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Characterization and identification of extracellular vesicles-coupled miRNA profiles in seminal plasma of fertile and subfertile rabbit bucks

OG. Sakr, A. Gad, K. Cañón-Beltrán, YN. Cajas, R. Prochazka, D. Rizos, PG. Rebollar

. 2023 ; 209 (-) : 76-88. [pub] 20230619

Jazyk angličtina Země Spojené státy americké

Typ dokumentu časopisecké články

Perzistentní odkaz   https://www.medvik.cz/link/bmc23016141

Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 1011 ± 1.04 × 1011 and 1.84 × 1012 ± 1.75 × 1011 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies.

Citace poskytuje Crossref.org

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$a Sakr, Osama G $u Dept. Animal Production, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt; Dept. Agrarian Production, Technical University of Madrid, 28040, Madrid, Spain
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$a Seminal plasma (SP) provides essential nutrients, transport, and protection to the spermatozoa during their journey through the male and female reproductive tracts. Extracellular vesicles (EVs) are one of the main components of the SP with several biomolecular cargoes, including miRNAs, that can influence spermatozoa functions and interact with the cells of the female reproductive tract. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SP-EVs isolated from fertile (F) and subfertile (S) rabbit bucks that could serve as fertility biomarkers. In this study, the methods to isolate and identify EVs including exosomes, from SP of 3 F and S bucks have been developed. Ultracentrifugation and size exclusion chromatography analysis were using to isolate EVs from SP of F and S males that were qualitative and quantitively characterised using transmission electron microscopy, nanoparticle tracking analysis and western blotting. In addition, total RNA, including miRNA, was isolated, sequenced and identified from SP-EVs samples. Different SP-EVs concentrations (8.53 × 1011 ± 1.04 × 1011 and 1.84 × 1012 ± 1.75 × 1011 particles/mL of SP; P = 0.008), with a similar average size (143.9 ± 11.9 and 115.5 ± 2.4 nm; P = 0.7422) in F and S males, respectively was observed. Particle size was not significantly correlated with any kinetic parameter. The concentration of SP-EVs was positively correlated with the percentage of abnormal forms (r = 0.94; P < 0.05) and with the percentage of immotile spermatozoa (r = 0.88; P < 0.05). Small-RNA-seq analysis identified a total of 267 and 244 expressed miRNAs in the F and S groups, respectively. Two miRNAs (let-7b-5p and let-7a-5p) were the top most abundant miRNAs in both groups. Differential expression analysis revealed that 9 miRNAs including miR-190b-5p, miR-193b-5p, let-7b-3p, and miR-378-3p, and another 9 miRNAs including miR-7a-5p, miR-33a-5p, miR-449a-5p, and miR-146a-5p were significantly up- and downregulated in the F compared to the S group, respectively. The SP from F and S rabbit males contains EVs with different miRNA cargo correlated with spermatogenesis, homeostasis, and infertility, which could be used as biomarkers for male fertility and potential therapies for assisted reproductive technologies.
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$a Gad, Ahmed $u Dept. Animal Production, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt; Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721, Liběchov, Czech Republic
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$a Cañón-Beltrán, Karina $u Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain; Department of Biochemistry and Molecular Biology, Veterinary Faculty, Complutense University of Madrid (UCM), Madrid, Spain
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$a Cajas, Yulia N $u Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain; Dept. de Ciencias de la Vida y la Agricultura, Universidad de las Fuerzas Armadas (ESPE), Sede, Santo Domingo, 171-5-231, Ecuador
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$a Prochazka, Radek $u Institute of Animal Physiology and Genetics, Czech Academy of Sciences, 27721, Liběchov, Czech Republic
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$a Rizos, Dimitrios $u Dept. Animal Reproduction, National Institute for Agriculture and Food, Research and Technology (INIA-CSIC), 28040, Madrid, Spain. Electronic address: drizos@inia.csic.es
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$a Rebollar, Pilar G $u Dept. Agrarian Production, Technical University of Madrid, 28040, Madrid, Spain
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