Advertisement

Cell and Tissue Research

, Volume 372, Issue 1, pp 149–159 | Cite as

Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm

  • Atefeh Najafi
  • Emmanuel Adutwum
  • Abazar Yari
  • Ensieh Salehi
  • Saideh Mikaeili
  • Fariba Dashtestani
  • Farid Abolhassani
  • Leila Rashki
  • Setareh Shiasi
  • Ebrahim Asadi
Regular Article

Abstract

Cryopreservation is known to induce oxidative stress in spermatozoa. Although melatonin has powerful antioxidant properties, little is known about its effects on human sperm quality during cryopreservation. The present study was undertaken to investigate the effects of melatonin treatment on human sperm parameters essential for fertilization. We first evaluated the effects of various concentrations of melatonin (0–15 mM) on human sperm parameters such as motility, viability and levels of intracellular reactive oxygen species during cryopreservation in order to identify an optimal dose with the greatest effects for further studies. Liquefied semen samples were then divided into three aliquots: cryopreserved without melatonin (control), cryopreserved with 3 mM melatonin and fresh groups. After being thawed, samples were evaluated for motility, viability, membrane integrity, intracellular reactive oxygen species levels, caspase-3 activity and AKT phosphorylation. Treatment of spermatozoa with the various concentrations of melatonin significantly increased their motility and viability and decreased their intracellular reactive oxygen species levels compared with the control group. The optimal melatonin concentration (3 mM) significantly decreased the intracellular reactive oxygen species levels, caspase-3 activity and the percentage of both dead and apoptotic-like sperm cells and increased the vitality, progressive motility and total motility and AKT phosphorylation compared with the control group. Thus, melatonin exerts protective effects against cryodamage during human spermatozoa cryopreservation and may exert its effects via the PI3K/AKT signaling pathway.

Keywords

Melatonin Sperm cryopreservation AKT phosphorylation Intracellular reactive oxygen species Caspase3 activity 

Notes

Funding

This study was supported by the Tehran University of Medical Sciences (grant no. 21614).

Compliance with ethical standards

Declaration of interest

The authors have no conflicts of interest to declare.

