Dept. Animal Production, Faculty of Agriculture, Cairo University, 12613, Giza, Egypt; Dept. Agrarian Production, Technical University of Madrid, 28040, Madrid, Spain.
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.
Theriogenology. 2023 Oct 1;209:76-88. doi: 10.1016/j.theriogenology.2023.06.020. Epub 2023 Jun 19.
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 × 10 ± 1.04 × 10 and 1.84 × 10 ± 1.75 × 10 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.
精液（SP）为精子在男性和女性生殖道中的运输和保护提供必需的营养物质。细胞外囊泡（EVs）是 SP 的主要成分之一，具有多种生物分子货物，包括 miRNA，可影响精子功能并与女性生殖道细胞相互作用。本研究旨在从有生育力（F）和生育力低下（S）的兔公兔中分离、表征和鉴定 SP-EVs 中的 miRNA 表达谱，这些 EVs 可作为生育力生物标志物。在这项研究中，开发了从 F 和 S 公兔 SP 中分离和鉴定包括外泌体在内的 EVs 的方法。超速离心和分子筛层析分析用于分离 F 和 S 雄性 SP 中的 EVs，并用透射电子显微镜、纳米颗粒跟踪分析和 Western blot 对其进行定性和定量表征。此外，从 SP-EVs 样品中分离、测序并鉴定了总 RNA，包括 miRNA。在 F 和 S 雄性中观察到不同的 SP-EVs 浓度（SP 中的 8.53×10±1.04×10和 1.84×10±1.75×10 颗粒/mL；P=0.008），平均粒径相似（143.9±11.9 和 115.5±2.4nm；P=0.7422）。颗粒大小与任何动力学参数均无显著相关性。SP-EVs 的浓度与异常形态的百分比呈正相关（r=0.94；P<0.05），与非运动精子的百分比呈正相关（r=0.88；P<0.05）。Small-RNA-seq 分析分别在 F 和 S 组中鉴定出总共 267 和 244 个表达的 miRNA。两组中最丰富的 miRNA 均为 let-7b-5p 和 let-7a-5p。差异表达分析显示，9 个 miRNA（包括 miR-190b-5p、miR-193b-5p、let-7b-3p 和 miR-378-3p）和另外 9 个 miRNA（包括 miR-7a-5p、miR-33a-5p、miR-449a-5p 和 miR-146a-5p）在 F 组中均显著上调和下调与精子发生、动态平衡和不育相关的 SP-EVs，可作为男性生育力的生物标志物和辅助生殖技术的潜在治疗方法。
