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Table 1 summarizes our observations of Sox9-positive cells in B6.Ytir embryos at various developmental stages. Although in 50% both gonads and in 25% one gonad showed pre-Sertoli cells at 13.5 dpc, in 25% both gonads and in 25% one gonad showed Sox9-positive Sertoli cells by 18.5 dpc. Taken together, Sox9-positive cells were absent in about half of the gonads studied between 13.5 and 18.5 dpc. This corresponds to the reported proportion of ovotestis and ovaries that form in B6.Ytir mice. Profiles of Sox9 Transcripts in B6.Ytir Ovaries and Ovotestis In contrast to the immunocytochemical detection of the Sox9 protein, RT-PCR detection of Sox9 transcripts showed that they were present at all stages and in all B6.Ytir fetal gonads (Fig. 5). A semiquantitative densitometric analysis revealed that the levels of Sox9 transcripts increased from 13.5 to 17.5 dpc in both B6 testes and B6.Ytir ovotestes. Nevertheless, statistical analysis indicated that B6.Ytir ovotestis had lower Sox9than B6 testis at 13.5-17.5 dpc, which was significantly lower (P < 0.001) at 17.5 dpc. In contrast, B6.Ytir ovaries showed significantly lower Sox9 earlier at 15.5 and 17.5 dpc (P < 0.001; Fig. 5). On the other hand, Sox9 transcripts were not detected in all B6 XX fetal ovaries at 13.5-17.5 dpc stages (not shown). Representative gels and semiquantitative densitometric analyses of Sox9 transcripts in fetal (18.5 dpc) and postnatal gonads (30 and 60 dpp) of two normal strains (CD1 and B6) and B6.Ytir gonads are shown in Figure 6. Although at 18.5 dpc and 30 dpp, Sox9 mRNA was detected in all fetal gonads with a Y chromosome from CD1, B6, or Ytir, the expression levels varied according to the strain and the type of gonad formed (ovotestis, testis, or ovary). Sox9 levels were not significantly different between testes of CD1 and B6 at all three stages. Comparing fetal gonads of B6 testes with B6.Ytir ovotestis at 18.5 dpc, no significant differences were found. In contrast, B6.Ytir ovaries had significantly lower levels than B6 testis at 18.5 dpc (Fig. 6A). FIG. 5. Semiquantitative densitometric analysis of Sox9 transcripts in B6.Ytir fetal gonads at 13.5, 15.5, and 17.5 dpc. Sox9 levels increase significantly from 13.5 to 17.5 dpc in normal developing testes (B6-T). In B6.Ytir ovotestes (Ytir-ot) at 13.5 and 15.5 dpc, the levels of Sox9 are not significantly different compared with normal B6 testes. However, at 15.5 dpc the levels of Sox9 transcripts of Ytir ovaries (Ytir-o) were lower, showing significant differences compared with B6 testes. At 17.5 dpc the levels of Sox9 transcripts of Ytir-ot and Ytir-o showed significant lower differences compared with B6 testes. Densitometric profiles are expressed as Sox9/ p-actin ratio per pair of gonads. Values are given as mean ± standard deviation of five independent assays (ANOVA followed by Tukey's test, *P < 0.001 versus values from bilateral testes from the B6 strain). FIG. 6. Expression of Sox9 transcripts in gonads from CD1, C57BL/6J (B6), and from litters of B6 females mated with B6.Ytir males (Ytir). Samples were analyzed at 18.5 dpc (A), 30 dpp (B), and 60 dpp (C). Top figures show representative ethidium bromide gel electrophoresis of testes (CD1-T, B6-T); ovaries (CD1-O, B6-O); B6.Ytir bilateral testes and bilateral ovaries (Ytir-T, Ytir-O); normal ovaries (XX-O); and unilateral testes and ovaries (Ytir-HT, Ytir-HO) from hermaphrodite mice. p-actin served as loading control. Columns below each gel show a compilation of semiquantitative data from densitometric profiles of the Sox9/p-actin ratio obtained in RT-PCR assay at 18.5 dpc and at 30 and 60 dpp. Bilateral testes (T), bilateral ovaries (O), unilateral testes (HT), and unilateral ovaries (HO) are again indicated. At 18.5 dpc, CD1, B6, and Ytir bilateral and unilateral testes show similar levels of Sox9, whereas they were significantly lower in B6.Ytir bilateral and unilateral ovaries. At 30 dpp, only significant differences were observed in unilateral ovaries compared with CD1, B6, and Ytir testes. No differences were detected in levels of Sox9 between CD1, B6, and B6.Ytir testes. Although Sox9 transcripts are absent in normal ovaries, they were detected in B6.Ytir ovaries at 18.5 dpc and 30 dpp, but not at 60 dpp. Values are given as mean ± standard deviation of least three independent determinations. (ANOVA followed by Tukey's test, *P < 0.001 versus values from bilateral testes from B6 strain).