Protective effects of nuclear factor erythroid 2-related factor 2 on whole body heat stress-induced oxidative damage in the mouse testis
© Li et al.; licensee BioMed Central Ltd. 2013
Received: 3 February 2013
Accepted: 17 March 2013
Published: 21 March 2013
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.
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.
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.
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.
In most mammals, the testes normally complete descent into a scrotum before birth to provide a lower temperature for spermatogenesis . If descent does not occur and testis remains in abdomen, it’s described as the cryptorchidism which present invariably sterile and spermatogenesis does not begin until testis is surgically moved into the scrotum . Although testes suspend in a scrotum outside the body cavity, spermatogenesis is still disturbed by exposure to high ambient temperature via weakening the ability of thermoregulatory system and inducing increased temperature in testis. Air temperature above 40°C caused scrotal temperature to rise to the value of deep body temperature in rams and rats exposed to an environment of 35°C also exhibited an higher intra-scrotal temperature closed to the core body temperature [3, 4]. In addition, whole body heat exposure resulted in high scrotal temperature and poor semen quality in boars, mice and human [5–7]. The correction or prevention of whole body heat stress-induced sterility is therefore a problem of major concern.
Nuclear factor erythroid 2-related factor 2 (Nrf2), a Cap'n'Collar basic leucine zipper transcription factor, plays an important role in preventing the development of oxidative stress through up-regulation of the Nrf2-related antioxidants [8, 9]. Under homeostatic conditions, the synthetic Nrf2 is captured by Kelch-like ECH-associated protein 1 (Keap1) and constitutively degraded via the ubiquitin-proteasome pathway in cytoplasm . The presence of oxidative or electrophilic stresses can modify cysteine residues on Keap1, thereby inactivating it . As a result, Nrf2 is stabilized and exported into the nuclei to stimulate the transcription of antioxidant genes through heterodimerization with a small musculoaponeurotic fibrosarcoma (Maf) protein and binding to the antioxidant response element (ARE) sequence . The up-regulated antioxidants reverse serve to scavenge the oxidative and electrophilic stressors.
A recent study found that scrotal heat stress induced severe oxidative stress in mouse testes, which consequently caused germ cell death . Moreover, Nrf2-knockout mouse presented an oxidative disruption in spermatogenesis . However, it's still unclear whether Nrf2 can protect germ cell against oxidative stress in testes via Nrf2-antioxidant pathway. In this study, male mice were exposed to the room temperature of 42°C to investigate the impact of whole body heat on the temperature parameters, relative testes weight, levels of MDA and GSH, as well as the activities of enzymatic antioxidants. We hypothesized that Nrf2 accumulated in nucleus and regulated the Nrf2-regulated genes against heat-induced oxidative stress.
Male adult Institute of Cancer Research (ICR) mice, 8 weeks old, were purchased from Nanjing Qinglongshan Experimental Animal Center. The animals were provided with food and water ad libitum and were maintained on a 12 h light–dark cycle in a controlled temperature (25°C) and humidity (50% ± 5%). The animal procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University.
Concentrations of cortisol and testosterone in serum were measured by double-antibody RIAs using 125I-cortisol and 125I-testosterone Radioimmunoassay Kits (Beijing North Institute of Biological Technology, Beijing, China) according to manufacturer’s protocol. The intra- and inter-assay coefficients of variation were 6.1% and 9.2% for cortisol, and 6.9% and 7.2% for testosterone, respectively.
Frozen testicular tissue was homogenized in ice-cold NaCl solution. The homogenate was centrifuged at 1,700 × g for 15 min at 4°C, and the MDA level in the supernatant was analyzed using the MDA assay kit (Jiancheng Bioengineering Institute, Nanjing, China) according to manufacture information. It was analyzed with thiobarbituric acid method by monitoring MDA-reactive products spectrophotometrically. The absorbance of the organic layer was measured at 532 nm. Data were expressed as nanomoles of MDA per milligram of testis protein.
Analysis of antioxidant status
The samples as prepared above were used to analyze parameters of oxidative stress including GSH concentration and the activities of enzymatic antioxidants: T-SOD, CuZn-SOD, glutathione peroxidase (GPX), and catalase (CAT). Reduced GSH was determined by measuring absorbance at 412 nm, while T-SOD activity was measured at 560 nm. GPX activity was measured using a method detedts a complex with a maximal absorbance at 412 nm formed by the reaction of GSH remaining after the action of GPX with 5, 5'-dithiobis-2-nitrobenoic acid. One unit of GPX activity was defined as the decrease amount of 1 μmol/L of GSH per min (except the effect of non-enzymatic reaction) in system of enzymatic reaction of 1 mg protein per minute. CAT activity was measured as absorbance at 405 nm by using an assay based on the consumption of H2O2. The parameters above were determined using commercial kits (Jiancheng Bioengineering Ltd., Nanjing, China) according to the manufacturer's instructions.
