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.     

Determination of dorso-ventral axis in early embryos of the sea urchin, Hemicentrotus pulcherrimus

To elucidate a relationship between early cleavage planes and dorso-ventral (DV)-axis of sea urchin embryos, a fluorescent dye, Lucifer Yellow CH, was iontophoretically introduced into one blastomere at the 2-cell stage, and the location of the progeny cells was determined in the half-labeled prism larvae by examining the embryos from the animal pole. The boundary plane which divides the embryonic tissue into the labeled and nonlabeled parts was (1) coincident with, (2) perpendicular to, or (3) obliquely crossing the larval plane of bilateral symmetry. The oblique boundaries took only two angles mutually symmetrical with regard to the DV-axis of embryos. Combining these labeling patterns, the tissue of prism larvae could be divided into 8 sectors around the animal-vegetal axis. When the 2-cell stage embryos with different diameters of sister blastomeres were labeled with the dye, one end of the boundary plane was again found at one of the 8 boundary points noticed in equally cleaved embryos, while the other was observed to fall in the middle of a sector. These results indicate that the DV-axis of the embryo is established according to the spatial arrangement of blastomeres during the 5-6th cleavage stages when blastomeres align in 8 rows in meridional direction. It was also suggested that intercellular communication takes part in the determination of the fate of individual founder blastomeres during the two subsequent cleavages, i.e., 7-8th cleavage stages.     

Effect of serum on cell-to-cell associations during in vitro development of preimplantation mouse embryos

We have identified an activity which alters the morphology and developmental timing of post-compaction mouse embryos. A 15-min exposure of 4- and 8-cell mouse embryos to sera containing this activity induced monolayer formation, changing the normal positions of blastomeres at the 16- to 64-cell stages. Recovered embryos form normal blastocysts, based on morphology and in vitro production of trophectoderm and inner cell mass derivatives. These results suggest that under certain circumstances blastomeres remain developmentally labile as late as the sixth or seventh cleavage cycle.     

Nucleolar and mitochondrial morphology in bovine embryos reconstructed by nuclear transfer

The nucleolar and mitochondrial morphology of developing reconstructed bovine nuclear transfer (NT) embryos and stage-matched in vivo-produced control embryos were examined under the electron microscope. Each reconstructed embryo at the one-cell (n = 12), two-cell (n = 5), three-cell (n = 3), four-cell (n = 5), 5–8 cell (n = 5) and blastocyst (n = 3) stages was produced by fusion of a 16–32-cell-stage blatomere with an aged enucleated bovine oocyte. The normal and reconstructed embryos showed similar mitochondrial morphology. However, NT embryos produced several pleiomorphic forms not seen in controls, and were more heterogenous at early stages of development. Control embryos exhibited nucleolar features considered indicative of rRNA synthesis from the eight-cell stage onwards. In contrast, the NT embryos presented nucleoli with morphology consistent with rRNA synthesis in all embryos examined, except in the three-cell and in two of the five four-cell embryos. From this nucleolar morphology, it was concluded that nuclear reprogramming does not occur immediately following nuclear transfer, but occurs gradually over the first two or three cell cycles. © 1996 Wiley-Liss, Inc.     

Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos

Isolated blastomeres from 8- to 16-cell-stage embryos were fused by standard micromanipulatory means with either unfertilized eggs or fertilized or haploid parthenogenetically activated pronuclear-stage embryos. The hybrid eggs/embryos were incubated overnight in the presence of Colcemid until they had entered the first cleavage division. Air-dried chromosome preparations were then stained with silver nitrate in order to detect active nucleolar organizing regions (NOR). While control unfertilized eggs and 1-cell-stage fertilized and parthenogenetically activated embryos showed no evidence of silver-staining NOR-positive regions, the metaphase plates from 8- to 16-cell embryos showed characteristic NOR-positive regions, while their interphase nuclei also showed a characteristic reticular staining appearance. When hybrids between blastomere nuclei and unfertilized eggs were examined, none of the blastomere nuclei entered mitosis. However, when hybrids between blastomere nuclei and fertilized embryos were examined, in two thirds of the embryos, a single blastomere-derived diploid metaphase plate was present in association with two pronuclear-derived haploid metaphase plates. In most instances, the blastomere-derived chromosomes did not display silver-nitrate-staining NOR. Similar findings were observed when the blastomere-derived chromosomes in hybrids between blastomere nuclei and haploid parthenogenetic embryos were analysed. In the majority of cases, when blastomere nuclei remained in interphase, the characteristic silver-nitrate-staining fine reticular material either was not seen, or the nuclear contents were dispersed into clumps of chromatin-like material. Occasionally, the diploid chromosomes in the hybrids displayed morphological abnormalities. Our findings suggest that the cytoplasm of activated (but not nonactivated) 1-cell embryos is capable of influencing the nucleolar activity of the introduced 8- to 16-cell nuclei, effectively erasing from their chromosomes the memory of at least three previous rounds of rRNA synthesis.     

Differentiation of 2-cell and 8-cell mouse embryos arrested by cytoskeletal inhibitors

Mouse embryos at the 2-cell stage were cultured in the presence of cytochalasin B (CB), cytochalasin D (CD), colchicine (COL) or colcemid (COM) for up to 72 h. Cleavage was arrested in the 2-cell and 8-cell embryos cultured in CB or CD but the blastomeres continued to differentiate, since chromosome replication occurred in the blastomeres at approximately the same time as control embryos underwent cleavage; an increase in the incorporation of [³H]uridine into RNA was also detected. Furthermore, the cleavage-arrested embryos acquired the necessary information to undergo morphogenesis; these embryos when explanted to fresh medium after 48 h culture in CB or CD underwent compaction within 15–60 min and started to cavitate to produce trophoblastic vesicles within 5–6 h at the same time as when the control embryos were undergoing compaction and beginning to form blastocoelic cavities. In contrast, the embryos arrested in the presence of COM or COL showed none of these differentiative, biochemical or morphogenetic changes. Hence, differentiation of blastomeres and morphogenesis is apparently coupled with nuclear divisions and the information does not reside within the blastomeres at the 2-cell or 8-cell stage. The trophoblastic vesicles produced after cleavage arrest subsequently gave rise to only trophoblast giant cells and no embryonic derivatives were detected.