Cytoplasmic maturation of bovine oocytes: Structural and biochemical modifications and acquisition of developmental competence

Oocyte maturation is a long process during which oocytes acquire their intrinsic ability to support the subsequent stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. This process involves complex and distinct, although linked, events of nuclear and cytoplasmic maturation. Nuclear maturation mainly involves chromosomal segregation, whereas cytoplasmic maturation involves organelle reorganization and storage of mRNAs, proteins and transcription factors that act in the overall maturation process, fertilization and early embryogenesis. Thus, for didactic purposes, we subdivided cytoplasmic maturation into: (1) organelle redistribution, (2) cytoskeleton dynamics, and (3) molecular maturation. Ultrastructural analysis has shown that mitochondria, ribosomes, endoplasmic reticulum, cortical granules and the Golgi complex assume different positions during the transition from the germinal vesicle stage to metaphase II. The cytoskeletal microfilaments and microtubules present in the cytoplasm promote these movements and act on chromosome segregation. Molecular maturation consists of transcription, storage and processing of maternal mRNA, which is stored in a stable, inactive form until translational recruitment. Polyadenylation is the main mechanism that initiates protein translation and consists of the addition of adenosine residues to the 3` terminal portion of mRNA. Cell cycle regulators, proteins, cytoplasmic maturation markers and components of the enzymatic antioxidant system are mainly transcribed during this stage. Thus, the objective of this review is to focus on the cytoplasmic maturation process by analyzing the modifications in this compartment during the acquisition of meiotic competence for development. (c) 2009 Elsevier Inc. All rights reserved.

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Towards an assessment of character interdependence in avian RNA phylogenetics: A general secondary structure model for the avian mitochondrial 16S rRNA

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ribosomal RNA gene insertions in the R2 site of Rhynchosciara (Diptera: Sciaridae)

Ribosomal RNA genes of most insects are interrupted by R1/R2 retrotransposons. The occurrence of R2 retrotransposons in sciarid genomes was studied by PCR and Southern blot hybridization in three Rhynchosciara species and in Trichosia pubescens. Amplification products with the expected size for non-truncated R2 elements were only obtained in Rhynchosciara americana. The rDNA in this species is located in the proximal end of the X mitotic chromosome but in the salivary gland is associated with all four polytene chromosomes. Approximately 50% of the salivary gland rDNA of most R. americana larval groups analysed had an insertion in the R2 site, while no evidence for the presence of R1 elements was found. In-situ hybridization results showed that rDNA repeat units containing R2 take part in the structure of the extrachromosomal rDNA. Also, rDNA resistance to Bal 31 digestion could be interpreted as evidence for nonlinear rDNA as part of the rDNA in the salivary gland. Insertions in the rDNA of three other sciarid species were not detected by Southern blot and in-situ hybridization, suggesting that rDNA retrotransposons are significantly under-represented in their genomes in comparison with R. americana. R2 elements apparently restricted to R. americana correlate with an increased amount of repetitive DNA in its genome in contrast to other Rhynchosciara species. The results obtained in this work together with previous results suggest that evolutionary changes in the genus Rhynchosciara occurred by differential genomic occupation not only of satellite DNA but possibly also of rDNA retrotransposons.

Evaluation of Quantitative RT-PCR Using Nonamplified and Amplified RNA

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a standard assay in molecular medicine for gene expression analysis. Samples from incisional/needle biopsies, laser-microdissected tumor cells and other biologic sources, normally available in clinical cancer studies, generate very small amounts of RNA that are restrictive for expression analysis. As a consequence, an RNA amplification procedure is required to assess the gene expression levels of such sample types. The reproducibility and accuracy of relative gene expression data produced by sensitive methodology as qRT-PCR when cDNA converted from amplified (A) RNA is used as template has not yet been properly addressed. In this study, to properly evaluate this issue, we performed 1 round of linear RNA amplification in 2 breast cell lines (C5.2 and HB4a) and assessed the relative expression of 34 genes using cDNA converted from both nonamplified (NA) and A RNA. Relative gene expression was obtained from beta actin or glyceraldehyde 3-phosphate dehydrogenase normalized data using different dilutions of cDNA, wherein the variability and fold-change differences in the expression of the 2 methods were compared. Our data showed that 1 round of linear RNA amplification, even with suboptimal-quality RNA, is appropriate to generate reproducible and high-fidelity qRT-PCR relative expression data that have similar confidence levels as those from NA samples. The use of cDNA that is converted from both A and NA RNA in a single qRT-PCR experiment clearly creates bias in relative gene expression data.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1-/- mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs. (C) 2010 Elsevier B.V. All rights reserved.

Differential expression of connexins during histogenesis of the chick retina

Gap junction (GJ) channels couple adjacent cells, allowing transfer of second messengers, ions, and molecules up to 1 kDa. These channels are composed by a multigene family of integral membrane proteins called connexins (Cx). In the retina, besides being essential circuit element in the visual processing, GJ channels also play important roles during its development. Herein, we analyzed Cx43, Cx45, Cx50, and Cx56 expression during chick retinal histogenesis. Cx exhibited distinct expression profiles during retinal development, except for Cx56, whose expression was not detected. Cx43 immunolabeling was observed at early development, in the transition of ventricular zone and pigmented epithelium. Later, Cx43 was seen in the outer plexiform and ganglion cell layers, and afterwards also in the inner plexiform layer. We observed remarkable changes in the phosphorylation status of this protein, which indicated modifications in functional properties of this Cx during retinal histogenesis. By contrast, Cx45 showed stable gene expression levels throughout development and ubiquitous immunoreactivity in progenitor cells. From later embryonic development, Cx45 was mainly observed in the inner retina, and it was expressed by glial cells and neurons. In turn, Cx50 was virtually absent in the chick retina at initial embryonic phases. Combination of PCR, immunohistochemistry and Western blot indicated that this Cx was present in differentiated cells, arising in parallel with the formation of the visual circuitry. Characterization of Cx expression in the developing chick retina indicated particular roles for these proteins and revealed similarities and differences when compared to other species. (C) 2008 Wiley Periodicals, Inc.

