Assignment of human X-linked genes to a zebra finch microchromosome by in situ hybridization of BAC clones.
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Animals↗
Biomedical subjects
A P Arnold
Publications and source records attributed to A P Arnold.
Sexually dimorphic expression of Usp9x is related to sex chromosome complement in adult mouse brain.
We found the expression of Usp9x, an X-linked gene which encodes a ubiquitin protease implicated in synaptic development, to be significantly higher in the adult female mouse brains than in male brains. The sex difference in expression of Usp9x was localized to specific brain regions such as neocortex. Furthermore, in gonadally intact and gonadectomized mice, XX mice expressed Usp9x mRNA and protein more highly than XY mice irrespective of their gonadal type. No sex difference was found in the neonatal brain or peripheral tissues such as the adult kidney. This finding implies that the difference in sex chromosome complement between XY males and XX females could potentially contribute to sexual differentiation of brain structure and function. The relation of genomic dose and Usp9x expression could help explain the neural and behavioural phenotype of women with XO Turner syndrome.
Animals↗
Sex differences in mouse cortical thickness are independent of the complement of sex chromosomes.
Although the morphology of the cerebral cortex is known to be sexually dimorphic in several species, to date this difference has not been investigated in mice. The present study is the first to report that the mouse cerebral cortex is thicker in males than in females. We further asked if this sex difference is the result of gonadal hormones, or alternatively is induced by a direct effect of genes encoded on the sex chromosomes. The traditional view of mammalian neural sexual differentiation is that androgens or their metabolites act during early development to masculinize the brain, whereas a feminine brain develops in the relative absence of sex steroids. We used mice in which the testis determination gene Sry was inherited independently from the rest of the Y chromosome to produce XX animals that possessed either ovaries or testes, and XY animals that possessed either testes or ovaries. Thus, the design allowed assessment of the role of sex chromosome genes, independent of gonadal hormones, in the ontogeny of sex differences in the mouse cerebral cortex. When a sex difference was present, mice possessing testes were invariably masculine in the morphology of the cerebral cortex, independent of the complement of their sex chromosomes (XX vs. XY), and mice with ovaries always displayed the feminine phenotype. These data suggest that sex differences in cortical thickness are under the control of gonadal steroids and not sex chromosomal complement. However, it is unclear whether it is the presence of testicular secretions or the absence of ovarian hormones that is responsible for the thicker male cerebral cortex.
Animals↗
Controlling factors in the synthesis of cucurbituril and its homologues.
The acid-catalyzed synthesis of cucurbit[n]urils from formaldehyde and glycoluril is poorly understood. In this paper, we examine a wide range of reaction conditions that include the effects of acid type, acid concentration, reactant concentrations, and temperature to both probe the mechanism and optimize the yields of isolated cucurbit[n]urils, where n = 5-10. A mechanism for the formation of these cucurbit[n]urils is presented. Individual cucurbit[n]urils were unambiguously identified in reaction mixtures using ESMS and (13)C NMR.
Journal Article↗
Primate DAX1, SRY, and SOX9: evolutionary stratification of sex-determination pathway.
The molecular evolution of DAX1, SRY, and SOX9, genes involved in mammalian sex determination, was examined in six primate species. DAX1 and SRY have been added to the X and Y chromosomes, respectively, during mammalian evolution, whereas SOX9 remains autosomal. We determined the genomic sequences of DAX1, SRY, and SOX9 in all six species, and calculated K(a), the number of nonsynonymous substitutions per nonsynonymous site, and compared this with the K(s), the number of synonymous substitutions per synonymous site. Phylogenetic trees were constructed by means of the DAX1, SRY, and SOX9 coding sequences, and phylogenetic analysis was performed using maximum likelihood. Overall measures of gene and protein similarity were closer for DAX1 and SOX9, but DAX1 exhibited nonsynonymous amino acid substitutions at an accelerated frequency relative to synonymous changes, similar to SRY and significantly higher than SOX9. We conclude that, at the protein level, DAX1 and SRY are under less selective pressure to remain conserved than SOX9, and, therefore, diverge more across species than does SOX9. These results are consistent with evolutionary stratification of the mammalian sex determination pathway, analogous to that for sex chromosomes.
Amino Acid Sequence↗
Interaction of BDNF and testosterone in the regulation of adult perineal motoneurons.
