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. 2021 Nov 24;9(1):coab087. doi: 10.1093/conphys/coab087

Detection of steroid and thyroid hormones in mammalian teeth

Justine M Hudson ✉, Cory J D Matthews 1, Cortney A Watt 1
Editor: Steven Cooke
PMCID: PMC8633673  PMID: 36439380

We determined that steroid and thyroid hormones can be measured in narwhal, walrus, beluga and killer whale teeth, indicating that teeth may be a suitable matrix for studying reproduction and stress in wild mammal populations.

Abstract

Endocrine tools can provide an avenue to better understand mammalian life histories and predict how individuals and populations may respond to environmental stressors; however, few options exist for studying long-term endocrine patterns in individual marine mammals. Here, we (i) determined whether hormones could be measured in teeth from four marine mammal species: narwhal (Monodon monoceros), beluga (Delphinapterus leucas), killer whale (Orcinus orca) and Atlantic walrus (Odobenus rosmarus rosmarus); (ii) validated commercially available enzyme immunoassay kits for use with tooth extracts; and (iii) conducted biological validations for each species to determine whether reproductive hormone concentrations in teeth correlated with age of sexual maturity. Tooth extracts from all species had measurable concentrations of progesterone, testosterone, 17β-estradiol, corticosterone, aldosterone and triiodothyronine (T3); however, cortisol was undetectable. Parallelism between the binding curves of assay kit standards and serially diluted pools of tooth extract for each species was observed for all measurable hormones. Slopes of accuracy tests ranged from 0.750 to 1.116, with r2 values ranging from 0.977 to 1.000, indicating acceptable accuracy. Biological validations were inconsistent with predictions for each species, with the exception of female killer whales (n = 2), which assumed higher progesterone and testosterone concentrations in mature individuals than immature individuals. Instead, we observed a decline in progesterone and testosterone concentrations from infancy through adulthood in narwhal (n = 1) and walruses (n = 2) and higher reproductive hormone concentrations in immature individuals than mature individuals in belugas (n = 8 and 10, respectively) and male killer whales (n = 1 and 2, respectively). While unexpected, this pattern has been observed in other taxa; however, further analytical and biological validations are necessary before this technique can be used to assess individual mammalian endocrine patterns.

Introduction

Conservation of wildlife populations requires an understanding of life history characteristics such as reproductive rates and timing of sexual maturity. Endocrine techniques can help us better understand mammalian life histories and predict how individuals and populations may respond to environmental threats (Wikelski and Cooke, 2006; McCormick and Romero, 2017). For example, monitoring reproductive hormones such as testosterone, progesterone and estradiol can be used to determine the age of sexual maturity, the number of sexually mature individuals in a population, the timing of reproductive cycles and breeding success (McCormick and Romero, 2017). Monitoring stress-related hormones such as glucocorticoids can provide information on potential stressors (Wikelski and Cooke, 2006; McCormick and Romero, 2017) and their impacts on survival and reproduction (Pride, 2005; Scarlata et al., 2012; Young et al., 2019). Endocrinology can thus be used to assess population dynamics (Wikelski and Cooke, 2006) and assist with setting realistic conservation and mitigation targets (e.g. identifying when and where reproduction occurs to conserve critical habitat).

Several matrices have been used to study the endocrine state of mammals, including blood (Weber et al., 1982; McKenzie et al., 2004), hair (Macbeth et al., 2010; Mastromonaco et al., 2014; Calamari et al., 2020), blubber (Kellar et al., 2014; Kershaw and Hall, 2016; Beaulieu-McCoy et al., 2017), skin (Bechshoft et al., 2015), faeces (Rolland et al., 2012), saliva (Lutz et al., 2000) and blow (Thompson et al., 2014; Burgess et al., 2018; Mingramm et al., 2019; Hudson et al., 2021). While these matrices can provide information on the endocrine state of an individual at a specific point in time and can be useful for population-level assessments, understanding long-term changes in the endocrine state of individuals can assist with setting realistic conservation targets. Obtaining multiple samples from targeted individuals over time, however, is difficult for most species and nearly impossible for wild free-ranging marine mammals (see Rolland et al., 2005; Hunt et al., 2006; Wasser et al., 2017).

