Temporal organization of gonadal and adrenal steroid fluctuations in male mice

Steroid hormones exhibit temporal fluctuations that influence physiology, yet these dynamics remain incompletely characterized in male mice, a cornerstone model in endocrine research. Using serial arterial sampling and high-sensitivity liquid chromatography-tandem mass spectrometry (LC-MS/MS), we performed the first longitudinal, multi-analyte quantification of steroid hormones (testosterone, progesterone, corticosterone, and deoxycorticosterone (DOC)), within the same mice across consecutive days and diurnal periods. Adult male C57BL/6J mice (n = 13) were sampled daily for 5 days and again at 7 AM and 5:30 PM on non-consecutive days (days 1, 3, and 5). Corticosterone and its adrenal precursor - DOC - exhibited coordinated diurnal patterns, with corticosterone showing consistent AM-PM increases across all sampling days and DOC reaching significance only on day 5. These findings confirm a circadian pattern of adrenal steroidogenesis dominated by corticosterone, with its faster-turnover intermediate displaying less stable rhythmicity. In contrast, testosterone fluctuated irregularly and without a consistent AM-PM pattern, varying up to 100-fold within individual mice. Progesterone showed minimal day-to-day or diurnal variation. Correlation analyses revealed tight coupling among corticosterone and DOC (r = 0.99, P < 0.001), but no association between adrenal steroids and testosterone, indicating independent regulation of adrenal and gonadal axes. These results define the natural temporal organization of steroid hormones in male mice, distinguishing coordinated adrenal rhythmicity from pulsatile gonadal variability, and provide a physiologic framework for experimental design and data interpretation in murine endocrine research.