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To determine why the Wt1B6 and Sf1B6 alleles are haploinsufficient and function as hypomorphs, we compared the coding sequences and expression levels of the B6 and D2 alleles. The results indicated that the Wt1B6 and Wt1D2 allele encode the same protein and that the ±KTS Wt1 isoform expression ratio was the same in B6 and D2 gonad/mesonephros complexes. However, we found that Wt1 expression throughout the initial stage of testis differentiation was lower in complexes from B6 fetuses, suggesting that in the B6 genetic background the Wt1B6 allele is an expression hypomorph. In contrast, we were unable to detect significantly lower Sf1 expression in complexes from B6 fetuses but we did identify one coding polymorphism in Sf1, suggesting that the Sf1B6 allele is haploinsufficient not because its expression is reduced but possibly because the SF1B6 protein variant has reduced function. It appears that two SF1 variants are present among the common inbred mouse strains: SF1A172 (present in B6) and SF1S172 (present in D2). However, we found that homozygosity for the Sf1A172 allele in F1 hybrids was not sufficient to cause sex reversal in the presence of the YPOS Chr [as in (NZB/BlNJ×B6 XYPOS) F1s] and that trans-heterozygosity for the Sf1A172 and Sf1S172 alleles was not sufficient to prevent sex reversal in the presence of the YPOS Chr [as in (BALB/cBy×B6 XYPOS) F1s]. These data show that the SF1A172 polymorphism alone does not account for sex reversal in the presence of the YPOS Chr. Therefore, if the SF1A172 protein variant does indeed have reduced function during testis determination, then homozygosity for B6 alleles at other loci likely is necessary to cause Sf1B6 to function as a hypomorph. Frigeri and colleagues [41] demonstrated that the SF1A172 and SF1S172 protein variants did not consistently differ in their ability to transactivate reporter genes containing tandem SF1 binding sites, in vitro. However, further experiments are needed to determine if the SF1A172 protein variant has reduced function during gonad differentiation in vivo. 2b1af7f3a8