References

  1. Acuña-Castroviejo D, Martin M, Macías M, Escames G, León J, Khaldy H, Reiter RJ (2001) Melatonin, mitochondria, and cellular bioenergetics. J Pineal Res 30:65–74CrossRefPubMedGoogle Scholar
  2. Adam JK, Lisa AM, Ping W, Minjie L (2011) Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 436:687–698CrossRefGoogle Scholar
  3. Agarwal A, Said TM (2005) Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach. BJU Int 95:503–507CrossRefPubMedGoogle Scholar
  4. Agarwal A, Gupta S, Sharma R (2016) Basic semen analysis. In: Agarwal A, Gupta S, Sharma R (eds) Andrological evaluation of male infertility. Springer, New York, pp 39–46CrossRefGoogle Scholar
  5. Asadi E, Najafi A, Moeini A, Pirjani R, Hassanzadeh G, Mikaeili S, Salehi E, Adutwum E, Soleimani M, Khosravi F (2017) Ovarian tissue culture in the presence of VEGF and fetuin stimulates follicle growth and steroidogenesis. J Endocrinol 232:205–219CrossRefPubMedGoogle Scholar
  6. Ashrafi I, Kohram H, Ardabili FF (2013) Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa. Anim Reprod Sci 139:25–30CrossRefPubMedGoogle Scholar
  7. Awad H, Halawa F, Mostafa T, Atta H (2006) Melatonin hormone profile in infertile males. Int J Androl 29:409–413CrossRefPubMedGoogle Scholar
  8. Balao da Silva CM, Macías-García B, Miró-Morán A, González-Fernández L, Morillo-Rodriguez A, Ortega-Ferrusola C, Gallardo-Bolaños JM, Stilwell G, Tapia JA, Peña FJ (2011) Melatonin reduces lipid peroxidation and apoptotic-like changes in stallion spermatozoa. J Pineal Res 51:172–179CrossRefGoogle Scholar
  9. Banihani S, Sharma R, Bayachou M, Sabanegh E, Agarwal A (2012) Human sperm DNA oxidation, motility and viability in the presence of l-carnitine during in vitro incubation and centrifugation. Andrologia 44:505–512CrossRefPubMedGoogle Scholar
  10. Bansal AK, Bilaspuri G (2010) Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2011:7Google Scholar
  11. Bateni Z, Azadi L, Tavalaee M, Kiani-Esfahani A, Fazilati M, Nasr-Esfahani MH (2014) Addition of Tempol in semen cryopreservation medium improves the post-thaw sperm function. Syst Biol Reprod Med 60:245–250CrossRefPubMedGoogle Scholar
  12. Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Vialard F, Wainer R, Bailly M, Selva J (2012) Cryopreservation of human spermatozoa decreases the number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl 33:1371–1378CrossRefPubMedGoogle Scholar
  13. Bolaños JMG, Silva CMB da, Muñoz PM, Rodriguez AM, Dávila MP, Rodriguez-Martinez H, Aparicio IM, Tapia JA, Ferrusola CO, Pena FJ (2014) Phosphorylated AKT preserves stallion sperm viability and motility by inhibiting caspases 3 and 7. Reproduction 148:221–235Google Scholar
  14. Brugnon F, Pons-Rejraji H, Artonne C, Janny L, Grizard G (2012) The limits for detection of activated caspases of spermatozoa by Western blot in human semen. Andrologia 44:265–272CrossRefPubMedGoogle Scholar
  15. Cardinali DP, Pagano ES, Bernasconi PAS, Reynoso R, Scacchi P (2013) Melatonin and mitochondrial dysfunction in the central nervous system. Horm Behav 63:322–330CrossRefPubMedGoogle Scholar
  16. Casao A, Mendoza N, Pérez-Pé R, Grasa P, Abecia JA, Forcada F, Cebrián-Pérez JA, Muino-Blanco T (2010) Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate. J Pineal Res 48:39–46CrossRefPubMedGoogle Scholar
  17. Castroviejo D, Escames G, Carazo A, León J, Khaldy H, Reiter RJ (2002) Melatonin, mitochondrial homeostasis and mitochondrial-related diseases. Curr Top Med Chem 2:133–151CrossRefGoogle Scholar
  18. Diedrich K, Fauser B, Devroey P (2011) Cancer and fertility: strategies to preserve fertility. Reprod BioMed Online 22:232–248CrossRefPubMedGoogle Scholar
  19. Durgadoss L, Nidadavolu P, Valli RK, Saeed U, Mishra M, Seth P, Ravindranath V (2012) Redox modification of Akt mediated by the dopaminergic neurotoxin MPTP, in mouse midbrain, leads to down-regulation of pAkt. FASEB J 26:1473–1483CrossRefPubMedGoogle Scholar
  20. Espino J, Bejarano I, Ortiz Á, Lozano GM, García JF, Pariente JA, Rodríguez AB (2010) Melatonin as a potential tool against oxidative damage and apoptosis in ejaculated human spermatozoa. Fertil Steril 94:1915–1917CrossRefPubMedGoogle Scholar
  21. Fujinoki M (2008) Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 136:533–541CrossRefPubMedGoogle Scholar
  22. Gadea J, Molla M, Selles E, Marco M, Garcia-Vazquez F, Gardon J (2011) Reduced glutathione content in human sperm is decreased after cryopreservation: effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 62:40–46CrossRefPubMedGoogle Scholar
  23. Galano A, Tan DX, Reiter RJ (2011) Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res 51:1–16CrossRefPubMedGoogle Scholar
