Ethical approval and animal care

Akdeniz University Experimental Animals local ethics committee approved this protocol with the decision numbered B.30.2.AKD.0.05.07.00/30. Experimental protocols applied for rats were determined in accordance with the Akdeniz University Faculty of Medicine, Animal Care and Use Committee standards. Male albino Wistar rats, 3 months old, weighing 200–250 g, were housed in stainless steel cages (four rats per cage) and given food and water ad libitum. Animals were maintained on 12-h light–dark cycles and at a constant temperature of 23 ± 1 °C.

Study design and radio frequency radiation application

Rats were randomly divided into five groups (n = 10 per group): group 1: Control; group 2: Short-term 2100 MHz RFR (1 week); group 3: Short-term sham (sham, 1 week); group 4: Long-term 2100 MHz RFR (10 week); group 5: Long-term sham (sham, 10 week). During the experiment, each rat was placed in perforated plexiglass tubes to facilitate breathing and reduce the increase in body temperature. The 1-week and 10-week groups were exposed to 2100 MHz RFR emitted from the generator for 2 h a day for 1 and 10 weeks, respectively. Sham rats were housed under the same conditions for an equal amount of time in separate rooms without exposure to RFR. All researchers participated blind to data collection, analysis, and experimental group formation.

A radio frequency (RF) generator (UMTS Simulator 2.1 GHz; Everest Company, Adapazarı, Turkey), which produces 2100 MHz RFR, was used to represent the exposure of the Universal Mobile Telecommunications System (UMTS). The output power of the generator was fixed at 1.5 W during exposure. The modulation frequency was 217 Hz, the pulse width was 0.577 ms, and the power range of the generator was 0–2 W. During exposure, rats were placed in specially designed plexiglass tubes with air holes to assist breathing and prevent the rise in body temperature.

Rats in plexiglass tubes were placed radially at equal distances around the antenna (rat noses were 10 cm away from the antenna) (Fig. 2). The carousel setup procedure in this study was applied as described previously (Burkhardt et al. 1997; Fritze et al. 1997; Schonborn et al. 2004). The tubes restrained the movement of the rats to such an extent as to follow well-defined exposure conditions, yet without immobilizing them. RFR application was held in a shielded room to protect the rats from the effects of other electrical sources. For the frequency, the output powers of the source were selected by considering the power values emitted by the mobile phones (the output power was adjusted to 1.5 W during the exposure), and the electric field values were measured. During the “signal on” experiment, the measured electric field strengths over the rat’s head positioned 10 cm away from the antenna were 35.2 V/m for 2.1 GHz.

Fig. 2

Setup of the electromagnetic exposure system

Electric field strengths were measured by EMR300 (Narda, Germany) with a suitable probe in the experiment. The electric field background level of the shielded room was between 0.02 and 0.2 V/m. Also, the background magnetic field was measured between 0.01 and 0.03 µT by Hioki 3470 Magnetic Field Hitester (Hioki E. E. Corp., Japan) with an appropriate probe. Dosimetry simulations were carried out using a finite integration technique (FIT) based commercial software, CST Microwave Studio (3DEXPERIENCE®, Dassault Systems, Hamburg). The FIT was introduced by Weiland (Weiland 1977). Although the gridding can be applied as a finite difference time domain (FDTD) method, the FIT uses the integral form of Maxwell’s equations (Razi-Kazemi 2018). In the present study, the rat model used in simulations consisted of voxels with a resolution of 1.827 × 1.827 × 2.015 mm3. The interaction between the incident electromagnetic wave and the biological tissue is explained by electrical properties that can be obtained by the dielectric properties of the tissue of interest (Gabriel et al. 1996; Abdilla et al. 2013). In this study, each tissue of the simulated rats had electrical properties at the operating frequencies. The average whole-body SAR value at 2100 MHz was 0.16 W/kg. The SAR value for the testes was an average of 0.0347 W/kg. Before and after all experimental sessions, the body temperatures of rats were monitored by rectal measurements. The RFR exposure did not cause elevation in rectal temperature.

Collection and preparation of biological samples

After RFR administration, rats were euthanized by cervical dislocation under ether anesthesia, and the testicles were removed. After washing with 0.9% heparinized saline, one testis of each rat was placed in bouin fixative and kept at +4 °C for 12 h, and a tissue follow-up procedure was performed. The other testis of the rats was divided transversally into two and placed in liquid nitrogen to use one half in quantitative real-time polymerase chain reaction (qPCR) and the other half in immunoblot analysis.

Tissue processing

Tissues taken into bouin fixative were trimmed during the fixation process. Following the fixation, testis samples were passed through ascending alcohol series as described (Okan et al. 2024), they were washed in tap water overnight. After the tissues became completely transparent in xylol, paraffinization was performed, and the tissues were embedded in paraffin. Then, 5-µm sections from paraffin blocks were taken on Superfrost slides using a rotary microtome (Leica, Nussloch, Germany).

Preparation of protein lysate

Testicular tissues removed from the nitrogen tank were placed in sterile +4 °C cold glass and mechanically dissected with a scalpel. Next, they were taken in a lysis buffer (0.1 M Tris pH 7.4, Sodium-orthovanadate, SDS) supplemented with a competent protease inhibitor cocktail tablet (P8340; Sigma Aldrich), and tissues were homogenized using homogenizer (Bandelin electronics HD 2070) for 1 min and sonicated using a sonicator (Bandelin sonopuls GM 2070) for 6 s. The samples were then centrifuged for 10 min at 25200g at +4 °C. Finally, the supernatant was collected for immunoblotting analysis.

