DHEA and estradiol levels in brain, gonads, adrenal glands, and plasma of developing male and female European starlings.

Traditionally, sexual differentiation of the brain was thought to be driven by gonadal hormones, particularly testosterone (T). However, recent studies in songbirds suggest that other steroids may also be important. For example, dehydroepiandrosterone (DHEA) can be synthesized by the gonads, adrenal glands, and/or brain and locally metabolized into T and 17ß-estradiol (E(2)). Here, we examined DHEA and E(2) levels in the brain, peripheral tissues, and plasma of wild European starlings (Sturnus vulgaris). In Study 1, samples were collected from males and females at P0 (day of hatch), P6, and P8. In Study 2, samples were collected at P4. At P0, DHEA levels in the diencephalon were higher in males than females. DHEA levels were generally high in the gonads and adrenals, and they were higher in testes than ovaries at P8. Further, E(2) levels were non-detectable in most brain samples, suggesting that DHEA was not metabolized to E(2) or that locally produced E(2) was rapidly inactivated. At P4, DHEA levels in telencephalic regions were lower in males than females. Taken together, these data suggest that sex differences in peripheral DHEA secretion and neural DHEA metabolism at specific ages during development might play a role in sexual differentiation of the songbird brain.

17beta-Estradiol levels in male zebra finch brain: combining Palkovits punch and an ultrasensitive radioimmunoassay.

Local aromatization of testosterone into 17beta-estradiol (E(2)) 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 E(2) 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. E(2) was then measured with an ultrasensitive, specific and precise radioimmunoassay. Our protocol leads to high recovery of E(2) (84%) and effectively removes interfering brain lipids. E(2) levels were high in aromatase-rich regions such as caudal medial nidopallium and hippocampus. E(2) 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. E(2) levels were largely non-detectable in the cerebellum, HVC, lateral nidopallium and optic lobes. Importantly, E(2) levels were significantly lower in plasma than in the caudal medial nidopallium. This protocol allows one to measure E(2) in discrete brain regions and potentially relate local E(2) concentrations to aromatase activity and behavior.

Cortisol and corticosterone in immune organs and brain of European starlings: developmental changes, effects of restraint stress, comparison with zebra finches.

Glucocorticoids (GCs) are produced in the adrenal glands and also in extra-adrenal sites, including immune organs and brain. Here, we examined regulation of systemic GC levels in plasma and local GC levels in immune organs and brain during development. We conducted two studies and examined a total of 462 samples from 70 subjects. In study 1, we determined corticosterone and cortisol levels in the plasma, immune organs, and brain of wild European starlings on posthatch day 0 (P0) and P10 (at baseline and after 45 min of restraint). Baseline corticosterone and cortisol levels were low in the immune organs and brain at P0 and P10, providing little evidence for local GC synthesis in starlings. At P0, restraint had no significant effects on corticosterone or cortisol levels in the plasma or tissues; however, there was a trend for restraint to increase both corticosterone and cortisol in the immune organs. At P10, restraint increased corticosterone levels in the plasma and all tissues, but restraint increased cortisol levels in the plasma, thymus, and diencephalon only. In study 2, we directly compared GC levels in European starlings and zebra finches at P4. In zebra finches but not starlings, cortisol levels were higher in the immune organs than in plasma. This difference in immune GC levels might be due to evolutionary lineage, life history strategy, or experiential factors, such as parasite exposure. This is the first study to measure immune GC levels in wild animals and one of the first studies to measure local GC levels after restraint stress.

Sexual differentiation of the spinal nucleus of the bulbocavernosus is not mediated solely by androgen receptors in muscle fibers.

