17β-Estradiol levels in male zebra finch brain: Combining Palkovits punch and an ultrasensitive radioimmunoassay

Abstract

Local aromatization of testosterone into 17β-estradiol (E2) is often required for the physiological and behavioral actions of testosterone. In most vertebrates, aromatase is expressed in a few discrete brain regions. While many studies have measured brain aromatase mRNA or activity, very few studies have measured brain E2 levels, particularly in discrete brain regions, because of technical challenges. Here, we used the Palkovits punch technique to isolate 13 discrete brain nuclei from adult male zebra finches. Steroids were extracted via solid phase extraction. E2 was then measured with an ultrasensitive, specific and precise radioimmunoassay. Our protocol leads to high recovery of E2 (84%) and effectively removes interfering brain lipids. E2 levels were high in aromatase-rich regions such as caudal medial nidopallium and hippocampus. E2 levels were intermediate in the medial preoptic area, ventromedial nucleus of the hypothalamus, lateral and medial magnocellular nuclei of anterior nidopallium, nucleus taeniae of the amygdala, and Area X. E2 levels were largely non-detectable in the cerebellum, HVC, lateral nidopallium and optic lobes. Importantly, E2 levels were significantly lower in plasma than in the caudal medial nidopallium. This protocol allows one to measure E2 in discrete brain regions and potentially relate local E2 concentrations to aromatase activity and behavior.

Rapid Effects of an Aggressive Interaction on Dehydroepiandrosterone, Testosterone and Oestradiol Levels in the Male Song Sparrow Brain: a Seasonal Comparison

“…E 2 was measured using an assay kit (#DSL-4800) from Beckman Coulter (inter-assay variation = 6.5%; intra-assay variation = 6.0%). Modifications to the instructions for the E 2 kit have been described elsewhere (27). Briefly, 100 ll of diluted primary antibody was added to 300 ll (150 ll of resuspended sample + 150 ll of PBSG) of standards and diluted samples and incubated for 4 h at room temperature.…”

Avian ecologists and physiologists have different sexual preferences

“…While there are commercial kits available for testosterone, nothing is available for measuring estradiol in birds. Ironically, an extremely sensitive method for assaying 17-estradiol has recently been described, but this was developed to measure estrogens in the male brain [25], which are critical for the activation of male reproductive behaviors through the local aromatization of testosterone [9]. Another complication related to female physiology is that the rapid maturation of the ovarian follicles, mainly occurring during egg-yolk deposition, is sudden and transient and can easily be missed during sampling.…”

Genome–brain–behavior interdependencies as a framework to understand hormone effects on learned behavior

“…All of the genes required for de novo steroidogenesis are expressed in the zebra finch brain from as early as the first day of posthatch life (Jacobs et al, 1999;Kim et al, 2004;London et al, 2003London et al, , 2006London and Clayton, 2010;London and Schlinger, 2007;Shen et al, 1994Shen et al, , 1995. Biochemical enzyme assays demonstrated that all enzymes are present and active in the zebra finch brain and micropunches confirm generally that regional differences in steroid concentrations can be established (Cam and Schlinger, 1998;Charlier et al, 2010;Freking et al, 1998;Saldanha et al, 2000;Schlinger et al, 1994Schlinger et al, , 1995Schlinger and Arnold, 1991;Schlinger et al, 1999;Tam and Schlinger, 2007;Vanson et al, 1996;Wade et al, 1995). Further, the distribution of steroidogenic enzymes changes across the lifespan.…”