Recently, continuously and incrementally growing matrices, such as whiskers, claws, earplugs and baleen, have been used to explore longitudinal hormone profiles in marine mammals, increasing our knowledge of their reproduction and stress physiology (Trumble et al., 2013, 2018; Hunt et al., 2016, 2018; Lysiak et al., 2018; Keogh et al., 2020, 2021; Crain et al., 2021). Lifetime hormone profiles in earplugs (Trumble et al., 2013) and long-term hormone profiles of baleen (Hunt et al., 2016, 2017a, 2018; Lysiak et al., 2018) have proven valuable for studying seasonal, annual and multi-year endocrine patterns, but are taxonomically limited to mysticete whales. Teeth in odontocetes (toothed whales) and pinnipeds (seals) are continuously accreting structures that, like earplugs, represent growth over an animal’s lifetime (Perrin and Myrick 1980; Klevezal, 1996; Fig. 1). Whole-tooth and sequential measurements of annually deposited growth layer groups (GLGs) have been used to reconstruct marine mammal foraging behaviour, movement histories and onset of reproductive maturity from stable isotopes (Hobson and Sease, 1998; Mendes et al., 2007; Matthews and Ferguson, 2015), as well as lifetime trace element and contaminant exposure (Evans et al., 1995; Clark et al., 2021). Hormone measurements in teeth (including tusks) could similarly allow for assessment of endocrine function and history of odontocetes and pinnipeds. However, it is unknown whether the inorganic (hydroxyapatite) and cellular components (e.g. lipid, protein) of dentine and cementum, which accrete on the interior and outer surfaces of marine mammal teeth, respectively (Klevezal, 1980, 1996; Perrin and Myrick, 1980), incorporate hormones during formation and whether hormones are present in high-enough concentrations to be detected by commercially available enzyme immunoassays (EIAs).

Figure 1.

Figure 1

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Schematic diagram of narwhal, killer whale, beluga and walrus tooth structure, including relative position and orientation of dentine GLGs.

Analytical validations are imperative when assessing the potential of a new matrix (Amaral, 2010) to ensure that the analyte in the standards and samples bind to the antibody in a comparable way (Grotjan and Keel, 1996) and that the assay can differentiate between high and low hormone concentrations (Hunt et al., 2017b). In addition, biological validations are needed to ensure that hormone concentrations measured in new matrices are biologically relevant. The objectives of this study were to (i) determine whether hormones could be measured in teeth of representative odontocete and pinniped species, including narwhal (Monodon monoceros), beluga (Delphinapterus leucas), killer whale (Orcinus orca) and Atlantic walrus (Odobenus rosmarus rosmarus); (ii) conduct immunoassay validations, including parallelism and accuracy tests; and (iii) conduct biological validations to determine whether reproductive hormone concentrations in teeth correlate with known life history events, such as sexual maturity.

Materials and methods

Study species and tooth samples

Narwhal, beluga and walrus teeth were provided to Fisheries and Oceans Canada (DFO) by Inuit hunters through marine mammal sampling programs or purchased through local co-ops in Nunavut. Killer whale teeth were collected opportunistically from stranded or ice-entrapped whales. Specimen sex was determined morphologically in the field or genetically (Rosel, 2003; Shaw et al., 2003), and ages were estimated by counting annual GLGs in teeth (Table 1). Ages could not be estimated for the narwhal embedded tusk (ARGF-87-02) due to cementum occlusion of the pulp cavity, and one walrus tusk (RB-OR-04) due to sawing effects; however, these individuals were likely sexually mature adults based on their body and tusk sizes. Teeth were stored frozen at −20°C (beluga and killer whale) or at room temperature (narwhal and walrus) after collection.

Table 1.

Location and biological data for teeth collected from narwhal, beluga, killer whale and walrus specimens

Species Tissue type Sample ID or tag Location Age Sex Collection year Sample preparation
Narwhal Embedded tusk ARGF-87-02 Grise Fiord, NU Adulta Malec 1987 Whole tooth, separate
Erupted tusk NHB-2018-055A Naujaat, NU 5 Malec 2018 Drilled entire length
Erupted tusk EB-2018-009-A Pangnirtung, NU 15 Malec 2018 Drilled each GLG
Beluga Tooth B95-545 Pangnirtung, NU 1 Femalec 1996 Whole tooth, combined
B95-59 Pangnirtung, NU 2 Malec 1996 Whole tooth, combined
AREP85-16 Arviat, NU 4 Malec 1985 Whole tooth, combined
AREP86-30 Arviat, NU 5 Maled 1986 Whole tooth, combined
AREP86-18 Arviat, NU 2 Femaled 1986 Whole tooth, combined
AREP86-25 Arviat, NU 2 Femaled 1986 Whole tooth, combined
AREP86-16 Arviat, NU 3 Femaled 1986 Whole tooth, combined
ARAR-XX-1039 Arviat, NU 5 Femalec 1999 Whole tooth, combined
ARAR-XX-1013 Arviat, NU 33 Malec 1999 Whole tooth, separate
ARAR-XX-1025 Arviat, NU 28 Malec 1999 Whole tooth, separate
B96-155 Pangnirtung, NU 42 Femalec 1996 Whole tooth, separate
ARPG-XX-1037 Pangnirtung, NU 27 Malec 2002 Whole tooth, separate
ARRE-XX-1109 Resolute Bay, NU 29 Maled 2003 Whole tooth, separate
ARRE-XX-1085 Resolute Bay, NU 42 Femalec 2002 Whole tooth, separate
AREP85-01 Arviat, NU 26 Femalec 1985 Whole tooth, separate
B95-539 Pangnirtung, NU 33 Femalec 1996 Whole tooth, separate
ARAR-XX-1006 Arviat, NU 36 Femalec 1999 Whole tooth, separate
ARAR-XX-1027 Arviat, NU 34 Femalec 1999 Whole tooth, separate
Killer Whale Tooth ARSQ-XX-1397 Sanikiluaq, NU 6 Femalec 2016 Whole tooth, separate
NWA-BB-2002 Bonavista Bay, NL 3 Maled 2002 Whole tooth, separate
ARSQ-XX-1379 Sanikiluaq, NU 34 Malec 2016 Whole tooth, separate
10 001 Boundary Bay, BC 24 Maled 1979 Whole tooth, separate
16 006 Port Renfrew, BC 31 Femaled 1986 Whole tooth, separate
Walrus Tusk ARHB-XX-2068 Sanirajak, NU Min. 20 Malec 2020 Drilled each GLG
ARIQ-DFO-2126 Coral Harbour, NU Min. 33 Femalec 2018 Drilled each GLG
RB-OR-04 Naujaat, NU Adultb - 2017 Drilled entire length
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Ages were estimated by counting growth layer groups in teeth. Sex was determined either in the field or genetically. Bolded sample IDs indicate samples that were used for assay validations and italicized IDs indicate samples that were used for both assay validations and biological validations.

