The 6-week-old C57BL/6 wild-type (WT) (REGγ^+/+) and REGγ-deficient (REGγ^−/−) male mice were obtained from the Minhang Laboratory Animal Center of East China Normal University (Shanghai, China) and housed under specific pathogen-free conditions at 21±2°C and 55±10% humidity under a 12 h light/dark cycle, and fed normal chow with ad libitum access to water. The C57BL/6 REGγ^−/− mice were originally acquired from Dr John J. Monaco (University of Cincinnati College of Medicine, Cincinnati, OH, USA) (11-12). A total of 36 REGγ^+/+ mice and 24 REGγ^−/− mice were used for the current study.

Primary Leydig cells were collected from mouse testes. TM3 cells were purchased from the Cell Bank of Type Culture Collection Chinese Academy of Sciences (Shanghai, China; cat. no. GNM24). The TM3 cell line is a mouse epithelial Leydig cell line. Primary Leydig cells and the TM3 cell line were grown in Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12) supplemented with 5% fetal bovine serum, 2.5% horse bovine serum (all Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), L-glutamine (150 mg/l), NaHCO₃ (1.5 g/l), penicillin (100 U/ml) and streptomycin (100 µg/ml). Cells were cultured in a 37°C incubator with 5% CO₂ and 95% air.

Based on preliminary experiments, 12 REGγ^+/+ mice were selected and 6 of them were injected intraperitoneally with LPS (20 mg/kg body weight) dissolved in double distilled (dd) water for 6 h. As a control, 6 mice were injected intraperitoneally with equal volumes of dd water for 6 h.

The optimal conditions for creating an inflammatory cell model were explored. Concentration (0, 1, 5 or 10 mg/ml) and time gradients (0, 10, 20, 40 or 60 min) were established to determine the optimal model conditions. TM3 cells were treated for 10 min with LPS at concentrations of 0, 1, 5 or 10 mg/ml. The optimal concentration of LPS was then selected to treat the cells for different times (0, 10, 20, 40 or 60 min) in order to select the optimal treatment time.

Upon LPS treatment, mice were sacrificed by CO₂ anesthesia (SMQ-II; Tianhuan Technology, Co., Ltd., Shanghai, China; final CO₂ concentration, 80-100%). The displacement rates were between 10-30% of the chamber volume per minute (cv/min), and death was confirmed by observation of loss of respiration, heart beat and reflexes. Then, cervical dislocation was performed as a secondary physical method of sacrifice.

The testes were then collected from the mice in a sterile hood. Sterile tweezers were used to remove the testicular capsule. The tissue was digested in 0.25% collagenase at 37°C for 10 min (1 ml per testis). Next, the tissue suspension was filtered with a 40 µm filter. Following centrifugation at 200 x g and 4°C for 5 min, the cell suspension was placed in culture dishes and cultured in an incubator for 4 h. The adherent cells that were attached to the dishes were considered to be primary Leydig cells. The purity of the Leydig cells, as assessed by immunocytochemical staining for 3β-hydroxysteroid dehydrogenase.

For immunocytochemical staining, after the cells adhered to the slip, the glass slips were washed three times with 1X PBS, fixed with 4% paraformaldehyde at room temperature for 20 min and again washed three times with PBS. Then the glass slips were put in 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 20 min and washed with PBS. Then, the slips were put in 3% H₂O₂ at room temperature for 15 min, washed with PBS and blocked with 5% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at room temperature for 1 h. The glass slips were then treated with anti-3β-hydroxysteroid dehydrogenase primary antibodies (cat. no. sc-515120; 1:50; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C, washed with PBS, treated with horseradish peroxidase-conjugated secondary antibodies (cat. no. K5007; 1:200; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) at room temperature for 1 h and again washed with PBS. Next, the slips were washed with PBS, stained with 0.5 ug/ml DAPI for 3 min at room temperature and washed with PBS. Then, the glass slips were observed by fluorescence microscopy at a magnification of ×600. In each field of vision, the total number of cells and positive cell were counted, positive cells accounted for >90% of total cells.

Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.) was used to knock down REGγ and IkBε expression in TM3 cells using small hairpin (sh)RNA: shREGγ and shIkBε. TM3 control (shN) and REGγ-knockdown (shR) cells were generated by retroviral shRNA plasmids specific for REGγ or control vectors, respectively, from OriGene Technologies, Inc. (Rockville, MD, USA). The plasmids were provided by Shanghai GenePharma Co., Ltd. (Shanghai, China). A total of 1 µg/µl pshRNAIkBε or pshRNAREGγ1 vectors were incubated with TM3 cells for 12 h to obtain shN or shR cells, respectively. Then, the expression levels of REGγ in the shN and shR groups were detected by western blot analysis.

