CEEPC/IPM/CMSC - Abstrakt prezentace

(CEEPC/IPM/CMSC 2022 - ThP-17)
Proteomic analysis of extracellular vesicles from porcine seminal plasma - Search for fertility markers

Jakub Červenka 1,2, Jaromír Novák 1,2, Božena Bohuslavová 1, Petr Vodička 1, Helena Kupcová Skalníková 1 *

  1. Institute of Animal Physiology and Genetics of the Czech Academy of Sciences, Libechov, CZ
  2. Charles University, Faculty of Science, Department of Cell Biology, Prague, CZ

Abstrakt

Domestic pig (Sus scrofa) is considerable animal model for biomedical and translational research of various diseases and cancer. Moreover, pig has an irreplaceable role as a livestock, covering more than 30% of global meat consumption. Since the majority of sows on farms is inseminated artificially, assuring of stable fertility of boars and healthy litters is essential.
Seminal plasma is rich in proteins and is necessary for development and fertilizing capacity of sperms, thus male fertility. Seminal plasma is also enriched in extracellular vesicles, which participate in spermatozoa maturation, affect their motility, capacitation and survival.
We aimed to develop technique for isolation and proteomic characterization of small extracellular vesicles (average size 30-150 nm) from boar seminal plasma. We isolated extracellular vesicles by differential centrifugation and ultracentrifugation and verified their morphology and size by electron microscopy, flow cytometry and nanoparticle tracking analysis, as well as their purity by immunoblotting for selected markers (e.g. Alix, Lamin, UQCRC1, etc.). Using various detergents with FASP protocol we optimized protein identification and quantification with MS.
We identified almost 1,800 proteins in small extracellular vesicles (including 82% of the most often identified exosomal proteins), compared to 460 proteins in seminal plasma. Using SWATH-MS approach, we quantified almost 1,500 proteins and analyzed their abundances in small extracellular vesicles, sperms and seminal plasma, including known fertility markers.
Our workflow for isolation and MS-based proteomic characterization of extracellular vesicles is reproducible, robust and applicable to extracellular vesicle analyses from various body fluids or cell culture media.

* Korespondující autor: skalnikova@iapg.cas.cz

Poděkování:

This research was funded by the Grant Agency of the Czech Republic (19-01747S) and Operational Programme Research, Development and Education (CZ.02.1.01/0.0/0.0/16_019/0000785).


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