Localization of ribosomal RNA (rRNA), rRNA genes and silver staining nucleolar proteins in in vitro produced bovine embryos

1998 ◽  
Vol 49 (1) ◽  
pp. 190
Author(s):  
D. Viuff ◽  
B. Avery ◽  
G. Vajta ◽  
T. Greve ◽  
H. Callesen ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Silver Staining ◽  
Rrna Genes ◽  
Bovine Embryos ◽  
Nucleolar Proteins

scholarly journals 212 HETEROGENEITY OF RIBOSOMAL RNA GENE ACTIVATION AMONG CELLS OF IN VITRO-PRODUCED PORCINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 256
Author(s):  
B. Bjerregaard ◽  
F. Strejcek ◽  
Z. Rasmussen ◽  
J. Laurincik ◽  
H. Niemann ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Silver Staining ◽  
Transcriptional Activity ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Cell Stage ◽  
Nucleolar Proteins

In vitro production (IVP) of porcine embryos by in vitro maturation of oocytes followed by fertilization and culture in vitro is hampered by great deficiencies. Initiation of at least the major embryonic genome transcription, which includes activation of ribosomal RNA (rRNA) genes and the associated formation of a fibrillo-granular nuclealus, is normally seen during the 4-cell stage in pigs. We have investigated the activation of rRNA synthesis and the presence of silver staining nucleolar proteins in porcine IVP embryos as a marker of transcriptional activity and, thus, developmental competence. A total of 205 porcine IVP embryos from the 2-cell to the blastocyst stage were examined using sequential fluorescent in situ hybridization (FISH) to the rRNA genes and their transcripts and silver staining of nucleolar proteins as previously described (Viuff et al. 2002 Biol. Reprod. 66, 629–634). Briefly, cumulus-oocyte complexes with at least three cumulus cell layers and evenly granulated ooplasm were isolated from 2–5 mm ovarian follicles with stereomicroscopic evaluation. Subsequently, oocytes were matured in NCSU-37 and mechanically denuded followed by fertilization using frozen-thawed epididymal semen. Presumptive zygotes were then cultured in NCSU-23 at 39°C, 5% CO2. Around the time of expected cleavage, the embryos were examined every second hour to determine the time of cleavage. Embryos at the 2-cell stage were harvested at 5 h post-cleavage (hpc), 4-cell embryos late during the third cell cycle at 30 hpc, and tentative 8- and 16-cell embryos at 10 hpc. Blastocysts were harvested at Day 5 post-insemination. In general, nuclei of 2-cell embryos displayed 4 small foci of FITC labelling (presumably the rDNA), but no specific silver staining, and were consequently categorized as transcriptionally inactive. At the late 4-cell stage, 58% of the embryos resembled the 2-cell stage. However, in the remaining embryos (42%), some or all nuclei displayed large areas of FISH labelling (presumptive rDNA and rRNA) co-localized with silver staining, and were catagorized as transcriptionally active. Among the 8-cell embryos, 64% displayed a majority of transcriptionally active nuclei, whereas this was the case in 83% and 92% of the embryos in the 16-cell embryos and the blastocysts, respectively. In general, the majority of the embryos contained a mixture of transcriptionally active and inactive cells. These findings show that the porcine IVP embryos are often delayed and asynchronous with respect to activation of the rRNA genes. Table 1. Categorization of nuclei according to transcriptional activity This work was supported by grants from “Disease models, disease prevention and animal welfare improvement: The pig embryo as a model.” Danish Research Agency (Grant: 9901178), NATO (Grant: 978658), and Deutsche Forschungsgemeinschaft (DFG).

scholarly journals UBF levels determine the number of active ribosomal RNA genes in mammals

2008 ◽  
Vol 183 (7) ◽  
pp. 1259-1274 ◽  
Author(s):  
Elaine Sanij ◽  
Gretchen Poortinga ◽  
Kerith Sharkey ◽  
Sandy Hung ◽  
Timothy P. Holloway ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
High Mobility ◽  
Rrna Genes ◽  
Rrna Synthesis ◽  
Rrna Gene ◽  
Extracellular Signal ◽  
Binding Factor ◽  
Upstream Binding Factor ◽  
Polymerase I

