p. 2: "Rats were daily administered orally 0, 5, or 10 mg/kg PFDoA."

"After 14 days, rats were killed by CO₂ on postnatal day 35. We weighed each testis and collected serum samples for testosterone, LH, and FSH assays. We collected one testis for immunohistochemical analysis after Bouin’s solution fixation and the other testis for gene and protein expression analysis after freezing.

Testosterone Measurement. We detected serum testosterone concentrations using a commercial RIA kit (IBL, U.S.A.) as described previously.¹⁶ This kit has the lowest testosterone concentration detection limit of 5 pg/mL. Interassay variation of testosterone was within 15%.

ELISA for Serum LH and FSH Levels. Serum levels of LH and FSH hormones were measured by commercial ELISA kits as previously described.¹⁷ Briefly, serum sample and an assay diluent were put to a 96-well plate and the mixture was incubated for 2 h at room temperature. After the washing step, peroxidase-conjugated IgG anti-LH or anti-FSH antibody was transferred to the mixture. After 2 h reaction and washing, each LH or FSH substrate solution was added and the reaction was processed for 30 min and then stopped. The plate was subjected to the detection of LH or FSH at a wavelength of 450 nm.

Progenitor Leydig Cell Isolation. Eighteen male Sprague−Dawley rats (21 days of age) were killed by asphyxiation with CO₂. Progenitor Leydig cell isolation procedure was adopted according to a previous study.¹⁶ Briefly, we digested the testis with the collagenase and DNase, removed the tissue impurity through 100 μm nylon mesh

and separated the cells after a centrifugation of Percoll gradient at a higher speed. Cell fraction between 1.07 and 1.088 g/mL was aspirated into a new tube. Leydig cell purity was performed using a histochemical staining of 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) as previously described.¹⁸ Progenitor Leydig cells (≥95% purity) was obtained.

CCK-8 Assay. Cell kit-8 (CCK-8) was purchased to detect cell viability. Briefly, 100 μL of 10⁴ cells/ml progenitor Leydig cells were added into a 96-well plate and incubated for 24 h. Then, cells were treated with a range of (0.1, 1, and 10 μM) of PFDoA for 24 h at 37 °C. Subsequently, 10 μL CCK-8 was added and the plate was incubated for 4 h at 37 °C. The absorbance at 450 nm was measured with a microplate reader (Thermo, MA).

Annexin V and PI Assay. Cells at a density of 10⁶ cells per well were added into a 6-well culture plate and cultured for 24 h and then treated with a range (0, 0.1, 1, and 10 μM) of PFDoA for 24 h. We applied the Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences) to judge early and lately apoptosis. Briefly, after the washing step, cells were resuspended in the annexin V-binding buffer and stained with FITC-labeled Annexin V and PI for 15 min and were then subjected to the Flow Cytometer (BD FACSAria, Rockville, MD) for analysis.

Measurement of Cellular ROS. ROS levels were detected using indirect measurement with DCFH-DA assay kit according to the manufacturer’s instructions. Briefly, cells (10⁶ cells) were pippetted into a 6-well plate and cultured for 24 h and then treated with a range of (0, 0.1, 1, and 10 μM) PFDoA for 24 h. Cells were incubated with

DCFH-DA in the dark room for 20 min at 37 °C and then subjected to the detection of fluorescence intensity in the Flow Cytometer.

Measurement of Mitochondrial Membrane Potential (ΔΨm). A JC-1 ΔΨm assay kit was used according to manufacturer’s instruction. Briefly, cells (10⁶ cells) were pippetted and cultured for 24 h and then treated with PFDoA (0, 0.1, 1, and 10 μM) for 24 h. After a washing step, cells were incubated with JC-1 (5 mM) for 30 min and subjected to the detection of fluorescence emissions in the flow cytometry. ΔΨm was calculated by red/green fluorescence ratio.

Immunohistochemical Staining of Testis. Testis fixation, tissue-array preparation, and immunohistochemical staining were performed as in the previous study.¹⁷ In brief, after embedding fixation of paraffin and dehydration, testis was subjected to sectioning at 6 μm. The sections were subjected to the antigen display in the microwave heating and then endogenous peroxidase blockade at 0.5% H₂O₂. Two Leydig cell biomarkers, cholesterol side chain cleavage enzyme (CYP11A1) and 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), were selected to identify Leydig cells. One Sertoli cell biomarker SOX9 was selected to identify Sertoli cells.20 CYP11A1, HSD11B1 or SOX9 polyclonal antibody diluted at 1:200 was used. Diaminobenzidine was selected to identify antigen−antibody complex (brown staining).