  24. García JJ, López-Pingarrón L, Almeida-Souza P, Tres A, Escudero P, García-Gil FA, Tan DX, Reiter RJ, Ramírez JM, Bernal-Pérez M (2014) Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review. J Pineal Res 56:225–237CrossRefPubMedGoogle Scholar
  25. Gavella M, Lipovac V (2000) Antioxidative effect of melatonin on human spermatozoa.Arch Androl 44:23–27CrossRefPubMedGoogle Scholar
  26. Gharagozloo P, Aitken RJ (2011) The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod 26:1628–1640CrossRefPubMedGoogle Scholar
  27. Han EK, Leverson J, McGonigal T, Shah O, Woods K, Hunter T, Giranda V, Luo Y (2007) Akt inhibitor A-443654 induces rapid Akt Ser-473 phosphorylation independent of mTORC1 inhibition. Oncogene 26:5655–5661CrossRefPubMedGoogle Scholar
  28. Hardeland R (2009) Neuroprotection by radical avoidance: search for suitable agents.Molecules 14:5054–5102CrossRefPubMedGoogle Scholar
  29. Hardeland R, Cardinali DP, Srinivasan V, Spence DW, Brown GM, Pandi-Perumal SR (2011) Melatonin—a pleiotropic, orchestrating regulator molecule. Prog Neurobiol 93:350–384CrossRefPubMedGoogle Scholar
  30. Izadpanah G, Shahneh AZ, Zhandi M, Yousefian I, Emamverdi M (2015) Melatonin has a beneficial effect on stallion sperm quality in cool condition. J Equine Vet Sci 35:555-559CrossRefGoogle Scholar
  31. Karbownik M, Reiter RJ (2000) Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc Soc Exp Biol Med 225:9–22CrossRefPubMedGoogle Scholar
  32. Karimfar M, Niazvand F, Haghani K, Ghafourian S, Shirazi R, Bakhtiyari S (2015) The protective effects of melatonin against cryopreservation-induced oxidative stress in human sperm. Int J Immunopathol Pharmacol 28:69–76CrossRefPubMedGoogle Scholar
  33. Koh PO (2008a) Melatonin prevents the injury-induced decline of Akt/forkhead transcription factors phosphorylation. J Pineal Res 45:199–203CrossRefPubMedGoogle Scholar
  34. Koh PO (2008b) Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14-3-3. J Pineal Res 44:101–106PubMedGoogle Scholar
  35. Kong P-J, Byun J-S, Lim S-Y, Lee J-J, Hong S-J, Kwon K-J, Kim S-S (2008) Melatonin induces Akt phosphorylation through melatonin receptor- and PI3K-dependent pathways in primary astrocytes. Korean J Physiol Pharmacol 12:37–41CrossRefPubMedPubMedCentralGoogle Scholar
  36. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  37. Leon J, Acuña-Castroviejo D, Sainz RM, Mayo JC, Tan D-X, Reiter RJ (2004) Melatonin and mitochondrial function. Life Sci 75:765–790CrossRefPubMedGoogle Scholar
  38. Leon J, Acuña-Castroviejo D, Escames G, Tan DX, Reiter RJ (2005) Melatonin mitigates mitochondrial malfunction. J Pineal Res 38:1–9CrossRefPubMedGoogle Scholar
  39. Levy DS, Kahana JA, Kumar R (2009) AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines. Blood 113:1723–1729CrossRefPubMedGoogle Scholar
  40. Li Z, Lin Q, Liu R, Xiao W, Liu W (2010) Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation. J Androl 31:437–444CrossRefPubMedGoogle Scholar
  41. López A, García JA, Escames G, Venegas C, Ortiz F, López LC, Acuña-Castroviejo D (2009) Melatonin protects the mitochondria from oxidative damage reducing oxygen consumption, membrane potential, and superoxide anion production. J Pineal Res 46:188–198CrossRefPubMedGoogle Scholar
  42. López LC, Acuña-Castroviejo D, Pino A del, Tejada M, Escames G (2010) Mitochondrial disorders therapy: the utility of melatonin. Open Biol J 3:53–65Google Scholar
  43. Mahfouz RZ, Plessis SS du, Aziz N, Sharma R, Sabanegh E, Agarwal A (2010) Sperm viability, apoptosis, and intracellular reactive oxygen species levels in human spermatozoa before and after induction of oxidative stress. Fertil Steril 93:814–821Google Scholar
  44. Martín M, Macías M, Escames G, León J, Acuña-Castroviejo D (2000a) Melatonin but not vitamins C and E maintains glutathione homeostasis in t-butyl hydroperoxide-induced mitochondrial oxidative stress. FASEB J 14:1677–1679CrossRefPubMedGoogle Scholar
  45. Martín M, Macías M, Escames G, Reiter R, Agapito M, Ortiz G, Acuña-Castroviejo D (2000b) Melatonin-induced increased activity of the respiratory chain complexes I and IV can prevent mitochondrial damage induced by ruthenium red in vivo. J Pineal Res 28:242–248CrossRefPubMedGoogle Scholar
  46. Martín M, Macías M, León J, Escames G, Khaldy H, Acuña-Castroviejo DO (2002) Melatonin increases the activity of the oxidative phosphorylation enzymes and the production of ATP in rat brain and liver mitochondria. Int J Biochem Cell Biol 34:348–357CrossRefPubMedGoogle Scholar
  47. Najafi A, Asadi E, Moawad AR, Mikaeili S, Amidi F, Adutwum E, Safa M, Sobhani AG (2016a) Supplementation of freezing and thawing media with brain-derived neurotrophic factor protects human sperm from freeze-thaw-induced damage. Fertil Steril 106:1658–1665CrossRefPubMedGoogle Scholar
