Complete male-to-female sex reversal in XY mice lacking themiR-17∼92cluster

In mammals, sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads¹². The expression of the Y-linked geneSRYis sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation³⁴⁵. 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 thatSryis heterochronically expressed, showing a delay in XYmiR-17∼92knockout 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 ofmiR-17∼92target 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 ofSryexpression and Sertoli cell differentiation.