ABSTRACT
BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.
Subject(s)
Antioxidants , Blastocyst , Cryopreservation , Oxidative Stress , Resveratrol , Vitrification , Animals , Cattle , Resveratrol/pharmacology , Vitrification/drug effects , Oxidative Stress/drug effects , Oxidative Stress/genetics , Cryopreservation/methods , Antioxidants/pharmacology , Antioxidants/metabolism , Blastocyst/drug effects , Blastocyst/metabolism , Gene Expression Regulation, Developmental/drug effects , Fertilization in Vitro/veterinary , Fertilization in Vitro/methods , Embryo, Mammalian/drug effects , Embryo, Mammalian/metabolism , Embryo Culture Techniques/methods , Embryonic Development/drug effects , Embryonic Development/genetics , Oocytes/drug effects , Oocytes/metabolism , FemaleABSTRACT
BACKGROUND: Extracellular vesicles (EVs) are a promising biomarker and play a vital role in cell-cell communication. This study aimed (I) to identify and characterize EVs from low volume uterine lavage (LVL) and serum in mares with endometritis, compared to healthy controls and (II) to measure serum levels of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2). Mares were divided into 30 sub-fertile (endometritis) and 20 fertile (controls). Serum and LVL was collected for EV isolation, and determination of serum levels of inflammatory mediators. Characterization and visualization of EVs were done by electron microscopy, dynamic light scattering and flow cytometry. RESULTS: Serial ultracentrifugation of LVL and use of a commercial kit for serum were strategies for EVs isolation. Mares with endometritis released higher amounts of larger size EVs. The EVs from mares with endometritis differentially expressed CD9 and CD63, compared to controls. Mares suffering from endometritis evoked higher levels of inflammatory mediators. CONCLUSIONS: Thus, EVs could be used for a better understanding the regulatory mechanisms associated with developing endometritis in mares.
Subject(s)
Endometritis , Extracellular Vesicles , Horse Diseases , Animals , Biomarkers , Dinoprostone , Endometritis/diagnosis , Endometritis/veterinary , Female , Horse Diseases/diagnosis , Horses , Therapeutic Irrigation/veterinaryABSTRACT
We report the chemo-enzymatic synthesis of three cellobiono-1,5-lactone azido derivatives, designed as building blocks for biomedical polymer scaffolds. The synthesis is based on regioselective protection of cellobiose or 1,6-O-anhydro-ß-d-cellobiose before azidation and subsequent deprotection. The oxidation to the corresponding cellobiono-1,5-lactones was investigated with 6'-azido-6'-deoxycellobiose (6'N3Clb, 5), 6-azido-6-deoxycellobiose (6N3Clb, 11) and 2-azido-2-deoxycellobiose (2N3Clb, 15) under the catalysis of cellobiose dehydrogenase (CDH) from the plant-pathogenic fungus Sclerotium rolfsii. Substrate binding characteristics and kinetics of CDH for the three cellobiose azido derivatives were studied employing computational docking, steady-state and presteady-state techniques. The process of enzymatic oxidation of the cellobiose azido intermediates was optimized by using the available kinetic information. Whereas the conversion of 15 by CDH is very slow, the conversion of 5 and 11 by a regenerated, bi-enzymatic process (CDH/redox mediator/laccase/O2) is fast, quantitative and produces azido derivatives of cellobiono-1,5-lactone in an environmentally friendly, oxygen-driven process.
