Human sperm motility: a molecular study of mitochondrial DNA, mitochondrial transcription factor A gene and DNA fragmentation

  • Fabiana Faja
  • Tania Carlini
  • Giulia Coltrinari
  • Federica Finocchi
  • Matteo Nespoli
  • Francesco Pallotti
  • Andrea Lenzi
  • Francesco Lombardo
  • Donatella PaoliEmail author
Original Article


Alterations affecting the mitochondrial genome and chromatin integrity of spermatozoa impair male reproductive potential. This study aimed to evaluate the impact of mitochondrial DNA (mtDNA) copy number alterations on sperm motility and on the molecular mechanism regulating the number of mtDNA copies, through analysis of mitochondrial transcription factor A (TFAM) gene expression. It also investigated any correlation between mtDNA copy number and sperm DNA fragmentation (SDF). Sixty-three asthenozoospermic semen samples (Group A) and 63 normokinetic semen samples (Group N) were analysed according to WHO (WHO laboratory manual for the examination and processing of human semen, World Health Organization, Geneva, 2010). Sperm mtDNA copy number and TFAM gene expression were quantified by real time quantitative polymerase chain reaction. SDF was evaluated using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. The mtDNA copy number was higher in asthenozoospermic semen samples and was negatively correlated with sperm concentration, total sperm number and total motile spermatozoa. The caseload showed a global negative correlation of TFAM gene expression with total motile sperm and a positive correlation with abnormal forms, SDF and mtDNA copy number, but this was not confirmed within each subgroup. SDF was significantly increased in asthenozoospermic samples and correlated with abnormal forms. No correlation was found between SDF and mtDNA copy number. Our results suggest a potential role of mtDNA content as an indicator of semen quality and support the hypothesis that dysregulation of TFAM expression is accompanied by a qualitative impairment of spermatogenesis. Since mtDNA copy number alterations and impaired chromatin integrity could affect reproductive success, these aspects should be evaluated in relation to assisted reproductive techniques.


Mitochondrial DNA copy number Spermatozoa Asthenozoospermia Mitochondrial transcription factor A Sperm DNA fragmentation 



Mitochondrial DNA


Real time quantitative polymerase chain reaction


Cytochrome C oxidase 2


Threshold cycle


Glyceraldehyde-3-phosphate dehydrogenase


Mitochondrial transcription factor A


Sperm DNA fragmentation


Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay



The authors wish to thank Marie-Hélène Hayles for the English translation of the manuscript.


