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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV130131	1/1	Equus caballus	Equine seminal fluid	(d)(U)C DG Gel filtration	Aalberts M	2013	38%
Study summary Full title Spermatozoa recruit prostasomes in response to capacitation induction All authors Aalberts M, Sostaric E, Wubbolts R, Wauben MW, Nolte-'t Hoen EN, Gadella BM, Stout TA, Stoorvogel W Journal Biochim Biophys Acta Proteins Proteom Abstract Seminal plasma contains various types of extracellular vesicles, including prostasomes. Prostasomes (show more...)Seminal plasma contains various types of extracellular vesicles, including prostasomes. Prostasomes are small vesicles secreted by prostatic epithelial cells that can be recruited by and fuse with sperm cells in response of progesterone that is released by oocyte surrounding cumulus cells. This delivers Ca(2+) signaling tools that allow the sperm cell to gain hypermotility and undergo the acrosome reaction. Conditions for binding of prostasomes to sperm cells are however unclear. We found that classically used prostasome markers are in fact heterogeneously expressed on distinct populations of small and large vesicles in seminal plasma. To study interactions between prostasomes and spermatozoa we used the stallion as a model organism. A homogeneous population of ~60nm prostasomes was first separated from larger vesicles and labeled with biotin. Binding of biotinylated prostasomes to individual live spermatozoa was then monitored by flow cytometry. Contrary to assumptions in the literature, we found that such highly purified prostasomes bound to live sperm only after capacitation had been initiated, and specifically at pH ?7.5. Using fluorescence microscopy, we observed that prostasomes bound primarily to the head of live sperm. We propose that in vivo, prostasomes may bind to sperm cells in the uterus, to be carried in association with sperm cells into oviduct and to fuse with the sperm cell only during the final approach of the oocyte. This article is part of a Special Issue entitled: An Updated Secretome. (hide) EV-METRIC 38% (50th 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 Equine seminal fluid Sample origin NAY Focus vesicles Prostasomes 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 (d)(U)C DG Gel filtration Protein markers EV: CD9 non-EV: Proteomics no EV density (g/ml) 1.21 Show all info Study aim Function Sample Species Equus caballus Sample Type Equine seminal fluid Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Between 10,000 g and 50,000 g Pelleting performed No Density gradient Lowest density fraction 0.4 Highest density fraction 2 Orientation Bottom-up Rotor type TLA55 Speed (g) 108000 Other Name other separation method Gel filtration Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins CD9 Characterization: Particle analysis EM EM-type transmission EM Image type Wide-field

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EV-TRACK ID	EV130131
species	Equus caballus
sample type	Equine seminal fluid
condition	NAY
separation protocol	(d)(U)C DG Gel filtration
Exp. nr.	1
EV-METRIC %	38