In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

Localization of the rRNA genes in in vitro produced bovine embryos

1997 ◽  
Vol 47 (1) ◽  
pp. 218 ◽  
Author(s):  
D. Viuff ◽  
B. Avery ◽  
B. Høyheim ◽  
P. Hyttel ◽  
P.D. Thomsen ◽  
...  
Keyword(s):  
Rrna Genes ◽  
Bovine Embryos

Activation of ribosomal RNA genes in pre-implantation In Vitro-produced and nuclear transfer bovine embryos

2000 ◽  
Vol 35 (6) ◽  
pp. 255-259 ◽  
Author(s):  
J Laurincik ◽  
V Zakhartchenko ◽  
B Avery ◽  
M Stojkovic ◽  
G Brem ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Nuclear Transfer ◽  
Ribosomal Rna Genes ◽  
Bovine Embryos ◽  
Rna Genes

scholarly journals Ribosomal RNA gene expression and chromosome aberrations in bovine oocytes and preimplantation embryos

Reproduction ◽  
2001 ◽  
pp. 21-30 ◽  
Author(s):  
P Hyttel ◽  
D Viuff ◽  
T Fair ◽  
J Laurincik ◽  
PD Thomsen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Aberrations ◽  
Rrna Genes ◽  
Preimplantation Embryos ◽  
Fertilization In Vitro ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Oocyte Growth

This review focuses on the key features of development of the bovine oocyte and embryo, with comparisons of the developmental characteristics of embryos produced in vivo and in vitro. The oocyte is transcriptionally quiescent in the primordial and primary follicle. In the secondary follicle transcription is initiated in the oocyte and a ribosome-synthesizing nucleolus is established in this cell. Transcription and nucleolar activity are enhanced in the tertiary follicle during oocyte growth. When the oocyte reaches approximately 110 microm in diameter, corresponding to a follicle of about 3 mm in diameter, transcription ceases and the nucleolus is inactivated, forming a dense spherical remnant. During the final phase of follicular dominance this remnant becomes vacuolated and, in conjunction with resumption of meiosis, it disperses. The rRNA genes are apparently re-activated during the four-cell stage, that is, the third cell cycle after fertilization, but a nucleolus is not formed. During the subsequent cell cycle, that is, during the eight-cell stage, ribosome-synthesizing nucleoli are again established. Bovine embryos produced in vitro apparently display the same pattern of nucleolus development as that in embryos developed in vivo. Examination of the ploidy of embryonic cells using fluorescence in situ hybridization has revealed that the production of bovine embryos in vitro is associated with increased chromosome aberrations in the embryos. Blastocysts produced in vitro display a significantly higher rate of mixoploidy, that is, when the embryo consists of both normal diploid and abnormal polyploid cells, than that in embryos developed in vivo. The rate of mixoploidy among embryos produced in vitro increases with increasing developmental stage. Moreover, after fertilization in vitro, initially there is a high rate of 'true' polyploidy, that is, when all cells of the embryos are polyploid. However, the polyploid embryos are eliminated before they cleave beyond the eight-cell stage, the stage at which major activation of the embryonic genome occurs in cattle.

scholarly journals Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

2019 ◽  
Vol 54 (10) ◽  
pp. 1357-1365
Author(s):  
Luiz Sergio Almeida Camargo ◽  
Fernanda Queiros Costa ◽  
Michele Munk ◽  
Sabine Wohlres‐Viana ◽  
Raquel Varela Serapião ◽  
...  
Keyword(s):  
Heat Shock ◽  
In Vitro Maturation ◽  
Bovine Embryos

Phylogeny of hymenolepidids (Cestoda: Cyclophyllidea) from mammals: sequences of 18S rRNA and COI genes confirm major clades revealed by the 28S rRNA analyses

2021 ◽  
Vol 95 ◽  
Author(s):  
B. Neov ◽  
G.P. Vasileva ◽  
G. Radoslavov ◽  
P. Hristov ◽  
D.T.J. Littlewood ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
18S Rrna ◽  
Phylogenetic Analyses ◽  
The Other ◽  
Rrna Genes ◽  
Host Switching ◽  
28S Rrna ◽  
Mitochondrial Cytochrome ◽  
Rapid Radiation ◽  
18S Rrna Genes