This study was funded by a Grant from the Italian Ministry of Education and Research (MIUR-PRIN 2015-2015XSNA83-002) and the University of Rome “Sapienza” Faculty of Medicine.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the Ethical Committee of “Sapienza” University of Rome: Azienda Ospedaliera Policlinico Umberto I. All procedures performed in studies involving human participants were in accordance with the Ethical Standards of the Institutional and/or National Research Committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Logan DC (2007) The mitochondrial compartment. J Exp Bot 58:1225–1243Google Scholar
  2. 2.
    Suen DF, Norris KL, Youle RJ (2008) Mitochondrial dynamics and apoptosis. Genes Dev 22:1577–1590CrossRefGoogle Scholar
  3. 3.
    Tait SW, Green DR (2012) Mitochondria and cell signalling. J Cell Sci 125:807–815CrossRefGoogle Scholar
  4. 4.
    Ramalho-Santos J, Amaral S (2013) Mitochondria and mammalian reproduction. Mol Cell Endocrinol 379:74–84CrossRefGoogle Scholar
  5. 5.
    Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457–465CrossRefGoogle Scholar
  6. 6.
    St John JC, Sakkas D, Barratt CL (2000) A role for mitochondrial DNA and sperm survival. J Androl 21:189–199Google Scholar
  7. 7.
    Rajender S, Rahul P, Mahdi AA (2010) Mitochondria, spermatogenesis and male infertility. Mitochondrion 10:419–428CrossRefGoogle Scholar
  8. 8.
    Larsson NG, Oldfors A, Holme E, Clayton DA (1994) Low levels of mitochondrial transcription factor A in mitochondrial DNA depletion. Biochem Biophys Res Commun 200:1374–1381CrossRefGoogle Scholar
  9. 9.
    Larsson NG, Oldfors A, Garman JD, Barsh GS, Clayton DA (1997) Down-regulation of mitochondrial transcription factor A during spermatogenesis in humans. Hum Mol Genet 6:185–191CrossRefGoogle Scholar
  10. 10.
    Poulton J, Morten K, Freeman-Emmerson C, Potter C, Sewry C, Dubowitz V et al (1994) Deficiency of the human mitochondrial transcription factor h-mtTFA in infantile mitochondrial myopathy is associated with mtDNA depletion. Hum Mol Genet 3:1763–1769CrossRefGoogle Scholar
  11. 11.
    Spelbrink JN, Van Galen MJ, Zwart R, Bakker HD, Rovio A, Jacobs HT et al (1998) Familial mitochondrial DNA depletion in liver: haplotype analysis of candidate genes. Hum Genet 102:327–331CrossRefGoogle Scholar
  12. 12.
    Siciliano G, Mancuso M, Pasquali L, Manca ML, Tessa A, Iudice A (2000) Abnormal levels of human mitochondrial transcription factor A in skeletal muscle in mitochondrial encephalomyopathies. Neurol Sci 21:S985–S987CrossRefGoogle Scholar
  13. 13.
    Clayton DA (2000) Transcription and replication of mitochondrial DNA. Hum Reprod 15:11–17CrossRefGoogle Scholar
  14. 14.
    Alam TI, Kanki T, Muta T, Ukaji K, Abe Y, Nakayama H, Takio K, Hamasaki N, Kang D (2003) Human mitochondrial DNA is packaged with TFAM. Nucleic Acids Res 31:1640–1645CrossRefGoogle Scholar
  15. 15.
    Garrido N, Griparic L, Jokitalo E, Wartiovaara J, van der Bliek AM, Spelbrink JN (2003) Composition and dynamics of human mitochondrial nucleoids. Mol Biol Cell 14:1583–1596CrossRefGoogle Scholar
  16. 16.
    Folgerø T, Bertheussen K, Lindal S, Torbergsen T, Oian P (1993) Mitochondrial disease and reduced sperm motility. Hum Reprod 8:863–868CrossRefGoogle Scholar
  17. 17.
    Kao SH, Chao HT, Wei YH (1995) Mitochondrial deoxyribonucleic acid 4977-bp deletion is associated with diminished fertility and motility of human sperm. Biol Reprod 52:729–736CrossRefGoogle Scholar
  18. 18.
    Kao SH, Chao HT, Wei YH (1998) Multiple deletions of mitochondrial DNA are associated with the decline of motility and fertility of human spermatozoa. Mol Hum Reprod 4:657–666CrossRefGoogle Scholar
  19. 19.
    Holyoake AJ, McHugh P, Wu M, O’Carroll S, Benny P, Sin IL, Sin FY (2001) High incidence of single nucleotide substitutions in the mitochondrial genome is associated with poor semen parameters in men. Int J Androl 24:175–182CrossRefGoogle Scholar
  20. 20.
    Ieremiadou F, Rodakis GC (2009) Correlation of the 4977 bp mitochondrial DNA deletion with human sperm dysfunction. BMC Res Notes 4:2–18Google Scholar
  21. 21.
    Spiropoulos J, Turnbull DM, Chinnery PF (2002) Can mitochondrial DNA mutations cause sperm dysfunction? Mol Hum Reprod 8:719–721CrossRefGoogle Scholar
  22. 22.
    Selvi Rani D, Vanniarajan A, Gupta NJ, Chakravarty B, Singh L, Thangaraj K (2006) A novel missense mutation C11994T in the mitochondrial ND4 gene as a cause of low sperm motility in the Indian subcontinent. Fertil Steril 86:1783–1785CrossRefGoogle Scholar
  23. 23.
    Díez-Sánchez C, Ruiz-Pesini E, Lapeña AC, Montoya J, Pérez-Martos A, Enríquez JA et al (2003) Mitochondrial DNA content of human spermatozoa. Biol Reprod 68:180–185CrossRefGoogle Scholar
  24. 24.
    May-Panloup P, Chrétien MF, Savagner F, Vasseur C, Jean M, Malthièry Y et al (2003) Increased sperm mitochondrial DNA content in male infertility. Hum Reprod 18:550–556CrossRefGoogle Scholar
  25. 25.
