Chronic scrotal heat stress causes testicular interstitial inflammation and fibrosis: An experimental study in mice


Background: Chronic heat stress is a risk factor that adversely affects the reproduction system. Inflammation and fibrosis are 2 important response processes to damaged tissues.

Objective: This study investigates the association of chronic scrotal heat stress with testicular interstitial inflammation and fibrosis in mice.

Materials and Methods: For all experiments, 8-10 wk old male Swiss mice (Mus musculus) (20-23 gr) were divided into 3 groups (n = 10/each). The heat-stress groups were submerged in a water bath at 37°C and 40°C, while the control group was treated at 25°C. The testicular tissues underwent hematoxylin and eosin staining, picro sirius red staining, and immunohistochemistry for intercellular adhesion molecule-1, fibroblast-specific protein 1, F4/80, collagen I, and Ki-67 staining to determine the testicular interstitial inflammation and fibrosis.

Results: Chronic scrotal heat stress impairs spermatogenesis and reverses testicular histological structure. In this study, heat stress significantly induced increased interstitial cell proliferation and upregulation of intercellular adhesion molecule-1 expression in the interstitial testicular tissue. In the interstitial testicular tissue, the number of F4/80-positive macrophages and the number of fibroblast-specific protein 1- positive fibroblasts were significantly increased in the heat-exposed groups compared to those in the control group. The heat exposed groups had substantially increased extracellular matrix collagen accumulation in their testicular interstitial tissues.

Conclusion: Heat stress adversely affects the testicular structure and spermatogenesis, causes inflammation, and leads to testicular interstitial fibrosis.

Key words: Heat stress, Testicular, Inflammation, Fibrosis.

[1] Rockett JC, Mapp FL, Garges JB, Luft JC, Mori C, Dix DJ. Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice. Biol Reprod 2001; 65: 229–239.

[2] Boni R. Heat stress, a serious threat to reproductive function in animals and humans. Mol Reprod Dev 2019; 86: 1307–1323.

[3] Abdelhamid MHM, Walschaerts M, Ahmad G, Mieusset R, Bujan L, Hamdi S. Mild experimental increase in testis and epididymis temperature in men: Effects on sperm morphology according to spermatogenesis stages. Transl Androl Urol 2019; 8: 651–665.

[4] Cammack KM, Antoniou E, Hearne L, Lamberson WR. Testicular gene expression in male mice divergent for fertility after heat stress. Theriogenology 2009; 71: 651– 661.

[5] Durairajanayagam D, Agarwal A, Ong Ch. Causes, effects and molecular mechanisms of testicular heat stress. Reprod Biomed Online 2015; 30: 14–27.

[6] Setchell BP. The effect of heat on the testes of mammals. Anim Reprod 2006; 3: 81–91.

[7] Mohajeri D, Kaffashi Elahi R. Effects of Nigella sativa on heat-induced testis damage in mouse. Bratisl Lek Listy 2015; 116: 264–269.

[8] Dos Santos Hamilton TR, Perez Siqueira AF, de Castro LS, Mendes CM, Delgado JdC, de Assis PM, et al. Effect of heat stress on sperm DNA: Protamine assessment in ram spermatozoa and testicle. Oxid Med Cell Longev 2018; 2018: 5413056.

[9] Tesarik J, Mendoza-Tesarik R, Mendoza C. Sperm nuclear DNA damage: Update on the mechanism, diagnosis and treatment. Reprod Biomed Online 2006; 12: 715–721.

[10] Cheng K, Song Zh, Li S, Yan E, Zhang H, Zhang L, et al. Effects of resveratrol on intestinal oxidative status and inflammation in heat-stressed rats. J Therm Biol 2019; 85: 102415.

[11] Sanchez-Lozada LG, Garcia-Arroyo FE, Gonzaga G, Silverio O, Blas-Marron MG, Munoz-Jimenez I, et al. Kidney injury from recurrent heat stress and rhabdomyolysis: Protective role of allopurinol and sodium bicarbonate. Am J Nephrol 2018; 48: 339–348.

[12] Abuajamieh M, Kvidera SK, Mayorga EJ, Kaiser A, Lei S, Seibert JT, et al. The effect of recovery from heat stress on circulating bioenergetics and inflammatory biomarkers. J Anim Sci 2018; 96: 4599–4610.

[13] Ganesan S, Reynolds C, Hollinger K, Pearce SC, Gabler NK, Baumgard LH, et al. Twelve hours of heat stress induces inflammatory signaling in porcine skeletal muscle. Am J Physiol Regul Integr Comp Physiol 2016; 310: 1288–1296.

[14] Montilla SIR, Johnson TP, Pearce SC, Gardan-Salmon D, Gabler NK, Ross JW, et al. Heat stress causes oxidative stress but not inflammatory signaling in porcine skeletal muscle. Temperature (Austin) 2014; 1: 42–50.

