A New Key to the Story of How the Sexes Have Come to Be

In the beginning, we are all male and female… More specifically, we are all in between male and female. So what makes our embryonic selves choose and follow a developmental path to becoming the sex that we are today? New research has dramatically changed our understanding of this process.

During early embryonic development, all mammals develop a single pair of gonads that are neither testes nor ovaries, but have the potential to become either. Likewise, the external genitalia at this early stage has the potential to become either a penis and scrotum or a clitoris, vagina, and labia. Two pairs of ducts develop to connect the gonads to the undifferentiated external genitalia: One set of ducts, the Wolffian ducts, would become the epididymis, vas deferens, and seminal vesicles if this animal becomes male. The other set of ducts, the Müllerian ducts, would become the oviducts, uterus and innermost part of the vagina if this animal becomes female. So what determines if a given animal will develop male or female reproductive anatomy?

Early in development, mammalian embryos have one set of gonads that has the potential to become either testes or ovaries (here labeled as "bipotential gonad"). These gonads are connected the the developing external genitalia by two sets of tubes: The Wolffian ducts become the reproductive tracts in males and the Müllerian ducts become the reproductive tracts in females. The ducts that do not become reproductive tracts typically disintegrate. However, XX female embryos that lack the COUP-TFII protein do not dismantle their male-like reproductive tracts. Figure from Swain, 2017.

The sex of a mammal is determined by the combination of sex chromosomes it has. If the mammal has two X chromosomes, it will likely become female, and if it has an X chromosome and a Y chromosome, it will likely become male. The story physiologists have been telling for decades is that there is a single gene located on the Y chromosome, called the SRY gene, that single-handedly makes an embryo become a male. When expressed, the SRY gene produces a protein, called testes-determining factor, which interacts with the cells of the undifferentiated gonads to turn them into testicular cells. These newly formed testicular cells produce two key hormones: testosterone, which causes the Wolffian ducts to become the epididymis, vas deferens, and seminal vesicles, and anti-Müllerian hormone (AMH), which causes the Müllerian ducts to degenerate. In other words, if an animal has a Y chromosome, it will typically have an SRY gene that will trigger the sequence of events that causes the animal to develop into a male. If the animal does not have the Y chromosome, it will typically become female. However, it is not just the lack of a Y chromosome that can make a female; Any disruption of this pathway (such as an SRY gene that is not expressed, or the lack of testicular hormones) typically causes the animal to develop into a female. For this reason, females in mammals have been called the default sex. The scientific understanding since the 1950s has been that, in mammals, the development of a male reproductive system is an active process and the development of a female reproductive system is a passive process. However, a new study reveals that the process of becoming female mammal is not as passive as we have thought.

Fei Zhao, Humphrey Yao and their research team at the National Institute of Environmental Health Sciences and Baylor College of Medicine discovered a critical role for a specific protein, called COUP-TFII, in the active process of becoming a mammalian female. The research team examined female mouse embryos (which lack a Y chromosome, and hence an SRY gene) that had been genetically modified to lack a particular protein called the COUP-TFII protein. They compared these genetically modified XX embryos to genetically typical XX mouse embryos. When the unmodified XX embryos had developed to have only Müllerian ducts (the “typical” female reproductive pathway), the XX embryos without COUP-TFII protein retained both Müllerian and Wolffian ducts! Unfortunately, these XX mice that lacked the COUP-TFII protein died shortly after birth, so it was difficult to tell if this developmental process would have continued. The research team cultured reproductive organs of XX mice with and without COUP-TFII protein and found that this developmental trajectory likely would have continued after birth.

Images A and B show the reproductive tract from the side (A) and as a cross-section (B) in a "typical" XX female mouse embryo. Images D and E show that XX females that lack the COUP-TFII protein retain both Müllerian (pink arrows) and Wolffian (blue arrows) ducts. Figure from Zhao et al., 2017.

We know that testosterone helps promote the development of Wolffian ducts in XY males, so the most likely explanation of what they witnessed is that the lack of COUP-TFII protein somehow increased action of testosterone in these genetically modified XX embryos. The researchers ran a number of tests to explore this possibility. Testosterone is mostly produced by the gonads, so they compared the gene expression and enzymes of ovaries of unmodified XX mice with the ovaries of XX mice that lacked the COUP-TFII protein, and they found no differences that pointed to differences in testosterone production. They then considered the possibility that testosterone was produced somewhere else in the body, but the XX mice that lacked the COUP-TFII protein did not have more masculine body features compared to the unmodified XX mice. Finally, the researchers gave extra testosterone to the mother mice that were pregnant with unmodified XX mice and XX mice that lacked the COUP-TFII protein. The extra testosterone did not affect any of the mouse pups; it did not cause the Wolffian ducts of the XX mice that lacked the COUP-TFII protein to regress. Together, the researchers found that no, XX embryos that lack COUP-TFII protein do not have any more testosterone-like activity than their non-genetically modified XX sisters. This means that testosterone alone is not enough to keep Wolffian ducts.

This research has shown us that for the Wolfian ducts to go away during the reproductive development of a mammalian female, they need to be actively dismantled using a biochemical process (similar to how AMH dismantles Müllerian ducts during male reproductive development). COUP-TFII protein appears to be the chemical in charge of triggering this process. Female mammals are not the passive result of simply not becoming male, as has been taught in physiology classes for decades. Becoming a female mammal requires a process all its own, and we are only now starting to learn what that is.


Want to know more? Check these out:

F. Zhao et al. Elimination of the male reproductive tract in the female embryo is promoted by COUP-TFII in mice. Science. Vol. 357, August 18, 2017, p. 717. doi: 10.1126/science.aai9136

A. Swain. Ductal sex determination. Science. Vol. 357, August 18, 2017, p. 648. doi: 10.1126/science.aao2630