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Testosterone histories from tusks reveal woolly mammoth musth episodes

An Author Correction to this article was published on 26 May 2023

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Abstract

Hormones in biological media reveal endocrine activity related to development, reproduction, disease and stress on different timescales1. Serum provides immediate circulating concentrations2, whereas various tissues record steroid hormones accumulated over time3,4. Hormones have been studied in keratin, bones and teeth in modern5,6,7,8 and ancient contexts9,10,11,12; however, the biological significance of such records is subject to ongoing debate10,13,14,15,16, and the utility of tooth-associated hormones has not previously been demonstrated. Here we use liquid chromatography with tandem mass spectrometry paired with fine-scale serial sampling to measure steroid hormone concentrations in modern and fossil tusk dentin. An adult male African elephant (Loxodonta africana) tusk shows periodic increases in testosterone that reveal episodes of musth17,18,19, an annually recurring period of behavioural and physiological changes that enhance mating success20,21,22,23. Parallel assessments of a male woolly mammoth (Mammuthus primigenius) tusk show that mammoths also experienced musth. These results set the stage for wide-ranging studies using steroids preserved in dentin to investigate development, reproduction and stress in modern and extinct mammals. Because dentin grows by apposition, resists degradation, and often contains growth lines, teeth have advantages over other tissues that are used as records of endocrine data. Given the low mass of dentin powder required for analytical precision, we anticipate dentin-hormone studies to extend to smaller animals. Thus, in addition to broad applications in zoology and palaeontology, tooth hormone records could support medical, forensic, veterinary and archaeological studies.

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Fig. 1: Male African elephant tusk record displaying musth episodes.
Fig. 2: Male woolly mammoth tusk record displaying evidence of musth.
Fig. 3: Female woolly mammoth tusk record.

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Data availability

Steroid and isotope data generated and analysed during the current study are included in this Article and its supplementary files. CT scan data are available on the University of Michigan library’s repository for digital data, Deep Blue Data. URLs where CT scan data have been deposited are listed in the Supplementary InformationSource data are provided with this paper.

Change history

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Acknowledgements

LC–MS/MS operators P. O’Day and D. G. Stouffer facilitated steroid analyses. The Toledo Zoo and Aquarium provided frozen samples from long-term storage of unused elephant serum. K. Lohmann and L. Wingate provided access to space and equipment for dentin sampling and provided stable isotope analyses. J. El Adli, M. Jones, M. Lynch and A. Clark contributed to CT scanning efforts. K. Sobczyk-Kojiro and J. Rege assisted with preliminary analyses. The contributions of G.G.B. were conducted within the framework of the state assignment of the Diamond and Precious Metals Geology Institute SB RAS. S.L.V. was supported by the Russian Science Foundation (project no. 22-27-00082). This study received support from the University of Michigan’s seed funding programme for innovative interdepartmental collaborations, Mcubed 3.0.

Author information

Authors and Affiliations

Authors

Contributions

M.D.C. and D.C.F. conceived the investigation and initiated collaborations. M.D.C., D.C.F., R.J.A. and P.S. established the initial research objectives and A.N.R., E.A.S. and S.G.B. also contributed to subsequent development of research questions. M.D.C. conducted tusk sampling, sample pretreatment (isotope analyses), method development (steroid extraction) and LC–MS/MS steroid analyses. M.D.C. and D.C.F. established connections for CT scanning and isotope analyses. R.J.A., D.C.F. and P.S. provided funding. R.J.A. and D.C.F. provided space, material resources, and laboratory equipment. D.C.F., B.B., D.M., G.G.B., S.L.V. and A.N.T. contributed to discovery and acquisition of specimens. D.C.F. and A.N.R. provided access to specimens. M.D.C., D.C.F., R.J.A., P.S., A.N.R. and E.A.S. contributed to interpretation of results. M.D.C. and E.A.S. analysed CT data. S.G.B. produced and analysed thin sections. M.D.C. organized data, created tables and figures, and took the lead in drafting the manuscript. All authors provided feedback that helped shape the composition of the manuscript.

