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Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

ABSTRACT: Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the applied EIA to measure TMs and assessed diurnal fluctuations in TM excretions in male and female mice and across strains. We found that males excreted significantly more radiolabeled TMs via the feces (59%) compared to females (49.5%). TM excretion patterns differed significantly between urinary and fecal samples and were affected by the daytime of ³H-testosterone injection. Overall, TM excretion occurred faster in urinary than fecal samples. Peak excretion of fecal TMs occurred after 8 h when animals received the 3H-testosterone in the morning, or after 4 h when they received the 3H-testosterone injection in the evening. Daytime had no effect on the formed TMs; however, males and females formed different types of TMs. As expected, males showed higher fecal TM levels than females. Males also showed diurnal fluctuations in their TM levels but we found no differences in the TM levels of C57BL/6J and B6D2F1 hybrid males. Finally, we successfully validated our applied EIA (measuring 17?-hydroxyandrostane) by showing that hCG (human chorionic gonadotropin) administration increased TM levels, whereas castration reduced them. In conclusion, our EIA proved suitable for measuring fecal TMs in mice. Our non-invasive method to assess fecal TMs can be widely used in various research disciplines like animal behavior, reproduction, animal welfare, ecology, conservation, and biomedicine.

SUBMITTER: Auer KE

PROVIDER: S-EPMC7023058 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

Auer Kerstin E KE Kußmaul Marius M Möstl Erich E Hohlbaum Katharina K Rülicke Thomas T Palme Rupert R

Animals : an open access journal from MDPI 20200118 1

Testosterone is the main reproductive hormone in male vertebrates and conventional methods to measure testosterone rely on invasive blood sampling procedures. Here, we aimed to establish a non-invasive alternative by assessing testosterone metabolites (TMs) in fecal and urinary samples in mice. We performed a radiometabolism study to determine the effects of daytime and sex on the metabolism and excretion pattern of radiolabeled TMs. We performed physiological and biological validations of the a ...[more]

PMID: 31963733

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Project description:ObjectiveLow testosterone in men (hypogonadism) is associated with obesity and type II diabetes. Testosterone replacement therapy has been shown to reverse these effects. However, the mechanisms by which testosterone regulates total fat mass, fat distribution, and metabolic health are unclear. In this study, we clarify the impact of hypogonadism on these parameters, as well as parse the role of testosterone from its downstream metabolites, dihydrotestosterone (DHT), and estradiol, in the regulation of depot-specific adipose tissue mass.MethodsTo achieve this objective, we utilized mouse models of male hypogonadism coupled with hormone replacement therapy, magnetic resonance imaging (MRI), glucose tolerance tests, flow cytometry, and immunohistochemical techniques.ResultsWe observed that castrated mice develop increased fat mass, reduced muscle mass, and impaired glucose metabolism compared with gonadally intact males. Interestingly, obesity is further accelerated in castrated mice fed a high-fat diet, suggesting hypogonadism increases susceptibility to obesogenesis when dietary consumption of fat is elevated. By performing hormone replacement therapy in castrated mice, we show that testosterone impedes visceral and subcutaneous fat mass expansion. Testosterone-derived estradiol selectively blocks visceral fat growth, and DHT selectively blocks the growth of subcutaneous fat. These effects are mediated by depot-specific alterations in adipocyte size. We also show that high-fat diet-induced adipogenesis is elevated in castrated mice and that this can be rescued by androgen treatment. Obesogenic adipogenesis is also elevated in mice where androgen receptor activity is inhibited.ConclusionsThese data indicate that hypogonadism impairs glucose metabolism and increases obesogenic fat mass expansion through adipocyte hypertrophy and adipogenesis. In addition, our findings highlight distinct roles for testosterone, DHT, and estradiol in the regulation of total fat mass and fat distribution and reveal that androgen signaling blocks obesogenic adipogenesis in vivo.

Project description:BACKGROUND: High systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation. METHODS: Fifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, ?-glucuronidase and ?-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, ?-glucuronidase and ?-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with ?=0.05. RESULTS: In men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R?0.50, P?0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57-0.70, P=0.03-0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal ?-glucuronidase (R=0.36, P=0.04). In contrast, fecal ?-glucuronidase correlated inversely with fecal total estrogens, both conjugated and deconjugated (R?-0.47, P?0.01). Premenopausal female estrogen levels, which were collected across menstrual cycles and thus highly variable, were completely unrelated to fecal microbiome and enzyme parameters (P?0.6). CONCLUSIONS: Intestinal microbial richness and functions, including but not limited to ?-glucuronidase, influence levels of non-ovarian estrogens via enterohepatic circulation. Thus, the gut microbial community likely affects the risk for estrogen-related conditions in older adults. Understanding how Clostridia taxa relate to systemic estrogens may identify targets for interventions.

Dataset Information

Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

Publications

Measurement of Fecal Testosterone Metabolites in Mice: Replacement of Invasive Techniques.

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