Sex Determination: It’s in the Genes—Sometimes

Since ancient times, scientists and philosophers have pondered the fundamental question of what makes some organisms male and some female. Some hypotheses were at least plausible—Greek philosopher Empedocles thought that different temperatures produced different sexes—while others were decidedly folkloric, such as the idea that placing parsley on a woman’s head would ensure that she gave birth to a child that was the same sex as the next person she spoke to.¹

Over the following millennia, many would wrestle with this problem, but little progress was made until the beginning of the 20^th century. In 1900, scientists rediscovered Gregor Mendel’s largely forgotten work on units of heredity, ushering in a new age of research on inheritance, chromosomes, and genes. For Nettie Stevens, a cytologist at Bryn Mawr College, this meant long hours of dissecting, staining, and examining insect testes under the microscope. In her seminal 1905 publication on mealworms, Stevens became the first to determine that inheriting the Y chromosome resulted in male offspring, an example of genetic sex determination.²

The only rule is that there are no rules.

—Judith Mank, University of British Columbia

From these early genetic studies arose the XY sex determination concept. According to this system, an animal’s DNA is sorted into several pairs of autosomes, in which each member of the pair has similar morphology, and one pair of sex chromosomes. Traditionally, two copies of the X chromosome (XX), which has several hundred protein-coding genes, results in a female organism that produces larger gametes, or egg cells. One X chromosome and one comparatively diminutive Y chromosome (XY), on the other hand, results in a male organism that produces smaller gametes, or sperm.

Almost immediately, however, scientists began to realize there was much more to the story. Just four years after Stevens’ discovery, Thomas Hunt Morgan, an evolutionary biologist at Columbia University, published his research on an alternative sex determination system in aphids, in which females had two sex chromosomes (XX), but males had only one (X0).³

But that was just the beginning. So far, researchers have uncovered a dizzying array of exceptions to the XX/XY “rule:” birds and butterflies that use the ZW system, in which males have a matching pair of sex chromosomes (ZZ) and females have a mismatched pair (ZW), a monotreme with ten sex chromosomes, and animals for which maleness or femaleness can each be encoded by multiple genotypes.^4–7 There are species whose sex is not written into the genome at all: Some turtles have temperature-dependent sex determination and some fish can change from one sex to another in a matter of days in response to social signals.^8,9 When it comes to sex determination, “The only rule is that there are no rules,” said Judith Mank, a comparative genomics researcher at the University of British Columbia.

Now, Mank and other researchers around the world are going spelunking through the genomes of an ever-expanding number of species, teasing apart how networks of genes can initiate male or female development in both heritable and environmentally responsive ways. This research is providing insights into fundamental evolutionary processes, but it also has concrete applications, such as informing conservation efforts for temperature-sensitive species in the face of climate change and deepening scientific understanding of genes that are important for human health.

A Bug’s Life: From Beetles to House Flies

In many ways, the foundational discoveries made by Stevens and her contemporaries were facilitated by the species they chose to study. Without electron microscopes or genome sequencing—even a basic understanding of DNA’s structure was still nearly 50 years in the future—researchers depended on organisms whose sex was predictably determined by highly heteromorphic, or differently shaped, sex chromosomes. While Stevens studied insects from several different orders, the relatively straightforward biology of beetles was crucial for her insights into chromosomes and sex determination: Like mammals, beetles largely use an XY sex determination system.

But while insects can often serve as simplified models of human biology, they can be much more complicated at times. Take the example of a house fly, which has six pairs of chromosomes. “What's unique about the house fly is that people have found male determiners on all six chromosomes,” said Rich Meisel, an evolutionary biologist at the University of Houston. “We know that in four of those six [cases], it's actually the same gene that's jumped around.” On the remaining two chromosomes, however, the identity of the male determiners has yet to be discovered.

If a fly has no male-determining genes, the expression of a gene called transformer will initiate female developmental processes; if a fly has one or more male determiners, the production of a functional transformer protein is disrupted, and male development proceeds.¹⁰ But the process became even more complicated when some flies evolved a transformer allele that is resistant to disruption by the male-determining genes. For these individuals, Meisel explained, “It doesn't matter whether she has male determiners or not, [this allele] causes them to develop as females.”

