Complimentary diagnostic tools for endometrosis in biopsies of mares with clinical subfertility

Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiologyand pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis withglandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary amongindividuals according to the disease progression. The aim of this research was to combine histology with new immune andhistochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples weresubjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specifictechniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were presentin those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods forthe detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order todetect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. Asa result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb andIII (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result wefound specific staining in 15 samples coming from higher endometrial damage using Masson-...

Complimentary diagnostic tools for endometrosis in biopsies of mares with clinical subfertility

Background: Endometrosis is a multifactorial disease and one of the main causes of infertility in mares, its etiologyand pathogenesis are not completely understood. It is defined as peri glandular and/or stromal endometrial fibrosis withglandular alterations. Due to the few clinical symptoms, besides anamnesis and fertility data, endometrosis requires histological confirmation. The histo-morphology and immune histochemical characteristics of the endometrium vary amongindividuals according to the disease progression. The aim of this research was to combine histology with new immune andhistochemical tools for a more precise detection of fibrotic changes of mares with endometrosis.Materials, Methods & Results: The endometrium of forty thoroughbred mares aged 5-18 years, that did not become pregnant during the last two breeding seasons in a Chilean commercial equine breeding center were biopsied. Samples weresubjected to conventional histopathology with hematoxylin-eosin as well as to specific histological staining using specifictechniques such as Alcian blue and Masson Fontana, aimed to ascertain what types of mucopolysaccharides were presentin those samples. In order to have a deeper picture of the progression of the pathology, immune histochemical methods forthe detection of vimentin, cytokeratin, progesterone receptor and lymphocyte marker CD3 were used. Finally in order todetect fibrillar collagen we used second harmonic generation (SHG) technique with detects fibrillar collagen without staining, due to intrinsic hyperpolarization ability of this type of collagen, which can be detected by atomic force microscopy. Asa result of our research samples were categorized according to the scale of Keeney and Doig into categories I, IIa, IIb andIII (45, 42, 7.5 and 5% respectively). These samples also were characterized by the methods listed earlier and a result wefound specific staining in 15 samples coming from higher endometrial damage using Masson-...(AU)

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.

Characterization of mesenchymal stem cells derived from adipose tissue of a cougar (Puma concolor)

Adipose derived mesenchymal stem cells (AMSCs) have been isolated from domestic and wild cats. For wild cats, the isolation of AMSCs has been reported in the black-footed cats (Felis nigripes) and guigna (Leopardus guigna). Stromal vascular fraction (SVF) isolated from cougar adipose tissue have been used to restore elbow functionality in the cougar (Puma concolor) but multipotent characteristics of these cells have not been described. The present study describes for the first time the isolation and characterization of mesenchymal stem cells derived from adipose tissue of cougar. AMSCs and fibroblasts from six months female cougar were isolated and cultured in DMEM/F12, supplemented with FBS 10% + 1% Antibiotic/Antifungal + 2.4 mM L-Glutamine + 2.4 mM pyruvate up to passage 5. Expression of pluripotent and surface marker genes was evaluated at mRNA level. Mesodermal differentiation (adipogenic, osteogenic and chondrogenic) was described. AMSCs expressed mRNA of pluripotent genes Oct4, Nanog, Sox2 and Klf4 and surface markers Cd44, Cd90, Cd105 and MHCII. Fibroblasts showed similar mRNA expression with the exception of Sox2. AMSCs obtained from cougar exhibit multipotency features similar to domestic cats MSC, nevertheless, other analyses are required. AMSCs from cougar could be a source of interest for treatment of individuals that remain in captivity or arrive to wildlife rehabilitation centers.(AU)

Effect of nucleus transfer on gene expression in bovine embryos during early development

