Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis

Abstract

Background: Whole body heat stress had detrimental effect on male reproductive function. It's known that the nuclear factor erythroid 2-related factor 2 (Nrf2) activates expression of cytoprotective genes to enable cell adaptation to protect against oxidative stress. However, it's still unclear about the exactly effects of Nrf2 on the testis. Here, we investigate the protective effect of Nrf2 on whole body heat stress-induced oxidative damage in mouse testis.

Methods: Male mice were exposed to the elevated ambient temperature (42°C) daily for 2 h. During the period of twelve consecutive days, mice were sacrificed on days 1, 2, 4, 8 and 12 immediately following heat exposure. Testes weight, enzymatic antioxidant activities and concentrations of malondialdehyde (MDA) and glutathione (GSH) in the testes were determined and immunohistochemical detection of Nrf2 protein and mRNA expression of Nrf2-regulated genes were analyzed to assess the status of Nrf2-antioxidant system.

Results: Heat-exposed mice presented significant increases in rectal, scrotal surface and body surface temperature. The concentrations of cortisol and testosterone in serum fluctuated with the number of exposed days. There were significant decrease in testes weight and relative testes weight on day 12 compared with those on other days, but significant increases in catalase (CAT) activity on day 1 and GSH level on day 4 compared with control group. The activities of total superoxide dismutase (T-SOD) and copper-zinc SOD (CuZn-SOD) increased significantly on days 8 and 12. Moreover, prominent nuclear accumulation of Nrf2 protein was observed in Leydig cells on day 2, accompanying with up-regulated mRNA levels of Nrf2-regulated genes such as Nrf2, heme oxygenase 1 (HO-1), γ-Glutamylcysteine synthetase (GCLC) and NAD (P) H: quinone oxidoreductase 1 (NQO1)) in heat-treated groups.

Conclusions: These results suggest that Nrf2 displayed nuclear accumulation and protective activity in the process of heat treated-induced oxidative stress in mouse testes, indicating that Nrf2 might be a potential target for new drugs designed to protect germ cell and Leydig cell from oxidative stress.

Figure 1

Body and scrotal surface temperatures of mice before and during heat treatment. The table showed the body and scrotal surface temperatures of each mouse before (A) and during (B) heat treatment, as measured one time using a thermal imaging camera. (C) Mouse body surface temperatures were measured before and during heat treatment on days 1, 2, 4, 8 and 12. Average body surface temperature was achieved by counting the average of body surface temperatures of five mice, paired t-test, body surface temperature during treatment vs. before treatment, * P < 0.05. (D) Mice scrotal surface temperatures were measured before and during heat treatment on days 1, 2, 4, 8 and 12. Average scrotal surface temperature was achieved by counting the average of scrotal surface temperatures of five mice, scrotal surface temperature during treatment vs. before treatment, * P < 0.05. T, testis. Each bar represents the mean ± SEM (n = 5).

Figure 2

Effects of heat treatment on average rectal temperature of mice before and during heat treatment. Mouse rectal temperature were measured before and during heat treatment (42°C) using a digital thermometer on days 1, 2, 4, 8 and 12, paired t-test, rectal temperature during treatment vs. before treatment, * P < 0.05. Each bar represents the mean ± SEM (n = 5).

Figure 3

Effects of heat treatment on serum cortisol and testosterone in mice. The concentrations of cortisol (A) and testosterone (B) in serum were measured on days 0, 1, 2, 4, 8 and 12. Each bar represents the mean ± SEM (n = 5). Different superscripts above bar differ significantly between each other (P < 0.05).

Figure 4

Effects of heat treatment on oxidative stress parameters in the testes of mice. The parameters (A, MDA; B, GSH; C, GPX; D, CAT; E, T-SOD and F, CuZn-SOD) were measured on days 0, 1, 2, 4, 8 and 12. Each bar represents the mean ± SEM (n = 5). Different superscripts above bar differ significantly between each other (P < 0.05).

Figure 5

Immunohistochemical detection of Nrf2 in the testes of mice. Sections from heat-treated mice on 0 d (A), 2 d (B), 4 d (C) and 12 d (D) were stained with anti-body Nrf2. The immunohistochemical signals appear brown and the counterstained background appears blue in color. →, Leydig cell; ↓, spermatocyte; ←, round spermatid; ↑, enlongate spermatid. Bar = 100 μm.

Figure 6

Effects of heat treatment on mRNA expression of Nrf2-regulated genes in the testes of mice. The mRNA levels of Nrf2-regulated genes (A, Nrf2; B, GCLC; C, HO-1 and D, NQO1), as measured by real-time PCR. Values were normalized using ACTB as the endogenous standard. Each bar represents the mean ± SEM (n = 5). Different superscripts above bar differ significantly between each other (P < 0.05).

Figure 7

Proposed model of Nrf2-mediated protection to whole body heat stress in mice testes. ROS production induced by heat stress was inhibited by the antioxidative system in cytoplasm. Increased ROS stimulated Nrf2-antioxidant system and up-regulated CuZn-SOD concentration in spermatogonia. Thereby, the antioxidative system was enhanced and efficiently protected testes against oxidative damage.