The detailed materials and methods were described in the Supplementary material S1. General toxicological parameters after treatment of vinclozolin were described in Table1. Chemicals, reagents, kits, software, and equipment were described in Table2. Antibody sources and information were described in Table3. Primer information was described in Table4.
Animals and experimental design
The animal model used in the current study was the Sprague-Dawley rat. Adult female and male Sprague-Dawley rats (56 days of age, weighed at 220 ± 10 g) were purchased from the Shanghai Laboratory Animal Center (Shanghai, China). Rats were allowed one week to acclimate to the experimental environment. Time-pregnant female rats were randomly assigned into different treatment groups: 0, 25, 50, and 100 mg/kg/day VCZ (n = 8 rats/group). The experimental protocol for animal toxicity experiment was approved by Wenzhou Medical University Laboratory Animal Ethics Committee. The experiment was performed following the procedures described in the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.
VCZ was suspended in corn oil (vehicle control). Daily gavage dosing of the dams started on GD 14 and continued until the initiation of parturition at doses of 0, 25, 50, or 100 mg/kg. Dose selection is based on the observation that VCZ significantly caused anomalies of the male reproductive tracts of rats at 200-400 mg/kg . Body weights of dams were daily recorded. Male pups were euthanized by CO2. Blood and testis of each male pup was collected for the following studies.
Measurement of serum T level
Serum T level was detected by the Siemens Healthcare Diagnostics Total Testosterone Kit as previously described . The inter-assay and intra-assay coefficients of variation were within 15%.
Histochemical hematoxylin staining
The incidence of multinucleated gonocytes (MNGs) in the fetal testis can be increased by some endocrine-disrupting chemicals [14-16]. The occurrence rate of MNGs after VCZ treatment was counted as previously described . In brief, cross-sections were sliced at 6 µm from a testis tissue-array by microtome and stained with hematoxylin solution as previously described . The percentage of the seminiferous cord containing MNGs was calculated.
CYP11A1 is used as the biomarker of the FLC . Transcription factor SOX9 is used as the biomarker of the SC . The total numbers of CYP11A1-positive cell and SOX9-positive cells were counted by a fractionator technique as previously described . In brief, fetal testes were embedded in paraffin block in a tissue-array block, cross-sections were sliced and stained immunohistochemically with CYP11A1 or SOX9. Immunohistochemical staining method was used as previously described .
Computer-assisted analysis of FLC metrics
The FLC metrics include cell, nuclear, and cytoplasmic sizes, which were measured as previously described . In brief, cell and nuclear sizes were measured by the Media Cybernetics Image-Pro 6 Plus software, with the measurement parameter of the average area.
Semi-quantitative immunohistochemical measurement of CYP11A1 and SOX9
Immunohistochemical staining of CYP11A1 and SOX9 were performed as described . Target protein density and surrounding area background density were measured by the Media Cybernetics Image-Pro 6Plus with the measurement parameter of the main density.
Measurement of FLC proliferation
The proliferation of FLCs was measured by immunofluorescent staining of proliferating cell nuclear antigen (PCNA) and CYP11A1 as previously described . In brief, the cross-sections in the tissue-array congregated above were utilized. Sections were double-labeled with the primary antibodies of CYP11A1 and PCNA followed by the fluorescent secondary antibody.
Measurement of stem Leydig cell number
Previous studies demonstrated that FLCs were developed from stem Leydig cells [8, 24]. Stem Leydig cells in the fetal testis are spindle-shaped and express NR2F2, also called COUP-TFII [8, 24-26]. We used NR2F2 as the biomarker to identify the stem Leydig cell and SOX9 as the biomarker to draw a boundary for the seminiferous cord. Double immunofluorescent staining was performed as above. The number of NR2F2-positive cells per square millimeter was calculated.
Preparation of RNA-seq library
Eight testes per dam were randomly selected for total RNA isolation. Total RNAs were extracted from testes using Invitrogen Trizol Kit as previously described . Total RNAs were enriched by oligo-dT magnetic beads and RNA-seq library was set up by Illumina KAPA Stranded RNA-Seq Library Prep Kit. The libraries were sequenced on the Illumina HiSeq 4000 instrument.
We performed RNA-seq of the testis and biological pathway analysis to address the pathway of VCZ-mediated action in vivo as previously described . Gene expression level, Gene Ontology, Pathway analysis, scatter plots, and volcano plots were performed.
Biological pathway analysis
Biological pathway analysis was performed as previously described . In brief, the GenMAPP2.1 software was used to create a map of signal pathways for the potential pathways.
Quantitative real-time PCR (qPCR)
We performed the qPCR of VCZ-treated samples to verify the sequencing data of the testes and to measure the levels of some mRNAs that were not detectable by RNA-seq as previously described . The mRNA levels of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, Nr5a1, Pcna, Pdgfa, Amh, Dhh, and Sox9 were analyzed using the SYBR Green qPCR Kit. Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, were Insl3 were FLC genes. Pdgfa, Amh, Dhh, and Sox9 are SC genes, and Nr5a1 is the critical transcription factor for the development of both FLCs and SCs. Pcna is the proliferating cell biomarker. The mRNA levels were determined by a standard curve method.
Western blot was performed as previously described . In brief, testis is lysed, denatured, electrophoresed and blotted with the primary antibody followed by the secondary antibody. Blots were stripped and incubated with a monoclonal GAPDH antibody served as the internal control.
Measurement of serum IGF-1
Insulin-like growth factor 1 (IGF-1) plays important roles in cell proliferation, differentiation and growth of many cells, including Leydig cells [11, 31]. The circulatory IGF-I is mainly produced by mammalian livers . IGF-1 ELISA kit was used for the measurement of serum IGF-I according to the manufacturer’s instruction.
Values for all intents and purposes are expressed as mean ± S.E.M., and data were analyzed by one-way ANOVA followed ad hoc Turkey’s analysis to specifically compare values from VCZ-treated rats to the control. GraphPad Prism was used.