    Song GJ, Lewis V (2008) Mitochondrial DNA integrity and copy number in sperm from infertile men. Fertil Steril 90:2238–2244CrossRefGoogle Scholar
  26. 26.
    Gabriel MS, Chan SW, Alhathal N, Chen JZ, Zini A (2012) Influence of microsurgical varicocelectomy on human sperm mitochondrial DNA copy number: a pilot study. J Assist Reprod Genet 29:759–764CrossRefGoogle Scholar
  27. 27.
    Bonanno O, Romeo G, Asero P, Pezzino FM, Castiglione R, Burrello N et al (2016) Sperm of patients with severe asthenozoospermia show biochemical, molecular and genomic alterations. Reproduction 152:695–704CrossRefGoogle Scholar
  28. 28.
    Kao SH, Chao HT, Liu HW, Liao TL, Wei YH (2004) Sperm mitochondrial DNA depletion in men with asthenospermia. Fertil Steril 82:66–73CrossRefGoogle Scholar
  29. 29.
    Paoli D, Gallo M, Rizzo F, Baldi E, Francavilla S, Lenzi A et al (2011) Mitochondrial membrane potential profile and its correlation with increasing sperm motility. Fertil Steril 95:2315–2319CrossRefGoogle Scholar
  30. 30.
    Scarlett JL, Murphy MP (1997) Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition. FEBS Lett 418:282–286CrossRefGoogle Scholar
  31. 31.
    Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM et al (1999) Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397:441–446CrossRefGoogle Scholar
  32. 32.
    Gandini L, Lombardo F, Paoli D, Caponecchia L, Familiari G, Verlengia C et al (2000) Study of apoptotic DNA fragmentation in human spermatozoa. Hum Reprod 15:830–839CrossRefGoogle Scholar
  33. 33.
    Irvine DS, Twigg JP, Gordon EL, Fulton N, Milne PA, Aitken RJ (2000) DNA integrity in human spermatozoa: relationships with semen quality. J Androl 21:33–44Google Scholar
  34. 34.
    Aitken RJ, Krausz C (2001) Oxidative stress, DNA damage and the Y chromosome. Reproduction 122:497–506CrossRefGoogle Scholar
  35. 35.
    Varum S, Bento C, Sousa AP, Gomes-Santos CS, Henriques P, Almeida-Santos T et al (2007) Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers. Fertil Steril 87:572–583CrossRefGoogle Scholar
  36. 36.
    Carlini T, Paoli D, Pelloni M, Faja F, Dal Lago A, Lombardo F et al (2017) Sperm DNA fragmentation in Italian couples with recurrent pregnancy loss. Reprod Biomed Online 34:58–65CrossRefGoogle Scholar
  37. 37.
    World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. World Health Organization, GenevaGoogle Scholar
  38. 38.
    Paoli D, Pelloni M, Gallo M, Coltrinari G, Lombardo F, Lenzi A et al (2017) Sperm glyceraldehyde 3-phosphate dehydrogenase gene expression in asthenozoospermic spermatozoa. Asian J Androl 19:409–413CrossRefGoogle Scholar
  39. 39.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) Method. Methods 25:402–408CrossRefGoogle Scholar
  40. 40.
    Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G (1999) Ubiquitin tag for sperm mitochondria. Nature 402:371–372CrossRefGoogle Scholar
  41. 41.
    Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G (2000) Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol Reprod 63:582–590CrossRefGoogle Scholar
  42. 42.
    Sutovsky P (2003) Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 61:88–102CrossRefGoogle Scholar
  43. 43.
    Thompson WE, Ramalho-Santos J, Sutovsky P (2003) Ubiquitination of prohibitin in mammalian sperm mitochondria: possible roles in the regulation of mitochondrial inheritance and sperm quality control. Biol Reprod 69:254–260CrossRefGoogle Scholar
  44. 44.
    Lee HC, Yin PH, Lu CY, Chi CW, Wei YH (2000) Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem J 348:425–432CrossRefGoogle Scholar
  45. 45.
    Liu CS, Tsai CS, Kuo CL, Chen HW, Lii CK, Ma YS et al (2003) Oxidative stress-related alteration of the copy number of mitochondrial DNA in human leukocytes. Free Radic Res 37:1307–1317CrossRefGoogle Scholar
  46. 46.
    Lee HC, Wei YH (2005) Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. Int J Biochem Cell Biol 37:822–834CrossRefGoogle Scholar
  47. 47.
    Perez-de-Arce K, Foncea R, Leighton F (2005) Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: modulation by antioxidants. Biochem Biophys Res Commun 338:1103–1109CrossRefGoogle Scholar
  48. 48.
    Thomas RR, Khan SM, Portell FR, Smigrodzki RM, Bennett JP Jr (2011) Recombinant human mitochondrial transcription factor A stimulates mitochondrial biogenesis and ATP synthesis, improves motor function after MPTP, reduces oxidative stress and increases survival after endotoxin. Mitochondrion 11:108–118CrossRefGoogle Scholar
  49. 49.
    Tourmen Y, Baris O, Dessen P, Jacques C, Malthièry Y, Reynier P (2002) Structure and chromosomal distribution of human mitochondrial pseudogenes. Genomics 80:71–77CrossRefGoogle Scholar
  50. 50.
    Amaral A, Ramalho-Santos J, St John JC (2007) The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm. Hum Reprod 22:1585–1596CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Laboratory of Seminology-Sperm Bank, “Loredana Gandini”, Department of Experimental MedicineUniversity of Rome “Sapienza”RomeItaly

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