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Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection?

  • Gamete Biology
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro.

Methods

Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level.

Results

The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed.

Conclusions

The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

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References

  1. Cottell E, Harrison RF, McCaffrey M, Walsh T, Mallon E, Barry-Kinsella C. Are seminal fluid microorganisms of significance or merely contaminants? Fertil Steril. 2000;74:465–70.

    Article  CAS  PubMed  Google Scholar 

  2. Moretti E, Capitani S, Figura N, Pammolli A, Federico MG, Giannerini V, et al. The presence of bacteria species in semen and sperm quality. J Assist Reprod Genet. 2009;26:47–56.

    Article  PubMed Central  PubMed  Google Scholar 

  3. Barraud-Lange V, Pont JC, Pocate K, Kunstmann JM, Chalas-Boissonas C, Ducot B, et al. Seminal leukocytes and clinical outcomes with donor sperm insemination. Fertil Steril. 2011;96:1320–4. e1.

    Article  PubMed  Google Scholar 

  4. Cavagna M, Oliveira JB, Petersen CG, Mauri AL, Silva LF, Massaro FC, et al. The influence of leukocytospermia on the outcomes of assisted reproductive technology. Reprod Biol Endocrinol. 2012;10:44. doi:10.1186/1477-7827-10-44.

    Article  PubMed Central  PubMed  Google Scholar 

  5. Villegas J, Schulz M, Soto L, Sanchez R. Bacteria induce expression of apoptosis in human spermatozoa. Apoptosis. 2005;10:105–10.

    Article  CAS  PubMed  Google Scholar 

  6. Allam JP, Fronhoffs F, Fathy A, Novak N, Oltermann I, Bieber T, et al. High percentage of apoptotic spermatozoa in ejaculates from men with chronic genital tract inflammation. Andrologia. 2008;40:329–34.

    Article  PubMed  Google Scholar 

  7. Domes T, Lo KC, Grober ED, Mullen JB, Mazzulli T, Jarvi K. The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters. Fertil Steril. 2012;97:1050–5.

    Article  PubMed  Google Scholar 

  8. La Vignera S, Condorelli R, D’Agata R, Vicari E, Calogero AE. Semen alterations and flow-citometry evaluation in patients with male accessory gland infections. J Endocrinol Investig. 2012;35:219–23.

    Google Scholar 

  9. Grassmé H, Jendrossek V, Gulbins E. Molecular mechanisms of bacteria induced apoptosis. Apoptosis. 2001;6:441–5.

    Article  PubMed  Google Scholar 

  10. Diemer T, Weidner W, Michelmann HW, Schiefer HG, Rovan E, Mayer F. Influence of Escherichia coli on motility parameters of human spermatozoa in vitro. Int J Androl. 1996;19:271–7.

    Article  CAS  PubMed  Google Scholar 

  11. Köhn FM, Erdmann I, Oeda T, el Mulla KF, Schiefer HG, Schill WB. Influence of urogenital infections on sperm functions. Andrologia. 1998;30:73–80.

    Article  PubMed  Google Scholar 

  12. Eley A, Hosseinzadeh S, Hakimi H, Geary I, Pacey AA. Apoptosis of ejaculated human sperm is induced by co-incubation with Chlamydia trachomatis lipopolysaccharide. Hum Reprod. 2005;20:2601–7.

    Article  CAS  PubMed  Google Scholar 

  13. Satta A, Stivala A, Garozzo A, Morello A, Perdichizzi A, Vicari E, et al. Experimental Chlamydia trachomatis infection causes apoptosis in human sperm. Hum Reprod. 2006;21:134–7.

    Article  PubMed  Google Scholar 

  14. Berktas M, Aydin S, Yilmaz Y, Cecen K, Bozkurt H. Sperm motility changes after coincubation with various uropathogenic microorganisms: an in vitro experimental study. Int Urol Nephrol. 2008;40:383–9.

    Article  PubMed  Google Scholar 

  15. Schulz M, Sánchez R, Soto L, Risopatrón J, Villegas J. Effect of Escherichia coli and its soluble factors on mitochondrial membrane potential, phosphatidylserine translocation, viability, and motility of human spermatozoa. Fertil Steril. 2010;94:619–23.

    Article  CAS  PubMed  Google Scholar 

  16. Fraczek M, Piasecka M, Gaczarzewicz D, Szumala-Kakol A, Kazienko A, Lenart S, et al. Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus. Andrologia. 2012;44:315–29.

    Article  CAS  PubMed  Google Scholar 

  17. Fraczek M, Wiland E, Piasecka M, Boksa M, Gaczarzewicz D, Szumala-Kakol A, et al. Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection. Fertil Steril. 2014;102:711–9. e1.

