Marine Biotechnology

, Volume 21, Issue 3, pp 310–319 | Cite as

Seminal Plasma Exosomes: Promising Biomarkers for Identification of Male and Pseudo-Males in Cynoglossus semilaevis

  • Bo Zhang
  • Na Zhao
  • Lei Jia
  • Kang Peng
  • Jinyuan Che
  • Kunming Li
  • Xiaoxu He
  • Jinsheng Sun
  • Baolong BaoEmail author
Original Article


In mammals, small RNAs enclosed in exosomes have been identified as appropriate signatures for disease diagnosis. However, there is limited information on exosomes derived from seminal plasma, and few studies have reported analyzed the composition of exosomes and enclosed small RNAs in fish. The half-smooth tongue sole (Cynoglossus semilaevis) is an economically important fish for aquaculture, and it exhibits sexual dimorphism: the female gender show higher growth rates and larger body sizes than males. Standard karyotype analysis and artificial gynogenesis tests have revealed that this species uses heterogametic sex determination (ZW/ZZ), and so-called sex-reversed pseudo-males exist. In this study, we successfully identified exosomes in the seminal plasma of C. semilaevis; to the best of our knowledge, this is the first report of exosomes in fish seminal plasma. Analysis of the nucleotide composition showed that miRNAs were dominant in the exosomes, and the miRNAs were sequenced and compared to identify signature miRNAs as sexual biomarkers. Moreover, target genes of the signature miRNAs were predicted by sequence matching and annotation. Finally, four miRNAs (dre-miR-141-3P, dre-miR-10d-5p, ssa-miR-27b-3p, and ssa-miR-23a-3p) with significant differential expression in the males and pseudo-males were selected from the signature candidate miRNAs as markers for sex identification, and their expression profiles were verified using real-time quantitative PCR. Our findings could provide an effective detection method for sex differentiation in fish.


Exosomes Seminal plasma MicroRNAs Sexual biomarker Cynoglossus semilaevis 



We thank Dr. Zhang Fan of OE Biotech Ltd. (Shanghai, China) for her critique of this manuscript. This work was supported by grants from the Tianjin Natural Science Foundation (17JCQNJC15000), transformation project of Tianjin Agricultural Achievements (201604090), special funding for Modern Agricultural Industrial Technology System (CARS-47-Z01), Modern Industrial Technology System in Tianjin (ITTFRS2017011), and National Natural Science Foundation of China (31872546, 31472262), the China-ASEAN Maritime Cooperation Fund through the project “China-ASEAN Center for Joint Research and Promotion of Marine Aquaculture Technology.”

Author Contributions

Bo Zhang, Baolong Bao, and Jinsheng Sun conceived and designed the study. Bo Zhang and Na Zhao performed the computational analysis and expression profiling. Lei Jia and Na Zhao performed the identification of exosome experiments. Bo Zhang and Na Zhao wrote the manuscript. All authors read and approved the final manuscript. Na Zhao and Kunming Li performed to the RT-qPCR analysis.

Compliance with Ethical Standards

All experiments were approved by the Animal Care Committee of Shanghai Ocean University.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10126_2019_9881_MOESM1_ESM.jpg (34 kb)
ESM 1 (JPG 34 kb)
10126_2019_9881_MOESM2_ESM.jpg (54 kb)
ESM 2 (JPG 54 kb)
10126_2019_9881_MOESM3_ESM.docx (21 kb)
ESM 3 (DOCX 20 kb)
10126_2019_9881_MOESM4_ESM.xls (21 kb)
ESM 4 (XLS 21 kb)
10126_2019_9881_MOESM5_ESM.xlsx (109 kb)
ESM 5 (XLSX 108 kb)
10126_2019_9881_MOESM6_ESM.xls (133 kb)
ESM 6 (XLS 133 kb)
10126_2019_9881_MOESM7_ESM.xls (1.5 mb)
ESM 7 (XLS 1514 kb)
10126_2019_9881_MOESM8_ESM.xls (953 kb)
ESM 8 (XLS 952 kb)
10126_2019_9881_MOESM9_ESM.xlsx (21 kb)
ESM 9 (XLSX 21 kb)
10126_2019_9881_MOESM10_ESM.xlsx (13 kb)
ESM 10 (XLSX 13 kb)
10126_2019_9881_MOESM11_ESM.xlsx (16 kb)
ESM 11 (XLSX 15 kb)
10126_2019_9881_MOESM12_ESM.xls (106 kb)
ESM 12 (XLS 106 kb)
10126_2019_9881_MOESM13_ESM.xls (1 kb)
ESM 13 (XLS 1 kb)