[15] Hagiwara S, Iwasaka H, Matsumoto S, Noguchi T, Yoshioka H. Association between heat stress protein 70 induction and decreased pulmonary fibrosis in an animal model of acute lung injury. Lung 2007; 185: 287–293.

[16] Shibaguchi T, Sugiura T, Fujitsu T, Nomura T, Yoshihara T, Naito H, et al. Effects of icing or heat stress on the induction of fibrosis and/or regeneration of injured rat soleus muscle. J Physiol Sci 2016; 66: 345–357.

[17] Roncal-Jimenez CA, Sato Y, Milagres T, Andres Hernando A, Garcia G, Bjornstad P, et al. Experimental heat stress nephropathy and liver injury are improved by allopurinol. Am J Physiol Renal Physiol 2018; 315: F726–F733.

[18] Mann ChJ, Perdiguero E, Kharraz Y, Aguilar S, Pessina P, Serrano AL, et al. Aberrant repair and fibrosis development in skeletal muscle. Skelet Muscle 2011; 1: 21.

[19] Thanh TN, Van PD, Cong ThD, Minh TL, Nguyen Vu QH. Assessment of testis histopathological changes and spermatogenesis in male mice exposed to chronic scrotal heat stress. J Anim Behav Biometeorol 2020; 8: 174–180.

[20] 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.

[21] Nguyen-Thanh T, Kim D, Lee S, Kim W, Park SK, Kang KP. Inhibition of histone deacetylase 1 ameliorates renal tubulointerstitial fibrosis via modulation of inflammation and extracellular matrix gene transcription in mice. Int J Mol Med 2018; 41: 95–106.

[22] Kanter M, Aktas C, Erboga M. Heat stress decreases testicular germ cell proliferation and increases apoptosis in short term: An immunohistochemical and ultrastructural study. Toxicol Ind Health 2013; 29: 99–113.

[23] Shiraishi K, Matsuyama H, Takihara H. Pathophysiology of varicocele in male infertility in the era of assisted reproductive technology. Int J Urol 2012; 19: 538–550.

[24] 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.

[25] Suthahar N, Meijers WC, Sillje HHW, de Boer RA. From inflammation to fibrosis-molecular and cellular mechanisms of myocardial tissue remodelling and perspectives on differential treatment opportunities. Current Heart Fail Rep 2017; 14: 235–250.

[26] Wynn ThA, Ramalingam ThR. Mechanisms of fibrosis: Therapeutic translation for fibrotic disease. Nat Med 2012; 18: 1028–1040.

[27] Delkhosh A, Shoorei H, Niazi V, Delashoub M, Gharamaleki MN, Ahani-Nahayati M, et al. Coenzyme Q10 ameliorates inflammation, oxidative stress, and testicular histopathology in rats exposed to heat stress. Hum Exp Toxicol 2021; 40: 3–15.

[28] Beigi Harchegani A, Dahan H, Tahmasbpour E, Bakhtiari Kaboutaraki H, Shahriary A. Effects of zinc deficiency on impaired spermatogenesis and male infertility: The role of oxidative stress, inflammation and apoptosis. Hum Fertil 2020; 23: 5–16.

[29] Azenabor A, Ekun AO, Akinloye O. Impact of inflammation on male reproductive tract. J Reprod Infertil 2015; 16: 123–129.

[30] Hua S. Targeting sites of inflammation: Intercellular adhesion molecule-1 as a target for novel inflammatory therapies. Front Pharmacol 2013; 4: 127.

[31] Figenschau SL, Knutsen E, Urbarova I, Fenton C, Elston B, Perander M, et al. ICAM1 expression is induced by proinflammatory cytokines and associated with TLS formation in aggressive breast cancer subtypes. Sci Rep 2018; 8: 11720.

[32] Aktas C, Kanter M. A morphological study on Leydig cells of scrotal hyperthermia applied rats in short-term. J Mol Histol 2009; 40: 31–39.

[33] Oka S, Shiraishi K, Fujimoto M, Katiyar A, Takii R, Nakai A, et al. Role of heat shock factor 1 in conserving cholesterol transportation in Leydig cell steroidogenesis via steroidogenic acute regulatory protein. Endocrinology 2017; 158: 2648–2658.

[34] Li Z, Tian J, Cui G, Wang M, Yu D. Effects of local testicular heat treatment on Leydig cell hyperplasia and testosterone biosynthesis in rat testes. Reprod Fertil Dev 2015; 28: 1424–1432.

[35] Rasooli A, Taha Jalali M, Nouri M, Mohammadian B, Barati F. Effects of chronic heat stress on testicular structures, serum testosterone and cortisol concentrations in developing lambs. Anim Reprod Sci 2010; 117: 55–59.

[36] Le Hir M, Hegyi I, Cueni-Loffing D, Loffing J, Kaissling B. Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys. Histochem Cell Biol 2005; 123: 335–346.