Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster

In mammals, sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads1 2. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation3 4 5. Despite this strong genetic component, the involvement of non-coding regulation in determining mammalian sex remains unclear6. Here we show that the deletion of a single microRNA cluster, miR-17[~]92, induces complete primary male-to-female sex reversal in XY mice. Time-course analyses revealed that Sry is heterochronically expressed, showing a delay in XY miR-17[~]92 knockout gonads, which subsequently activate the ovarian genetic program. Bulk and single cell RNA-seq analyses showed that Sertoli cell differentiation is reduced, delayed and unable to sustain the testicular fate. This disrupted differentiation results from a transient state of sex ambiguity in pre-supporting cells, which is later resolved towards the ovarian fate. Consistent with known mechanisms of miRNA-mediated gene regulation, the expression of miR-17[~]92 target genes is not stabilized in undifferentiated XY mutant gonads, affecting concomitantly the fine regulation of gene networks with critical roles in developing gonads. Our results demonstrate that microRNAs are key components for mammalian sex determination, controlling the timing of Sry expression and Sertoli cell differentiation.