Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway Stephanie M. Correa1, Linda L. Washburn2, Ravi S. Kahlon 1 , Michelle C. Musson 1 , Gerrit J. Bouma2,3, Eva M. Eicher2, Kenneth H. Albrecht1,2* 1 Department of Medicine, Biomedical Genetics, Boston University School of Medicine, Boston, Massachusetts, United States of America, 2 The Jackson Laboratory, Bar Harbor, Maine, United States of America, 3 Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America Abstract Sex reversal can occur in XY humans with only a single functional WT1 or SF1 allele or a duplication of the chromosome region containing WNT4. In contrast, XY mice with only a single functional Wt1, Sf1, or Wnt4 allele, or mice that over-express Wnt4 from a transgene, reportedly are not sex-reversed. Because genetic background plays a critical role in testis differentiation, particularly in C57BL/6J (B6) mice, we tested the hypothesis that Wt1, Sf1, and Wnt4 are dosage sensitive in B6 XY mice. We found that reduced Wt1 or Sf1 dosage in B6 XYB6 mice impaired testis differentiation, but no ovarian tissue developed. If, however, a YAKR chromosome replaced the YB6 chromosome, these otherwise genetically identical B6 XY mice developed ovarian tissue. In contrast, reduced Wnt4 dosage increased the amount of testicular tissue present in Sf1+/2 B6 XYAKR, Wt1+/2 B6 XYAKR, B6 XYPOS, and B6 XYAKR fetuses. We propose that Wt1B6 and Sf1B6 are hypomorphic alleles of testis- determining pathway genes and that Wnt4B6 is a hypermorphic allele of an ovary-determining pathway gene. The latter hypothesis is supported by the finding that expression of Wnt4 and four other genes in the ovary-determining pathway are elevated in normal B6 XX E12.5 ovaries. We propose that B6 mice are sensitive to XY sex reversal, at least in part, because they carry Wt1B6 and/or Sf1B6 alleles that compromise testis differentiation and a Wnt4B6 allele that promotes ovary differentiation and thereby antagonizes testis differentiation. Addition of a ‘‘weak’’ Sry allele, such as the one on the YPOS chromosome, to the sensitized B6 background results in inappropriate development of ovarian tissue. We conclude that Wt1, Sf1, and Wnt4 are dosage-sensitive in mice, this dosage-sensitivity is genetic background-dependant, and the mouse strains described here are good models for the investigation of human dosage-sensitive XY sex reversal. Citation: Correa SM, Washburn LL, Kahlon RS, Musson MC, Bouma GJ, et al. (2012) Sex Reversal in C57BL/6J XY Mice Caused by Increased Expression of Ovarian Genes and Insufficient Activation of the Testis Determining Pathway. PLoS Genet 8(4): e1002569. doi:10.1371/journal.pgen.1002569 Editor: David R. Beier, Harvard Medical School, United States of America Received February 15, 2011; Accepted January 16, 2012; Published April 5, 2012 Copyright: 2012 Correa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by grants from the National Institutes of Health (HD042779 to KHA, GM20919 to EME, and CA34196 to The Jackson Laboratory) and American Cancer Society (RGS-06-033-01-DDC to KHA). Additional support was provided by a National Institutes of Health training grant (DK007053 for SMC). National Institutes of Health website: http://www.nih.gov/. American Cancer Society website: http://www.cancer.org/Research/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kha@bu.edu Introduction Testis differentiation in humans is gene dosage-sensitive. For example, heterozygosity for WT1 or SF1 (NR5A1) mutations and duplications containing WNT4 has been associated with XY sex reversal (SR) [1–10]. Testis differentiation in humans also appears to be genetic background-sensitive because fathers who are heterozygous for SF1 mutations or hemizygous for SRY mutations can pass these mutations to their heterozygous/ hemizygous XY daughters [4,11,12]. In contrast, initial studies in mice suggested testis differentiation is dosage-insensitive because heterozygosity for Wt1 or Sf1 alleles or transgenic Wnt4 over-expression did not cause XY SR [13–17]. This apparent human/mouse species difference led to the hypothesis that some genetic events leading to testis development differed between these species [18]. However, given the evidence that in mice genetic background plays a critical role in testis differen- tiation [19,20], we examined the possibility that Wt1, Sf1 and/or Wnt4 would be haploinsufficient for normal testis determination on specific genetic backgrounds. Work by our group and others has shown that testis determination in C57BL/6J (B6) inbred mice is particularly sensitive to genetic perturbation and a number of inherited gonadal sex reversal conditions have been identified in which B6 XY mice develop ovarian tissue [19–23]. The founding member of this group, B6-YPOS sex reversal, occurs when the Y Chromosome (Chr) from Mus domesticus poschiavinus (YPOS) is transferred to the B6 genetic background, replacing the Mus musculus-derived YB6 Chr [19]. B6 XYPOS fetal gonads develop as either ovotestes (gonads that contain both ovarian and testicular tissue) or ovaries, but not normal testes. The YPOS Chr is not inherently defective, however, because it is testis-determining when transferred to most other inbred strain backgrounds, including DBA/2J (D2), BALB/cBy, C3H/HeJ and 129, or when present in F1 hybrids, such as in (D26B6)F1 mice [24–27]. Furthermore, not all M. domesticus- derived Y Chrs (i.e., Sry alleles) behave like YPOS (SryPOS) when PLoS Genetics | www.plosgenetics.org 1 April 2012 | Volume 8 | Issue 4 | e1002569