Anabolic-androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly, the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion, high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Resistance to ultraviolet-induced apoptosis in DNA repair deficient growth arrested human fibroblasts is not related to recovery from RNA transcription blockage

The impact of ultraviolet (UV-C) photoproducts on apoptosis induction was investigated in growth arrested (confluent) and proliferating human primary fibroblasts. Confluent fibroblasts were more resistant to UV-C-induced apoptosis than proliferating cells, and this was observed for normal human cells and for cells from patients with Cockayne and trichothiodystrophy syndromes, deficient in transcription coupled repair. This resistance was sustained for at least seven days and was not due to DNA repair efficiency, as the removal of CPDs in the genome was similar under both growth conditions. There was no correlation between reduced apoptosis and RNA synthesis recovery. Following UV-C treatment, proliferating and confluent fibroblasts showed a similar level of RNA synthesis inhibition and recovery from transcription blockage. These results support the hypothesis that the decrease of DNA replication, in growth arrested cells, protects cell from UV-C-induced apoptosis, even in the presence of DNA lesions. (C) 2007 Elsevier B.V. All rights reserved.

Prevalence and microbiological diversity of Archaea in peri-implantitis subjects by 16S ribosomal RNA clonal analysis

Background and Objective: This study evaluated the prevalence and the molecular diversity of Archaea in the subgingival biofilm samples of subjects with peri-implantitis. Material and Methods: Fifty subjects were assigned into two groups: Control (n = 25), consisting of subjects with healthy implants; and Test (n = 25), consisting of subjects with peri-implantitis sites, as well as a healthy implant. In the Test group, subgingival biofilm samples were taken from the deepest sites of the diseased implant. In both groups, subgingival biofilm was collected from one site with a healthy implant and from one site with a periodontally healthy tooth. DNA was extracted and the 16S ribosomal RNA gene was amplified with universal primer pairs for Archaea. Amplified genes were cloned and sequenced, and the phylotypes were identified by comparison with known 16S ribosomal RNA sequences. Results: In the Control group, Archaea were detected in two and three sites of the implant and the tooth, respectively. In the Test group, Archaea were detected in 12, 4 and 2 sites of diseased implants, healthy implants and teeth, respectively. Diseased implants presented a significantly higher prevalence of Archaea in comparison with healthy implants and natural teeth, irrespective of group. Over 90% of the clone libraries were formed by Methanobrevibacter oralis, which was detected in both groups. Methanobacterium congelense/curvum was detected in four subjects from the Test group and in two subjects from the Control group. Conclusion: Although M. oralis was the main species of Archaea associated with both healthy and diseased implant sites, the data indicated an increased prevalence of Archaea in peri-implantitis sites, and their role in pathogenesis should be further investigated.

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4-6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4-6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC(50) = 1 x 10 (6) M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC(50) = 4.3 x 10 (6) M). (C) 2009 Elsevier Ltd. All rights reserved.

Modifications and Alternatives to the Tests of Levene and Brown & Forsythe for Equality of Variances and Means

The usual tests to compare variances and means (e. g. Bartlett`s test and F-test) assume that the sample comes from a normal distribution. In addition, the test for equality of means requires the assumption of homogeneity of variances. In some situation those assumptions are not satisfied, hence we may face problems like excessive size and low power. In this paper, we describe two tests, namely the Levene`s test for equality of variances, which is robust under nonnormality; and the Brown and Forsythe`s test for equality of means. We also present some modifications of the Levene`s test and Brown and Forsythe`s test, proposed by different authors. We analyzed and applied one modified form of Brown and Forsythe`s test to a real data set. This test is a robust alternative under nonnormality, heteroscedasticity and also when the data set has influential observations. The equality of variance can be well tested by Levene`s test with centering at the sample median.

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

Insights into the mechanism of progressive RNA degradation by the archaeal exosome

Initially identified in yeast, the exosome has emerged as a central component of the RNA maturation and degradation machinery both in Archaea and eukaryotes. Here we describe a series of high-resolution structures of the RNase PH ring from the Pyrococcus abyssi exosome, one of them containing three 10-mer RNA strands within the exosome catalytic chamber, and report additional nucleotide interactions involving positions N5 and N7. Residues from all three Rrp41-Rrp42 heterodimers interact with a single RNA molecule, providing evidence for the functional relevance of exosome ring-like assembly in RNA processivity. Furthermore, an ADP-bound structure showed a rearrangement of nucleotide interactions at site N1, suggesting a rationale for the elimination of nucleoside diphosphate after catalysis. In combination with RNA degradation assays performed with mutants of key amino acid residues, the structural data presented here provide support for a model of exosome-mediated RNA degradation that integrates the events involving catalytic cleavage, product elimination, and RNA translocation. Finally, comparisons between the archaeal and human exosome structures provide a possible explanation for the eukaryotic exosome inability to catalyze phosphate-dependent RNA degradation.