Sex-swapping Zebrafish Hold Clues for Reproductive Development Diseases in Humans

Zebrafish swim in a tank. David Slipher/UC Davis
In a study appearing in Genetics, UC Davis researchers relay new discoveries about the molecular mechanisms behind sexual determination. Using zebrafish, they highlighted the gene responsible for determining whether the fish will develop into a female or a male. David Slipher/UC Davis

When Associate Professor Bruce Draper, Department of Molecular and Cellular Biology, looks at an animal, he sees two things: the gonad and the rest of the body, or what he calls “the gonad protector.”

“Everything else—our eyes, our ears, our mouths, our hands—are all there to protect the gonad,” said Draper. “If we pass on our genes, then any advantageous mutation that has occurred will be passed on as well.”

Part of what Draper studies is how the development of the vertebrate gonad—the organ responsible for producing eggs and sperm—influences sexual determination. In a study appearing in Genetics, Draper, together with graduate student Michelle Kossack, Pharmacology and Toxicology Graduate Group, and their colleagues at the University of Oregon, relay new discoveries about the molecular mechanisms behind sexual determination.

Using zebrafish (Danio rerio), they highlighted the gene responsible for determining whether the fish will develop into a female or a male. The findings hold implications for mammalian sexual development, as mutations to a similar gene in humans can lead to various diseases affecting reproductive development, including polycystic ovary syndrome—a hormonal disorder that causes cysts—and abnormal development of the kidneys and lungs, among other problems.   

“A lot is known about how sex is determined in mammals and most of the genes that mammals use for sex determination are conserved in fish,” said Draper. Conserved genes remain unchanged across species throughout evolution. “This is just starting to dissect what the conserved genes are so that we can start to put a pathway together.” 

Sex-swapping gonads

In fish, the gonad is pivotal to sexual determination, releasing hormones that tell the rest of the body to be male or female. And a slight percentage of fish have the ability to change their sexes.

“About two percent of all fish species will normally at some point in their lives switch sex,” said Draper. “So they’ll start off as males and at some point, as an adult, they’ll switch from a male to a fertile female, which is remarkable.”

Zebrafish aren’t one of these species. But through investigations, Draper and his colleagues discovered that if they killed off all oocytes—the developing eggs—in an adult female zebrafish, the fish transformed into a male.       

“There was something that the oocytes, these immature eggs, some factor that they were producing that was required for the fish to stay female,” said Draper.

Draper and his colleagues eventually identified that factor, a protein called Bone Morphogenetic Protein 15 (Bmp15). Mutations to the gene resulted in zebrafish that initially developed as females but changed into males at a certain stage during adulthood.

Zebrafish
A live, 35-day old, transgenic (ziwi:EGFP) juvenile zebrafish expresses green fluorescent protein in all germ cells. Bruce Draper

“That got us more generally interested in how sex is regulated in zebrafish because it was unknown,” said Draper. “Even though people have been using zebrafish as a sort of really hardcore model system since the 1980s, no one has figured out how sex is really determined in zebrafish.”

Identifying the female factor

In mammals, the Wnt gene family member 4 (Wnt4) is required for female development. This gene is conserved in zebrafish, but it’s duplicated. According to Draper, wnt4a is predominantly involved in gonad development, while wnt4b is involved in neural development. It’s theorized that wnt4b was present in the last common ancestor of humans and zebrafish, roughly 450 million years ago, but that it was eventually lost in mammals. Genetically, mammals with XX sex chromosomes should be females that develop ovaries. However, XX WNT4 mammal mutants die during the embryo’s development, develop testis-like gonads instead of ovaries and have neural defects.    

The Genetics study focused on mutations to wnt4a in zebrafish. Through experiments, the researchers determined that developing zebrafish gonads express wnt4a up until roughly 25 days after fertilization. At that point, the gene is only expressed by the somatic cells in the ovaries of female zebrafish. To confirm wnt4a’s role in female development, the team created and observed the development of zebrafish with wnt4a mutations.  

“We made this mutation and as expected, the vast majority of wnt4a mutants in zebrafish are male,” said Draper.

Approximately 95 percent of the zebrafish mutants developed as male and 5% developed as female, suggesting that wnt4a plays a role in female differentiation but is not required. Regardless, both males and females with wnt4a mutations were sterile due to defects in reproductive duct development.

“This research provides further support that all vertebrates utilize a core molecular program for sex determination and differentiation and shows that zebrafish can serve as a useful model to study the functions of genes that in humans are responsible for disorders of sexual development,” said Draper.

It’s also further evidence that WNT4/wnt4a is a conserved regulator of female determination in both mammals and non-mammalian vertebrates. 

The research was supported the National Science Foundation and a T32 predoctoral training grant from the National Institute of Environmental Health Science.

Bruce Draper stands in the zebrafish facility
“A lot is known about how sex is determined in mammals and most of the genes that mammals use for sex determination are conserved in fish,” said Draper. David Slipher/UC Davis

 

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