Determination of tissue and plasma concentrations of PTHrP in fish: development and validation of a radioimmunoassay using a teleost 1-34 N-terminal peptide.

A specific and sensitive radioimmunoassay (RIA) for the N-terminus of sea bream (Sparus auratus) and flounder (Platichthys flesus) parathyroid hormone-related protein (PTHrP) was developed. A (1-34) amino-terminal sequence of flounder PTHrP was synthesized commercially and used as the antigen to generate specific antiserum. The same sequence with an added tyrosine (1-35(Tyr)) was used for iodination. Human (1-34) parathyroid hormone (PTH), human (1-34) PTHrP, and rat (1-34) PTHrP did not cross-react with the antiserum or displace the teleost peptide. Measurement of PTHrP in fish plasma was only possible after denaturing by heat treatment due to endogenous plasma binding activity. The minimum detectable concentration of (1-34) PTHrP in the assay was 2.5 pg/tube. The level of immunoreactive (1-34) PTHrP in plasma was 5.2+/-0.44 ng/ml (mean+/-SEM, n=20) for flounder and 2.5+/-0.29 ng/ml (n=64) for sea bream. Dilution curves of denatured fish plasma were parallel to the assay standard curve, indicating that the activity in the samples was indistinguishable immunologically from (1-34) PTHrP. Immunoreactivity was present, in order of abundance, in extracts of pituitary, oesophagus, kidney, head kidney, gills, intestine, skin, muscle, and liver. The pituitary gland and oesophagus contained the most abundant levels of PTHrP, 37.7+/-6.1 ng/g wet tissue and 2.3+/-0.7 ng/g wet tissue, respectively. The results suggest that in fish PTHrP may act in a paracrine and/or autocrine manner but may also be a classical hormone with the pituitary gland as a potential major source of the protein.

Glucocorticoid receptor immunoreactivity in neurons and pituitary cells implicated in reproductive functions in rainbow trout: a double immunohistochemical study.

In order to identify the nature of the glucocorticoid receptor (GR)-expressing neurons and pituitary cells that potentially mediate the negative effects of stress on reproductive performance, double immunohistochemical stainings were performed in the brain and pituitary of the rainbow trout (Oncorhynchus mykiss). To avoid possible cross-reactions during the double staining studies, combinations of primary antibodies raised in different species were used, and we report here the generation of an antibody raised in guinea pig against the rainbow trout glucocorticoid receptor (rtGR). The results obtained in vitellogenic females showed that GnRH-positive neurons in the caudal telencephalon/anterior preoptic region consistently exhibited rtGR immunoreactivity. Similarly, in the anterior ventral preoptic region, a group of tyrosine hydroxylase-positive neurons, known for inhibiting gonadotropin (GTH)-2 secretion during vitellogenesis, was consistently shown to strongly express GR. Finally, we show that a large majority of the GTH-1 (FSH-like) and GTH-2 (LH-like) cells of the pituitary exhibit rtGR immunoreactivity. These results indicate that cortisol may affect the neuroendocrine control of the reproductive process of the rainbow trout at multiple sites.

Immunohistochemical localization and biochemical characterization of two novel decapeptides derived from POMC-A in the trout hypothalamus.