Immunofluorescent staining of PCNA in the cells was performed for investigating cell proliferation. Sections were sequentially treated with CYP11A1 and PCNA antibody for each 90 min at room temperature. After washing, fluorescent secondary antibody (Alexa-conjugated antirabbit IgG, 1:500; Alexa-conjugated antirat IgG, 1:500) was added and then the sections were costained with DAPI. Sections were subjected to the detection of fluorescence in a microscope (Olympus, Japan).

Counting Leydig and Sertoli Cell Number by a Stereological Method. We performed a fractionator method to enumerate each cell types according to a previous study.²¹ Each testis of a rat was sampled into about 10 testis sections. The histochemical staining was executed as above. We multiplied the number of cells counted in a known fraction of the testis by the reciprocal of the sampling probability to calculate total number of cells.

Real-time PCR (RT-qPCR). Total RNA extraction was done using a TRIzol kit (Invitrogen, CA). The concentration of total RNA was read at 260 nm. RNAs were reversely transcribed into the cDNAs by a reverse transcription reagent kit (Invitrogen, CA). A SYBR Green qPCR Kit (Takara, Otsu, Japan) was used to detect RNA levels. Genes representing Leydig cell specific genes are membrane receptor gene (LH receptor, Lhcgr), cholesterol-transporting genes (high-density lipoprotein receptor, Scarb1; steroidogenic acute regulatory protein, Star; translocator protein, Tspo), steroidogenic enzyme genes (CYP11A1, Cyp11a1; HSD3B1, Hsd3b1; 17α-hydroxylase/C17,C20lyase, Cyp17a1; HSD11B1, Hsd11b1; 17β-hydroxysteroid dehydrogenase, Hsd17b3; nuclear receptor 5a1, Nr5a1). Genes representing Sertoli cell-related genes are SOX9 (Sox9) and dessert hedgehog (DHH, Dhh). The qPCR reaction mixture includes10 μL of SYBR Green Mix, 1.6 μL of forward and reverse primer mix, 1 μg of diluted cDNAs, and 5−8 μL of RNase-free water. The qPCR was set as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, and 59 °C for 30 s. The Ct value was collected, and the expression level of the target mRNA was calculated using a standard method as previously described.²² The relative expression level was standardized by Rps16, the house-keeping gene. Our previous studies have shown that Rps16 is stable during the development and under the stimulation of chemicals.^14,23 Melting curve was examined for the quality of qPCR amplification for each sample. The primers were listed in SI Table S1.

Western Blotting. Testis was homogenized at the ice-cold PBS to obtain a total protein samples. A BCA Assay Kit (Takara, Japan) was used to measure the protein concentration. Protein samples were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane. After being blocked with 5% fat-free milk, the membrane was incubated with the primary antibodies against the following antigens: LHCGR, STAR, SCARB1, CYP11A1, CYP17A1, SIRT1, PGC-1α, pAMPK, AMPK, pAKT2, AKT2, and ACTB (antibody information was listed in SI Table S2). After washing, HRP-conjugated antirabbit or antigoat IgG secondary antibody (1:2000, Bioword, U.S.A.) was added and incubated and the immunoreactive bands were identified by an ECL chemiluminescence kit (Amersham, Arlington Heights, IL). The intensity of protein was quantified by Image Lab software and adjusted to the ACTB, the internal control protein. Our previous studies have shown that ACTB is stable during the Leydig cell development and under the stimulation of chemicals.^14,23

Quantitative Immunohistochemical Measurement of CYP11A1, HSD11B1, and SOX9. The level of a protein in the tissue not only relies on its expression level but also on the cell number. Quantitative immunohistochemical staining was used to detect the intensity of CYP11A1 and HSD11B1 per Leydig cell and SOX9 per Sertoli cell. Image-Pro 6 Plus software was adopted to measure the intensity of the target protein and background area according to a previous study.²⁴

Statistical Analysis. The mean ± standard errors (SE) were used for data presentation. Statistical analysis was performed using GraphPad Prism (version 6, GraphPad Software, San Diego, CA) with one-way ANOVA followed by ad-hoc Dunnett’s multiple comparison. The p < 0.05 was considered a significant difference."

Male Sprague Dawley Rat