Following fixation of the testes, the fixed samples were passed through a graded series of ethanol and xylene solutions and embedded in paraffin wax. Serial sections (5 μm thick) cut from the paraffin-embedded tissues were transferred onto 3-aminopropyl-triethoxysilane (APES)-coated slides (S8441, MATSUNAMI, Tokyo, Japan). Sections were deparaffinized in xylene and rehydrated through a graded ethanol series. To increase epitope exposure, the sections were heated in sodium citrate buffer (0.01 M, pH 6.0) at 121°C for 15 min in an autoclave. After incubating in 3% H2O2 in methanol (v/v) at 32°C for 30 min, the sections were blocked with normal goat serum for 1 h and then incubated overnight at 4°C with rabbit polyclonal antibodies specific for Nrf2 (Santa Cruz Biotechnology, California, USA), diluted 1: 200 in phosphate-buffered saline (PBS, 0.01 M, pH 7.2). Control sections were incubated with blocking serum alone. The specific protein immunoreactivity was visualized with the VECTASTAIN ABC Kit (Histofine, Tokyo, Japan) and a DAB kit (Histofine, Tokyo, Japan). To identify structural features in tissue and assess cell morphology, the sections were counterstained with hematoxylin and mounted with coverslips. Immunostaining was evaluated digicalized images snaped with an Olympus camera.
RNA extraction and quantitative real-time PCR
Total RNA was isolated from frozen testes using TRIzol reagent (Invitrogen, Carlsbad, CA, US) and treated with DNase І (RNase-free) (TaKaRa, Dalian, China) to remove genomic DNA. The total RNA concentration and purity were determined by a spectrophotometer (SMOIF, Shanghai, China). For each sample, 5 μg of total RNA was reverse transcribed to cDNA with M-MLV reverse transcriptase (TaKaRa, Dalian, China) and oligonucleotide primers.
Primers used for real-time PCR
GenBank accession no.
Sequence (5' → 3')
Length of DNA product (bp)
Nuclear factor erythroid 2-related factor 2 (Nrf2)
Heme oxygenase 1 (HO-1)
NAD(P)H: quinine oxidoreductase 1 (NQO1)
γ-Glutamylcysteine synthetase (GCLC)
The results are presented as the mean ± standard error of the mean (SEM). Temperature parameters were analyzed by paired t-tests to identify significant differences between mice before heat treatment and during heat treatment. Data from control and treated mice were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical analysis was performed using GraphPad Prism Version 5.0 software program (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered to indicate a statistically significant result.
Assessment of heat treatment
Serum concentrations of cortisol and testosterone
Body and testes weights
Effects of heat treatment on body weight and testes weight in mice
Body weight (g)
Testes weight (mg)
Testis/body weight (mg/g)
33.92 ± 0.36ab
204.0 ± 4.9ab
6.02 ± 1.06ab
36.65 ± 0.97a
243.4 ± 9.3a
35.54 ± 1.36a
239.8 ± 11.6a
6.76 ± 1.08a
34.10 ± 1.31ab
247.2 ± 12.2a
7.28 ± 3.44a
29.98 ± 1.48b
173.4 ± 10.1b
5.84 ± 4.83ab
32.85 ± 1.03ab
167.4 ± 13.8b
5.09 ± 3.94b
Oxidative stress parameter
Heat stress-induced nuclear Nrf2 accumulation
Heat stress-induced Nrf2-regulated genes mRNA expression
The objective of the present study is to reveal the role of Nrf2 on suppressing the process of oxidative stress after whole body heat treatment, by which we might indentify new targets in diagnosed cases of oxidative stress-induced infertility diagnosis.