Several vertebrate species which underwent duplication of their genome, such as trout, salmon and Xenopus, possess two proopiomelanocortin (POMC) genes. In the trout, one of the POMC molecules, called POMC-A, exhibits a unique C-terminal extension of 25 amino acids which has no equivalent in other POMCs characterized so far. This C-terminal peptide contains three pairs of basic residues, suggesting that it may be the source of novel regulatory peptides. The aim of the present study was to investigate the occurrence of these peptides in the brain of the trout Oncorhynchus mykiss by using specific antibodies raised against two epitopes derived from the C-terminal extension of POMC-A, i.e., EQWGREEGEE and YHFQ-NH2. Immunohistochemical labeling of brain sections revealed the presence of EQWGREEGEE- and YHFQ-NH2-immunoreactive cell bodies in the anterior part of the nucleus lateralis tuberis of the hypothalamus. Immunoreactive fibers were observed in the dorsal hypothalamus, the thalamus, the telencephalon, the optic tectum and the medulla oblongata. In contrast, no labeling was detected using antibodies against the non-amidated peptide YHFQG. Biochemical characterization was performed by combining high-performance liquid chromatography (HPLC) analysis with radioimmunoassay (RIA) quantification. Two peptides exhibiting the same retention time as synthetic EQWGREEGEE and ALGERKYHFQ-NH2 were resolved. However, no peptide co-eluting with YHFQ-NH2 or YHFQG could be detected. These results demonstrate that, in the trout brain, post-translational processing of POMC-A generates the two decapeptides EQWGREEGEE and ALGERKYHFQ-NH2. The wide distribution of immunoreactive fibers in the diencephalon, telencephalon, optic tectum and medulla oblongata suggests that these peptides may exert neurotransmitter and/or neuromodulator activities.

Immunohistochemical localization of glucocorticoid receptors in the forebrain of the rainbow trout (Oncorhynchus mykiss).

The distribution of glucocorticoid receptor-expressing cells was studied in the forebrain of the rainbow trout by means of antibodies produced against a fusion protein made of the NH2-terminal fragment of the rainbow trout glucocorticoid receptor fused in frame with glutathione-S-transferase. The results indicate that glucocorticoid receptor-expressing cells are located in many brain regions from the telencephalon to the spinal cord, with the highest density in the neuroendocrine component of the brain, the preoptic region and the mediobasal hypothalamus, and in the periventricular zone of the optic tectum. In virtually all cases, the labeling was located in the nucleus of the cells, although on very rare occasions, a slight labeling of the cytoplasm was detected. Concerning the preoptic region, the most striking feature was the high density of glucocorticoid receptors in the magnocellular preoptic nucleus, known to contain corticotrophin-releasing factor (CRF)-, vasotocin-, and isotocin-expressing cells. Colocalization experiments showed that 100% of the CRF-immunoreactive neurons in the preoptic nucleus express glucocorticoid receptors. In the mediobasal hypothalamus, the highest expression was found in the nucleus lateralis tuberis and parts of the nucleus recessus lateralis. Concerning the pituitary, the glucocorticoid receptor was consistently found in the rostral pars distalis, with the exception of the prolactin cells, and in the proximal pars distalis, which in trout contains thyrotrophs, gonadotrophs, and somatotrophs. In the hindbrain, expression of glucocorticoid receptors were localized mainly in the periventricular regions.

Distribution and expression of glucocorticoid receptor mRNA in the forebrain of the rainbow trout.

The expression and distribution of glucocorticoid receptor mRNA was studied in the forebrain of mature female and immature undifferentiated rainbow trout (Oncorhynchus mykiss) by means of Northern blotting and in situ hybridization. A single mRNA species of 7.5 kb was detected in mRNA polyA+ prepared from the anterior brain. In situ hybridization was carried out using a 35S-labelled riboprobe corresponding to the A/B-domain (between nucleotides 1224 and 1763) of the recently cloned rainbow trout glucocorticoid receptor cDNA. Comparison of adjacent sections hybridized with the sense and antisense probes allowed detection of a specific signal with a similar distribution pattern in all animals studied. In the telencephalon, a specific hybridization was detected in scattered cells of the dorsal telencephalic hemisphere, but the stronger signal was consistently observed in the dorsal nucleus, and to a lesser degree in the ventral nucleus of the ventral telencephalon. Heavy hybridization staining was consistently observed in all subdivisions of the preoptic nucleus and the nucleus lateralis tuberis, which are the main hypophysiotrophic regions in fish. A weaker signal was detected in the nucleus anterioris periventricularis, nucleus suprachiasmaticus and thalamic region. The presence of a strong signal in virtually all magnocellular neurons of the nucleus preopticus, known for producing vasotocin, isotocin and corticotropin-releasing factor favors a direct function of cortisol in regulating its own secretion.