Impairments in Embryonic Genome Activation in Rhesus Monkey Somatic Cell Nuclear Transfer Embryos

Abstract

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%) was markedly (p < 0.05) impaired compared with those derived from in vitro fertilization (IVF) (42.1%) in rhesus monkey. Also, SCNT embryos were abnormal in their time course of embryonic development. SCNT produced embryos reached the eight-cell stage faster than did IVF produced embryos. We compare the transcription patterns of five nucleolar-related proteins-nucleolin, nucleophosmin, fibrillarin, PAF53, and UBF-in single IVF and SCNT blastocysts by RT-PCR. The SCNT embryos showed abnormal gene transcription. Immunolocalization of fibrillarin was undetectable in 8-cell and 16-cell SCNT embryos, indicating embryonic genomic activation was delayed in monkey embryos produced by SCNT compared to their IVF-derived counterparts. Some of SCNT embryos appeared to relative higher developmental potential and fibrillarin expression by prolonged exposure of incoming nuclei to a cytoplasm. Thus, our data show that SCNT embryos are characterized by abnormal cleavage and the timely onset of embryonic genome transcription, deficits that may explain their reduced pre- and postimplantation developmental capacity.

DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer

“…46 This phenomenon may be a consequence of aberrant function of DNMT1, 47,48 and/or impaired embryonic genome activation. 49 On the other hand, the level of normalized DNA demethylation in at least a certain proportion of SCNT embryos was comparable to that seen in IV embryos, as reported previously. 37,38 From the eight-cell to the blastocyst stage, normalized DNA methylation levels continuously increased in IV, PA and partially preserve its methylation pattern even though the structural remodeling of the nucleus to a pronucleus-like structure has already occurred.…”

Importance of the pluripotency factor LIN28 in the mammalian nucleolus during early embryonic development

“…As functional nucleoli are assembled when the embryonic genome has been activated (Kopecny, 1989; Svarcova et al, 2009), the transformation of NPBs to mature nucleoli also reflects the transition from the maternal to embryonic control. In contrast to mice, EGA in non-human primate embryos occurs between the 8- and 16-cell stages (Niu et al, 2008). It is therefore noteworthy that LIN28 appeared at nucleoli of marmoset embryos following EGA, whereas it was still enriched in the nucleoplasm at the 11-cell stage.…”

Transgenic rhesus monkeys produced by gene transfer into early-cleavage–stage embryos using a simian immunodeficiency virus-based vector

“…In the rhesus monkey, embryonic genome activation takes place at the six-to eight-cell stage (8,26). We thus infected preimplantation embryos at the one-or two-cell stage and at the four-to eight-cell stage to compare the rates of gene transfer and of development to the blastocyst stage when proviral integration occurs before or during embryonic genome activation.…”

“…The efficacy of IVF methods is critically dependent on the protocols used for ovarian stimulation, in vitro embryo culture, embryo transfer, and uterine-embryo synchrony. During the past decade, we and others have developed and improved these protocols in the rhesus monkey (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Recently, IVF and intracytoplasmic sperm injection also were achieved in the marmoset (19) and in the cynomolgus monkey (20).…”