aBased on size (440 cm) and weight (1350 kg), this individual was likely a mature adult (Hay, 1984).

bBased on the size of the tusk (47.7 cm), this was likely a mature adult.

cSexed genetically.

dSexed morphologically.

Tooth pulverization and hormone extraction

Teeth were cleaned with three washes of 100% ethanol (EtOH) and then air-dried. For the narwhal embedded tusk (n = 1) and beluga (n = 18, one from each of 18 animals) and killer whale teeth (n = 5, one each from 5 animals), small tooth pieces were placed into a TissueLyser II (Qiagen, Hilden, Germany) chamber. The chamber was filled with liquid nitrogen several times to ensure the tooth pieces were completely frozen. Teeth were pulverized for 30+ seconds at 30 Hz. The walrus and narwhal erupted tusks (n = 3 and 2, respectively) were sectioned lengthwise along the midline using a waterjet saw (Shape Industries, Winnipeg, Canada). Dentine was drilled from one half of each sectioned tusk using a high-resolution micromill system (New Wave Research, Freemont, California) fitted with a 1000-μm drill bit at a depth of 250–600 μm. The walrus tusk used in the assay validation study (RB-OR-04) was only drilled from the area closest to the pulp cavity, due to sawing effects, which based on tusk size, likely represented years of sexual maturity (Table 1). For the biological validations, each individual GLG was drilled along the walrus and narwhal erupted tusks. Whole tooth (beluga, killer whale and narwhal embedded tusk) and dentine (walrus tusk and narwhal embedded tusk) powder were frozen at −20°C until extraction.

Hormones were extracted using a modified protocol originally developed by Hunt et al. (2014) for baleen. Tooth powder (150 mg) was combined with 6 ml of 100% high-performance liquid chromatography grade methanol (MeOH) in a 15-ml glass centrifuge tube and agitated on a plate shaker for 24 hours. Samples were centrifuged at 3500 rpm for 5 minutes and 5 ml of the supernatant was isolated and dried under nitrogen for ~ 2 hours. After the initial evaporation, an additional 1 ml of MeOH was added as a rinse and dried again. Dried samples were reconstituted in 150 μl of assay buffer (Catalogue number X065, Arbor Assay, Ann Arbor, MI, USA) and vortexed for 60 seconds. For the cortisol assay, dried samples were reconstituted in the cortisol assay buffer (Catalogue number K003, Arbor Assay, Ann Arbor, MI, USA). Due to the large volume of extract needed to conduct assay validations, 10 different 150-mg aliquots from the same sample were made for each species and pooled to measure all seven hormones. The reconstituted samples were frozen at −20°C until validations.

Preliminary assay validations indicated low hormone concentrations in some tooth extracts; therefore, we extracted new aliquots of tooth powder and concentrated the extracts two-fold by either extracting 150 mg of tooth powder and reconstituting in 75 μl of assay buffer (killer whale) or extracting 300 mg of tooth powder using 12 ml of MeOH and reconstituting in 150 μl of assay buffer (narwhal erupted tusk, beluga), depending on how much tooth powder was available.

Hormone assays

Hormones were measured using commercially available EIA kits (Arbor Assay, Ann Arbor, MI, USA). Manufacturer’s procedures were followed for all hormones except T3 and aldosterone. For T3 and aldosterone (overnight protocol) assays, 50 μl of standard/sample per well (rather than 100 μl) was used to conserve limited sample, and the amount of assay buffer in non-specific binding (NSB) wells and maximum binding (zero) wells was reduced to 75 μl and 50 μl, respectively (Hunt et al., 2017b). An additional standard was added to the upper-end of the binding curve to increase the assay range for all hormones, except for aldosterone. All samples and standards, along with NSB and zero wells, were assayed in duplicate and were re-assayed if the coefficient of variation was larger than 10% between duplicate samples (n = 0). Final hormone concentrations are presented as ng/g of tooth powder, hereafter ng/g.

