Histomorphometric evaluation of mice testicular tissue following short- and long-term effects of lipopolysaccharide-induced endotoxemia

Document Type : Original Article


1 Graduate Student of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

2 Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

3 Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran


Objective(s):Lipopolysaccharide (LPS)-induced endotoxemia is known to cause male infertility. This study was designed to explore the effects of bacterial LPS on histomorphometric changes of mice testicular tissues.   
Materials and Methods: In experiment 1, a pilot dose responsive study was performed with mice that were divided into five groups, receiving 36000, 18000, 9000, and 6750 µg/kg body weight (B.W) of LPS or only saline (control). White blood cells (WBC) were observed for 3 days after LPS inoculation. In experiment 2, two groups of mice were treated with 6750 µg/kg B.W of LPS or only saline (control). Five cases from each experimental group were sacrificed at 3, 30, and 60 days after LPS inoculation. Left testes were fixed in Bouin’s solution, and stained for morphometrical assays.
Results: Time-course changes of WBC obtained from different doses of LPS-treated mice showed that inoculation of 6750 µg/kg B.W produced a reversible endotoxemia that lasts for 72 hr and so it was used in the second experiment. In experiment 2, during the first 3 days, no significant changes were observed in the evaluated parameters instead of seminiferous tubules diameter. Spermatogenesis, Johnsen’s score, meiotic index, and epithelial height were significantly affected at 30th day. However, complete recovery was only observed for the spermatogenesis at day 60. Interestingly, deleterious effects of LPS on spermatogonia were only seen at 60th day (P<0.05).    
Conclusion: Endotoxemia induced by LPS has long-term detrimental effects on spermatogonia and later stage germ cells, which are reversible at the next spermatogenic cycle.


