Daniel J. Bernard

Androgen Receptor, Estrogen Receptor α, and Estrogen Receptorβ Show Distinct Patterns of Expression in Forebrain Song Control Nuclei of European Starlings1

In songbirds, singing behavior is controlled by a discrete network of interconnected brain nuclei known collectively as the song control system. Both the development of this system and the expression of singing behavior in adulthood are strongly influenced by sex steroid hormones. Although both androgenic and estrogenic steroids have effects, androgen receptors (AR) are more abundantly and widely expressed in song nuclei than are estrogen receptors (ERα). The recent cloning of a second form of the estrogen receptor in mammals, ERβ, raises the possibility that a second receptor subtype is present in songbirds and that estrogenic effects in the song system may be mediated via ERβ. We therefore cloned the ERβ complementary DNA (cDNA) from a European starling preoptic area-hypothalamic cDNA library and used in situ hybridization histochemistry to examine its expression in forebrain song nuclei, relative to the expression of AR and ERα messenger RNA (mRNA), in the adjacent brain sections. The starling ERβ cDNA...

Properties of inhibin binding to betaglycan, InhBP/p120 and the activin type II receptors.

Activin-stimulated FSH synthesis and release by the pituitary gonadotrope is antagonized by gonadally derived inhibins. The two isoforms of inhibin, inhibin A and B, bind to the activin type II receptors, though at a lower affinity than the activins, but do not stimulate intracellular signaling. Theoretically, therefore, inhibins can prevent activin signaling through competitive binding if present at higher concentrations than the activins. In reality, the inhibins have been shown to antagonize activin signaling when the two ligand types are present at equimolar concentrations. These observations led to the hypothesis that inhibin binding proteins or co-receptors exist that either increase the affinity of the inhibins for the activin receptors or propagate inhibin-specific intracellular signals. Two candidate inhibin co-receptors, betaglycan and InhBP/p120, interact with activin receptors and augment inhibin antagonism of activin action. Here, we report the effect of betaglycan and InhBP/p120 on both inhibin A and inhibin B binding to the activin receptors ActRIIA and ActRIIB2. InhBP/p120 did not bind inhibin A or B when expressed alone or in combination with activin receptors, requiring a re-examination of the role of this protein in inhibin biology. Both inhibins bound the activin type II receptor, ActRIIB2. Inhibin B had a higher affinity for this receptor than inhibin A but an approximately 10-fold lower affinity than that of activin A. Inhibin A and B bound betaglycan with high affinity; however, only inhibin A binding to ActRIIB2 was significantly enhanced in the presence of betaglycan. Both inhibin isoforms showed slight but significant binding to ActRIIA, yet this binding was potentiated in the presence of betaglycan. Additionally, the complex formed between the inhibins, ActRIIA, and betaglycan was resistant to disruption by activin A, whereas activin A potently competed for inhibin binding to ActRIIB2 and betaglycan. Collectively, these data show that the inhibin isoforms have different affinities for the activin type II receptors but bind betaglycan with high affinity. A recently developed model of inhibin action proposes that inhibins form a high affinity, activin-resistant ternary complex with activin type II receptors and betaglycan, thereby providing a mechanism for inhibin antagonism of activin signaling. Importantly, the results presented here clearly show that this model does not apply equally to both forms of inhibin nor to the different activin type II receptor isoforms. Thus, it appears that the mechanisms of inhibin action may vary depending on the ligand and receptor types involved.

Gonadal steroid receptor mRNA in catecholaminergic nuclei of the canary brainstem

Steroid actions in the song system may be modulated by ascending inputs from catecholaminergic (CA) brain nuclei; however, whether these nuclei contain steroid receptors is unknown. Here, we compared the distribution of androgen receptor (AR) and estrogen receptor-alpha (ER-alpha) mRNA with that of tyrosine hydroxylase immunoreactivity (TH-IR) in the brainstems of male canaries. Areas containing AR and ER-alpha mRNA overlapped with areas containing TH-IR cell bodies in the locus ceruleus and the area ventralis of Tsai. The substantia nigra and the midbrain central gray contained both TH-IR and AR mRNA. The presence of AR and ER-alpha within CA cell groups suggests that sex steroid hormones may modulate song production at the site of CA synthesis.

