Brain Gonadotropin-releasing Hormone Research Articles

An Enzyme Immunoassay for Salmon Gonadotropin-Releasing Hormone and Its Application to the Study of the Effects of Diet on Brain and Pituitary GnRH in the Sea Bass, Dicentrarchus labrax

The effects of two different diets [diet 1 (D1), high protein-low carbohydrate; diet 2 (D2), low protein-high carbohydrate] on brain and pituitary gonadotropin-releasing hormone (GnRH) contents, as well as circulating steroids and vitellogenin, were studied over the reproductive period of the sea bass. Salmon GnRH was measured using a newly developed competitive enzyme immunoassay with an enzymatic tracer made of sGnRH covalently coupled to actetylcholinesterase from the electric organ of the eel Electrophorus electricus. The pituitary GnRH content of animals of both sexes fed D1 was significantly reduced at the time of spawning compared with the pre- and postspawning stages, whereas fish fed D2 did not exhibit such changes. In the brain only minor differences in the GnRH content were observed between the two diets. It is concluded that GnRH release rather than synthesis is affected in fish fed a low protein-high carbohydrate regimen. Plasma sex steroids and vitellogenin were not greatly affected by the diet.

Immunoreactive GnRH Suggesting a Third Form of GnRH in Addition to cIIGnRH and sGnRH in the Brain and Pituitary Gland of Prochilodus lineatus (Characiformes)

Molecular variants of GnRH (gonadotropin-releasing hormone) in brain and pituitary extracts of the South American characiforme Prochilodus lineatus were studied using a combination of reverse-phase high-performance liquid chromatography and radioimmunoassay with different antisera. In brain extracts our study revealed that this fish has at least two different types of GnRH: cIIGnRH (chicken II) and sGnRH (salmon), and possibly a third variant of this molecule. In pituitary extracts we could find only two immunoreactive peaks corresponding to sGnRH and the possible third form.

Differential distribution and response to experimental sexual maturation of two forms of brain gonadotropin-releasing hormone (GnRH) in the European eel, Anguilla anguilla

Using specific radioimmunoassays for the two GnRH molecular forms present in the European eel, Anguilla anguilla, (mGnRH and cGnRH II), we compared their distributions in the pituitary and different parts of the brain of female silver eels, as well as the modifications of their levels in experimentally matured female eels (treated with carp pituitary extract). In control eels, mGnRH levels were higher than cGnRH II levels in the pituitary, olfactory lobes and telencephalon, di- and mesencephalon, while the opposite was found in the posterior part of the brain (met- and myelencephalon). Experimental sexual maturation of the gonads significantly increased mGnRH levels in the pituitary and anterior parts of the brain; such a positive effect was not observed on the low cGnRH II levels, which were, in contrast, reduced. These data indicate that the positive feedback of gonadal hormones on GnRH, that we previously demonstrated, would specifically affect the mGnRH form. The differential distribution and control of mGnRH and cGnRH II suggest that these two forms have different physiological roles in the eel. The large increase in mGnRH during sexual maturation suggests the prime implication of this form in the neuroendocrine control of reproduction.

Changes in brain gonadotropin-releasing hormone, plasma estradiol 17-beta, and progesterone during the final reproductive cycle of the female sea lamprey, Petromyzon marinus.

Changes in ovarian morphology, brain gonadotropin-releasing hormone (GnRH), plasma estradiol, and progesterone were examined during the 1988 and 1989 spawning migrations of the adult female sea lamprey, Petromyzon marinus. There were significant increases through time in brain GnRH (1989) and plasma estradiol (1988 and 1989), with progesterone levels fluctuating (1988 and 1989) during the freshwater phase of the spawning migrations. In 1989, brain GnRH and plasma estradiol levels gradually increased through time until just prior to spawning when levels decreased. During 1988, there were no significant changes in GnRH, which may reflect lower temperatures in that year. These data provide new information on brain GnRH during the final maturational processes in the female sea lamprey.