  48. Najafi A, Amidi F, Gilani S, Moawad A, Asadi E, Khanlarkhni N, Fallah P, Rezaiian Z, Sobhani A (2016b) Effect of brain-derived neurotrophic factor on sperm function, oxidative stress and membrane integrity in human. Andrologia 49:e12601.  https://doi.org/10.1111/and.12601
  49. Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJ, Glander H-J, Agarwal A (2004) Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biol Reprod 71:1828–1837CrossRefPubMedGoogle Scholar
  50. Peyrot F, Ducrocq C (2008) Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species. J Pineal Res 45:235–246CrossRefPubMedGoogle Scholar
  51. Pujianto DA, Curry BJ, Aitken RJ (2010) Prolactin exerts a prosurvival effect on human spermatozoa via mechanisms that involve the stimulation of Akt phosphorylation and suppression of caspase activation and capacitation. Endocrinology 151:1269–1279CrossRefPubMedGoogle Scholar
  52. Reiter RJ (1995) Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J 9:526–533CrossRefPubMedGoogle Scholar
  53. Reiter RJ, Tan D-x, Osuna C, Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 7:444–458CrossRefPubMedGoogle Scholar
  54. Reiter RJ, Tan D-X, Manchester LC, Paredes SD, Mayo JC, Sainz RM (2009) Melatonin and reproduction revisited. Biol Reprod 81:445–456CrossRefPubMedGoogle Scholar
  55. Said TM, Gaglani A, Agarwal A (2010) Implication of apoptosis in sperm cryoinjury. Reprod BioMed Online 21:456–462CrossRefPubMedGoogle Scholar
  56. Sherman J (1973) Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 24:397CrossRefPubMedGoogle Scholar
  57. Siu AW, Maldonado M, Sanchez-Hidalgo M, Tan DX, Reiter RJ (2006) Protective effects of melatonin in experimental free radical-related ocular diseases. J Pineal Res 40:101–109CrossRefPubMedGoogle Scholar
  58. Succu S, Berlinguer F, Pasciu V, Satta V, Leoni GG, Naitana S (2011) Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner. J Pineal Res 50:310–318CrossRefPubMedGoogle Scholar
  59. Succu S, Pasciu V, Manca ME, Chelucci S, Torres-Rovira L, Leoni GG, Zinellu A, Carru C, Naitana S, Berlinguer F (2014) Dose-dependent effect of melatonin on postwarming development of vitrified ovine embryos. Theriogenology 81:1058–1066CrossRefPubMedGoogle Scholar
  60. Tan DX, Manchester LC, Terron MP, Flores LJ, Reiter RJ (2007) One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species? J Pineal Res 42:28–42CrossRefPubMedGoogle Scholar
  61. Thomson L, Fleming S, Aitken R, De Iuliis G, Zieschang J-A, Clark A (2009) Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod 24:2061–2070CrossRefPubMedGoogle Scholar
  62. Tremellen K (2008) Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 14:243–258CrossRefPubMedGoogle Scholar
  63. Wang F, Tian X, Zhang L, Gao C, He C, Fu Y, Ji P, Li Y, Li N, Liu G (2014) Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1. J Pineal Res 56:333–342CrossRefPubMedGoogle Scholar
  64. Wang X, Sharma RK, Sikka SC, Thomas AJ, Falcone T, Agarwal A (2003) Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 80:531–535CrossRefPubMedGoogle Scholar
  65. World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. WHO Press, GenevaGoogle Scholar
  66. Yoon SJ, Kwon WS, Rahman MS, Lee JS, Pang MG (2015) A novel approach to identifying physical markers of cryo-damage in bull spermatozoa. PLoS One 10:e0126232.  https://doi.org/10.1371/journal.pone.0126232 CrossRefPubMedPubMedCentralGoogle Scholar
  67. Yoon SJ, Rahman MS, Kwon WS, Park YJ, Pang MG (2016a) Addition of cryoprotectant significantly alters the epididymal sperm proteome. PLoS One 11:e0152690.  https://doi.org/10.1371/journal.pone.0152690
  68. Yoon SJ, Rahman MS, Kwon WS, Ryu DY, Park YJ, Pang MG (2016b) Proteomic identification of cryostress in epididymal spermatozoa. J Anim Sci Biotechnol 7:67CrossRefPubMedPubMedCentralGoogle Scholar
  69. Yu D, Simon L, Lewis SE (2011) The impact of sperm processing and cryopreservation on sperm DNA integrity. In: Zini A, Agarwal A (eds) Sperm chromatin. Springer, New York, pp 397–409CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  • Atefeh Najafi
    • 1
  • Emmanuel Adutwum
    • 2
  • Abazar Yari
    • 3
  • Ensieh Salehi
    • 1
  • Saideh Mikaeili
    • 1
  • Fariba Dashtestani
    • 4
  • Farid Abolhassani
    • 1
  • Leila Rashki
    • 5
  • Setareh Shiasi
    • 1
  • Ebrahim Asadi
    • 1
  1. 1.Department of Anatomical Sciences, School of MedicineTehran University of Medical ScienceTehranIran
  2. 2.Department of Medicine, School of MedicineTehran University of Medical ScienceTehranIran
  3. 3.Department of Anatomical Sciences, School of MedicineAlborz University of Medical ScienceAlborzIran
  4. 4.Institute of Biochemistry and BiophysicsTehran UniversityTehranIran
  5. 5.Department of Embryology at Reproductive Biomedicine Research CenterRoyan InstituteTehranIran

Personalised recommendations