In mammals, heat stress is defined as an environment which acts to drive body temperature above set-point temperature . If the ambient temperature exceeds 40°C, the scrotal temperature will rise to the values in the abdomen . Heat stress can induce disruption in internal secretion system such as hypothalamic-pituitary-adrenal axis [17, 18]. In this study, mice rectal temperature and body surface temperature were significantly increased after 2-h heat exposure (Figure 1 and 2). The fluctuant cortisol level in serum also confirmed the occurrence of heat stress induced by whole body heat exposure (Figure 3A). The scrotal surface temperature rose close to rectal temperature during heat treatment (Figure 1D). In addition, heat stress led to an elevated scrotal surface temperature, consistent with previous researches in boar and human .
Most mammal testes suspend in a scrotum outside the body cavity and are more susceptible to heat than other organs [2, 16]. Scrotal temperature above the normal range can disrupt spermatogenesis and increase rates of sub or infertility in rodents, domestic animals, monkey and human [7, 19–21]. In mice, acute or chronic testicular heat exposure led to a fall in testes weight [22, 23]. Additionally, heat stress has been found to cause germ cell loss and poor quality semen [12, 19]. In this study, both reduced testes weight and germ cell loss on day 12 post heat treatment (Table 2 and Figure 5D) indicated germ cell loss might be a potential reason partly responsible for the low testes weight. The stable concentration of mammal testosterone was maintained by the hypothalamic - pituitary - Leydig cell (HPL) axis . Elevated scrotal temperature induced up-regulation of testosterone in patients, which might contribute to attenuate testicular oxidative stress-mediated apoptosis . The testosterone concentration in serum was reduced after 14 days of chronic body heat in boars . In present study, the testosterone concentration in serum was fluctuated, accompanying with increased temperature in scrotal and body surface, indicating that elevation of whole body heat disturbed the functions of Leydig cells and testosterone concentration was determined by heat-treated intension and exposed term. The peak value of testosterone concentration at 4th day suggested that mice might promote HPL axis to protect germ cells against heat-induced damage. However, chronic heat failed to maintain persistent activation of HPL axis and might inhibited the function of HPL axis after 12 days of whole body heat.
Nrf2-antioxidant system has been already recognized as a prime molecular target against oxidative and electrophilic stresses via accumulating in nucleus and inducing expression of cytoprotective enzymes and related proteins . Previous studies have reported that Nrf2-knockout mouse had high testicular and epididymal lipid peroxidation levels and low antioxidants levels which resulted in lower epididymal sperm motility than wild-type males . In this study, it's obvious that 2-day consecutive heat stress induced Nrf2 accumulation in the nuclei of Leydig cells and germ cells. Our findings are now well documented that a strong nuclear Nrf2 accumulation exists in mouse testes. However, the decreased nuclear Nrf2 accumulation on day 12 in heated testes suggested Nrf2 nuclear export was inhibited even a high Nrf2 expression in cytoplasm. A recent study indicated failure to Nrf2-nuclear export, mediated by exportin-1, might be prevented by the failure to phosphorylate Tyr568 during oxidative stress . Moreover, there are a variety of transcription factors antagonizing the Nrf2 activity such as nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptor (PPAR), short form estrogen-related receptor β, estrogen receptor α and so on . Therefore, decreased nuclear Nrf2 accumulation might be attributed to other involved pathways and impaired nuclear export caused by heat stress. To confirm activation of the Nrf2-ARE system in the present study, we measured the mRNA expression of Nrf2 gene and three antioxidant genes containing ARE at their promoter regions (Nrf2, contains two ARE sequences ; HO-1, a well-characterized Nrf2 target gene ; NQO1, reduces quinones to hydroquinones to protect against oxidative stress ; GCLC, combines Glu and Cys as the first step in GSH production ). Previous studies have been reported that the activation of Nrf2 induced increased expression of Nrf2, HO-1, NQO1 and GCLC [37, 38]. Consistent with the results above, our founding showed an increase in mRNA expression of Nrf2, HO-1and GCLC during consecutive heat stress which may reversely promote the expression of oxidative-mediated antioxidant genes via activation of Nrf2 transcription factor (Figure 7).
In conclusion, the present study demonstrates that the whole body heat treatment triggers oxidative stress in mice testes, which acts as partners to induce cell death and reduced relative weight of testis. Nrf2 and its attended anti-oxidative enzyme systems are obviously up-regulated in the process of heat-mediated oxidative stress.
The authors are thankful to Prof. Peishi Yan for technical assistance. Financial support came from the National Nature Science Foundation of China (No. 31272485), Qing Lan Project, the National “Twelfth-Five Year” Research Program of China (2012BAD39B02), and #44120, Research and Development Projects for Application in Promoting New Policy of Agriculture, Forestry and Fisheries, Japan.
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