Female-biased sex difference in vasotocin-immunoreactive neural structures in the developing quail brain.

The bed nucleus of the stria terminalis pars medialis (BSTM), medial preoptic nucleus (POM), and lateral septal region (LS) exhibit more vasotocin-immunoreactive (VT-ir) neural structures in male than in female adult quail. VT-ir cells and fibers in these regions are sensitive to gonadal steroids only in males. The insensitivity of adult female VT-ir neural structures to sex steroids is attributed to estradiol exposure during a critical period in embryonic life. Although the VT-ir system has been intensively examined in adult quail, information is limited in embryos and juveniles. Therefore, we herein investigated the development of VT-immunoreactive neural structures from embryonic day (E) 9 to adulthood with a particular focus on the BSTM, POM and LS of both sexes. VT-ir neural structures were more evident in female than in male embryos from E9 (BSTM and POM) and E11 (LS). This sex difference disappeared between E15 and post-hatch day 1 in the BSTM and POM, and during the first week of life in the LS. Male-biased sex differences in VT-ir structures appeared at puberty. Female-biased sexual dimorphism in the density of the VT-ir structures of BSTM was reflected by the stronger expression of VT mRNA in females than in males. However, the density of VT mRNA somata was comparable in the two sexes. The exposure of male embryos to estradiol resulted in the feminization of VT-ir neural structures in the BSTM, but not in the POM or LS at E11. Collectively, these results suggest that sex differences in VT-ir neural structures changes drastically throughout quail life. In embryos, endogenous estradiol may stimulate the expression of VT in females, resulting in a robust sex difference in VT-ir cells and fibers in favor of this sex.

Immunolocalization of aquaporin-4 in the brain, kidney, skeletal muscle, and gastro-intestinal tract of chicken.

Aquaporin-4 (AQP4), a member of the water channel family, has high water permeability and multi-functional potentiality. Although an avian AQP4 homolog has recently been identified, its overall localization is still largely unknown. This study demonstrates the presence of AQP4 in several organs of chicken by using a specific chicken AQP4 antibody. Western blot analysis has revealed two bands of chicken AQP4 (30 and 32 kDa) in the brain, proventriculus, pectoral muscle, kidney, and ureter. The brain is the primary expression site of AQP4 in chicken. Immunohistochemical analysis of the brain has shown the highest AQP4 immunoreactivity around the cerebral ventricles, blood vessels, and the Purkinje cells. In peripheral organs, AQP4-immunoreactive elements have been observed in the ureter, glandular cells of the proventriculus, sarcolemma of the pectoral muscle, and the epithelium of the ceca and the rectum. Moreover, a heavily stained network of AQP4-immunoreactive fibers has been detected within the enteric plexuses.

Distribution and sex differences in aromatase-producing neurons in the brain of Japanese quail embryos.

The biochemical properties, neuroanatomical location, and function of aromatase (ARO), the enzyme that converts testosterone to 17beta-estradiol, have been studied extensively in the adult quail brain. Conversely, very little is known about ARO in quail embryos. This study investigated the distribution of ARO in quail prosencephalon at embryonic days (E) 9, 11, and 15 by immunocytochemistry. ARO-immunoreactive cells were observed within the walls of the cerebral ventricles, the ventral striatum, medial preoptic nucleus (POM), medial part of the bed nucleus of the stria terminalis (BSTM), lateral part of the BST, and in the tuberal region. The BSTM and to a lesser extent the POM showed transient, female-biased sex-differences. In the BSTM, the number of the ARO-immunoreactive cells, the fractional area covered by ARO-immunoreactive structures, and the overall extension of ARO-immunoreactivity were greater in females at E9 and E11, but these differences largely disappeared at E15 and post-hatch day 1. The sex differences were confirmed at the transcriptional level by in situ hybridization. In the lateral part of the POM, females showed slightly more ARO-immunoreactivity than males at E11. Treatment of E9 male embryos with estradiol completely feminized ARO-immunoreactivity at E11. The origins and the functional significance of these sex differences remain unknown.

TonEBP regulates hyperosmolality-induced arginine vasotocin gene expression in the chick (Gallus domesticus).

Arginine vasotocin (AVT) is expressed mainly in the paraventircular and supraoptic nuclei of the hypothalamus in chicken. This peptide is known to act as an antidiuretic hormone and its gene expression is stimulated by hyperosmolality. However, the transcription factors that regulate the AVT gene expression induced by hyperosmolality are still unknown. In this study, we examined the role of hyper-tonicity enhancer binding protein (TonEBP) in the transcriptional regulation of AVT gene in chicken. TonEBP mRNA expression levels increased at 1h after salt-loading treatment in the hypothalamus. This increase preceded that in AVT and c-fos mRNA expression. Intracerebroventricular injections of TonEBP antisense oligonucleotides, before the salt-loading treatment, prevented the increase in AVT gene expression. These results, all together, suggest that the transcription factor TonEBP may be involved in the regulation of AVT genes expression in response to a hyperosmotic environment in chicken.

Sex- and age-related variation in neurosteroidogenic enzyme mRNA levels during quail embryonic development.

Brain can synthesize steroids de novo from cholesterol and this biochemical feature is a conserved property of vertebrates. There is growing evidence indicating that neurosteroids might participate in sexual differentiation of the brain. Therefore, in this study we investigated the presence, the sex differences, and the development-dependent variation of mRNAs coding for key neurosteroidogenic enzymes, namely cytochrome P450 side-chain cleavage enzyme (P450scc), 3beta-hydroxysteroid-dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450c17), and aromatase in embryonic prosencephali. Our results indicated that 3beta-HSD mRNA levels were sexually dimorphic and developmental age-dependent. In particular, 3beta-HSD mRNA levels were higher in females than in males at E7, whereas, this dimorphism was reversed at E9 and E15. In females, the relative levels of 3beta-HSD mRNA were highest at E7, whereas, in males they were significantly higher at E9 and E15 than at E7 and at E11. This sexual dimorphism was a peculiar feature of the prosencephalon, it could not be observed before gonadal sexual differentiation and it was not paralleled by a dimorphism in the brain content of progesterone. The level of mRNA coding for P450scc and for P450c17 did not show obvious developmental- or sex-related variation. Aromatase mRNA varied as a function of the embryonic age but not of the sex. These results, taken together, are suggestive of a potential role of some neurosteroidogenic enzymes in the development of quail brain and suggest that sexual differences in the hormonal environment may occur during brain development.