Birth of rats following nuclear exchange at the 2-cell stage

Abstract

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

Reprogramming after Chromosome Transfer into Mouse Blastomeres

“…Reprogramming and embryonic development can occur in animals after transfer of somatic nuclei into oocytes and zygotes in metaphase of the cell cycle, but fails after transfer during interphase [6–8]. Nuclear transfer into embryonic blastomeres enucleated in interphase has also been attempted, but failed to demonstrated reprogramming activities [9, 10]. In particular, no development was observed after transfer of inner cell mass nuclei into 2-cell stage embryos enucleated in interphase [10].…”

Mouse cloning and somatic cell reprogramming using electrofused blastomeres

“…These failures most likely are due to the tetraploid nature of the reconstructed embryos [21]. The full-term developmental potential of blastomeres has been demonstrated in mouse [22][23][24] or rat [25]; however, whether the electrofusion of blastomeres affects the full-term development is still not clear. Here, we have shown that if the polyploidy of these embryos is rectified, full-term development can be recovered, even though the transcripts present in the cytoplast of blastomeres are different from zygotes [26].…”

A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

“…In the present study, we compared the developmental abilities of rat oocytes activated by four different treatments (ethanol, electrical pulses, strontium and ionomycin), which have been reported as popular agents for oocyte activation in other species (Cuthbertson et al ., 1983; Ito et al ., 2004; Méo et al ., 2005; Jellerette et al ., 2006). We also examined the developmental abilities of oocytes collected from two different outbred types of rat, SD and Wistar, that have been widely used in studies on reproductive technologies (Roh et al ., 2003; Shinozawa et al ., 2004; Hirabayashi et al ., 2005; Ross et al ., 2006). The oocytes collected from SD rats showed a significantly higher developmental ability than those collected from Wistar rats independent of activation treatment.…”

“…Moreover, ethanol treatment is also well known as a popular regent that induces oocyte activation in mice (Cuthbertson et al ., 1983), cattle (Nagai et al ., 1987) and pigs (Yamauchi et al ., 1996). It is also known that, after the use of these stimuli, additional treatment with 6-dimethylaminopurine (6-DMAP) (Méo et al ., 2005), cycloheximide (Yamanaka et al ., 2007) or puromycin (Roh et al ., 2003) improves the development of parthenogenetic or nuclear-transferred embryos in vitro .…”