Dataset Information

Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles.

ABSTRACT: Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 proteins as present in all OC-EVs samples. The functional enrichment analysis using Gene Ontology terms and an Ingenuity Pathway Analysis revealed that the identified proteins were involved in several cellular metabolic processes, including translation, synthesis, expression, and protein metabolism. Notably, the proteins were also involved in critical canonical pathways with essential functions in oocyte and embryo development, such as ERK/MAPK, EIF2, PI3K/AKT, and mTOR signaling. These data would be an important resource for studying canine reproductive physiology and establishing a successful in vitro embryo production system in dogs.

SUBMITTER: Lee SH

PROVIDER: S-EPMC7926305 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles.

Lee Seok Hee SH Lira-Albarrán Saúl S Saadeldin Islam M IM

Animals : an open access journal from MDPI 20210223 2

Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 prot ...[more]

PMID: 33672125

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Project description:Coronavirus (CoV) has persistently become a global health concern causing various diseases in a wide variety of hosts, including humans, birds, and companion animals. However, the virus-mediated responses in animal hosts have not been studied extensively due to pathogenesis complexity and disease developments. Extracellular vesicles (EVs) are widely explored in viral infections for their intercellular communication, nanocarrier, and immunomodulatory properties. We proposed that coronavirus hijacks the host exosomal pathway and modulates the EV biogenesis, composition, and protein trafficking in the host. In the present study, Crandell-Rees feline kidney (CRFK) cells were infected with canine coronavirus (CCoV) in an exosome-free medium at the multiplicity of infection (MOI) of 400 infectious units (IFU) at various time points. The cell viability was significantly decreased over time, as determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Post-infection EVs were isolated, and transmission electron microscopy (TEM) showed the presence of small EVs (sEVs) after infection. NanoSight particle tracking analysis (NTA) revealed that EV sizes averaged between 100 and 200 nm at both incubation times; however, the mean size of infection-derived EVs was significantly decreased at 48 h when compared to uninfected control EVs. Quantitative analysis of protein levels performed by dot blot scanning showed that the expression levels of ACE-2, annexin-V, flotillin-1, TLR-7, LAMP, TNF-α, caspase-1, caspase-8, and others were altered in EVs after infection. Our findings suggested that coronavirus infection impacts cell viability, modulates EV biogenesis, and alters cargo composition and protein trafficking in the host, which could impact viral progression and disease development. Future experiments with different animal CoVs will provide a detailed understanding of host EV biology in infection pathogenesis and progression. Hence, EVs could offer a diagnostic and therapeutic tool to study virus-mediated host responses that could be extended to study the interspecies jump of animal CoVs to cause infection in humans.

Project description:BackgroundClinical applications have shown extracellular vesicles (EVs) to be a major paracrine effector in therapeutic responses produced by human mesenchymal stromal/stem cells (hMSCs). As the regenerative capacity of EVs is mainly ascribed to the transfer of proteins and RNA composing its cargo, and to the activity attributed by the protein surface markers, we sought to profile the protein composition of small EVs released from hMSCs to identify hMSC-EV biomarkers with potential clinical relevance.MethodsSmall EVs were produced and qualified from five human bone marrow MSC donors at low passage following a 48-h culture in exosome-depleted medium further processed by steps of centrifugation, filtration, and precipitation. Quantitative proteomic analysis comparing the protein profile of the EVs released from hMSCs and their parental cell was conducted using tandem mass tag labeling combined to mass spectrometry (LC-MS/MS) to identify enriched EV protein markers.ResultsNanoparticle tracking analysis showed no differences in the EV concentration and size among the five hMSC donors (1.83 × 1010 ± 3.23 × 109/mL), with the mode particle size measuring at 109.3 ± 5.7 nm. Transmission electron microscopy confirmed the presence of nanovesicles with bilayer membranes. Flow cytometric analysis identified commonly found exosomal (CD63/CD81) and hMSC (CD105/CD44/CD146) markers from released EVs in addition to surface mediators of migration (CD29 and MCSP). Quantitative proteomic identified 270 proteins significantly enriched by at least twofold in EVs released from hMSCs as compared to parental hMSCs, where neuropilin 1 (NRP1) was identified among 21 membrane-bound proteins regulating the migration and invasion of cells, as well as chemotaxis and vasculogenesis. Validation by western blot of multiple batches of EVs confirmed consistent enrichment of NRP1 in the nanovesicles released from all five hMSC donors.ConclusionThe identification and verification of NRP1 as a novel enriched surface marker from multiple batches of EVs derived from multiple hMSC donors may serve as a biomarker for the assessment and measurement of EVs for therapeutic uses.

Project description:BackgroundIn vitro production of bovine embryos is a well-established technology, but the in vitro culture (IVC) system still warrants improvements, especially regarding embryo quality. This study aimed to evaluate the effect of extracellular vesicles (EVs) isolated from oviductal (OF) and uterine fluid (UF) in sequential IVC on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented with either BSA or EVs-depleted fetal calf serum (dFCS) in the presence (BSA-EV and dFCS-EV) or absence of EVs from OF (D1 to D4) and UF (D5 to D8), mimicking in vivo conditions. EVs from oviducts (early luteal phase) and uterine horns (mid-luteal phase) from slaughtered heifers were isolated by size exclusion chromatography. Blastocyst rate was recorded on days 7-8 and their quality was assessed based on lipid contents, mitochondrial activity and total cell numbers, as well as survival rate after vitrification. Relative mRNA abundance for lipid metabolism-related transcripts and levels of phosphorylated hormone-sensitive lipase (pHSL) proteins were also determined. Additionally, the expression levels of 383 miRNA in OF- and UF-EVs were assessed by qRT-PCR.ResultsBlastocyst yield was lower (P < 0.05) in BSA treatments compared with dFCS treatments. Survival rates after vitrification/warming were improved in dFCS-EVs (P < 0.05). EVs increased (P < 0.05) blastocysts total cell number in dFCS-EV and BSA-EV compared with respective controls (dFCS and BSA), while lipid content was decreased in dFCS-EV (P < 0.05) and mitochondrial activity did not change (P > 0.05). Lipid metabolism transcripts were affected by EVs and showed interaction with type of protein source in medium (PPARGC1B, LDLR, CD36, FASN and PNPLA2, P < 0.05). Levels of pHSL were lower in dFCS (P < 0.05). Twenty miRNA were differentially expressed between OF- and UF-EVs and only bta-miR-148b was increased in OF-EVs (P < 0.05).ConclusionsMimicking physiological conditions using EVs from OF and UF in sequential IVC does not affect embryo development but improves blastocyst quality regarding survival rate after vitrification/warming, total cell number, lipid content, and relative changes in expression of lipid metabolism transcripts and lipase activation. Finally, EVs miRNA contents may contribute to the observed effects.

Dataset Information

Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles.

Publications

Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles.

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