Changes in brain gonadotropin-releasing hormone, pituitary and plasma gonadotropins, and plasma thyroxine during smoltification in chinook salmon ( Oncorhynchus tschawytscha)

Concentrations of brain salmon gonadotropin-releasing hormone (sGnRH), plasma gonadotropin I (GTH I), and pituitary GTH I and GTH II were determined in yearling chinook salmon ( Oncorhynchus tschawytscha) during the parr-smolt transformation in two successive seasons. There were significant elevations in brain sGnRH content from February to March in 1988, and from February to April in 1989. Increases in brain sGnRH content coincided with elevations in plasma thyroxine levels that occurred from February to March, 1988 and 1989. Plasma GTH levels were relatively constant (1–2 ng/ml) throughout the period of sampling. However, during 1988, plasma concentrations of GTH I decreased significantly between late March and early April. During 1989, plasma GTH I levels appeared to reach a peak (2 ng/ml) in mid-February, but otherwise remained near 1 ng/ml. Previous studies have shown that GTH II was not detectable in plasma at this stage. During 1989, pituitary GTH I concentrations were 50- to 70-fold higher than that of GTH II, and increased, though not significantly, from February through April. Although GTH II was detected in the pituitary by RIA, it is likely that the measurable levels are due to GTH I cross-reaction in the GTH II RIA. Histological examination of the gonads indicated that throughout smoltification the oocytes remained in the perinucleolar stage of oogenesis and the testes were in the spermatogonial stage of spermatogenesis. Although no observable changes in gametogenesis occurred, the changes in brain sGnRH content, plasma GTH I levels, and pituitary GTH content suggest that some changes in the hypothalamic-pituitary axis may occur during smoltification.

Effects of courtship on brain gonadotropin hormone-releasing hormone and plasma steroid concentrations in a female amphibian ( Taricha granulosa)

Courtship-induced changes in plasma steroid and brain gonadotropin-releasing hormone (GnRH) concentrations in Taricha granulosa were determined with respect to changes in female sexual receptivity. Females were sacrificed at several times after courtship initiation. Concentrations of GnRH (determined by RIA) in the anterior telencephalon were high at courtship initiation (females unreceptive), but decreased by sperm transfer (females receptive). Courtship had no affect on GnRH concentrations in any other brain region examined. Furthermore, courted, receptive females had higher plasma levels of estradiol than did uncourted controls, and estradiol levels remained elevated above control levels 24 hr after courtship initiation. Courtship had no influence on plasma progesterone or corticosterone levels. To determine if the observed changes in GnRH concentrations in the telencephalon were localized to the nervus terminalis, courted females and controls were sacrificed after 5, 20, or 60 min of courtship. Nervus terminalis GnRH concentrations were higher in courted females than in uncourted controls. These results may represent the first documentation of a naturally occurring physiological change in the nervus terminalis.

Changes in brain levels of gonadotropin-releasing hormone and serum levels of gonadotropin and growth hormone in goldfish during spawning

Changes in serum levels of gonadotropin (GtH) and growth hormone (GH) and in levels of gonadotropin-releasing hormone (GnRH) in the pituitary and discrete brain areas were studied in male goldfish during spawning with spontaneously ovulating females. Spontaneous ovulation in females was induced by raising water temperature from 12 to 20 °C and providing spawning substrate of artificial floating vegetation. Spawning occurs naturally in sexually mature male goldfish in the presence of ovulating females. Serum GtH levels in spawning male goldfish exposed to ovulatory females increased markedly in synchrony with the ovulatory GtH surge in females. There was also a significant increase in serum GH levels in the spawning males. GnRH levels in the olfactory bulbs, telencephalon, hypothalamus, and pituitary of the spawning males showed marked decreases at the same time as serum levels of both GtH and GH peaked; these events in the males corresponded to the approximate time of ovulation, and similar changes in serum GtH and GH levels and brain GnRH levels, in the females. This temporal correlation between changes in GnRH levels in the brain and pituitary and increases in serum levels of GtH and GH in males as well as females supports the idea that activation of the GnRH neuronal system may be a common pathway for the stimulation of pituitary GtH and GH secretion in goldfish during spawning.

