Skip to main content
SpringerLink
Log in

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

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Acuña-Castroviejo D, Martin M, Macías M, Escames G, León J, Khaldy H, Reiter RJ (2001) Melatonin, mitochondria, and cellular bioenergetics. J Pineal Res 30:65–74

    Article  PubMed  Google Scholar 

  • 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 436:687–698

    Article  Google Scholar 

  • Agarwal A, Said TM (2005) Oxidative stress, DNA damage and apoptosis in male infertility: a clinical approach. BJU Int 95:503–507

    Article  CAS  PubMed  Google Scholar 

  • 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 39–46

    Chapter  Google Scholar 

  • 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 232:205–219

    Article  CAS  PubMed  Google Scholar 

  • Ashrafi I, Kohram H, Ardabili FF (2013) Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of cryopreserved bull spermatozoa. Anim Reprod Sci 139:25–30

    Article  CAS  PubMed  Google Scholar 

  • Awad H, Halawa F, Mostafa T, Atta H (2006) Melatonin hormone profile in infertile males. Int J Androl 29:409–413

    Article  CAS  PubMed  Google Scholar 

  • 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 51:172–179

    Article  CAS  Google Scholar 

  • 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 44:505–512

    Article  PubMed  Google Scholar 

  • Bansal AK, Bilaspuri G (2010) Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2011:7

    Google Scholar 

  • 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 60:245–250

    Article  CAS  PubMed  Google Scholar 

  • 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 33:1371–1378

    Article  PubMed  Google Scholar 

  • 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 148:221–235

  • 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 44:265–272

    Article  CAS  PubMed  Google Scholar 

  • Cardinali DP, Pagano ES, Bernasconi PAS, Reynoso R, Scacchi P (2013) Melatonin and mitochondrial dysfunction in the central nervous system. Horm Behav 63:322–330

    Article  CAS  PubMed  Google Scholar 

  • 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 48:39–46

    Article  CAS  PubMed  Google Scholar 

  • Castroviejo D, Escames G, Carazo A, León J, Khaldy H, Reiter RJ (2002) Melatonin, mitochondrial homeostasis and mitochondrial-related diseases. Curr Top Med Chem 2:133–151

    Article  CAS  Google Scholar 

  • Diedrich K, Fauser B, Devroey P (2011) Cancer and fertility: strategies to preserve fertility. Reprod BioMed Online 22:232–248

    Article  CAS  PubMed  Google Scholar 

  • 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 26:1473–1483

    Article  CAS  PubMed  Google Scholar 

  • 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 94:1915–1917

    Article  CAS  PubMed  Google Scholar 

  • Fujinoki M (2008) Melatonin-enhanced hyperactivation of hamster sperm. Reproduction 136:533–541

    Article  CAS  PubMed  Google Scholar 

  • 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 62:40–46

    Article  CAS  PubMed  Google Scholar 

  • Galano A, Tan DX, Reiter RJ (2011) Melatonin as a natural ally against oxidative stress: a physicochemical examination. J Pineal Res 51:1–16

    Article  CAS  PubMed  Google Scholar 

  • 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 56:225–237

    Article  PubMed  Google Scholar 

  • Gavella M, Lipovac V (2000) Antioxidative effect of melatonin on human spermatozoa.Arch Androl 44:23–27

    Article  CAS  PubMed  Google Scholar 

  • Gharagozloo P, Aitken RJ (2011) The role of sperm oxidative stress in male infertility and the significance of oral antioxidant therapy. Hum Reprod 26:1628–1640

    Article  PubMed  Google Scholar 

  • 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 26:5655–5661

    Article  CAS  PubMed  Google Scholar 

  • Hardeland R (2009) Neuroprotection by radical avoidance: search for suitable agents.Molecules 14:5054–5102

    Article  CAS  PubMed  Google Scholar 

  • Hardeland R, Cardinali DP, Srinivasan V, Spence DW, Brown GM, Pandi-Perumal SR (2011) Melatonin—a pleiotropic, orchestrating regulator molecule. Prog Neurobiol 93:350–384

    Article  CAS  PubMed  Google Scholar 

  • 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 35:555-559

    Article  Google Scholar 

  • Karbownik M, Reiter RJ (2000) Antioxidative effects of melatonin in protection against cellular damage caused by ionizing radiation. Proc Soc Exp Biol Med 225:9–22

    Article  CAS  PubMed  Google Scholar 

  • 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 28:69–76

    Article  CAS  PubMed  Google Scholar 

  • Koh PO (2008a) Melatonin prevents the injury-induced decline of Akt/forkhead transcription factors phosphorylation. J Pineal Res 45:199–203

    Article  CAS  PubMed  Google Scholar 

  • Koh PO (2008b) Melatonin attenuates the focal cerebral ischemic injury by inhibiting the dissociation of pBad from 14-3-3. J Pineal Res 44:101–106

    CAS  PubMed  Google Scholar 

  • 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 12:37–41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Leon J, Acuña-Castroviejo D, Sainz RM, Mayo JC, Tan D-X, Reiter RJ (2004) Melatonin and mitochondrial function. Life Sci 75:765–790

