Effect of vitamin D on apoptotic marker, reactive oxygen species and human sperm parameters during the process of cryopreservation

Document Type : Original Article

Authors

1 Cellular and Molecular Research center, Ahvaz Jundishapur University of Medical Sciences

2 Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Fertility, Infertility and Perinatology center, Imam Khomeini Hospital, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

Abstract

Objective(s): Sperm cryopreservation plays an undeniable role in assisted reproductive technology. However, this process significantly reduces the motility, viability, morphology and nuclear integrity of sperm. Reasons of these changes were oxidative stress and apoptosis. The aim of this study was to evaluate the influence of vitamin D on the survival and integrity of fertile sperm after cryopreservation.
Materials and Methods: Semen sample of 18 males with normal parameters was used. After swimming up, each sample was divided into two parts. 20 µmol vitamin D was added to one part as experimental group and the other part was left untreated as control group. The samples in all groups were frozen for 14 days. Post-thawing, the groups were evaluated for sperm motility, and viability using eosin staining, morphology using the Diff-Quick staining and apoptosis by TUNEL, Annexin-V and caspase-3 activity assay. By using nitrobluetetraxolium test and thiobarbituric acid, the reactive oxygen species (ROS) and lipid peroxidation of sperms were measured, respectively.
Results: In comparison with control groups, motile and viable sperm concentration was substantially higher in treated groups (P-value<0.05); however, morphological analysis did not show any remarkable changes. Also, ROS and lipid peroxidation values were dramatically reduced by vitamin D (P-value<0.05). TUNEL and Annexin assay for apoptosis were considerably lower in treated groups (P-value<0.05), but caspase activity assay revealed no significant difference between groups.
Conclusion: The results have shown that the addition of vitamin D to a freezing medium leads to higher quality and function of human sperm.