In androgen-sensitive motoneurons of the spinal nucleus of the bulbocavernosus (SNB), we investigated the interaction of BDNF (brain-derived neurotrophic factor) and testosterone to understand whether each factor gates the ability of the other to regulate androgen receptor expression and soma size, and whether each factor requires the presence of the other for its action. We axotomized SNB motoneurons and applied BDNF or PBS (phosphate-buffered saline) to the cut ends of the axons in rats that were castrated and treated with either testosterone or placebo. Control groups were either not castrated or not axotomized, or had intact SNB axons and were castrated and treated with testosterone or placebo. We found that testosterone determined the expression of nuclear androgen receptor, and this effect was enhanced by both BDNF and contact with the target muscles. The effect of BDNF on androgen receptor expression was seen only when testosterone was present. In the regulation of soma size, BDNF dominated. The application of BDNF completely compensated for the loss of testosterone in castrated males so that the testosterone effect on soma size was seen only in intact SNB motoneurons and in axotomized motoneurons treated with PBS. Moreover, testosterone increased androgen receptor and soma size in axotomized SNB motoneurons, indicating that testosterone can act on sites other than the target muscles of the SNB to regulate each of these. These results indicate that the regulation of androgen receptor by testosterone does not require BDNF, but the regulation of androgen receptor by BDNF does require testosterone. The regulation of soma size by BDNF does not require high expression of nuclear androgen receptor.
Animals↗
Distribution and regulation of telencephalic aromatase expression in the zebra finch revealed with a specific antibody.
In songbirds, aromatase (estrogen synthase) activity and mRNA are readily detectable in the brain. This neural aromatization presumably provides estrogen to steroid-sensitive targets via autocrine, paracrine, and synaptic mechanisms. The location of immunoreactive protein, however, has been difficult to describe completely, particularly in distal dendrites, axons, and terminals of the forebrain. Here we describe the neuroanatomical distribution of aromatase in the zebra finch by using a novel antibody raised specifically against zebra finch aromatase. The distribution of aromatase-positive somata in the zebra finch brain is in excellent agreement with previous reports. Additionally, this antibody reveals elaborate, spinous dendritic arbors, fine-beaded axons, and punctate terminals of telencephalic neurons that may synthesize estrogen. Some of these axon-like fibers extend into the high vocal center (HVC) and the robust nucleus of the archistriatum (RA) in males and females, suggesting a role for presynaptic aromatization in cellular processes within these loci. Adult males have more aromatase-positive fibers in the caudomedial neostriatum (NCM) and the preoptic area (POA) compared to females, despite the lack of detectable sex differences in the number of immunoreactive somata at these loci. Thus, the compartmentalization of aromatase in dendrites and axons may serve a sexually dimorphic function in the songbird. Finally, in adult males, aromatase expression is down-regulated by circulating estradiol in the hippocampus, but not in the NCM or POA. The distribution of aromatase suggests a role for aromatization in the regulation of pre- and postsynaptic function in steroid sensitive areas of the songbird forebrain.
Age Factors↗
BDNF regulation of androgen receptor expression in axotomized SNB motoneurons of adult male rats.
Brain-derived neurotrophic factor (BDNF) prevents the axotomy-induced loss of androgen receptor-like immunoreactivity (AR-LI) in the spinal nucleus of the bulbocavernosus (SNB) motoneurons of adult male rats. In this report, we investigated the dose-response effect of BDNF on androgen receptor expression in axotomized SNB motoneurons, and examined whether delayed application of BDNF to the cut SNB axons can completely reverse the axotomy-induced loss of androgen receptor expression. We also used autoradiography to test whether axotomy decreases the ability of SNB motoneurons to accumulate androgens. SNB motoneurons were axotomized bilaterally and BDNF or PBS was applied to the proximal ends of the axons. The percentage of SNB motoneurons expressing medium or high AR-LI was the major measure of androgen receptor expression. AR-LI was significantly higher on the BDNF-treated side than on the contralateral side treated with phosphate-buffered saline (PBS) for all three doses of BDNF (1.45, 2.9, and 5.8 mg/ml) and was higher than in rats treated bilaterally with PBS. Moreover, AR-LI at the highest dose of BDNF was not different from that in intact SNB motoneurons. Delayed application of BDNF to the axotomized SNB motoneurons restored the AR-LI to the intact level. The AR-LI decreased by axotomy started to increase significantly 4 days after BDNF application and returned to the intact level by 10 days. Furthermore, axotomy significantly decreased the percentage of SNB motoneurons to accumulate tritiated testosterone or its metabolites. In conclusion, our data demonstrate that BDNF completely prevents and reverses the axotomy-induced loss of AR-LI. Moreover, decrease of AR-LI by axotomy reflects the decrease in the ability of SNB motoneurons to accumulate androgens.