Isolation of total RNA and preparation of cDNA

After the testicular tissues were removed from the nitrogen tank, they were placed in +4 °C cold glass under sterile conditions and quickly mechanically dissected using a scalpel. Testis tissues were then placed in Trizol reagent (Life Technologies, Darmstadt, Germany) for total RNA isolation. The RNA concentration was calculated by measuring absorbance at 260 and 280 nm using the EPOCH nanodrop system. After applying DNase I (Ambion, Austin, TX) to 10 μg RNA to eliminate genomic DNA contamination, complementary DNA (cDNA) was generated using the RETROScript kit (Ambion, Austin, TX).

Immunohistochemistry

The streptavidin–biotin peroxidase method was used as previously described (Okan et al. 2021). Briefly, paraffin sections (5 µm) were deparaffinized and then rehydrated. The sections were boiled with sodium citrate buffer pH 6.0 for heat-mediated antigen retrieval in a microwave. Then, the slides were placed in 3% hydrogen peroxide (H2O2) (Sigma-Aldrich) for blocking of endogenous peroxidase activity. Thereafter, ultra V block (#TA-125-UB; Thermo Scientific/Lab Vision) solution was applied for protein blocking. Sections were then incubated with primary antibodies (respectively; Grp78, ab21685 Abcam,1:600; p-Perk, bs-3330R Bioss, 1:100; caspase 12, PA5-19,963 Thermo Fisher Scientific, 2 μg/ml; Chop (Ddit3), ab179823 Abcam, 1:50) at +4 °C overnight. To test secondary-dependent specificity, primer-free antibody diluent was used in negative control staining After several rinses in PBS, secondary antibody (BA-1000 Vector Laboratories, 1:400) was applied for 1 h and followed by several rinses in PBS; the slides were then incubated in streptavidin–peroxidase complex (TS-125-HR, THERMO) for 30 min at room temperature. After several washes in PBS, 3,3′-diaminobenzidine (DAB) substrate (TA-012-HDC- TA-125-HDS) was used to visualize the peroxidase reaction. Counterstaining of sections with Mayer’s hematoxylin solution (#1.09249.1000; Merck), was followed by dehydration and mounting with Entellan (#1.07961.0100; Merck). Images were captured using ZEN 2.5 lite program in a bright field microscopy (Zeiss-Primostar), and expression levels of each protein in all groups were analyzed using ImageJ software.

Immunoblotting

The sodium dodecyl sulfate–polyacrylamide gel prepared in this study was 15% for Chop and 10% for other proteins. After electrophoresis, proteins were electrotransferred to the nitrocellulose membrane. The target protein is labeled with the following antibodies as follows: rabbit anti-Grp78 antibody (ab21685 Abcam,1: 500 in 5% skim milk powder-TBS-t), rabbit polyclonal Chop (Ddit3) antibody (ab179823 Abcam, 1: 250 in TBS-t), rabbit anti-p-Perk antibody (bs-3330R Bioss, 1: 250 in TBS-t), rabbit polyclonal anti-caspase 12 antibody (PA5-19963 Thermo Fischer Scientific, 1 μg/ml in TBS-t), and beta-actin (4970 Cell Signaling Technology, 1: 1000 in 5% skim milk powder-TBS-t). Immunodetection was performed using HRP-labeled goat anti-rabbit secondary antibody (PI-1000 Vector Laboratories), and then protein levels were detected using an enhancer chemiluminescent (ECL) substrate (THERMO-32106).

Quantitative real-time PCR (qPCR)

PCR reactions (25 μl) mix contained 12.5 μl 2 × SYBR green superMix (Qiagen), 0.5 μl of each specific primer (10 μM), 10 ng first-strand cDNA, and nuclease-free water. Gene-specific primers for Hspa5 (Grp78) and Ddit3 (Chop) are given in Table 1. The qPCR reactions were performed on a Rotor-Gene (Corbett Research). Beta-actin (β-actin) was used as an internal control housekeeping gene to normalize the expression of target genes.

Table 1 List of primers used in qPCR analysis

The qPCR program included an initial denaturation at 95 °C for 5 min followed by 30 cycles set at 92 °C for 30 s for denaturation, 65 °C for 20 s for annealing, and 72 °C for 60 s for elongation, and the subsequent 35 cycles were set at as 92 °C for 20 s, 55 °C for 15 s, and 72 °C for 60 s. A dissociation step cycle (55 °C for 10 s and then 0.5 °C for 10 s until 95 °C) was added for melting curve analysis. Three technical replicates and two biological replicates were analyzed for each specimen. As a result of the analysis of the samples, qPCR was created with reference DNA in the standard range. Hspa5, Ddit3, and β-actin expression levels were quantitatively determined in the threshold cycle and other cycles obtained by reading on the instrument (Rotor-Gene-QIAGEN) and using the software program. Delta Ct (ΔCt) indicates the difference between the expression levels of the target gene and the housekeeping gene. ΔCt values were determined by subtracting the Ct value given by the endogenous control from the Ct value of the tested gene separately for each sample. The relative mRNA levels were calculated using 2−ΔΔCTvalues.

Statistical analysis

The normalization test was performed using the Kolmogorov–Smirnov test. A one-way analysis of variance (ANOVA) and post hoc Tukey test was applied using GraphPad (Prisms10) program to determine statistical significance between the groups. Values with P < 0.05 were considered statistically significant. Results are presented as mean ± standard error of the mean (SEM).