A new study on the evolution of steroid hormones shows that two tiny, one-letter gene mutations in our distant past had a 70,000-fold impact on the hormones’ behavior.

In the course of evolution, sometimes a small change can make a big difference.

Researchers at the University of Chicago have traced a crucial step in the evolution of modern sex hormones to a pair of small gene mutations that occurred about half a billion years ago.

Evolution occurs gradually, as many small mutations accumulate until they produce a new physical feature or ability. Today, scientists can observe evolution in action by examining animals that are at midway points in their development, including a reptile that is in the process of evolving from egg laying to live birth.

Such a development would require hundreds of new incremental mutations in order for the animal to grow whole new organs. But can just a few mutations have such a sweeping effect? The University of Chicago researchers decided to find out.

They examined steroid hormones, a group that includes estrogen, testosterone, progesterone, and cortisol. Steroid hormones have widespread effects on the body, regulating reproduction, development, stress, and other vital functions. A change in how steroid hormones behave could trigger a cascade of other changes throughout the body, making them a perfect candidate for an evolutionary scavenger hunt.

“Changes in just two letters of the genetic code in our deep evolutionary past caused a massive shift in the function of one protein and set in motion the evolution of our present-day hormonal and reproductive systems,” said lead study author Joe Thornton, PhD, a professor of human genetics and ecology and evolution at the University of Chicago, in a press release.

“If those two mutations had not happened, our bodies today would have to use different mechanisms to regulate pregnancy, libido, the response to stress, kidney function, inflammation, and the development of male and female characteristics at puberty,” Thornton added.

To trace these steroid hormones into the distant past, Thornton’s team examined the DNA that codes for the steroid receptor, a protein that sits at the edge of a cell and allows hormones in the bloodstream to communicate with the cell.

Using a computational method called ancient sequence reconstruction, they compared this DNA from humans with this same DNA sequence from hundreds of other animal species. The less similar an animal is to modern humans, the further back in time our species parted ways.

Modern humans emerged around 100,000 years ago. About 12 million years ago, our distant ancestors began to break away from the other great apes. Thornton traced functional changes in steroid receptors all the way back to 500 million years ago, long before the first mammal, long before animals learned to walk on land, and when the first fish-like vertebrates were just making their debut.

At that point, estrogen was the only steroid hormone in use, bonding to a single receptor. But then, two tiny point mutations changed everything.

A point mutation occurs when a single “letter” in a DNA sequence gets replaced with a different letter. Often, this change has no effect at all on what protein the DNA will go on to build, but other times, the mutation will cause the DNA to code for a different amino acid, the building blocks of proteins. If the replacement amino acid is a different size, has a different electric charge, or differs in other ways, it can alter how the protein behaves.

By using computer models and building the actual proteins in the lab, Thornton reconstructed the ancient estrogen-only steroid receptor. Then, he exposed it to an array of steroid hormones, mapping their effects and seeing how their behavior differed from that of the modern receptor.

In order for a hormone to fit with its receptor, it has to have a number of “sticky” bonding points that match the receptor’s exactly, like a key in a lock. With the wrong key, a lock won’t turn—with the wrong hormone, a receptor won’t activate, and the hormone won’t affect the target cell.

Even though steroid hormones are all very similar, they can each only bond to their own receptor type because they must match up perfectly. Drugs like hormonal birth control mimic hormones by being similar enough that they can bond with the hormone’s receptor.

The first mutation Thornton found swapped in an amino acid that added a new bonding point to the receptor. Estrogen doesn’t use this extra point, but the other steroid hormones do. For them to evolve, they’d need a receptor able to receive them.

The second mutation produced an even bigger change, altering the bonding points that estrogen was using. Estrogen was no longer able to properly fit into the receptor, causing it to shift constantly and letting water leak in.

Together, these two changes made the steroid receptor 70,000 times less likely to bond properly with an estrogen molecule. This new estrogen-hating steroid receptor would eventually combine with other mutations to create all the steroid receptor types that human bodies have today. This paved the way for the rise of testosterone, among other hormones.

Thornton’s research is not just a glimpse into our evolutionary past, but it also lays the groundwork for other scientists to better understand how cells communicate with one other. Using this information, they can develop new, targeted drugs to seek out the exact binding sites they need.