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410PubMedPubMedCentralCrossRefGoogle Scholar
  2. Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355PubMedPubMedCentralCrossRefGoogle Scholar
  3. Anders S, Huber W (2010) Differential expression analysis for sequence count data. Genome Biol 11(10):R106.
  4. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116(2):281–297CrossRefGoogle Scholar
  5. Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–216PubMedPubMedCentralCrossRefGoogle Scholar
  6. Bartel DP (2018) Metazoan microRNAs. Cell 173(1):20–26PubMedPubMedCentralCrossRefGoogle Scholar
  7. Bizuayehu TT, Babiak J, Norberg B, Fernandes JM, Johansen SD, Babiak I (2012) Sex-biased miRNA expression in Atlantic halibut (Hippoglossus hippoglossus) brain and gonads. Sex Dev 6(5):257–266PubMedCrossRefGoogle Scholar
  8. Booth AM, Fang Y, Fallon JK, Yang JM, Hildreth JE, Gould SJ (2006) Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane. J Cell Biol 172(6):923–935PubMedPubMedCentralCrossRefGoogle Scholar
  9. Chen S, Li J, Deng S, Tian Y, Wang Q, Zhuang Z, Sha Z, Xu J (2007) Isolation of female-specific AFLP markers and molecular identification of genetic sex in half-smooth tongue sole (Cynoglossus semilaevis). Mar Biotechnol 9(2):273–280PubMedCrossRefGoogle Scholar
  10. Chen S, Zhang G, Shao C, Huang Q, Liu G, Zhang P, SongW AN, Chalopin D, Volff JN et al (2014) Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat Genet 46(3):253–260PubMedCrossRefGoogle Scholar
  11. Cui Y, Wang W, Ma L, Jie J, Zhang Y, Wang H, Li H (2018) New locus reveals the genetic architecture of sex reversal in the Chinese tongue sole (Cynoglossus semilaevis). Heredity 121(4):319–326PubMedPubMedCentralCrossRefGoogle Scholar
  12. Enright AJ, John B, Gaul U, Tuschl T, Sander C, Marks DS (2003) MicroRNA targets in Drosophila. Genome Biol 5(1):R1–R9PubMedPubMedCentralCrossRefGoogle Scholar
  13. Eslamloo K, Inkpen SM, Rise ML, Andreassen R (2018) Discovery of microRNAs associated with the antiviral immune response of Atlantic cod macrophages. Mol Immunol 93:152–161PubMedCrossRefGoogle Scholar
  14. Fabian MR, Sonenberg N, Filipowicz W (2010) Regulation of mRNA translation and stability by microRNAs. Annu Rev Biochem 79(1):351–379PubMedCrossRefGoogle Scholar
  15. Fahlgren N, Carrington JC (2010) MiRNA target prediction in plants. Methods Mol Biol 592:51–57PubMedCrossRefGoogle Scholar
  16. Fang M, Huang W, Wu X, Gao Y, Ou J, Zhang X, Li Y (2018) MiR-141-3p suppresses tumor growth and metastasis in papillary thyroid cancer via targeting Yin Yang 1. Anat Rec (Hoboken, N.J.: 2007).
  17. Flynt AS, Thatcher EJ, Burkewitz K, Li N, Liu Y, Patton JG (2009) Mir-8 microRNAs regulate the response to osmotic stress in zebrafish embryos. J Cell Biol 185(1):115–127PubMedPubMedCentralCrossRefGoogle Scholar
  18. Friedländer MR, Mackowiak SD, Li N, Chen W, Rajewsky N (2012) MiRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res 40(1):37–52PubMedCrossRefGoogle Scholar
  19. Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy SR (2003) Rfam: an RNA family database. Nucleic Acids Res 31(1):439–441PubMedPubMedCentralCrossRefGoogle Scholar
  20. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ (2008) MiRBase: tools for microRNA genomics. Nucleic Acids Res 36(Database issue):D154–D158PubMedGoogle Scholar
