Propolis Protects GC-1spg Spermatogonial Cells against Tert-Butyl Hydroperoxide-Induced Oxidative Damage

Abstract

Propolis is a natural resin produced by honeybees with plenty of pharmacologic properties, including antioxidant activity. Oxidative stress disrupts germ cell development and sperm function, with demonstrated harmful effects on male reproduction. Several natural antioxidants have been shown to reduce oxidative damage and increase sperm fertility potential; however, little is known about the effects of propolis. This work evaluated the role of propolis in protecting spermatogonial cells from oxidative damage. Propolis' phytochemical composition and antioxidant potential were determined, and mouse GC-1spg spermatogonial cells were treated with 0.1-500 µg/mL propolis (12-48 h) in the presence or absence of an oxidant stimulus (tert-butyl hydroperoxide, TBHP, 0.005-3.6 µg/mL, 12 h). Cytotoxicity was assessed by MTT assays and proliferation by Ki-67 immunocytochemistry. Apoptosis, reactive oxygen species (ROS), and antioxidant defenses were evaluated colorimetrically. Propolis presented high phenolic and flavonoid content and moderate antioxidant activity, increasing the viability of GC-1spg cells and counteracting TBHP's effects on viability and proliferation. Additionally, propolis reduced ROS levels in GC-1spg, regardless of the presence of TBHP. Propolis decreased caspase-3 and increased glutathione peroxidase activity in TBHP-treated GC-1spg cells. The present study shows the protective action of propolis against oxidative damage in spermatogonia, opening the possibility of exploiting its benefits to male fertility.

Keywords: antioxidant; oxidative stress; propolis; spermatogonial cells; tert-butyl hydroperoxide.

Figure 1

Viability of GC-1spg cells in the presence of different concentrations of propolis (0.1, 1, 10, 100, 250, and 500 µg/mL) for 12 h, 24 h, and 48 h, evaluated by MTT assay. Results are expressed as % of the control group (C). Ethanol in a concentration of 0.61% was used as a vehicle control (V). Data are presented as mean ± S.E.M. (6 replicates/group, mean of two independent assays). (*) Statistically significant differences to control (above error bars) and between consecutive concentrations (above bounding lines). ** p < 0.01; **** p < 0.0001.

Figure 2

Propolis effect on tert-butyl hydroperoxide (TBHP) cytotoxicity in GC−1spg cells. (A) Viability of GC-1spg cells in the presence of different concentrations of TBHP (0.005, 0.009, 0.018, 0.045, 0.09, 0.9, 1.8 and 3.6 µg/mL) for 12 h. (B) Viability of GC-1spg cells in the presence of 0.1 or 1 µg/mL propolis alone for 36 h, and after a propolis’ 24 h-pre-treatment followed by 12 h exposure to 1.8 µg/mL TBHP (with or without propolis). Cell viability was evaluated by MTT assay. Results are expressed as % of control group. Data are presented as mean ± S.E.M. (6 replicates/group, mean of two independent assays). (*) Statistically significant difference when compared to control; (^$) Statistically significant difference when compared to TBHP plus propolis respective group. ** p < 0.01; **** p < 0.0001; ^$$ p < 0.01; ^$$$ p < 0.001. (C) Representative images of GC-1spg cells from each study condition (control, 1.8 µg/mL TBHP, 0.1 µg/mL propolis, and both). Micrographs were taken by Olympus CKX41, with an ampliation of 100×. The scale bar represents 200 µm.

Figure 3

Proliferative status (A) and caspase-3 activity (B) of GC−1spg cells cultured for 24 h in the presence of 0.1 µg/mL propolis followed by 12 h in the presence of 1.8 µg/mL tert-butyl hydroperoxide (TBHP, with or without propolis). Proliferative index was determined by counting the number of K-i67-positive cells normalized with Hoechst-stained cells. Results are expressed as fold variation to the control group. Data are presented as mean ± S.E.M. (triplicates/group, two independent assays). (*) Statistically significant difference when compared to control; (^#) Statistically significant difference when compared to TBHP group. * p < 0.05, *** p < 0.001; ^# p < 0.05; ^#### p < 0.0001. (C) Representative confocal microscopy images showing Ki-67 labeling in the different groups. Images were taken by Zeiss LMS 710 laser scanning confocal microscope under 400× magnification. The scale bar represents 50 µm. Ki-67 positive cells are shown as red, and the nuclei are stained blue (Hoechst 33342). Negative controls were obtained by omission of the primary antibody and provided as insert panels (-).

Figure 4

ROS levels (A), glutathione peroxidase (GPx, B), and superoxide dismutase (SOD, C) activity in GC-1spg cells cultured for 24 h in the presence of 0.1 µg/mL propolis followed by 12 h in the presence of 1.8 µg/mL tert-butyl hydroperoxide (TBHP, with or without propolis). Dihydroethidium (DHE) fluorescence normalized with Hoechst, and results are expressed as % of the control group. Data are presented as mean ± S.E.M. (triplicates/group, two independent assays). (*) Statistically significant difference when compared to control; (^#) Statistically significant difference when compared to TBHP group. (^$) Statistically significant difference when compared to TBHP plus propolis group. * p < 0.05; **** p < 0.0001; ^#### p < 0.0001; ^$ p < 0.05; ^$$ p < 0.01.

Figure 5

Experimental layout for propolis and tert-butyl hydroperoxide (TBHP) treatments. GC-1spg cells were first exposed to propolis for 24 h, followed by a 12 h exposure to either TBHP alone or propolis and TBHP.