    Article  CAS  PubMed  Google Scholar 

  • Leon J, Acuña-Castroviejo D, Escames G, Tan DX, Reiter RJ (2005) Melatonin mitigates mitochondrial malfunction. J Pineal Res 38:1–9

    Article  CAS  PubMed  Google Scholar 

  • Levy DS, Kahana JA, Kumar R (2009) AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines. Blood 113:1723–1729

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Lin Q, Liu R, Xiao W, Liu W (2010) Protective effects of ascorbate and catalase on human spermatozoa during cryopreservation. J Androl 31:437–444

    Article  CAS  PubMed  Google Scholar 

  • 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 46:188–198

    Article  PubMed  Google Scholar 

  • López LC, Acuña-Castroviejo D, Pino A del, Tejada M, Escames G (2010) Mitochondrial disorders therapy: the utility of melatonin. Open Biol J 3:53–65

  • 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 93:814–821

  • 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 14:1677–1679

    Article  PubMed  Google Scholar 

  • 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 28:242–248

    Article  PubMed  Google Scholar 

  • 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 34:348–357

    Article  PubMed  Google Scholar 

  • 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 106:1658–1665

    Article  CAS  PubMed  Google Scholar 

  • 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

  • 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 71:1828–1837

    Article  CAS  PubMed  Google Scholar 

  • 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 45:235–246

    Article  CAS  PubMed  Google Scholar 

  • 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 151:1269–1279

    Article  CAS  PubMed  Google Scholar 

  • Reiter RJ (1995) Oxidative processes and antioxidative defense mechanisms in the aging brain. FASEB J 9:526–533

    Article  CAS  PubMed  Google Scholar 

  • Reiter RJ, Tan D-x, Osuna C, Gitto E (2000) Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 7:444–458

    Article  CAS  PubMed  Google Scholar 

  • Reiter RJ, Tan D-X, Manchester LC, Paredes SD, Mayo JC, Sainz RM (2009) Melatonin and reproduction revisited. Biol Reprod 81:445–456

    Article  CAS  PubMed  Google Scholar 

  • Said TM, Gaglani A, Agarwal A (2010) Implication of apoptosis in sperm cryoinjury. Reprod BioMed Online 21:456–462

    Article  PubMed  Google Scholar 

  • Sherman J (1973) Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 24:397

    Article  CAS  PubMed  Google Scholar 

  • 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 40:101–109

    Article  CAS  PubMed  Google Scholar 

  • 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 50:310–318

    Article  CAS  PubMed  Google Scholar 

  • 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 81:1058–1066

    Article  CAS  PubMed  Google Scholar 

  • 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 42:28–42

    Article  CAS  PubMed  Google Scholar 

  • 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 24:2061–2070

    Article  CAS  PubMed  Google Scholar 

  • Tremellen K (2008) Oxidative stress and male infertility—a clinical perspective. Hum Reprod Update 14:243–258

    Article  CAS  PubMed  Google Scholar 

  • 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 56:333–342

    Article  CAS  PubMed  Google Scholar 

  • 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 80:531–535

    Article  PubMed  Google Scholar 

  • World Health Organization (2010) WHO laboratory manual for the examination and processing of human semen, 5th edn. WHO Press, Geneva

    Google Scholar 

  • 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

    Article  PubMed  PubMed Central  Google Scholar 

  • 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

  • Yoon SJ, Rahman MS, Kwon WS, Ryu DY, Park YJ, Pang MG (2016b) Proteomic identification of cryostress in epididymal spermatozoa. J Anim Sci Biotechnol 7:67

    Article  PubMed  PubMed Central  Google Scholar 

  • 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 397–409

    Chapter  Google Scholar 

Download references

Funding

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

Author information

Authors and Affiliations

  1. Department of Anatomical Sciences, School of Medicine, Tehran University of Medical Science, Building number 2, 16 Azar Street, Keshavarz Street, Tehran, 14155-6447, Iran

    Atefeh Najafi, Ensieh Salehi, Saideh Mikaeili, Farid Abolhassani, Setareh Shiasi & Ebrahim Asadi

  2. Department of Medicine, School of Medicine, Tehran University of Medical Science, Tehran, Iran

    Emmanuel Adutwum

  3. Department of Anatomical Sciences, School of Medicine, Alborz University of Medical Science, Alborz, Iran

    Abazar Yari

  4. Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran

    Fariba Dashtestani

  5. Department of Embryology at Reproductive Biomedicine Research Center, Royan Institute, Tehran, Iran

    Leila Rashki

Authors
  1. Atefeh Najafi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Emmanuel Adutwum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Abazar Yari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ensieh Salehi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saideh Mikaeili
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Fariba Dashtestani
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Farid Abolhassani
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Leila Rashki
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Setareh Shiasi
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ebrahim Asadi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ebrahim Asadi.

Ethics declarations

Declaration of interest

The authors have no conflicts of interest to declare.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Najafi, A., Adutwum, E., Yari, A. et al. Melatonin affects membrane integrity, intracellular reactive oxygen species, caspase3 activity and AKT phosphorylation in frozen thawed human sperm. Cell Tissue Res 372, 149–159 (2018). https://doi.org/10.1007/s00441-017-2743-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-017-2743-4

Keywords

Search

Navigation