Main Subjects

1. Sarkar O, Bahrainwala J, Chandrasekaran S, Kothari S, Mathur PP, Agarwal A. Impact of inflammation on male fertility. Front Biosci (Elite Ed) 2011; 3:89-95.
2. Lynn WA, Cohen J. Adjunctive therapy for septic shock: a review of experimental approaches. Clin Infect Dis 1995; 20:143-158.
3. Shakoory B, Fitzgerald SM, Lee SA, Chi DS, Krishnaswamy G. The role of human mast cell-derived cytokines in eosinophil biology. J Interferon Cytokine Res 2004; 24:271-281.
4. Hailman E, Vasselon T, Kelley M, Busse LA, Hu MC, Lichenstein HS, et al. Stimulation of macrophages and neutrophils by complexes of lipopolysaccharide and soluble CD14. J Immunol 1996; 156:4384-4390.
5. Zhou J, Schmidt M, Johnston B, Wilfart F, Whynot S, Hung O, et al. Experimental endotoxemia induces leukocyte adherence and plasma extravasation within the rat pial microcirculation. Physiol Res 2011; 60:853-859.
6. Turnbull AV, Rivier C. Regulation of the HPA axis by cytokines. Brain Behav Immun 1995; 9:253-275.
7. Knotek M, Rogachev B, Wang W, Ecder T, Melnikov V, Gengaro PE, et al. Endotoxemic renal failure in mice: Role of tumor necrosis factor independent of inducible nitric oxide synthase. Kidney Int 2001; 59:2243-2249.
8. Turnbull AV, Lee S, Rivier C. Mechanisms of hypothalamic-pituitary-adrenal axis stimulation by immune signals in the adult rat. Ann N Y Acad Sci 1998; 840:434-43.
9. O'Bryan MK, Schlatt S, Phillips DJ, de Kretser DM, Hedger MP. Bacterial lipopolysaccharide-induced inflammation compromises testicular function at multiple levels in vivo. Endocrinology 2000; 141:238-246.
10. Hales KH, Diemer T, Ginde S, Shankar BK, Roberts M, Bosmann HB, et al. Diametric effects of bacterial endotoxin lipopolysaccharide on adrenal and Leydig cell steroidogenic acute regulatory protein. Endocrinology 2000; 141:4000-4012.
11. Reddy MM, Mahipal SV, Subhashini J, Reddy MC, Roy KR, Reddy GV, et al. Bacterial lipopolysaccharide-induced oxidative stress in the impairment of steroidogenesis and spermatogenesis in rats. Reprod Toxicol 2006; 22:493-500.
12. Kajihara T, Okagaki R, Ishihara O. LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice. Med Mol Morphol 2006; 39:203-208.
13. Bosmann HB, Hales KH, Li X, Liu Z, Stocco DM, Hales DB. Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells. Endocrinology 1996; 137:4522-4525.
14. Brecchia G, Cardinali R, Mourvaki E, Collodel G, Moretti E, Dal Bosco A, et al. Short- and long-term effects of lipopolysaccharide-induced inflammation on rabbit sperm quality. Anim Reprod Sci 2010; 118:310-316.
15. Collodel G, Castellini C, del Vecchio MT, Cardinali R, Geminiani M, Rossi B, et al. Effect of a bacterial lipopolysaccharide treatment on rabbit testis and ejaculated sperm. Reprod Domest Anim 2012; 47:372-378.
16. Johnsen SG. Testicular biopsy score count--a method for registration of spermatogenesis in human testes: normal values and results in 335 hypogonadal males. Hormones 1970; 1:2-25.
17. Azizollahi S, Babaei H, Derakhshanfar A, Oloumi MM. Effects of co-administration of dopamine and vitamin C on ischaemia-reperfusion injury after experimental testicular torsion-detorsion in rats. Andrologia 2011; 43:100-105.
18. Kheirandish R, Askari N, Babaei H. Zinc therapy improves deleterious effects of chronic copper administration on mice testes: histopathological evaluation. Andrologia 2014; 46:80-85.
19. Inoue T, Aoyama-Ishikawa M, Kamoshida S, Nishino S, Sasano M, Oka N, et al. Endogenous interleukin 18 regulates testicular germ cell apoptosis during endotoxemia. Reproduction 2015; 150:105-114.
20. Watanabe-Fukunaga R, Brannan CI, Itoh N, Yonehara S, Copeland NG, Jenkins NA, et al. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J Immunol 1992; 148:1274-1279.
21. Suda T, Takahashi T, Golstein P, Nagata S. Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75:1169-1178.
22. Palladino MA, Savarese MA, Chapman JL, Dughi MK, Plaska D. Localization of Toll-like receptors on epididymal epithelial cells and spermatozoa. Am J Reprod Immunol 2008; 60:541-555.
23. Sahnoun S, Sellami A, Chakroun N, Mseddi M, Attia H, Rebai T, et al. Human sperm Toll-like receptor 4 (TLR4) mediates acrosome reaction, oxidative stress markers, and sperm parameters in response to bacterial lipopolysaccharide in infertile men. J Assist Reprod Genet 2017; 34:1067-1077.
24. Bhushan S, Schuppe HC, Fijak M, Meinhardt A. Testicular infection: microorganisms, clinical implications and host-pathogen interaction. J Reprod Immunol 2009; 83:164-167.
25. Jegou B, Cudicini C, Gomez E, Stephan JP. Interleukin-1, interleukin-6 and the germ cell-Sertoli cell cross-talk. Reprod Fertil Dev 1995; 7:723-730.
26. Lin T, Wang D, Stocco DM. Interleukin-1 inhibits Leydig cell steroidogenesis without affecting steroidogenic acute regulatory protein messenger ribonucleic acid or protein levels. J Endocrinol 1998; 156:461-467.
27. Troiano L, Fustini MF, Lovato E, Frasoldati A, Malorni W, Capri M, et al. Apoptosis and spermatogenesis: evidence from an in vivo model of testosterone withdrawal in the adult rat. Biochem Biophys Res Commun 1994; 202:1315-1321.
28. Wang RS, Yeh S, Tzeng CR, Chang C. Androgen receptor roles in spermatogenesis and fertility: lessons from testicular cell-specific androgen receptor knockout mice. Endocr Rev 2009; 30:119-132.
29. Sadasivam M, Ramatchandirin B, Ayyanar A, Prahalathan C. Bacterial lipopolysaccharide differently modulates steroidogenic enzymes gene expressions in the brain and testis in rats. Neurosci Res 2014; 83:81-88.
30. Tsai MY, Yeh SD, Wang RS, Yeh S, Zhang C, Lin HY, et al. Differential effects of spermatogenesis and fertility in mice lacking androgen receptor in individual testis cells. Proc Natl Acad Sci U S A 2006; 103:18975-18980.
31. Jegou B, Risbridger GP, de Kretser DM. Effects of experimental cryptorchidism on testicular function in adult rats. J Androl 1983; 4:88-94.
32. Haywood M, Spaliviero J, Jimemez M, King NJ, Handelsman DJ, Allan CM. Sertoli and germ cell development in hypogonadal (hpg) mice expressing transgenic follicle-stimulating hormone alone or in combination with testosterone. Endocrinology 2003; 144:509-517.
33. Turner TT, Lysiak JJ. Oxidative stress: a common factor in testicular dysfunction. J Androl 2008; 29:488-498.
34. Rao F, Tian H, Li W, Hung H, Sun F. Potential role of punicalagin against oxidative stress induced testicular damage. Asian J Androl 2016; 18:627-632.
35. Mates JM. Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology. Toxicology 2000; 153:83-104.
36. Hsu HY, Wen MH. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J Biol Chem 2002; 277:22131-22139.
37. Manna SK, Aggarwal BB. Lipopolysaccharide inhibits TNF-induced apoptosis: role of nuclear factor-kappaB activation and reactive oxygen intermediates. J Immunol 1999; 162:1510-1518.
38. Celino FT, Yamaguchi S, Miura C, Ohta T, Tozawa Y, Iwai T, et al. Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase. PLoS One 2011; 6:e16938.
39. Paul C, Murray AA, Spears N, Saunders PT. A single, mild, transient scrotal heat stress causes DNA damage, subfertility and impairs formation of blastocysts in mice. Reproduction 2008; 136:73-84.
40. Paul C, Teng S, Saunders PT. A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death. Biol Reprod 2009; 80:913-919.
41. Fridovich I. Superoxide radical and superoxide dismutases. Annu Rev Biochem 1995; 64:97-112.