BPA (chemical purity 99%) was obtained from Sigma-Aldrich (St. Louis, MO, USA). DMEM/HIGH GLUCOSE medium, penicillin, and streptomycin were purchased from HyClone (Logan, Utah, USA). Fetal bovine serum (FBS) was purchased from ZETA™ (Shanghai, China). RNAiso Plus and PrimeScript™RT Reagent Kit with gDNA Eraser were obtained from TaKaRa (Osaka, Japan). SYBR^® Green Realtime PCR Master Mix were obtained from TOYOBO (Osaka, Japan). Dimethyl sulfoxide (DMSO; cell culture grade), polyvinylidene difluoride (PVDF), Oil Red O Saturated Solution 0.5%, Mayer’s Hematoxylin stain, Annexin V-FITC Apoptosis Detection Kit, Free Cholestenone Content Assay Kit and Horseradish peroxidase-linked secondary antibodies were purchased from Solarbio (Beijing, China). Enhanced Cell Counting Kit-8 (CCK-8), BeyoECL Plus, Enhanced BCA Protein Assay Kit, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer were obtained from Beyotime (Shanghai, China). APOA1 antibody was obtained from Proteintech (Chicago, Kentucky, USA). Anti-antibody coated bacteria test (ACTB) mouse monoclonal antibody was purchased from Sangon Biotech (Shanghai, China). Goat Anti-Rabbit IgG H&L/AF488 antibody was obtained from Bioss (Beijing, China). The immunostaining permeabilization buffer containing Triton X-100, bovine albumin, DAPI staining solution, and antifade mounting medium for fluorescence (with DAPI) were obtained from Biosharp (Anhui, China). Testosterone, HDL cholesterol, and total cholesterol (TC) assay kits were obtained from Jiancheng (Nanjing, China). 22-hydroxycholesterol and filipin III were obtained from Cayman Chemical (Ann Arbor, MI, USA).

Twelve 8-week-old male Kunming mice were obtained from The 2^nd Affiliated Hospital of Harbin Medical University (Harbin, China). Before the formal experiment, the mice were acclimatized for one week in a standard environment (25°C, 50% humidity, 12 h/12 h light-dark cycle) with food and water ad libitum. All procedures for the care and use of animals followed the guidelines of Northeast Agricultural University (NEAUEC20).

After adaptive feeding, the mice were randomly classified into two groups including control (CON) and BPA-treated group, with 6 mice in each group. The mice in the treatment group were injected intraperitoneally with BPA at a dose of 20 mg/kg of body weight daily for seven consecutive days (BPA group). The mice in the CON group were intraperitoneally injected with the same volume of corn oil. BPA was dissolved in corn oil by intraperitoneal injection. The chosen dose of 20 mg/kg BPA and 7 days treatment duration were based on previous studies (38, 39), which reported no significant signs of toxicity at this concentration, although adverse effects on the reproductive system were observed (39). Finally, the mice were euthanized by cervical dislocation, and the bilateral testes of each animal were collected. The left testis tissues were fixed with 4% paraformaldehyde overnight, the right testis tissues were stored at –80°C immediately after collection. Before euthanasia, blood samples were drawn by extirpating the eyeballs and centrifuged for serum preparation at 2000 xg for 15 min to assess serum testosterone levels.

TM3 cell, a mouse Leydig cell line, was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which were seeded in 35-mm collagen-coated dishes and cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and maintained at 37°C in an incubator humidified with 5% CO₂ for 24 h before treatment.

TM3 cells were seeded at a density of 5×10³ per well in 96-well plates and cultured for 24 h. BPA was solubilized by DMSO and then diluted to the appropriate concentration with cell culture solution according to the BPA concentration range reported by Li and colleagues (39), cells were treated with 0, 5, 10, 20, 40, 60, 80 and 100 μmol/L of BPA for 12 h and 24 h. The final DMSO concentration in all cell cultures was adjusted to 0.1% (v/v). Cell viability was determined using a CCK-8 commercial kit. Finally, the optical density was measured at 450 nm using a BioTek Epoch (BioTek Instruments, USA). The highest concentration of BPA that did not significantly affect cell viability was selected as the optimal concentration for subsequent experiments.