  21. Guo H, Wei M, Liu Y, Zhu Y, Xu W, Meng L, Wang N, Shao C, Lu S, Gao F, Cui Z, Wei Z, Zhao F, Chen S (2017a) Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole (Cynoglossus semilaevis). PLoS One 12(4):e0175033PubMedPubMedCentralCrossRefGoogle Scholar
  22. Guo D, Ye Y, Qi J, Tan X, Zhang Y, Ma Y, Li Y (2017b) Age and sex differences in microRNAs expression during the process of thymus aging. Acta Biochim Biophys Sin Shanghai 49(5):1–11Google Scholar
  23. Hutcheon K, Mclaughlin EA, Stanger SJ, Bernstein IR, Dun MD, Eamens AL, Nixon B (2017) Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa. RNA Biol 14(12):00–00Google Scholar
  24. Iliev D, Jorgensen S, Rode M, Krasnov A, Harneshaug I, Jorgensen J (2010) CpG-induced secretion of MHCII beta and exosomes from salmon (Salmo salar) APCs. Dev Comp Immunol 34(1):29–41PubMedCrossRefGoogle Scholar
  25. Jing J, Wu J, Liu W, Xiong S, Ma W, Zhang J, Wang W, Gui JF, Mei J (2014) Sex-biased miRNAs in gonad and their potential roles for testis development in yellow catfish. PLoS One 9(9):e107946PubMedPubMedCentralCrossRefGoogle Scholar
  26. Keller S, Sanderson MP, Stoeck A, Altevogt P (2006) Exosomes: from biogenesis and secretion to biological function. Immunol Lett 107(2):102–108PubMedCrossRefGoogle Scholar
  27. Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12(9):735–739PubMedCrossRefGoogle Scholar
  28. Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75(5):843–854PubMedPubMedCentralCrossRefGoogle Scholar
  29. Li H, Xu W, Zhang N, Shao C, Zhu Y, Dong Z, Wang N, Jia X, Xu H, Chen S (2016) Two Figla homologues have disparate functions during sex differentiation in half-smooth tongue sole (Cynoglossus semilaevis). Sci Rep 6:28219PubMedPubMedCentralCrossRefGoogle Scholar
  30. Li J, Zhang Z, Du M, Guan Y, Yao J, Yu H, Wang B, Wang X, Wu S, Li Z (2018) MicroRNA-141-3p fosters the growth, invasion, and tumorigenesis of cervical cancer cells by targeting FOXA2. Arch Biochem Biophys 657:23–30PubMedCrossRefGoogle Scholar
  31. Liao X, Xu G, Chen S (2014) Molecular method for sex identification of half-smooth tongue sole (Cynoglossus semilaevis) using a novel sex-linked microsatellite marker. Int J Mol Sci 15(7):12952–12958PubMedPubMedCentralCrossRefGoogle Scholar
  32. Liu J, Zhang W, Du X, Jiang J, Wang C, Wang X, Zhang Q, He Y (2016) Molecular characterization and functional analysis of the GATA4 in tongue sole (Cynoglossus semilaevis). Comp Biochem Physiol B 193(2):1–8PubMedCrossRefGoogle Scholar
  33. Ma K, Yu S, Du Y, Feng S, Qiu L, Ke D, Luo M, Qiu G (2019) Construction of a genomic bacterial artificial chromosome (BAC) library for the prawn Macrobrachium rosenbergii and initial analysis of ZW chromosome-derived BAC inserts. Mar Biotechnol.
  34. Malla B, Aebersold DM, Pra AD (2018) Protocol for serum exosomal miRNAs analysis in prostate cancer patients treated with radiotherapy. J Transl Med 16(1):223–238PubMedPubMedCentralCrossRefGoogle Scholar
  35. Mei J, Yue H, Li Z, Chen B, Zhong J, Dan C, Zhou L, Gui J (2014) C1q-like factor, a target of mir-430, regulates primordial germ cell development in early embryos of Carassius auratus. Int J Biol Sci 10(1):15–24CrossRefGoogle Scholar
