Oviductal microvesicles and their effect on in vitro maturation of canine oocytes.

The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 g and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO2 and 5% of O2 in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 µg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25-50-75-100-150 × 106 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 106 MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies.

Isolation, molecular characterization, and in vitro differentiation of bovine Wharton jelly-derived multipotent mesenchymal cells.

Extrafetal tissues are a noncontroversial and inexhaustible source of mesenchymal stem cells that can be harvested noninvasively at low cost. In the veterinary field, as in man, stem cells derived from extrafetal tissues express plasticity, reduced immunogenicity, and have high anti-inflammatory potential making them promising candidates for treatment of many diseases. Umbilical cord mesenchymal cells have been isolated and characterized in different species and have recently been investigated as potential candidates in regenerative medicine. In this study, cells derived from bovine Wharton jelly (WJ) were isolated for the first time by enzymatic methods, frozen/thawed, cultivated for at least 10 passages, and characterized. Wharton jelly-derived cells readily attached to plastic culture dishes displaying typical fibroblast-like morphology and, although their proliferative capacity decreased to the seventh passage, these cells showed a mean doubling time of 34.55 ± 6.33 hours and a mean frequency of one colony-forming unit fibroblast like for every 221.68 plated cells. The results of molecular biology studies and flow cytometry analyses revealed that WJ-derived cells showed the typical antigen profile of mesenchymal stem cells and were positive for CD29, CD44, CD105, CD166, Oct-4, and c-Myc. They were negative for CD34 and CD14. Remarkably, WJ-derived cells showed differentiation ability. After culture in induced media, WJ-derived cells were able to differentiate into osteogenic, adipogenic, chondrogenic, and neurogenic lines as shown by positive staining and expression of specific markers. On polymerase chain reaction analysis, these cells were negative for MHC-II and positive for MHC-I, thus reinforcing the role of extrafetal tissue as an allogenic source for bovine cell-based therapies. These results provide evidence that bovine WJ-derived cells may have the potential to differentiate to repair damaged tissues and reinforce the importance of extrafetal tissues as stem cell sources in veterinary regenerative medicine. A more detailed evaluation of their immunologic properties is necessary to better understand their potential role in cellular therapy.

Microvesicles secreted from equine amniotic-derived cells and their potential role in reducing inflammation in endometrial cells in an in-vitro model.

BACKGROUND: It is known that a paracrine mechanism exists between mesenchymal stem cells and target cells. This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication. METHODS: In this context, this study aims to understand the efficacy of MVs in in-vitro endometrial stressed cells in view of potential healing in in-vivo studies. For this purpose, the presence and type of MVs secreted by amniotic mesenchymal stem cells (AMCs) were investigated and the response of endometrial cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract the in vitro stress in endometrial cells induced by lipopolysaccharide was studied by measuring the rate of apoptosis and cell proliferation, the expression of some pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin 1ß (IL-1ß), and metalloproteinases (MMP) 1 and 13, and the release of some pro- or anti-inflammatory cytokines. RESULTS: MVs secreted by the AMCs ranged in size from 100 to 200 nm. The incorporation of MVs was gradual over time and peaked at 72 h. MVs reduced the apoptosis rate, increased cell proliferation values, downregulated pro-inflammatory gene expression, and decreased the secretion of pro-inflammatory cytokines. CONCLUSION: Our data suggest that some microRNAs could contribute to counteracting in-vivo inflammation of endometrial tissue.

Effects of platelet-rich plasma in a model of bovine endometrial inflammation in vitro.

BACKGROUND: Endometritis reduces fertility and is responsible for major economic losses in beef and dairy industries. The aim of this study was to evaluate an alternative therapy using platelet-rich plasma (PRP). PRP was tested in vivo, after bovine intrauterine administration, and in vitro on endometrial cells. METHODS: Bovine endometrial cells were cultured until passage (P) 10 with 5 % or 10 % PRP. Effect of PRP on endometrial cell proliferation and on the expression of genes [prostaglandin-endoperoxide synthase 2 (COX2), tumor protein p53 (TP53), oestrogen receptors (ER-α and ER-ß), progesterone receptor (PR) and c-Myc] involved in the regulation of oestrus cycle and fetal-maternal interaction were evaluated. Moreover, to evaluate the ability of PRP to counteract inflammation, 10 and 100 ng/ml of bacterial endotoxin lipopolysaccharide (LPS) were used to inflame endometrial cells in vitro for 1, 6, 12, 24 and 48 h. The expression of genes such as interleukin 1ß (IL-1ß), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and the release of PGE-2, IL-1ß and IL-8 were evaluated. RESULTS: In vivo treatment with PRP increased the detection of PR. In vitro, 5 % PRP at passage 5 increased proliferation rate and induced a significant increase in the expression of all studied genes. Furthermore, the results revealed that 10 ng/ml of LPS is the most effective dose to obtain an inflammatory response, and that PRP treatment significantly down regulated the expression of pro-inflammatory genes. CONCLUSION: This study lays the foundations for the potential treatment of endometritis with PRP in vivo.

Does the Bovine Pre-Ovulatory Follicle Harbor Progenitor Stem Cells?

Recent studies have revealed the presence of a mesenchymal stem cell (MSC) population in human and in gilt granulosa cells (GCs), thus increasing the interest in identifying the same population in the bovine species. We first isolated GCs by scraping from bovine preovulatory follicles and then tested several different media to define the ideal conditions to select granulosa-derived stem cells. Although expressing MSC-associated markers, none of the media tested proven to be efficient in selecting MSC-like cells that were able to differentiate into mesodermic or ectodermic lineages. We performed another experimental approach exposing cells to a chemical stress, such as lowering of pH, as a system to select a more plastic population. Following the treatment, granulosa-specific granulose markers [follicle-stimulating hormone receptor (FSHR), follistatin (FST), and leukemia inhibitory factor receptor (LIFR)] were lost in bovine GCs, whereas an increase in multi- (CD29, CD44, CD73) and pluripotent (Oct-4 and c-Myc) genes was noticed. The stress allowed up-regulation of tumor necrosis factor-α and interleukin-1ß expression and the dedifferentiation of GCs, which was demonstrated by differentiation studies. Indeed, pH-treated cells were able to differentiate into the mesodermic and ectodermic lineages, thus suggesting that the chemical stress allows for the selection of cells that are more prone to adjust and respond to the environmental changes.

Microwave processing and ethanol-based fixation in forensic pathology: an addendum of further scanning electron microscope observations.

Scanning electron microscope microscopy on samples of tissue fixed with alcohol-based fixative and processed using a microwave device confirmed the validity of the fixation procedure. The details are clearer with respect to those obtainable with formalin fixatives. It was interesting to work on sections prepared for normal histologic processing because the metallization indispensable for scanning electron microscope occurred without difficulty. All together, the procedure seems to be very flexible and adapted to the complexity of forensic investigation, above all, when the tissue is altered by autolytic phenomena.