Relationship between brain gonadotropin-releasing hormone and final reproductive period of the adult male sea lamprey, Petromyzon marinus

Gonadotropin-releasing hormone (GnRH) concentrations were measured in brains of adult male sea lamprey, Petromyzon marinus, during their final reproductive period. The lampreys were collected during their upstream migration in coastal New Hampshire rivers and sampled at the trap (referred to as group A) or they were transferred to an artificial spawning channel (referred to as Group B). Plasma estradiol and progesterone were also measured, and histological examination of the gonadal stages was done as well. The concentrations of brain GnRH and plasma estradiol fluctuatedsignificantly through time. There was a rise in brain concentrations of GnRH coincident with an increase in temperature just prior to spawning. In addition, there was a significant progressive correlation between increasing plasma estradiol and temperature in lampreys from Group B during the period studied. These studies provide evidence for progressive seasonal relationships between changes in brain GnRH and gametogenic and steroidogenic activity of the gonads in adult male sea lampreys during their final reproductive period.

Gonadotropin releasing hormone binding sites in rat hippocampus: Different structure/binding relationships compared to the anterior pituitary

In vitro autoradiography and radioreceptor assays were used to assess the binding characteristics of the gonadotropin releasing hormone (GnRH) binding sites in rat hippocampus. Competition studies with various GnRH agonists, antagonists, and fragments of GnRH indicate that the structure/binding requirement of the brain GnRH receptors is similar, but not identical, to that described in the pituitary. In the hippocampas, GnRH and the agonist d-Ala 6-GnRH exhibit an IC 50 for displacement of 125I-[ d-Ser(tBu) 6-Pro 9-NHEt]-GnRH (Buserelin, Hoechst) similar to that of the anterior pituitary; in contrast, the antagonist [ d- p-Glu 1- d-Phe 2- d-Trp 3,6]-GnRH bound only to the anterior pituitary GnRH receptor. Similarly, [His 5-Trp 7-Tyr 8]-GnRH (chicken II) recognized the pituitary GnRH receptor, but did not bind to hippocampal membranes. The difference in the binding affinities is likely not due to differential metabolism because of the stability of the tracer and of the antagonist and the addition of several enzyme inhibitors to the incubation buffer. The GnRH fragments [4–10]GnRH, [5–10]-GnRH, and [Ac5–10]-GnRH and [Gln 8]GnRH (chicken I), [Trp 7-Leu 8]-GnRH (salmon), and [Tyr 3-Leu 5-Glu 6-Trp 7-Lys 8]-GnRH (lamprey) did not displace the radiolabeled agonist binding in either the rat brain or the pituitary. Similarly, GTP and GTPγS had no effect on the binding of the GnRH agonist 125I-[ d-Ser(tBu) 6-Pro 9-NHEt]-GnRH (buserelin) to hippocampal or pituitary membrane preparations. Photoaffinity labeling of pituitary and hippocampal GnRH binding sites with 125I-[azidobenzoyl- d-Lys 6]-GnRH followed by one-dimensional gel electrophoresis in sodium dodecyl sulfate and by autoradiography revealed, in both tissues, two similar bands of proteins with apparent molecular weights of 29,000 and 60,000 Da. The results suggest that GnRH acts in the brain through specific binding sites that share some but not all of the characteristics of the pituitary receptor. Brain GnRH receptors are likely the site through which the neuropeptide can act to modify behavioral, sensory, and endocrine events.

Chromatographic and immunological evidence for mammalian GnRH and chicken GnRH II in eel (Anguilla anguilla) brain and pituitary

Gonadotropin-releasing hormone (GnRH) peptides in the brain and pituitary of the European eel (Anguilla anguilla) were investigated by reverse phase high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. Two GnRH molecular forms were demonstrated in brain and pituitary extracts. One form eluted in the same position as synthetic mammalian GnRH on HPLC and was recognized by antibodies directed against the NH 2 and COOH termini of mammalian GnRH as well as by antibodies to the middle region. The second form eluted in the same position as synthetic chicken GnRH II and was recognized by specific antibodies to this molecule. Salmon GnRH and chicken GnRH I were not detected. The occurrence of mammalian GnRH in teleost fish suggests that this molecular form is more ancient than was previously suspected and arose earlier than in primitive tetrapods, or that it has arisen in the eel through random mutation of salmon GnRH. The lack of salmon GnRH in the eel brain indicates that this molecular form is not common to all teleost species. The finding in eel brain of chicken GnRH II, which has previously been described in species of Mammalia, Aves, Reptilia, Amphibia, Osteichthyes, and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.

