Icariin Improves Age-Related Testicular Dysfunction by Alleviating Sertoli Cell Injury via Upregulation of the ER α/Nrf2-Signaling Pathway

Abstract

Sertoli cells play crucial roles in spermatogenesis and are impaired by aging. Icariin, a flavonoid from Epimedium, has been reported to exhibit anti-aging effects and improve testicular dysfunction in the clinical setting. However, whether icariin improves age-related degeneration of testicular function via protection from Sertoli cell injury remains unclear. In the present study, we evaluated the protective effect of icariin on Sertoli cell injury and explored the possible mechanism(s) in vivo and in vitro. Dietary administration of icariin for 4 months significantly ameliorated the age-related decline in testicular function by increasing testicular and epididymal weights and indices, sperm count and sperm viability, testicular testosterone and estradiol concentrations, and seminiferous tubule diameters and heights. In addition, icariin protected age-related Sertoli cells from injury as evidenced by an analysis of Sertoli cell number, ultrastructure, and function. Such changes were accompanied by upregulation of ERα and Nrf2 signaling in Sertoli cells. Parallel in vitro studies also demonstrated that icariin inhibited untoward effects on the TM4 mouse Sertoli cell line with concomitant upregulation of ERα and Nrf2 signaling. Conversely, ERα siRNA reversed icariin-mediated protection of Sertoli cell injury. Our data suggest that icariin effectively ameliorates age-related degeneration of testicular function by alleviating Sertoli cell injury via the ERα/Nrf2 signal-transduction pathway. Thus, mitigating Sertoli cell damage via the ERα/Nrf2 signaling pathway likely represents a promising strategy for the prevention of age-related testicular dysfunction.

Keywords: ERα/Nrf2; Sertoli cell; aging; icariin; testis.

Figure 1

Icariin ameliorates the decline in testicular function in aging rats. (A) Testicular weight. (B) Testicular index. (C) Epididymal weight. (D) Epididymal index. (E) Sperm count. (F) Sperm viability. (G) Testicular testosterone concentrations. (H) Testicular histology. Representative images of haematoxylin and eosin staining. Upper panels indicate the alterations of several seminiferous tubules (original magnification, 100×; scale bar = 500 μm). Lower panels correspond to magnified boxed areas (original magnification, 400×; scale bar = 100 μm). (I) Seminiferous tubule diameter. (J) Seminiferous tubule height. Seminiferous tubule diameters and seminiferous tubule heights were measured using Image J software for more than 200 tubules per animal, with 5–8 rats in each group. Data are presented as means ± SEM. ^#P< 0.05, ^##P < 0.01, ^###P < 0.001 versus adult control group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus aging model group.

Figure 2

Icariin protects against testicular Sertoli cell injury in aging rats. (A) The numbers of Sertoli cells in testicular tissue. Anti-SOX9 rabbit-polyclonal antibody (red) was used to detect Sertoli cell numbers, and DAPI (blue) was used for nuclear staining (original magnification, 400×). Sertoli cell numbers were measured using Image J software for more than 80 tubules per animal, with 5–8 rats in each group. (B) The ultrastructure of Sertoli cell in testicular tissues was examined using a Hitachi H-7500 transmission electron microscopy (original magnification, 20,000×; scale bar = 800 nm). These electronmicrographs are representative of three independent experiments. Nu, nuclei; M, mitochondria. Black arrowheads indicate endoplasmic reticulum. (C) The relative protein expression levels of GDNF, PLZF, BMP4, and SCF in testicular tissues were detected using western immunoblotting analysis. All values are expressed as means ± SEM of three independent experiments. ^#P < 0.05, ^###P < 0.001 versus adult control group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus aging model group.

Figure 3

Icariin alleviates TM4 cells injury induced by D-galactose (D-gal). (A) Cellular viability was detected using MTT assay. TM4 cells at a concentration of 3 × 10⁴ cells/well in 96-well plates were pretreated with icariin (0.01–8 μM) or DMSO (0.1%) for 20 h and then incubated with 100 mM D-gal for 60 h. (B–D) TM4 cells at 5 × 10⁵/well in 6-well plates were pretreated with icariin (0.5–1 μM) for 20 h and then incubated with 100 mM of D-gal for 60 h. (B) Representative images of SA-β-gal staining of cells (scale bar = 50 μm). (C) The percentages of SA-β-gal-positive cells from a total of 500 cells. (D) The relative protein expression levels of GDNF, PLZF, BMP4, and SCF were measured using western blotting analysis. All values are expressed as means ± SEM of three independent experiments. ^##P < 0.01 versus control; ^*P < 0.05, ^**P < 0.01 versus D-gal-stimulated group.

