Musth, a time of heightened testosterone levels and aggression in male elephants related to reproduction, has now been identified in woolly mammoth tusks. Remarkably, this is the first time hormones have been seen in the extant or the extinct. And it opens up an exciting new field of paleontology that the team behind the discovery calls ‘palaeoendocrinology’—the study of hormones in ancient species.

A paper published on Wednesday in Nature describes that work, in which an international team of scientists studied African elephant and woolly mammoth tusks. Elephants and woolly mammoths are distantly related, and both belong to a group of animals known as proboscideans.

Testing tusks

Michael Cherney is the lead author and research affiliate at the University of Michigan Museum of Paleontology. He said his team started by testing elephant tusks. “We wanted to start with something that provided the best chance of recovering data, because we didn’t know that we’d see any,” Cherney told Ars in a video interview. Nobody knew for sure whether hormonal signatures existed in modern elephant tusks prior to this study, but the team was able to identify testosterone in the tusks.

Once the researchers identified hormones in male elephant tusks, they could compare them to hormonal signatures, if any existed, in extinct proboscideans.

He described searching for musth as “low-hanging fruit because we had male [woolly] mammoth tusks in our possession. We had discovered that testosterone provided a record that looked really clean in the modern elephant tusk.” By contrast, they couldn’t test for female hormones to test “for pregnancy, for instance,” because they didn’t have a modern female elephant tusk to compare.

Exceptionally well-preserved fossils were a good place to start. Woolly mammoths in Arctic regions such as Siberia and Wrangel Island off the coast of Russia (which is where the tusks they sampled were found) are better preserved than remains from other Pleistocene animals such as mastodons, another elephant-like animal.

The right time to mate

The team found similar spikes of testosterone recorded within the adult years of the male African elephant and the male woolly mammoth tusks, consistent with those that occur during musth. These spikes were absent in the sub-adult years of that same male woolly mammoth, evidence that supports a lack of musth in the years before sexual maturity, and they were completely absent in the female woolly mammoth.

The overall levels of testosterone were lower in both mammoth specimens compared to the elephant, something the team posits might be due to limits of preservation. The fact that hormones were preserved at all, however, is astounding after thousands of years (over 33,000 years for the male woolly mammoth).

Given their close relation to Asian elephants, is it surprising that musth has been discovered?

“It’s not a surprise that woolly mammoths were experiencing musth,” Cherney admitted. “What is a surprise is that we can see it. And what is powerful is the ability to not just know that they had musth, but to see when they were going into musth. We could potentially see what age they were, we can see how frequent it was, and see how extreme the testosterone increases were compared to the inter-musth period, for instance.”

There’s a lot we can potentially correlate the hormone levels with, because proboscidean tusks record the life of that animal. Its rings record days and years of growth, for example, seasons in which it experienced famine or abundance, from when it was born to when it died. So if further information, such as additional hormones, can be detected, this opens up a new window into the lives of extinct species.

Tracking hormones

How do you go about tracking a single hormone within the complex mix of chemicals present in a tusk? The team relied on a technique called liquid chromatography-tandem mass spectrometry (LC-MS/MS). “We started with the method that others have used to extract hormones from hair, but we modified it,” Cherney said. “And then we used the method of isolating hormones from the serum samples, and we modified it a little bit.”

LC-MS/MS involves a two-step process. The first step, liquid chromatography, separates molecules based on how well they dissolve in a mix of solvents. Different fractions of the solvent contain a subset of the full mixture of molecules originally present. Those fractions can then be separated further by the next step, mass spectroscopy.

As its name implies, mass spectroscopy involves identifying molecules based on their mass. The molecules are first broken into fragments, and then those fragments are spread out based on their mass so that each molecule creates a distinct “fingerprint” of fragments.

Cherney said the approach offers several advantages when identifying the molecules present. “One is that it’s highly precise,” he told Ars. “Another is that it’s hard to fool. And then another is that we can look at several things simultaneously.”

It certainly worked well here. Co-author Richard Auchus is a professor of translational medicine and pharmacology and internal medicine at the University of Michigan. “There are few times in my scientific career when I’ve been just floored with a piece of data,” he said. “When Mike [Cherney] showed me the elephant results, it was pretty black-and-white what was going on. I never thought we’d be able to see the testosterone changes with such clarity.”