Analytical validations

To test for parallelism, a pool of tooth extract was serially diluted to make six dilutions (1:1, 1:2, 1:4, 1:8, 1:16 and 1:32) and the curve of the serially diluted pool was statistically compared to the standard curve for each hormone and species. To test for accuracy, kit standards of known concentrations (progesterone: 100, 200, 400 and 800 pg/ml; testosterone: 256, 640, 1600 and 4000 pg/ml; 17β-estradiol: 39.06, 156.25, 625 and 2500 pg/ml; corticosterone: 312.5, 625, 1250 and 2500 pg/ml; aldosterone: 15.625, 62.5, 250 and 1000 pg/ml; T3: 156.25, 312.5, 625 and 1250 pg/ml) were spiked with equal amounts of pooled tooth extract with a low concentration (80–100% binding), determined from the parallelism test. These spiked standards were then assayed alongside the same volumes of kit standards and the pooled extract to determine the amount of endogenous hormone present. Results were graphed as observed concentration (spiked standard concentration minus pooled extract concentration) versus expected concentration (standard concentration divided by two) (Brown et al., 2008).

Biological validations

Biological validations were conducted to determine whether reproductive hormone patterns in teeth corresponded with age of sexual maturity. Progesterone and testosterone have been shown to increase at the onset of sexual maturation (Burgess et al., 2012; Kinoshita et al., 2012; Trumble et al., 2013; Beaulieu-McCoy et al., 2017; Carlitz et al., 2019), and we predicted similar patterns would be observed across the four marine mammal species in our study. For the narwhal (n = 1) and walrus tusks (n = 2), we collected dentine from each GLG, and assessed progesterone concentrations in the female and testosterone concentrations in males, from infancy to adulthood. Beluga tooth extracts from 8 immature (<5 years) males (n = 3) and females (n = 5) were combined to create a single sample representative of immature belugas. Beluga teeth from mature (>25 years) males (n = 4) and females (n = 6) were extracted as 10 individual samples. Prior to extraction, the five earliest GLGs were removed, as the youngest age of sexual maturity was estimated to be 6 years of age for belugas (Robeck et al., 2005; Ferguson et al., 2020). Progesterone concentrations from the combined immature beluga sample were compared to progesterone concentrations of mature female belugas, while testosterone concentrations from the combined immature beluga sample were compared to testosterone concentrations from mature male belugas. Lastly, for the killer whale biological validations, progesterone and testosterone concentrations were examined in whole teeth of six individuals, two immature (3–6 years) and three mature (24–34 years). Progesterone concentrations from an immature female were compared to a mature female and testosterone concentrations from an immature male were compared to mature males.

Statistical analysis

Hormone concentrations and parallelism results were calculated using GraphPad Prism (Version 8.1.2; San Diego, CA, USA). For parallelism, F-tests were used to determine whether the binding curves of the standards were parallel to those of serially diluted samples. To assess accuracy, linear regressions of observed versus expected doses were calculated in R Studio (Version 1.1.463; Boston, MA, USA). Acceptable accuracy results have a slope between 0.7 and 1.3 and an r2 that is >0.95 (Grotjan and Keel, 1996). No statistical analyses were performed for the biological validations due to the small sample sizes; however, qualitative comparisons were conducted using summary statistics.

Results

Analytical validations

Parallelism between the binding curves of the assay kit standards and serially diluted pools of tooth extract was observed for each hormone, in each species, except for cortisol (Figs 1 and 2; Table 2). The slopes of the accuracy tests ranged from 0.750 to 1.116 and the r2 values ranged from 0.977 to 1.000 (Figs 2 and 3; Table 3).

Figure 2.

Figure 2

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Parallelism (left) and accuracy (right) results for progesterone (a), testosterone (b) and 17β-estradiol (c) using a serially diluted pool of extract from narwhal (embedded: light blue; erupted: orange), beluga (yellow), killer whale (red) and walrus (red) teeth. Dotted lines represent samples that were concentrated two-fold. Sample concentrations falling outside the assay binding curves are not shown. Note different scales of axes for observed and expected dose.

Table 2.

F-tests and P-values (α = 0.05) from parallelism tests for progesterone, testosterone, 17β-estradiol, corticosterone, aldosterone and T3 EIA kits using extracts from narwhal, beluga, killer whale and walrus teeth