We view this variation within the species as a window into the evolutionary process.

—Rich Meisel, University of Houston

Thus, within the same species, there are multiple genetic ways to end up female or male. Even more strangely, the presence of specific male- or female-determining factors is not distributed evenly across populations; instead, Meisel and others have demonstrated that distribution is correlated with climate factors like temperature variability.¹¹

As researchers investigate these deceptively complex creatures, they hope to learn lessons that can be applied on a broader scale. “We view this variation within the species as a window into the evolutionary process,” said Meisel. “If two things differ between species, and you want to know how they got to be different, you can find one species where both things exist, which gives you a snapshot of how these differences could occur.”

Temperature-Dependent Sex Determination and the Threat of a Warming World

For many species, the trigger that initiates male or female development is encoded in the genome, for others, environmental factors during embryogenesis send an organism down one pathway or another.

Take crocodiles, for example, said Mank. “Their male and female genomes are exactly the same; they determine sex based on temperature. They have no genetic differences between the sexes, it's all based on how the genes are regulated…You can have sex without sex chromosomes, no problem.” Understanding exactly how temperature regulates sex determination could be crucial for the conservation of many types of reptiles.

Sea turtles are a particularly salient example of this. In the 1960s and 1970s, countries around the world began conservation programs for imperiled sea turtles, whose populations had dropped precipitously in the preceding decades. One major element of sea turtle conservation efforts involved collecting the eggs and incubating them in artificial nests to keep them safe from predators and poachers. At first, this appeared to be a successful strategy for producing healthy baby turtles. But in the early 1980s, scientists began to express concerns that this practice might actually be hastening the turtles’ extinction instead of preventing it.¹²

Sea turtle embryos, they discovered, were extremely sensitive to temperature. In the green turtle, for example, eggs kept at 28°C or below almost exclusively hatched into male turtles, while at 29.5°C, almost all the hatchlings were female.¹² Subsequently, scientists determined that the Styrofoam artificial nests kept eggs slightly cooler than their natural incubation conditions buried in the sand; at a nature reserve in Suriname, artificial incubation sometimes produced clutches of leatherback turtles without any females at all, a dire problem for an already-endangered species.¹³

Sea turtles today face a different challenge: As the global temperature creeps upward, nesting beaches in warmer regions are producing almost exclusively female hatchlings. And it’s not just the sea turtles that are in trouble: Hundreds of other species, including many types of freshwater turtles, crocodilians, lizards, and fish are also at risk.¹⁴ “There is a worry that with climate change, they’ll end up being one sex all of the time,” said Melissa Wilson, an evolutionary biologist at Arizona State University. “Maybe there will be a clutch that develops genetic sex determination, or maybe not, and the species will go extinct.”

Evaluating the effects of temperature on reptile sex determination could have important conservation implications, helping scientists predict which species will be most imperiled by even a few degrees of warming. Identifying the genetic mechanisms by which reptile embryos sense temperature and initiate female or male developmental pathways could also provide key information: Allelic variation in these genes could help define a species’ capacity to quickly adapt to a warming climate.

Indeed, there is some evidence that within-species variation in developmental response to temperature exists in certain species. In particular, species that live over a wide range of latitudes may need to develop different pivotal temperatures (the tipping point at which embryos switch from mostly male to mostly female, or vice versa) in the north versus the south in order to maintain optimal sex ratios in places with different climates. In snapping turtles, at least, there is some evidence that variation in the cold-inducible RNA-binding protein (CIRBP) gene may contribute.¹⁵ Researchers found that, at a given temperature, turtles with two copies of the CIRBP A allele were more likely to be female than turtles with two copies of the C allele; the frequency of these alleles appeared to be different in turtles from Minnesota compared to those from Texas, suggesting a mechanism by which genetic variation may contribute to different thermal set points for sex determination.