    Article  PubMed  Google Scholar 

  18. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO Press; 2010.

    Google Scholar 

  19. Endtz AW. A rapid staining method for differentiating granulocytes from germinal cells in Papanicolau-stained semen. Acta Cytol. 1974;18:2–7.

    CAS  PubMed  Google Scholar 

  20. Fraczek M, Szumala-Kakol A, Jedrzejczak P, Kamieniczna M, Kurpisz M. Bacteria trigger oxygen radical release and sperm lipid peroxidation in in vitro model of semen inflammation. Fertil Steril. 2007;88:1076–85.

    Article  PubMed  Google Scholar 

  21. Beutin L, Miko A, Krause G, Pries K, Haby S, Steege K, et al. Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes. Appl Environ Microbiol. 2007;73:4769–75.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Piasecka M, Kawiak J. Sperm mitochondria of patients with normal sperm motility and with asthenozoospermia: morphological and functional study. Folia Histochem Cytobiol. 2003;41:125–39.

    PubMed  Google Scholar 

  23. Mitchell LA, De Iuliis GN, Aitken RJ. The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology. Int J Androl. 2011;34:2–13.

    Article  CAS  PubMed  Google Scholar 

  24. Fraczek M, Szumala-Kakol A, Dworacki G, Sanocka D, Kurpisz M. In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation. J Reprod Immunol. 2013;100:76–85.

    Article  CAS  PubMed  Google Scholar 

  25. Muratori M, Porazzi I, Luconi M, Marchiani S, Forti G, Baldi E. AnnexinV binding and merocyanine staining fail to detect human sperm capacitation. J Androl. 2004;25:797–810.

    CAS  PubMed  Google Scholar 

  26. Martin G, Sabido O, Durand P, Levy R. Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction. Hum Reprod. 2005;20:3459–68.

    Article  CAS  PubMed  Google Scholar 

  27. Marchetti P, Ballot C, Jouy N, Thomas P, Marchetti C. Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome. Andrologia. 2012;44:136–41.

    Article  CAS  PubMed  Google Scholar 

  28. Marchetti C, Jouy N, Leroy-Martin B, Defossez A, Formstecher P, Marchetti P. Comparison of four fluorochromes for the detection of the inner mitochondrial membrane potential in human spermatozoa and their correlation with sperm motility. Hum Reprod. 2004;19:2267–76.

    Article  PubMed  Google Scholar 

  29. Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti P. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod. 2002;17:1257–65.

    Article  PubMed  Google Scholar 

  30. Wang X, Sharma RK, Gupta A, George V, Thomas AJ, Falcone T, et al. Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study. Fertil Steril. 2003;80:844–50.

    Article  PubMed  Google Scholar 

  31. Barroso G, Taylor S, Morshedi M, Manzur F, Gaviño F, Oehninger S. Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations. Fertil Steril. 2006;85:149–54.

    Article  CAS  PubMed  Google Scholar 

  32. Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin EA, Aitken RJ. Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab. 2008;93:3199–207.

    Article  CAS  PubMed  Google Scholar 

  33. Espinoza JA, Schulz MA, Sánchez R, Villegas JV. Integrity of mitochondrial membrane potential reflects human sperm quality. Andrologia. 2009;41:51–4.

    Article  CAS  PubMed  Google Scholar 

  34. Aitken RJ, Baker MA. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int J Dev Biol. 2013;57:265–72.

    Article  CAS  PubMed  Google Scholar 

  35. Burrello N, Salmeri M, Perdichizzi A, Bellanca S, Pettinato G, D’Agata R, et al. Candida albicans experimental infection: effects on human sperm motility, mitochondrial membrane potential and apoptosis. Reprod Biomed Online. 2009;18:496–501.

    Article  PubMed  Google Scholar 

  36. Collodel G, Baccetti B, Capitani S, Moretti E. Necrosis in human spermatozoa. I. Ultrastructural features and FISH study in semen from patients with uro-genital infections. J Submicrosc Cytol Pathol. 2005;37:67–73.

    CAS  PubMed  Google Scholar 

  37. Lu Y, Bhushan S, Tchatalbachev S, Marconi M, Bergmann M, Weidner W, et al. Necrosis is the dominant cell death pathway in uropathogenic Escherichia coli elicited epididymo-orchitis and is responsible for damage of rat testis. PLoS One. 2013;8:e52919.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Boguen R, Uribe P, Treulen F, Villegas JV. Distinct isolates of uropathogenic Escherichia coli differentially affect human sperm parameters in vitro. Andrologia. 2013;46:943–7.

    Article  PubMed  Google Scholar 

  39. Rodin DM, Larone D, Goldstein M. Relationship between semen cultures, leukospermia, and semen analysis in men undergoing fertility evaluation. Fertil Steril. 2003;79:1555–8.

