Ultrastructural localization of silver-staining nuclear proteins at the onset of transcription in early bovine embryos.

Argyrophilic nuclear proteins, known to be functionally associated with ribosomal genes, were localized, in four-, eight-, and 16-cell bovine embryo blastomere nuclei using two different silver-staining procedures. Within the eight-cell cleavage stage by the process of embryonal nucleologenesis in the cow embryo the full-capacity ribosome-producing machinery is established. In the four-cell embryo, many patches and islands of argyrophilic (Ag+) material were detected in the nucleoplasm. The nucleolus-precursor bodies (NPBs), composed uniformly of a homogeneous compact mass, were completely devoid of any silver staining. On the other hand, clear-cut localization of argyrophilic proteins was detected during the eight-cell stage either inside the transforming NPBs or in the close vicinity, or in the already differentiated nucleolus. In compact, nonvacuolated NPB, an intensive Ag+ area was detected, in the form of a lenticle, at the periphery of the NPB. During and following vacuolation of the NPB, no Ag+ was detected inside these vacuoles. It was seen, however, in the dense fibrillar nucleolar component surrounding the smaller vacuoles formed at the time of the establishment of nucleolar structure. Ag+ areas were seen repeatedly in the vicinity of NPBs, probably a part of the nucleolus-associated chromatin or, alternatively, representing the extranucleolar bodies. In blastomere nuclei of 16-cell embryos, already possessing reticulated nucleoli known from intensively synthesizing somatic cells, the silver-staining pattern corresponded to the usual situation in differentiated cells: slight staining of fibrillar centers, heavy labelling in the dense fibrillar component, and absence of silver deposits in the granular component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Architectural reorganization of the nuclei upon transfer into oocytes accompanies genome reprogramming

The ability of cloned embryos to sustain full-term development depends on the ability of the recipient ooplasm to reprogram the donor cell genome. As the nuclear architecture has recently emerged as a key-factor in the regulation of gene expression, we questioned whether early embryos obtained from transfer of ES metaphasic chromosomes into mouse ooplasm would adopt the somatic or embryonic type of nuclear organization. We have particularly focused on the arrangement of chromosomal territories with respect to the nucleolar compartment, and the pericentric heterochromatin domains called chromocenters. We found that nuclear transfer triggers profound chromatin rearrangements including the dispersion of the donor cell chromocenters components. These rearrangements lead to a typical 1-cell pronuclear organization, namely a radial arrangement of the chromosome territories with centromeres attached to the nucleoli, which adopt the compact fibrillar structure of nucleolar precursor bodies (NPBs). Subsequently, during the second cycle, the cloned embryos undergo further reorganization with the establishment of new chromocenters, clustered in one part of the nucleus, as during normal embryogenesis. We could also establish that the adequate distribution of chromosomal territories at the pronuclear stage seems important for the development until blastocyst.     

Nucleolar changes in bovine nucleotransferred embryos.

This study focused on nucleolar changes in bovine embryos reconstructed from enucleated mature oocytes fused with blastomeres of morulae or with cultured, serum unstarved bovine fetal skin fibroblasts (embryonic vs. somatic cloning). The nucleotransferred (NT) embryos were collected and fixed at time intervals of 1-2 h (early 1-cell stage), 10-15 h (late 1-cell stage), 22-24 h (2-cell stage), 37-38 h (4-cell stage), 40-41 h (early 8-cell stage), 47-48 h (late 8-cell stage), and 55 h (16-cell stage) after fusion. Immunocytochemistry by light and electron microscopy was used for structure-function characterization of nucleolar components. Antibodies against RNA, protein B23, protein C23, and fibrillarin were applied. In addition, DNA was localized by the terminal deoxynucleotidyl transferase (TdT) technique, and the functional organization of chromatin was determined with the nick-translation immunogold approach. The results show that fully reticulated (active) nucleoli observed in donor cells immediately before fusion as well as in the early 1-cell stage after fusion were progressively transformed into nucleolar bodies displaying decreasing numbers of vacuoles from the 2- to 4-cell stage in both types of reconstructed embryos. At the late 8-cell stage, morphological signs of resuming nucleolar activity were detected. Numerous new small vacuoles appeared, and chromatin blocks reassociated with the nucleolar body. During this period, nick-translation technique revealed numerous active DNA sites in the periphery of chromatin blocks associated with the nucleolar body. Fully reticulated nucleoli were again observed as early as the 16-cell stage of embryonic cloned embryos. In comparison, the embryos obtained by fetal cloning displayed a lower tendency to develop, mainly during the first cell cycle and during the period of presumed reactivation. Correlatively, the changes in nucleolar morphology (desegregation and rebuilding) were at least delayed in many somatic NT embryos in comparison with the embryonic NT group. It is concluded that complete reprogramming of rRNA gene expression is part of the general nuclear reprogramming necessary for development after NT.     

Activation of nucleolar and extranucleolar RNA synthesis and changes in the ribosomal content of human embryos developing in vitro

RNA synthetic activity of human 2-16-cell embryos developing in vitro was studied by [3H]uridine light-microscope autoradiography. Parallelly cut thin sections were examined in the electron microscope. The first extranucleolar RNA synthesis was detected in 4-cell embryos, but nucleoli were never labelled until the 3rd cleavage (6-8-cell embryos). In 6-cell embryos the nucleolar labelling was mostly confined to a narrow peripheral zone. In later cleavage stages most of the blastomeres showed intensive labelling of nucleoli and extranucleolar chromatin. However, rather low levels of extranucleolar RNA synthesis and the absence of nucleolar activity were often seen even in blastomeres of fully compacted morulae. The activation of nucleolar RNA synthesis entailed a noticeable increase in the number of ribosomes (estimated by electron microscope morphometry) that followed a marked drop during the period between the 2-cell and 8-cell stages. The results indicate that the concentration of ribosomes in the preovulatory oocyte is a major factor of its developmental potential.