While many reptiles display rapid evolution of sex-determining mechanisms, and the evolutionary histories of some groups may have even involved multiple transitions between genetic and temperature-dependent sex determination, it’s not yet clear if they can adapt quickly enough to keep pace with climate change.¹⁶ Identifying the genetic and epigenetic mechanisms that govern whether sex is inherited or environmentally determined as well as the forces that drive either stability or flexibility in these systems could help scientists predict the adaptive potential of these ecologically important species and potentially inform conservation strategies.

Something Fishy: Stability vs. Flexibility in Fish Sex Determination

While reptile sex determination systems might seem highly dynamic compared to mammalian systems—which have remained fairly stable for the last 180 million years or so—fish take things to a whole new level.¹⁷ “Fish have this sort of ridiculous diversity; it boggles the mind,” said Mank. “Whether you make big gametes or small gametes, that's conserved, and it has been for about a billion years. And the traits that are associated with that, like the testis and the ovary, and all sorts of other things, those are pretty conserved. So why would you rewire sex determination, even between closely [related species]? It made no sense to me.”

This was particularly perplexing given that changing the system could lead to a loss of reproductive abilities—in other words, an evolutionary dead end. In 2014, Mank and a group of researchers in the Tree of Sex Consortium published a paper entitled, “Sex Determination: Why So Many Ways of Doing It?”¹⁷ Now, more than a decade later, Mank said, “We still don’t have the answer, which is good. It means we're still in business.”

We know the ‘why’ of sex change pretty well. The ‘how’ we know a lot less well… Exactly how they transduce a social cue into a change in the gonads remains somewhat mysterious.

—John Godwin, North Carolina State University

To understand the forces that drive the evolution of sex chromosomes, including how a perfectly matched pair of autosomes begins to diverge into an X and a Y, and why a sex chromosome might degenerate or expand after this divergence, Mank studies several species of closely related guppies. Her work has highlighted the importance of sexual selection and sexual antagonism in Y chromosome evolution.

Sexual antagonism occurs when a trait is beneficial to one sex but deleterious to the other. For male guppies, being brightly colored is a great way to attract females, rendering it a beneficial trait even though it also increases their visibility to predators. For female guppies, however, bright colors don’t seem to increase their odds of mating, so this conspicuousness only makes them more likely to become something’s dinner.

Mank studied the genomes of guppy populations with different levels of sexual conflict: Upstream, where there were fewer predators, male guppies were brighter, meaning greater conflict with the dull-colored females.¹⁸ Downstream, where predation pressures were higher, males were more muted, and thus there was a lesser degree of sexual conflict. Mank and her team found that upstream guppies had greater divergence in the X and Y chromosomes, indicating that sexual conflict may contribute to the pace of change in sex chromosomes. These studies could help shed light on the evolutionary pressures that could drive either stability or flexibility in sex determining systems.

For Mank’s guppies, and many other species of fish with genetic or temperature-dependent sex determination, an individual is committed to a male or female fate early in development. But other fish, like the bluehead wrasse, keep their options open, and can change sex in response to changes in the social environment.

John Godwin, a neurogenomics researcher at North Carolina State University, has been studying the wrasse for the past three decades, trying to understand this fascinating little fish. Female bluehead wrasse are generally yellow and nonaggressive; intermediate phase males are similar to females in color and behavior, whereas terminal phase males are blue-green and highly protective of a particular territory for spawning. But if a reef’s terminal phase male is eaten by a predator—or kidnapped by an experimenter—the largest female can take his place.¹⁹ Within hours of this change in her social environment, she becomes more aggressive, and in about 10 days, her ovaries become functional testes.

Although this strategy may seem unusual from a human perspective, scientists think they understand the evolutionary pressures that favored the female-to-male change. Females, explained Godwin, spawn about once a day. “But if they can change sex and secure one of the spawning sites, they’ll spawn 20, 30, 40 times a day. The record is [held by] one male in Panama who spawned, on average, about 180 times a day. So, there’s a big reproductive payoff.”

“We know the ‘why’ of sex change pretty well,” said Godwin. “The ‘how’ we know a lot less well…Exactly how they transduce a social cue into a change in the gonads remains somewhat mysterious.”

