The rRNA gene activation and the associated nucleolus formation may be used as a marker for the activation of the embryonic genome in mammalian embryos and, thus, serve to evaluate the
developmental potential of embryos originating from different embryo technological procedures. By this approach, we have demonstrated that in vitro produced porcine embryos display a lack of
localization of nucleolar proteins to the nucleolar anlage as compared with in vivo developed counterparts. Similarly, bovine embryos produced by nuclear transfer from morulae display such
deviations as compared with in vitro produced counterparts. Collectively, this information may help to explain the appearance of abnormalities seen in a certain proportion of offspring derived
from in vitro produced embryos and after cloning. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Embryo; Cattle; Swine; rRNA genes; Nucleolus; Gene activation
1. Introduction
The initial development of mammalian pre-implantation embryos is governed by gene transcripts and polypeptides produced by, and stored in, the oocyte during its develop-
Ž .
ment Schultz, 1993 . However, following one to three cleavage divisions, control of development is taken over by the expression of portions of the embryonic genome as the
Ž maternally derived transcripts and proteins are gradually degraded Telford et al., 1990;
. De Sousa et al., 1998; Watson et al., 1996, 1999 . This transition from maternal to
embryonic control of development is a gradual phenomenon. In the mouse and rabbit, it has been clearly demonstrated that a minor transcriptional activation occurring at the G2
stage of the first post-fertilization cell cycle precedes a major activation taking place at the G2 stage of the second cell cycle in mouse and at the fourth to fifth cell cycle in
Ž .
rabbit for review see Thompson, 1996 . Accordingly, using long-term incubation with
3
Ž .
H-uridine we have observed transcription during the first Hay-Schmidt et al., 1997 Ž
. and second cell cycle Plante et al., 1994; Hyttel et al., 1996; Viuff et al., 1996 in cattle,
a stage which is well before the major transcriptional activation which takes place Ž
. during the fourth cell cycle Camous et al., 1986 . In swine, the major transcriptional
Ž .
activation is seen during the fourth cell cycle Tomanek et al., 1989 , but whether this activation is preceded by an earlier minor one has not been investigated.
It is a common phenomenon that the major transcriptional activation includes Ž
. Ž
activation of the ribosomal RNA rRNA genes King et al., 1988; Kopecny et al., 1989; .
Tomanek et al., 1989 . The rRNA genes are located at the nucleolus organizer regions Ž
. NORs of the chromosomes, and the transcription of these genes is a prerequisite for
association with the formation of the nucleolus, which is the most prominent nuclear organelle and the site of formation of the ribosomal subunits. The subunits subsequently
leave the nucleus through the pores of the nuclear envelope and associate to form ribosomes in conjunction with translation of mRNAs. Thus, rRNA gene transcription is
a prerequisite for protein synthesis. The developing pre-implantation embryo has a profound need for synthesis of proteins for both housekeeping functions and cell
specialization. With this in mind, it is logical that the rRNA genes are found among the genes activated in the major cascade, and, moreover, it is clear that a proper activation
of these genes is crucial for continued embryonic development.
From the presented data, it is evident that the activation of the rRNA genes can be visualized ultrastructurally through the formation of the nucleolus in the blastomeres of
the embryo. However, the application of different cell biological techniques in embryo technology during the past few years has allowed for a more molecular understanding of
this process by the use of immunocytochemistry for the localization of specific proteins
Ž .
of importance for nucleolar function, and fluorescence in situ hybridization FISH for the localization of the rRNA genes and transcripts. On the following pages, the
ultrastructure and the molecular composition of the developing nucleolus in pre-implan- tation cattle and swine embryos will be discussed.
2. Ultrastructure of rRNA gene activation
