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. 2022 Aug 8;20(7):569-580.
doi: 10.18502/ijrm.v20i7.11559. eCollection 2022 Jul.

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

Tung Nguyen-Thanh  1   2 , Phuoc Dang-Van  3 , Phuc Dang-Ngoc  4 , Won Kim  5 , Tam Le-Minh  6 , Quoc-Huy Nguyen-Vu  6
Affiliations

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

Tung Nguyen-Thanh et al. Int J Reprod Biomed. .

Abstract

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.

Keywords: Fibrosis.; Inflammation; Testicular; Heat stress.

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Conflict of interest statement

The authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1
Testicular structure and spermatogenesis in the normal and heat-stress-exposed male mice. a-b: Testicular structure and spermatogenesis in the normal male mice (control). c-d: Testicular structure and spermatogenesis in the 37 C heat-stress-exposed male mice (H37 C). e-f: Testicular structure and spermatogenesis in the 40 C heat-stress-exposed male mice (H37 C). Le: Leydig cells, Se: Sertoli cells, Sg: Spermatogonia, Sc: Spermatocytes, rs: Round spermatids, es: Elongated spermatids, S: Spermatozoa, Gc: Multinucleated giant cells.
Figure 2
Figure 2
Heat stress induces histopathology and spermatogenesis changes in the testis. a: Johnsen score of seminiferous tubular cross-sections in the normal and heat-stress-exposed male mice. b: Frequency distribution of the Johnsen scores of seminiferous tubular cross-sections in the normal and heat-stress-exposed male mice. c: Spermatozoa number per tubular cross-section in the normal and heat-stress-exposed male mice. CON: Control group, H37: 37 C heat exposure group, 40 C heat exposure group.
Figure 3
Figure 3
Expression of cell proliferation marker Ki-67, intercellular adhesion molecule-1, and macrophage marker F4/80 in the interstitial tissue of testes after heat stress. a-c: Immunohistochemical staining for Ki-67 in the normal and heat-stress-induced testicular tissue. d-f: Intercellular adhesion molecule-1 expression in the testicular interstitial tissue. g-i: F4/80 macrophage marker detected by immunohistochemical staining. Control: Control group, H37 C: 37 C heat exposure group, H40 C: 40 C heat exposure group, ICAM-1: Intercellular adhesion molecule-1.
Figure 4
Figure 4
Immunohistochemical analysis of the expression of Ki-67, intercellular adhesion molecule-1, and F4/80 in the heat-stress-induced testicular interstitial tissue. a: The number of Ki-67-positive interstitial cells per high-power field. b: Histogram analysis of the area fraction of intercellular adhesion molecule-1. c: The number of F4/80-positive cells per high-power field. HPF: High-power field, NS: Not significant, CON: Control group, H37: 37 C heat exposure group, H40: 40 C heat exposure group, ICAM-1: Intercellular adhesion molecule-1.
Figure 5
Figure 5
Evidence of fibrosis in testicular interstitial tissue after heat stress exposure. a-c: Immunohistochemical staining for fibroblast-specific protein 1. d-f: Immunohistochemical detection of collagen I deposition following scrotal heat stress. g-i: Collagen network detected by picro sirius red staining. Control: Control group, H37 C: 37 C heat exposure group, H40 C: 40 C heat exposure group, FSP-1: Fibroblast-specific protein 1.
Figure 6
Figure 6
Interstitial fibrosis measurement of heat-stress-induced testicular tissue. a: Histogram analysis of the number of FSP-1-positive cells per high-power field. b: Histogram analysis of area fraction of collagen I. c: Picro sirius red-positive area analysis. HPF: High-power field, NS: Not significant, CON: Control group, H37: 37 C heat exposure group, H40: 40 C heat exposure group, FSP-1: Fibroblast-specific protein 1.
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