Figure 4

Icariin alleviates Sertoli cell injury via ERα signaling. (A) Testicular estradiol concentrations were measured using ELISA kits. Each value represents the mean ± SEM with 5–6 rats per group. ^#P < 0.05, versus adult control group; ^**P < 0.01 versus aging model group. (B) Co-localization of Alexa Fluor 488-labeled ERα (green) with Alexa Fluro 594-labeled SOX9 (red) in the testis was detected with a Nikon A1R+ confocal microscope. Upper panels indicate one intact seminiferous tubule (original magnification, 400×). Lower panels correspond to magnified boxed areas (original magnification, 800×). White arrows indicate colocalization of ERα and SOX9 in Sertoli cells. These photomicrographs are representative of three independent experiments. (C) The relative protein expression levels of ERα in D-gal-stimulated TM4 cells were measured by western blotting. TM4 cells at 5 × 10⁵/well in 6-well plates were pretreated with or without icariin (0.5–1 μM) or DMSO (0.1%) for 20 h, and then incubated with 100 mM D-gal for 60 h. All values are expressed as means ± SEM of three independent experiments. (D, E) TM4 cells at 1 × 10⁶/well in 6-well plates transferred with or without ERα siRNA were incubated with icariin (1 μM) or DMSO (0.1%) for 20 h, followed by treatment with 100 mM D-gal for 60 h. (D) Representative images of SA-β-gal staining of cells (left; scale bar = 50 μm), and the percentages of SA-β-gal-positive cells from a total of 500 cells (right). (E) The relative protein expression levels of ERα, GDNF, PLZF, BMP4, and SCF in TM4 cells were measured by western blotting analysis. All values are expressed as means ± SEM of three independent experiments. ^#P < 0.05, ^##P < 0.01 versus control; ^*P < 0.05, ^**P < 0.01 versus D-gal-stimulated group; ^{^}P < 0.05, ^{^^}P < 0.01 versus icariin-treated group.

Figure 5

Icariin activates Nrf2 signaling in Sertoli cells. (A) Colocalization of Alexa Fluor 488-labeled Nrf2 (green) with Alexa Fluro 594-labeled SOX9 (red) in testis was examined with a Nikon A1R+ confocal microscope. Upper panels indicate one intact seminiferous tubule (original magnification, 400×). Lower panels correspond to magnified boxed areas (original magnification, 800×). White arrows indicate co-localization of Nrf2 and SOX9. These photomicrographs are representative of three independent experiments. (B) Activity of SOD. (C) Content of MDA. Each value represents the means ± SEM, with 5–6 rats in each group. ^##P < 0.01, ^###P < 0.001 versus adult control group; ^**P < 0.01, ***P < 0.001 versus aging model group. (D–F) TM4 cells were pretreated with icariin (0.5–1 μM) or DMSO (0.1%) for 12 h, and then incubated with 100 mM D-gal for 60 h. (D) The relative protein expression levels of Nrf2, HO-1, and NQO-1 in TM4 cells were measured by western blotting analysis. (E) Representative images of Nrf2 using immunofluorescence (scale = 50 μm). (F) Intracellular ROS levels were measured using flow cytometry. All values are expressed as means ± SEM of three independent experiments. ^#P < 0.05, ^##P < 0.01 versus control; ^*P < 0.05, ^**P < 0.01 versus D-gal-stimulated group.

Figure 6

Icariin protects against TM4 cells injury via the ERα/Nrf2 signaling pathway. TM4 cells at 1 × 10⁶/well in 6-well plates transferred with or without ERα siRNA were incubated with icariin (1 μM) or DMSO (0.1%) for 12 h, followed by treatment with 100 mM D-gal for 60 h. (A) The relative protein expression levels of ERα, Nrf2, HO-1, and NQO-1 in TM4 cells as described above were measured by western blotting analysis. All values are expressed as means ± SEM of three independent experiments. ^#P < 0.05, ^##P < 0.01 versus control; ^*P < 0.05, ^**P < 0.01 versus D-gal-stimulated group; ^^P < 0.01 versus icariin-treated group. (B) Representative images of Nrf2 using immunofluorescence (scale = 50 μm). These photomicrographs are representative of three independent experiments.