Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. 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. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Adequate nutrient supply is crucial for the proper development of the embryo. Although nutrient supply is determined by maternal diet, the gut microbiota also influences nutrient availability. While currently there is no cure for neural tube defects (NTDs), their prevention is largely amenable to maternal folic acid and inositol supplementation. The gut microbiota also contributes to the production of these nutrients, which are absorbed by the host, but its role in this context remains largely unexplored. In this study, we performed a functional and morphological analysis of the intestinal tract of loop-tail mice (Vangl2 mutants), a mouse model of folate/inositol-resistant NTDs. In addition, we investigated the changes in gut microbiota using 16S rRNA gene sequencing regarding (1) the host genotype; (2) the sample source for metagenomics analysis; (3) the pregnancy status in the gestational window of neural tube closure; (4) folic acid and (5) D-chiro-inositol supplementation. We observed that Vangl2^+/Lp mice showed no apparent changes in gastrointestinal transit time or fecal output, yet exhibited increased intestinal length and cecal weight and gut dysbiosis. Moreover, our results showed that the mice supplemented with folic acid and D-chiro-inositol had significant changes in their microbiota composition, which are changes that could have implications for nutrient absorption.

Neural tube defects (NTDs) are the second most common cause of congenital malformations and are often studied in animal models. Loop-tail (Lp) mice carry a mutation in the Vangl2 gene, a member of the Wnt-planar cell polarity pathway. In Vangl2+/Lp embryos, the mutation induces a failure in the completion of caudal neural tube closure, but only a small percentage of embryos develop open spina bifida. Here, we show that the majority of Vangl2+/Lp embryos developed caudal closed NTDs and presented cellular aggregates that may facilitate the sealing of these defects. The cellular aggregates expressed neural crest cell markers and, using these as a readout, we describe a systematic method to assess the severity of the neural tube dorsal fusion failure. We observed that this defect worsened in combination with other NTD mutants, Daam1 and Grhl3. Besides, we found that in Vangl2+/Lp embryos, these NTDs were resistant to maternal folic acid and inositol supplementation. Loop-tail mice provide a useful model for research on the molecular interactions involved in the development of open and closed NTDs and for the design of prevention strategies for these diseases.

In species with seasonal breeding, male specimens undergo substantial testicular regression during the nonbreeding period of the year. However, the molecular mechanisms that control this biological process are largely unknown. Here, we report a transcriptomic analysis on the Iberian mole, Talpa occidentalis, in which the desquamation of live, nonapoptotic germ cells is the major cellular event responsible for testis regression. By comparing testes at different reproductive states (active, regressing, and inactive), we demonstrate that the molecular pathways controlling the cell adhesion function in the seminiferous epithelium, such as the MAPK, ERK, and TGF‐β signaling, are altered during the regression process. In addition, inactive testes display a global upregulation of genes associated with immune response, indicating a selective loss of the “immune privilege” that normally operates in sexually active testes. Interspecies comparative analyses using analogous data from the Mediterranean pine vole, a rodent species where testis regression is controlled by halting meiosis entry, revealed a common gene expression signature in the regressed testes of these two evolutionary distant species. Our study advances in the knowledge of the molecular mechanisms associated to gonadal seasonal breeding, highlighting the existence of a conserved transcriptional program of testis involution across mammalian clades.

In mammals, sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads ^{1 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 differentiation ^{3 4 5} . Despite this strong genetic component, the involvement of non-coding regulation in determining mammalian sex remains unclear ⁶ . 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.