Abstract The aim of the study is to test a hypothesis for the phylogenetic relationships among mammalian hymenolepidid tapeworms, based on partial (D1–D3) nuclear 28S ribosomal RNA (rRNA) genes, by estimating new molecular phylogenies for the group based on partial mitochondrial cytochrome c oxidase I (COI) and nuclear 18S rRNA genes, as well as a combined analysis using all three genes. New sequences of COI and 18S rRNA genes were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analyses confirmed the major clades identified by Haukisalmi et al. (Zoologica Scripta 39: 631–641, 2010): Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade. While the Ditestolepis clade is associated with soricids, the structure of the other three clades suggests multiple evolutionary events of host switching between shrews and rodents. Two of the present analyses (18S rRNA and COI genes) show that the basal relationships of the four mammalian clades are branching at the same polytomy with several hymenolepidids from birds (both terrestrial and aquatic). This may indicate a rapid radiation of the group, with multiple events of colonizations of mammalian hosts by avian parasites.

scholarly journals Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Karina Cañón-Beltrán ◽  
Yulia N. Cajas ◽  
Serafín Peréz-Cerezales ◽  
Claudia L. V. Leal ◽  
Ekaitz Agirregoitia ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Mitochondrial Activity ◽  
Bovine Embryos ◽  
Akt Signaling Pathway ◽  
Cell Stage ◽  
Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

scholarly journals Characterization, Comparative Analysis and Phylogenetic Implications of Mitogenomes of Fulgoridae (Hemiptera: Fulgoromorpha)

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1185
Author(s):  
Wenqian Wang ◽  
Huan Zhang ◽  
Jérôme Constant ◽  
Charles R. Bartlett ◽  
Daozheng Qin
Keyword(s):  
Control Region ◽  
Ribosomal Rna ◽  
Phylogenetic Analyses ◽  
Start Codon ◽  
Rrna Genes ◽  
Rich Region ◽  
Protein Coding ◽  
Phylogenetic Implications ◽  
Rna Genes ◽  
Unpaired Base

The complete mitogenomes of nine fulgorid species were sequenced and annotated to explore their mitogenome diversity and the phylogenetics of Fulgoridae. All species are from China and belong to five genera: Dichoptera Spinola, 1839 (Dichoptera sp.); Neoalcathous Wang and Huang, 1989 (Neoalcathous huangshanana Wang and Huang, 1989); Limois Stål, 1863 (Limois sp.); Penthicodes Blanchard, 1840 (Penthicodes atomaria (Weber, 1801), Penthicodes caja (Walker, 1851), Penthicodes variegata (Guérin-Méneville, 1829)); Pyrops Spinola, 1839 (Pyrops clavatus (Westwood, 1839), Pyrops lathburii (Kirby, 1818), Pyrops spinolae (Westwood, 1842)). The nine mitogenomes were 15,803 to 16,510 bp in length with 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs) and a control region (A + T-rich region). Combined with previously reported fulgorid mitogenomes, all PCGs initiate with either the standard start codon of ATN or the nonstandard GTG. The TAA codon was used for termination more often than the TAG codon and the incomplete T codon. The nad1 and nad4 genes varied in length within the same genus. A high percentage of F residues were found in the nad4 and nad5 genes of all fulgorid mitogenomes. The DHU stem of trnV was absent in the mitogenomes of all fulgorids sequenced except Dichoptera sp. Moreover, in most fulgorid mitogenomes, the trnL2, trnR, and trnT genes had an unpaired base in the aminoacyl stem and trnS1 had an unpaired base in the anticodon stem. The similar tandem repeat regions of the control region were found in the same genus. Phylogenetic analyses were conducted based on 13 PCGs and two rRNA genes from 53 species of Fulgoroidea and seven outgroups. The Bayesian inference and maximum likelihood trees had a similar topological structure. The major results show that Fulgoroidea was divided into two groups: Delphacidae and ((Achilidae + (Lophopidae + (Issidae + (Flatidae + Ricaniidae)))) + Fulgoridae). Furthermore, the monophyly of Fulgoridae was robustly supported, and Aphaeninae was divided into Aphaenini and Pyropsini, which includes Neoalcathous, Pyrops, Datua Schmidt, 1911, and Saiva Distant, 1906. The genus Limois is recovered in the Aphaeninae, and the Limoisini needs further confirmation; Dichoptera sp. was the earliest branch in the Fulgoridae.

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