Nicotine-induced damages in testicular tissue of rats; evidences for bcl-2, p53 and caspase-3 expression

Document Type : Original Article

Authors

Department of Comparative Histology & Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

Abstract

Objective(s): Present study was performed in order to uncover new aspects for nicotine-induced damages on spermatogenesis cell lineage.
Materials and Methods: For this purpose, 36 mature male Wistar rats were divided into three groups as; control-sham (0.2 ml, saline normal, IP), low dose (0.2 mg/kg BW-1, IP) nicotine-received and high dose (0.4 mg/kg BW-1, IP) nicotine-received groups. Following 7 weeks, the expression of bcl-2, p53 and caspase-3 at mRNA and protein levels were investigated by using reverse-transcriptase PCR (RT-PCR) and immunohistochemical (IHC) analyses, respectively. Moreover, the serum level of FSH, LH and testosterone were evaluated. Finally, the mRNA damage was analyzed by using special fluorescent staining.
Results: Nicotine, at both dose levels, decreased tubular differentiation, spermiogenesis and repopulation indices and enhanced cellular depletion. Animals in nicotine-received groups exhibited a significant (P<0.05) reduction at mRNA and protein levels of bcl-2. More analyses revealed a remarkable (P<0.05) enhancement in expression of p53 and caspase-3 in comparison to control-sham animals. Finally, nicotine resulted in a significant (P<0.05) reduction in serum level of testosterone and elevated mRNA damage.
Conclusion: Our data showed that, nicotine by suppressing the testosterone biosynthesis, reducing mRNA and protein levels of bcl-2 and up regulating the p53 and caspase-3 mRNA and protein levels adversely affects the spermatogenesis and results in cellular depletion.

