Nucleolar substructures of rabbit cleaving embryos: An immunocytochemical study

Abstract

The structure‐function relationships of the nucleolar substructures were studied in preimplantation rabbit embryos, where nucleologenesis is extending over the first four cell cycles and may not be synchronous in each blastomere. Immunocytochemical methods using light and electron microscopy were applied for protein and RNA localization as well as nick translation and terminal deoxynucleotidyl transferase techniques for DNA detection. DNA was gradually associated with the periphery of the compact nucleolar precursor bodies (NPBs) but was never found inside NPBs at the four‐cell stage. In 16‐cell embryos, some NPBs displayed a reticulated periphery forming the branching network of the dense fibrillar component (DFC) surrounding the “residual body” (remnant of NPB) in the process of activation. At the 32‐cell stage, fully reticulated nucleoli were observed in each blastomere. DNA was then associated with the DFC of reticulated nucleoli. RNA was first detected at the 16 cell‐stage in close contact with the DFC as well as inside the “residual body” which was not immunolabeled with the DNA antibodies used. When observed by light microscopy, fibrillarin, nucleolin, and protein B23 displayed a changing distribution pattern during nucleologenesis. At early stages (up to the 16‐cell stage), small dot‐ and spot‐like structures were distributed within the whole nuclei. In 16‐cell embryos, these proteins started to accumulate in an irregular thin layer around the NPBs in the process of activation. The reorganization process described may be in relation with the redistribution of chromatin and nuclear/nucleolar matrix components during the activation of rDNA transcription localized in the NPB shell. In conclusion, nucleologenesis is only achieved at the fourth cell cycle in the cleaving rabbit embryo at the corresponding time when the first detectable nucleolus‐associated RNA is detectable. Our results show a good correlation between the establishment of structure and function. Mol. Reprod. Dev. 48:34–44, 1997. © 1997 Wiley‐Liss, Inc.

Impairments in Embryonic Genome Activation in Rhesus Monkey Somatic Cell Nuclear Transfer Embryos

“…The onset of fibrillarin expression mirrored that of tritiated uridine incorporation into the nucleolus, first appearing in sixto eight-cell stage macaque embryos (Schramm and Bavister, 1999). It was also reported that fibrillarin acts as a genome activation marker in mice (Baran et al, 1995;Cuadros-Fernandez and Esponda, 1996), rabbits (Baran et al, 1997;PintoCorreia et al, 1995), and cows (Schram and Paprocki, 2000). In the present study, transcripts of fibrillarin, nucleolin, nucleophosmin, PAF53, and UBF were detected in all blastocysts.…”

Onset of Nucleolar and Extranucleolar Transcription and Expression of Fibrillarin in Macaque Embryos Developing In Vitro1

“…In no case was fibrillarin detected in 4-or 5-cell embryos. In similar studies in mice [43,44], rabbits [45,46], and cows (unpublished results), expression of fibrillarin at the periphery of the nucleus was initially detected at the species-specific time of genome activation, coincident with expression of nucleolin [43], protein B23 [47], and RNA polymerase I [44]. Following genome activation, the fluorescent pattern progressively changed into either a continuous layer of label in the cortex (mouse [43,44]) or into branch-shaped speckles in reticulated nucleoli (rabbit [45]; cow, unpublished results).…”

“…In the present study, fibrillarin was also localized to the perimeter of the nucleolar precursor bodies from the 6-to 8-cell through the morula stage, forming a ring that completely encapsulated these structures, before changing into a reticulated-like pattern at the blastocyst stage. Although the specific functions of fibrillarin in embryonic transcription have not been completely defined, its expression at the periphery of the nucleolar precursor body along with nucleolin, protein B23, and RNA polymerase I [43][44][45]47], which coincides with the onset of nucleolar transcription in mouse [43,44], cattle [47], and rabbit [46] embryos, suggests a role of this protein in rRNA gene transcriptional activity. Moreover, lymphocytes, which have transcriptionally inactive nucleoli, exhibit weak labeling with fibrillarin antibodies [48].…”

Three-dimensional analysis of nuclear heterochromatin distribution during early development in the rabbit

“…In rabbit, Baran et al (1997) identified by electron microscopy NPBs as round shape and compact structures from the 1- to 8-cell stages. At the 16-cell stage, some NPBs (called A-NPB) displayed a less compact structure associated with RNA detection but fully functional nucleoli were only detected at the morula and blastocyst stages (Baran et al 1997).…”