Gonadotropin-inhibitory hormone neurons interact directly with gonadotropin-releasing hormone-I and -II neurons in European starling brain

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic dodecapeptide (SIKPSAYLPLRF-NH(2)) that directly inhibits gonadotropin synthesis and release from quail pituitary. The action of GnIH is mediated by a novel G-protein coupled receptor. This gonadotropin-inhibitory system may be widespread in vertebrates, at least birds and mammals. In these higher vertebrates, histological evidence suggests contact of GnIH immunoreactive axon terminals with GnRH neurons, thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study we investigated the interaction of GnIH and GnRH-I and -II neurons in European starling (Sturnus vulgaris) brain. Cloned starling GnIH precursor cDNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini. Starling GnIH was further identified as SIKPFANLPLRF-NH(2) by mass spectrometry combined with immunoaffinity purification. GnIH neurons, identified by in situ hybridization and immunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus. GnIH immunoreactive fiber terminals were present in the external layer of the median eminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons were shown by GnIH and GnRH double-label ICC. Furthermore, the expression of starling GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by in situ hybridization combined with GnRH ICC. The cellular localization of GnIH receptor has not previously been identified in any vertebrate brain. Thus, GnIH may regulate reproduction of vertebrates by directly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing the pituitary gland.     

FMRFamide-like immunoreactive nervus terminalis innervation to the pituitary in the catfish, Clarias batrachus (Linn.): demonstration by lesion and immunocytochemical techniques.

Certain thick FMRFamide-like immunoreactive fibers arising from the ganglion cells of nervus terminalis in the olfactory bulb of Clarias batrachus can be traced centripetally through the medial olfactory tract, telencephalon, lateral preoptic area, tuberal area, and hypothalamohypophysial tract to the pituitary. Following 6 days of bilateral olfactory tract transection, the immunoreactivity in the thick fibers, caudal to the lesion site, was partially eliminated, whereas after 10 and 14 days, it was totally abolished in the processes en route to the pituitary. The results indicate a direct innervation of the pituitary gland by the FMRFamide-like peptide containing fibers of the nervus terminalis.     

Thyroid hormone and estrogen regulate brain region-specific messenger ribonucleic acids encoding three gonadotropin-releasing hormone genes in sexually immature male fish, Oreochromis niloticus

The present study was undertaken to determine whether T3, estrogen, and 11-ketotestosterone could alter a specific population of GnRH-containing neurons, as indicated by a change in messenger RNA (mRNA) levels in sexually immature male tilapia, Oreochromis niloticus. Two weeks after castration, fish were assigned to four treatment groups. One group served as the control (sesame oil); a single ip injection of (T3; 5 microg/g), estradiol benzoate (EB; 5 microg/g), or 11-ketotestosterone (KT; 5 microg/g) was administered to the remaining three groups. Twenty-four hours after the injection, brains were collected and processed for in situ hybridization histochemistry using 35S-labeled 30-mer antisense oligonucleotide probes complementary to the GnRH-coding region of chicken II, salmon, and seabream GnRH. Computerized image analysis was performed to quantify mRNA concentrations, neuronal numbers, and neuronal size of the terminal nerve-nucleus olfactoretinalis, preoptic, and midbrain GnRH neurons. KT had no effect on any of the above neuronal parameters examined for salmon or seabream GnRH. Neither T3, EB, nor KT was effective to induce changes in midbrain chicken GnRH II mRNA concentrations, neuronal numbers, and neuronal size, indicating that an as yet unknown regulatory mechanism may operate midbrain GnRH neurons. T3 specifically suppressed the concentration of terminal nerve salmon GnRH mRNA, and EB significantly increased preoptic seabream GnRH neuronal numbers. These results are consistent with the hypothesis that thyroid hormone, by suppressing terminal nerve GnRH expression, promotes inhibition of sexual maturation. Furthermore, the failure of KT, a nonaromatizable androgen, to influence preoptic GnRH neurons emphasizes that an estrogenic pathway, at the onset of sexual maturation, is responsible for the recruitment of additional preoptic GnRH neurons that are fundamental to reproduction and behavior.     