Keywords


1. Ibukun Oyeyipo IP, Raji Y, Benjamin Emikpe O, Bolarinwa AF. Effects of nicotine on sperm characteristics and fertility profile in adult male rats: a possible role of cessation.  J Reprod Infertil 2011; 12:201-207.
2. Armitage AK, Dollery CT, George CF, Houseman TH, Lewis PJ, Turner DM.  Absorption and  metabolism of nicotine from cigarettes. Br Med J 1975; 4:313-316.
3.Russell MA, Jarvis MJ, Devitt G, Feyerabend C. Nicotine intake by snuff users. Br Med J 1981; 283:814-817.
4. Blake CA. Paradoxical effects of drugs acting on the CNS on the preovulatory release of pituitary LH in porestrous rats. Endocrinology1978; 79:319-326.
5. Yeh J, Barbieri RL, Friedman AJ. Nicotine and cotinine inhibit rat testis androgen biosynthesis in vitro. J Steroid Biochem 1989; 33:627–630.
6. Mostafa T, Tawadrous G, Roaia MM,  Amer MK, Kader RA, Aziz A. Effect of smoking on seminal plasma ascorbic acid in infertile and fertile males. Andrologia 2006;38:221–224.
7. Ahmadnia H, Ghanbari M, Moradi MR, Khaje-Dalouee M. Effect of cigarette smoke on spermatogenesis in rats. Urol J 2007; 4:159–163.
8. Hassan A, Abo-Azma SM, Fayed S, Mostafa T. Seminal plasma cotinine and insulin-like growth factor-I in idiopathic oligoasthenoteratozoospermic smokers. Br J Urol Int2009; 103: 108–111.
9. Husain K, Scott BR, Reddy SK, Somani SM. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 2001; 25:89–97.
10. Nesseim WH, Haroun HS, Mostafa E, Youakim MF, Mostafa T. Effect of nicotine on spermatogenesis in adult albino rats. Andrologia 2010; 43:398–404.
11. Gambo IM, Galam NZ, Adamu G, Ayaka LO, Mohammed MB, Ahmed MR, et al. The effect of aqueous leave extract of Nicotiana tabacum(tobacco) on some reproductive parameters and micro anatomy of the testis in male albino wistar rats.  J Natl Sci Res 2013; 3:137-143.
12. Russell LD, Chiarini-Garcia H, Korsmeyer SJ,Knudson CM. Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis. Biol Peprod 2002; 66:950–958.
13. Yin Y, Stahl DE, Wolf BC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. J Androl 2002; 23:64–70.
14. Shukla KK, Mahdi AA, Rajender S. Apoptosis, spermatogenesis and male infertility. Front Biosci2012; 1:746-754.
15. Aitken RJ, Mark AB. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int J Dev Biol 2012; 57:265-272.
16. Hakem R, Duncan GS,  Henderson JT, Woo M, Soengas MS, et al. Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell1998;94:339–352.
17. Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, et al. Apoptosis initiated by Bcl‑2‑regulated caspase activation independently of the cytochrome c/Apaf‑1/caspase‑9 apoptosome.Nature 2002;419:634–637.
18. Borner C. The bcl-2 protein family: sensors and checkpoints for life-or-death decisions. Mol Immunol 2003; 39:615-647.
19. Richard J, Youle AS. The bcl-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 2008; 9:47-59.
20. Agarwall ML, Agarwall A, Taylore WR, Stark GR. P53 controls both the G2/M and the G1 cell cycle checkpoints and mediates reversible growth arrest in human fibroblasts. ProcNatl Acad Sci U S A 1995; 92:8493-8497.
21. Puzio-Kuter, AM. The Role of p53 in metabolic regulation. Genes Cancer 2011; 2:385-391.
22. Li M, He Y, Dubois W, Wu X, Shi J, Huang J. Distinct regulatory mechanisms and functions for p53-activated and p53-repressed DNA damage response genes in embryonic stem cells. Mol Cell 2012;46:30-42.
23. Mc Ilwain DR, Berger T, Mak TW. Caspase functions in cell death and disease.ColdSpring Harb Perspect Biol2013;5:a008656.
24.Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C, et al. Catalytic activity of the caspase-8-FLIP (L) complex inhibits RIPK3-dependent necrosis. Nature2011; 471:363–367.
25. Darzynkiewicz Z. Differential staining of DNA and RNA in intact cells and isolated cell nuclei with acridine-orange. Methods Cell Biol 1990; 33:285-298.
26. Li Q, Falsey RR, Gaitonde S, Sotello V, Kislin K,  Martinez JD. Genetic analysis of p53 nuclear importation. Oncogene 2007; 26:7885–7893.
27. Soleimani Asl S, Farhadi MH, Moosavizadeh K, Samadi Kuchak Saraei A, Soleimani M, Jamei SB, et al. Evaluation of Bcl-2 family gene expression in hippocampus of 3, 4-methylenedioxymethampheta-mine treated rats. Cell J2012;13:275-280.
28. Kermera P, Klöckera N, Labesa M, Thomsen S, Srinivasan A, Bähr M, et al. Activation of caspase-3 in axotomized rat retinal ganglion cells in vivo. FEBS Lett 1999; 453:361-364.   
29. Walker WH, Cheng J. FSH and testosterone signaling in Sertoli cells. Reproduction2005; 130:15-28.
30. Zhang QX, Zhang XY, Zhang ZM, Lu W, Liu L, Li G, et al. Identification of testosterone-/androgen receptor-regulated genes in mouse Sertoli cells. Asian J Androl 2012; 14:294–300.
31. Erster S, Moll UM. Stress-induced p53 runs a transcription-independent death program. Biochem Biophys Res Commun 2005;331:843-850.
32. Petros AM, Gunasekera A, Xu N, Olejniczak ET, Fesik SW. Defining the p53 DNA-binding domain/Bcl-x (L)-binding interface using NMR. FEBS  Lett 2004; 559:71-174.
33. Talos F, Petrenko O, Mena P, Moll UM. Mitochondrially targeted p53 has tumor suppressor activities in vivo. Cancer Res 2005; 65:9971-9981.
34. Sakai W, Suqasawa K. FANCD2 is a target for caspase 3 during DNA damage-induced apoptosis. FEBS Lett 2014; 588:3778-3785.
35. Huang Y, Nakada S, Ishiko T, Utsugisawa T, Datta R, Kharbanda S, et al. Role for caspase- mediated cleavage of Rad51 in induction of apoptosis byDNA damage. Mol Cell Cell Biol 1999; 4:2986-2997.
36. Elmore S. Apoptosis: A review of programmed cell death. Toxicol Pathol 2007;35:495-516. 
37. Tani H, Mizutani R, Salam KA, Tano K, Ijiri K, Wakamatsu A, et al. Genome-wide determination of RNA stability reveals hundreds of short-lived noncoding transcripts in mammals. Genome Res 2012; 22:947–956.
38. Shen B, Goodman HM. Uridine addition after microRNA-directed cleavage. Science2004; 306:997.
39. Thomas M, Liu X, Whangbo J, McCrossan G, Sanborn KB, Basar E, et al. Apoptosis Triggers Specific, Rapid, and Global mRNA Decay with 3′ Uridylated Intermediates Degraded by DIS3L2. Cell Report 2015;11:1079-1089.
40. Saleh RA, Agrawal A, Sharma RK, Nelson DR, Thomas AJ Jr, et al. Effect of cigarette smoking on levels of seminal oxidative stress in infertile men: a prospective study. FertilSteril 2002;78:491-499.
41. Jana K, Samanta PK, Kumar D. Nicotine diminishes tresticular gametogenesis, steroidogenesis, and steroidogenic acute regulatory protein expression in adult albino rats: possible influence on pituitary gonadotropins and alteration of testicular antioxi-dant status. Toxicol Sci 2010; 116:647-659.
42. Khosravanian H,  Razi M, Farokhi F, Khosravanian N. Simultaneous administration of dexamethasone and vitamin E reversed experimental varicocele-induced impact in testicular tissue in rats; correlation with Hsp70-2 chaperone expression. Int Braz J Urol 2015;41:773-790.