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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV220412	1/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Control condition Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 137.75 EV concentration Yes Particle yield as numer of particles per mililiter: 1.02E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 137.75

	EV220412	2/1	Bos taurus	ovary-derived granulosa cells	(d)(U)C Filtration	Menjivar, Nico G	2023	67%
Study summary Full title Granulosa cell-derived extracellular vesicles mitigate the detrimental impact of thermal stress on bovine oocytes and embryos All authors Nico G. Menjivar, Ahmed Gad, Samuel Gebremedhn, Soham Ghosh and Dawit Tesfaye Journal Front Cell Dev Biol Abstract Climate change-induced global warming results in rises in body temperatures above normal physiologic (show more...)Climate change-induced global warming results in rises in body temperatures above normal physiological levels (hyperthermia) with negative impacts on reproductive function in dairy and beef animals. Extracellular vesicles (EVs), commonly described as nano-sized, lipid-enclosed complexes, harnessed with a plethora of bioactive cargoes (RNAs, proteins, and lipids), are crucial to regulating processes like folliculogenesis and the initiation of different signaling pathways. The beneficial role of follicular fluid-derived EVs in inducing thermotolerance to oocytes during in vitro maturation (IVM) has been evidenced. Here we aimed to determine the capacity of in vitro cultured granulosa cell-derived EVs (GC-EVs) to modulate bovine oocytes’ thermotolerance to heat stress (HS) during IVM. Moreover, this study tested the hypothesis that EVs released from thermally stressed GCs (S-EVs) shuttle protective messages to provide protection against subsequent HS in bovine oocytes. For this, sub-populations of GC-EVs were generated from GCs subjected to 38.5°C (N-EVs) or 42°C (S-EVs) and supplemented to cumulus-oocyte complexes (COCs) matured in vitro at the normal physiological body temperature of the cow (38.5°C) or HS (41°C) conditions. Results indicate that S-EVs improve the survival of oocytes by reducing ROS accumulation, improving mitochondrial function, and suppressing the expression of stress-associated genes thereby reducing the severity of HS on oocytes. Moreover, our findings indicate a carryover impact from the addition of GC-EVs during oocyte maturation in the development to the blastocyst stage with enhanced viability. (hide) EV-METRIC 67% (94th percentile of all experiments on the same sample type) Reported Not reported Not applicable EV-enriched proteins Protein analysis: analysis of three or more EV-enriched proteins non EV-enriched protein Protein analysis: assessment of a non-EV-enriched protein qualitative and quantitative analysis Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected. electron microscopy images Particle analysis: inclusion of a widefield and close-up electron microscopy image density gradient Separation method: density gradient, at least as validation of results attributed to EVs EV density Separation method: reporting of obtained EV density ultracentrifugation specifics Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps antibody specifics Protein analysis: antibody clone/reference number and dilution lysate preparation Protein analysis: lysis buffer composition Study data Sample type Cell culture supernatant Sample origin Heat stress Focus vesicles extracellular vesicle Separation protocol Separation protocol Gives a short, non-chronological overview of the different steps of the separation protocol. dUC = (Differential) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture (Differential) (ultra)centrifugation Filtration Protein markers EV: CD63/ CD81/ TSG101 non-EV: CYCS Proteomics no Show all info Study aim Function/Mechanism of uptake/transfer Sample Species Bos taurus Sample Type Cell culture supernatant EV-producing cells ovary-derived granulosa cells EV-harvesting Medium EV-depleted medium Cell count 250000 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Between 50,000 g and 100,000 g Pelleting performed Yes Pelleting: rotor type SW 55 Ti Pelleting: speed (g) 120000 Wash: volume per pellet (ml) 3.5 Wash: time (min) 70 Wash: Rotor Type SW 55 Ti Wash: speed (g) 120000 Filtration steps 0.2 or 0.22 µm Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? No Detected EV-associated proteins CD63/ CD81/ TSG101 Not detected contaminants CYCS Characterization: RNA analysis RNA analysis Type (RT)-(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer) Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Median Reported size (nm) 147.95 EV concentration Yes Particle yield as numer of particles per mililiter: 1.51E+11 EM EM-type Transmission-EM Image type Close-up Report size (nm) 147.95

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EV-TRACK ID	EV220412
species	Bos taurus
sample type	Cell culture
cell type	ovary-derived granulosa cells
condition	Control condition	Heat stress
separation protocol	dUC/ Filtration	dUC/ Filtration
Exp. nr.	1	2
EV-METRIC %	67	67