Daidzein (CAS# 486-66-8; purity ≥98%) was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Daidzein stock solution was generated using dimethyl sulfoxide (DMSO; Amresco, LLC, Cleveland, OH, USA) and was diluted in culture medium.

Male neonatal mice (postnatal day 4) Kunming mice were obtained from the Experimental Animal Center of Sichuan University (SYXK 2009-045; Chengdu, China) and 10–15 mice were used. The animals were maintained at 25°C, with access to food and water ad libitum and a 12-h light/dark cycle. All animal studies were conducted in accordance with the principles and procedures for the care and use of laboratory animals. The present study was approved by the ethics committee of West China School of Public Health, Sichuan University (Chengdu, China).

Organ culture was performed as described in our previous studies (23,24). The mice were anesthetized and sacrificed by decapitation. Testes obtained from neonatal mice [postnatal day 4 (PND4), 10–15 mice] were isolated and cut into six to eight pieces. The pieces were pooled and transferred into 50 ml bottles containing 6 ml culture medium; six or more pieces were randomly put into each bottle. The bottles were attached to a rotator at 30 rpm and were incubated at 34°C for 72 h. Organ culture of the testes was performed in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Hali Biotech, Chengdu, China) supplemented with 10% calf serum (Hali Biotech), 15 mmol/l HEPES (pH 7.4), 5 µg/ml transferrin, 10 µg/ml insulin, 2 mmol/l glutamine, 100 U/ml penicillin G and 100 µg/ml streptomycin. The culture medium was collected and changed daily. Mixed gas comprised of 50% O2, 45% N2 and 5% CO2 was injected into the bottles to maintain a fresh atmosphere, as previously stated (24,25). Testes were assigned to five groups: Control group and daidzein-treated groups (0.03, 0.3, 3 and 30 µmol/l). At the end of the 72 h culture, the testes were fixed for 12 h in Bouin's fluid (trinitrophenol:methanal:ethanoic acid =15:5:1) at 4°C, embedded in paraffin and cut into 5 µm sections. The media were collected and stored at −80°C for the subsequent testosterone radioimmunoassay. For mRNA analysis, testes were collected and immediately frozen in liquid nitrogen. Furthermore, 30 µg/ml 5′-bromo-2′-deoxyuridine (BrdU) was mixed into the culture system 3 h prior to testis harvesting to determine proliferation of Sertoli cells. The data were obtained from at least three independently repeated culture bottles.

Leydig cells were obtained from the testes of male preadolescent mice (10–15 mice; age, 18–21 days) following euthanasia by decapitation under anesthesia. Subsequently, Leydig cells were isolated by a combination of collagenase digestion and Percoll density centrifugation. After digestion with 8 ml collagenase II with 1.5% bovine serum albumin (BSA; Sigma-Aldrich; Merck Millipore) at 35°C for 25 min, and prior to Percoll density centrifugation, seminiferous tubules were removed by passage of testicular fractions through a 200-mesh filter. The dispersed cells were washed with DMEM/F12 and layered over a Percoll gradient (70, 58, 30 and 5%; Pharmacia Biotech; GE Healthcare, Uppsala, Sweden). The gradient was centrifuged for 30 min at 1,500 × g, and cells localized between Percoll gradient 70 and 58% were isolated. This step ensured the removal of heavier red blood cells and lighter germ cells.

To determine the purity of the target cells, enzyme histochemical and immunocytochemical methods were used to detect 3β-HSD. After a 24 h culture period, purified Leydig cells were incubated for 60 min at 34°C in a phosphate buffer solution containing 10% nitroblue tetrazolinum (Amresco, LLC), 10% nicotanamide adenosine dinucleotide (Amresco, LLC), 6% DHEA (Merck Millipore) and 6% DMSO (26). Positive cells (containing blue granules) were identified as Leydig cells. In addition, Leydig cell slides were fixed with 4% paraformaldehyde, and were then immunostained according to standard protocol. Briefly, fixed cell slides were treated with 5% Triton X-100 and sealed with 5% BSA. Subsequently, the slides were incubated with 3β-HSD antibodies (1:800; BIOSS, Beijing, China; cat. no. bs-3906R) overnight at 4°C, then incubated with a secondary antibody working solution from an immunohistochemistry kit (SP-9000; ZSGB-BIO) for a further 15 min at room temperature, followed by incubation with avidin-biotin peroxidase complex for 15 min. DAB was used as the chromogen and slides were observed under a light microscope. Leydig cells were typically 90% pure as assessed by these two staining methods.

Leydig cells were cultured in the same DMEM/F12 medium as the organ culture. Leydig cells (3×10⁵/1 ml medium) were plated into six-well plates and were cultured at 34°C in a humidified atmosphere containing 95% air and 5% CO₂. A total of 1 day after plating, fresh medium was added, and treatments were initiated. Doses of daidzein were added to wells in triplicate, and cells were cultured at 34°C in a humidified atmosphere containing 5% CO₂ for 24 h. Subsequently, the medium was collected and stored at −20°C until further analysis.

