Configurations of germinal vesicle (GV) chromatin in the goat differ from those of other species

Configuration of germinal vesicle (GV) chromatin has been studied and found correlated with the developmental competence of oocytes in several mammalian species. A common feature in the configuration of GV chromatin in the species studied so far is that the diffuse chromatin (the so called "NSN" pattern) condenses into a perinucleolar ring (the so called "SN" configuration) with follicular growth. However, no study has been published on the configuration of GV chromatin in the goat. Nor is it known whether the perinucleolar ring of condensed chromatin (CC) in an oocyte represents a step toward final maturation or atresia. Changes in configurations of GV chromatin and RNA synthesis during goat oocyte growth, atresia and maturation in vivo and in vitro were investigated in this study. Based on both the size of nucleoli and the degree of chromatin condensation, the GV chromatin of goat oocytes was classified into GV1 characterized by large nucleoli and diffuse chromatin, GV2 with medium-sized nucleoli and condensed net-like (GV2n) or clumped (GV2c) chromatin, GV3 with small nucleoli and net-like (GV3n) or clumped (GV3c) chromatin, and GV4 with no nucleolus but clumped chromatin. The results showed that (i) the configurations of GV chromatin in the goat differ from those of other species in that the chromatin did not condense into a perinucleolar ring; (ii) most of the goat oocytes are synchronized at the GV3n configuration before GVBD; (iii) the GVn pattern might represent a healthy state, but the GVc an atretic state; (iv) in both goats and mice, the GC-specific (Chromomycin A3, CMA3) and the AT-specific (Hoechst 33342) fluorochromes followed the same pattern of distribution in GV chromatin; (v) the nucleolar size decreased significantly with oocyte growth and maturation in vivo and in vitro; and (vi) goat oocytes began GVBD at 8 hr and had completed it by 20 hr after onset of estrus. The peculiar configuration of GV chromatin of goat oocytes can be a useful model for studies of morphological and functional changes of different nuclear compartments during the cell cycle and cell differentiation, and the functional differentiation between GV3n and GV3c might be used for reference to the question whether the "SN" configuration in other species inclines toward ovulation or atresia.     

Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification

In the present study, we examined a novel lipid removal method, centrifugation in solutions made hypertonic by adding 0.27 M sugar. This allowed the lipid to be extruded and removed without the loss of active mitochondria or extra cytoplasm. The type of sugar influenced the proportion of oocytes that could be stratified by centrifugation. Glucose induced the highest extrusion rate of lipid droplets. After vitrification the rates of survival, germinal vesicle breakdown and metaphase II were 30, 26, and 7%, respectively, for lipid-removed GV oocytes; this was significantly higher (P<0.05) than for corresponding vitrified lipid-intact oocytes (2, 0, and 0%, respectively). These results indicated that this method is useful to remove whole lipid droplets without losing mitochondria and improves cryotolerance of porcine GV oocytes.     

Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.     

粘虫核型多角体病毒和颗粒体病毒侵染粘虫前胸腺的病理观察

观察比较了粘虫核型多角体病毒(Pseudelatia separata NPV,PsNPV)、粘虫颗粒体病毒(Pseudelatia separata GV,PsGV)感染粘虫,以及两种病毒混合感染粘虫后,粘虫(Pseudelatia separata)前胸腺的形态特征和前胸腺细胞的超微结构。结果表明,不同感染组粘虫前胸腺腺体都有不同程度的组织病变,PsNPV感染组在感染晚期与前胸腺相连的气管严重病变,出现大量白色颗粒状物累积,被伊红染成紫黑色,腺体细胞被挤压变形;PsGV感染组的前胸腺腺体变小,单个细胞也小,细胞界限不十分明显;两种病毒混合感染组的腺体细胞小,能被伊红染色的细胞极少。同时,前胸腺细胞的超微结构也有不同程度的病变。     

Presence of N-methyl-D-aspartate-like and kainate-like activities in bovine brain

Searching for the natural ligands interacting with brain excitatory amino acid receptors, we have isolated from cow brain a low-Mr ampholyte fraction containing molecules with excitatory properties similar to those of N-methyl-D-aspartate and kainate, which cannot be accounted for by any of the known brain excitants. This finding supports the hypothesis of the existence of excitatory neurotransmitters other than L-glutamate and L-aspartate.     

