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Molecular Medicine

, Volume 21, Issue 1, pp 109–122 | Cite as

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress

  • Monica Muratori
  • Lara Tamburrino
  • Sara Marchiani
  • Marta Cambi
  • Biagio Olivito
  • Chiara Azzari
  • Gianni Forti
  • Elisabetta Baldi
Research Article

Abstract

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine (8-OHdG) and malondialdehyde (MDA)), apoptosis (caspase activity and cleaved poly(ADP-ribose) polymerase (cPARP)) and sperm immaturity (creatine phosphokinase (CK) and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.

Notes

Acknowledgments

We are grateful to D Manganaro (Becton Dickinson, Milan, Italy) for precious technical assistance in the experiments of sorting spermatozoa. We also thank E Filimberti, S Degl’Innocenti and MG Fino (Azienda Ospedaliera-Universitaria Careggi), for evaluation of semen parameters. This study was supported by Regione Toscana (grant to G Forti), Ministry of Education and Scientific Research (PRIN 2009 project to E Baldi and FIRB project to S Marchiani).

Supplementary material

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Authors and Affiliations

  • Monica Muratori
    • 1
  • Lara Tamburrino
    • 1
  • Sara Marchiani
    • 1
  • Marta Cambi
    • 1
  • Biagio Olivito
    • 2
  • Chiara Azzari
    • 2
  • Gianni Forti
    • 1
  • Elisabetta Baldi
    • 1
  1. 1.Sexual Medicine and Andrology Unit, Department of Experimental, Clinical and Biomedical Sciences, Center of Excellence DeNotheUniversity of FlorenceFlorence, FirenzeItaly
  2. 2.Pediatric Section, Department of Health SciencesUniversity of Florence and Anna Meyer Children’s University HospitalFlorenceItaly

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