Keywords

Main Subjects

References

1. Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. J Assist Reprod Genet 2008; 25:403-411.
2. Holt W. Alternative strategies for the long-term preservation of spermatozoa. Reprod Fertil Dev 1997; 9:309-319.
3. Said T, Gaglani A, Agawal A. Implication of apoptosis in sperm cryoinjury. Reprod Biomed Online 2010; 21:456-462.
4. Di Santo M, Tarozzi N, Nadalini M, Borini A. Human sperm cryopreservation: update on techniques, effect on DNA integrity, and implications for ART. Adv Urol 2012 ;2012:3.
5. Serviddio G, Bellanti F, Vendemiale G. Free radical biology for medicine: learning from nonalcoholic fatty liver disease. Free Radic Biol Med 2013; 65: 952-968.
6. Hess RA, Renato De, Franca L. Spermatogenesis and cycle of the seminiferous epithelium. Adv Exp Med Biol 2008; 636:1-15.
7. Glander HJ, Schaller J. Hidden effects of cryopreservation on quality of human spermatozoa. Cell Tissue Bank 2000; 1:133-142
8. Sinclair S.  Male infertility nutritional and environmental considerations. Altern Med Rev 2000;5:28-38
9. Kalthur G, Adiga SK, Upadhya D, Rao S, Kumar P. Effect of cryopreservation on sperm DNA integrity in patients with teratozoospermia. Fertil Steril 2008; 89:1723-1727.
10. Galatioto GP, Gravina GL, Angelozzi G, Sacchetti A, Innominato PF, Pace G, et al. May antioxidant therapy improve sperm parameters of men with persistent oligospermia after retrograde embolization for varicocele? World J Urol 2008; 26: 97-102.
11. Inpanbutr N, Reiswig J, Bacon W, Slemons R, Iacopino A. Effect of vitamin D on testicular CaBP28K expression and serum testosterone in chickens. Biol Reprod 1996; 54:242–248.
12. Blomberg Jensen M, Jorgensen A, Nielsen JE, Bjerrum PJ, Skalkam M, Petersen JH, et al. Expression of the vitamin D metabolizing enzyme CYP24A1 at the an-nulus of human spermatozoa may serve as a novel marker of semen quality. Int J Androl. 2012; 35:499-510.
13. Esteves S, Sharma R, Thomas A, Agarwal A. Cryopreservation of human spermatozoa with pentoxifylline improves the post-thaw agonist induced acrosome reaction rate. Hum Reprod 1998. 13: 3384-3389.
14. WHO. WHO laboratory manual for the examination and processing of human semen. Geneva: WHO Press; 2010.
15. Oral E, Yetis O, Elibol F, Senol H, Irez T, Aksu FM. Assessment of human sperm morphology by strict criteria: comparison of wet preparation versus stained with the modified Diff-Quick method. Arch Androl 2002; 48:307-314.
16. Fanaei H, Khayat S, Halvaei I, Ramezani V, Azizi Y, Kasaeian A, et al. Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples. Iran j reprod med 2014; 12:103-110.
17. Bjorndahl L, Söderlund I, Kvist U. Evaluation of the one-step Eosin–nigrosin staining technique for human sperm vitality assessment. Hum Reprod 2003; 4:813–816.
18. Tahmineh P, Gholamhossein F. Vitrification induced apoptosis in spermatozoa from fertile and subfertile men. Iran J Repod Med 2007; 5: 117-120.
19. Muratori M, Tamburrino L, Tocci V, Costantino A, Marchiani S, Giachini C, et al. Small variations in crucial steps of TUNEL assay coupled to flowcytometry greatly affect measures of sperm DNA fragmentation. J Androl 2013; 31: 45-336.
20. Armstrong R, Aja T, Hoang K, Gaur S, Bai X, Alnemri E, et al. Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J Neurosci 1997; 17: 553- 562.
21. Esfandiari N, Sharma RK, Saleh RA, Thomas AJ, Agarwal A. Utility of the nitrobluetetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa. J Androl 2003; 24:862-870.
22. Bilodeau JF, Chatterjee S, Sirard MA, Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev 2000; 55: 282–288.
23. Bucak MN, Atessahin A, Yuce A. Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Ruminant Res 2008; 75: 128–134.
24. Bansal AK, Bilaspuri GS. Impacts of oxidative stress and antioxidants on semen functions. Vet Med Int 2010.
25. Athayde KS, Cocuzza M, Agarwal A, Krajcir N, Lucon AM, Srougi M, et al. Development of normal reference values for seminal reactive oxygen species and their correlation with leukocytes and semen parameters in a fertile population. J Androl 2007; 28:613-620.
26. Surai P, Kostjuk I, Wishart G. Effect of vitamin E and selenium supplementation of cockerel diets on glutathione peroxidase activity and lipid peroxidation susceptibility in sperm, testes, and liver. Biol Trace Elem Res 1998; 64:119–132.
27. Luo HL, Jia ZH, Zhu SE. Effect of vitamin E on the qualities of fresh and frozen thawed ram semen. China Herbivores 2004; 24:14–16.
28. Labudzynskyi DO, Zaitseva OV, Latyshko NV, Gudkova O, Veliky M. Vitamin D3 contribution to the regulation of oxidative metabolism in the liver of diabetic mice. Ukr Biochem J 2015; 87:75-90.
29. Gorlach A, Brandes R, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res 2000; 87:26-32.
30. Zhong W, Gu B, Gu Y, Groome LJ, Sun J, Wang Y. Activation of vitamin d receptor promotes VEGF and CuZn-SOD expression in endothelial cells. J Steroid Biochem Mol Biol 2014; 140:56-62.
31. Glander HJ, Schaller J. Binding of Annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. Mol Hum Reprod 1999; 5:109–115.
32. Garner DL, Johnson LA. Viability assessment of mammalian sperm using Sybr-14 and propidium iodide. Biol Rep 1995; 53:276–284.
33. Hu JH, Zhao XL, Tian WQ, Zan LS, Li QW. Effects of vitamin E supplementation in the extender on frozen- thawed bovine semen preservation. Animal 2011; 5: 107–112
34. Mokhtar Z, Hekmatdoost A, Nourian M. Antioxidant efficacy of vitamin D. J Parathyr Dis  2017;5:11–16
35. Wiseman H. Vitamin D is a membrane antioxidant. Ability to inhibit iron-dependent lipid peroxidation in liposomes compared to cholesterol, ergosterol and tamoxifen and relevance to anticancer action. FEBS Lett 1993; 326:285-288.
36. Moustafa MH, Sharma RK, Thornton J, Mascha E, Abdel-Hafez MA, Thomas AJ Jr, et al. Relationship between ROS production, apoptosis and DNA denaturation in spermatozoa from patients examined for infertility. Hum Reprod 2004; 19:129–138.
37. Smith S, Zwart S, Block G, Rice B. The nutritional status of astronauts is altered after long-term space flight aboard the International Space Station. J Nutr 2005; 135: 437–443.
38. Eghbali M, Alavi-Shoushtari SM, Asri-Rezaei S, Khadem Ansari MH. Calcium, magnesium and total antioxidant capacity (TAC) in seminal plasma of water buffalo (BubalusBubalis) bulls and their relationships with semen characteristics. Veter Res Forum 2012; 1: 12-20.
39. Manion MK, Hockenbery D. Targeting BCL-2-related proteins in cancer therapy. Cancer Biol Ther 2003; 2:105–114.
40. Wundrich K, Paasch U, Leicht M. Activation of caspases in human spermatozoa during  cryopreservation – an immunoblot study. Cell Tissue Bank 2006; 7: 81–90.
41. Kalthur G, Adiga SK, Upadhya D, et al. Effect of cryopreservation on sperm DNA integrity in patients with teratospermia. Fertil Steril 2008; 89: 1723-1727.
42.Isachenko E, Isachenko V, Weiss J. Acrosomal status and mitochondrial activity of human spermatozoa vitrified with sucrose. Reproduction 2008; 136:167-173.
43. Satirapod C, Treetampinich C, Weerakiet S, Wongkularb A, Rattanasiri S, Choktanasiri W. Comparison of cryopreserved human sperm from solid surface vitrification and standard vapor freezing method: on motility, morphology, vitality and DNA integrity. Andrologia 2012; 44:786-790.
44. Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant supplementation of cryopreservation medium on post-thaw integrity of human spermatozoa. Reprod Biomed Online 2009; 18: 184–189.
45. Ghiuru FV, Ioan L, Roman I, Hettig A, Marius Z, Miclea V. Antioxidant medium for mangalita boar semen cryopreservation. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Anim Sci Biotechnol 2010; 67: 1-2.
46. Breininger E, Beorlegui NB, O’Flaherty CM, Beconi MT. Alpha-tocopherol improves biochemical and dynamic parameters in cryopreserved boar semen. Theriogenology 2005; 63: 2126–2135.
47. Pena FJ, Johannisson A, Wallgren M, Rodriguez Martinez H. Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture. Zygote 2004; 12: 117–124.
Iranian Journal of Basic Medical Sciences
Volume 22, Issue 9
September 2019
Pages 1036-1043

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