Apocynin Ameliorates Monosodium Glutamate Induced Testis Damage by Impaired Blood-Testis Barrier and Oxidative Stress Parameters

Abstract

Background: the aim of this study was to investigate the effects of apocynin (APO) on hormone levels, the blood-testis barrier, and oxidative biomarkers in monosodium glutamate (MSG) induced testicular degeneration.

Methods: Sprague Dawley male rats (150-200 g; n = 32) were randomly distributed into four groups: control, APO, MSG, and MSG + APO. MSG and MSG + APO groups were administered MSG (120 mg/kg) for 28 days. Moreover, the APO and MSG + APO groups received APO (25 mg/kg) during the last five days of the experiment. All administrations were via oral gavage. Finally, biochemical analyses were performed based on the determination of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD), as well as light and transmission electron microscopic examinations, assessment of sperm parameters, ZO-1, occludin, NOX-2, and TUNEL immunohistochemistry were evaluated.

Results: MSG increased both the oxidative stress level and apoptosis, decreased cell proliferation, and caused degeneration in testis morphology including in the blood-testis barrier. Administration of apocynin reversed all the deteriorated morphological and biochemical parameters in the MSG + APO group.

Conclusions: apocynin is considered to prevent testicular degeneration by maintaining the integrity of the blood-testis barrier with balanced hormone and oxidant/antioxidant levels.

Keywords: NOX-2; apocynin; blood-testis barrier; monosodium glutamate; ultrastructure.

Figure 1

Testosterone (A), FSH (B), LH (C), MDA (D), GSH (E) levels, and SOD (F) activity in the experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; ⁺ p < 0.05 vs. MSG group; ^αα p < 0.01, ^ααα p < 0.001 vs. APO group.

Figure 2

Representative light micrographs (A–D) of sperm morphology and graphs of total sperm count (E) and motility (F) in the experimental groups. Normal sperm morphology in the control (A) and APO groups (B). Spermatozoa with disturbed morphology with tailless sperm head (black arrow) and neck (black arrowhead) in MSG group (C). Inset: tail abnormality (*). Spermatozoa with normal morphology (white arrow) and disturbed morphology with head structure of tailless spermatozoa (black arrow) were observed in MSG + APO group (D). Inset: spermatozoa with normal morphology (white arrow) and tail abnormality (*). Scale bar: 50 μm. Diff-Quik staining. Total sperm count (E) and motility (F) graphs in the experimental groups. ** p < 0.01, *** p < 0.001 vs. control group; ⁺⁺ p < 0.01 vs. MSG group; ^ααα p < 0.001 vs. APO group.

Figure 3

Representative light micrographs of testis tissue (A1–D3), testis/body weight (E), histopathological Johnsen’s score (F) and thickness of seminiferous tubules (G) in the experimental groups. The regular morphology of the germinal epithelium is observed in the control (A1) and APO (B1) groups. Vacuolization (black arrowhead) in basement membranes and few spermatozoa were observed in the lumen of degenerative seminiferous tubules in the MSG (C1) group. Inset: cell division (white arrowhead) and immature germ cells (asterisk) were observed in the seminiferous tubule epithelium. Improved morphology of seminiferous tubules was observed in the MSG + APO (D1) group. Inset: vacuolization (black arrow) was observed in a small number of tubules. d: degenerative tubule, n: normal tubule; H & E staining. Regular PAS-positive stained basement membrane morphology was observed in the control (A2), APO (B2), and MSG + APO (D2) groups, but irregular PAS-positive stained basement membrane morphology (arrow) was observed in the MSG group (C2). PAS reaction. The morphology of the testicular tubules and tunica albuginea was normal in the control (A3), APO (B3), and MSG + APO (D3) groups. However, in the MSG group, fat tissue (arrow) was observed in the tunica albuginea, and normal morphology of the tubular stroma was also observed. Masson’s trichrome. Scale bar: 50 μm. Testis/body weight ratio (E), histopathological Johnsen’s score (F), and thickness of seminiferous tubules (G) in the experimental groups. ** p < 0.01, *** p < 0.001 vs. control group; ⁺ p < 0.05, ⁺⁺⁺ p < 0.001 vs. MSG group; ^ααα p < 0.001 vs. APO group.

