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
From a molecular perspective, the nucleolus consists of the rRNA genes and their transcripts associated with a long list of proteins that play different roles in rRNA
transcription and processing to the final pre-ribosomal particles. The location of several important nucleolar proteins is well established and their function is to some degree
Ž .
known for review see Wachtler and Stahl, 1993 . Likewise, the localization of the rRNA genes within the nucleolus has been established although the actual site of rRNA
transcription is still a controversial issue: some reports suggest that transcription occurs in the DFC, others that it takes place in the FCs, while a third and tempting model
Ž suggests that the process occurs at the interface between the DFC and the FCs Hozak et
. al., 1994 .
In order to elucidate the molecular composition of the nucleolus before and during Ž
the activation of the rRNA genes, we have analyzed in vitro produced cattle embryos as .
Ž .
for TEM, see above and in vivo developed swine embryos as for TEM, see above by immunocytochemistry and FISH. For immunocytochemistry, antibodies for six impor-
Ž .
tant nucleolar proteins see later were applied on whole mounts of embryos that were Ž
subsequently analyzed by confocal laser scanning microscopy Laurincik et al., 2000; .
Fair et al., unpublished data . For FISH, probes recognizing the rRNA genes and their transcripts were applied on RNase- or non-RNase-treated spread preparations of cattle
embryos that were subsequently processed for silver staining; a staining technique that
localizes certain nucleolar proteins characterized by silver affinity. The resultant speci- Ž
mens were analyzed by both bright field and fluorescence microscopy Viuff et al., .
1998 . The nucleolar proteins against which antibodies were applied were: Topoisomerase I,
Ž .
RNA polymerase I, upstream binding factor UBF , fibrillarin, nucleolin and nucle- ophosmin. The nucleolar proteins investigated in the present study may be divided into
two categories: proteins such as topoisomerase I, RNA polymerase I and UBF that are characterized by a well-defined action during transcription and processing, while others,
such as fibrillarin, nucleolin and nucleophosmin, are characterized by their spatial localization within the nucleolus. The putative role of each of these proteins in relation
to rRNA transcription and processing is addressed in the following paragraph.
As a prerequisite for transcription of the rRNA genes, the supercoiled DNA must be Ž
. uncoiled. This process is mediated by topoisomerase I Muller et al., 1985 which has
Ž .
also been localized to the DFC and FCs Wachtler and Stahl, 1993 . The actual
transcription of the rRNA genes is dependent on the activity of RNA polymerase I Ž
which also has been localized mainly to the FCs and to some degree to the DFC Raska .
et al., 1989 . The binding of RNA polymerase I to the DNA requires several transcrip- Ž
. tion factors, one of which is UBF Schnapp et al., 1994 . UBF is thought to bind to the
Ž promoter and to recruit another transcription factor, the promotor selectivity factor SL1,
. Maeda et al., 1992 , thus forming a pre-initiation complex to which RNA polymerase I
Ž .
can bind and initiate transcription Jordan et al., 1996 . The roles of the remaining three nucleolar proteins investigated is more putative and they are primarily characterized by
their nucleolar localization. Fibrillarin was first identified by human autoimmune sera Ž
from patients with scleroderma and was localized to the DFC and the FCs Ochs et al., .
Ž .
1985; Raska et al., 1989 . Fibrillarin is a small nucleolar ribonucleoprotein snoRNP Ž
associated with U3 small nucleolar RNA snoRNA; Lischwe et al., 1985; Lapeyere et .
Ž .
al., 1990 and with U8 and U13 snoRNAs Tyc and Steitz, 1989; Baserga et al., 1991 . SnoRNAs are believed to be involved in the processing of the primary rRNA transcript
Ž .
Jansen et al., 1991; Aris and Bloberg, 1991; Tollervey et al., 1991, 1993 . Nucleolin is a phosphorylated protein present in large amounts in nucleoli with active ribosomal
Ž .
Ž biogenesis Lapeyere et al., 1987 . It has RNA-binding properties Ghisolfi-Nieto et al.,
. 1996 and, furthermore, it is identical to the human DNA helicase IV, which unwinds
Ž .
RNA–RNA, DNA–DNA and DNA–RNA duplexes Teteja et al., 1995 . The protein is associated with the primary rRNA transcripts. Nucleolin may act in promoting the
functional secondary structure of the rRNA that is necessary for the assembly of the Ž
. pre-ribosomal particles Ghisolfi et al., 1990 . However, it is not part of the final product
Ž .
Hernandez-Verdun, 1991 . Nucleolin has been localized to the DFC and the GC Ž
. Biggiogera et al., 1990 . Nucleophosmin may be involved in shuttling other proteins,
Ž .
Ž .
such as nucleolin Li et al., 1996 and the nucleolar protein p120 Valdez et al., 1994 , into the nucleolus. However, nucleophosmin also has DNA and RNA binding properties
Ž .
Ž .
Wang et al., 1994 , ribonuclease activity Herrera et al., 1995 and associates with the Ž
. most mature nucleolar pre-ribosomal RNP Dumbar et al., 1989 . Therefore, it has been
proposed that nucleophosmin, together with nucleolin, functions in the assembly of pre-ribosomal particles. Nucleophosmin has been localized to the DFC and the GC
Ž .
Biggiogera et al., 1990 .
In the following, the immunocytochemical labelling of structural nuclear entities by antibodies directed against the above mentioned proteins is presented together with
observations on the localization of the rRNA genes and their transcripts in order to elucidate the gradual assembly of the nucleolus from a molecular point of view.
