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

Alicia Hurtado, Irene Mota-Gómez, Miguel Lao, Francisca M. Real, Johanna Jedamzick, Miguel Burgos, Darío G. Lupiáñez, Rafael Jiménez, Francisco J. Barrionuevo

Posted on: 7 May 2023 , updated on: 10 May 2023

Preprint posted on 23 March 2023

Small but mighty! miRNA cluster miR-17-92 knock out results in complete testis to ovary sex reversal

Selected by Martin Estermann

Categories: bioinformatics, cell biology, developmental biology, genetics, molecular biology

Background:

During embryogenesis, the mammalian bipotential gonads differentiate into ovaries in XX females and testis in XY males. The supporting cell lineage is the first to sexually differentiate into their ovarian (pre-granulosa) or testicular (Sertoli) fates. Once these cells are committed, they regulate the differentiation of the rest of the gonadal cells. In XY males, the expression of the Y-linked gene Sry is required to activate the testicular differentiation program, including Sox9. Sox9 then will cooperate with Fgf9 to achieve Sertoli cell differentiation, while inhibiting the expression of pre-granulosa differentiation genes. In XX females, in the absence of Sry, the gonads upregulate Foxl2, initiating the differentiation towards the pre-granulosa fate and inhibiting Sertoli cell genes.

In mouse, the activation of Sry occurs in a precise and narrow time window (Embryonic day 11.0 to 11.25). A delay in Sry expression (6 hours) results in the improper activation of Sox9. As a result, these gonads will differentiate into ovaries as the pre-granulosa cell program is not repressed.

Most of the research in the field of gonadal sex determination has focused on the coding genome, whereas the non-coding regions remain unexplored. In this preprint, the researchers evaluated the role of the microRNA (miRNA) cluster miR-17-92 in gonadal sex determination. miRNAs are small non-coding RNA molecules (21-23 nucleotides) that bind to complementary regions of mRNAs, targeting them for degradation and inhibiting their expression. The miR-17-92 cluster contains six miRNA genes (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1) and is known to be involved in several developmental processes. In this research, the authors knocked out (KO) this cluster in mice, resulting in a complete male to female sex reversal: ovarian differentiation in XY males.

Key findings:

1) miR-17-92 KO causes ovarian development and FOXL2 expression.

The authors evaluated gonadal development and supporting cell differentiation in knockout mice for the miR-17-92 cluster. As can be seen in Figure 1, control male gonads showed normal expression of SOX9 in the testicular cords, and specifically in the Sertoli cells. In female controls, FOXL2 expression was detected in the pre-granulosa cells, as expected. Surprisingly, XY gonads lacking the miRNA cluster developed ovaries, having FOXL2 positive pre-granulosa cells instead of SOX9 positive Sertoli cells. This indicates that by E17.5 there was a complete male to female sex reversal in the XY knockouts.

Figure 1. Immunofluorescence for Sertoli (SOX9, red) and pre-granulosa (FOXL2, green) cell markers in E17.5 XY and XX, wild type or miR-17-92 cluster KO gonads.

2) miR-17-92 KO results in delayed SRY expression and SOX9 activation.

To gain insight into the mechanism behind the sex reversal in the XY knockout gonads, SRY and SOX9 immunofluorescence was performed during the process of sex determination (E11.0 to E12.5). As shown in Figure 2A, there was a delay in SRY expression in the knockout compared with the control. This delay prevented the activation of SOX9 and therefore the commitment into the Sertoli fate (Figure 2B), resulting in pre-granulosa cell differentiation.

Figure 2. Immunofluorescence for SRY (A, red) and SOX9 (B, green) in control or miR-17-92 KO XY differentiating gonads, from E11.0 to E12.5.

3) Removal of miRNA binding site in Foxl2 UTR does not alter gonadal development.

As miRNAs have been reported to inhibit gene expression, the authors wanted to evaluate how these miRNAs regulate sex differentiation. Since Foxl2 carries a miR-17 binding site in its 3’ untranslated region (UTR) and FOXL2 was upregulated in the XY knockout gonads, the authors deleted this binding site to evaluate if the miRNAs are inhibiting FOXL2 activation in male gonads. Unfortunately, the loss of the binding site was not sufficient to induce FOXL2 expression in E12.5 XY gonads, suggesting an indirect effect or the involvement of other factors.

