Breed influences on in vitro development of abattoir-derived bovine oocytes

ABSTRACT: BACKGROUND: There is a discrepancy in the reproductive performance between different cattle breeds. Using abattoir-derived ovaries and data base information we studied the effects of breed on in vitro fertilization and early embryo development. METHODS: The in vitro developmental competence of oocytes from cattle (n = 202) of Swedish Red (SR), Swedish Holstein (SH) and mixed beef breeds was compared, retrospectively tracing donors of abattoir-derived ovaries using a combination of the national animal databases and abattoir information. Age was significantly lower and carcass conformation score was higher in the beef breeds than in the dairy breeds. Cumulus oocyte complexes (n = 1351) were aspirated from abattoir-derived ovaries from animals of known breed (visual inspection confirmed through databases), age (databases), and abattoir information. Oocytes were matured, fertilized (frozen semen from two dairy bulls) and cultured according to conventional protocols. On day 8, blastocysts were graded and the number of nuclei determined. RESULTS: Cleavage rate was not different between the breeds but was significantly different between bulls. The percentage of blastocysts on day 8 was significantly higher when the oocyte donor's breed was beef or SR than SH. There was no significant difference in blastocyst grades or stages between the breeds, but the number of nuclei in day 8 blastocysts was significantly lower in SH compared to the beef. CONCLUSIONS: The use of abattoir-derived ovaries from animals whose background is traceable can be a valuable tool for research. Using this approach in the present study, oocyte donor breed was seen to affect early embryo development during in vitro embryo production, which may be a contributing factor to the declining fertility in some dairy breeds seen today.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Lipid droplets interact with mitochondria using SNAP23

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.     

Few alterations in clinical pathology and histopathology observed in a CYP2C18&;19 humanized mice model

Background  

This study was performed to characterize a gene-addition transgenic mouse containing a BAC (bacterial artificial chromosome) clone spanning the human CYP2C18&19 genes (tg-CYP2C18&19).     

Large scale identification of proteins, mucins, and their O-glycosylation in the endocervical mucus during the menstrual cycle

The mucus filling the human cervical opening blocks the entry to the uterus, but this has to be relative and allow for the sperm to penetrate at ovulation. We studied this mucus, its content of proteins and mucins, and the mucin O-glycosylation in cervical secretions before, during, and after ovulation. Cervical mucosal secretions from 12 subjects were collected, reduced-alkylated, separated with polyacrylamide or agarose/polyacrylamide gel electrophoresis, and stained with silver, Alcian blue, or Coomassie Blue stain. Protein and mucin bands from before and during ovulation were digested and subsequently analyzed by nano-LC-FT-ICR MS and MS/MS. We identified 194 proteins after searches against the NCBI non-redundant protein database and an in-house mucin database. Three gel-forming (MUC5B, MUC5AC, and MUC6) and two transmembrane mucins (MUC16 and MUC1) were identified. For the analysis of mucin O-glycosylation, separated mucins from six individuals were blotted to PVDF membranes, and the O-glycans were released by reductive beta-elimination and analyzed with capillary HPLC-MS and -MS/MS. At least 50 neutral, sialic acid-, and sulfate-containing oligosaccharides were found. An increase of GlcNAc-6GalNAcol Core 2 structures and a relative decrease of NeuAc residues are typical for ovulation, and NeuAc-6GalNAcol and NeuAc-3Gal- epitopes are typical for the non-ovulatory phases. The cervical mucus at ovulation is thus characterized by a relative increase in neutral fucosylated oligosaccharides. This comprehensive characterization of the mucus during the menstrual cycle suggests mucin glycosylation as the major alteration at ovulation, but the relation to the altered physicochemical properties and sperm penetrability is still not understood.     

Crystal structure of bifunctional aldos-2-ulose dehydratase/isomerase from Phanerochaete chrysosporium with the reaction intermediate ascopyrone M

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn²⁺ and Mg²⁺ located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn²⁺-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn²⁺ in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn²⁺ acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.     

The significance of host-fungus combinations in ectomycorrhizal symbioses for the chemical quality of willow foliage

The majority of plants interacts with mycorrhizal fungi, which predominantly provide mutual benefits, but also costs. We tested the hypothesis that specific combinations of host plants (four commercial varieties of Salix spp.) and ectomycorrhizal partners (species of Laccaria, Paxillus, Tricholoma and Hebeloma) differ in their effects on the host foliar chemistry. Twenty specific host - mycorrhiza combinations were pot-grown outdoors under low-N conditions. Foliar concentrations of total phenolics and salicylic acid were decreased by mycorrhizas in S. schwerinii x S. viminalis and S. x dasyclados genotypes, but increased in S. viminalis. Mycorrhiza effects on host biomass production were positive, indifferent or negative, depending on the specific host—mycorrhiza combination. The host plant genotype influenced mainly the direction of mycorrhizal effects on foliar chemistry, whereas the magnitude of mycorrhizal effects varied mostly among the fungal genotypes. The results are relevant with respect to the possible interactions between mycorrhizas, plants and leaf herbivores.     

Study of antimicrobial activity amongLactobacillus helveticus strains using three different assays

The antimicrobial activity of 18Lactobacillus helveticus strains as well as one control strain, the bacteriocin producingLactobacillus helveticus 481, was tested with three different inhibition assays. FourLactobacillus helveticus strains had antimicrobial activity against seven otherLactobacillus helveticus strains and twoLactobacillus delbrueckil strains while the remaining sevenLactobacillus helveticus strains were indifferent. Inhibition was also observed againstLactococcus andLeuconostoc species, due to production of organic acids or hydrogen peroxide. The strains with the highest antimicrobial activity produced a heat sensitive proteinacious bacteriocin with a narrow species activity spectrum against only thermophilicLactobacillus strains.     

Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.