A novel method to measure steroid hormone concentrations in walrus bone from archaeological, historical, and modern time periods using liquid chromatography/tandem mass spectrometry

Abstract

RationaleA liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was validated and utilized to measure and analyze four steroid hormones related to stress and reproduction in individual samples from a novel tissue, Pacific walrus (Odobenus rosmarus divergens, herein walrus) bone. This method determines steroid hormone concentrations in the remote walrus population over millennia from archaeological (>200 bp), historical (200–20 bp), and modern (2014–2016) time periods.MethodsLipids were extracted from walrus bone collected from these periods using methanol before LC/MS/MS analysis. Isotopically labeled internal standards for each target hormone were added to every sample. Analytical and physiological validations were performed. Additionally, a tissue comparison was done among paired walrus bone, serum, and blubber samples. A rapid resolution liquid chromatography system coupled to a QqQ mass spectrometer was used to analyze all samples after derivatization for progesterone, testosterone, cortisol, and estradiol concentrations. Multiple reaction monitoring was used for MS analysis and data were acquired in positive electrospray ionization mode.ResultsProgesterone, testosterone, cortisol, and estradiol were linear along their respective standard calibration curves based on their R2 values (all > 0.99). Accuracy ranged from 93–111% for all hormones. The recovery of extraction, recovery of hormones without matrix effect, was 92–101%. The overall process efficiency of our method for measuring hormones in walrus bone was 93–112%. Progesterone and testosterone concentrations were not affected by reproductive status among adult females and males, respectively. However, estradiol was different among pregnant and non‐pregnant adult females. Overall, steroid hormones reflect a long‐term reservoir in cortical bone. This method was also successfully applied to walrus bone as old as 3585 bp.ConclusionsLC/MS/MS analysis of bone tissue (0.2–0.3 g) provides stress and reproductive data from elusive walruses that were alive thousands of years ago. Based on physiological validations, tissue comparison, and published literature, steroid hormone concentrations measured in walrus cortical bone could represent an accumulated average around a 10–20‐year time span. By investigating how stress and reproductive physiology may have changed over the past ~3000 years based on bone steroid hormone concentrations, this method will help answer how physiologically resilient walruses are to climate change in the Arctic.

Endocrine profiling of reproductive status and evidence of pseudopregnancy in the Pacific walrus (Odobenus rosmarus divergens)

“…Interestingly, unbred females yielded the highest progesterone levels, even compared with the pregnant females. The unbred female used in Charapata et al [18] (G150005) yielded among the greatest progesterone concentrations for all females in their sample; in her blubber and serum, progesterone levels were 110.87 ng/g and 18.04 ng/ml respectively. Recognizing that LC/MS/MS and EIAs are not necessarily comparable methods for measuring progesterone concentrations, these levels in the blubber and serum are still much lower than the concentration measured in her CL (1533.01 ng/g).…”

“…The mean concentration for total estrogens of the pregnant females was nominally much smaller than that of unbred females. Female G150005 has been used in another study that analyzed progesterone and estradiol in bones, blubber and serum using liquid chromatography/tandem mass spectrometry (LC/MS/MS) [18]. The estradiol concentrations in the female from that study were low; 0.97 ng/g and 1.34 ng/ml in her blubber and serum respectively.…”

“…This "false pregnancy" has been termed pseudopregnancy and occurs primarily in copulation-induced ovulators [33]. Few published studies have profiled reproductive hormones in the tissues of free-ranging walruses [18]. The Pacific walrus has a unique reproductive cycle among pinnipeds, most of which have an annual breeding cycle which includes a postpartum estrus in spring or summer followed by an embryonic diapause [30].…”

“…Fewer studies exist for pinnipeds and many of them have been carried out on animals in human care, which may not follow the life history timing or seasonal reproductive patterns witnessed in conspecifics in their native environments [ 14 – 17 ]. Studies on progesterone and estrogen concentrations in pinnipeds have shown that these endocrine markers are able to detect pregnancy and sometimes estrus in Pacific walruses ( Odobenus rosmarus divergens ) [ 18 ], Hawaiian monk seals ( Monachus schauinslandi ) [ 19 ], harbor seals ( Phoca vitulina) [ 20 ], and Steller ( Eumetopias jubatus ) [ 21 ], California ( Zalophus californianus ) [ 22 ], and Galapagos sea lions ( Zalophus wollebaeki ) [ 23 ]. In addition to reproductive hormones, lipids play an important role in reproduction.…”

Determining sex of adult Pacific walruses from mandible measurements

“…Female and male walruses have different physiological needs and display strong sexual dimorphism ( Fay 1982 ; Noren et al 2012 , 2014 , 2015 ). Determining sex from mandibles also may allow past research on Pacific walruses to be revisited to increase sample size and improve the interpretations of analyses (e.g., stable isotope, trace element, and hormone analyses) carried out on bones of unknown-sex walruses (e.g., Charapata et al 2018 ; Clark et al 2019 ).…”