Species Progesterone Testosterone 17β-Estradiol Corticosterone Aldosterone T3
Narwhal Embedded F 1,10 = 1.793 F 1,10 = 4.813 F 1,8 = 4.158 F 1,10 = 2.381 F 1,8 = 0.147 F 1,10 = 1.115
P = 0.210 P = 0.053 P = 0.076 P = 0.154 P = 0.712 P = 0.316
Narwhal Erupted F 1,9 = 2.090 F 1,9 = 2.163 F 1,8 = 0.826 F 1,10 = 2.50 F 1,8 = 0.095 F 1,9 = 2.721
P = 0.182 P = 0.176 P = 0.390 P = 0.145 P = 0.766 P = 0.134
Beluga F 1,9 = 1.420 F 1,8 = 2.123 F 1,6 = 5.829 F 1,8 = 0.309 F 1,6 = 5.329 F 1,8 = 3.891
P = 0.264 P = 0.183 P = 0.052 P = 0.594 P = 0.060 P = 0.084
Killer whale F 1,10 = 4.007 F 1,7 = 0.751 F 1,6 = 1.009 F 1,8 = 3.680 F 1,6 = 1.012 F 1,8 = 2.404
P = 0.073 P = 0.415 P = 0.354 P = 0.091 P = 0.353 P = 0.160
Walrus F 1,10 = 0.968 F 1,9 = 1.246 F 1,6 = 2.236 F 1,8 = 3.635 F 1,6 = 1.666 F 1,7 = 1.336
P = 0.348 P = 0.293 P = 0.185 P = 0.093 P = 0.244 P = 0.286
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Results were calculated by comparing the linear slopes of the standard curve to the slopes of serially diluted pools of tooth extract (1:1, 1:2, 1:4, 1:8, 1:16 and 1:32). Degrees of freedom vary due to the removal of dilutions falling below the limit of detection. Cortisol results are not shown due to all dilutions falling below the limit of detection.

Figure 3.

Figure 3

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Parallelism (left) and accuracy (right) results for corticosterone (a), aldosterone (b) and triiodothyronine (T3; c) using a serially diluted pool of extract from narwhal (embedded: light blue; erupted: orange), beluga (yellow), killer whale (red) and walrus (red) teeth. Dotted lines represent samples that were concentrated two-fold. Sample concentrations falling outside the assay binding curves are not shown. Note different scales of axes for observed and expected dose.

Table 3.

Dilutions and accuracy results for progesterone, testosterone, 17β-estradiol, corticosterone, aldosterone and T3 using extracts from narwhal, walrus, beluga and killer whale teeth

Progesterone Testosterone 17β-Estradiol Corticosterone Aldosterone T3
Narwhal Embedded 1:32 1:32 1:32 1:16 1:16 1:16
Slope = 1.028 Slope = 0.868 Slope = 0.883 Slope = 0.845 Slope = 0.890 Slope = 0.963
SE = 0.053 SE = 0.024 SE = 0.057 SE = 0.015 SE = 0.003 SE = 0.074
r  2 = 0.992 r  2 = 0.998 r  2 = 0.991 r  2 = 0.999 r  2 = 1.000 r  2 = 0.988
Narwhal Erupted 1:16 1:32 1:8 1:4 1:8 1:4
Slope = 1.084 Slope = 1.015 Slope = 0.935 Slope = 0.896 Slope = 0.960 Slope = 0.866
SE = 0.014 SE = 0.007 SE = 0.049 SE = 0.012 SE = 0.006 SE = 0.013
r  2 = 0.999 r  2 = 0.999 r  2 = 0.994 r  2 = 0.999 r  2 = 0.999 r  2 = 0.999
Beluga 1:8 1:8 1:4 1:2 1:4 1:4
Slope = 0.750 Slope = 1.022 Slope = 0.814 Slope = 0.883 Slope = 0.870 Slope = 0.872
SE = 0.059 SE = 0.008 SE = 0.045 SE = 0.016 SE = 0.019 SE = 0.040
r  2 = 0.987 r  2 = 0.999 r  2 = 0.993 r  2 = 0.999 r  2 = 0.998 r  2 = 0.995
Killer whale 1:8 1:16 1:8 1:2 1:4 1:4
Slope = 0.946 Slope = 1.038 Slope = 0.937 Slope = 0.891 Slope = 0.990 Slope = 1.061
SE = 0.009 SE = 0.060 SE = 0.053 SE = 0.036 SE = 0.028 SE = 0.018
r  2 = 0.999 r  2 = 0.993 r  2 = 0.993 r  2 = 0.996 r  2 = 0.998 r  2 = 0.999
Walrus 1:32 1:32 1:16 1:4 1:2 1:2
Slope = 1.116 Slope = 0.963 Slope = 0.921 Slope = 0.817 Slope = 1.043 Slope = 1.096
SE = 0.023 SE = 0.038 SE = 0.051 SE = 0.054 SE = 0.024 SE = 0.116
r  2 = 0.999 r  2 = 0.996 r  2 = 0.993 r  2 = 0.991 r  2 = 0.999 r  2 = 0.977
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Species and hormone comparisons

Progesterone, testosterone, 17β-estradiol, corticosterone, aldosterone and T3 were detected in tooth extracts from each of the four species. In general, progesterone was present in the highest concentrations in all pooled tooth extracts, followed by T3, testosterone, estradiol, corticosterone and aldosterone (Table 4). Cortisol was not detected in any pooled tooth extract, even after concentrating the extracts two-fold (results not shown). Extract from the narwhal embedded tusk consistently had the highest hormone concentrations, followed by the narwhal erupted tusk, walrus tusk, beluga teeth and finally killer whale teeth (Table 4).

