Nerve growth factor in human semen: Effect of nerve growth factor on the normozoospermic men during cryopreservation process

Document Type : Original Article


1 Department of Anatomy, School of Medicine, Baqiyatallah Medical Science University, Tehran, Iran

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

3 Department of Anatomy, School of Medicine, Tehran Medical Science University, Tehran, Iran. Urology and Nephrology Research Center (UNRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Department of Infertility, Shareati Hospital, Tehran, Iran


Objective(s):Althoughroutinely applied in assisted reproductive technology, human sperm cryopreservation is not a completely successful procedure. Adverse effects of cryopreservation on the fertilization capacity, motility, morphology, and viability of spermatozoa have been proven; cryopreservation has also shown a role in sperm DNA fragmentation and infertility. The post-thaw survival of spermatozoa improved after addition of supplementation of antioxidant molecules to freezing media. Nerve growth factor (NGF) as one of the prosurvival substances has gained great attention in recent years. The aim of this study was the usage of NGF as prosurvival factor after cryopreservation process of human semen samples to assess the motility and viability of sperm, nitric oxide (NO) concentration, and DNA fragmentation in normozoospermic men.
Materials and Methods: Semen samples were collected from 25 normozoospermic men and were divided into fresh semen samples as control group, frozen–thawed semen samples without addition of exogenous NGF, and three groups of semen samples cryopreserved with addition of exogenous NGF (0.5, 1, and 5 ng/ml) in freezing medium. Viability was assessed by eosin-negrosin staining technique. Motility was evaluated with inverted microscope.  NO concentration and apoptosis content were measured with flow cytometry.
Results: Results showed that exogenous NGF at 0.5 ng/ml could significantly (P-value <0.05) influence viability, motility, nitric oxide, and DNA fragmentation content.
Conclusion: Exogenous NGF as cryoprotectant improved sperm viability and motility, increased intracellular NO concentration, and decreased apoptosis content in normal human spermatozoa.


