The first cell cycle after transfer of somatic cell nuclei in a non-human primate

Abstract

Production of genetically identical non-human primates through somatic cell nuclear transfer (SCNT) can provide diseased genotypes for research and clarify embryonic stem cell potentials. Understanding the cellular and molecular changes in SCNT is crucial to its success. Thus the changes in the first cell cycle of reconstructed zygotes after nuclear transfer (NT) of somatic cells in the Long-tailed Macaque (Macaca fascicularis) were studied. Embryos were reconstructed by injecting cumulus and fibroblasts from M. fascicularis and M. silenus, into enucleated M. fascicularis oocytes. A spindle of unduplicated premature condensed chromosome (PCC spindle) from the donor somatic cell was formed at 2 hours after NT. Following activation, the chromosomes segregated and moved towards the two PCC spindle poles, then formed two nuclei. Twenty-four hours after activation, the first cell division occurred. A schematic of the first cell cycle changes following injection of a somatic cell into an enucleated oocyte is proposed. Ninety-three reconstructed embryos were transferred into 31 recipients, resulting in 7 pregnancies that were confirmed by ultrasound;unfortunately none progressed beyond 60 days.

Dynamic Changes in Microtubules and Early Development of Reconstructed Embryos after Somatic Cell Nuclear Transfer in a Non-Human Primate

“…It was reported that primates were different from other animals, as disarrayed abnormal mitotic spindles with misaligned chromosomes were formed in all SCNT embryos (Simerly et al, 2003). However, in our earlier report, 14.8% of SCNT embryos formed normal spindle and clinical pregnancies were achieved in cynomolgus monkey after SCNT embryos were transferred into the recipients (Ng et al, 2004). There is little information available on the timing of chromatin condensation and dynamic changes in microtubules after SCNT in non-human primate.…”

“…Procedures for manipulating monkey oocytes have been described (Ng et al, 2004). Briefly, a very small opening on zona pellucida was made by acidic Tyrode solution (pH ϭ 1.8), followed by aspiration of the meiotic spindle with small volume cytoplasm (Ͻ2% of oocyte) under polarized microscopy (SpindleView; CRI, Woburn, MA).…”

“…LoTM fresh cumulus and starved adult skin fibroblast cells were used as donor cells for nuclear transfer. Donor cell preparation has been described previously (Ng et al, 2004). Single donor cell collected from a 10% PVP droplet was ruptured by repeated gentle aspiration and expulsion out of the injection needle, then directly microinjected into the enucleated oocyte.…”

“…After nuclear transfer, when a stimulus for activation is applied, MPF levels in oocytes decrease, resulting in chromosome decondensation and formation of pronuclear-like structures (Choi et al, 2004)-single or multiple (Ng et al, 2004). But as to why different numbers of pronuclei were formed is not clear and the relationship between the number of pronuclei formed after nuclear transfer and the developmental potential is not clear (Tani et al, 2001).…”

Transgenic Nonhuman Primate Models for Human Diseases: Approaches and Contributing Factors

“…However, this method, when applied to NHPs, has not yet produced any live monkeys. This failure may be related to their complicated genome reprogramming process (Ng et al, 2004;Zhou et al, 2006;.…”