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Correspondence to Michael D. Cherney.

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Extended data figures and tables

Extended Data Fig. 1 LC-MS/MS Integration peaks for testosterone standard and blank.

a, Testosterone plots for calibration standard 5 (expected concentration = 390.625 pg ml−1). b, Testosterone plots for a representative process blank sample. The left plots for each sample show the “quantifier” ions (mass-to-charge ratio = 97.0) that were produced from the fragmentation of the dentin testosterone and the deuterated testosterone internal standard. These were used to quantify testosterone in each injection, based on a calibration curve measured with every batch of samples. Plots on the right compare the quantifier ion with the “qualifier” ion (mass-to-charge ratio = 109.0) for the dentin testosterone and the deuterated testosterone internal standard. When the analyte is present and measurable, quantifier and qualifier ion plots should overlap and match the expected ratio based on previous empirical tests. When sample testosterone results 1) did not match the acquisition time (x-axis) for the deuterated internal standard, 2) lacked matching peaks in quantifier and qualifier ions with a qualifier/quantifier ratio within the expected range [73, 109], or 3) had a signal/noise ratio less than 3.0, they were considered unreliable. Plot for the calibration standard (a) shows reliable signals of sample testosterone and internal standard testosterone. The upper plots for the process blank (b) show the “noise” typical of a sample with no measurable testosterone. The lower plots for the process blank (b) show a reliable signal of deuterated testosterone indicating successful extraction and proper LC-MS/MS function.

Extended Data Fig. 2 LC-MS/MS Integration peaks for tusk dentin testosterone.

a, Testosterone plots for a representative sample of dentin from the modern African elephant tusk, UMMP 118186. b, Testosterone plots from a representative sample of dentin from the approximately 35,000 year-old male woolly mammoth tusk, UMMP 118711. The left plots for each sample show the “quantifier” ions (mass-to-charge ratio = 97.0) that were produced from the fragmentation of the dentin testosterone and the deuterated testosterone internal standard. These were used to quantify testosterone in each injection, based on a calibration curve measured with every batch of analyses. Plots on the right compare the quantifier ion with the “qualifier” ion (mass-to-charge ratio = 109.0) for the dentin testosterone and the deuterated testosterone internal standard. When the analyte is present and measurable, quantifier and qualifier ion plots should overlap and match the expected ratio based on previous empirical tests. When sample testosterone results 1) did not match the acquisition time (x-axis) for the deuterated internal standard, 2) lacked matching peaks in quantifier and qualifier ions with a qualifier/quantifier ratio within the expected range [73, 109], or 3) had a signal/noise ratio less than 3.0, they were considered unreliable. Plots show reliable signals for sample testosterone and internal standard testosterone in representative dentin samples milled from the African elephant tusk, UMMP 118186 (a) and the permafrost woolly mammoth tusk, UMMP 118711 (b).

Extended Data Fig. 3 UMMP 118186.

a, Complete right tusk of African elephant from Botswana. The location where the core was removed can be seen on the ventral aspect of the tusk. b, Preparing to collect the core sample using a standard 1.25 inch coring bit. c, 3D surface model of the core showing internal surface exposed via a cut transverse to the tusk axis and perpendicular to the appositional surface of dentin. Curved growth increments can be seen on the cut surface. The curved bottom edge of the core is the pulp cavity surface. d, Virtual slice from microCT data showing curved growth features and flatbed scanner image of tusk block with sample boundaries superimposed. e, Records of testosterone, progesterone, androstenedione and cortisol in dentin powder samples. Vertical lines mark annual boundaries in the tusk growth record. Gaps in plotted data represent analyses that produced unreliable results, most likely due to concentrations being below the lower limit of quantitation. Scale bars are 20 cm (a) and 1 cm (c, d).

