Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes

Background

Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes.

Methods

Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20°C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO₂ in air at 38°C and 100% humidity in the presence of 5 × 10⁶ fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM).

Results

Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger.

Conclusion

In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20°C are less suitable for use in feline ZBA.     

Taxonomy of primycin producing actinomycetes. I. Description of the type strain of thermomonospora galeriensis.

Strain FBUA 1274 (=strain 28/650707, Hungarian National Collection of Medical Bacteria, National Institute of Public Health, Budapest) is designated the type strain of Thermonospora galeriensis (Vályi-Nagy et al.) comb. nov., and the morphological characteristics of this strain are described.     

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.     

Wing morph-related physiological differences in adults of temperate population of Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae)

The reproductive and diapausing adult females of brachypterous morph and macropterous females with reproductive arrest of non-diapause type, originating from the laboratory cultures of Pyrrhocoris apterus, were studied for their feeding and drinking behaviour, digestive enzyme activities, and carbohydrate and lipid contents. The highest feeding and drinking activities were observed in reproductive brachypters, the lowest in macropters. Macropters also differed from brachypters by lower activities of gut lipase, peptidase and protease, lower concentration of haemolymph sugars, and lower weight of fat body, which probably reflects their low feeding activity. The total content of fat body lipids was also lower in macropters (0.6 mg) than in reproductive and diapausing brachypters (4.6 and 7.5 mg, respectively) on day 14. A very high amount of glycogen was found in the fat body of diapausing brachypters, 363 μg on day 14, as opposed to 15 and 80 μg in macropterous and reproductive brachypterous females, respectively. The obtained data indicate that the most important difference between macropterous and brachypterous females with different types of reproductive arrest consists of an enhanced mobilization of lipids for dispersal in macropters and accumulation of energetic reserves for hibernation in brachypters.     

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D₂O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d₁₀. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.     

Species diversity in Gymnogeophagus (Teleostei: Cichlidae) and comparative biogeography of cichlids in the Middle Paraná basin,an emerging hotspot of fish endemism

Hydrobiologia - We address the diversity of two species groups of the cichlid genus Gymnogeophagus in the Middle Paraná basin using molecular phylogeography and traditional morphological...     

Crystal structure of the cytokine interleukin-1 beta.

The crystal structure of human recombinant interleukin-1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R-factor of 42.3%. The structure is composed of 12 beta-strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta-strands. The interior of this structure is filled with hydrophobic side chains. There is a 3-fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin-1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557-563, for soybean trypsin inhibitor.     

Tyrosine Phosphorylation of the Rho Guanine Nucleotide Exchange Factor Trio Regulates Netrin-1/DCC-Mediated Cortical Axon Outgrowth

The chemotropic guidance cue netrin-1 mediates attraction of migrating axons during central nervous system development through the receptor Deleted in Colorectal Cancer (DCC). Downstream of netrin-1, activated Rho GTPases Rac1 and Cdc42 induce cytoskeletal rearrangements within the growth cone. The Rho guanine nucleotide exchange factor (GEF) Trio is essential for Rac1 activation downstream of netrin-1/DCC, but the molecular mechanisms governing Trio activity remain elusive. Here, we demonstrate that Trio is phosphorylated by Src family kinases in the embryonic rat cortex in response to netrin-1. In vitro, Trio was predominantly phosphorylated at Tyr²⁶²² by the Src kinase Fyn. Though the phospho-null mutant Trio^Y2622F retained GEF activity toward Rac1, its expression impaired netrin-1-induced Rac1 activation and DCC-mediated neurite outgrowth in N1E-115 neuroblastoma cells. Trio^Y2622F impaired netrin-1-induced axonal extension in cultured cortical neurons and was unable to colocalize with DCC in growth cones, in contrast to wild-type Trio. Furthermore, depletion of Trio in cortical neurons reduced the level of cell surface DCC in growth cones, which could be restored by expression of wild-type Trio but not Trio^Y2622F. Together, these findings demonstrate that Trio^Y2622 phosphorylation is essential for the regulation of the DCC/Trio signaling complex in cortical neurons during netrin-1-mediated axon outgrowth.     

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.