Table 4.

Progesterone, testosterone, estradiol, aldosterone, corticosterone and T3 concentrations from a single pooled extract from narwhal, walrus, beluga and killer whale teeth

Number of individuals Progesterone (ng/g) Testosterone (ng/g) Estradiol (ng/g) Aldosterone (ng/g) Corticosterone (ng/g) T3 (ng/g)
Narwhal embedded 1 2.150 0.959 0.967 0.283 0.519 2.593
Narwhal erupted 1 0.451 0.236 0.212 0.175 0.210 0.770
Walrus 1 0.796 0.254 0.104 0.054 0.129 0.227
Beluga 3 0.761 0.118 0.050 0.013 0.137 0.166
Killer whale 1 0.343 0.045 0.064 0.037 0.065 0.293
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Biological validations

Testosterone concentrations in the male narwhal erupted tusk ranged from 0.630 to 1.236 ng/g. Testosterone was high in GLGs 1 and 2, followed by a steady decrease until GLG 14, after which concentrations increased (Fig. 4a). Testosterone concentrations in the male walrus tusk ranged from 0.071 to 0.204 ng/g. Concentrations were high in GLGs 1–3, followed by a decrease between GLGs 3 and 12, and subsequent increase from GLGs 13 to 17 (Fig. 4b). Progesterone concentrations in the female walrus tusk ranged from 0.456 to 1.437 ng/g. Fluctuations in progesterone were pronounced between GLGs 1 and 16 (0.567–1.123 ng/g) and GLGs 29 and 33 (0.486–1.436 ng/g) and relatively constant (0.458–0.680 ng/g) between GLGs 17 and 29 (Fig. 4c).

Figure 4.

Figure 4

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Progesterone (blue) and testosterone (orange) concentrations measured in individual growth layers from a male narwhal tusk (a) and male (b) and female (c) walrus tusks. Note that GLGs of walrus tusks do not correspond to age, as occlusal wear removed an unknown number of GLGs and prevented assigning absolute ages.

Pooled tooth extract from immature belugas had a progesterone concentration of 0.361 ng/g, whereas individual concentrations from six mature females ranged from 0.079 to 0.885 ng/g. Only two mature female belugas had higher progesterone concentrations than the pooled extract from immature belugas (Fig. 5a). Pooled extract from immature belugas had a testosterone concentration of 0.142 ng/g, which was higher than the testosterone concentrations from all individual mature male belugas (range: 0.072–0.088 ng/g; Fig. 5b). A mature female killer whale (n = 1) had higher progesterone (0.211 ng/g) than an immature female (n = 1; 0.136 ng/g; Fig. 5c), while an immature male killer whale (n = 1) had higher testosterone concentration (0.131 ng/g) than mature males (n = 2; 0.036–0.061 ng/g; Fig. 5d).

Figure 5.

Figure 5

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Progesterone and testosterone concentrations measured in immature and mature belugas (a and b) and killer whales (c and d). The boxplot shows the median (midline), the lower and upper quartiles (top and bottom of the box), 1.5× the interquartile range (whiskers) and point estimates for hormone concentrations.

Discussion

Hormone measurement in teeth offers a potential means to reconstruct long-term mammalian endocrine patterns, but this matrix has not been the focus of previous endocrine studies. With the exception of cortisol, which was not detected in the teeth of any species, our measurement of progesterone, testosterone, 17β-estradiol, corticosterone, aldosterone and T3 in tooth extracts from each of the four species shows promise for using tooth hormone measurements to assess an animal’s lifetime reproductive activity and stress response.

Conducting assay and biological validations is the first step in determining the potential of utilizing teeth as a tool for evaluating the endocrine state of marine mammals. Parallelism between the binding curves of the assay kit standards and serially diluted pools of tooth extract, accuracy slopes between 0.7 and 1.3 and r2 values of >0.95 (Grotjan and Keel, 1996) indicate that EIAs used in this study are suitable for use with tooth extracts. To fully validate the use of EIAs with tooth extracts, the chemical identities of the immunoreactive hormones in teeth should be determined using high-performance liquid chromatography, as the EIAs may be detecting hormone metabolites and/or conjugates in tooth extracts (Hunt et al., 2014).

High concentrations of progesterone observed in all tooth extracts in the assay validations may reflect its important role in mammal reproduction, since progesterone increases during specific life history events, such as pregnancy, ovulation and seasonal mating, and remains elevated for extended periods in sexually mature animals (e.g. pregnancy) (Sawyer-Steffan et al., 1983; Gardiner et al., 1996; Kinoshita et al., 2012; Ahuja-Aguirre et al., 2017). Although progesterone is often referred to as the ‘female reproductive hormone’, it also influences the biosynthesis of testosterone in Leydig cells, spermiogenesis, sperm capacitation and sexual behaviour in males (Oettel and Mukhopadhyay, 2004; Xia et al., 2018) and it is possible that the high progesterone concentrations observed here are due to the role that progesterone plays in these processes. Additionally, unidentified compounds in tooth extracts may be binding to the assay antibodies, potentially causing an overestimation of progesterone concentrations or progesterone may be preferentially incorporated into teeth. In baleen, reproductive hormones were present in higher concentrations than the adrenal and thyroid hormones (Hunt et al., 2017b) and our results followed a similar pattern with the exception of T3, which was present in higher concentrations than both testosterone and estradiol. This observation may be due to the importance of thyroid hormones in tooth development (Vucic et al., 2017).

