"dams were ranked by weight and randomly divided into 4 groups (10 rats per group): 0, 10, 50, and 200 mg/kg/d BPAF. A volume of 2 ml per kilogram of corn oil containing BPAF were orally gavaged to dams from GD 14 to GD 21. The dose range was selected based on a reproductive toxicity study, in which BPAF significantly reduced T production in male adult rats after oral exposure to 50 and 200 mg/kg/d BPAF"

"After the neonates were delivered, the male pups were euthanized and anogenital distance (AGD) was gauged. Blood samples of male offspring were collected and centrifuged at 3000 ×g for 10 min for sera, which were pulled together for each litter. The body weight of the male offspring was recorded. The testis was stored at -80 ◦C for subsequent analysis of mRNA and protein levels. The other testes were fixed in Bouin's solution for subsequent immunohistochemistry."

"Serum T concentration in the pooled male pups per litter was measured by immunoassay using the Immulite 2000 Immunoassay System and total Testosterone Kit as previously described (Zhu et al., 2021). Minimal detection limit of T was 0.1 ng per ml. The intra and interassay variation coefficients of T evaluation were within 10%."

"The fetal testis is an ellipsoid with “a”, “b”, and “c” for half length of the x-axis, y-axis, and z-axis, respectively. One testis in each litter was randomly selected. The image was taken by a digital camera. The image was displayed by ImagePro Plus software 6.0 (Media Cybernetics, Rockville, MD) and the size of each testis (the length of “a”, “b”, and “c”). The volume of the ellipsoid (V) was calculated as 4/3πabc."

"The tissue samples for immunohistochemistry were dehydrated after Bouin's fixation, and embedded in paraffin. To achieve an equivalent target protein density after immunohistochemistry, the testis was assembled into the tissue-array as described (Ye et al., 2020). A series of five-micron-thick cross sections were prepared under a microtome and some of which were stained by immunohistochemistry. CYP11A1, the first steroidogenic enzyme in the fetal Leydig cell lineage, was used to identify fetal Leydig cells. The transcription factor SOX9, existing in Sertoli cells, was used to identify Sertoli cells. Samples stained with CYP11A1 and SOX9 were antigen unmasked using 10 mM sodium citrate (pH 6) and ABC vector kit with DAB substrate (Vector Labs) was used to generate the signal according to manufacturer's protocol. The slides were scanned to digital files by NanoZoomer-XR (Hamamatsu, Japan) by the 0.23 μm/pixel mode."

"The scanned image was displayed by Image-Pro plus software, and then CYP11A1-positive fetal Leydig cells or SOX9-positive Sertoli cells were identified and calculated as the specific cell density. The number of fetal Leydig cells and Sertoli cells per testis was calculated by the testicular volume. Fetal Leydig cells appear as a single cell or a cluster, in which two or more fetal Leydig cells gathered. We defined the cluster size of fetal Leydig cells as single (1 cell per cluster), small (2–4 cells per cluster), medium (5–16 cells per cluster), and large (>16 cells per cluster). The cluster distribution was calculated as a percentage as previously described (Li et al., 2014). Since some endocrine disruptors can affect cell size and the cytoplasm/nucleus ratio of a fetal Leydig cell, these parameters were measured by Image-Pro plus software as previously described"

"RNA was extracted from the testis with Trizol (Invitrogen, Waltham, MA) according to the manufacturer's instructions. The cDNA was produced using random primers and a Reverse Transcription Kit (Promega Madison, WI). The expression of fetal Leydig cell genes (Lhcgr, Star, Scarb1, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, and Nr5a1), Sertoli cell genes (Fshr, Amh, Dhh, and Sox9), and house-keeping genes (Rps16, Gapdh and Actb) was determined by a SYBR Green qPCR Kit according to the previously described standard curve method (Ge et al., 2005)."

"Frozen testis was placed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) and homogenized. The supernatant was separated from the pellet after centrifugation at 14,000 rpm at 4 ◦C for 10 min. The protein concentration was determined by the BCA (Beyotime, Shanghai, China) method using bovine serum albumin (BSA) as the standard. The sample was denatured and loaded onto a 10% sodium dodecyl sulfatepolyacrylamide gel. The proteins were separated by electrophoresis and then were transferred onto a polyvinylidene difluoride membrane. The membrane was placed in an incubation box containing 5% BSA to block non-specific binding for 2 h, and the membrane was incubated overnight at 4 ◦C with primary antibodies for each of the following antigens: SCARB1, STAR, CYP17A1, HSD17B3, DHH, SIRT1, PGC1α, AMPK, pAMPK, and RPS16. After washing, the membrane was incubated with the corresponding HRP-conjugated anti-rabbit (1:2000) or anti-mouse IgG secondary antibody (1:2000, MultiSciences Biotechnology, Hangzhou, China) at room temperature for 2 h, and then subjected to ECL display using a kit (Amersham, Little Chalfont, UK). Bio-Rad Lab software (Hercules, CA) was used to calculate the gray intensity of the protein. The test protein intensity was normalized to housekeeping protein RPS16."

"The scanned testis image was displayed by Image-Pro plus software, and then CYP17A1-positive fetal Leydig cells were identified. The CYP17A1 staining and the background were selected to calculate the mean density. Fifty cells per sample were counted and averaged as previously described (Li et al., 2014)."

"Malondialdehyde (MDA) is the product of lipid peroxidation, reflecting the oxidative stress level. An MDA kit (Solarbio Science and Technology, Beijing, China) was used to measure the MDA levels in the male pup's testicular homogenates as previously described (Li et al.,

2020b). Five-six testes (about 10 mg) per litter were randomly selected and the MDA contents were extracted by 500 μl MDA extraction buffer. 100 μl extraction suspension was reacted with reaction agent and the solution was read at the absorbance at 532, 450, and 600 nm."

"HE staining was carried out on the testicular tissue-array sections. The multinucleated gonocyte (MNG)-positive testicular cord was counted and calculated as a percentage of the total cords counted as previously described (Li et al., 2014)."

"To best describe the results, all data were listed with mean ± standard errors (SEM). Statistical significance was done by GraphPad software (version 8, San Diego, CA) to analyze differences using one-way ANOVA followed by post hoc Dunnett's multiple comparisons to the control. p < 0.05 was set as statistical significance."

F1 Male Sprague-Dawley Rats