While researchers are still working on mapping the pathways that govern this change, Godwin, along with collaborator Neil Gemmell, an evolutionary geneticist at the University of Otago, has identified some of the alterations in gene expression, DNA methylation, and hormone levels that play key roles in this process.

Their research suggests that the stress hormone cortisol helps kick off changes in the brain and the gonads at the beginning of the female-to-male sex change process.²⁰ The gonads undergo extensive epigenetic reprogramming, and genes involved in the biosynthesis of estrogens are silenced.

Indeed, estrogens—or lack thereof—seem to be essential for this sex change: When Godwin’s team gave fish an estradiol-releasing implant, they didn’t develop the characteristically male aggression.²¹ But while the ovaries may be the best-known source of estrogens in females, they don’t seem to be necessary for the female-to-male behavior changes, as female fish developed male-like behaviors even when scientists removed their gonads.¹⁹

Instead, Godwin’s team explores the role of estrogens that fish—similarly to other vertebrates, including mammals—synthesize in their brains.²² Interestingly, the team did not observe extensive changes in whole-brain gene expression as the fish changed from female to male. But instead of deterring him, this observation prompted Godwin to study the brain on a finer scale.

“This is where single-cell approaches are really exciting,” said Godwin. “Even a tiny chunk of brain might have 100 different kinds of cells in it, so it [can be difficult to separate] the signal from the noise from all these cell types, but now we can ask individual cells what they're doing…[With these new technologies], all sorts of questions that would have been difficult or even impossible to ask not long ago are, all of a sudden, quite feasible. I think we're in for an exciting time.” Godwin hopes that creating transcriptomic profiles of individual cells and measuring estrogen in small, well-defined brain regions will help bring the neural processes driving sex change into focus.

Moreover, studying animals from many different classes can offer new perspectives about what it means to be male or female. “Because we're mammals and we’re most familiar with our own species, we have this idea of different sexes and genders being these tightly integrated suites of traits. But if you zoom out a little bit, phylogenetically, this is [just one potential] reproductive strategy.” Mixing and matching male and female characteristics can be an equally successful strategy, said Godwin. “It's more flexible than we previously thought, and that's pretty fascinating.”

Mammal Sex Determination: SRY, Not SRY

Although Stevens’ work in insects at the beginning of the 1900s laid the foundations for sex determination by X and Y chromosomes, it would take almost a century for scientists to begin identifying the key genes involved in this process in mammals. Interestingly, studying the chromosomes of individuals who broke the typical patterns of sex determination was a crucial part of these genetic discoveries. In XX individuals who were phenotypically male, researchers identified sequences of DNA usually found on the Y chromosome; in XY individuals who were phenotypically female, they identified deletions in these same sequences.²³

This allowed researchers to substantially narrow down the region of the Y chromosome where the gene that initiated male development might be, and in 1990, a team of British geneticists pinned down the responsible gene, which was christened SRY (sex-determining region of the Y chromosome).²⁴ The following year, scientists created a phenotypically male mouse with XX chromosomes by introducing the Sry gene.²⁵

While mammalian female and male development may at first appear clear-cut, the careful study of human and mouse genomes, along with an ever-expanding list of weird and wonderful mammals from around the world, has revealed surprising levels of complexity and flexibility in these sex-determination systems. While SRY is undoubtedly important, scientists have discovered numerous other genes that play important roles in male or female developmental cascades and suppress the expression of genes in the alternate pathway.²⁶ In XY mice, for example, inactivation of Sox9, a transcription factor necessary for testis development, produces phenotypically female mice.²⁷ Conversely, in XX mice, inactivation of Wnt4 and Foxl2, which are crucial for the development of ovaries and other female reproductive structures, results in phenotypic males.²⁸

While researchers once assumed that gonadal fates were set in stone once animals reached adulthood, some studies now suggest that a structure’s identity as an ovary or testis may need to be actively maintained throughout the lifespan. When researchers deleted Foxl2 in adult female mice, for example, their ovaries quickly began to change: In a matter of weeks, they became testes-like in terms of both structure and gene expression.²⁹

“There aren't quite the barriers that we previously thought there were, even in groups like mammals,” remarked Godwin.

