Early oocyte penetration can predict the efficiency of bovine embryo production in vitro

The aim of this work was to characterize oocyte fertilization and embryo cleavage in nine AI bulls to find parameters suitable for prediction of in vitro fertility. According to the d8 blastocysts rate, they were categorized as high, medium and low productive (HP, MP and LP, mean: 25.4, 21.0 and 13.6% respectively) bulls. For these categories, oocyte penetration and fertilization efficiency were assessed at 6 and 18 hours post insemination (hpi), respectively. Some presumptive zygotes were cultured and cleaved and fast-cleaved embryo rates were checked at 44 hpi. The penetration rate was significantly higher for HP bulls than for MP and LP bulls (67.9 versus 50.3 and 33.1%; p<0.01). The syngamy rate was significantly higher for HP bulls than for MP and LP bulls (21.4 versus 10.2 and 5.7%; p<0.05). Conversely, no significant differences in fertilization rates were found among HP, MP and LP bulls. The cleavage rate was significantly higher for HP than LP bulls (82.4 versus 74.4%; p<0.01). The fast cleavage rate was significantly higher for both HP and MP bulls, as compared with LP bulls (82.1 and 84.7 versus 73.5%; p<0.01). A strong correlation was found between blastocyst production and penetration (r=0.803), syngamy (r=0.826), cleavage (r=0.635) and fast cleavage (r=0.709). In conclusion, all the evaluated parameters showed a predictive value, the most significant being early penetration and syngamy.     

Computational neurogenetic modelling: a pathway to new discoveries in genetic neuroscience

The paper presents a methodology for using computational neurogenetic modelling (CNGM) to bring new original insights into how genes influence the dynamics of brain neural networks. CNGM is a novel computational approach to brain neural network modelling that integrates dynamic gene networks with artificial neural network model (ANN). Interaction of genes in neurons affects the dynamics of the whole ANN model through neuronal parameters, which are no longer constant but change as a function of gene expression. Through optimization of interactions within the internal gene regulatory network (GRN), initial gene/protein expression values and ANN parameters, particular target states of the neural network behaviour can be achieved, and statistics about gene interactions can be extracted. In such a way, we have obtained an abstract GRN that contains predictions about particular gene interactions in neurons for subunit genes of AMPA, GABAA and NMDA neuro-receptors. The extent of sequence conservation for 20 subunit proteins of all these receptors was analysed using standard bioinformatics multiple alignment procedures. We have observed abundance of conserved residues but the most interesting observation has been the consistent conservation of phenylalanine (F at position 269) and leucine (L at position 353) in all 20 proteins with no mutations. We hypothesise that these regions can be the basis for mutual interactions. Existing knowledge on evolutionary linkage of their protein families and analysis at molecular level indicate that the expression of these individual subunits should be coordinated, which provides the biological justification for our optimized GRN.     

Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca²⁺ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)₂ or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)₂ fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.     

Mitochondrial chloride channels: electrophysiological characterization and pH induction of channel pore dilation

Physiological and pathological functions of mitochondria are highly dependent on the properties and regulation of mitochondrial ion channels. There is still no clear understanding of the molecular identity, regulation, and properties of anion mitochondrial channels. The inner membrane anion channel (IMAC) was assumed to be equivalent to mitochondrial centum picosiemens (mCS). However, the different properties of IMAC and mCS channels challenges this opinion. In our study, we characterized the single-channel anion selectivity and pH regulation of chloride channels from purified cardiac mitochondria. We observed that channel conductance decreased in the order: Cl^? > Br^? > I^? > chlorate ≈ formate > acetate, and that gluconate did not permeate under control conditions. The selectivity sequence was Br^? ≥ chlorate ≥ I^? ≥ Cl^? ≥ formate ≈ acetate. Measurement of the concentration dependence of chloride conductance revealed altered channel gating kinetics, which was demonstrated by prolonged mean open time value with increasing chloride concentration. The observed mitochondrial chloride channels were in many respects similar to those of mCS, but not those of IMAC. Surprisingly, we observed that acidic pH increased channel conductance and that an increase of pH from 7.4 to 8.5 reduced it. The gluconate current appeared and gradually increased when pH decreased from pH 7.0 to 5.6. Our results indicate that pH regulates the channel pore diameter in such a way that dilation increases with more acidic pH. We assume this newly observed pH-dependent anion channel property may be involved in pH regulation of anion distribution in different mitochondrial compartments.     

1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

Background  

Quantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify.     

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.     

Efficiency and kinetics of the in vitro fertilization process in bovine oocytes with different meiotic competence

The aim of the study was to investigate the efficiency and kinetics of fertilization in oocytes with different meiotic competence, as defined by the phase of the follicular wave and follicle size. Oocytes were recovered from cows with synchronized estrus cycles, slaughtered in either the growth (day 3) or the dominant (day 7) phase, separately from large, medium and small follicles. The oocytes were matured and fertilized by a standard protocol. Twenty-four hours after fertilization, the oocytes were denuded from cumulus cells, fixed and stained with bisbensimid Hoechst-PBS. Fertilization was more efficient and the first cleavage was accelerated in growth phase-derived oocytes, as shown by significantly higher (p < or = 0.01) proportions of both normally fertilized and cleaved oocytes (68.8 and 25.1%), in comparison with dominant phase-derived oocytes (44.2 and 10.3%). In the growth-phase derived oocytes, proportions of normally fertilized and cleaved oocytes were significantly higher (p < or = 0.01) in oocytes from large (100.0 and 36.4%) and medium (83.3 and 36.5%) follicles than in those from small (54.8 and 14.6%) follicles. The dominant phase-derived oocytes showed higher proportions of normally fertilized and cleaved oocytes in the populations recovered from small (51.5 and 10.0%) and medium (43.1 and 12.0%) follicles than in those from large (25.0 and 0%) follicles; however, the differences were not significant. It can be concluded that: (i) efficiency and kinetics of fertilization differ in relation to oocyte's meiotic competence; (ii) improved development of embryos from oocytes with greater meiotic competence is associated with a more effective fertilization process.