Structural divergence among genomes of closely related baculoviruses and its implications for baculovirus evolution

Baculoviruses are members of a large, well-characterized family of dsDNA viruses that have been identified from insects of the orders Lepidoptera, Hymenoptera, and Diptera. Baculovirus genomes from different virus species generally exhibit a considerable degree of structural diversity. However, some sequenced baculovirus genomes from closely related viruses are structurally very similar and share overall nucleotide sequence identities in excess of 95%. This review focuses on the comparative analysis of partial and complete nucleotide sequences from two groups of closely related baculoviruses with broad host ranges: (a) group I multiple nucleopolyhedroviruses (MNPVs) from a cluster including Autographa californica (Ac)MNPV, Rachiplusia ou (Ro)MNPV, and Plutella xylostella (Plxy)MNPV; and (b) granuloviruses (GVs) from a cluster including Xestia c-nigrum (Xecn)GV and Helicoverpa armigera (Hear)GV. Even though the individual viruses in these clusters share high nucleotide sequence identities, a significant degree of genomic rearrangement (in the form of insertions, deletions, and homologous recombination resulting in allelic replacement) is evident from alignments of their genomes. These observations suggest an important role for recombination in the early evolution and biological characteristics of baculoviruses of these two groups.     

Spectrum of variations in dog-1/FANCJ and mdf-1/MAD1 defective Caenorhabditis elegans strains after long-term propagation

Background

Whole and partial chromosome losses or gains and structural chromosome changes are hallmarks of human tumors. Guanine-rich DNA, which has a potential to form a G-quadruplex (G₄) structure, is particularly vulnerable to changes. In Caenorhabditis elegans, faithful transmission of G-rich DNA is ensured by the DOG-1/FANCJ deadbox helicase.

Results

To identify a spectrum of mutations, after long-term propagation, we combined whole genome sequencing (WGS) and oligonucleotide array Comparative Genomic Hybridization (oaCGH) analysis of a C. elegans strain that was propagated, in the absence of DOG-1 and MDF-1/MAD1, for a total of 470 generations, with samples taken for long term storage (by freezing) in generations 170 and 270. We compared the genomes of F₁₇₀ and F₄₇₀ strains and identified 94 substitutions, 17 InDels, 3 duplications, and 139 deletions larger than 20 bp. These homozygous variants were predicted to impact 101 protein-coding genes. Phenotypic analysis of this strain revealed remarkable fitness recovery indicating that mutations, which have accumulated in the strain, are not only tolerated but also cooperate to achieve long-term population survival in the absence of DOG-1 and MDF-1. Furthermore, deletions larger than 20 bp were the only variants that frequently occurred in G-rich DNA. We showed that 126 of the possible 954 predicted monoG/C tracts, larger than 14 bp, were deleted in unc-46 mdf-1 such-4; dog-1 F₄₇₀ (JNC170).

Conclusions

Here, we identified variants that accumulated in C. elegans’ genome after long-term propagation in the absence of DOG-1 and MDF-1. We showed that DNA sequences, with G₄-forming potential, are vulnerable to deletion-formation in this genetic background.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1402-y) contains supplementary material, which is available to authorized users.     

How viruses access the nucleus

Many viruses depend on nuclear proteins for replication. Therefore, their viral genome must enter the nucleus of the host cell. In this review we briefly summarize the principles of nucleocytoplasmic transport, and then describe the diverse strategies used by viruses to deliver their genomes into the host nucleus. Some of the emerging mechanisms include: (1) nuclear entry during mitosis, when the nuclear envelope is disassembled, (2) viral genome release in the cytoplasm followed by entry of the genome through the nuclear pore complex (NPC), (3) capsid docking at the cytoplasmic side of the NPC, followed by genome release, (4) nuclear entry of intact capsids through the NPC, followed by genome release, and (5) nuclear entry via virus-induced disruption of the nuclear envelope. Which mechanism a particular virus uses depends on the size and structure of the virus, as well as the cellular cues used by the virus to trigger capsid disassembly and genome release. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.