  36. Mishima Y, Giraldez AJ, Takeda Y, Fujiwara T, Sakamoto H, Schier AF, Inoue K (2006) Differential regulation of germline mRNAs in soma and germ cells by zebrafish miR-430. Curr Biol 16(21):2135–2142PubMedPubMedCentralCrossRefGoogle Scholar
  37. Pan Z, Li X, Zhou F, Qiang X, Gui J (2015) Identification of sex-specific markers reveals male heterogametic sex determination in Pseudobagrus ussuriensis. Mar Biotechnol 17(4):441–451PubMedCrossRefGoogle Scholar
  38. Pisitkun T, Shen RF, Knepper MA (2004) Identification and proteomic profiling of exosomes in human urin. Proc Natl Acad Sci U S A 101(36):13368–13373PubMedPubMedCentralCrossRefGoogle Scholar
  39. Shao CW, Chen SL, Scheuring CF, Xu JY, Sha ZX, Dong XL, Zhang HB (2010) Construction of two BAC libraries from half-smooth tongue sole Cynoglossus semilaevis, and identification of clones containing candidate sex-determination genes. Mar Biotechnol 12(5):558–568PubMedCrossRefGoogle Scholar
  40. Shao C, Li Q, Chen S, Zhang P, Lian J, Hu Q, Sun B, Jin L, Liu S, Wang Z, Zhao H, Jin Z, Liang Z, Li Y, Zheng Q, Zhang Y, Wang J, Zhang G (2014) Epigenetic modification and inheritance in sexual reversal of fish. Genome Res 24(4):604–615PubMedPubMedCentralCrossRefGoogle Scholar
  41. Skaftnesmo KO, Edvardsen RB, Furmanek T, Crespo D, Andersson E, Kleppe L, Taranger GL, Bogerd J, Schulz RW, Wargelius A (2017) Integrative testis transcriptome analysis reveals differentially expressed miRNAs and their mRNA targets during early puberty in Atlantic salmon. BMC Genomics 18(1):801PubMedPubMedCentralCrossRefGoogle Scholar
  42. Song J, Li Q, Yu Y, Wan S, Han L, Du S (2018) Mapping genetic loci for quantitative traits of golden shell color, mineral element contents, and growth-related traits in Pacific oyster (Crassostrea gigas). Mar Biotechnol 20(5):666–675PubMedCrossRefGoogle Scholar
  43. Sun Z, Hao T, Tian J (2017) Identification of exosomes and its signature miRNAs of male and female Cynoglossus semilaevis. Sci Rep 7(1):860PubMedPubMedCentralCrossRefGoogle Scholar
  44. Tani S, Kusakabe R, Naruse K, Sakamoto H, Inoue K (2010) Genomic organization and embryonic expression of miR-430 in medaka (Oryzias latipes): insights into the post-transcriptional gene regulation in early development. Gene 449(1):41–49PubMedCrossRefGoogle Scholar
  45. Tao W, Sun L, Shi H, Cheng Y, Jiang D, Fu B, Conte MA, Gammerdinger WJ, Kocher TD, Wang D (2016) Integrated analysis of miRNA and mRNA expression profiles in tilapia gonads at an early stage of sex differentiation. BMC Genomics 17(1):328PubMedPubMedCentralCrossRefGoogle Scholar
  46. Tino P (2009) Basic properties and information theory of Audic-Claverie statistic for analyzing cDNA arrays. BMC Bioinformatics 10(1):310PubMedPubMedCentralCrossRefGoogle Scholar
  47. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9(6):654–659PubMedCrossRefGoogle Scholar
  48. van der Pol E, Böing AN, Harrison P, Sturk A, Nieuwland R (2012) Classification, functions, and clinical relevance of extracellular vesicle. Pharmacol Rev 64(3):676–705PubMedCrossRefGoogle Scholar
  49. Wang X, Reyes JL, Chua NH, Gaasterland T (2004) Prediction and identification of Arabidopsis thaliana microRNAs and their mRNA targets. Genome Biol 5(9):1–15Google Scholar
  50. Wang K, Zhang H, Hu Q, Shao C, Chen S (2014) Expression and purification of half-smooth tongue sole (Cynoglossus semilaevis) CSDAZL protein. Protein Expr Purif 102:8–12PubMedCrossRefGoogle Scholar