Why I chose this preprint:

This study is the first to report miRNAs associated with a complete sex reversal in mammals. This manuscript is a great example of the importance of the non-coding genome in the regulation of sex determination. In addition, complete male to female sex reversals is rare in the literature with ovotestis development or gonadal agenesis being the most prominent phenotypes. This makes the results described in this preprint even more fascinating.

Future directions / questions for the authors:

What controls the expression of these mRNAs? Are they sexually dimorphic?
Do you think that all the miRNAs in from clusters are important for sex determination? Or do you think that one miRNA is regulating this process?
Are you planning to evaluate the effect of single miRNA knockout on the differentiation process?

Tags: dsd

doi: https://doi.org/10.1242/prelights.34595

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Author's response

Rafael Jiménez, Darío Lupiáñez and Francisco Barrionuevo shared

Q1: What controls the expression of these mRNAs? Are they sexually dimorphic?

A: Expression studies revealed that members of the miR-17∼92 cluster are detectable in many different tissues [Ventura, A. et al. Cell 132, 875–886 (2008)]. However, little is known on how the the expression of this miRNA cluster is regulated [e.g see Zhao wt al., Cancer Treatment and Research Communications 33, 100647 (2022)]. This, per se, is an interesting question that remains to be answered

Nevertheless, we previously found that the members of the cluster were expressed in the developing gonad of both sexes, albeit they were not sexually dimorphic [Real, et al. A. Biology of Reproduction 89(4):78, 1–11, (2013)]. In agreement with this observation, we have noted that miR-17∼92 KO gonads of both sexes are reduced in size at E11.5 and, more importantly, that their transcriptomic profile is remarkably similar (there are 12 differentially expressed genes, most of them X- or Y- linked ones) but very different from the control counterparts. Further analyses revealed that several molecular pathways necessary for testis development are compromised in XY miR-17∼92 KO gonads. Overall, our results indicate that miR-17~92 primes the bipotential gonadal primordium to ensure proper Sry expression timing and subsequent testicular differentiation. Thus, these miRNAs act similarly in both XY and XX embryos and no sexually dimorphic expression is necessary as their action is required for testis but not for ovary differentiation.

Q2: Do you think that all the miRNAs in from clusters are important for sex determination? Or do you think that one miRNA is regulating this process?

A: Crosses between mice harboring heterozygous deletions of single-seed families of the cluster [miR-17∼92Δ17 (deletion of Mir17 and Mir20a), miR-17∼92Δ18 (deletion of Mir18), miR-17∼92Δ19 (deletion of Mir19a and Mir19b-1) and miR-17~92Δ92 (deletion of Mir92-1)] yielded viable homozygous adults at expected mendelian ratios. Further crosses between homozygous mice even produced viable offspring [Han, Y.-C. et al. Nat Genet 47, 766–775 (2015)]. Our own analyses revealed that miR-17∼92 target genes belonging to the 4 seed families are differentially expressed between XY control and mutant mice, and many of those play important roles in sex determination. Overall, this evidence points to a synergistic cooperation between the 4 seed families for proper testicular differentiation. For some reason, natural selection has kept these miRNAs bound together in the genomes of so many different animal taxa.

Q3: Are you planning to evaluate the effect of single miRNA knockout on the differentiation process?

A: Although mice carrying single-seed deletions of the miR-17∼92 cluster are viable and can breed normally, we think that it might be very informative to evaluate potential effects at the transcriptome levels. Such analysis might help to delineate what are the functional targets for different seed regions. Furthermore, the generation of mice carrying combined deletions of these families might reveal those that play a more critical role in sex determination. These are experiments that we are surely interested in pursuing in the future.

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

Figure 1. Immunofluorescence for Sertoli (SOX9, red) and pre-granulosa (FOXL2, green) cell markers in E17.5 XY and XX, wild type or miR-17-92 cluster KO gonads.

Figure 2. Immunofluorescence for SRY (A, red) and SOX9 (B, green) in control or miR-17-92 KO XY differentiating gonads, from E11.0 to E12.5.

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