Our inability to detect cortisol in any tooth extract is interesting, as cortisol is often considered the dominant glucocorticoid in most mammals, while corticosterone is considered the dominant glucocorticoid in birds and reptiles (Ortiz and Worthy, 2000; Möstl et al., 2005; Touma and Palme, 2005; Sheriff et al., 2011). This is the case with odontocetes, as serum of killer whale and bottlenose dolphins have cortisol:corticosterone ratios of 8:1 and 5:1, respectively (Thomson and Geraci, 1985; O’Brien et al., 2017). However, glucocorticoid ratios differ between matrices from the same species of mysticete whales. For example, in North Atlantic right whales, cortisol is the dominant glucocorticoid in serum (Rolland et al., 2017), while corticosterone is the dominant glucocorticoid in baleen (Hunt et al., 2017a). Our inability to measure cortisol in teeth from each species in this study suggests that either corticosterone is preferentially incorporated into teeth, cortisol is not deposited in teeth or the extraction process is less effective at extracting cortisol.

While tooth hormone concentrations differed among and within species, the consistently high concentrations in the narwhal embedded tooth (embedded in the upper jaw bone) may reflect hormone diffusion from the surrounding tissue that encases the tooth, or conversely, less diffusion of hormones out of the tooth. Although recent studies suggest that hormones remain relatively stable once they are deposited into tissues (Keogh et al., 2020, 2021), the relatively low hormone concentrations in the beluga and killer whale teeth and the narwhal erupted tusk could be the result of leaching into ambient water (Li et al., 2012). Following the same rationale, the intermediate hormone concentrations in the walrus tusk may reflect a balance between immersion in water and time spent hauled out on sea ice or land. However, the high hormone concentrations in the oldest GLGs (1–2) of the narwhal and (1–3 for the male and 1–4 for the female) walrus tusks are inconsistent with this explanation, as those GLGs are the oldest in the tusk and closest to the surface. Both of these factors would be expected to lead to lower hormone concentrations, due to leaching, and not the relatively high concentrations we observed. We also note that these general patterns were not observed for all hormones, and biological factors, such as age and sex, are also likely to explain observed differences among and within species.

Reproductive state is often assessed using endocrine techniques, as most reproductive activities are regulated by hormones (Burgess et al., 2012). An elevation in reproductive hormones can indicate the onset of sexual maturity and pregnancy in individuals (Beehner et al., 2009; Burgess et al., 2012; Kinoshita et al., 2012; Beaulieu-McCoy et al., 2017; Carlitz et al., 2019; Inoue et al., 2019). For example, Trumble et al. (2013) measured lifetime patterns of testosterone and cortisol deposited in the earplug of a male blue whale (Balaenoptera musculus) and found increases in testosterone at ~10 years, which corresponded with previous estimates of age of maturation. We therefore expected to see low progesterone and testosterone concentrations before the onset of sexual maturity, which was only observed in female killer whales. Instead, we observed higher concentrations of progesterone and testosterone during infancy and in immature animals. Although these results were unexpected, similar patterns have been observed in other species. For example, in walrus bone, higher steroid hormone concentrations were observed in subadults than adults (Charapata et al., 2018) and a surge of hormones during infancy were reported in baboons (Papio cynocephalus; Gesquiere et al., 2005), common marmoset monkeys (Callithrix jacchus; (Abbott et al., 1978; Pryce et al., 2002), cotton-top tamarins (Saguinus oedipus oedipus; Ginther et al., 2002) and humans (Reyes et al., 1974; Kuiri-Hänninen et al., 2014). In humans, this postnatal activation of the hypothalamic–pituitary–gonadal axis and consequential surge in hormones is often referred to as minipuberty and is thought to play a role in reproductive and neurobehavioural development (Kuiri-Hänninen et al., 2014). Although our results were contrary to predictions, the observed patterns may be capturing a biologically relevant change that has been observed in other taxa.

