Gustavo M. Somoza

Chromatographic and immunological identification of GnRH (Gonadotropin-releasing hormone) variants. Occurrence of mammalian and a salmon-like GnRH in the forebrain of an eutherian mammal : Hydrochaeris hydrochaeris (Mammalia, Rodentia)

The molecular variants of Gonadotropin releasing hormone (GnRH) in brain extracts of the eutherian mammal Hydrochaeris hydrochaeris (Mammalia, Rodentia) were characterized. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) with different antisera was used. Two different forebrain regions (olfactory bulbs and preoptic-hypothalamic region) were analyzed. Characterization of RP-HPLC fractions from preoptic-hypothalamic extracts with three different RIA systems revealed two immunoreactive GnRH (ir-GnRH) peaks coeluting with mammalian GnRH (mGnRH) and salmon GnRH (sGnRH) synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. Similar results were obtained from olfactory bulb extracts with the same methodology. However, a third ir-GnRH peak in a similar position to that of chicken GnRH II (cIIGnRH) synthetic standard was revealed. As far as we know, this is the first report showing chromatographic and immunological evidences for the presence of a second GnRH variant in the forebrain of an eutherian mammal.

Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia).

Abstract1.In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic–hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested.2.The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents.3.In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve–septopreoptic and the midbrain systems.4.An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera.5.To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.

Effects of light and temperature conditions on the expression of GnRH and GtH genes and levels of plasma steroids in Odontesthes bonariensis females.

In this study we examined the endocrine mediation between environmental factors (temperature and photoperiod) and the brain–pituitary–gonadal axis in females of pejerrey Odontesthesxa0bonariensis. Changes in the expression of brain gonadotropin-releasing hormones (GnRHs) and gonadotropin (GtH) subunit [follicle stimulating-β (FSH-β), luteinizing hormone-β (LH-β), glycoprotein hormone-α (GPH-α)] genes, plasma gonadal steroids [estradiol (E2) and testosterone (T)], gonadal histology, and gonadosomatic index (GSI) in adult females exposed to combinations of short-day (8xa0h) or long-day (16xa0h) photoperiods and low (12°C) or high (20°C) temperatures after winter conditions (8xa0h light, 12°C) were analyzed. Pejerrey females kept under the short photoperiod had low GSIs, and their ovaries contained only previtellogenic oocytes regardless of the experimental temperature. In contrast, females exposed to the long photoperiod had high GSIs and ovaries with vitellogenic oocytes at both temperatures. These fish also showed a significantly higher expression of sGnRH, pjGnRH, cGnRH-II (the three different GnRH variants found to date in the pejerrey brain), FSH-β, LH-β and GPH-α genes and plasma E2 levels than those at the shorter photoperiod. No significant changes were observed in plasma T levels. Based on these results, we concluded that the increase in day length but not that of temperature triggers the maturation of pejerrey females after the winter period of gonadal rest and that this occurs by an integrated stimulation of the various components of the brain–pituitary–gonad axis.

Gonadotropin-releasing hormone (GnRH) neuronal systems in the pejerrey, Odontesthes bonariensis (Atheriniformes)

The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.

Close association of gonadotropin-releasing hormone fibers and gonadotropin, growth hormone, somatolactin and prolactin expressing cells in pejerrey, Odontesthes bonariensis

The purpose of this study was to determine if there is any association between immunoreactive (ir) gonadotropin-releasing hormone (GnRH) fibers with different pituitary endocrine cell types in the pejerrey, Odontesthes bonariensis. Using a monoclonal antibody raised against mammalian GnRH (mGnRH) (LRH13), ir-GnRH fibers were observed passing through the pituitary stalk and reaching the three areas of the pituitary gland: rostral (RPD) and proximal pars distalis (PPD) and pars intermedia (PI). Double labeled immunocytochemistry showed ir-GnRH fibers in close association with prolactin (PRL)-producing cells in the RPD, growth hormone (GH)-producing cells in the PPD, gonadotropin (GtH)-producing cells in the PPD and the external border of the PI, and with somatolactin (SL)-producing cells in the PI. Our results show, direct morphological evidences of a close association of GnRH fibers with GH, PRL, GtH and SL-expressing cells. These results would suggest that GnRH has a broad role in the regulation of the secretion of different pituitary hormones.