  51. Wang X, Yin D, Li P, Yin S, Wang L, Jia Y, Shu X (2015) MicroRNA-sequence profiling reveals novel osmoregulatory microRNA expression patterns in catadromous eel Anguilla marmorata. PLoS One 10(8):e0136383PubMedPubMedCentralCrossRefGoogle Scholar
  52. Wang W, Liu W, Liu Q, Li B, An L, Hao R, Zhao J, Liu S, Song J (2016) Coordinated microRNA and messenger RNA expression profiles for understanding sexual dimorphism of gonads and the potential roles of microRNA in the steroidogenesis pathway in nile tilapia (Oreochromis niloticus). Theriogenology 85(5):970–978PubMedCrossRefGoogle Scholar
  53. White RJ, Collins JE, Sealy IM, Wali N, Dooley CM, Digby Z, Stemple DL, Murphy DN, Billis K, Hourlier T, Füllgrabe A, Davis MP, Enright AJ, Busch-Nentwich EM (2017) A high-resolution mRNA expression time course of embryonic development in zebrafish. eLife 6:e30860PubMedPubMedCentralCrossRefGoogle Scholar
  54. Williams AE (2008) Functional aspects of animal microRNAs. Cell Mol Life Sci 65(4):545–562PubMedCrossRefGoogle Scholar
  55. Yang Q, Lin J, Liu M, Li R, Tian B, Zhang X, Xu B, Liu M, Zhang X, Li Y, Shi H, Wu L (2016) Highly sensitive sequencing reveals dynamic modifications and activities of small RNAs in mouse oocytes and early embryos. Sci Adv 2(6):e1501482PubMedPubMedCentralCrossRefGoogle Scholar
  56. Ye Y, Li SL, Ma YY, Diao YJ, Yang L, Su MQ, Li Z, Ji Y, Wang J, Lei L, Fan WX, Li LX, Xu Y, Hao XK (2017) Exosomal miR-141-3p regulates osteoblast activity to promote the osteoblastic metastasis of prostate cancer. Oncotarget 8(55):94834–94849PubMedPubMedCentralCrossRefGoogle Scholar
  57. Yu Y, Zhang X, Yuan J, Wang Q, Li S, Huang H, Li F, Xiang J (2017) Identification of sex-determining loci in Pacific white shrimp Litopeneaus vannamei using linkage and association analysis. Mar Biotechnol 19(3):277–286PubMedCrossRefGoogle Scholar
  58. Zhang L, Liu W, Shao C, Zhang N, Li H, Liu K, Dong Z, Qi Q, Zhao W, Chen S (2014) Cloning, expression and methylation analysis of piwil2 in half-smooth tongue sole (Cynoglossus semilaevis). Mar Genomics 18:45–54PubMedCrossRefGoogle Scholar
  59. Zhang Y, Miao G, Fazhan H, Waiho K, Zheng H, Li S, Ikhwanuddin M, Ma H (2018) Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab (Charybdis feriatus). Physiol Genomics 150(5):393–405CrossRefGoogle Scholar
  60. Zhu T, Corraze G, Plagnes-Juan E, Quillet E, Dupont-Nivet M, Skiba-Cassy S (2017a) Regulation of genes related to cholesterol metabolism in rainbow trout (Oncorhynchus mykiss) fed plant-based diet. Am J Physiol Regul Integr Comp Physiol 314(1):R58–R70PubMedCrossRefGoogle Scholar
  61. Zhu Y, Hu Q, Xu W, Li H, Guo H, Meng L, Wei M, Lu S, Shao C, Wang N, Yang G, Chen S (2017b) Identification and analysis of the β-catenin1 gene in half-smooth tongue sole (Cynoglossus semilaevis). PLoS One 12(5):e0176122PubMedPubMedCentralCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources (Shanghai Ocean University, Ministry of Education; International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology; National Demonstration Center for Experimental Fisheries Science EducationShanghai Ocean UniversityShanghaiChina
  2. 2.Tianjin Bohai Sea Fisheries Research InstituteTianjinChina
  3. 3.Tianjin Sheng Fa Biotechnology Co, LtdTianjinChina

Personalised recommendations