Age- and behavior-related variation in the volume of song control nuclei in male European starlings

There is considerable interindividual variation in the volumes of song control nuclei. Sex and physiological condition appear to contribute to these differences; however, these factors alone do not account for all of the variation. Studies have attempted to relate differences in song behavior (i.e., song repertoire size) to variation in song nucleus volume, but have met with mixed success. In this article, two studies are presented that used male European starlings (Sturnus vulgaris) to explore the relationship between song nuclei volumes and age-related differences in song behavior and interindividual variation in song behavior in adults. The results of the first study showed that song repertoire size and song bout length were significantly greater in older adult than in yearling males. In addition, the volumes of the high vocal center (HVC) and nucleus robustus archistriatalis (RA) were significantly larger in older adults than yearlings. Area X of the parolfactory lobe did not differ significantly in volume between the two age classes. In the second study, both HVC and RA volume correlated positively with song bout length but not repertoire size among adult birds. Based on these results a new hypothesis is presented that states that variation in song nuclei volumes in starlings relates more to the amount of song produced than to the number of song types stored in memory.

STEROID SENSITIVE SITES IN THE AVIAN BRAIN : DOES THE DISTRIBUTION OF THE ESTROGEN RECEPTOR ALPHA AND BETA TYPES PROVIDE INSIGHT INTO THEIR FUNCTION ?

Studies in avian species have often been useful in elucidating basic concepts relevant to the regulation of reproductive behaviors by sex steroid hormones. Once a link between a steroid hormone and a behavioral response has been established, one can use the localization of steroid hormone receptors in the brain to facilitate the identification of neural circuits that control behavior. The recent identification of a second type of estrogen receptor called estrogen receptor β or ERβ has raised new issues about the action of steroid hormones in the brain. A hypothesis has been proposed by Kuiper et al. [1998] based on studies in mammalian species suggesting that ERα (the name given to the ER that was previously described) is important for reproduction while ERβ is more important for non-reproductive functions. In this paper we apply this hypothesis more generally by examining possible functions of ERβ in avian species. We have initiated studies of the ERβ in the brain of two avian species, the Japanese quail (Coturnix japonica) and the European starling (Sturnus vulgaris). ERβ was cloned in both species and the mRNA for this receptor type was localized in the brain employing in situ hybridization histochemistry methods. In both species ERβ was found to be diffusely present in telencephalic areas consistent with a role for this receptor subtype in cognitive functions. However, ERβ mRNA was also found in many brain areas that are traditionally thought to be important in the regulation of reproductive functions such as the preoptic region, the bed nucleus of the stria terminalis and the nucleus taeniae. Of the two receptor types, only mRNA for ERα was observed in the telencephalic vocal control nucleus HVc of male starlings. Steroid receptors in this nucleus are thought to be an example of an evolutionary specialization that has evolved to coordinate the production of courtship vocalizations with other aspects of reproduction. The lack of ERβ mRNA expression in HVc is consistent with the hypothesis that ERα is preferentially involved in reproductive behaviors while ERβ is involved in the steroid regulation of other neural functions. However, the widespread occurrence of ERβ in other nuclei involved in reproductive function suggests that one must be cautious about the general applicability of the above hypothesis until more is known about ERβ function in these other nuclei.

Normal reproductive function in InhBP/p120-deficient mice.

ABSTRACT The inhibins are gonadal transforming growth factor β superfamily protein hormones that suppress pituitary follicle-stimulating hormone (FSH) synthesis. Recently, betaglycan and inhibin binding protein (InhBP/p120, also known as the product of immunoglobulin superfamily gene 1 [IGSF1]) were identified as candidate inhibin coreceptors, shedding light on the molecular basis of how inhibins may affect target cells. Activins, which are structurally related to the inhibins, act within the pituitary to stimulate FSH production. Betaglycan increases the affinity of inhibins for the activin type IIA (ACVR2) receptor, thereby blocking activin binding and signaling through this receptor. InhBP/p120 may not directly bind inhibins but may interact with the activin type IB receptor, ALK4, and participate in inhibin Bs antagonism of activin signaling. To better understand the in vivo functions of InhBP/p120, we characterized the InhBP/p120 mRNAs and gene in mice and generated InhBP/p120 mutant mice by gene targeting in embryonic stem cells. InhBP/p120 mutant male and female mice were viable and fertile. Moreover, they showed no alterations in FSH synthesis or secretion or in ovarian or testicular function. These data contribute to a growing body of evidence indicating that InhBP/p120 does not play an essential role in inhibin biology.

An emerging role for co-receptors in inhibin signal transduction.

While many transforming growth factor-beta (TGFbeta) superfamily ligands such as TGFbeta, activin, and the bone morphogenic proteins (BMPs) are critical to the control of growth, differentiation, and cell fate, inhibin has a more limited role and is primarily responsible for the regulation of one hormone from one cell-type in the anterior pituitary. Inhibin is an endocrine hormone, produced by the gonads, that inhibits follicle stimulating hormone (FSH) release from the pituitary gonadotrope. The other hormones in the superfamily do not appear to act in an endocrine fashion, but rather control cell function in a paracrine or autocrine manner. Many components of the TGFbeta/activin/BMP signal transduction pathway have been elegantly defined; however, the mechanism of inhibin action has not been completely dissected. Several cell surface proteins that associate with inhibin have been identified recently, and these molecules may provide the clues necessary to understand how inhibin regulates reproductive function.