Background: Somatic cell nuclear transfer, commonly known as cloning is a powerful tool for the production of identical animals for productive, biomedical or conservation purposes. Today several species of mammals has been cloned using this technology. Nevertheless, the efficiency of the process is still low in terms of newborn animals. The causes for such inefficiencies are mainly the lack of knowledge about biological processes underlying this complex technology particularly nuclear reprogramming to allow the establishment of embryonic gene expression pattern. The abnormal phenotypes observed in clones during pregnancy and even after birth, have been associated with an aberrant gene expression during early embryo development. A better understanding about the molecular process that governs the early bovine embryo development could help to improve animal cloning efficiency. This paper reviewed the effect of somatic cell nuclear transfer on gene expression in bovine embryos at both pre and peri-implantation stages. Review: This paper reviewed the effect of somatic cell nuclear transfer on early bovine embryo development. During the early developmental period and prior to implantation, a bovine embryo passes through proliferation and differentiation stages in order to assure implantation and foetation. Two stages have been described: pre-implantation stage when the embryo reaches the blastocyst stage and peri-implantation also known as elongation stage. Those periods are the result of a coordinated and robustly regulated gene expression patterns. In cloned bovine embryos as well as in other species, several genes have been found deregulated not only during pre-implantation stages, but also during elongation. Many of those genes such as OCT4, SOX2, NANOG and FGF4 are related with maintain of cell pluripotence, while others such as INFtau, EOMES, CDX2 and TKDP1 are markers of trophoblastic function. Aberrant gene expression pattern is not morphologically evident at blastocyst stage. However a deregulation of the expression of trophoblastic markers during the peri-implantation period could be related with an incomplete morphological elongation. The pattern of gene expression established as consequence of the reprogramming of the donor nucleus could change depending on different factors inherent to the technique. Those factors include the donor cell, the cytoplast receptor and also the technical process associated to nuclear transfer such as enucleation, cell fusion, and activation protocol. Despite the multiples studies performed in order to improve cloned embryo competence in terms of gene expression pattern, today this topic is still the milestone of many research groups. Conclusion: Identification of deregulated genes may yield insights into mechanisms underlying the high mortality rate of cloned bovine embryos, and lead to strategies to reduce embryonic losses.

All roads lead to milk: transgenic and non-transgenic approaches for expression of recombinant proteins in the mammary gland

Background: With the advent of transgenic technology to farm animals, it became possible to express recombinant proteins of high complexity in the body compartments and fluids of these animals; the term "pharming" was coined. This was a tremendous achievement taking into consideration the high costs associated with conventional (cell-based) production methods and the incapacity of lower organisms to adequately process complex proteins. The mammary gland had been the organ of choice and milk the appropriate vector for successful expression of many recombinant drugs of high added value. Review: While theoretically the mammary gland is able to carry out all the complex post-translational changes related with glycosylation or others, in the practice, not all proteins can be actually processed in a way that closely remembers the wild protein, thus making difficult the production of some proteins in full biologically active form. This is especially true for complex (branched) forms of glycosylation as it is the case of human erythropoietin (hEPO), or gamma carboxylation of blood clotting factors, to mention a few examples. These cases are discussed in this review, with special emphasis in the glycosylation of hEPO. In spite of the imperfectness of the mammary gland to accurately add some sugar residues, it continues to be the most desirable organ to which target gene expression, due to its potent biosynthetic machinery and the possibilities to amend the said incapacity to glycosylate appropriately all kinds of proteins. In line with this, the European Medicines Agency first (in 2006) and the Food and Drug Administration later (2009) approved the first milk-derived recombinant protein for human use, (ATryn; human anti-thrombin-III) after more than two decades of thorough reviews and test, thus opening the way for future massive production of blockbuster drugs using the mammary gland as bioreactor. In this job we reviewed briefly the state of the art of mammary gland-based production of recombinant proteins with emphasis in two different systems to target it. In the first approach, a transgenic mammal carrying appropriate mammary specific gene promoter linked to a transgene is made, then grown, mated, its milk tested for the presence of the protein, if expression levels and biological activity of the proteins meet the requirements, then a production flock is created from the founder(s) and milk collected and processed. In this way, most of the recombinant proteins produced in the milk had been created, including the leading drug ATryn. We developed an alternative method for transient viral vectors-mediated transduction of the mammary gland, using constitutive viral promoters linked to the transgene, thus producing very quickly high amounts of the desired protein. The drawback of this method is its transient nature; the advantage is the fastness and easiness to produce grams of recombinant proteins in the milk of otherwise non-transgenic mammals. In this way several drugs had been produced. Notably one of them, the E2 antigen of classic swine fever (CSF) had been secreted in biologically active form at high levels in goats´ milk; a veterinary vaccine formulation was established and tested successfully in clinical trials that included viral challenging with CSF. It is foreseen that this vaccine could be in the market this year and became the first recombinant drug produced in the milk of non-transgenic animals to get regulatory approval. In this article, we also reviewed the state of the art of different body fluids as vectors for recombinant protein production Conclusions: At least for the next coming years, all the animal-based recombinant protein production ways will lead to the milk.