1.   Aitken RJ, Koppers AJ. Apoptosis and DNA damage in human spermatozoa. Asian J Androl 2011; 13:36–42.
2.   Lassalle B, Testart J. Human zona pellucida recognition associated with removal of sialic acid from human sperm surface. J Reprod Fertil 1994; 101:703-711.
3.   Cheema RS, Bansal AK, Bilaspuri GS. Manganese provides antioxidant protection for sperm cryopreservation that may offer new consideration for clinical fertility. Oxid Med Cell Longev 2009; 2:152-159.
4.   Du Plessis SS, Makker K, Desai NR, Agarwal A. The impact of oxidative stress on in vitro fertilization. Expert Rev Obstet Gynecol 2008; 3:539–554.
5.   Du Plessis SS,  McAllister DA, Luu A, Savia J, Agarwal A, Lampiao F. Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels. Andrologia 2010; 42:206–210.
6.   Gadea J,  Molla M, Selles E, Marco MA, Garcia-Vazquez FA,  Gardon JC. Reduced glutathione content in human sperm is decreased after cryopreservation: Effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 2011; 62:40-46.
7.   Branco CS, Garcez ME, Pasqualotto FF, Erdtman B, Salvador M. Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen. Cryobiology 2010; 60:235–237.
8.   Kalthur G, Raj S, Thiyagarajan A, Kumar S, Kumar P, Adiga SK. Vitamin E supplementation in semen-freezing medium improves the motility and protects sperm from freeze-thaw–induced DNA damage. Fertil Steril 2011; 95:1149-1151.
9.Talaei T, Esmaeelpour T, Aekiyash F, Bahmanpour S. Effects of cryopreservation on plasma membrane Glycoconjugates of human spermatozoa. Iran J Reprod Med 2010; 8:119-124.
10. Critser JK, Arneson BW, Aaker DV, Huse-Benda AR, Ball GD. Cryopreservation of human spermatozoa. II. Post-thaw chronology of motility and zona-free hamster ova penetration. Fertil Steril 1987; 47:980-984.
11. McLaughlin EA, Ford WC, Hull MG. Motility characteristics and membrane integrity of cryopreserved human spermatozoa. J Reprod Fertil 1992; 95:527–534.
12. Alvarez JG, Storey BT. Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerols and other polyols as sole cryoprotectant. J Androl 1993; 14:199–208.
13. Mack SR, Zaneveld LJ. Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res 1987; 18:375–383.
14. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online2009; 8:184-189.
15. Snider WD. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 1994; 77:627–638.
16. Barbacid M. Structural and functional properties of the TRK family of neurotrophin Receptors. Ann N Y Acad Sci 1995; 766:442–458.
17. Levine E, Cupp AS, Skinner MK. Role of neurotrophins in rat embryonic testis morphogenesis (cord formation). Biol Reprod 2000; 62:132–142.
18.   Abir R, Fisch B, Jin S, Barnnet M, Ben-Haroush A, Felz C, et al. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod 2005; 11:229–236.
19. Muller D, Paust HJ, Middendorff R, Davidoff MS. Nerve growth factor (NGF) receptors in male reproductive organs. Adv Exp Med Biol 1997; 424:157–158.
20. Reichardt LF. Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci 2006; 361:1545-1564.
21. Li C, Zheng L, Wang C, Zhou X. Absence of nerve growth factor and comparison of tyrosine kinase receptor A levels in mature spermatozoa from oligoasthenozoospermic, asthenozoospermic and fertile men. Clinica Chimica Acta 2010; 411:1482–1486.
22. Mutter D, Middendorff R, Davidoff MS. Neurotrophic factors in the testis. Biomed Reviews 1999; 10:25-30.
23. Lipps BV. Isolation of nerve growth factor (NGF) from human body fluid; saliva, serum and urine: comparison between cobra venom and cobra serum NGF. J Nat Toxins 2000; 9:349–356.
24. Li C, Sun Y, Yi K, Ma Y, Sun Y, Zhang W, Zhou X. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function. Theriogenology 2010; 74:1615–1622.
25. Graiani G, Emanueli C, Desortes E, Van Linthout S, Pinna A, Figueroa CD, et al. Nerve growth factor promotes reparative angiogenesis and inhibits endothelial apoptosis in cutaneous wounds of type 1 diabetic mice. Diabetologia 2004; 47:1047–1054.
26. Gross SS, Wolin MS. Nitric oxide: pathophysiological mechanisms. Annu Rev Physiol 1995; 57:737–769.
27. Rosselli M, Keller PJ, Dubey RK. Role of nitric oxide in the biology, physiology and pathophysiology of Reproduction. Hum Reprod Update 1998; 4:3–24.
28. Roessner C, Paasch U, Glander HJ, Grunewald S. Activity of nitric oxide synthase in mature and immature human spermatozoa. Andrologia 2010; 42:132–137.
29. Herrero MB, Cebral E, Boquet M, Viggiano JM, Vitullo A, Gimeno MA. Effect of nitric oxide on mouse sperm hyperactivation. Acta Physiol Pharmacol Ther Latinoam 1994; 44:65–69.
30. Yeoman RR, Jones WD, Rizk BM. Evidence for nitric oxide regulation of hamster sperm. hyperactivation. J Androl 1998; 19:58–64.
31. Hellstrom WJG, Bell M, Wang R, Sikka SC. Effects of sodium nitroprusside on sperm motility, viability, and lipid Peroxidation. Fertil Steril 1994; 61:1117–1122.
32. Herrero MB, Viggiano JM, Perez-Martinez S, De Gimeno MF. Evidence that nitric oxide synthase is involved in progesterone-induced acrosomal exocytosis in mouse spermatozoa. Reprod Fertil Dev 1997; 9:433–439.
33. Zamir N, Barkan D, Keynan N, Naor Z, Breitbart H. Atrial natriuretic peptide induces acrosomal exocytosis in bovine spermatozoa. Am J Physiol 1995; 269:E216–E221.
34. Sengoku K, Tamate K, Yoshida T, Takaoka Y, Miyamoto T, Ishikawa M. Effects of low concentrations of nitric oxide on the zona pellucida binding ability of human spermatozoa. Fertil Steril 1998; 69:522–527.
35. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva, Switzerland: WHO Press; 2010.
36. Kläver R, Bleiziffer A, Redmann K, Mallidis C, Kliesch S, Gromoll J. Routine cryopreservation of spermatozoa is safe — Evidence from the DNA methylation pattern of nine spermatozoa genes. J Assist Reprod Genet 2012; 29:943–950.
37. Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA, Clark AM. Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis. Hum Reprod 2009; 24:2061–2070.
38. Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res 1989; 23:77–90.
39. Martinez-Soto JC, De DiosHourcade J, Gutierrez-Adan A, Landeras JL, Gadea J. Effect of genistein supplementation of thawing medium on characteristics of frozen human spermatozoa. Asian J Androl 2010; 12:431–441.
40. Lemma A. Effect of cryopreservation on sperm quality and fertility. Artificial Insemination Farm Animals 2011; 12:191–216.
41. Chen Y, Dicou E, Djakiew D. Characterization of nerve growth factor precursor protein expression in rat round spermatids and the trophic effects of nerve growth factor in the maintenance of Sertoli cell viability. Mol Cell Endocrinol 1997; 127:129–136.
42. Abram M, Wegmann M, Fokuhl V, Sonar S, Luger EO, Kerzel S, et al. Nerve growth factor and neurotrophin-3 mediate survival of pulmonary plasma cells during the allergic airway inflammation. J Immunol 2009; 182:4705-4712.
43. Jin WZ, Taya K. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro. J Reprod Dev 2010; 56:437–443.
44. Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J Androl 2003; 24:621–628.
45. Shi CG, Lin K, Xu XB, Zhang SC, Wang N, Fan M. Evidence for the involvement of NGF in human sperm motility. J Biomed Sci Eng 2012; 5:534-541.
46. Miraglia E, De Angelis F, Gazzano E, Hassanpour H, Bertagna A, Aldieri E, et al. Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling  pathway. Reproduction 2011; 141:47–54.
47. Nguyen TL, Kim CK, Cho JH, Lee KH, Ahn JY.  Neroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7 cells. Exp Mol Med 2010; 42:583-595.
48. Kawamoto K, Okada T, Kannan Y, Ushio H, Matsumoto M, Matsuda H. Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor. Blood 1995; 86:4638-4644.
49. Hirata Y, Meguro T, Kiuchi K. Differential effect of nerve growth factor on dopaminergic neurotoxin-induced apoptosis. J  Neurochem 2006; 99:416-425.