Following treatment, Leydig and TM3 cells were lysed in 1X loading buffer (5X loading buffer: 250 mM Tris-HCl pH 6.8, 10% SDS, 10% bromphenol blue, 50% glycerin and 5% β-5 Mercaptoethanol) for total protein collection. Protein concentrations were determined using the bicinchoninic acid method. The buffer, including all the extracted proteins, was then heated at 100°C for 30 min. Protein samples (50 µg/lane) were subsequently separated using SDS-PAGE on 11% gels and transferred onto nitrocellulose membranes. Upon blocking with 7% BSA dissolve in PBS at room temperature for 1 h, the membranes were incubated with primary antibodies (1:1,000) at 4°C for 12 h and then with IRDye^® 800CW-conjugated donkey anti-rabbit secondary antibodies (cat. no. 925-32213; 1:5,000; LI-COR Biosciences, Lincoln, NE, USA). β-actin (1:5,000) was used as a loading control. Finally, the membranes were scanned using an Odyssey imaging system (LI-COR Biosciences). Image-Pro Plus software (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA) was used for the densitometric analysis. The following antibodies were employed: Anti-IkBε (cat. no. sc-7155; Santa Cruz Biotechnology, Inc.), anti-IkBα (cat. no. 4812), anti-IkBβ (cat. no. 94101; both Cell Signaling Technology, Inc., Danvers, MA, USA), anti-β-actin (cat. no. a2228; Sigma-Aldrich; Merck KGaA) and anti-p-p65 (cat. no. ab16502; Abcam, Cambridge, UK).

Total RNA was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol, and reverse transcribed into complementary DNA using the Mx3005P qPCR system (Agilent Technologies, Inc.) under the following thermocycling conditions: Denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 55-58°C for 30 sec, 72°C for 30 sec and 70°C for 1 min, and a final step at 4°C for 10 min. Each PCR mixture contained 10 µl SYBR-Green Premix Ex Taq polymerase (Takara Biotechnology Co., Ltd., Dalian, China). For each sample, the mRNA expression levels of the genes of interest were normalized to those of GAPDH. The 2^−ΔΔCq method was used for quantification (13). The primers used for the qPCR were as follows: Tumor necrosis factor-α (TNF-α) forward (F), 5′-GAC GTG GAA CTG GCA GAA GAC-3′ and reverse (R), 5′-TCG CAC AAG CAG GAA TGA GA-3′; interleukin (IL)-6 F, 5′-CCA CGG CCT TCC CTA CTT C-3′ and IL-6 R, 5′-CTG TTG GGA GTG GTA TCC TCT GT-3′; IL-1β F, 5′-GAT GAT AAC CTG CTG GTG TGT GA-3′ and R, 5′-GTT GTT CAT CTC GGA GCC TGT AG-3′; and GAPDH F, 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and R, 5′-GGG GTC GTT GAT GGC AAC A-3′.

Tissues were fixed with Bouin's solution (Sigma-Aldrich; Merck KGaA) overnight at room temperature. The tissues were transferred to 70% ethanol for 48 h prior to treatment with a solution of lithium carbonate (1.25%). Then the samples were dehydrated through a graded series of ethanol and embedded in paraffin. The inflamed testes were sectioned into 5-µm-thick slices, and stained with 5% hematoxylin at room temperature for 10 min and 0.5% eosin for at room temperature 3 min to observe structural changes. Other slices were subjected to immunohistochemical (IHC) analysis to observe specific expression patterns of REGγ, IkBε and p-p65 in testicular tissue upon LPS treatment. IHC was performed using the Histostain-Plus (DAB) kit (PH0723; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The slices were stained with anti-REGγ, anti-IkBε and anti-p-p65 antibodies (dilution, 1:100) overnight at 4°C and observed under a light microscope (Eclipse E100; Nikon Corporation, Tokyo, Japan) to evaluate histological changes. The following antibodies were employed: Anti-IkBε (cat. no. sc-7155; Santa Cruz Biotechnology, Inc.) and anti-p-p65 (cat. no. ab16502; Abcam). The slides were observed by a fluorescence microscope at a magnification of ×200.

Venous blood was collected from the tail vein of mice, coagulated for 2 h at room temperature and then centrifuged at 1,800 x g for 15 min at 4°C (Centrifuge 5408R; Eppendorf, Hamburg, Germany). The upper serum layer was collected to determine the testosterone levels using the F-TESTO ELISA kit (cat. no. JL10895; Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China), according to the manufacturer's protocol.

Leydig and TM3 cells were stained with fluo-rescein isothiocyanate and propidium iodide, and then analysed with a BD™ LSR II flow cytometer (all BD Biosciences, San Jose, CA, USA) using FlowJo software (version 10; FlowJo LLC, Ashland, OR, USA).

TM3 shN and shR cells were seeded into a six-well plate at a density of 1×10⁶ cells/well. Cells were then treated with 50 µg/ml cycloheximide (Sigma-Aldrich; Merck KGaA) for 0, 20, 40 and 60 min. For this treatment, cells were culture in DMEM/F-12 supplemented with 5% fetal bovine serum, 2.5% horse bovine serum, L-glutamine (150 mg/l), NaHCO₃ (1.5 g/l), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO₂.

