Lycopene protects against ionizing radiation-induced testicular damage by inhibition of apoptosis and mitochondrial dysfunction

Abstract

Ionizing radiation (IR) is one of the key contributors that cause male infertility by disturbing spermatogenesis. Lycopene, a carotenoid with strong antioxidant properties, was shown to protect against oxidative damage induced by IR in several experimental models. The present study was designed to explore the possible protective effects of lycopene against IR-induced testicular damage in C57BL/6 mice. Mice were administered lycopene (20 mg/kg) by oral gavage for seven consecutive days prior to a single dose of whole-body X-ray irradiation (4 Gy, 1 Gy/min). We observed that lycopene remarkably augmented sperm motility and reduced sperm abnormalities in mice following IR exposure. Histopathological analyses also revealed that lycopene ameliorated the structural damage of seminiferous tubules and enhanced the regeneration of seminiferous epithelium following IR stress. Moreover, lycopene attenuated IR-induced oxidative stress, as evidenced by a decreasing lipid peroxidation level and an increase in the antioxidant enzyme superoxide dismutase activity. In addition, lycopene reduced the γH2AX expression and the number of TUNEL-positive cells in the germinal epithelium, as well as restoring the imbalance of Bax/Bcl-2 expression induced by IR exposure. Furthermore, lycopene prevented mitochondrial membrane potential depolarization and ATP reduction and preserved the activities of mitochondrial complexes I-IV in the testes of mice after exposure to IR. Lycopene also improved mitochondrial biogenesis in testes of mice exposed to IR, presenting as restored expressions of PGC-1α, Nrf1, and Tfam. Taken together, our results suggest that lycopene alleviates IR-induced testicular damage, and the underlying mechanism involves at least in part the inhibition of the mitochondrial apoptotic pathway and the maintenance of mitochondrial respiration and biogenesis. The beneficial effect of lycopene highlights the therapeutic potential of this plant-derived antioxidant against impaired spermatogenesis and male infertility induced by IR.

Keywords: apoptosis; ionizing radiation; lycopene; oxidative stress; testis.

FIGURE 1

Effects of lycopene on IR‐induced sperm parameters changes. Sperm parameters, including sperm count (a) sperm motility (b) and progressive motility (c) and sperm abnormalities (d) were detected in mice 4 weeks after IR exposure with or without lycopene. Results are expressed as the mean ± SD (n = 6). *p < .05, **p < .01 for the comparison with the IR exposure group, ^# p < .05 for the comparison with the control group. (e) Representative images of eosin‐stained sperm after IR exposure. Sperm abnormalities were described as two‐head, two‐tail, folded, banana‐Like head, amorphous, etc. Bar = 5 μm.

FIGURE 2

Effects of lycopene on IR‐induced testicular histopathological alterations. (a) Representative images of hematoxylin/eosin stained transverse sections of testis from different groups of mice. Scale bar: up 50 μm, down 20 μm. Quantitative evaluation of the seminiferous epithelium thickness (b) and seminiferous tubule diameter (c) from different groups of mice. (d) Johnsen's score following IR exposure and/or lycopene treatment. Results are expressed as the mean ± SD (n = 6). *p < .05, **p < .01 for the comparison with the IR exposure group, ^# p < .05 for the comparison with the control group.

FIGURE 3

Effects of lycopene on oxidative damage in the testes of mice exposed to IR. Changes in MDA level (a) and SOD activity (b) were determined in mice 24 h after IR exposure with or without lycopene. Results are expressed as the mean ± SD (n = 6). *p < .05, **p < .01 for the comparison with the IR exposure group, ^# p < .05 for the comparison with the control group.

FIGURE 4

Effects of lycopene on DNA damage and apoptosis in the testes of mice exposed to IR. (a) Representative immunofluorescence images of γH2AX expression in testicular sections from different groups of mice at 24 h after IR exposure. Scale bar: 50 μm. (b) Quantitative analysis of γ‐H2AX expression. (c) Representative images of TUNEL‐stained transverse sections of testis from different groups of mice at 24 h after IR exposure. The black arrow indicates apoptotic cells, which have brown staining within their nuclei. The apoptotic cells are primarily located in the outer layer of the seminiferous tubules. Scale bar: 50 μm. (d) The number of TUNEL‐positive cells per seminiferous tubule in the testis sections of different groups of mice. (e) Representative Western blot images of Bax and Bcl‐2 protein expression in testis from different groups at 24 h after IR exposure. (f) The bar graph represents the densitometric quantification of the Bax/Bcl‐2 ratio. β‐tublin was used as a protein loading control. Results are expressed as the mean ± SD (n = 6). **p < .01 for the comparison with the IR exposure group, ^# p < .05, ^## p < .01 for the comparison with the control group.

FIGURE 5

Effects of lycopene on the reduced ΔΨM, ATP contents, and mitochondrial complexes I‐IV activities in the testes of mice exposed to IR. Changes in ΔΨM (a) ATP contents (b) and mitochondrial complex I–IV activities (c–f) were detected in mice 24 h after IR exposure with or without lycopene. Results are expressed as the mean ± SD (n = 6). *p < .05, **p < .01 for the comparison with the IR exposure group, and ^# p < .05 for the comparison with the control group.

FIGURE 6

Effects of lycopene on the decreased PGC‐1α, Nrf1, and Tfam expression in the testes of mice exposed to IR. (a) Representative Western blot images of PGC‐1α, Nrf1, and Tfam protein expression in testis from different groups at 24 h after IR exposure. The bar graph represents densitometric quantification of PGC‐1α (b) Nrf1 (c) and Tfam (d) β‐tublin was used as a protein loading control. Results are expressed as the mean ± SD n = 6 for each group. **p < .01 for the comparison with the IR exposure group, ^# p < .05, and ^## p < .01 for the comparison with the control group.

FIGURE 7

Proposed mechanisms underlying the radioprotective effect of lycopene in mice testes. Lycopene protects against ionizing radiation‐induced testicular damage in mice. The possible mechanism is involved in blocking the mitochondrial apoptotic pathway, inhibiting mitochondrial respiratory dysfunction, and improving mitochondrial biogenesis.