ABOVE: Deletion of a microRNA cluster in mice causes a complete sex reversal. An XY mouse embryo lacking the microRNA cluster expressed the male development factor sex-determining region Y (red) and the ovarian cell marker Forkhead Box L2 (green). Alicia Hurtado Madrid

In mammals, the chromosomes that an individual is endowed with influence sex determination. This process occurs during embryonic development and relies on a balance between two very different sets of genes: one promotes ovarian development, while the other causes testes to develop. 1  

For decades, scientists have explored the genes that regulate sex determination. However, whether noncoding elements, which make up a majority of the mammalian genome, played a role in orchestrating this process remained unclear.

In a recent study, researchers found that embryonic deletion of a cluster of microRNA—noncoding small molecules that control gene expression—transformed male mice into female mice.2 The findings, reported in Nature Communications, revealed that non-coding molecules play a fundamental role in mammalian sex determination.

“We were looking for new actors in the process [of sex determination],” said study coauthor Francisco Barrionuevo, a geneticist at the University of Granada. Despite previous evidence suggesting that microRNA do not influence sex determination, his team remained skeptical given the crucial roles of microRNA in organ development.3,4

The team narrowed their investigation to a cluster of six microRNA that scientists previously identified as major player in developmental processes like formation of neurons and blood vessels. Using algorithms that search for likely targets of these microRNA, Barrionuevo and his team identified several target genes that are involved in ovarian development. To further probe the involvement of the microRNA cluster in mammalian development, the researchers used one of two different approaches to delete the genetic sequences encoding this microRNA cluster. In some experiments, they removed the noncoding elements directly from mouse embryonic stem cells. For other experiments, they generated mutant mouse embryos by breeding mice that lacked this microRNA cluster.

When they analyzed the developing mice towards the end of gestation when sex differentiation has already occurred, they found that wild type fetuses with XY chromosomes exhibited descended testes. In contrast, testes-specific structures were absent in XY fetuses lacking the microRNA cluster. Moreover, these mutant fetuses showed ovarian development that was similar to that observed in wild type and mutant XX embryos.

To confirm this phenotype, the researchers turned to immunofluorescence to capture molecular markers of gonadal development. SRY-box transcription factor 9 (SOX9), an essential factor for testicular development, showed up in the gonads of wild type, but not mutant XY fetuses. In contrast, XY mutant gonads, like XX gonads, expressed the ovarian marker Forkhead box L2.   

Barrionuevo said that the team was surprised by the complete sex reversal. He added, “We expected that [these microRNA] play a role in sex determination, but we did not expect such a result.” 

Darío Lupiáñez, a study coauthor and geneticist at the Andalusian Center for Development Biology, agreed, and added that such a phenotype is uncommon.

Their finding was so rare that the team used several methods to confirm that XY mice developed a female phenotype. Once they confirmed that XY mice developed female traits, the team profiled the embryonic cells to understand which cells were involved in this process. They found similar cell populations in wild type and mutant samples irrespective of sex, with the exception of Sertoli cells, which support developing sperm. The researchers detected Sertoli cells only in wild type XY gonads. Their absence from mutant XY embryos indicated that the microRNA cluster plays a crucial role in Sertoli cell development. 

When Barrionuevo and his team explored the effect of microRNA cluster ablation on the cells that give rise to Sertoli cells, they found that these progenitors lacked Sox9. They then investigated the levels of the sex-determining region Y (SRY) protein, encoded by a gene located on the Y chromosome. SRY protein activates Sox9, which leads to testis development in the gonads of XY embryos, whereas its absence in embryos with XX chromosomes induces ovarian development.5 

Proper testis development in XY animals requires timely expression of Sry.6  RNA sequencing analyses revealed that mutant gonads expressed this gene half a day later. Immunofluorescence analyses confirmed delayed SRY protein expression and lower protein quantities compared to those in wild type gonads.  

“It is a striking phenotype,” said Roberta Migale, an ovarian developmental biologist at The Francis Crick Institute who was not involved in the study. “Complete sex reversal is quite remarkable.” 

The microRNA cluster implicated in this study is evolutionarily well-conserved, suggesting that a similar system for sex determination likely exists in humans, Migale added. She hopes that these results put noncoding elements on the radar of clinicians who are screening patients with differences of sexual development. However, the study does not shed light on what regulates the expression of these microRNA, or how they control Sry or Sox9 expression, Migale noted. 

Barrionuevo and Lupiáñez expressed similar concerns. Although Sox9 expression is reduced when the microRNA cluster is deleted, the gene is not a direct target of the microRNA, making it difficult to determine the relationship between the two genomic elements. They would like to explore this aspect, but understanding the exact regulation would be technically challenging, Barrionuevo said. 

“Science is continuously developing, and new technologies come out every day,” said Lupiáñez. “Maybe this is a question we would be able to revisit in the future.”