Cellular viability was evaluated using the MTT proliferation assay. Briefly, cells were plated in a 96-well plate at a density of 10,000 cells/well. Following 24 or 48 h incubation at 37°C with various concentrations of daidzein, 20 µl MTT was added to each well and the cells were incubated for 4 h at 37°C. Subsequently, the medium was replaced with 150 µl DMSO and the cells were oscillated for 15 min. Finally, absorbance was measured at 490 nm. Results were presented as a percentage of the control values from untreated cells.

Testosterone secreted into the culture medium was determined in duplicate by Iodine [125I] Testosterone Radioimmunoassay kit (Beijing North Institute of Biological Technology, Beijing, China), according to the manufacturer's protocol.

Histopathological evaluation was conducted using hematoxylin and eosin (H&E) staining. Following fixation in Bouin's fixative and dehydration, the testes were embedded in paraffin and cut into 5 µm sections. Sections from the testes were stained with H&E for histopathological evaluation, hematoxylin was applied for 5 min and eosin for 2 min (both at room temperature). Protein expression in tissue was detected by immunohistochemical staining. Serial sections (5 µm) were then mounted on slides, deparaffinized with xylene twice for 15 min and rehydrated in an alcohol gradient. The sections were then immunostained with antibodies according to manufacturer's protocol. For antigen retrieval, sections were microwaved at 450 W in 10 mmol/l citrate buffer solution. For all immunohistological procedures, slides were treated with Triton X-100 for 1 min, and were then incubated in 0.3% H2O2 for 10 min and in 5% normal goat serum albumin (ZSGB-BIO, Beijing, China) in PBS for 1 h, in order to block nonspecific antigen-binding. Subsequently, the slides were incubated with the following primary antibodies: Anti-3β-HSD (1:800), anti-cholesterol side chain cleavage enzyme (P450scc; 1:200; Wuhan Boster Biological Technology Co., Ltd., Wuhan, China; cat. no. BA3699), anti-17α-hydroxylase/20-lyase (P450C17α; 1:200; BIOSS; cat. no. bs-6695R), anti-vimentin (1:400; BIOSS; cat. no. bs-8533R) and anti-BrdU (1:100; Sigma-Aldrich; Merck Millipore; cat. no. B2531) overnight at 4°C. The primary antibodies were detected by incubation with a secondary antibody working solution from a immunohistochemistry kit (SP-9000; ZSGB-BIO) for a further 15 min at room temperature, followed by incubation with avidin-biotin peroxidase complex (Vector Laboratories, Inc., Burlingame, USA) for 15 min. 3′,3′-Diaminobenzidine (ZSGB-BIO) was used as the chromogen and hematoxylin as the nuclear counterstain. Negative control refers to samples in which the primary antibody was omitted. Staining was observed under a light microscope.

Testes were collected and homogenized, followed by total RNA extraction from the testes and cells using the MicroElute Total RNA kit (Omega Bio-tek, Norcross, GA, USA), and 0.8 µg total RNA was reverse transcribed using Oligo (dT) 18 primers and RevertAid M-MuLV reverse transcriptase (Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a 20 µl reaction mixture, according to the manufacturer's protocol. To determine the expression levels of mRNAs that code for proteins implicated in the steroidogenic pathway (StAR, P450scc, P450C17α and 3β-HSD), qPCR amplification was performed using a Bio-Rad CFX96 Detector system and SsoFast EvaGreen Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). A reaction volume of 10 µl was used containing 2 µl cDNA, 5 µl supermix, 0.2 µl each primer and 2.6 µl RNase/DNase-free water. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec; followed by 35 cycles of 95°C for 5 sec and Tm for 5 sec. Tm was 60°C for StAR and p450c17α, 55°C for P450scc and 3β-HSD, 56.3°C for inhibin B, 57.4 for vimentin and 57.4 for β-actin. In testes, the expression levels of inhibin B and vimentin were also detected. The primers used were as follows: StAR, forward (f) 5′-cgg gtg gat ggg tca agt tc-3′, reverse (r) 5′-cca agc gaa aca cct tgcc-3′; p450scc, f 5′-aca tgg cca aga tgg tac agt tg-3′, r 5′-acg aag cac cag gtc att cac-3′; 3β-HSD, f 5′-tgg aca aag tat tcc gac caga-3′, r 5′-ggc aca ctt gct tga aca cag-3′; p450c17α, f 5′-tga cca gta tgt agg ctt cag tcg-3′, r 5′-tcc ttc ggg atg gca aac tctc-3′; vimentin f 5′-cgt cca cac gca cct acag-3′, r 5′-ggg gga tga gga ata gag gct-3′; and inhibin B, f 5′-ctt cgt ctc taa tga agg caa cc-3′, and r 5′-ctc cac cac att cca cct gtc-3′. Gene expression was normalized to the housekeeping gene β-actin: F 5′-ggc tgt att ccc ctc cat cg-3′ and R 5′-cca gtt ggt aac aat gcc atgt-3′, and expression levels are presented relative to vehicle control (DMSO) at the same time point. All samples were run together in triplicate. Quantification cycle (Cq) values obtained for triplicates were averaged and normalized to β-actin for each RNA sample. Analyses were performed using the 2^−ΔΔCq method (27).

