimplantation Meishan and Yorkshire Conceptuses1

“…We have previously shown that there are no obvious differences between Meishan and Yorkshire females in the time from onset of estrus to ovulation in our herds [16,28]. These studies [16,28] also demonstrated no difference in stage (i.e., cell numbers) between Meishan and Yorkshire oviductal conceptuses collected 48-54 h after first exhibition of estrus, a stage when conceptuses are still relying on maternal mRNA for their programming [29]. Once the conceptuses reach the 4-cell stage, the embryonic genome is turned on [29], and the conceptuses enter the uterus [30].…”

“…These studies [16,28] also demonstrated no difference in stage (i.e., cell numbers) between Meishan and Yorkshire oviductal conceptuses collected 48-54 h after first exhibition of estrus, a stage when conceptuses are still relying on maternal mRNA for their programming [29]. Once the conceptuses reach the 4-cell stage, the embryonic genome is turned on [29], and the conceptuses enter the uterus [30]. Although the conceptuses of both Meishan and Yorkshire breeds reach the blastocyst stage synchronously 3 to 4 days later, Meishan blastocysts contain markedly fewer trophectoderm cells than do Yorkshire blastocysts when compared at the same number of inner cell mass cells [18], indicating that even early in the preimplantation period Meishan trophectoderm cells are mitosing at a reduced rate.…”