Differential Regulation of Pituitary Gonadotropin Subunit Messenger Ribonucleic Acid Levels in Photostimulated Siberian Hamsters

Abstract FSH levels begin to rise 3–5 days after male Siberian hamsters are transferred from inhibitory short photoperiods to stimulatory long photoperiods. In contrast, LH levels do not increase for several weeks. This differential pattern of FSH and LH secretion represents one of the most profound in vivo examples of differential regulation of the gonadotropins. The present study was undertaken to characterize the molecular mechanisms controlling differential FSH and LH synthesis and secretion in photostimulated Siberian hamsters. First, we cloned species-specific cDNAs for the three gonadotropin subunits: the common α subunit and the unique FSHβ and LHβ subunits. All three subunits share high nucleotide and predicted amino acid sequence identity with the orthologous cDNAs from rats. We then used these new molecular probes to examine the gonadotropin subunit mRNA levels from pituitaries of short-day male hamsters transferred to long days for 2, 5, 7, 10, 15, or 20 days. Short-day (SD) and long-day (LD) controls remained in short and long days, respectively, from the time of weaning. We measured serum FSH and LH levels by RIA. FSHβ, LHβ, and α subunit mRNA levels were measured from individual pituitaries using a microlysate ribonuclease protection assay. Serum FSH and pituitary FSHβ mRNA levels changed similarly following long-day transfer. Both were significantly elevated after five long days (2.3- and 3.6-fold, respectively; P < 0.02) and declined thereafter, but they remained above SD control values through 20 long days. Alpha subunit mRNA levels also increased significantly relative to SD control values (maximum 2-fold increase after seven long days; P < 0.03), although to a lesser extent than FSHβ. Neither serum LH nor pituitary LHβ mRNA levels changed significantly following long-day transfer. The results indicate that long-day-associated increases in serum FSH levels in Siberian hamsters reflect an underlying increase in pituitary FSHβ and α subunit mRNA accumulation.

Lesions of glucose-responsive neurons impair synchronizing effects of calorie restriction in mice

Calorie restriction can induce phase-advances of daily rhythms in rodents exposed to light-dark cycles. To test whether glucose-responsive neurons are involved in the synchronizing effects of calorie restriction, C57BL/6J mice were injected with gold-thioglucose (GTG; 0.6 g/kg) which damages glucose-responsive neurons, primarily located in the ventromedial hypothalamus. From the day of injection, GTG-treated and control mice received a hypocaloric diet (66% of ad libitum food intake) 2 h after lights on. When mice were transferred to constant darkness after 4 weeks and fed ad libitum, the onset of circadian rhythm of locomotor activity was phase-advanced by 1 h in control but not in GTG-treated mice. Therefore, glucose-responsive neurons in the ventromedial hypothalamus may play a role in the synchronizing effects of calorie restriction on circadian rhythmicity.

Gold-thioglucose-induced hypothalamic lesions inhibit metabolic modulation of light-induced circadian phase shifts in mice

The circadian clock located in the suprachiasmatic nuclei is entrained by the 24-h variation in light intensity. The clocks responses to light can, however, be reduced when glucose availability is decreased. We tested the hypothesis that the ventromedial hypothalamus, a key area in the integration of metabolic and hormonal signals, mediates the metabolic modulation of circadian responses to light by injecting C57BL/6J mice with gold-thioglucose (0.6 g/kg) which damages glucose-receptive neurons, primarily located in the ventromedial hypothalamus. Light pulses applied during the mid-subjective night induce phase delays in the circadian rhythm of locomotor activity in mice kept in constant darkness. As previously observed, light-induced phase delays were significantly attenuated in fed mice pre-treated with 500 mg/kg i.p. 2-deoxy-D-glucose and in hypoglycemic mice fasted for 30 h, pre-treated with 5 IU/kg s.c. insulin or saline, compared to control mice fed ad libitum. In contrast, similar metabolic challenges in mice with gold-thioglucose-induced hypothalamic lesions did not significantly affect light-induced phase delays compared to mice treated with gold-thioglucose and fed ad libitum. These results indicate that destruction of gold-thioglucose-sensitive neurons in the ventromedial hypothalamus prevent metabolic regulation of circadian responses to light during shortage of glucose availability. Therefore, the ventromedial hypothalamus may be a central site coordinating the metabolic modulation of light-induced phase shifts of the circadian clock.