Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry

Abstract

Accurate measurement of sex steroid concentrations in rodent serum is essential to evaluate mouse and rat models for sex steroid-related disorders. The aim of the present study was to develop a sensitive and specific gas chromatography-tandem mass spectrometry (GC-MS/MS) method to assess a comprehensive sex steroid profile in rodent serum. A major effort was invested in reaching an exceptionally high sensitivity for measuring serum estradiol concentrations. We established a GC-MS/MS assay with a lower limit of detection for estradiol, estrone, T, DHT, progesterone, androstenedione, and dehydroepiandrosterone of 0.3, 0.5, 4.0, 1.6, 8, 4.0, and 50 pg/mL, respectively, whereas the corresponding values for the lower limit of quantification were 0.5, 0.5, 8, 2.5, 74, 12, and 400 pg/mL, respectively. Calibration curves were linear, intra- and interassay coefficients of variation were low, and accuracy was excellent for all analytes. The established assay was used to accurately measure a comprehensive sex steroid profile in female rats and mice according to estrous cycle phase. In addition, we characterized the impact of age, sex, gonadectomy, and estradiol treatment on serum concentrations of these sex hormones in mice. In conclusion, we have established a highly sensitive and specific GC-MS/MS method to assess a comprehensive sex steroid profile in rodent serum in a single run. This GC-MS/MS assay has, to the best of our knowledge, the best detectability reported for estradiol. Our method therefore represents an ideal tool to characterize sex steroid metabolism in a variety of sex steroid-related rodent models and in human samples with low estradiol levels.

Elevated Serum Levels of Estradiol Induce Endometrial Hyperplasia Rather than Carcinoma in a Mouse Model Using a Carcinogen

“…In the present study, the serum E2 levels were classified as follows: high E2 level: >10 pg/ml, intermediate E2 level: 1.0~10 pg/ml, low E2 level: <1.0 pg/ml. The mean serum E2 level in all estrous stages was reportedly 2.7 ± 1.0 pg/ml (mean±SEM) with the highest level being observed in the proestrus stage (approximately 8.0 ± 3.0 pg/ml) [12]. Therefore, moderate E2 levels roughly corresponded to that of the normal E2 range in mice.…”

“…Another important factor is the concentration of E2. Physiological serum levels of E2 in mice reportedly range between from 1 and 10 pg/ml [12]. However, none of the above-described animal studies examined the concentration of E2 in each mouse used.…”

Old age causes de novo intracortical bone remodeling and porosity in mice

“…18), the marrow begins to expand, and additional bone is slowly added to the periosteum; however, the former exceeds the latter, leading to a thinner and more fragile cortex (19)(20)(21)(22). Aging female mice do not experience menopause but become acyclic while retaining functional levels of estrogens (17,23), and androgen levels in aged male mice are maintained at a 20-fold higher level than in females (24). However, both female and male mice exhibit all of the major features of skeletal aging, including the decline of cortical bone mass and the development of cortical porosity by 18 months of age, independently of sex steroid deficiency and by mechanisms that are distinct from those of sex steroid deficiency (23).…”

Sex and regional differences in microglia morphology and complement receptor 3 are independent of constitutive neuroinflammatory protein concentrations in healthy mice

“…The female estrus cycle has four phases that occur in a particular order (estrus, metaestrus, diestrus, and proestrus). The relative serum sex hormone concentrations (Nilsson et al, 2015) and sex hormone receptors fluctuate according to cycle phase (Mitterling et al, 2010) and vary between brain locations (Kato et al, 2013;Saito et al, 2016). The cessation of ovarian function, cyclic release of sex hormones, and termination of menses are among the elements that define the post-menopause phase of life for women.…”