While these studies required genetic manipulations in the lab, there are also examples of naturally occurring creative sex determination systems in mammals. The Amami spiny rat for instance, has lost the Sry gene along with its entire Y chromosome and has evolved an alternative system for producing males and females.³⁰

While the spiny rat only has one sex chromosome (X), the African pygmy mouse has three: X, Y, and X*.³¹ Frédéric Veyrunes, who currently researches sex chromosomes at the Institute of Evolutionary Science of Montpellier, has devoted his career to the study of this remarkable mammal.

Veyrunes didn’t begin his scientific journey with the intention of studying sex determination; during graduate school, he was interested in pygmy mice chromosomal number variation—they can have anywhere from 18 to 34. While trapping mice in South Africa, he noticed that many of the females possessed something that looked suspiciously similar to a Y chromosome. He was immediately intrigued, and performed further testing that eventually confirmed his suspicions. But what could be causing these XY mice to be functionally female?

“At first, we thought that it was mutation on the Y chromosome,” said Veyrunes. “Sry, of course, was our first candidate. But when we sequenced the full length of the Sry gene in the males and the females, we didn't detect any mutations.” These females could also pass their Y chromosomes on to their offspring, resulting in typical XY males, further suggesting a perfectly functional Y chromosome. The researchers eventually found that all the females with Y chromosomes also possessed a different version of the X chromosome, dubbed X*, from the X found in typical females. Something about X* was overriding the male developmental pathways normally initiated by Sry.

“It was very exciting,” Veyrunes recalled. “It’s one of the very rare cases in mammals where there are these bizarre sex chromosomes.”

Despite years of study, the researchers still haven’t been able to pin down the X* gene that’s responsible for this male-to-female switch, although Veyrunes said that they have identified regions of the X and X* chromosomes that do not recombine. “We believe that the mutation is in this region, but it is quite large—it comprises hundreds and hundreds of genes.”

“We first analyzed all the genes that are known to be involved in sex reversal, [including those] in humans and mice, and we discarded all these genes. So, that means it's something new…a gene on the X chromosome with an unknown function in sex determination,” said Veyrunes.

He and his team are currently analyzing gene expression in embryonic pygmy mouse gonads around the time in development where these structures become committed to life as an ovary or a testis. Identifying which genes are expressed during this crucial time will help them narrow down the X* gene that tips the scale in favor of ovaries, even in the presence of the Sry gene. “We’re getting closer and closer to identifying the mutation,” he said.

“The genes that I'm looking for in my pygmy mice are also interesting to many [scientists engaged] in human research,” added Veyrunes. Specifically, Veyrunes’s research is relevant for geneticists and clinicians studying disorders of sex development (DSDs), a group of congenital disorders characterized by atypical development of the reproductive system. Identifying the genetic etiology in each patient is important for developing personalized treatment plans, evaluating risk of gonadal tumors, and determining fertility potential.³² However, at present, testing can only identify the genetic cause in about half of DSD patients.³³

That means it's something new…a gene on the X chromosome with an unknown function in sex determination.

—Frédéric Veyrunes, the Institute of Evolutionary Science of Montpellier

“This means that there are still many genes in the sex determining pathway in humans that are missing, that we don't know about,” said Veyrunes. “So maybe my pygmy mice will help to identify new genes [in this pathway], which could ultimately help some patients with disorders of sex development. And I think that's very cool.”

Overall, explorations of sex determination mechanisms across the animal kingdom showcase an incredibly diverse array of ways to become male or female. This field of research could help inform conservation strategies for many fish and reptile species, as well as have important implications for human health. Understanding this diversity could also help researchers and the general public expand their thinking beyond strict boundaries between XX and XY, between male and female, between stereotypically masculine and stereotypically feminine profiles of hormones and behavior. The natural world is rarely black and white, and systems that initially seem simple can grow more and more complex the closer that scientists look.