Gadd45g is required for timely Sry expression independently of RSPO1 activity.

Sex determination in mammals is controlled by the dominance of either pro-testis (SRY-SOX9-FGF9) or pro-ovary (RSPO1-WNT4-FOXL2) genetic pathways during early gonad development in XY and XX embryos, respectively. We have previously shown that early, robust expression of mouse Sry is dependent on the nuclear protein GADD45g. In the absence of GADD45g, XY gonadal sex reversal occurs, associated with a major reduction of Sry levels at 11.5 dpc. Here, we probe the relationship between Gadd45g and Sry further, using gain- and loss-of-function genetics. First, we show that transgenic Gadd45g overexpression can elevate Sry expression levels at 11.5 dpc in the B6.YPOS model of sex reversal, resulting in phenotypic rescue. We then show that the zygosity of pro-ovarian Rspo1 is critical for the degree of gonadal sex reversal observed in both B6.YPOS and Gadd45g-deficient XY gonads, in contrast to that of Foxl2. Phenotypic rescue of sex reversal is observed in XY gonads lacking both Gadd45g and Rspo1, but this is not associated with rescue of Sry expression levels at 11.5 dpc. Instead, Sox9 levels are rescued by around 12.5 dpc. We conclude that Gadd45g is absolutely required for timely expression of Sry in XY gonads, independently of RSPO1-mediated WNT signalling, and discuss these data in light of our understanding of antagonistic interactions between the pro-testis and pro-ovary pathways.

Protection Against XY Gonadal Sex Reversal by a Variant Region on Mouse Chromosome 13.

XY C57BL/6J (B6) mice harboring a Mus musculus domesticus-type Y chromosome (Y POS ), known as B6.Y POS mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.Y POS males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.Y POS colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.Y POS sex reversal. Here. we show that a B6.Y POS colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call Mod13, protects against B6.Y POS sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss Mod13's influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.Y POS sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.

Gadd45γ and Map3k4 interactions regulate mouse testis determination via p38 MAPK-mediated control of Sry expression.

Loss of the kinase MAP3K4 causes mouse embryonic gonadal sex reversal due to reduced expression of the testis-determining gene, Sry. However, because of widespread expression of MAP3K4, the cellular basis of this misregulation was unclear. Here, we show that mice lacking Gadd45γ also exhibit XY gonadal sex reversal caused by disruption to Sry expression. Gadd45γ is expressed in a dynamic fashion in somatic cells of the developing gonads from 10.5 days postcoitum (dpc) to 12.5 dpc. Gadd45γ and Map3k4 genetically interact during sex determination, and transgenic overexpression of Map3k4 rescues gonadal defects in Gadd45γ-deficient embryos. Sex reversal in both mutants is associated with reduced phosphorylation of p38 MAPK and GATA4. In addition, embryos lacking both p38α and p38ß also exhibit XY gonadal sex reversal. Taken together, our data suggest a requirement for GADD45γ in promoting MAP3K4-mediated activation of p38 MAPK signaling in embryonic gonadal somatic cells for testis determination in the mouse.