Figure 4

Representative photomicrographs of PCNA immunostained (A1–D1) and TUNEL stained (A2–D2) testis tissues, proliferative (E), and apoptotic (F) indexes in the experimental groups. Numerous PCNA-positive (arrow) spermatogenic cells are seen in seminiferous tubules of the control (A1) and APO (B1) groups. Decreased PCNA-positive cells (arrow) in the seminiferous tubules and PCNA-positive spermatogenic cells (asterisk) in the lumen of seminiferous tubules are seen in the MSG group (C1). Numerous PCNA-positive spermatogenic cells (arrow) are seen in the seminiferous tubules of the MSG + APO group (D1). A few TUNEL-positive spermatogenic cells (arrow) are seen in the control (A2) and APO (B2) groups. An increased number of TUNEL-positive cells (arrow) in the seminiferous tubules were observed in the MSG group (C2). A decreased number of TUNEL-positive cells (arrow) was observed in the seminiferous tubules of the MSG + APO group (D2). Scale bar: 50 μm. Proliferative (E) and apoptotic (F) indexes of the experimental groups. ** p < 0.01, *** p < 0.001 vs. control group; ⁺⁺⁺ p < 0.001 vs. MSG group; ^α p < 0.05, ^ααα p < 0.001 vs. APO group.

Figure 5

Immunofluorescence analysis of NOX-2 in experimental groups (A–L). The nuclei were labeled with DAPI (blue). NOX-2 expression was similar in the control, APO, and MSG + APO groups. An increased number of NOX-2-positive cells (red) were observed in the MSG group. White arrows indicate NOX-2-positive cells. Scale bar: 50 μm.

Figure 6

Immunofluorescence analysis of ZO-1 in experimental groups (A–L). Representative photomicrographs showing immunodetection of ZO-1 (red) in the experimental groups. Cell nuclei were labeled with DAPI (blue). ZO-1-positive (white arrow) basolateral and apical regions of Sertoli cells were observed in each experimental group. ZO-1-positive cells were decreased in the MSG group. Scale bar: 50 μm.

Figure 7

Immunofluorescence analysis of occludin in experimental groups (A–L). Representative photomicrographs showing immunodetection of occludin (red) in the experimental groups. Cell nuclei were labeled with DAPI (blue). Occludin-positive (white arrow) basolateral and apical regions of Sertoli cells were observed in each experimental group. Occludin-positive cells were decreased in the MSG group. Scale bar: 50 μm.

Figure 8

Representative photomicrographs of ZO-1 (A1–D1) and occludin (A2–D2) immunostained testis tissue samples. Similar ZO-1 and occludin immunostaining in the basolateral regions of Sertoli cells was observed in the control (A1,B1) and APO (A2,B2) groups. Decreased ZO-1 (C1) and occludin (C2) immunostaining were observed in the basolateral regions of Sertoli cells in the MSG group. Increased ZO-1 (D1) and occludin (D2) immunostaining in the basolateral regions of Sertoli cells were observed in the MSG + APO group. Black arrows show ZO-1 and occludin-positive regions of the cells. Scale bar: 50 μm.

Figure 9

NOX-2 (A), ZO-1 (B), and occludin (C) immunoreactivity densities in the experimental groups. * p < 0.05, *** p < 0.001 vs. control group. ⁺⁺ p <0.01; ⁺⁺⁺ p <0.001 vs. MSG group; ^ααα p < 0.001 vs. APO group. Values are given as the mean ± SD.

Figure 10

Representative electron micrographs of testis samples in the experimental groups. The regular morphology of tight junctions (arrow) between adjacent Sertoli cells and numerous spermatozoa (s) were observed in the control (A) and APO (B) groups. Degenerated tight junctions (arrowhead) and lipid droplets (LD) in the cytoplasm were observed in the MSG group (C). Normal organization of seminiferous tubules and tight junction morphology (arrowhead) with few lipid droplets (LD) in the cytoplasm were observed in MSG + APO (D) group. St: Sertoli cell, s: spermatozoa, LD: lipid droplets. Ultrathin sections were contrasted with uranyl-acetate-lead citrate.