Ontogeny of GnRH-like immunoreactive neuronal systems in the forebrain of the Indian major carp, Cirrhinus mrigala

GnRH immunoreactivity appeared in the medial olfactory placode very early in the development of Cirrhinus mrigala. The immunoreactive elements were divisible into distinct migratory and non-migratory components. The migratory component appeared as a patch of intensely immunoreactive cells located close to the olfactory epithelium in day 6 post-fertilization larvae. Subsequently, these neurons migrate caudally along the ventromedial aspect of the developing forebrain and enroute give rise to GnRH immunoreactive neurons in the (1) nervus terminalis located in ventral and caudal part of the olfactory bulb (day 8), and (2) basal telencephalon (day 9). The non-migratory GnRH immunoreactive component appeared in the olfactory placode of day 1 post-fertilization larvae. It consisted of few olfactory receptor neuron (ORN)-like cells with distinct flask-shaped somata, dendrites that communicate with the periphery and a single axon on the basal side; GnRH immunoreactivity was seen throughout the neuron. Considerable increase in the number of immunoreactive ORNs was encountered in day 2 post-fertilization larvae. On day 3, the dendrites of ORNs sprout bunches of apical cilia, while on the basal side the axonal outgrowths can be traced to the olfactory bulb. GnRH immunoreactive fibers were distributed in the olfactory nerve layer in the periphery of the bulb and glomeruli-like innervation was clearly established in 5 days old larvae. The innervation to the olfactory bulb showed a considerable increase in GnRH immunoreactivity in 9 and 19 days old larvae. However, GnRH immunoreactivity in non-migratory as well as migratory components gradually diminished and disappeared altogether by the age of 68 days. Results of the present study suggest that GnRH may serve a neurotransmitter role in the ORNs during early stages of development in C. mrigala.     

beta-endorphin and gonadotropin-releasing hormone in the forebrain and pituitary of the female catfish, Clarias batrachus: double-immunolabeling study

The role of beta-endorphin in modulating the gonadotropic action of gonadotropin-releasing hormone (GnRH) is well established in mammals. Although the information from teleosts also suggests that endogenous opioids modulate GnRH secretion and influence gonadotropic hormone release, the anatomical substrate in which opiate peptides and GnRH may interact has not been studied. Herein we describe the mammalian GnRH- and beta-endorphin-like immunoreactivities in the olfactory system, forebrain, and pituitary of the teleost, Clarias batrachus, using the double immunocytochemical method. While several olfactory receptor neurons showed beta-endorphin- or GnRH-like immunoreactivity, some neurons with dual immunoreactivities were also seen. GnRH- and/or beta-endorphin-like immunolabeled fascicles were seen in the olfactory nerves as they run caudally to the olfactory bulb and spread in the periphery. Several fascicles branch profusely to form tufts organized as spherical neuropils in the glomerular layer. Frequently, the innervation of the glomeruli showed a distinct pattern. While the fascicles on the medial side showed a predominance of beta-endorphin-like fibers, the majority of the fascicles on the lateral side of the bulb showed dual immunoreactivities. Several GnRH- and beta-endorphin-like immunoreactive fibers were seen in the medial olfactory tract as it extends through the telencephalon in the area ventralis telencephali/pars supracommissuralis; individual fibers with dual staining were also seen. The nucleus lateralis tuberis showed beta-endorphin- as well as GnRH-like immunoreactive neurons. While GnRH-containing cells were seen in the proximal pars distalis and pars intermedia, beta-endorphin-like cells were located throughout the pituitary; some cells in the pars intermedia showed dual immunoreactivity. The high degree of overlapping suggests the possibility of profound interplay between GnRH- and beta-endorphin-like immunoreactive systems at different levels of the neuraxis.     