TM3 cells were plated in 6-well plates at a density of 3×10⁵ cells/well and incubated for 24 h prior to treatment. Then, the cells were treated with 0, 20 and 40 μmol/L of BPA for 24 h, then gently digested with 0.5% trypsin (EDTA-free) and rinsed with 0.01 M phosphate buffer solution (PBS, 1×) twice. An Annexin V-FITC Apoptosis Detection Kit was used to characterize apoptosis. Cell pellets were re-suspended to a concentration of 5×10⁵ cells/mL at 500 μL binding buffer. One hundred microliters of the cell suspension was placed into a 5 mL flow-through tube, to which 5 µL of Annexin V/FITC was added and mixed well. The cells were then incubated in the dark at 25°C for 5 min, 5 μL propidium iodide and 400 µL PBS was added within 1 min before testing. Finally, the samples were assayed by flow cytometry (FACSCelesta, Becton Dickinson, USA). All data were analyzed using Flow.joX.10.0.7 software.

TM3 cells were plated in 6-well plates at a density of 3×10⁵ cells/well and incubated for 24 h prior to treatment. The cells were divided into two groups and treated with 0, 20 μmol/L BPA for 24 h. Total RNA from TM3 cell samples and mouse testis tissues was extracted using TRIzol reagent. Total RNA was reverse transcribed into cDNA using the PrimeScript™RT Reagent Kit according to the manufacturer’s instructions, with gDNA Eraser, reverse transcription was performed using a 20 μL system set at 37°C for 15 min, 85°C for 5 s. The primer sets used for RT-qPCR is summarized in Table 1 , which were constructed as span introns to avoid genomic DNA amplification. Gapdh was used as an internal reference, and mRNA expression was detected using the SYBR^® Green Realtime PCR Master Mix. The predegeneration was 95°C for 30 s with 1 cycle, and circular reaction was 95°C for 5 s, 55°C for 10 s, 72°C for 15 s for 40 cycles. Melting curve analysis was performed to determine the melting peaks to ensure that only a single product was amplified. All reactions were performed in triplicate. The 2^-△△Ct method was implemented for the quantitative analysis of the relative levels of mRNA expression, which were further normalized to the average level of the steadily expressed caretaker gene Gapdh (40).

TM3 cells were plated in 6-well plates at a density of 3×10⁵ cells/well and incubated for 24 h prior to treatment. The cells were divided into two groups and treated with 0, 20 μmol/L BPA for 24 h. Cellular and testicular tissue proteins were extracted using lysates containing protease and phosphatase inhibitors, and the protein concentration in the extracts was measured using the Enhanced BCA Protein Assay Kit according to the manufacturer’s instruction. Dual Color SDS-PAGE Protein Sample Loading Buffer (6×) was added proportionally. The same amounts of protein were separated individually on a 10% SDS-PAGE gel and then transferred to PVDF membranes. QuickBlock™ Blocking Buffer was incubated at 25°C for 15 min, the membranes were incubated with primary antibodies at 4°C overnight and horseradish peroxidase-conjugated (1:5000) secondary antibodies at 25°C for 1 h sequentially. The two antibodies were diluted in the appropriate diluents as follows: APOA1 (1:1000) and ACTB (1:3000). The target bands were detected using BeyoECL Plus reagent. A Tanon 5200 luminescence imaging system (Tanon 5200, Yuanpinghao Biotechnology, China) and ImageJ 1.53a software (National Institute of Health, Bethesda, MD, USA) were used to visualize and digitize the immunoreactive bands, respectively (41). ACTB was used as an internal reference protein. Relative expression was derived from the grey value of the target gene and protein grey value/inner reference gene.

The testes were embedded in paraffin, and 6 μm thick paraffin sections were cut with a rotary microtome. Sections were dewaxed in xylene and rehydrated in a graded alcohol series. After blocking with 5% bovine serum albumin (BSA), the sections were incubated with anti-APOA1 (1:200) antibody and the corresponding secondary antibody (1:500). As a negative control, the anti-APOA1 antibody was replaced with PBS (1×). Microscopic examination was performed using a fluorescence microscope (Leica DM IL LED; Leica Camera, Germany). Three different fields of view were randomly selected for each group, and three images were quantitatively analyzed using ImageJ 1.53a software (41). For cellular immunofluorescence, TM3 cells were inoculated in 6-well plates with cell crawlers in advance, and 2×10⁵ cells were assessed per well and incubated for 24 h prior to treatment. The cells were divided into two groups and treated with 0, 20 μmol/L BPA for 24 h. The cells were rinsed three times with PBS (1×), fixed, stained, photographed, and analyzed as described above.