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Zebra finch aromatase gene expression is regulated in the brain through an alternate promoter.
The learned singing behavior in songbirds is sex-steroid-dependent and sexually dimorphic. Estrogen plays a major role in masculinizing the song system in these songbirds. The songbird brain synthesizes large amounts of estrogen, which, in the case of zebra finches, have been found to enter the systemic circulation. Aromatase cytochrome P450 is the key enzyme catalyzing the conversion of androgens to estrogens. We have cloned a novel alternatively spliced form of aromatase cDNA expressed predominantly in the zebra finch brain. We have also isolated and characterized the gene coding for zebra finch aromatase which spans 20kb in length. The alternate forms of aromatase mRNA (ARO) differ in their 5'-untranslated regions encoded by either exon 1a or 1b. The putative promoter sequences controlling the regulation of the alternate forms of ARO in zebra finches contain consensus binding sites for various transcription factors. While both the promoters have binding sites for SRY-like transcription factor, a binding site for SF-1 is present only in the promoter 1b active in the ovary. Intriguingly, a 55bp segment within the promoter 1a sequence appears to be highly conserved among zebra finch, mouse and human aromatase promoters active in the brain.
5' Untranslated Regions↗
Effects of embryonic treatment with fadrozole on phenotype of gonads, syrinx, and neural song system in zebra finches.
Previous studies have found that treatment of zebra finch embryos with an aromatase inhibitor on Day 5 or 8 of incubation caused partial sex reversal of gonadal phenotype in females. These females possessed both testicular and ovarian tissue, and the development of the neural circuit for song remained feminine. The present study attempted more complete gonadal reversal by treating zebra finch embryos earlier, on Day 3 of incubation, with Fadrozole (CGS 16949A), an aromatase inhibitor, or with saline. We examined the phenotype of the syrinx (androgen-dependent vocal organ), the gonads, and the telencephallic neural song system in 100-day-old birds. Treated females typically possessed a left ovotestis and a right testis, and significantly larger syringes than control females. The histology and steroid synthetic enzyme activity of the testicular tissue in treated females were quite masculine and similar to that of control males. At the time of sacrifice, the plasma concentrations of testosterone and estradiol for fadrozole-treated females did not differ from those of control females, but dihydrotestosterone was lower in treated females. Despite the large amount of functional testicular tissue and a masculine syrinx, the volumes and soma sizes of song system nuclei (HVC, RA) in treated females remained feminine. These results suggest that testicular secretions masculinize the syrinx, but are not sufficient to masculinize the song system in zebra finches.
Animals↗
Developmental regulation of the distribution of aromatase- and estrogen-receptor- mRNA-expressing cells in the zebra finch brain.
Strong evidence exists for the masculinizing effects of estrogen on the neural network that controls song learning and behavior in zebra finches. However, the mechanisms by which estrogen acts to influence the development of this circuitry are not well understood. In this study, we used in situ hybridization to detect the distribution of cells expressing mRNAs for AROM and ERalpha at postnatal days 5-25 (P5-25). Our findings revealed developmental regulation of both mRNAs in the neostriatum, archistriatum, hippocampus, diencephalon and midbrain. Within the vocal control circuitry, cells expressing ERalpha mRNA were found in the medial HVC (P10-25), archistriatum lateral to the RA (Ad; P25), in the ICo (P5-25), and along the fiber tract containing efferents from the RA. High levels of AROM mRNA were found in the neostriatum, including both the lateral and mMAN and along their projections to the RA and HVC, respectively, (P5-25), in the archistriatum (P18-25) and around RA (P18). Codistribution of the two mRNAs occurred along the border of the HVC suggesting that in this region, local synthesis of estrogen may be acting through its nuclear receptor to regulate gene transcription. Taken together, our findings show that the neural circuitry controlling song may be exposed to the effects of estrogen during early postnatal development. However, in most of the song control regions, these mRNAs were not expressed together either temporally or spatially, indicating that AROM may have a role in the development of the song system independent of ERalpha.