Dopaminergic Regulation of Brain Gonadotropin-Releasing Hormone in Male Goldfish during Spawning Behavior

Dopaminergic regulation of gonadotropin-releasing hormone (GnRH) concentrations in discrete brain areas of male goldfish was studied using the centrally active dopaminergic agonist apomorphine and antagonist pimozide. Pimozide caused time- and dose-dependent increases in serum gonadotropin levels and accumulation of GnRH in the olfactory bulbs, telencephalon and pituitary. The effects of pimozide were partially antagonized by apomorphine, which, when given alone, did not influence basal levels of brain GnRH and serum gonadotropin, but inhibited the increases in brain GnRH and serum gonadotropin levels that normally occur in response to spawning stimuli from prostaglandin-treated females. To study the physiological significance of the accumulation of brain GnRH due to central dopaminergic receptor blockade, the influences of pimozide on the brain GnRH and serum gonadotropin responses of male goldfish to spawning stimuli were tested. Pimozide pretreatment potentiated the serum gonadotropin increases and caused a marked reduction in the GnRH levels in olfactory bulbs, telencephalon and pituitary in response to spawning stimuli. These findings demonstrate that the central dopaminergic system is an important inhibitory component in the regulatory circuitry of brain GnRH levels in normal and behaviorally stimulated male goldfish.

Combined immunohistochemistry for gonadotropin-releasing hormone (GnRH) and pro-GnRH, and in situ hybridization for GnRH messenger ribonucleic acid in rat brain.

Experiments were performed to explore the distribution of neurons containing pro-GnRH and GnRH in the rat brain and to determine the correspondence of immunoreactive peptides and pro-GnRH mRNA. Using avidin-biotin immunohistochemistry on free floating vibratome sections it was found that pro-GnRH- and GnRH-containing cells exhibited a similar distribution within the preoptic area-basal hypothalamus region. Within individual neurons pro-GnRH was primarily detected in the cell soma and proximal fibers, whereas the decapeptide was present in cells, fibers, and nerve terminals. Combined avidin-biotin immunohistochemistry for GnRH or pro-GnRH peptides and in situ hybridization for pro-GnRH mRNA using a cRNA probe revealed that the peptides and mRNA could be detected in the same cells. In both male and female rats pro-GnRH mRNA was localized primarily in GnRH-containing cells; however, not all immunoreactive GnRH neurons contained detectable levels of pro-GnRH mRNA, and not all neurons containing pro-GnRH mRNA contained GnRH peptides. In proestrous females a close correlation existed between the total number of neurons containing GnRH and those containing pro-GnRH mRNA (r = 0.84-0.9), while in intact male rats the correlation was not as high (r = 0.56). These results document the distribution of pro-GnRH and GnRH in the rat preoptic area-basal hypothalamus and describe the extent of colocalization of GnRH peptide and pro-GnRH mRNA in proestrous females and intact male rats. Further work will determine how each of the molecular components is regulated during different reproductive states.

Effects of estrogen on hypothalamic gonadotropin-releasing hormone release in castrated male rhesus macaques

