DMRT1 is a Testis Determining Gene in Rabbits and is Also Essential for Female Fertility

Reviewer #1 (Public Review):

DMRT1 is essential in testis development in different species. While Dmrt1 is the testis-determining factor in chicken and deletion encompassing this gene lead to gonadal dysgenesis in human, the role of DMRT1 in testis development remains to be clarified. Despite an early expression of Dmrt1 in the mouse gonad and a potential function as a pioneer factor, DMRT1 is only required for the maintenance of the Sertoli cell identity in the postnatal testis. The use of a new animal model could provide new insights into the role of this factor in humans. Here the authors have generated a knockout model of DMRT1 in rabbits. They show that the XY mutant gonads differentiate as ovary indicating that DMRT1 is required for testis differentiation in rabbits. In addition, most of the germ cells remain pluripotent as evidenced by the maintenance of POU5F1 in both XY and XX mutant gonads. These are very important results potentially explaining gonadal dysgenesis associated with the DMRT1 locus in disorders of sex development in humans.

The experiments are meticulous and convincing. I find the arguments of the authors about the role of DMRT1 in germ cells in addition to its function in Sertoli cell differentiation, both comprehensible and compelling. Clearly, this is an important insight in sex determination and gametogenesis.

Reviewer #2 (Public Review):

It is well known that DMRT proteins and more specifically, DMRT1 plays a key role in sex determination processes of many species. While DMRT1 has been shown to be critical for the sex determination of fish, birds, and reptiles, it seems less crucial at the sex determination stages of the mice. It is important though for adult sex maintenance in mice.

Unlike its minor role in mouse sex determination, it seems that variants in DMRT1 in humans cause 46, XY DSD and sex reversal.

The paper by Dujardin et al., is a beautiful study that provides an answer to this long-lasting discrepancy of the difference between the two common mammal species: human and mouse. It is a really nice example of how working with other mammal species, like the rabbit, could serve as a nice model for understanding mammalian sex determination.

In this study the researchers first described the expression patterns of DMRT1 in the rabbit XY and XX gonads throughout the window of sex determination.

They then used CRISPR/Cas9 to generate DMRT1 KO rabbits and analysed the phenotype in XY and XX rabbits. They show that XY rabbits present with complete XY male-to-female sex reversal, very similar to what was observed in human 46, XY DSD patients (but not the mice model). They further show that in the XY sex-reversed gonads, germ cells fail to enter meiosis. They next analysed XX gonads and while there is no major effect on sex determination (as expected), the germ cells in these ovaries fail to enter meiosis, highlighting the critical role that DMRT1 has in germ cells.

I think it is really important that we start to embrace other mammal models that are not the mouse as we find many instances that the mouse is not the optimal system for understanding human sex determination. The study is well explained and presented. The data is clear, and the paper is fluent to read.

Reviewer #3 (Public Review):

This manuscript deals with the sex-related gene, DMRT1, showing that it has a testis-promoting function in the rabbit. In loss-of-function studies in the mouse and human, DMRT1 has a role in testis maintenance after birth, although forced expression in the mouse can induce testis formation.

The authors used CRISPR/Cas9 genome editing to generate DMRT1-/- rabbit embryos. The gonads of these embryos developed as ovaries. Interestingly, in addition Y-linked SRY, DMRT1 is required for timely up-regulation of SOX9 during Sertoli cell differentiation in the male gonad. This is quite different to the situation in mice, where Dmrt1 is not required in the testis until after birth (and Sry induced up-regulation of Sox9 hence does not require Dmrt1).

The work adds to the field of sex determination by further broadening our understanding of the DMRT1 gene and the evolution of gonadal sex determination.

In the Discussion section, it is suggested that DMRT1 could act as a pioneering factor to allow SRY action upon Sox9 in the rabbit model. The data show that DMRT1 may be more central to testis formation in mammals than previously considered. The work supports the notion that our understanding that the genetics of gonadal development (and indeed development more generally) should not rest solely on findings in the mouse.