Leydig cells were washed with PBS and lysed in cell lysis/extraction reagent (Nanjing KeyGen Biotech, Co., Ltd., Nanjing, China), including phenylmethanesulfonyl fluoride. Protein concentration was quantified using the Bradford method. Proteins (40 µg) were separated by 10% SDS-PAGE, and were then electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were incubated at 4°C overnight with specific primary antibodies against StAR (1:100; cat. no. sc-25806), P450scc (1:200; cat. no. 18043), P450C17α (1:200; cat. no. 66850) and 3β-HSD (1:200; cat. no. sc-28206), all obtained from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (1:1,000; ZSGB-BIO; cat. no. TA-09). Subsequently, membranes were incubated with secondary antibodies (1:5,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc-2004 and sc-2020) for 1 h at room temperature, according to the manufacturer's protocol. The membranes were then washed with TBS-Tween 20, and immunoreactivity was visualized using an enhanced chemiluminescence reagent and analyzed with ChemiDoc MP system and ImageLab 4.0 (Bio-Rad Laboratories, Inc.).

Inhibin B secreted into the medium by testes was detected by ELISA [Human/Mouse/Rat Inhibin B (β B subunit); RayBiotech, Norcross, GA, USA] according to the manufacturer's protocol. Absorbance was measured using an ELISA plate reader at a wavelength of 450 nm.

All values are expressed as the mean ± standard error. Statistical analysis was performed using one-way analysis of variance followed by Dunnett's t-test with SPSS 20.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

Testicular histopathology was observed 72 h following daidzein administration, and testosterone secreted by neonatal testes was collected every 24 h and analyzed. No obvious histopathological alterations were observed in the daidzein-treated testes (Fig. 1A). However, testosterone secretion by PND4 testes during 3 days of culture was reduced following incubation with 30 µmol/l daidzein (Fig. 1B).

To determine the effects of daidzein on steroidogenic-related protein and enzyme expression in neonatal testes, qPCR and immunohistochemical analysis were performed. After 72 h culture with or without daidzein, the mRNA expression levels of StAR, P450scc and 3β-HSD, which are involved in the steroidogenic process, were downregulated following treatment with 30 µmol/l daidzein compared with the control (P<0.05; Fig. 2A). No obvious changes in the mRNA expression levels of P450C17α were detected.

Corresponding with the findings of qPCR, the protein expression levels of P450scc and 3β-HSD in the testes were reduced following treatment with 30 µmol/l daidzein (Fig. 2B). No marked change in P450C17α staining intensity was detected.

To further verify the effects of daidzein on testosterone production in testes, the present study investigated testosterone secretion, and StAR, P450scc, 3β-HSD and P450C17α expression in Leydig cells in vitro. Cell viability was analyzed by MTT assay, and no changes were observed (Fig. 3A). Consistent with organ culture, incubation with 30 µmol/l daidzein induced a marked suppression of testosterone production by Leydig cells compared with in the control cells (P<0.05; Fig. 3B). The qPCR results indicated that the mRNA expression levels of StAR, P450scc, 3β-HSD and P450C17α were lower following treatment with 30 µmol/l daidzein compared with the control (P<0.05; Fig. 3C).

The present study also examined the protein expression levels of StAR, P450scc, 3β-HSD and P450C17α in Leydig cells. StAR, P450scc, 3β-HSD and P450C17α protein expression levels were decreased in Leydig cells exposed to daidzein (Fig. 3D). Collectively, these effects on mRNA and protein expression indicate that a general downregulation of the steroid synthesis pathway leads to the observed low testosterone levels.

To investigate the effects of daidzein exposure on Sertoli cells in mouse testes, cell proliferation was analyzed with BrdU staining. A total of 3 hours after BrdU was mixed into the culture system, some labeled cells were observed in the seminiferous tubules and in the interstitium of the testes. The number of labeled Sertoli cells was reduced in the testes following treatment with 30 µmol/l daidzein (Fig. 4A and B).

To further determine the effects of daidzein on Sertoli cells in neonatal testes, the expression levels of vimentin were analyzed. Vimentin mRNA expression levels were markedly lower following treatment with 30 µmol/l daidzein compared with the control (P<0.05; Fig. 5A). In addition, immunostaining of Sertoli cells in daidzein-treated (30 µmol/l) testes was weaker compared with the control (Fig. 5B).

The effects of daidzein on the expression levels of inhibin B, which is a regulator involved in steroidogenesis, were also analyzed. As shown in Fig. 6A, the mRNA expression levels of inhibin B exhibited no alteration following treatment with daidzein. Consistent with this result, the levels of inhibin B secreted by testes exhibited no difference between daidzein-treated testes and controls (Fig. 6B).