The evidence that hypothalamic gonadotropin-releasing hormone (GnRH) release increases during the estrogen-induced luteinizing hormone (LH) surge in castrated female macaques and that estrogen induces a similar LH surge in the male suggests that either sexual differentiation of GnRH secretion does not exist or that changes in brain GnRH are not critical for ovulation. We tested this hypothesis by using the push-pull perfusion (PPP) technique to monitor GnRH release in the mediobasal hypothalamus continuously from 10 h before to 50 h after estrogen injection in 9 castrated male rhesus macaques. Blood sampling and PPP were performed with monkeys freely moving in their own cage by using a specially designed swivel/tethering system. PPP samples were collected every 10 min and analyzed for GnRH concentration by radioimmunoassay. Blood samples were collected every hour and plasma LH was measured by bioassay. Estradiol benzoate (EB, 42 μg/kg, b. wt.) was subcutaneously injected after 10 h of initial PPP. The PPP was continued for 10 h after EB in 4 and 50 h after EB in 5 monkeys. Hypothalamic GnRH patterns were analyzed by the Pulsar algorithms. The results show that during the 10 h after EB, plasma LH declined rapidly, reaching low or non-detectable levels by 7–9 h, while hypothalamic GnRH releasing patterns did not change during this period ( n = 9). In contrast, estrogen enhanced GnRH level and pulse amplitude, but not pulse frequency, several hours before mean peripheral plasma LH increased from suppressed values ( n = 4). These data suggest that the immediate negative feedback effect of estrogen on LH release occurs mainly in the pituitary, and that estrogen subsequently stimulates hypothalamic release of GnRH in advance of the initial rise in plasma LH. The results further support the hypothesis that the gonadotropin-releasing mechanism in primates is functionally non-differentiated, and that increased release of hypothalamic GnRH is actively involved in regulating the estrogen-induced gonadotropin surge in both sexes.

Immunocytochemical Localization of Gonadotropin and Gonadotropin Releasing Hormone in Pituitary and Brain of the Gilthead Seabream, Sparusaurata L. (Teleost)

Characterization of gonadotropic cells in the pituitary gland of male gilthead seabream (Sparus aurata) was performed at the light- and electron microscopic level with the peroxidase-anti peroxidase and the immunogold technique, respectively. The cells were identified with antibodies raised against three different teleost gonadotropins (carp α, β-GTH, carp β-GTH, catfish α, β-GTH). Immunostained cells were mainly located in the ventral and dorsal areas of the proximal pars distalis (PPD), with a few cells in the pars intermedia (PI). At the light microscopic level the gonadotrops showed a variable granulation and they were often strongly vacuolated. In the electron microscope the latter appeared to be caused by strongly dilated rough endoplasmic reticulum (RER). Immunogold labeling was restricted to all secretory granules and most of the larger globules. Different fixation and embedding procedures were tested: an optimal combination of good ultrastructure and intense immunogold labeling was obtained when osmium tetroxide postfixation was omitted and with embedding in 'Epon. The distribution of gonadotropin-releasing hormone (GnRH) immunoreactivity was studied with antibodies against mammalian LHRH and salmon GnRH. Only with anti-mammalian LHRH immunostained perikarya were observed in the ventral preoptic area. With both antisera immunostained fibers were seen directed towards the pituitary stalk, as well as in the neurohypophysis, whereas no such fibers were present in the PPD. Immunostained fibers were also found in several other regions of the brain.

Brain distribution of radioimmunoassayable gonadotropin-releasing hormone in female goldfish: Seasonal variation and periovulatory changes

A radioimmunoassay (RIA) for [Trp 7, Leu 8]gonadotropin-releasing hormone (sGnRH) was developed to determine the gonadotropin-releasing hormone (GnRH) content in discrete brain areas of female goldfish at different stages of ovarian development. Temporal changes in serum gonadotropin (GtH) and GnRH concentrations in discrete brain areas were measured during spontaneous ovulation. There were no clear parallel changes in brain GnRH with seasonal ovarian development in goldfish. However, under a 10° temperature acclimation regimen, the GnRH content in the hypothalamus and pituitary decreased as the ovary progressed from the regressed to the mature condition; on the other hand, GnRH content in the spinal cord increased in sexually mature fish compared with that in regressed fish. Significant decreases in GnRH concentration were observed in certain brain areas (olfactory bulbs, telencephalon, hypothalamus, and pituitary) of fish undergoing spontaneous ovulation compared with those of nonovulatory fish. The simultaneous changes of GnRH concentration in these brain areas suggested that the GnRH neuronal system may function as an integrated unit for the activation of GtH secretion during ovulation in goldfish.