While the observed patterns in the narwhal and walrus tusks may indicate minipuberty, it does not explain the lack of increase in reproductive hormones at the estimated age of sexual maturity. Male narwhals and walruses reach sexual maturity between the ages of 12 and 20 (Garde et al., 2015) and 7 and 12 (Born, 2003), respectively, whereas the average age of sexual maturity for female walruses is estimated to be 6 years (Born, 2001). In the male narwhal tusk, a slight increase in testosterone was observed in GLG 15 (age 15 years), which may indicate the onset of sexual maturity; however, this increase was small and did not follow similar patterns seen in other studies where multi-fold increases were observed (Burgess et al., 2012; Trumble et al., 2013). An increase in testosterone in the male walrus tusk was observed in GLG 13, which is close to the estimated age of sexual maturity for Atlantic walruses (Born, 2003). However, we note that the walrus tusk had substantial wear on the tip of the tusk; therefore, the age estimate of 20 years is a minimum age estimate. The progesterone peak observed in the female walrus tusk occurred in GLG 30, which is markedly higher than the estimated average of 6 years old for sexual maturity (Born, 2001). This peak may indicate a pregnancy event; however, it could also be an artefact of the hormone extraction process, since GLGs at the base of a tusk (i.e. most recent growth; Fig. 1) are compressed and thinner than GLGs at the tip and less tooth powder can be collected and extracted. It is therefore possible that this peak in progesterone is a consequence of extracting a small sample (Fernández Ajó et al., 2021), despite the fact that tooth powder to solvent ratios were kept the same and all hormone concentrations were corrected for sample mass.

Continuously growing matrices, including whiskers, earplugs, claws and baleen, have been used to explore longitudinal hormone profiles in marine mammals (Trumble et al., 2013, 2018; Hunt et al., 2016, 2018; Lysiak et al., 2018; Keogh et al., 2020, 2021; Crain et al., 2021), and baleen in particular has proven to be an especially valuable tool. For example, elevated levels of progesterone measured along baleen plates correlated with known pregnancies in North Atlantic right whales (Hunt et al., 2016), while elevated corticosterone levels were observed in an entangled North Atlantic right whale (Lysiak et al., 2018). Additionally, testosterone concentrations along baleen plates of three mysticete species were found to fluctuate seasonally, indicating annual testosterone cycles consistent with seasonal mating (Hunt et al., 2018). Cyclic patterns in progesterone have also been observed in whiskers from Steller sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus), potentially indicating pregnancy or embryonic diapause (Keogh et al., 2021). Since breeding intervals are seasonal (Sjare and Stirling, 1996; Kelley et al., 2015) and gestational periods range from 11 to 14 months (Best and Fisher, 1974; COSEWIC, 2006) in narwhal and walrus, our measurements of reproductive hormones in entire, single GLGs, coupled with uncertainty in dentine deposition rates within annual GLGs (Klevezal, 1996), likely obscured our ability to capture temporal patterns. Sampling GLGs to match relevant timing and duration of reproductive events would help determine whether hormone concentrations in teeth are biologically meaningful. Further biological validations using larger datasets with known individuals (e.g. captive animals with known reproductive histories) are necessary to better understand long-term endocrine patterns in teeth and how they correlate with known life history events.

Arctic marine mammals, which are regularly hunted for subsistence, require a sustainable management framework that depends on information on basic life history characteristics, such as reproductive rates and age of sexual maturity (Eberhardt and Siniff, 1977; Taylor et al., 1987). Teeth are commonly collected during necropsies and are readily stored for extended periods. Our ability to measure hormones in a narwhal tusk from 1987 and beluga teeth from 1986 indicates the potential for comparing modern samples with those from historical museum collections to assess the impacts of changing environmental and anthropogenic conditions on reproductive parameters and stress in populations. Given the continual accretion of dentine and cementum in annual growth layers, teeth would add to the limited types of structures available for reconstructions of lifetime endocrine histories, such as ear plugs, baleen, whiskers and claws (Trumble et al., 2013; Hunt et al., 2016; Crain et al., 2021; Keogh et al., 2021). However, further methodological refinement and validations are needed before tooth hormone analysis can be used to study the long-term endocrine function of both marine and terrestrial mammals.

Funding

This work was supported by Fisheries and Oceans Canada.

Contributions

All authors have contributed significantly to study design and planning, data collection and analyses and writing the manuscript.

Compliance with Ethical Standards

The authors have no conflicting interests to declare. Ethics approval was not required for sampling of dead animals by beneficiaries.

Acknowledgments

The authors would like to thank local hunters and Hunters and Trappers Organizations in the communities of Grise Fiord, Arviat, Pangnirtung, Naujaat and Sanirajak, NU, Canada, for providing narwhal, beluga and walrus samples. We also thank the Sanikiluaq Hunters and Trappers Association in Sanikiluaq, NU, for organizing killer whale sample collection by J. Kavik, L. Takatak and N. Arragutainaq. Thanks to the Royal British Columbia Museum and W. Ledwell (Whale Release and Strandings Group) for providing killer whale teeth. We thank C. MacNichol and the Inuit Co-op in Sanirajak, NU, for setting aside tusks for DFO’s walrus research program. We would also like to thank S.-T. Zhao (University of Manitoba), for assistance in the laboratory; Shape Industries, Winnipeg, Canada, for sectioning tusks; and two anonymous reviewers whose comments improved the final version of this manuscript. Narwhal and walrus tusks were collected under Fisheries and Oceans Canada permits and tags (Marine Mammal Transportation Licence: C08923, C08209; Tag: NHB-2018-055A, EB-2018-009-A).

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