Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Abstract Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.

Nanoparticles from culture media are internalized by in vitro-produced bovine embryos and its depletion affect expression of pluripotency genes

Extracellular vesicles are nanoparticles secreted by cell and have been proposed as suitable markers to identify competent embryos produced in vitro. Characterizing EVs secreted by individual embryos is challenging because culture medium itself contributes to the pool of nanoparticles that are co-isolated. To avoid this, culture medium must be depleted of nanoparticles that are present in natural protein source. The aim of this study was to evaluate if the culture medium subjected to nanoparticle depletion can support the proper in vitro development of bovine embryos. Zygotes were cultured in groups on depleted or control medium for 8 days. Nanoparticles from the medium were characterized by their morphology, size and expression of EVs surface markers. Isolated nanoparticles were labelled and added to depleted medium containing embryos at different developmental stages and evaluated after 24 hours at 2, 8-16 cells, morula and blastocyst stages. There were no statistical differences on blastocyst rate at day 7 and 8, total cell count neither blastocyst diameter between groups. However, morphological quality was better in blastocysts cultured in non-depleted medium and the expression of SOX2 was significantly lower whereas NANOG expression was significantly higher. Few nanoparticles from medium had a typical morphology of EVs but were positive to specific surface markers. Punctuated green fluorescence near the nuclei of embryonic cells was observed in embryos from all developmental stages. In summary, nanoparticles from culture medium are internalized by in vitro cultured bovine embryos and their depletion affects the capacity of medium to support the proper embryo development.(AU)

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)