Loris Commin

Follicle development in cryopreserved bitch ovarian tissue grafted to immunodeficient mouse

The present study evaluated: (1) in vivo follicular development in canine ovarian tissue after slow freezing and xenotransplantation; and (2) the use of erythropoietin (EPO) as an angiogenic factor to optimise the transplantation procedure. Frozen-thawed ovarian tissue from five bitches was grafted into severe combined immunodeficient (SCID) mice (n=47) treated with or without EPO (500 IU kg(-1), once daily for 3 days) (Groups A and B, respectively) and analysed after 0, 1, 8 or 16 weeks. Follicle grade, follicle density, follicle morphology and stromal cells density were assessed by histological analysis, whereas vascularisation of the graft was quantified by immunohistochemistry with anti-α-smooth muscle actin antibody. Despite a massive loss of follicles after grafting, secondary follicle density was higher at 8 and 16 weeks than at 1 week regardless of EPO treatment. EPO significantly improved early follicle morphology and stromal cell density after 8 weeks and blood vessel density at 16 weeks after transplantation (P<0.05). Intact secondary follicles with more than three granulosa cells layers were observed 16 weeks after transplantation. The results suggest that canine ovarian tissue can be successfully preserved by our slow-freezing protocol because the tissue showed follicular growth after xenotransplantation. EPO treatment did not lessen the massive loss of follicles after transplantation.

VEGF overexpression in the astroglial cells of rat brainstem following ozone exposure.

Ozone, a major photochemical pollutant, produces rapid damages in the pulmonary airway tract and in the central nervous system. This study focused on the neural mechanisms underlying the adaptive responses to an acute ozone exposure. Vascular endothelial growth factor (VEGF) is a factor associated with cellular recovery following brain injury. The aim of this study was to assess and localize the cellular expression of VEGF, since the central respiratory areas show a neuroplasticity in response to ozone. Adult rats were subjected to 0.5ppm ozone for 3h and then recovered for further 3h. The expression of VEGF was evaluated by immunocytochemistry in the central respiratory areas, i.e., the nucleus tractus solitarius (NTS) and the ventrolateral medulla (VLM). The data show a VEGF overexpression at the end of ozone exposure, which persisted during the 3-h recovery. Interestingly, using confocal analysis the bulk of VEGF labeling was observed in astroglial cell bodies and branches, while neuronal labeling was hardly noticed. Moreover, VEGF colocalized with IL-6 and TNFalpha in astrocytes closely apposed to blood vessel walls. The vasculature area was markedly increased (+58%) during post-ozone recovery. The data show that an acute ozone exposure affects primarily glial cells in the central nervous system. The VEGF up-regulation which persists after ozone exposure may contribute to brain repair and consecutive functional adaptations.

Genomic Rearrangements and Functional Diversification of lecA and lecB Lectin-Coding Regions Impacting the Efficacy of Glycomimetics Directed against Pseudomonas aeruginosa

LecA and LecB tetrameric lectins take part in oligosaccharide-mediated adhesion-processes of Pseudomonas aeruginosa. Glycomimetics have been designed to block these interactions. The great versatility of P. aeruginosa suggests that the range of application of these glycomimetics could be restricted to genotypes with particular lectin types. The likelihood of having genomic and genetic changes impacting LecA and LecB interactions with glycomimetics such as galactosylated and fucosylated calix[4]arene was investigated over a collection of strains from the main clades of P. aeruginosa. Lectin types were defined, and their ligand specificities were inferred. These analyses showed a loss of lecA among the PA7 clade. Genomic changes impacting lec loci were thus assessed using strains of this clade, and by making comparisons with the PAO1 genome. The lecA regions were found challenged by phage attacks and PAGI-2 (genomic island) integrations. A prophage was linked to the loss of lecA. The lecB regions were found less impacted by such rearrangements but greater lecB than lecA genetic divergences were recorded. Sixteen combinations of LecA and LecB types were observed. Amino acid variations were mapped on PAO1 crystal structures. Most significant changes were observed on LecBPA7, and found close to the fucose binding site. Glycan array analyses were performed with purified LecBPA7. LecBPA7 was found less specific for fucosylated oligosaccharides than LecBPAO1, with a preference for H type 2 rather than type 1, and Lewisa rather than Lewisx. Comparison of the crystal structures of LecBPA7 and LecBPAO1 in complex with Lewisa showed these changes in specificity to have resulted from a modification of the water network between the lectin, galactose and GlcNAc residues. Incidence of these modifications on the interactions with calix[4]arene glycomimetics at the cell level was investigated. An aggregation test was used to establish the efficacy of these ligands. Great variations in the responses were observed. Glycomimetics directed against LecB yielded the highest numbers of aggregates for strains from all clades. The use of a PAO1ΔlecB strain confirmed a role of LecB in this aggregation phenotype. Fucosylated calix[4]arene showed the greatest potential for a use in the prevention of P. aeruginosa infections.