Source Data

Extended Data Fig. 4 UMMP 118711.

a, Field photograph of tusk segment prior to cutting a ~2-cm-thick slab from the proximal end of the segment as marked (disc 4 – museum specimen displayed in inset). The tusk had been cut into segments before the UMMP specimen was collected. b, The UMMP specimen consists of ten discs removed from the complete right tusk of a male woolly mammoth. The spacing of discs was reconstructed based on the lengths of segments that had been cut prior to inspection by UMMP representatives. In most cases, the disc was removed from the end of a segment. c, MicroCT projection of the surface and flatbed scan of the disc 10 block with mill paths overlain. Samples from disc 10 represent pre-musth years of growth. d, MicroCT projection of the surface and flatbed scan of the disc 2 block with mill paths overlain. Samples from disc 2 represent mature adult years of growth. e, MicroCT projection of the surface and flatbed scan of the disc 1 block with mill paths overlain. Samples from disc 1 represent mature adult years of growth. The bottom edge of each disc 1 block image is the pulp cavity surface. Scale bar for ce is 1 cm. f, g, Records of testosterone, progesterone, androstenedione and cortisol in dentin powder samples from the pre-musth growth record contained in disc 10 (f) and the adult record contained in disc 2 (g). Vertical lines mark annual boundaries in the tusk growth record. Gaps in plotted data represent analyses that produced unreliable results, most likely due to concentrations being below the lower limit of quantitation.

Source Data

Extended Data Fig. 5 UMMP 118710.

a, Lateral view of UMMP 118710 (ventral surface up), a proximal end of a woolly mammoth tusk from Wrangel Island. Based on the girth, shallow pulp cavity, and proximally decreasing diameter, the specimen appears to be from a female that was advanced in age at death. Scale bar is 1 cm. b, A longitudinal plane projected from microCT data reveals clear annual growth increments, each of which begins with an abrupt transition to low-density growth (dark) followed by a gradual change to higher density (lighter colour) that continues until the beginning of the next annual increment. c, Dentin powder samples for UMMP 118710 were collected from an interior surface exposed via a longitudinal cut through the entire specimen. d, Records of testosterone, progesterone, androstenedione and cortisol in dentin powder samples are similar to those from the pre-musth record in UMMP 118711 (see Extended Data Fig. 4) but have the lowest overall testosterone and androstenedione concentrations of the three tusks analysed. Progesterone concentrations are higher than in the UMMP 118711 pre-musth samples, but lower than in the adult male records. Vertical lines mark annual boundaries in the tusk growth record. Gaps in plotted data represent analyses that produced unreliable results, most likely due to concentrations being below the lower limit of quantitation.

Source Data

Extended Data Fig. 6 Chronological correlation of growth layers in UMMP 118711.

Thin sections made from each disc (1 – 10, numbered by owner from the proximal to the distal end of the tusk (see Fig. 2) photographed under ultraviolet light. Annual growth features are identified with arrows, and lines connect corresponding features visible in adjacent discs. Corresponding features in thin sections of adjacent discs were identified from matching qualities such as thickness, spacing and fine-scale intra-feature patterning of light and dark bands (many annual ‘features’ are made up of multiple growth lines). Years are identified as ‘x’ through ‘x+31.’ The precise number of years lost due to wear at the distal end of the tusk is unknown, but year x is estimated to be when the animal was in its early 20s. Based on these correlations, the growth record spans 32 years. Air bubbles between the slides and histological thin sections for discs 1, 2, 3, 7 and 9 appear as irregularly shaped zones of lighter colour.

Extended Data Fig. 7 Tusk slab sampling procedure.

The slab featured here is the one removed from disc 2 of UMMP 118711. a, Diagram of tusk cross section showing growth layers in a longitudinal plane aligned with the tusk axis. The grey box shows the orientation and approximate position of the tusk block pictured in b – g. b, Using a 1-mm cylindrical diamond grinding bit, powder samples were collected using a computer-controlled Merchantek Micromill. c, Flatbed scan of the polished surface of the tusk block. d, Projection of microCT scan data, showing average luminosity for a 1-mm-thick slice parallel to and 0.5 mm below the surface used for sampling. e, Sample boundaries were planned based on annually recurring features visible in microCT. Each year of growth was divided into ~0.5-mm-wide increments. The number of increments ranged from 7 to 15 based on the thickness of annual growth layers. f, Growth features visible in the flatbed scan of the cut surface were used to fine-tune sample paths to follow growth increments. These sample paths were programmed into the computer-automated milling station. g, Flatbed scan of the block after sampling. Scale bar for cg is 1 cm.

