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
The active ribosome-synthesizing nucleolus contains three main ultrastructural com- Ž
. ponents: the fibrillar components consisting of the fibrillar centres FCs , and the dense
Ž .
Ž . Ž
fibrillar component DFC , and the granular component GC for review see Wachtler
. and Stahl, 1993 . These components of the so-called fibrillo-granular nucleolus reflect
the steps in the biosynthesis of ribosomes according to the following model: the FCs house the enzymatic apparatus for the transcriptional process, the DFC carries the
primary unprocessed transcripts, while the GC represents processed transcripts associ- ated with proteins in the form of pre-ribosomal particles.
2.1. Cattle It is known that active fibrillo-granular nucleoli are established in cattle embryos
Ž during the fourth cell cycle under in vivo conditions Camous et al., 1986; King et al.,
. 1988; Kopecny et al., 1989 . In order to elucidate the chronology of this event in greater
detail, we analyzed in vitro produced cattle zygotes and embryos fixed at different Ž
. Ž
intervals during the one-cell stage first cell cycle , the two-cell stage second cell .
Ž .
Ž .
cycle , the four-cell stage third cell cycle , the eight-cell stage fourth cell cycle and Ž
. the 16-cell stage fifth cell cycle; Laurincik et al., 2000 by transmission electron
Ž .
microscopy TEM . The embryos were produced by in vitro oocyte maturation, in vitro fertilization and subsequent culture.
The structurally most prominent nuclear entities observed during the initial three cell Ž
. cycles were large electron-dense fibrillar spheres Fig. 1 . These spheres typically
developed one or several vacuoles with the progression of the cell cycles. A central primary vacuole developed first, followed by the formation of several peripheral
secondary vacuoles.
At the onset of the fourth cell cycle, the fibrillar spheres were again observed. With the progression of the cell cycle, the fibrillar spheres first displayed a central primary
vacuole and later several peripheral secondary vacuoles. Towards the end of the cell cycle, a DFC and tiny FCs developed in the peripheral zone of the vacuolated fibrillar
sphere. The FCs were closely associated with heterochromatin. We assume that this chromatin arrangement allows for the rRNA genes to be localized at the FCs. Addition-
ally, the formation of DFC at the inner lining of the large primary vacuole was noted.
Fig. 1. Schematic illustration of the ultrastructural aspects of nucleolus development in cattle embryos. During the first, second and third cell cycles, the nuclei display spherical fibrillar spheres that undergo vacuolization
Ž .
during the cell cycle. During the fourth cell cycle, the formation of dense fibrillar component DFC and Ž
. fibrillar centres FCs occurs in the periphery of the fibrillar spheres. DFC also forms at the rim of the central
vacuole. Later during the cell cycle, the substance of the sphere is gradually displaced by the granular Ž
. component GC . During the fifth cell cycle, a fibrillo-granular nucleolus emerges from the start.
Subsequently, the DFC and the FCs developed and a GC consisting of pre-ribosomal particles emerged and occupied the remaining portion of the former fibrillar sphere.
Thus, through this process a functional fibrillo-granular nucleolus had emerged. At the fifth cell cycle, fibrillo-granular nucleoli were formed already from the start of the cycle
indicating that the meiotically induced inactivation of the rRNA genes had ceased. In addition to the precise chronological mapping, this study established that the DFC and
the FCs were formed initially at the periphery of the fibrillar sphere, which is a new finding. Previously, this event was thought to occur at the rim of the primary vacuole
Ž .
inside the fibrillar sphere Kopecny et al., 1989 .
2.2. Swine It has been documented that swine embryos display the first signs of development of
FCs towards the end of the third cell cycle, i.e. one cell cycle earlier than in cattle Ž
. Tomanek et al., 1989 . In order to elucidate the chronology of this event in greater
detail, we analyzed in vivo developed swine zygotes and embryos by TEM. Following flushing from the oviducts, the zygotes and embryos were cultured into the subsequent
Ž cell cycle in vitro to be fixed at different intervals during the one-cell stage first cell
. Ž
. cycle; the zygotes were not cultured in vitro , the two-cell stage second cell cycle , the
Ž .
Ž .
four-cell stage third cell cycle , the eight-cell stage fourth cell cycle and the 16-cell Ž
. stage fifth cell cycle; Laurincik et al., 1996; Hyttel et al., unpublished data .
The structurally most prominent nuclear entities observed during the initial two cell cycles were electron-dense fibrillar spheres that were markedly larger than in cattle
Ž .
embryos Fig. 2 . In contrast to the observations in cattle, the spheres did not undergo vacuolization.
Fig. 2. Schematic illustration of the ultrastructural aspects of nucleolus development in swine embryos. During the first and second cell cycle, the nuclei display spherical fibrillar spheres. During the third cell cycle, dense
Ž .
Ž .
Ž .
fibrillar component DFC , fibrillar centres FCs and granular component GC form on the surface of the sphere. During the fourth and fifth cell cycle, a fibrillo-granular nucleolus emerges from the start.
At the onset of the third cell cycle, the fibrillar spheres were again observed. Around the midpoint of the third cell cycle, the nuclei of the blastomeres displayed different
stages of nucleolus formation, ranging from the inactive fibrillar spheres to fibrillo-gran- ular nucleoli presenting a DFC, FCs and a GC. Different stages of nucleolus develop-
ment were observed within the same nucleus, i.e. fibrillar spheres and fibrillo-granular nucleoli were found side by side. Apparently, nucleolus formation was initiated by the
formation of the DFC and GC and later the FCs on the surface of, but not in, the fibrillar spheres. Throughout this process, semilunar fibrillo-granular nucleoli more or less
encapsulated the fibrillar sphere. At the fifth cell cycle, fibrillo-granular nucleoli, as described for the previous cycle were already formed at the start of the cycle, indicating
that the meiotically induced inactivation of the rRNA genes had ceased. Again, the fibrillo-granular nucleoli were found together with inactive fibrillar spheres.
2.3. ComparatiÕe aspects In cattle and swine embryos, fibrillo-granular ribosome-synthesizing nucleoli become
structurally recognizable towards the end of the fourth and third cell cycle, respectively. In cattle, the formation of DFC and FCs occurs in the periphery of pre-existing nucleolar
anlages appearing as fibrillar spheres, whereas in the swine the DFC and the FCs appear on the surface of these anlages instead.
3. Molecular aspects of rRNA gene activation
