Assay temperature affects corticosteroid-binding globulin and free corticosterone estimates across species.

Glucocorticoid hormones are often measured to assess how organisms physiologically respond to challenges in their environment. In plasma, glucocorticoids circulate in two forms: bound to corticosteroid-binding globulins (CBG) or unbound (free). Measuring CBG allows us to estimate the amount of free glucocorticoids present in a plasma sample. However, free glucocorticoid estimates are affected by the assay temperature used when measuring CBG, with colder temperatures maximizing specific binding but likely underestimating glucocorticoid's affinity for CBG. Here, we test how a biologically relevant incubation temperature (41 °C) changes the disassociation constant (Kd; used to estimate free glucocorticoid levels) when compared to the traditional 4 °C incubation temperature, across four commonly studied avian species. We then apply the new Kd's calculated at 41 °C to existing data sets to examine how the change in Kd affects free corticosterone estimates and data interpretation. Kd's were generally higher (lower affinity for CORT) at warmer incubation temperatures which resulted in higher levels of estimated free CORT in all four species but differed among subspecies. This increase in free CORT levels did not qualitatively change previously reported statistical relationships, but did affect variance and alpha (P) values. We suggest that future assays be run at biologically relevant temperatures for more accurate estimates of free CORT levels in vivo and to increase the chances of detecting biological patterns of free-CORT that may not be revealed with the classic methodology that tends to underestimate free CORT levels.

Periodic Cooling during Incubation Alters the Adrenocortical Response and Posthatch Growth in Zebra Finches.

AbstractIn birds, incubation temperature is critically deterministic for a range of traits. When parents leave the nest to forage, developing embryos can be exposed to cooling events that represent thermal stress. To investigate the consequences of periodic cooling on offspring development and physiology, we exposed zebra finch embryos to cooling events throughout the incubation period. We then compared embryonic survival, egg mass change, incubation duration, posthatch growth, and adrenocortical response of these individuals with embryos reared at a constant optimal temperature of 37.4°C and embryos reared at a constant suboptimal temperature of 36.4°C, the mean incubation temperature of periodically cooled embryos. There were no differences in embryonic survival or egg mass change during incubation, but individuals exposed to periodic cooling had longer incubation periods than those from the 37.4°C treatment and shorter incubation periods than those from the 36.4°C treatment. Periodically cooled individuals showed slower posthatch growth in comparison with both constant-temperature treatments, but this did not impact adult body size. Treatment groups did not differ in their adrenocortical response, but embryos exposed to periodic cooling and a constant temperature of 37.4°C were able to habituate to repeated capture and restraint stress, while individuals exposed to the constant temperature of 36.4°C were not. These results point to the differential impacts of cooling events versus constant low temperatures during incubation on posthatch growth and physiology and may represent a way for parents to devote less energy toward incubation while still ensuring offspring success.

Corticosteroid-binding globulins: Lessons from biomedical research.

Glucocorticoids (GCs) circulate in the plasma bound to corticosteroid-binding globulin (CBG). Plasma CBG may limit access of glucocorticoids to tissues (acting as a sponge: the free hormone hypothesis), or may solely serve as a transport molecule, releasing GCs to tissues as the plasma moves through capillaries (the total hormone hypothesis). Both biomedical (focused on human health) and comparative (focused on ecological and evolutionary relevance) studies have worked to incorporate CBG in glucocorticoid physiology, and to understand whether free or total hormone is the biologically active plasma fraction. The biomedical field, however, has been well ahead of the comparative physiologists, and have produced results that can inform comparative research when considering the import of total vs. free plasma hormone. In fact, biomedical studies have made impressive strides regarding the function of CBG in tissues as well as plasma; we, however, focus solely on the plasma functions in this review as this is the primary area of disagreement amongst comparative physiologists. Here we present 5 sets of biomedical studies across genomics, pharmacology, cell culture, whole animal research, and human medicine that strongly support a role for CBG limiting hormone access to tissue. We also discuss three areas of concern across comparative researchers. In contrast to former publications, we are not suggesting that all comparative studies in glucocorticoid physiology must measure CBG, or that only free corticosterone levels are valid. However, we propose that comparative physiologists be aware of biomedical results as they investigate glucocorticoids and interpret how total hormone may or may not impact behavior and physiology of free-living vertebrates.