Extended Data Fig. 8 Effects of soaking time on methanol extraction of steroids from dentin powder.

a, In the first controlled experiment, subsamples of a pooled dentin powder sample from the woolly mammoth tusk (UMMP 118710) were soaked in methanol for varying amounts of time. Five batches of triplicates were soaked for 0, 1.5, 3, 6 and 24 h prior to analysis of methanol supernatant. b, In the second controlled experiment, subsamples from a pooled powder sample of dentin from the African elephant tusk (UMMP 118186) were soaked in methanol for varying amounts of time. Six batches of duplicates were soaked for 0, 0.5, 1, 1.5, 2 and 2.5 h prior to analysis of methanol supernatant. For all dentin powders, soaking time refers to the amount of time powder samples were soaked in methanol at 4 °C. At the beginning of methanol extraction, all tubes were vortexed for 60 seconds at 10 krpm. Prior to transferring supernatant to clean vials for evaporation and subsequent supported liquid extraction, all tubes were centrifuged at 12.7 krpm for 8 min. Thus, 0-minute samples of dentin powder were actually submerged in the methanol extraction solution for up to 15 min. Power trendlines are displayed for concentrations of each analyte measured after different soaking times.

Source Data

Extended Data Fig. 9 Absolute testosterone measurements and concentrations relative to local minima.

a, The same sequence of powder samples was collected on two different occasions from the disc-1 block of UMMP 118711. The two sampling episodes were several weeks apart, and the slab was exposed to moderate heat when removing it from the milling stage after the first iteration. Testosterone was lower in the second set of analyses, likely a consequence of heat exposure. b, When values are normalised to the average of the five lowest values in the batch, plots match closely. Thus, although testosterone recovery appears to have been affected by exposure to heat, relative changes associated with musth remained unchanged. This suggests that relative peak heights could provide useful comparisons even when absolute measurements are affected by diagenetic alteration. Vertical lines mark annual boundaries in tusk growth record.

Source Data

Extended Data Fig. 10 Absolute testosterone measurements and concentrations relative to androstenedione.

Absolute measurements of testosterone from UMMP 118186 (Fig. 1) are plotted in black. The ratio of testosterone to androstenedione for each sample is plotted in grey. Both plots display increases associated with musth. In addition, testosterone/androstenedione is elevated to musth-like levels in the years prior to musth, when absolute testosterone remains low. Androstenedione was the most consistent steroid in analyses reported here. As such, androstenedione provides a potential indicator of diagenetic effects and may be useful for normalising degraded testosterone results. Vertical lines mark annual boundaries in tusk growth record.

Source Data

Supplementary information

Supplementary Information

A pdf containing a supplementary discussion (including Musth in mammoths and other proboscideans, Evaluating the reliability of testosterone in tusk records, Dentin steroids, Comparing dentin steroid records, Sample mass considerations) and notes (including Steroid survey, Links to CT data, Metadata for AMS dates, and Specimen acquisition).

Reporting Summary

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Supplementary Data

A Microsoft Excel spreadsheet containing minimum steroid and stable isotope data. It contains six sheets: UMMP 118186 Steroid Data, UMMP 118711 Steroid Data, UMMP 118710 Steroid Data, Elephant Serum Data, Pooled Serum Data (control), UMMP 118186 Stable Isotope Data, Time Trial1 Steroid Data, Time Trial2 Steroid Data.

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Cherney, M.D., Fisher, D.C., Auchus, R.J. et al. Testosterone histories from tusks reveal woolly mammoth musth episodes. Nature 617, 533–539 (2023). https://doi.org/10.1038/s41586-023-06020-9

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