3.1. Cattle During the first cell cycle, topoisomerase I, RNA polymerase I, UBF, fibrillarin,
nucleolin and nucleophosmin may be localized to discrete foci in the pronuclei. We believe that the foci are identical with the fibrillar spheres observed by TEM. During the
second cell cycle, only fibrillarin and UBF could be localized to such foci, suggesting that the proteins observed during the first cycle were of maternal origin. During the third
cell cycle, fibrillarin and UBF displayed the same labelling pattern as during the previous cycle and, in addition, nucleolin and nucleophosmin were localized to foci or
spherical bodies presenting a central vacuole, i.e. appearing shell-like in the confocal sections.
During the fourth cell cycle, fibrillarin was initially localized to spherical bodies that later during the cell cycle developed a central vacuole. Towards the end of the cell
cycle, this protein was localized to more complex bodies showing several distinct intensely labelled foci embedded in a less intensely labelled matrix. During this cell
cycle, UBF was initially localized to small discrete foci which later during the cycle appeared to cluster. Towards the end of the cell cycle, UBF was localized to complexes
similar to those described for fibrillarin. Nucleolin and nucleophosmin were initially localized to small discrete foci that during the cell cycle developed a central vacuole.
Towards the end of the cell cycle, these entities developed into large shell-like bodies as the vacuole expanded in size. Topoisomerase I was detected for the first time around the
midpoint of the cell cycle. It was localized to discrete foci which appeared to cluster towards the end of the cycle. RNA polymerase I was detected for the first time towards
the end of the cycle in clusters of discrete foci. The gradual localization of this palette of proteins to more complex entities is compatible with the formation of fibrillo-granular
nucleoli within the fibrillar spheres as observed by TEM towards the end of the fourth cell cycle. In this context, topoisomerase I, UBF, RNA polymerase I and fibrillarin are
thought mainly to be confined to the fibrillar components and nucleolin and nucleophos- min to the GC. During the fifth cell cycle, the same labelling pattern as observed at the
end of the fourth was observed throughout the cell cycle.
The FISH analyses demonstrated that the rRNA genes were already associated with silver staining nucleolar proteins from at least the second cell cycle. During the fourth
cell cycle, however, they became co-localized with large silver deposits, substantiating their localization within the nucleoli developing during this cell cycle. Interestingly, the
non-RNase-treated embryos processed during the third cell cycle displayed a granular labelling pattern of the probe associated with the stronger labelling of the presumptive
rRNA genes. This pattern was not identified in the RNase-treated specimens. This feature signals that rRNA may be transcribed already during the third cell cycle before
nucleoli are structurally assembled, which is in accordance with results demonstrating a small but repeatable increase in the rRNA hybridization signal on Northern blots of
Ž RNA from two- to four-cell embryos as compared with zygotes Bilodeau-Goeseels and
. Schultz, 1997 . On the other hand, it is also possible that the phenomenon is caused by
the association of rRNA transcripts of maternal origin with the rRNA genes as has been Ž
. observed in the Xenopus Verheggen et al., 1998 .
In conclusion, the labelling patterns observed by immunocytochemistry and FISH are compatible with the formation of fibrillo-granular nucleoli towards the end of the fourth
cell cycle in which topoisomerase I, UBF, RNA polymerase I and fibrillarin are mainly confined to the fibrillar components and nucleolin and nucleophosmin to the GC, and in
which the rRNA genes are located.
3.2. Swine During the first cell cycle, labelling of nucleophosmin was localized to large
shell-like bodies. It is tempting to suggest that these bodies are identical with the electron-dense spheres observed by TEM. The remaining proteins were not localized to
nuclear entities. During the second and early third cell cycles, none of the proteins were localized to nuclear entities. This feature indicates that the nucleophosmin observed
during the first cell cycle is probably of maternal origin. However, towards the end of the third cell cycle, RNA polymerase I was localized to discrete foci arranged in a
shell-like pattern. None of the remaining proteins were localized to nuclear entities. This observation is in accordance with the observation by TEM of the first fibrillo-granular
nucleoli established as lentiform structures on the surface of the fibrillar spheres towards the end of this cell cycle. Early during the fourth cell cycle, labelling of topoisomerase I,
RNA polymerase I, UBF and fibrillarin were localized to small foci arranged in a shell-like pattern while labelling for nucleolin and nucleophosmin was localized to more
or less shell-like bodies. This labelling pattern is compatible with the presence of fibrillo-granular nucleoli as lentiform structures on the surface of the fibrillar sphere
with topoisomerase I, UBF, RNA polymerase I and fibrillarin mainly being confined to the fibrillar components and nucleolin and nucleophosmin to the GC. Similar observa-
tions were made during the fifth cell cycle.
In conclusion, the labelling patterns observed by immunocytochemistry are compati- ble with the formation of fibrillo-granular nucleoli towards the end of the third cell cycle
in which topoisomerase I, UBF, RNA polymerase I and fibrillarin are mainly confined to the fibrillar components and nucleolin and nucleophosmin to the GC.
3.3. ComparatiÕe aspects In both cattle and swine, nucleolar proteins become gradually localized to the
nucleolar anlage. In cattle, the process occurs over several cell cycles with a full compliment of the investigated nucleolar proteins being established towards the end of
the fourth cell cycle. In swine, the localization of the proteins to the anlage is more synchronous and occurs towards the end of the third and from the onset of the fourth
cell cycle when the full compliment of the investigated proteins is established.
4. Conclusions and perspectives
