Nuclear transplantation of ectodermal cells in pig oocytes: Ultrastructure and radiography

Abstract

This study investigated the changes in nucleolar fine structure and the synthesis of both heterogeneous nuclear RNA (hnRNA) and ribosomal RNA (rRNA) in pig reconstructed embryos obtained by electrofusion of a single pig ectodermal cell to an enucleated metaphase‐II oocyte. The nucleolar morphological changes and the pattern of transcription were examined in the ectodermal cells before fusion and in the nuclear transfer reconstructed embryos 16–18 hr after fusion. Before fusion the ectodermal cells exhibited a reticulated nucleolus with active RNA synthesis. In the reconstructed embryos, modifications of the nucleolar structure were observed, as assessed by the presence of either round‐shaped, compact, dense nucleolar precursor bodies, or reticulated nucleoli. However, in both cases there was no RNA synthesis. Blebbing activity of the nuclear envelope was not observed. These results indicate that the nucleus of pig ectodermal cells exhibited either a complete or an incomplete remodelling when transferred to an enucleated metaphase‐II oocyte, with no detectable RNA synthesis. Cell‐cycle synchronization of ectodermal cell donor nuclei may play an important role in nuclear reprogramming after fusion. © 1996 Wiley‐Liss, Inc.

Nuclear Remodeling and Reprogramming in Transgenic Pig Production

“…In contrast, a cell undergoing active rRNA synthesis at and beyond the four-cell stage in the pig has nucleoli with a reticulated or vacuolated appearance and a very rough or granular periphery. When nuclei with nucleoli that have an "active" nucleolar morphology are transferred to an enucleated oocyte and subsequently activated, the nucleolar morphology is reversed to be similar to the nucleoli in pronuclei (i.e., compact, agranular, and without reticulations or vacuoles) (24,25). Such observations indirectly indicate that rRNA synthesis has ceased.…”

Essential roles of the nucleolus during early embryonic development: a regulatory hub for chromatin organization

“…In theory, following SCNT, the reconstructed embryos need to undergo proper nuclear architecture reorganization (including nucleolar remodelling) to ensure the establishment of totipotency [86][87][88]. However, in early cloned embryos, somatic cell-like nucleoli frequently appear in pseudo-pronuclei, without the typical NPBs that appear in early fertilized embryos, and this may correspond to Dux silencing [20,21]. Moreover, cloned embryos exhibit multiple nucleoli maintenance in pseudo-pronuclei that is caused by an insufficient volume of the nucleolus, while in fertilized embryos, a few small nucleoli are fused into a single NPB [89].…”

“…In particular, ZGA is incomplete, and two-cell-specific genes are not properly activated in cloned embryos [ 16 – 19 ]. Concomitant with these defects, incomplete NPB architecture remodelling also exists in cloned embryos [ 20 , 21 ]. We propose that NPBs may be associated with the establishment of totipotency and ZGA by organizing chromatin around the nucleolar periphery.…”

Parthenogenetic Activation of Porcine Oocytes After Nuclear Transfer

“…Electroporation was used to activate reconstructed embryos in other studies as well where it stimulated (although at fairly low rates) development to the blastocyst stage (Terlouw, 1993;Nagashima et al, 1992Nagashima et al, , 1997. When ectodermal cells were fused with enucleated oocytes using three DC pulses, extensive remodeling of the nucleus was observed in about 50% of the cases (Ouhibi et al, 1996). Electrical activation was also reported to induce activation and subsequent cleavage of nuclear transfer embryos that were produced by transferring primordial germ cells into enucleated oocytes (Liu et al, 1995).…”