Melatonin alleviates oxidative stress damage in mouse testes induced by bisphenol A

Abstract

We investigated the effect of melatonin on bisphenol A (BPA)-induced oxidative stress damage in testicular tissue and Leydig cells. Mice were gavaged with 50 mg/kg BPA for 30 days, and concurrently, were injected with melatonin (10 mg/kg and 20 mg/kg). Leydig cells were treated with 10 μmol/L of BPA and melatonin. The morphology and organ index of the testis and epididymis were observed and calculated. The sperm viability and density were determined. The expressions of melatonin receptor 1A and luteinizing hormone receptor, and the levels of malonaldehyde, antioxidant enzymes, glutathione, steroid hormone synthases, aromatase, luteinizing hormone, testosterone, and estradiol were measured. TUNEL assay was utilized to detect testicular cell apoptosis. The administration of melatonin at 20 mg/kg significantly improved the testicular index and epididymis index in mice treated with BPA. Additionally, melatonin promoted the development of seminiferous tubules in the testes. Furthermore, the treatment with 20 mg/kg melatonin significantly increased sperm viability and sperm density in mice, while also promoting the expressions of melatonin receptor 1A and luteinizing hormone receptor in Leydig cells of BPA-treated mice. Significantly, melatonin reduced the level of malonaldehyde in testicular tissue and increased the expression of antioxidant enzymes (superoxide dismutase 1, superoxide dismutase 2, and catalase) as well as the content of glutathione. Moreover, melatonin also reduced the number of apoptotic Leydig cells and spermatogonia, aromatase expression, and estradiol level, while increasing the expression of steroid hormone synthases (steroidogenic acute regulatory protein, cytochrome P450 family 17a1, cytochrome P450 17α-hydroxylase/20-lyase, and, 17β-hydroxysteroid dehydrogenase) and the level of testosterone. Melatonin exhibited significant potential in alleviating testicular oxidative stress damage caused by BPA. These beneficial effects may be attributed to melatonin's ability to enhance the antioxidant capacity of testicular tissue, promote testosterone synthesis, and reduce testicular cell apoptosis.

Keywords: bisphenol A; melatonin; oxidative stress; testis; testosterone.

FIGURE 1

Analysis of testicular and epididymal morphology and organ index. (A) The gross morphology of mouse testes and epididymides. Black arrows indicate the epididymal head, while red arrows indicate the epididymal tail. (B) The testis index (ratio of testis weight to body weight) and the epididymis index (ratio of epididymis weight to body weight) (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 2

HE staining of mouse testicular tissue. (A) HE staining images of the control group, 20MT group, BPA group, BPA+10MT group, and BPA+20MT group are presented (Scale = 100 μm). (B) The thickness of germinal epithelium was measured (N = 5). The bidirectional arrows indicate the thickness of the germinal epithelium. Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05, compared with the BPA+10MT group, ^△ p < 0.05.

FIGURE 3

Assessment of viability of epididymal sperm in mice. (A) Sperm from the control group, 20MT group, BPA group, BPA+10MT group, and BPA+20MT group after eosin staining. White arrows indicate live sperm and red arrows indicate dead sperm (Scale = 100 μm). (B) Statistical analysis of the sperm viability in the epididymal tail of mice in each group (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05, compared with the BPA+10MT group, ^△ p < 0.05.

FIGURE 4

Analysis of epididymal sperm density in mice. (A) Sperm from the control group, 20MT group, BPA group, BPA+10MT group, and BPA+20MT group was observed under a microscope (Scale = 200 μm). (B) Statistical analysis of sperm density in each group of mice (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 5

Expression of MTNR1A in mouse testicular tissue. (A) Immunohistochemical staining of MTNR1A in mouse testicular tissue (Scale bar = 100 μm). The black arrow indicates the expression of MTNR1A. (B) Expression of MTNR1A in mouse testicular tissue analyzed by grayscale value (N = 3). (C) Western blotting analysis of MTNR1A expression in mouse testicular tissue. (D) Relative level of MTNR1A expression (N = 3). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 6

Assessment of antioxidant capacity in mouse testicular tissues. The contents of MDA, SOD1, SOD2, CAT, and GSH in mouse testicular tissues were measured (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 7

Detection of cell apoptosis in mouse testicular tissue and Leydig cells. (A) Cell apoptosis in mouse testicular tissue was detected using the TUNEL assay. Cells showing green fluorescence represent apoptotic cells (Scale = 100 μm). The white arrow indicates apoptotic Leydig cells. The red arrow indicates apoptotic spermatogonia. (B) The number of apoptotic cells was statistically analyzed in each group (N = 3). (C) The apoptosis of Leydig was determined using a TUNEL assay (Scale = 50 μm). (D) The ratio of TUNEL-positive cells was compared (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 8

Expressions of steroid hormone synthesizing enzymes and aromatase in mouse testicular tissue and Leydig cells. (A) The expression of steroid hormone synthesizing enzymes StAR, CYP11A1, CYP17A1, 17β-HSD, and aromatase CYP19 in mouse testicular tissue was detected by ELISA (N = 5). (B) The expression of steroid hormone synthesizing enzymes StAR, CYP11A1, CYP17A1, and 17β-HSD in Leydig cells was detected by ELISA (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.

FIGURE 9

Detection of LHR expression in mouse testicular tissue. (A) LHR expression in mouse testicular tissue was detected using immunofluorescence. Green fluorescence represents the expression of LHR protein (Scale = 100 μm). The white arrow indicates the LHR expression. (B) LHR expression in each group of testicular tissue was represented by mean fluorescence intensity (N = 3). Compared with the control group, ^* p < 0.05.

FIGURE 10

Levels of LH and E2 in mouse testicular tissue and levels of testosterone in mouse testicular tissue and Leydig cells. (A) The contents of LH, E2, and testosterone were measured in mouse testicular tissue using ELISA (N = 5). (B) The content of testosterone was measured in Leydig cells using ELISA (N = 5). Compared with the control group, ^* p < 0.05; compared with the BPA group, ^# p < 0.05.