Sexing Dinos: Paleontologists Seek Fossilized Hormones

How can you tell if a dinosaur is female or male? It’s one of the most basic aspects of biology and yet, for the most part, it continues to be a mystery in paleontology. We don’t yet know the sex of most extinct species, even if those in museum displays have gender-specific names like Sue the T.rex or Cliff the Triceratops. But this past October at the annual meeting of the Society of Vertebrate Paleontologists, one scientist offered insight into the work he and his team are doing to uncover hormones in fossil bones. Their initial results indicate that trace amounts of sex hormones may indeed survive the fossilization process. If so, this could be a game-changer, offering phenomenal insight into ancient animals and their evolution on this planet.

Evan Saitta presented that work during an SVP poster session. Currently a postdoctoral paleontologist at the University of Chicago, he’s been pondering the possibility of uncovering fossil hormones since 2018, when he read a number of papers that found cholesterol in fossils, some dating as far back as 720 million years ago. Cholesterol, he noted, is “another type of steroid,” and “sex hormones are biosynthesized from cholesterol.”

In other words, the body creates hormones such as estrogen and testosterone by modifying existing cholesterol molecules. Therefore, Saitta explained, “these hormones have a similar basic structure” to cholesterol. That structure is stable, which makes cholesterol more conducive to surviving millions of years of geologic pressure. This led Saitta to wonder if hormones such as estrogen and testosterone might survive as well, even if in an altered form.

“Cholesterol is in every cell membrane in your body, whereas these hormones are in much lower concentrations,” he said. “They’re concentrated and produced in the gonads, which don’t fossilize. They travel through the bloodstream, which doesn’t fossilize. And so your hope is that, when an organism dies, there’s enough residual hormone left behind in the bone that you can detect it.”

But first, he and his team needed to understand what they were looking for. So they put pure estrogen, in the form of estradiol, a powdered extract, through experiments designed to simulate the fossilization process. These artificial maturation experiments enabled them to imperfectly replicate the heat and pressure that occurs over millions of years.

“When you heat up a system,” Saitta said in a phone interview, “the chemical reactions can happen more quickly, and so that’s the sort of trick that you’re going for: You’re using heat to not only directly simulate some of the geothermal heat when [a fossil is] buried underground, but also to account for time.”

Estradiol survived the artificial maturation experiment, meaning that, in theory, it might survive fossilization. Putting it through gas chromatography-mass spectrometry—a process that separates molecules out from complex mixtures—gave them a road map: a way to compare potential estrogen “fingerprints” in bones and to understand how and when that fingerprint should appear in that process.

Instead of jumping directly to fossils, however, the team tested the bones of extant animals, including a hawk and a goose. They found that same estrogen fingerprint. Saitta went a step further and tested the goose bone surrounded in clay. Would hormones leech out of the bone and into surrounding sediment? In this case, they did not, a finding with implications for future fossil research.

With examples from pure estrogen and modern bones, the team moved to fossils. These were not found in formations known for exceptional preservation. They were simply various chunks of bone from different dinosaurs—known and unknown at the time of the experiments. The results were mixed. Four of them suggested trace amounts of estrogen; four had none; and one was uncertain.

For Saitta, the potential hormone traces in fossils are not as important as the results from the experiments themselves. “This work is exciting and it’s proof of concept, more than anything else,” he said, because it indicates that hormones might just survive fossilization and they can be detected.

“We know estrogen is stable. We know you can detect it in bone beyond just in gonad or blood,” he continued. “And we know that if you pressure cook it, either as a purified extract itself or as residual hormone within bones, you can detect it and you can detect it separate from the surrounding environment. All of that is, I think, the key result.”

Jasmina Wiemann, also at the University of Chicago, was not involved in this research. She is a molecular geo-, astro-, paleobiologist and Agouron Institute Fellow at the Field Museum of Natural History. In an email to Gizmodo, she noted that “the authors tackle a question here that has not been rigorously investigated in the past.”

But she also wrote that “it can be quite challenging to reliably detect traces of biomolecules that occurred originally in very high concentrations.” She points to heme, “the porphyrin compound that is responsible for the red color of blood: heme is a small and very stable molecule that occurs bound to a protein in the blood of vertebrates. Despite its abundance in vascularized bone of living vertebrates, the detection of traces of heme from fossil bone has proven very challenging and preserved quantities barely exceed detection limits. Sex hormones are distributed through the bloodstream, but occur at roughly one billionth of the concentration of heme.”

She added that “trying to extract compounds that are chemically stable, but occur in very low concentrations in fluids that do not leave fossil traces, from adjacent bone matrix, opens up the following question: Given that diagenetic fluids percolate through bone during the fossilization process, what is the chance that a detectable quantity of estrogen traces would survive over millions of years?”

Saitta agrees that detecting heme is challenging, but notes that “porphyrins also have an added challenge (which hormones do not): they can also derive from abundant exogenous/environmental sources such as algae in the water in which a body fossil is deposited.”

And, he said, in terms of diagenetic fluids–a term that refers to water within sediments and how liquid impacts sediment over time–“this is certainly a hurdle to overcome,” noting that “these molecules are not very soluble in water. We know steroids such as cholesterol can remain in bone without total loss into the matrix over millions of years, so hormones likely should as well.”

He points to the quantity of bones he and his colleagues have analyzed (“up to ~50 g in some cases”), stating that “one way to search for low-concentration molecules is to be willing to destroy big chunks of fossil!”

But he maintains that “it does appear to be possible to detect estrogen in dried-out bone both before and after maturation,” pointing to the detection of estrogen in the duck and goose bone artificial maturation experiments.

Holly Latta is second author on this current work and an organic chemist. She performed the experiments on all of the bones the team has worked on thus far. “Obviously, more work needs to be done to confirm the results and the full extent of what they mean,” she wrote in an email. “But the promise of these results is incredibly exciting.”

One might wonder if the absence of estrogen from their results could indicate that the bone tested came from a male. Saitta explained that both females and males contain both estrogen and testosterone; the key to determining one sex over another is in the quantities contained therein. If estrogen can be detected in a fossil at all, he said, that might indicate high levels of estrogen, therefore suggesting a female.

Part of their future work will also involve testing for testosterone, along with testing much larger sample sizes and testing fossils from formations that are known for better preservation.

“While the question is incredibly exciting,” Wiemann agreed, “there is a lot of fundamental work to be done by both biologists, paleontologists, and geochemists: We first need to know if estrogen indeed can fossilize. And if we are successful in finding traces of estrogen, we need to understand what factors drive differences in the preserved quantities (different depositional settings, different tissues, different taxa, etc.)”

But Saitta believes that the question is, “‘Can it be reliably detected at low concentration?’ All the evidence and logic points towards it being able to fossilize.” He conceded that “hormone concentrations are extremely low, but the reasonable expectation is that mass spectrometers will improve over time and detection limits will be lowered. As such, I see only future potential for the study of ancient hormones.”

“These hormones,” Saitta said, “they are the driving agents of sexual development and of sexual dimorphism.” That the hormones proved to be stable in their experiments and that trace elements of these hormones might be detected in fossils with little, if any, organics expected to survive, “is all very encouraging. You’re really getting at the direct drivers of sexual development by searching for the hormones themselves.”

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