ABSTRACT
Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40-400 nm) and concentrations (2.4 ± 0.2 × 1012-1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.
Subject(s)
Embryo Culture Techniques , Embryonic Development/genetics , Extracellular Vesicles/metabolism , Oogenesis/drug effects , Animals , Blastocyst/drug effects , Cattle , Centrifugation, Density Gradient , Culture Media, Conditioned , Female , Follicular Fluid/chemistry , Follicular Fluid/metabolism , Hair Cells, Ampulla/chemistry , Hair Cells, Ampulla/metabolism , Humans , Oviducts/drug effectsABSTRACT
Polarized Fourier Transform Infrared (FTIR) spectroscopy is employed to study structure-property relationships in major ampullate spider silk being exposed to an external mechanical strain. From the measured infrared dichroism of aminoacid-residue - specific bands the molecular order parameter, the frequency width at half-maximum (FWHM) and the spectral position of the absorption maximum are determined in dependence on the external strain. For the highly ordered alanine-rich beta sheets a change in the vibrational potential is found for macroscopic strains as low as a few percent. It can be quantitatively described by a quantum-mechanical approach in which the mechanical strain is treated as a weak external perturbation. The immediate microscopic response to the external field proves that beta -sheeted crystals are tightly interconnected by pre-stretched chains as suggested recently (Y. Liu et al., Nat. Mater. 4, 901 (2005)).