Animals↗
Effects of embryonic estrogen on differentiation of the gonads and secondary sexual characteristics of male zebra finches.
Male zebra finches sing to court females, whereas females do not normally sing. In parallel, the telencephalic brain regions that control song are larger in volume and contain larger cells in males than in females. The vocal control organ (syrinx) is also larger in males. Some evidence suggests that the sexual differentiation of both anatomy and behavior is under the regulation of gonadal hormones during early development, yet recent data conflict with the idea that the sole source of masculinization of the neural song system is the testes. In the present experiment, we treated genetic males with estradiol benzoate on embryonic day 5 and measured the volume of and neuron soma size in robust nucleus of the archistriatum (RA) and the high vocal center (HVC), two telencephalic song control nuclei. We also weighed the syrinx, the muscles of which are the target of the motor pathway containing the two brain regions. The estrogen treatment disrupted testicular morphology, and induced an oviduct in six of seven animals, but it had no effect on any of four measures of masculinization of the neural song system. These results suggest that normal testicular tissue is not required for masculine development of the neural song system.
Animals↗
A putative 5 alpha-reductase inhibitor demasculinizes portions of the zebra finch song system.
One model of the sexual differentiation of the zebra finch song system holds that both major metabolites of testosterone, dihydrotestosterone (DHT) and estradiol (E2), act together to masculinize the song system. To test this model, we administered a putative inhibitor of 5 alpha-reductase (MK-434) to decrease the synthesis of DHT from testosterone (T) in hatchling zebra finches. We tested MK-434's inhibition of 5 alpha-reductase, 5 beta-reductase, and aromatase in vivo and in vitro. In vivo, MK-434 significantly inhibited 5 alpha-reductase activity but also reduced the activities of 5 beta-reductase and aromatase. In vitro, MK-434 was extremely effective in inhibiting 5 alpha-reductase in the rat prostate but only slightly inhibited 5 alpha-reductase in the zebra finch telencephalon, where it also reduced aromatase and 5 beta-reductase activities. These results suggest that MK-434 might differentially influence the availability of androgenic and estrogenic substrates, depending on the relative abundance of these enzymes in brain. MK-434 demasculinized (decreased) the number and decreased the density of RA neurons but did not significantly affect any other sexually dimorphic aspect of the song system, including the volumes of RA, HVC, and Area X; the size of neural somata in IMAN, HVC, and RA; and the number of neurons in HVC and IMAN. The differential influence of MK-434 on sexually dimorphic characteristics suggests that the various sexually dimorphic characteristics of the song system (1) are sensitive to different hormones, depending on the characteristic; or (2) have different sensitivities to hormone levels, some being easily affected by slightly reduced hormone levels whereas others are not; or (3) have markedly different critical periods depending on the characteristic. Regardless of the reason(s) for differential effects on the sexually dimorphic characteristics of the song system, the data clearly suggest that steroid hormones play a role in the normal masculine development of the song system.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Brain-derived neurotrophic factor regulates expression of androgen receptors in perineal motoneurons.
Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) express androgen receptors and innervate striated muscles attached to the penis. Previous studies indicated that androgen receptor immunoreactivity in the SNB motoneurons decreases after axotomy and returns to normal only in motoneurons allowed to reinnervate their muscle targets, suggesting that neuron-target interactions play a role in regulating steroid receptor expression in the central nervous system. This study demonstrates that (i) silencing the SNB neuromuscular system with tetrodotoxin did not affect androgen receptor expression in these motoneurons, suggesting that the regulation of androgen receptor is activity-independent; (ii) disruption of axonal transport with vinblastine caused a down-regulation of androgen receptor expression in the SNB motoneurons; and (iii) treatment with brain-derived neurotrophic factor, but not ciliary neurotrophic factor, neurotrophin-4, or glial cell line-derived neurotrophic factor, reversed the axotomy-induced down-regulation of androgen receptor expression. These findings demonstrate neurotrophin regulation of steroid receptor expression in the central nervous system in vivo.
Animals↗
Song lateralization in the zebra finch.
