When a sperm fertilizes an egg, the newly formed cell, or zygote, begins rapidly dividing into a ball of cells. As it divides, it becomes an embryo. At week six, tubes, dents and bulges of cells begin to take shape into early organs. 

 

In terms of sex, this six-week-old embryo is a blank state. It contains what is called an undifferentiated gonad, an early organ structure that will eventually mature. Seven days later, it will differentiate to become more testis-like, ovary-like or somewhere in between, depending on the activity of genes such as SRY.  

 

SRY, located on the Y-chromosome, encodes the sex-determining region Y protein. This protein functions as a transcription factor. In the world of gene expression, transcription factors are like dimmer switches. By binding to DNA in specific ways, they can either dial up or down – called “upregulating” or “downregulating” – the expression of other genes. 

 

The SRY protein controls a cascade of genes and their protein products that promote testis development, most notably a protein called SOX9. SOX9 is a transcription factor that helps make the cells that support sperm and testis development, called Sertoli cells.  

 

“SRY is the trigger, but SOX9 does all the hard work,” said Robin Lovell-Badge, one of the scientists who discovered SRY and SOX9’s functions. Lovell-Badge now works at the Francis Crick Institute in London. 

 

In the absence of SRY, typical female development proceeds via the production of ovarian supporting cells, or granulosa cells, with the help of other transcription factors. One of these is FOXL2, whose primary function is to repress SOX9. The gene encoding SOX9 is present in all cells, not just those in males, so repressing its activity is key for female sex development to proceed.  

 

In 2009, Lovell-Badge collaborated with researchers at the European Molecular Biology Laboratory and University of Cologne to turn off FOXL2, thereby upregulating SOX9, in six-week-old female mice. In a study published in Cell, they found that this caused the supporting cells in the ovary to turn into male Sertoli-like cells. The gonads also looked more testicular. Despite the female XX mice not having the SRY gene, the simple upregulation of SOX9 led to male gonad development. 

 

“Basically, you don't need SRY. All you need to do is be able to upregulate SOX9,” Lovell-Badge said. In a 2018 Science study, alongside researchers from Northwestern University, Lovell-Badge’s team showed male-to-female mouse sex reversal by downregulating SOX9.  

 

Andrew Sinclair, developmental geneticist at the Murdoch Children’s Research Institute in Melbourne, found similar results in humans. His team studied genetic samples from four DSD patients born with either duplications or deletions of a tiny regulatory region which controlled the expression of SOX9. Their study, published in Nature Communications in 2018, found that duplications of this SOX9 regulatory region, would amp up SOX9 expression, leading to female-to-male sex reversal and the presence of male gonads in the XX DSD patients. Deletions of the SOX9 regulatory region in XY DSD individuals had led to male-to-female sex reversal.  

 

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These fascinating results from SOX9 have shown that the regulatory elements –– the dimmer switches –– are key in sex determination. Scientists have since found dozens more genes and transcription factors involved.  

 

“We are working with people to try to really map all those parts of that dark, you know, unstudied DNA to understand what’s important in the gonads,” said Katie Ayers, a developmental geneticist at Murdoch Children’s Research Institute and one of Sinclair’s colleagues.  

 

With all these new findings, there needs to be a way to observe their impact on gonadal development in the embryo in real time.

 

To model this, developmental biologists use stem cells, specifically human induced pluripotent stem cells, or hiPSCs. Stem cells are biological empty canvases. When exposed to chemical signals in the lab, scientists can model how specific cell types differentiate from their embryonic precursors. Embryonic stem cells make up the early embryo and can differentiate into many different cell types, such as testis- or ovary-supporting cells. Thus, they are considered pluripotent.  

 

Using hiPSCs involves restoring a differentiated cell to its pluripotent cell state. After gathering cells from a patient, such as through a skin sample, scientists can reprogram those cells by exposing them to specific signals until they resemble their pluripotent embryonic precursors.  

 

“It feels like it's an embryo and then you can drive it down different pathways to produce different tissue types,” Sinclair said.  

 

hiPSCs are great models for studying gonadal development and DSD, especially because they are patient-specific. For example, Lovell-Badge’s team collaborated with scientists at the Pasteur Institute in Paris to produce hiPSCs from an XY male, XX female and XY female patient. They found that the XY male’s hiPSCs could form Sertoli cells and the XX female’s hiPSCs could form granulosa cells as expected. However, the XY female’s hiPSCs could not produce Sertoli cells but did produce some more granulosa-like cells, suggesting a genetic hiccup along the testis development pathway.  

 

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There are close clinical ties between the research and clinical spaces. Ayers published a study in Nature Communications in 2023 identifying how a variation in both copies of an individual’s SART3 gene, a variation that was not thought to be involved in any organ disorder let alone gonadal development, led to a similarly presenting syndromic condition including DSD in nine individuals. Syndromic means that the DSD was one of multiple different conditions associated with the same cause. The project started when two families of children with a then-mysterious condition approached the researchers’ clinical collaborators.  

 

“The families would come to us seeking answers,” Ayers said. After 12 years of searching for individuals with a similar DSD, her team finally found nine total patients. Normal stem cells introduced with the patients’ SART3 variant showed the gene’s involvement. 

 

In the clinic, physicians say that learning from research is something both they and their patients value.  

 

“Most families and patients are very receptive to discussions and information and, you know, future progress,” said a pediatric endocrinologist specializing in DSD care who chose to stay anonymous given the current volatility around this subject. 

 

Experts also said that understanding these genetic and developmental factors will help better inform young patients about their health needs, which is especially important for navigating adult health care in the future.  

 

“There’s a lot of adult health care providers that don’t really understand DSD, and it’s sometimes on the young adult or adult to kind of explain, ‘This is how my body works,’” said Jackie Papadakis, a clinical psychologist at Lurie Children’s Hospital in Chicago.  

 

Developmental genetics research will also provide more specific diagnoses for various DSD conditions, particularly through genetic counseling, which is the focus of the next post. This will help identify potential health concerns, such as medically necessary hormone therapy, earlier on and better prepare patients and families for puberty or conversations about fertility.