GnRH Molecular Variants in the Brain and Pituitary Gland of Pejerrey, Odontesthes bonariensis (Atheriniformes). Immunological and Chromatographic Evidence for the Presence of a Novel Molecular Variant

Gonadotropin-releasing hormone (GnRH) molecular variants in the brain and pituitary gland of pejerrey, Odontesthes bonariensis (Atheriniformes), were characterized by gradient reverse phase high performance liquid chromatography (RP-HPLC). Eluted fractions were tested in radioimmunoassays with different antisera. The results show that the brain extract contains three forms of GnRH: one is immunologically and chromatographically similar to cIIGnRH (chicken II), and another is similar to sGnRH (salmon). A third GnRH appears to be chromatographic and immunologically different from the nine other known forms of the vertebrate hormone. This is the only variant present in the pituitary gland.

Identification of immunoreactive mammalian gonadotropin-releasing hormone in the brain of metamorphic larvae of Bufo arenarum hensel (Amphibia: Anura)

Gonadotropin‐releasing hormone (GnRH) immunoreactivity in brain extracts of Bufo arenarum tadpoles were investigated by high‐performance liquid chromatography, followed by radioimmunoassay analysis using two different antisera raised against different GnRH variants. Only one immunoreactive peak was identified, eluting in the same position as synthetic mammalian GnRH. This result was further confirmed by serial dilution studies using more specific mammalian GnRH antisera. Our results suggest that mammalian GnRH is most likely an endogenous peptide in the brain of the developing larvae and froglets of Bufo arenarum and quite likely it is the only GnRH variant present during those development stages. The distribution and density of cell bodies and fibers were analysed by immunocytochemical procedures. Immunoreactive cell bodies appeared in the olfactory epithelium and across the olfactory nerve at late prometamorphic larval stages. Near the metamorphic climax and in froglets, perikarya and fibers were detected in basal forebrain, preoptic and hypothalamic areas. No immunoreaction was observed at midbrain, hindbrain and spinal cord levels. This study suggests that mammalian GnRH is most likely an endogenous peptide and is probably the only GnRH variant in the brain of the developing larvae and froglets of Bufo arenarum.

Characterization of brain gonadotropin-releasing hormone (GnRH) molecular variants in brain extracts from different perciform fishes from Antarctic waters

Abstract Gonadotropin-releasing hormone (GnRH) is the hypothalamic hormone that regulates the reproductive system by stimulating release of gonadotropins from the anterior pituitary gland. The molecular variants of the reproductive neuropeptide GnRH were characterized from brain tissue of three perciform species from Antarctic waters: Pseudochaenichthys georgianus, Chaenocephalus aceratus, and Notothenia rossi. The study involved reverse phase high-performance liquid chromatography (RP-HPLC) followed by radioimmunoassay (RIA) with two antisera that recognize all GnRH variants already identified: PBL 45 and PBL 49. The results showed that brain extracts of P. georgianus, C. aceratus, and N. rossi contain, like those of other perciform fish, three forms of GnRH likely to be: sbGnRH (seabream GnRH), cGnRH-II (chicken GnRH II) and sGnRH (salmon GnRH). They also showed evidence for the presence of a fourth GnRH variant, chromatographically and immunologically different from the other known forms of the vertebrate hormone. Although final conclusions will require isolation, purification, and sequencing of these molecules, these results offer encouraging possibilities of further advances in the characterization of a multiplicity of GnRH molecular variants.

Immunocytochemical study of pituitary FSHß and LHß cells during the ontogeny of the toad Bufo arenarum

The ontogeny of gonadotrophs in the pituitary of Bufo arenarum was studied by immunocytochemical techniques using monoclonal antibodies against bullfrog lutropin s-subunit and bullfrog follitropin s-subunit. The first appearance of immunoreactive gonadotropic cells was at midprometamorphosis in the caudal zone of the pars distalis. During metamorphosis, in juveniles and especially in adult animals, follicle stimulating hormone (FSH) and luteinizing hormone (LH) producing cells were distributed throughtout the gland. The number of these cells was greatly increased in older stages. In all the stages studied colocalization of FSH and LH was observed. At least three types of gonadotrophs were identified: the first cell type containing only FSH; the second only LH; and the third, FSH and LH in the same cell.