    Article  PubMed  Google Scholar 

  40. Stovall DW, Bailey LE, Talbert LM. The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates. Fertil Steril. 1993;59:197–201.

    CAS  PubMed  Google Scholar 

  41. Eggert-Kruse W, Probst S, Rohr G, Aufenanger J, Runnebaum B. Screening for subclinical inflammation in ejaculates. Fertil Steril. 1995;64:1012–22.

    CAS  PubMed  Google Scholar 

  42. Krzymińska S, Szczuka E, Kaznowski A. Staphylococcus haemolyticus strains target mitochondria and induce caspase-dependent apoptosis of macrophages. Antonie Van Leeuwenhoek. 2012;102:611–20.

    Article  PubMed Central  PubMed  Google Scholar 

  43. Balmelli T, Stamm J, Dolina-Giudici M, Peduzzi R, Piffaretti-Yanez A, Balerna M. Bacteroides ureolyticus in men consulting for infertility. Andrologia. 1994;26:35–8.

    Article  CAS  PubMed  Google Scholar 

  44. Aitken RJ, Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl. 2011;13:36–42.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Villegas J, Schulz M, Soto L, Iglesias T, Miska W, Sánchez R. Influence of reactive oxygen species produced by activated leukocytes at the level of apoptosis in mature human spermatozoa. Fertil Steril. 2005;83:808–10.

    Article  CAS  PubMed  Google Scholar 

  46. Zorn B, Ihan A, Kopitar AN, Kolbezen M, Sesek-Briski A, Meden-Vrtovec H. Changes in sperm apoptotic markers as related to seminal leukocytes and elastase. Reprod Biomed Online. 2010;21:84–92.

    Article  CAS  PubMed  Google Scholar 

  47. Mupfiga C, Fisher D, Kruger T, Henkel R. The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa. Syst Biol Reprod Med. 2013;59:304–11.

    Article  CAS  PubMed  Google Scholar 

  48. Said T, Agarwal A, Grunewald S, Rasch M, Baumann T, Kriegel C, et al. Selection of nonapoptotic spermatozoa as a new tool for enhancing assisted reproduction outcomes: an in vitro model. Biol Reprod. 2006;74:530–7.

    Article  CAS  PubMed  Google Scholar 

  49. Muratori, M, Tamburrino L, Marchiani S, Azzari C, Forti G, Baldi E. Investigation of the origin of sperm DNA fragmentation: the role of apoptosis, immaturity and oxidative stress. Proceedings of the 8th Congress of the European Academy of Andrology; 2014 October 15–17; Barcelona, Spain. American Society of Andrology and European Academy of Andrology.

  50. Fourie J, Loskutoff N, Huyser C. Elimination of bacteria from human semen during sperm preparation using density gradient centrifugation with a novel tube insert. Andrologia. 2012;44:513–7.

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors wish to thank Anna Czernikiewicz, M.Sc. for blood sample processing. Tomasz Kolanowski is a scholarship recipient of EU 8.2.2 OP- Innovative Economy.

Grant support

This work was financed by Ministry of Science and Higher Education grant No. NN 407283539 and National Centre for Research and Development grant No. NR 13006606.

Ethical statement

This study was approved by the Local Bioethical Committee of Medical University of Poznan, Poland. Informed consent was obtained from all the participants included to the study.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Reproductive Biology and Stem Cells, Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-685, Poznan, Poland

    Monika Fraczek, Tomasz J. Kolanowski & Maciej Kurpisz

  2. Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Dojazd 11, 60-632, Poznan, Poland

    Magdalena Hryhorowicz

  3. Department of Animal Reproduction, Biotechnology and Environmental Hygiene, West Pomeranian University of Technology, Doktora Judyma 6, 71-446, Szczecin, Poland

    Dariusz Gaczarzewicz

  4. Unit of Microbiology, Hospital Medical College, Polna 33, 60-535, Poznan, Poland

    Anna Szumala-Kakol

  5. National Reference Laboratory for Escherichia Coli, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277, Berlin, Germany

    Lothar Beutin

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  1. Monika Fraczek
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  2. Magdalena Hryhorowicz
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  3. Dariusz Gaczarzewicz
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Correspondence to Maciej Kurpisz.

Additional information

Capsule Ejaculated human spermatozoa undergo complete apoptosis and necrosis during semen bacterial infection; it is possible to induce sperm apoptosis/necrosis in the presence of conditionally pathogenic bacterial strains.

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Fraczek, M., Hryhorowicz, M., Gaczarzewicz, D. et al. Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection?. J Assist Reprod Genet 32, 771–779 (2015). https://doi.org/10.1007/s10815-015-0462-x

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  • DOI: https://doi.org/10.1007/s10815-015-0462-x

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