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Two microliters of each DNA sample was processed for PCR amplification in a total volume of 20 |xl with 0.25 |xM of each of the following primers: Zfy1, 5'-AAG ATA AGC TTA CAT AAT CAC ATG GA-3'; Zy2, 5'-CCT ATG AAA TCC TTT GCT GCA CAT GT-3' for Zfy; Actin 1, GGG TCA GAA GGA TTC CTA TG; and Actin 2, GGT CTC AAA CAT GAT CTG GG for p-actin used as an internal control. The primers were in 10 mM Tris-HCl pH 8.3, 50 mM KCl, 0.01% gelatin, 2.0 mM MgCl2, 0.2 mM of each dNTP and 1.0 U Taq DNA polymerase. The amplification program was 94°C/10 min (once), 94°C/15 sec, 60°C/15 sec, 72°C/30 sec (35 cycles), and 72°C/10 min (once). The PCR reactions were electrophoresed on a 1% agarose gel in TAE 1X buffer and visualized with ethidium bromide fluorescence in UV light. Immunocytochemical Detection of SOX9 Antigen-affinity antibody raised to the human SOX9 C-terminal 24-amino-acid epitope (VPSIPQTHSPQHWEQPVYTQLTRP) was prepared according to the previously described protocol. For immunocyto-chemical detection of Sox9, B6-Ytir gonads were dissected from embryos from 11.5 to 18.5 dpc and fixed in 4% paraformaldehyde in PBS for 10 min, washed in PBS, and incubated in 30% sucrose overnight.