The neural mechanisms for bird song commonly are lateralized, depending greatly on the integrity of the left song system and responding much less to manipulations of the system on the right. These results suggest that it is advantageous for the mechanisms controlling song to be lateralized and in this direction. In this context, it is of special interest to study likely exceptions to these rules, for the light they can shed on the functions and mechanisms of song lateralization. Accordingly, we have tested the extent and manner of song lateralization in the zebra finch (Taeniopygia guttata), a species previously identified with an atypical pattern of lateralization. Song by male finches was observed before and after sham operations or transections of the left or right tracheosyringeal nerve. Sham operations failed to affect song. In contrast, males with cuts of the right nerve experienced a depression in fundamental frequency to an apparent baseline of approximately 500 Hz. This effect is consistent with the dominance of the right song system in zebra finches. On the other hand, males with cuts of the left nerve also showed consistent changes, exhibiting frequency increases, especially toward the ends of syllables. The source of these changes is not completely clear. However, rather than challenging the emerging view of lateralization in zebra finches, they may reflect a combination of right dominance and an atypical mechanism for the gating of syringeal airflow and vocalization during song.
Animals↗
Experimental analysis of sexual differentiation of the zebra finch brain.
Classical theories of sexual differentiation of brain and behavior hold that sex differences in the brain arise because of the action of gonadal steroid hormones. In mammals, testosterone secretion by the testes stimulates a masculine pattern of neural differentiation, whereas feminine patterns of development occur in the absence of testicular secretions. In some bird species, estrogen secreted by the ovary is thought to trigger feminine patterns of neural development, whereas masculine development occurs in the absence of ovaries. Sexual differentiation of the neural circuit for song in zebra finches is not easily explained by these theories. Although female zebra finches can be masculinized by treatments with estrogen, it has proven difficult to prevent masculine neural development in genetic males by treating them with inhibitors of estrogen synthesis. Moreover, when genetic female embryos are treated with inhibitors of estrogen synthesis, they develop significant amounts of testicular tissue that causes little or no masculinization of the song system. Thus, testicular secretions alone appear to be insufficient to cause masculine neural differentiation, and other factors need to be invoked. These factors may include ovarian secretions that inhibit masculine development, or direct genetic (nonhormonal) effects on neural differentiation.
Animals↗
Developmental plasticity in neural circuits for a learned behavior.
The neural substrate underlying learned vocal behavior in songbirds provides a textbook illustration of anatomical localization of function for a complex learned behavior in vertebrates. The song-control system has become an important model for studying neural systems related to learning, behavior, and development. The song system of zebra finches is characterized by a heightened capacity for both neural and behavioral change during development and has taught us valuable information regarding sensitive periods, rearrangement of synaptic connections, topographic specificity, cell death and neurogenesis, experience-dependent neural plasticity, and sexual differentiation. The song system differs in some interesting ways from some well-studied mammalian model systems and thus offers fresh perspectives on specific theoretical issues. In this highly selective review, we concentrate on two major questions: What are the developmental changes in the song system responsible for song learning and the restriction of learning to a sensitive period, and what factors explain the highly sexually dimorphic development of this system? We discuss the important role of sex steroid hormones and of neurotrophins in creating a male-typical neural song circuit (which can learn to produce complex vocalizations) instead of a reduced, female-typical song circuit that does not produce learned song.
Animals↗
Sexual differentiation of the zebra finch song system: positive evidence, negative evidence, null hypotheses, and a paradigm shift.
Permanent sex differences in the brain are found in many vertebrates, and are thought to be induced by sex differences in secretion of gonadal steroid hormones during critical periods of early development. This theory has received support primarily from many experiments conducted on mammals, but also from studies on other vertebrate classes, including birds. The only avian neural dimorphism that has allowed extensive tests of this hypothesis is the neural circuit for song in passerine birds, which is much larger in males than in females. Experiments in zebra finches have yielded contradictory results. Although it is relatively easy to induce masculine patterns of development in genetic females with estrogen, it has not been possible to induce feminine patterns of development in males with any treatments, including antiestrogens and inhibitors of estrogen synthesis. Moreover, genetic females that develop with large amounts of functional testicular tissue but with virtually no ovarian tissue nevertheless have a feminine song circuit. The latter studies fail to support the idea of steroid induction of sexual differentiation. An alternative to the steroidal control hypothesis is that nonhormonal gene products expressed in the brain early in development trigger sexually dimorphic patterns of development. Although current evidence in several neural and nonneural systems indicates that sexual differentiation of some somatic phenotypes cannot be explained by the actions of gonadal steroids, the idea of direct genetic (nonhormonal) induction of sexual differentiation has yet to be proved.
Animals↗