This study focuses on the structure and expression of the mesotocin (MT) gene in the chicken hypothalamus. Using an anchored and nested RT-PCR strategy, combined with circular RACE-PCR, the full length sequence of the chicken MT cDNA was obtained. The cDNA and derived amino acid sequences conformed to the structure of the oxytocin-like gene family. However, unlike most mammalian species, there does not appear to be frequent gene conversion between the MT and AVT cDNA sequences. A single specific hybridization signal of 1.2 kb was detected by Southern analysis of chicken genomic DNA, indicating only a single gene copy in the chicken genome. Northern analysis of hypothalamic RNA revealed a single band at approximately 0.6 kb. Using the same probe for in situ hybridization histochemistry, MT-mRNA was demonstrated to be predominantly localized in the parvocellular, magnocellular and periventricular subgroups of the paraventricular nucleus and, when compared to the distribution of neurons containing arginine-vasotocin (AVT)-mRNA in the same region, with far fewer neurons expressing the MT gene in the lateral subgroups. Only few and scattered neurons expressing the MT gene were found in the ventral and external subgroups of the supraoptic nucleus in which many neurons contain AVT transcripts, as demonstrated in consecutive sections. In all nuclei investigated, the intensity of AVT and MT hybridization signals per cell was approximately equal. No specific labelling for MT-mRNA was found in the bed nucleus of the stria terminalis, nor the nucleus accumbens. Using immunocytochemical detection of AVT and in situ hybridization for neurons expressing MT-mRNA, some neurons were found to contain both AVT and MT gene products in the paraventricular nucleus but not in the supraoptic nucleus.

A specific homologous radioligand receptor assay for thyroid-stimulating hormone (TSH) using bovine thyroid membranes was adapted for use with human thyroid. Specific 125I-labelled TSH binding was detected in the 3000 g membrane pellet from bovine thyroid but predominantly in the 3000 g supernatant of the human thyroid homogenate. Both assays required incubation in the presence of 10% serum, whilst the assay using human thyroid could only be precipitated using polyethylene glycol (PEG). The serum requirement transcended a possible role as carrier protein and unmasked specific TSH binding. Molecular sieving determined that the active fraction of the serum had an apparent size of 30,000-100,000. The requirement for PEG-assisted precipitation of the TSH receptor assay was a consequence of the TSH-binding entity from Graves' thyroid behaving like a soluble 'receptor': it did not sediment with the membranes, passed a 0.2 microns filter and, upon molecular sieving, had an apparent size of 300,000-1,000,000. A full-length TSH receptor cDNA was cloned from a human Graves' thyroid library and stably transfected cell lines expressing the TSH-receptor protein were constructed using human HeLa and murine 3T3 cells. Specific TSH binding was unmasked by serum in the human cell lines, as observed for the human thyroid TSH receptor, whereas serum hindered TSH binding in the murine cell lines. A soluble form of the receptor was not released from the cells and was not produced in conditions which demonstrated a soluble receptor-like binding component in human thyroid tissue.

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The development of the anterior pituitary gland involves the proliferation and differentiation of ectodermal cells in Rathke's pouch to generate distinct cell types, each of which produces its corresponding trophic hormone. Studying pituitary development will therefore reveal novel aspects of organogenesis. In the present study, we examined by in situ hybridization the expression of genes for anterior pituitary hormones during development of the fetal pig pituitary. We found that the beta-subunit gene of thyroid-stimulating hormone (TSH beta) was first expressed at E40, (E = day of embryonal/fetal life), growth hormone (GH) mRNA appeared between E40 and E50, and the gonadotrophin genes (LH beta and FSH beta) were expressed at E50. The transcripts for TSH beta, LH beta and FSH beta were abundantly expressed at about E80, while GH mRNA continued to be richly expressed until after birth. The GH gene was first expressed in the mantle layer of the anterior lobe, while the TSH beta and gonadotrophin (LH beta and FSH beta) mRNAs were found in the central and the basal regions of the anterior lobe, respectively. All of these mRNAs (GH, TSH beta, LH beta, and FSH beta) remained concentrated until the end of gestation in the area where they first appeared. The distinctive pattern of developmental expression of these hormone genes in the fetal pig anterior pituitary makes this tissue an excellent system in which to study tissue-specific gene activation and regulation.

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