Fluorescent polystyrene microplastics (PS-MPs) (10 mg/mL) with different sizes (0.5 μm, 4 μm, 10 μm) were purchased from Tianjin Baseline ChromTech Research Centre (Tianjin,
China). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM)-F12 were obtained from Gibco (Grand Island, NY). Penicillin-streptomycin were gained from Sigma-Aldrich (St.Louis, MO).
Animals and Treatment
Specifc pathogen-free (SPF) male BALB/c mice aged six-weeks were obtained from Medical School of Yangzhou University (Yangzhou, China). Mice were divided into 7 groups with 15 mice in each group. Mice in each group were randomly placed in three cages (n = 5/cage). Mice of PS-MPs exposure group were given drinking water containing 100 μg/L and 1000 μg/L PS-MPs with sizes of 0.5 μm, 4 μm, and 10 μm for 180 continuous days. For control group, mice were provided the blank water without PS-MPs. After the exposure period, mice were anesthetized to gain blood and testes. All experimental protocols were approved by the Animal Care and Use Committee of Nanjing University according to the animal protocol number SYXK (Su) 2009-0017.
Cell Culture and PS-MPs Exposure
Primary Leydig cells were isolated from 3-week-old BALB/c male mouse testes, as described previously (27884405). Cells were cultured in DMEM-F12 supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.1% human chorionic gonadotropin (hCG). After adherence for 48 h, cells were cultured in DMEM-F12 containing various concentrations of PS-MPs with a diameter of 0.5 μm for 24 h. The concentration of PS-MPs was diluted to 0.1, 0.15, 0.2 mg/mL.
Cells were cultured in DMEM-F12 supplemented with 10% FBS for 96 h, and then cultured in serum-free medium in the presence of 0.1% hCG and 0.2 mg/mL PS-MPs for 1 h. Subsequently, 20 mM forskolin (MedChemExpress, Monmouth Junction, USA) or 0.1 mM 8-bromo-cAMP (MedChemExpress, Monmouth Junction, USA) were added in medium for 24 h.
Sperm Viability and Sperm Abnormality Assessment
Sperm viability and sperm abnormality assessment were performed as previously described .
H&E staining was carried out as previously described .
Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using Trizol reagent (Vazyme, Nanjing, China) according to the manufacturer’s protocol. The cDNA was synthesized by using HiScript Q RT SuperMix (Vazyme, Nanjing, China). QRT-PCR were performed as previously described . The primer sequences were exhibited in Supplementary Table S1.
Western Blotting and ELISA
Proteins were purified from testis tissues or Leydig cells by using the RIPA buffer (Beyotime, Shanghai, China). Western blotting was carried out as previously mentioned . Proteins were separated using 12% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. Transferred blots were incubated with rabbit anti-CYP11A (P450scc) (Proteintech Group, Rosemont, IL, USA) , rabbit anti-CYP17A (P450c17) (Proteintech Group, Rosemont, IL, USA), rabbit anti-StAR (Cell Signaling Technology, USA), mouse anti-HSD3β (Proteintech Group, Rosemont, IL, USA), rabbit anti-HSD17β2 (Proteintech Group, Rosemont, IL, USA), rabbit anti-LHR (Proteintech Group, Rosemont, IL, USA), mouse anti-GAPDH (Proteintech Group, Rosemont, IL, USA) overnight at 4℃. The specific information of antibodies was shown in Supplementary Table S2. The secondary antibody horseradish peroxidase-conjugated goat anti-rabbit/mouse IgG (Boster, Wuhan, China) was used in present study. In addition, the levels of testosterone, LH and FSH in serum were measured with ELISA kits (Elabscience Biotechnology Co., Ltd) according to manufacturer's instructions.
Immunofluorescence staining were performed as previously described . The primary antibody rabbit anti-StAR (Cell Signaling Technology, USA) was employed. The secondary antibody Alexa Fluor 594-conjugated goat anti-rabbit IgG was used in the study. Nuclei were stained with DAPI (Sigma). The images were captured using FV10i microscope (Olympus, Japan).
GraphPad Prism 8 (USA) was applied for statistical analysis. Oneway ANOVA with Dunnett’s t-test was used to analyze differences between groups. The data were shown as mean values ± SD. The value P < 0.05 was regarded as statistically significant.