Reagent and Animal treatments
BPA stock solution at 50 mg/ml was prepared using solid BPA (purity ≥ 99%, Sigma, US), and stored at 4℃. When used, the stock solution was fixed to the desired concentration with distilled water containing 0.5 mol/L NAOH and 25% ethanol.
The experiments were conducted with 8-week-old Kunming mice weighing 20 ± 2 g, supplied by the Beijing Sibefu Experimental Animal Center (ID number: SCXK, Beijing 2016-0002). Two females were caged overnight, with one male after one week of acclimatization. Confirmation of a vaginal plug in the following morning was designated as gestation day 0 (GD 0). Pregnant mice were then randomly divided into 7 groups of 20 mice each and given different doses of BPA by drinking water from GD 0 to the end of the lactation period. Group A was the control group in which the mice received BPA at 0.00 mg/kg/d; Mice in groups B-G were treated with BPA at 0.05, 0.5, 5, 10, 20, 50 mg/kg/d in drinking water, respectively. After weaning at postnatal day 21 (PND 21), the F1 male mice continued to drink BPA at the same dosages as their mothers until day 45 (PND 45). The total BPA exposure duration was 63 days. To ensure the accurate intake amount of BPA solution, water daily intake by each group of mice was accurately measured. The concentration of BPA solution was adjusted accordingly, as described by Zhang and his colleagues (2019). Animal using procedures were approved by the Animal Welfare Committee of the Agricultural University of Hebei, China (approval No. IACECHEBAU20171017).
Measurement of BPA contents in serum and testicular tissues
BPA levels in serum and testes homogenates of the F1 mice were detected using the BPA assay kit (Shanghai Runyu Biotechnology Co., Ltd., Lot No: RY-12919) following the manufacturer’s instructions. Briefly, serum or testicular homogenate of 50 μl was added to the ELISA plate in duplicates and incubated at 37 °C for 30 min. Then 50 μl enzyme-labeled reagents were added after washing and incubated at 37 °C for 30 min. The optical density (OD) values were read at 450 nm using a plate reader (H4MFPTAD, BioTek).
H&E staining of testes
Testes samples were fixed with 4% paraformaldehyde and embedded in paraffin. 5 μm thickness was prepared and stained with hematoxylin-eosin (H&E) to observe histopathological damages and immunohistochemistry.
Immunohistochemistry for synaptonemal complex protein 3 (Scp3) in testes
The paraffin sections of testicular samples were xylene-dewaxed and rehydrated with graded ethanol. The sections were then incubated with 3 % H2O2 at 37 °C for 10 min to quench the endogenous peroxidase. After washing with phosphate-buffered saline (PBS) three times, the slides were incubated with bovine serum albumin (BSA,5%) at 37 °C for 10 min to reduce the non-specific staining. After that, the sections were covered with rabbit anti-mouse SCP-3 antibody (bs-3509R, Bioss, China) and incubated overnight at 4 ℃. The sections were then incubated with goat anti-rabbit antibody (bs-0259G-Bio, Bioss, China) at 37 ℃ for 30 min. The sections were covered with horseradish peroxidase-labeled streptavidin (bs-0437P-HRP, Bioss, China) at 37 ℃ for 30 min after being washed three times with PBS. Finally, the sections were washed with PBS and then visualized with 3,3′-diaminobenzidine (DAB; CWBIO). The positive cells were stained in brown color. The negative controls were processed in the same procedure except that the primary antibody was replaced with PBS.
Sperm apoptosis in testes
Testes were chopped and mixed with RMPI 1640 medium (37℃), then pipette 10 μl of the sample and mixed it with 120 μl low melting point agarose (0.5%, 37℃). Then, the mixture was transferred to the glass slide and let to solidify. The finished slides were placed in lysate solution for digestion (4℃, 1h; Lysate solution: 2.5 mol/L NaCl, 100 mmol/L MTA, 10 mmol/L Tris, aqueous solution, pH10.0, 89 ml; Triton X- Mix 100 1 ml and DMSO 10 ml). After that, electrophoresis (4℃, 1.2 V/cm, 300 mA, 20 min) was performed. Finally, the slides were stained with ethidium bromide (20 μg/ml) and observed using fluorescence microscopic imaging system (Olympus BX43, US). The tail formed by DNA fragments represents the degree of DNA damage. The damage was calculated by the Olive Tail Moment (OTM) value, which is the percentage of tail DNA multiplied by the distance between the tail and the head.
Sperm count and abnormality
The cauda epididymis of mice was collected and macerated in 1 mL of phosphate-buffered saline. The obtained suspension was filtered to remove tissue debris. Sperm count was conducted under a light microscope using a haemocytometer . In measuring sperm abnormality, the separated cauda epididymis was placed in 1 ml phosphate-buffered saline and minced to obtain the suspension as mentioned above. Then the suspension was filtered through a fine mesh cloth to remove tissue debris. After the staining with 1% eosin Y for 20 minutes, a suspension drop was transferred onto a clean slide and air-dried. One thousand sperms per animal were scored from each group for the presence of sperm shape abnormalities .
Total RNA from the testicular tissues was extracted using Eastep Super Total RNA Extraction Kit (LS1040, Promega, USA). Then it was reversed using the Advantage RT-for-PCR Kit (Code No. 639505, TAKARA, Japan) for the cDNA synthesis. The testis of the BPA 50 mg/kg/d group and the control group were subjected to transcriptome sequencing using the Illumina HiSeq Illumina sequencing platform. The GO categories and KEGG pathways were clustered using the DAVID Functional Annotation Clustering tool .
RT-qPCR for splicesome
All 7 groups of differentially expressed genes in the splicing were verified by RT qPCR, and the primer sequences are listed below (Table 1). RT qPCR method: RT qPCR mixture10 μl; forward primer 1 μl; reverse primer 1 μl; cDNA of samples 2 μl; ddH2O 6 μl. Amplification procedure: 95℃ for 600 s; 95℃ for 30 s, 60℃ for 10 s, 45 cycles. Melting curve program: 95℃ for 10 s, 65℃ for 60 s and then ramped to 97℃ at a rate of 0.2℃/ s, and finally cooled at 37℃ for 30 s.
Immunohistochemistry results were analyzed using Image J, and the results of the comet assay were detected using the comet assay software project (CASP) software. The test data were analyzed with SPSS 19.0 software, and the results were expressed as mean ± standard error (`x ± SEM). Data were analyzed by one-way ANOVA and Chi-square test (χ2), P < 0.05 indicates significant difference.