Estrogen profiling in blood and brain: effects of season and an aggressive interaction in a songbird

Abstract

Neuroestrogens are synthesized in the brain and regulate social behavior and cognition. In the song sparrow (Melospiza melodia), 17{beta}-estradiol (17{beta}-E2) promotes aggression, even during the non-breeding season, when circulating 17{beta}-E2 levels are low. Measuring estrogens is challenging due to their low concentrations and the limited sensitivity of many existing assays. Moreover, estrogens other than 17{beta}-E2 are often overlooked. Here, we developed a method for simultaneous measurement of eleven estrogens that is highly specific, sensitive, accurate, and precise. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) with derivatization of estrogens by 1,2-dimethylimidazole-5-sulfonyl-chloride (DMIS) to enhance sensitivity. We included four 13C-labeled internal standards, which corrected for matrix effects when measuring catecholestrogens and methoxyestrogens. This method greatly improves upon our prior protocol, which could measure only four estrogens and included only one deuterated internal standard. Then, the new method was applied to samples from male song sparrows exposed to either a simulated territorial intrusion (STI) or a control condition during the breeding season or non-breeding season. Only estrone and 17{beta}-E2 were present in blood and brain, while the other nine estrogens in the panel were non-detectable. As expected, there was large regional variation in neuroestrogen levels and very low estrogen levels in blood. There was large seasonal variation, and estrogen levels were lower in the non-breeding season. Despite robust aggressive responses to the STI, estrogen levels did not differ between STI and control subjects in either season. In sum, our novel method enables ultrasensitive measurement of eleven estrogens and will be useful for studies of songbirds and other animals.