EXTRACTION OF HORMONES FROM PLEISTOCENE MAMMOTH TUSK DENTIN TO UNDERSTAND PALEOECOLOGY

Hormones in multicellular organisms are critical intercellular signaling molecules responsible for physiological control of coordinated actions such as digestion and lactation. Through complex interactions between physiological and cognitive processes, hormones also influence certain multifaceted behaviors, such as those relating to reproduction. In mammals, many hormones are produced in endocrine organs like the adrenal gland, ovaries and testes, and then circulate throughout the body to different organs. Hormones are found in excreta and can be incorporated in tissue during growth. Measurements of hormone concentrations in feces, urine, and tissues are used to monitor aspects of behavior (such as reproductive activity and stress) that are difficult to observe directly. Here, we describe the extraction of hormones preserved in late Pleistocene mammoth tusk dentin and two independent methods of quantifying hormones in this ancient tissue. Following procedures adapted from those used to study steroids in hair and baleen, we soaked powder dentin samples in methanol to extract steroid hormones for measurement. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) provides an efficient way of measuring small quantities of multiple steroid hormone targets in a small sample volume. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), are cost-effective and widely available independent analyses for measuring targeted single-hormone measurements but are generally less specific than LC-MS/MS. Consistent with interpretations of sex based on tusk size, male tusk specimens analyzed have consistently higher average concentrations of the steroid hormone testosterone (T) than females. In addition, fine-scale serial sampling of a male woolly mammoth tusk reveals annual surges in T that match those experienced during musth in modern elephants. In addition to T, we find androstenedione, progesterone, and cortisol, although often in lower concentrations than T, in permafrost and modern remains of mammoths and elephants. Future work with similar methods might help understand behaviors beyond male reproduction in permafrost mammoths, including those in non-permafrost proboscidean specimens, along with behaviors in other animals and older fossil material.