Ontogeny of gonadotropin releasing hormone and gonadotropin immunoreactivity in brain and pituitary of normal and estrogen-treated guppies, Poecilia reticulata Peters

Gonadotropin releasing hormone (GnRH) and gonadotropic hormone (GTH) were identified by immunohistochemistry in the brains and pituitaries of neonate, juvenile and adult guppies. GTH was present in some cells of the pars intermedia (pi) and proximal pars distalis (ppd) of all animals. GnRH was found in the perikarya of the nucleus olfactoretinalis. In the pituitaries of juvenile 30-day-old guppies, GnRH-immunoreactive cells existed in a "juvenile pattern", whereas in adult animals GnRH was recognized in only a few cells. GnRH-immunoreactive fibers were seen in the pituitaries of animals that were 30 days or older. In adult guppies, the ventral and lateral ppd (the gonadotropic region) contained a dense network of GnRH-immunoreactive fibers. Pituitary cells staining for either GnRH or GTH were located in different places. After immunohistochemical double staining of adult pituitaries, none of the GnRH-immunoreactive cells were LH-immunoreactive, although both cell types were often found in close proximity. After 20 days or more of ethinylestradiol treatment, less immunoreactive GnRH was detected in the pituitary cells of juvenile guppies, and fewer animals exhibited the "juvenile pattern" of GnRH-immunoreactive pituitary cells, when compared with untreated controls. The results indicate that GnRH-immunoreactive pituitary cells in the guppy are distinct from gonadotropes and that these cells are involved in regulatory processes along the juvenile brain-pituitary-gonad axis.

Identification of diverse molecular forms of GnRH in reptile brain

Gonadotropin-releasing hormone (GnRH) molecular forms in the brains of three reptiles, Alligator mississippiensis (alligator), Calcides ocellatus tiligugu (skink) and Podarcis s. sicula (lizard) were characterized by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera, and by assessment of luteinizing (LH)-releasing activity in chicken dispersed pituitary cells. In alligator brain two GnRHs had identical properties to the two known forms of chicken hypothalamic GnRH (Gln 8-GnRH and His 5,Trp 7,Tyr 8-GnRH) in their elution on two reverse phase HPLC systems, cross-reaction with region-specific GnRH antisera, and ability to release LH. In skink brain, one immunoreactive and bioactive GnRH form, which eluted in the same position as His 5,Trp 7,Tyr 8-GnRH on reverse phase HPLC, was identified. Three bioactive and immunoreactive GnRHs were detected in lizard brain. One form had similar properties to salmon brain GnRH (Trp 7,Leu 8-GnRH). The other two GnRH-like peptides are novel forms. One of these forms eluted in the same position as Gln 8-GnRH on HPLC but had different immunological properties, while the third form was a rather hydrophobic species which appeared to be modified in the middle region of the molecule.

Multiple forms of gonadotropin-releasing hormone in amphibian brains

Several forms of gonadotropin-releasing hormone (GnRH)-like molecules were found in brains of both anurans (frogs) and urodeles (salamanders). The presence of the mammalian-like GnRH molecule was confirmed by HPLC and cross-reactivity studies. Small amounts of salmonid-like GnRH molecules in the brains of frogs ( Rana pipiens, Hyla regilla) and salamanders ( Taricha granulosa, Ambystoma gracile) were detected by comparing the HPLC chromatographic pattern and immunological reactivity of the brain extracts with native trout and synthetic salmon GnRH. This nonmammalian form of GnRH in the amphibian brain is similar and perhaps identical, at least by indirect evidence, to a form of GnRH reported earlier to be in sympathetic ganglion, retina, chromaffin tissue, and tadpole brain. If two of the amphibian GnRH molecules prove to be mammalian and salmon GnRH, then it is likely that two separate genes in amphibians code for the distinct primary structures of the molecules. The most parsimonious interpretation of the presence of both mammalian- and salmon-like GnRH in anurans and urodeles is that a common phylogenetic ancestor also possessed the two forms of GnRH. Thus the mammalian form of GnRH may well have been present in labyrinthodont amphibians. Independent of evolutionary origin, the functions of the different GnRH molecules in amphibians are unknown.

