Comprehensive adrenal steroid profiling during frog metamorphosis

Measurement of adrenal hormones in amphibians is important in studies on stress, development, osmoregulation, endocrine disruption, and conservation to help reveal mechanisms within amphibians and evolution among vertebrates. Corticosteroids measured in frogs are typically corticosterone and/or aldosterone, but steroid intermediates that may activate hormone receptors are thus far not quantified. Also, steroidogenesis in frogs has been examined in vitro but little work has been done using tadpoles with mutations affecting steroidogenesis. To advance such studies, we developed a comprehensive liquid chromatography - tandem mass spectrometry (LC-MS/MS) method to quantify 13 corticosteroid hormones and intermediates and 5 sex steroids in plasma and tail during natural metamorphosis and in pomc and cyp21a2 mutant tadpoles. Four of these steroids were consistently quantified in plasma and tail during development of wild-type tadpoles, namely 11-deoxycorticosterone (11-DOC), corticosterone (CORT), aldosterone (ALDO), and 11-dehydrocorticosterone (11-dehydroCORT). During metamorphosis, each of these steroids increased 4- to 5-fold in plasma, whereas in tail they were high during premetamorphosis and climax but lower in prometamorphosis. pomc mutant tails had only 2-fold less CORT, but cyp21a2 mutants also had an accumulation of progesterone and 11β-hydroxyprogesterone. This study revealed the in-vivo presence of 11-DOC, 11-dehydroCORT, and adrenal steroidogenic capacity in tadpoles and provided a comprehensive LC-MS/MS method for quantifying steroids relevant for a wide variety of studies.