This paper reports the results of in vivo and in vitro experiments on the feedback effects of corticosterone on the hypothalamo-pituitary-adrenal axis in embryos at day 18 of incubation and in 9-day-old chickens. In vivo, a significant negative feedback was detected on the levels of corticotropin-releasing factor (CRF) precursor (proCRF) mRNA and on the plasma concentration of corticosterone, two hours after a single intravenous injection with 40 µg corticosterone. In contrast, the levels of CRF peptide in the hypothalamic area, the CRF receptor type 1 (CRF-R1) mRNA and pro-opiomelanocortin (POMC) mRNA levels in the pituitary were not affected by the in vivo administration of corticosterone. In vitro, incubation with 1 µM corticosterone did not affect the CRF-R1 mRNA levels in the pituitary, but significant feedback inhibition was observed on the POMC mRNA levels. These in vitro effects were the same at the two ages studied. The in vitro feedback effect on the proCRF gene expression, however, differed with age. In 9-day-old animals a decrease in gene expression was observed which was not detectable in embryonic tissue at day 18 of the ontogeny.

We have characterized the structure of the chicken corticotropin-releasing factor (CRF) gene through cDNA cloning and genomic sequence analysis, and we analyzed the expression of CRF mRNA and peptide in the diencephalon of the chick throughout embryonic development. The structure of the chicken CRF gene is similar to other vertebrate CRF genes and contains two exons and a single intron. The primary structure of the mature chicken CRF peptide is identical to human and rat CRF. This is the first archosaurian CRF gene to be characterized. We used RIAs to analyze CRF peptide content in the diencephalon and the median eminence and plasma corticosterone during the last week of embryonic development. We also developed a semiquantitative RT-PCR method to analyze the expression of CRF mRNA during the same period. CRF peptide content in the diencephalon increased, whereas peptide content in the ME decreased just before hatching, suggesting that release and biosynthesis are coupled. Plasma corticosterone concentration significantly increased between embryonic d 20 and the first day post hatch. By contrast, CRF mRNA levels in the diencephalon decreased just before hatching. Changes in CRF production just before hatching may be causally related to the regulation of the thyroid and interrenal axes at this stage of chicken development.

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In this paper we report the cloning of the chicken preprothyrotropin-releasing hormone (TRH) cDNA and the study of its hypothalamic distribution. Chicken preproTRH contains five exact copies of the TRH progenitor sequence (Glu-His-Pro-Gly) of which only four are flanked by pairs of basic amino acids. In addition, the amino acid sequence contains three sequences that resemble the TRH progenitor sequence but seem to have lost their TRH-coding function during vertebrate evolution. The amino acid sequence homology of preproTRH between different species is very low. Nevertheless, when the tertiary structures are compared using hydrophobicity plots, the resemblance between chicken and rat prepro-TRH is striking. The cloning results also showed that the chicken preproTRH sequence includes neither a rat peptide spacer 4 (Ps4) nor a Ps5 connecting peptide.Comparison of the cDNA sequence with the chicken genome database revealed the presence of two introns, one in the 5 untranslated region, and another downstream from the translation start site. This means that the gene structure of chicken preproTRH resembles the gene stucture of this precursor in mammals. Based on the cDNA sequence, digoxigenin-labelled probes were produced to study the distribution of preproTRH in the chicken brain. By means of in situ hybridization, preproTRH mRNA was detected in the chicken paraventricular nucleus (PVN) and in the lateral hypothalamus (LHy).

Because iodothyronine deiodinases play a crucial role in the regulation of the available intracellular T(3) concentration, it is important to determine their cellular localization. In brain, the presence of type III iodothyronine deiodinase (D3) seems to be important to maintain homeostasis of T(3) levels. Until now, no cellular localization pattern of the D3 protein was reported in chicken brain. In this study polyclonal antisera were produced against specific peptides corresponding to the D3 amino acid sequence. Their use in immunocytochemistry led to the localization of D3 in the Purkinje cells of the chicken cerebellum. Both preimmune serum as well as the primary antiserum exhausted with the peptide itself were used as negative controls. Extracts of chick cerebellum and liver were made in the presence of Triton X-100 to solubilize the membrane-bound deiodinases. Using these extracts in Western blot analysis, a band of the expected molecular weight ( approximately 30 kDa) could be detected in both tissues. Using a full-length (32)P-labeled type III deiodinase cRNA probe, we identified a single mRNA species in the cerebellum that was of the exact same size as the hepatic control mRNA (+/-2.4 kb). RT-PCR, followed by subcloning and sequence analysis, confirmed the expression of D3 mRNA in the chicken cerebellum. In this study we provide the first evidence of the presence of the D3 protein in a neuronal cell type, namely Purkinje cells, by means of immunocytochemical staining. We were able to detect a protein fragment corresponding to the expected molecular mass (30 kDa) for type III deiodinase by means of Western blot analysis. RT-PCR as well as Northern blot analysis confirmed the presence of D3 mRNA in the cerebellum.