Frozen sections of 6 µm testis were fixed in 4% paraformaldehyde for 15 min and washed with distilled water thrice. The sections were stained with Oil Red solution for 10 min and washed with distilled water again. The sections were rinsed with 60% isopropanol for 2–30 s until the stroma was clear. It was then washed a further 2–5 times with water until there is no excess dye runoff. The sections were stained with hematoxylin for 5 min, washed with tap water until the nuclei turned blue, dried, and sealed with glycerol gelatin. The sections were visualized using an optical microscope (SOPTOP EX21, Ningbo Sunny Instruments, China). Three images were randomly selected from each group and quantitatively analyzed using ImageJ 1.53a software (41). For Oil Red O staining, TM3 cells were plated in 6-well plates at a density of 3×10⁵ cells per well and incubated for 24 h prior to treatment. The cells were divided into two groups and treated with 0, 20 μmol/L BPA for 24 h. Then, the culture medium was discarded and washed three times with PBS (1×), fixed, stained with oil red O, washed, re-stained with hematoxylin, washed, photographed, and analyzed as described above.

For Filipin III staining, the cells were treated in the same manner as in the Oil Red O experiment. Processed cell samples were fixed in 4% paraformaldehyde, washed with PBS (1×) and incubated with glycine (1.5 mg/mL PBS) at 25°C for 10 min to quench paraformaldehyde, and then rinsed again three times with PBS (1×). Afterwards, the cells were stained with 50 μg/mL Filipin III at room temperature for 30 min. The samples were rinsed three times with PBS (1×), and the fluorescence intensity of Filipin III was visualized using fluorescence microscopy. Filipin III had excitation maxima at 338 and 357 nm. In each experiment, three randomly selected fields per sample were visualized and captured. The resulting fluorescence was quantified using ImageJ 1.53a software and expressed as relative fluorescence intensity per cell (41).

Testicular tissue samples were collected and homogenized using phosphate buffer (0.1 mol/L, pH 7.4), the samples were then pulverized and homogenized using a high throughput tissue grinder. The processed testes were cryo-centrifuged at 2500 rpm for 10 min at 4°C and the supernatant was retained for subsequent testing. The testis tissue supernatant was used to evaluate the levels of TC (detection limits: 0–19.39 mmol/L), HDL-C (detection limits: 0.09–2.50 mmol/L), and FC levels (detection limits: 0.055–4 mmol/L).

TM3 cells were plated in 6-well plates at a density of 3×10⁵ cells/well and incubated for 24 h prior to treatment. The cells were divided into 4 groups, specifically the CON, BPA (20 μmol/L), 22-OH-Chol (30 μmol/L), and 22-OH-Chol (30 μmol/L) and BPA (20 μmol/L) co-treatment groups, with 3 replicates in each group, and were treated for 24 h. Mouse serum, TM3 cell culture supernatant, and testicular supernatant was measured using a highly sensitive ELISA detection kit according to the manufacturer’s instructions. The detection limit was less than 0.1 ng/mL, and the intra-assay coefficient of variation (CV%) was less than 10%. Absorbance was measured at 450 nm using a BioTek Epoch.

The data was shown with mean ± standard error mean (S.E.M) of at least three independent experiments and were performed in triplicate. For in vivo experiments, statistical analysis was conducted using data from 6 mice. GraphPad Prism (version 10.0) was used for graph visualization and data analysis. Differences between groups were analyzed using Student’s t-test or single-factor analysis of variance (one-way ANOVA). Differences with P-values of less than 0.05 were considered statistically significant.

To investigate the effects of BPA exposure on serum and testicular testosterone synthesis in mice, testosterone levels were measured using ELISA. The results showed dramatic declines in serum and testicular testosterone levels in BPA-treated mice compared to those in the CON group (P < 0.01, Figures 1A, B ). These results indicate that BPA exposure significantly inhibits testosterone synthesis and secretion in mice.

Lipid droplets are the primary storage sites for neutral lipids in cells. Because the accumulation of lipid droplets in testicular tissue is correlated with cholesterol levels, Oil Red O staining was used to study the accumulation of lipid droplets in mouse testicular tissue. Oil Red O staining of testicular tissue revealed that lipid droplet accumulation was significantly reduced in the BPA-treated group compared to that in the CON group (P < 0.0001, Figures 2A, B ). Compared with the CON group, testicular TC and FC levels were significantly decreased following BPA exposure (P < 0.01 and P < 0.001, respectively, Figures 2C, D ). Testicular HDL-C levels significantly increased following BPA exposure (P < 0.05, Figure 2E ). These results suggest that the exposure to BPA may enhance RCT, reduce cholesterol levels and lipid droplet accumulation, and ultimately inhibit testosterone synthesis and secretion in mouse testes.

