The sex of the first-trimester samples was determined by polymerase chain reaction (PCR) analysis of genomic DNA using primers directed against the SRYgene. Briefly, 100 |xl of 25 mM NaOH/0.2 M EDTA were added to approximately 50 mg of tissue and heated at 95°C for 20 min before the addition of 100 |xl of 40 mM Tris-HCl. The samples were then mixed by vortex and centrifuged briefly. Five microliters of supernatant were used in a standard 25-|xl PCR reaction with Qiagen HotStar Taq. The SRY primers used were those detailed by Friel et al. : forward, 5′-ACAGTAAAGGCAACGTCCAG, and reverse, 5′-ATCTGCGGGA-AGCAAACTGC].
Details of all antibodies used in the present study and the dilutions used for immunohistochemistry with a single antibody are shown in Table 1. Sections (thickness, 5 |xm) of Bouin-fixed, paraffin-embedded fetal testes were mounted on charged glass slides (BDH Chemicals, Poole, U.K.), dewaxed, rehydrated, and subjected to heat-induced antigen retrieval in 0.1 M citrate (pH 6) if required (Table 1). Endogenous peroxidase activity was blocked by incubation in 3% (v/v) H2O2 in methanol for 30 min.