Nucleolar re‐activation is delayed in mouse embryos cloned from two different cell lines

Abstract

Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer of mouse embryonic fibroblast (MEF) and mouse HM1 embryonic stem cells (HM1), were processed for autoradiography following (3)H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF and nucleophosmin, B23). All early 2-cell embryos showed transcriptional activity only in nucleoplasm, not over nucleolar precursor bodies (NPBs). UBF was diffusely localized to cytoplasm and B23 to cytoplasm and nucleoplasm. Late 2-cell IVF and PG embryos displayed transcription over nucleoplasm and NPBs. Ultrastructurally, the latter were developing into functional nucleoli. NT-MEF and NT-HM1 embryos displayed transcription over nucleoplasm, but not over NPBs. Development of NPBs into nucleoli was lacking. UBF was in both groups localized to nucleoplasm or distinctly to presumptive NPBs. B23 was distinctly localized to NPBs. All 4-cell embryos presented nucleoplasmic transcription and developing fibrillo-granular nucleoli. UBF and B23 were distinctly localized to nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli were found in IVF and PG embryos, NT-MEF and -HM1 embryos displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both of these processes were delayed.

Oxamflatin Improves Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

“…In a previous experiment, HDAC2 showed a low level of expression at the four-cell stage, with a dramatic decrease from the two-cell stage and an increase reaching a maximum at the blastocyst in IVF and in vivo porcine embryos, whereas steady changes were observed in nuclear transfer embryos (Kumar et al, 2007). In addition, there are reports that embryonic activated genes are suppressed (Suzuki et al, 2006;Vassena et al, 2007) and nucleolar reactivation is delayed in SCNT-generated embryos (Svarcova et al, 2009). We observed that oxamflatin treatment enhances in vitro development competence and cloning efficiency along with decreased HDAC1 and HDAC2 expression at the four-cell stage.…”

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

“…Because LIN28 is deposited in the nucleoplasm of zygotes and early 2-cell embryos, we envision a translocation of the maternal protein from the nucleoplasm to the periphery of active NPBs at the late 2-cell stage. 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).…”

“…Blastocysts continue to expand, while differentiating into trophectoderm (TE) and inner cell mass (ICM). As functional nucleoli are only assembled when the embryonic genome has been activated (Kopecny, 1989; Svarcova et al, 2009), it is speculated that early protein synthesis uses maternally stored ribosomal proteins and RNAs. The primary function of the nucleolus involves ribosome-subunit biogenesis in eukaryotic cells.…”

rRNA Genes Are Not Fully Activated in Mouse Somatic Cell Nuclear Transfer Embryos

“…However, researchers demonstrated that mouse MEFNT embryos had poor nucleolus development at the 4-cell stage, as the fibrillo-granular structure only appeared at the surface of the nucleolus (18). This phenomena could be partially explained by our results that MEFNT embryos had the least active NORs and the functional nucleoli formation was based on the active NORs (4).…”

“…In pig, only half late 4-cell fibroblast NT embryos had transcriptionally active nucleoli, whereas in in vivo embryos the portion was 92% (17). Moreover, in mouse embryonic fibroblast or stem cellcloned embryos, the activation of functional nucleoli was also one cell cycle-delayed (18).…”