Immunoreactive gonadotropin-releasing hormone-like material in the brain and the pituitary gland during the periovulatory period in the brown trout ( Salmo trutta L.): Relationships with the plasma and pituitary gonadotropin

In fish there are few data on the gonadotropin-releasing hormone (Gn-RH) neurosecretory activity, which could explain long- and short-term variations of the gonadotropin secretion. There is no biological species specificity between mammal and fish Gn-RH; although there is a structural difference, they are, on the contrary, characterized by a high immunological specificity which does not allow measurement of fish Gn-RH using radioimmunoassay for LH-RH. We have synthesized salmon Gn-RH according to the formula recently proposed by Sherwood ( N. Sherwood, L. Eiden, M. Brownstein, J. Spies, J. Rivier, and W. Vale, 1983. Proc. Natl. Acad. Sci. USA 80, 2794–2798). Its activity has been tested by its ability to stimulate the gonadotropin hormone (GtH) secretion in vivo in testosterone-implanted juvenile rainbow trout, and for the recognition of synthesized Gn-RH (s-Gn-RH) perykaria by a specific antibody raised against the s-Gn-RH in regions of the brain described as containing LH-RH immunoreactive-like material. A radioimmunoassay has been developed for the salmon Gn-RH, and its specificity to measure trout Gn-RH has been tested. Using this assay, the brain and pituitary Gn-RH contents have been measured throughout the final phases of maturation and ovulation. Brain Gn-RH increases from the end of vitellogenesis (8.9 ± 0.76 ng/brain) to ovulation (more than 15 ng/brain). Pituitary Gn-RH is lower (1.58 ± 0.69 ng/pituitary) at the end of vitellogenesis and follows a similar profile as in the brain, except for a significant decrease just prior the beginning of oocyte maturation. The correlations between Gn-RH levels and GtH pituitary and plasma levels show that total brain Gn-RH is never correlated to the GtH, suggesting that the increase in the brain Gn-RH content is related to a Gn-RH system closely related to maturation and ovulation, which remains to be investigated. On the contrary, pituitary Gn-RH levels are well correlated with pituitary and plasma GtH levels, indicating that pituitary Gn-RH levels might represent a good index of the Gn-RH neurosecretory activity in the fish hypothalamohypophysial complex, given the absence of a portal system in teleost.

Multiple molecular forms of gonadotropin-releasing hormone in teleost fish brain

Gonadotropin-releasing hormone (GnRH) immunoreactive peptides in extracts of hake ( Merluccius capensis) and tilapia ( Tilapia sparrmanii) brain were investigated by high performance liquid chromatography (HPLC) and radioimmunoassay with region-specific antisera. In hake brain, content and concentration of GnRH was higher in the pituitary gland than in the hypothalamic lobes or extrahypothalamic brain. Hake pituitary gland GnRH was purified by six consecutive HPLC systems. The major GnRH molecular form co-eluted with salmon brain GnRH (Trp 7, Leu 8-GnRH) in four different HPLC systems which were specifically designed to separate the four natural vertebrate GnRHs (mammalian, salmon, chicken I and II). The immunoreactive peak in the final purification step had a retention time identical to that of Trp 7, Leu 8-GnRH and an UV absorbance (280 nm) peak appropriate for two tryptophan residues in the peptide, as in Trp 7, Leu 8-GnRH. Six additional less hydrophobic forms of GnRH were detected. Tilapia brain extract contained two major GnRH molecular forms which had identical retention times to chicken GnRH I (Gln 8-GnRH) and Trp 7, Leu 8-GnRH in an HPLC system which separates the natural vertebrate GnRHs. The immunological properties of these two immunoreactive peaks, determined by relative interaction with four region-specific GnRH antisera raised against vertebrate GnRHs, were identical to those of Gln 8-GnRH and Trp 7, Leu 8-GnRH. Additional GnRH molecular forms were also detected. In summary, these findings indicate that a major GnRH molecule in hake pituitary gland is Trp 7,Leu 8-GnRH, while tilapia brain contains both Trp 7,Leu 8-GnRH and Gln 8-GnRH. Additional GnRH molecular forms were detected in both species. The presence of multiple GnRH-related molecules in teleost fish brain suggests that the GnRH molecule has experienced gene duplication during evolution and the different structures co-opted for as yet undetermined physiological functions.