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system is a highly conserved and well-studied model of sexual differentiation of the vertebrate nervous system. Sexual differentiation of the SNB is currently thought to be mediated by the direct action of perinatal testosterone on androgen receptors (ARs) in the bulbocavernosus/levator ani muscles, with concomitant motoneuron rescue. This model has been proposed based on surgical and pharmacological manipulations of developing rats as well as from evidence that male rats with the testicular feminization mutation (Tfm), which is a loss of function AR mutation, have a feminine SNB phenotype. We examined whether genetically replacing AR in muscle fibers is sufficient to rescue the SNB phenotype of Tfm rats. Transgenic rats in which wild-type (WT) human AR is driven by a human skeletal actin promoter (HSA-AR) were crossed with Tfm rats. Resulting male HSA-AR/Tfm rats express WT AR exclusively in muscle and nonfunctional Tfm AR in other tissues. We then examined motoneuron and muscle morphology of the SNB neuromuscular system of WT and Tfm rats with and without the HSA-AR transgene. We observed feminine levator ani muscle size and SNB motoneuron number and size in Tfm males with or without the HSA-AR transgene. These results indicate that AR expression in skeletal muscle fibers is not sufficient to rescue the male phenotype of the SNB neuromuscular system and further suggest that AR in other cell types plays a critical role in sexual differentiation of this system.

Neurosteroids, immunosteroids, and the Balkanization of endocrinology.

Traditionally, the production and regulation of steroid hormones has been viewed as a multi-organ process involving the hypothalamic-pituitary-gonadal (HPG) axis for sex steroids and the hypothalamic-pituitary-adrenal (HPA) axis for glucocorticoids. However, active steroids can also be synthesized locally in target tissues, either from circulating inactive precursors or de novo from cholesterol. Here, we review recent work demonstrating local steroid synthesis, with an emphasis on steroids synthesized in the brain (neurosteroids) and steroids synthesized in the immune system (immunosteroids). Furthermore, recent evidence suggests that other components of the HPG axis (luteinizing hormone and gonadotropin-releasing hormone) and HPA axis (adrenocorticotropic hormone and corticotropin-releasing hormone) are expressed locally in target tissues, potentially providing a mechanism for local regulation of neurosteroid and immunosteroid synthesis. The balance between systemic and local steroid signals depends critically on life history stage, species adaptations, and the costs of systemic signals. During particular life history stages, there can be a shift from systemic to local steroid signals. We propose that the shift to local synthesis and regulation of steroids within target tissues represents a "Balkanization" of the endocrine system, whereby individual tissues and organs may become capable of autonomously synthesizing and modulating local steroid signals, perhaps independently of the HPG and HPA axes.

Sex differences in DHEA and estradiol during development in a wild songbird: Jugular versus brachial plasma.

Sexual differentiation of the brain has traditionally been thought to be driven by gonadal hormones, particularly testosterone (T). Recent studies in songbirds and other species have indicated that non-gonadal sex steroids may also be important. For example, dehydroepiandrosterone (DHEA)--a sex steroid precursor that can be synthesized in the adrenal glands and/or brain--can be converted into active sex steroids, such as 17beta-estradiol (E(2)), within the brain. Here, we examine plasma DHEA and E(2) levels in wild developing European starlings (Sturnus vulgaris), from hatch (P0) to fledging (P20). Blood samples were collected from either the brachial vein (n=143) or the jugular vein (n=129). In songbirds, jugular plasma is enriched with neurally-synthesized steroids and, therefore, jugular plasma is an indirect measure of the neural steroidal milieu. Interestingly, brachial DHEA levels were higher in males than females at P4. In contrast, jugular DHEA levels were higher in females than males at P0 and P10. Brachial E(2) levels were higher in males than females at P6. Surprisingly, jugular E(2) levels were not high and showed no sex differences. Also, we calculated the difference between brachial and jugular steroid levels. At several ages, jugular steroid levels were lower than brachial levels, particularly in males, suggesting greater neural metabolism of circulating DHEA and E(2) in males than females. At a few ages, jugular steroid levels were higher than brachial levels, suggesting neural secretion of DHEA or E(2) into the general circulation. Taken together, these data suggest that DHEA may play a role in brain sexual differentiation in songbirds.