"The maternal and pup effects (body weight and birth rate of dams, as well as body weight, AGD, and male number from each dam for male pups) were studied. The trunk blood of each male pup was harvested and blood samples from all males in each litter were pooled and stored at room temperature for 30 min. The pooled blood samples were then centrifuged at 2000 × g at 4_C for 10 min. Serum was collected for T measurement. Fetal testes were collected for morphological examination and molecular biology study.

2.3 | Determination of serum T levels

Serum T concentrations from pooled samples were determined using the Immulite2000 Total T Kit (Sinopharm, Hangzhou, China) in Department of Clinical Chemistry of our hospital as previously described.²⁰ The lowest detectable level of T was 0.1 ng/ml. The coefficient of variation of intraassay was less than 15%.

2.4 | Testis tissue-array setup and immunohistochemistry

One male pup testis per litter was randomly selected and fixed in Bouin's solution for 24 h. The fixed testis was embedded in paraffin, and then assembled in a tissue array tray as described previously.²¹ CYP11A1, a key enzyme for T synthesis, was used as a biomarker²¹ to show FLCs in the fetal testes. SOX9, a key transcription factor of SCs,²² was used to show SCs. Antibody-derived immunohistochemistry was performed using a Vector kit (Burlingame, CA) to identify FLCs and SCs as described.²¹ In brief, the testis tissue-array was cut into a series of 6 μm sections by a microtome. The sections were dewaxed and subjected to sequential procedures of immunohistochemistry, including antigen presentation, endogenous peroxidase inhibition, primary antibody incubation, secondary antibody ligation, color staining, and counterstaining. The cell with a brown staining in the cytoplasm after CYP11A1 antibody incubation was called the FLC, and cell with a brown staining in the nucleus after SOX9 antibody treatment was called the SC. The number of CYP11A1-positive FLCs and SOX9-positive SCs were counted using a fractionation technique as described previously.²³

P0 Female Sprague–Dawley rats