To investigate the relationship between the inhibition of testosterone synthesis and RCT in mice following BPA exposure, the mRNA expression of Apoa1, Apoa2, and Apoc3 in the testicles was detected using RT-qPCR. Following BPA exposure, Apoa1 was significantly increased (P < 0.001, Figure 3A ), whereas there was no significant difference in Apoa2 and Apoc3 compared with that of the CON group ( Figures 3B, C ). Western blotting revealed that APOA1 protein levels were significantly upregulated (P < 0.001, Figures 3D, E ). The IF results showed that the fluorescence intensity of APOA1 in the BPA-treated group was significantly higher than that in the CON group (P < 0.001, Figures 3F, G ). These results suggest that exposure to BPA enhances the RCT process in testicular tissues by up-regulating Apoa1, which in turn inhibits testosterone synthesis and secretion.

The CCK-8 assay was performed using different BPA concentrations for 12 and 24 h to verify the toxicity of BPA in TM3 cells. The data indicated that 40 μmol/L BPA significantly reduced the cell viability at 12 or 24 h compared to the group that received 0 μmol/L (P < 0.05 and P < 0.01, respectively; Figures 4A, B ), cell viability was reduced by 2.11% after 12 h of BPA treatment and by 2.7% after 24 h of exposure. Hence, BPA exhibited a concentration-dependent inhibitory effect on the cell viability of TM3 cells at 12 and 24 h ( Figures 4A, B ). In addition, the status of TM3 cells cultured in 0, 20, and 40 μmol/L BPA for 24 h was observed. The cells in the 0 and 20 μmol/L BPA groups grew in a shuttle or polygonal shape with uniform distribution and good adherence to the wall, the 40 μmol/L BPA group showed no significant difference in cell density compared to the other two groups, although a few cells exhibited morphological changes ( Figure 4C ).

In order to further screen the optimal concentration of BPA, this study examined the apoptosis of cells treated with 0, 20, and 40 μmol/L BPA for 24 h. The apoptosis rate was not significantly affected in the 20 μmol/L BPA group compared to that of the CON group, and apoptosis was significantly increased (5.3%) in the 40 μmol/L BPA group compared to that of the CON group, and the type of apoptosis observed was primarily late apoptosis (P < 0.0001, Figures 4D, F ); the percentage of viable cells in the 40 μmol/L BPA group was significantly lower than that in the CON group (P < 0.001, Figures 4D, E ). The above results indicated that 20 μmol/L BPA treatment for 24 h had no significant effect on the cell viability of TM3 cells and was selected for subsequent experiments.

To explore the effect of 20 μmol/L BPA on testosterone synthesis in TM3 cells, the testosterone secretion level was examined in the cellular supernatant. The results showed that testosterone levels in the cellular supernatant were significantly lower in the BPA-treated group than in the CON group (P < 0.05; Figure 5 ). The results suggest 20 μmol/L BPA treatment significantly inhibited testosterone secretion level in TM3 cells.

To investigate the relationship between the inhibition of testosterone synthesis and RCT after BPA exposure, this study detected changes in the mRNA expression of Apoa1, Apoa2, and Apoc3 in TM3 cells exposed to BPA for 24 h, using RT-qPCR. The mRNA expression level of Apoa1 gene was significantly increased in the BPA-treated group compared to that of the CON group (P < 0.01, Figure 6A ), whereas there was no significant difference in the mRNA expression of Apoa2 and Apoc3 genes compared to that of the CON group ( Figures 6B, C ). Western blotting results showed that the APOA1 protein level was significantly increased after BPA exposure (P < 0.01, Figures 6D, E ), and the IF results showed that the intracellular APOA1 fluorescence intensity was significantly stronger after 24 h of BPA exposure than in the CON group (P < 0.01, Figures 6F, G ). Taken together, these results suggested that upregulated Apoa1 gene expression level may be associated with decreased testosterone synthesis and secretion in 20 μmol/L BPA treatment TM3 cells.

This study investigated whether BPA inhibits testosterone synthesis and secretion by affecting cholesterol levels. After the TM3 cells were exposed to BPA for 24 h, cholesterol levels were detected by Filipin staining. Compared to that of the CON group, the level of FC was significantly reduced (P < 0.01, Figures 7A, B ). Since the accumulation of lipid droplets in the cells correlated with a reduction in the available cholesterol pool, the accumulation of lipid droplets in TM3 cells after BPA treatment was further investigated. Oil Red O staining revealed that the number of lipid droplets was significantly lower than that in the CON group (P < 0.05, Figures 7C, D ). To further validate whether changes in FC content mediates the effect of BPA on synthesis of testosterone in TM3 cells, the cells were treated with 22-OH-Chol (30 μmol/L) in the presence or absence of BPA for 24 h. The exogenous addition of 22-OH-Chol promoted testosterone synthesis (P < 0.05) and alleviated the inhibitory effect of BPA (P < 0.05, Figure 7E ). These results suggest that BPA inhibits testosterone synthesis by reducing FC levels in TM3 cells.