All data analyses were conducted using the Student's t-test or one-way analysis of variance followed by a Fisher's Least Significant Difference post-hoc test for multiple comparisons with SPSS v12.0 software (SPSS, Inc., Chicago, IL, USA). Values are presented as the mean ± standard error of the mean. P<0.05 was considered to indicate a statistically significant difference.

Testes were collected from C57BL/6 WT mice upon LPS treatment for hematoxylin and eosin (H&E) staining and western blot analysis. The results of the western blot analysis indicated that the p-p65 levels were significantly increased in the inflammatory group (Fig. 1A). H&E staining revealed that the testicular structure of the inflammatory group was damaged compared with the control group (Fig. 1B). These results confirmed the successful establishment of a mouse model of testicular inflammation. The western blot analysis results of primary Leydig cells demonstrated that the p-p65 expression levels in the Leydig cells in the inflammation group were increased compared with those in the control group (Fig. 1C). For the TM3 cell line, the inflammation was most pronounced in cells treated with LPS at a concentration of 5 mg/ml (Fig. 1D) for 10 min (Fig. 1E).

In addition, the ELISA results demonstrated that the serum testosterone levels in the inflammatory group were decreased compared with in the control group (Fig. 1F). Leydig cells are the primary cells that secrete testosterone, which indicated that the function of these cells was impaired.

Control and inflammatory groups were established in 6-week-old C57 WT male mice and REGγ^−/− mice. Then, testes from these mice were collected for western blot analysis, qPCR assays and H&E staining. The western blot analysis results revealed that the expression level of p-p65 was significantly decreased in REGγ^−/− mice (Fig. 2B) compared with WT mice. These results were confirmed by H&E staining (Fig. 2A). The results of qPCR analyses demonstrated that the expression levels of TNF-α, IL-1β and IL-6 were significantly increased in the C57 WT mice testes compared with the REGγ^−/− mice testes following LPS treatment (Fig. 2C).

Primary Leydig cells were extracted from C57 WT and REGγ^−/− mice testes and divided into LPS^- or LPS⁺ groups. Then, western blot analysis and qPCR assays were performed. The western blot analysis results revealed that p-p65 expression was significantly decreased in REGγ^−/− mice compared with the WT group (Fig. 3A). The qPCR results indicated that the expression levels of TNF-α were significantly decreased in REGγ^−/− mice compared with the WT group (Fig. 3B).

Western blot analysis confirmed that REGγ was successfully knocked down (Fig. 3C). shN and shR cells were treated with LPS (5 mg/ml) for 0, 10, 20, 40 and 60 min. The western blot analysis results revealed that the expression level of p-p65 in the shN groups was increased compared with in the shR groups, with increasing incubation time (Fig. 3D).

Flow cytometry was used to detect levels of cell apoptosis. Briefly, shN and shR cells were treated with 5 mg/ml LPS for 10 min. The results indicated that the number of apoptotic cells was significantly increased in the shR cells compared with the shN cells (Fig. 3E).

Inflammation models were established in two groups of REGγ^+/+ and REGγ^−/− mice. The testes of these mice were collected, sliced and stained with an anti-REGγ antibody. The immunohistochemical staining results revealed no REGγ expression in REGγ^−/− mice testes, whereas REGγ was highly expressed in REGγ^+/+ mouse testis (Fig. 4A). The expression level of p-p65 was markedly decreased in the REGγ^−/− mice compared with the REGγ^+/+ mice following LPS treatment (Fig. 4B). LPS treatment increased the expression level of REGγ in Leydig cells of REGγ^+/+ mice (Fig. 4C). In addition, LPS treatment upregulated the expression levels of REGγ in TM3 WT cells compared with the control group (Fig. 4D).

To explore the mechanism behind the association between REGγ and NF-κβ in Leydig cells, several upstream signaling pathways were identified from previous studies (5-7,12). Of these, the present study focused on the IkB family proteins. Western blot analysis of IkBα, IkBβ and IkBε were performed in shN and shR cells. The results demonstrated significant differences in IkBε expression levels between shN and shR cells (Fig. 5A). The results of the immunohistochemical staining also revealed that IkBε expression levels were increased in the testicular tissues of REGγ^−/− mice compared with REGγ^+/+ mice (Fig. 5B). Based on these results, cycloheximide degradation analyses were conducted. The results revealed that IkBε degradation was increased in shN cells compared with in shR cells treated for the same time interval. These results demonstrated that the degradation of IkBε increased with increased expression of REGγ (Fig. 5C).

shRNA was used to knock down the REGγ and IkBε genes in the TM3 cell line. shN, shR (IkBε knockdown) and dKD (REGγ and IkBε knockdown) cells were treated with 5 mg/ml LPS for 10 min. The western blot analysis results demonstrated that the expression levels of REGγ and IkBε were significantly decreased in the dKD cells compared with the shN cells (Fig. 5D). In addition, p-p65 was highly expressed in dKD cells, which was similar to the expression levels observed in WT cells (Fig. 5D).