Characterization and Localization of Mouse Hypothalamic Growth Hormone‐Releasing Factor and Effect of Gold Thioglucose‐Induced Hypothalamic Lesions

Abstract

Hypothalamic growth hormone-releasing factor (GRF) in higher mammals, including human GRF, is a 44 amino acid residue peptide and is highly homologous in structure. By contrast, mouse GRF (mGRF) recently deduced by cDNA cloning consists of only 42 residues and shows relatively low homology to the GRFs of higher mammals and the same rodent species, rat. To characterize and localize the predicted mature mGRF peptide in the hypothalamus, we have generated its antiserum and developed a homologous radioimmunoassay. lmmunoreactive mGRF in the acid hypothalamic extract was eluted as a single peak at a position identical to that of synthetic peptide on both gel filtration chromatography and reverse-phase high-performance liquid chromatography (HPLC). Secretion of immunoreactive mGRF from incubated hypothalami increased several fold in response to 50 mM K' , and this rise was abolished in the absence of medium Ca'+. Only a single peak of irnmunoreactive mGRF that coeluted with synthetic replicate was observed after the K+-stimulated medium was extracted on Bond Elut C18 cartridges and applied on reverse-phase HPLC. lmmunohistochemistry identified many mGRF-positive cell bodies in the arcuate nucleus and dense bundles of immunoreactive fibers in the median eminence. Treatment of mice with gold thioglucose (GTG), a chemical agent known to cause hypothalamic lesions, markedly depleted both content and in vitro secretion of immunoreactive mGRF. The decline in mGRF secretion was greater in GTG obese than in nonobese mice, whereas somatostatin secretion was not affected by GTG treatment. AS compared to the previous findings obtained on rat GRF, mouse had approximately the same content of hypothalamic GRF, but retained only one third the capacity to release GRF in response to the same depolarizing stimulus.These findings demonstrate that mouse hypothalamus contains and secretes a mGRF-like molecule which has immunological and chromatographic characteristics identical to that predicted by molecular cloning. The depolarization-evoked, Ca' ' -dependent release of immunoreactive mGRF and its neuronal localization in the arcuate nucleus-median eminence region indicate that it functions as a hypophysiotropic hormone of physiological importance. GTG produces hypothalamic GRF deficiency, probably sparing hypophysiotropic somatostatin neurons, and could be used to generate a mouse model for human GRF-deficient dwarfism.

Immunohistochemical evidence of neurons with GHRH or LHRH in the arcuate nucleus of male mice and their possible role in the postnatal development of adenohypophysial cells

“…Our morphological results showed that GH cells in the anterior lobe of the treated male mice were hypofunctioning (i.e., there was a significant decrease in both the cell size and number). A first report of GHRH-immunoreactive neurons in the arcuate nucleus in mice, as in rats, has already appeared (Miki et al, 1994). We found here that the destruction of GHRH neurons by neonatal MSG treatment occurs in the mouse, as it is known to in the rat (Bloch et al, 1984;Merchenthaler et al, 1984;Meister et al, 1989).…”

“…With these control procedures immunostaining did not occur. Absorption tests of the GH, PRL (Sasaki and Iwama, 1988), and GHRH antisera (Miki et al, 1994) were done as described elsewhere. Absorption tests of the FSH, LH, and LHRH antisera were carried out by the addition of 50-100 pgiml hFSH (BiosPacific, CAI, hLH (BiosPacific, CA), and 100-200 pgiml LHRH (Cambridge Research Biochemicals, Northwich, UK) to the corresponding antiserum.…”

“…In 0 1994 WILEY-LISS, INC the mouse, the question about the existence of LHRHimmunoreactive neurons in the arcuate nucleus has not been explored immunohistochemically, to our knowledge. There are GHRH-immunoreactive neurons in the arcuate nucleus of the mouse (Miki et al, 1994). Olney (1969) reported that the administration of MSG to neonatal mice destroys the perikarya in the arcuate nucleus, and that pituitary and gonadal atrophy and stunted growth follow.…”

Distribution of galaninergic immunoreactivity in the brain of the mouse

“…Immunohistochemical studies have revealed the presence of gonadotropin releasing factor hormone (GnRH) and growth hormonereleasing factor (GHRF) in cells of the arcuate nucleus and in fibers of the median eminence of mice (Miki et al, 1994;Sasaki et al, 1994). The presence of GAL and GHRF in neurons of the arcuate nucleus and their axonal projections to the median eminence was demonstrated in rat (Niimi et al, 1990).…”

Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects

“…Hypothalamic peptides from wild-type (wt), heterozygous, and null animals of 8 weeks of age were extracted and assayed, as described (19,20). The final dilution of antiserum (kindly provided by L. Frohman, Univ.…”