Image of a turtle
Enlarge / A red-eared slider.

There’s a global pandemic happening on a scale that hasn’t been seen in roughly a century. So we decided it would be the perfect time to talk about turtle sex. Not turtles having sex, which is undoubtedly an interesting geometry problem, but rather the process by which turtles develop as male or female.

That process is interesting because it seems, at least from our XY chromosomal perspective, to be a bit haphazard: turtles and many other reptiles determine their sex based on ambient temperature. In elevated temperatures, most of the eggs will develop as female; at lower temperatures, most of the eggs will develop as males. We don’t really know how they register the temperature and somehow translate it to a complex program of anatomical development. But a new paper in Science fills in some of our gaps.

Sex, of the less interesting sort

If pressed, most of us could remember that human sex determination involves the X and Y chromosomes. But it’s often overlooked how things get very complicated downstream of this simple signal. A gene on the Y chromosome turns out to be critical to registering which chromosome combination someone has. A specific tissue interprets the presence or absence of that gene to start a cascade of hormones that reshape how tissues develop and continue to influence things throughout a person’s life.

(This is ignoring all the many things that can happen during this process and produce a non-binary result.)

So it’s not enough for a signal to simply indicate which sex to develop as. The signal has to be significant enough that it can trigger a large program of changes in response to it.

Despite that seemingly rigorous requirement, biology has found a bewildering variety of ways for handling sex. To give just one example, fruit flies also have X and Y chromosomes, but you can get rid of the Y chromosome and males will still develop mostly normally. And rather than having a hormone to get every cell in the body pulling in the same direction, each individual cell in the fly figures out which chromosomes it has without consulting its neighbors.

Compared to chromosomes, which are pretty stable, it’s hard to see how temperature can work to set a binary state. Turtle eggs are left in the environment, where the temperatures vary over time. That variation occurs over the long term, as incubation may take place during changing seasons; medium term, due to day-to-day changes in temperature; and short term, with temperatures rising in the morning and falling at night. Somehow, all that has to be converted to a reasonably binary decision. For turtles, that conversion happens in a way that does have some strong parallels to humans. The signal is read by cells that go on to form the animal’s gonads, which go on to produce hormones that direct the turtle’s development.

So how does this tissue register the temperature? Within the last few years, we started to get a hint thanks to studies of a turtle species named the red-eared slider. Above 30°C, most red-eared slider eggs will develop as females, while at 26°C and below, a clutch of eggs will mostly develop as males. These eggs can be raised in a laboratory, allowing researchers to start poking at the pathway that determines their sex.

Some like it hot

The key to figuring this out turned out to be evolution. This is a bit surprising, given that we just mentioned above that evolution has produced animals that handle sex determination in a huge variety of ways. Despite that variety, however, many of these animals use part of a core set of genes during the sex determination process. One of those is called doublesex, after the fruit fly version of the gene. In 2017, researchers found that the version of the doublesex gene in the red-eared slider was able to promote male development.

But this gene was only translated into protein at low temperatures and was silent when temperatures were elevated. That suggests it’s not responding to the temperature directly, as a true temperature sensor would be translated into protein at both high and low temperatures, while the protein would only be active at one of these. Further searches found a gene called Kdm6b that regulates the turtle version of doublesex, but that too was made into proteins at only one temperature, indicating it was also downstream of the sensor.

At this point, however, the researchers got a bit lucky. Kdm6b is involved in a lot of processes in other species. In one case, the researchers found two papers that identified a regulator of Kdm6b. But that regulation takes place in cancer cells and immune cells in mammals. There’s no reason to think that the same relationship would hold in turtle sex determination.

Yet amazingly, it did.

More information, stat

The researchers identified the turtle version of the regulator, Stat3, and showed that it could block the activity of the male-determining pathway. And they found it was made into proteins in the gonads regardless of the temperature. But it was converted into an active form (by the addition of chemical links to phosphates) more often at the high temperatures that trigger female development. Blocking the activity of Stat3 led to activation of the male-specific pathway, even at elevated temperatures.

Somehow, Stat3 is activated specifically by high temperatures and, once active, blocks the pathway that triggers male development. That activation is driven by chemically linking the Stat3 protein to phosphates. So what regulates the phosphate addition?

Here again, evolution provided the answer. In some contexts, having more calcium in cells will cause Stat3 to be activated. Again, there’s no reason to expect that should also happen in turtles. But the authors discovered that it does. And they went on to show that the levels of calcium in the gonad change with differences in temperature. Ultimately, it looks like the developing turtle registers its temperature through changes in the amount of calcium present.

While this work goes a long way toward piecing together the pathway that turns temperature into sex determination, it still leaves a critical step a mystery: what causes cells to accumulate calcium at certain temperatures? Fortunately, there are a number of candidates—proteins that control the flow of ions into and out of cells. It’s probably a matter of time before the full pathway is worked out.

Obviously, understanding this pathway isn’t going to help us cure cancer or anything like that. But it is an intriguing example of how a variable environmental signal can be converted to an essentially permanent alteration in gene activity—something that seems to happen in humans, such as when they’re subjected to persistent stress. And as climate change begins to alter the temperatures of turtle habitats, understanding this pathway could potentially inform conservation approaches. So it isn’t exactly esoteric science for science’s sake, either.

Science, 2020. DOI: 10.1126/science.aaz4165  (About DOIs).