A Chemically Defined Medium for Rabbit Embryo Cryopreservation

This study evaluates a new synthetic substitute (CRYO3, Ref. 5617, Stem Alpha, France) for animal-based products in rabbit embryo cryopreservation solutions. This evaluation was performed using two approaches: a thermodynamic approach using differential scanning calorimetry and a biological approach using rabbit embryo slow-freezing. During the experiment, foetal calf serum (FCS) was used as a reference. Because FCS varies widely by supplier, three different FCS were selected for the thermodynamic approach. The rabbit embryo slow-freezing solutions were made from Dulbeccos phosphate buffer saline containing 1.5 M Dimethyl Sulfoxide and 18% (v.v−1) of CRYO3 or 18% (v.v−1) of FCS. These solutions were evaluated using four characteristics: the end of melting temperature, the enthalpy of crystallisation (thermodynamic approach) and the embryo survival rates after culture and embryo transfer (biological approach). In the thermodynamic approach, the solutions containing one of the three different FCS had similar mean thermodynamic characteristics but had different variabilities in the overall data with aberrant values. The solution containing CRYO3 had similar thermodynamic properties when compared to those containing FCS. Moreover, no aberrant value was measured in the solution containing CRYO3. This solution appears to be more stable than the solutions containing a FCS. In the biological approach, the in vitro embryo survival rates obtained with the solution containing CRYO3 (73.7% and 81.3%) and with the solution containing a FCS (77.6% and 71.9%) were similar (p = 0.7). Nevertheless, during the in vivo evaluation, the implantation rate (21.8%) and the live-foetuses rate (18.8%) of the CRYO3 group were significantly higher than the implantation rate (7.1%, p = 0.0002) and the live-foetuses rate (5.3%, p = 0.0002) of the FCS group. The pregnancy rate was also higher in the CRYO3 group compared to the FCS group (81.3% and 43.8%, respectively, p = 0.066). We conclude that CRYO3 can be used as a chemically defined substitute for animal-based products in rabbit embryo cryopreservation solutions.

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species

However, each variant of the reproductive cells has introduced their own problems, and it has been realized that many aspects of the particular physiology of the cells will dictate how they respond to cryopreservation. Both male and female gametes have acquired highly specialized structural components (essential to fertilization and development) that may respond to the freezing process in ways different from that of basic cell structures.

Ex vivo Recovery of Preimplantatory Embryos in Bitches

The present study was conducted to investigate the timing of preimplantatory development in the dog and to evaluate the efficiency of flushing oviducts and uterine horns to collect embryos. Among the embryonic structures collected between day 8 and day 12 after ovulation, 43 % were at the 1-16 cells stage, 23% were at the morula stage and 34% at the blastocyst stage. Our collection method yielded to a recovery rate of 61.3 %, and 7.1 ± 0.7 embryos were harvested per bitch. In addition, the ovulation rate reached 11.6 ± 0.8 per bitch. The first morulae were observed from day 9 post-ovulation, while the first blastocyst appeared from day 10. Two-thirds of the collected morulae-blastocysts were obtained between the 11th and the 12th day after ovulation. To the moment, we suggest this is the best period to harvest canine embryo for cryopreservation.