Transplanted gonadotropin-releasing hormone neurons promote pulsatile luteinizing hormone secretion in congenitally hypogonadal (hpg) male mice

Congenitally hypogonadal (hpg) male mice are unable to synthesize biologically active gonadotropin-releasing hormone (GnRH). Implantation of normal fetal preoptic area tissue containing GnRH neurons into the third ventricle of adult hpg males significantly elevates pituitary levels of luteinizing hormone (LH) and corrects their hypogonadism. In all responding animals, immunoreactive GnRH neurons within the transplant innervate the median eminence of the host. To assess whether gonadal recovery in hpg hosts results from pulsatile secretion of GnRH from grafted neurons, we compared the pattern of variation in plasma LH levels in 19 hpg graft recipients with testicular growth to that of 10 normal adult mice. All animals were castrated prior to receiving an indwelling catheter in the jugular vein. Sequential blood samples were collected (t = 10 min) and assayed for LH. Pulsatile LH secretion was seen in 11 of 19 hpg hosts and in all control mice. While there was great variability between individual animals, measures of baseline LH, LH pulse amplitude and duration, interpulse interval, and LH pulse frequency revealed no difference between hpg graft recipients and normal castrates in their LH pulse pattern. Immunocytochemical analysis of the brain in hpg hosts suggested no correlation between any parameter of pulse activity and individual differences in GnRH cell number or GnRH fiber outgrowth into the median eminence. Sources of variation in LH secretion among graft recipients, and between hpg hosts and normal mice, are discussed. We suggest that transplanted GnRH neurons are capable of integration into a GnRH 'pulse generator' which can support a near-normal pattern of pulsatile LH secretion, leading to testicular growth and steroid production.     

Molecular cloning and tissue-specific expression of a gonadotropin-releasing hormone receptor in the Japanese eel

Gonadotropin-releasing hormone (GnRH) is a key regulatory neuropeptide involved in the control of reproduction in vertebrates. In the Japanese eel, one of the most primitive teleost species, two molecular forms of GnRH, mammalian-type GnRH and chicken-II-type GnRH (cGnRH-II), have been identified. This study has isolated a full-length cDNA for a GnRH receptor from the pituitary of the eel. The 3233-bp cDNA encodes a 380-amino acid protein which contains seven hydrophobic transmembrane domains and N- and C-terminal regions. The exon/intron organization of the open reading frame of the eel GnRH receptor gene was also determined. The open reading frame consists of three exons and two introns. The exon-intron splice site is similar to that of the GnRH receptor genes of mammals reported so far. Expression of the eel GnRH receptor was detected in various parts of the brain, pituitary, eye, olfactory epithelium, and testis. This result suggests that GnRH has local functions in these tissues in addition to its actions on gonadotropin synthesis and release in the pituitary. This tissue-specific expression pattern is similar to that of the eel cGnRH-II. Furthermore, the present eel receptor shows very high amino acid identity with the catfish and goldfish GnRH receptors, which are highly selective for the cGnRH-II. These results suggest that the cGnRH-II acts through binding to the present receptor in the eel.     

Immunohistochemical demonstration of the presence and localization of diverse molecular forms of gonadotropin-releasing hormone in the lizard (Podarcis s. sicula) brain.

The immunohistochemical presence and the distribution pattern of four different molecular forms of gonadotropin-releasing hormone (GnRH) were investigated in the brain of both sexes of the lizard, Podarcis s. sicula. Animals used in this study were collected in November and April, representing two different periods of the reproductive cycle. The antisera used were those raised against synthetic mammalian GnRH, chicken GnRH-I and II, and salmon GnRH. Strong immunoreaction was obtained for salmon, chicken-I, and chicken-II GnRHs, whereas a very weak reaction was seen for the mammalian form of GnRH. The distribution of immunoreactive-GnRH perikarya and fibers did not vary with the sex, the reproductive condition of the animals, or the antiserum used. Also, the intensity of immunoreaction with any one antiserum was quite similar in both periods of the year and in all brains examined. The immunoreactive perikarya was seen as two distinct groups, one in the mesencephalon and the other in the infundibulum. Immunoreactive fiber endings were seen in the telencephalon, the optic tectum, the anterior preoptic area, the median eminence, the central grey matter, the rhombencephalon, and the cerebellum. No immunoreactive perikarya were seen in the telencephalon or the anterior preoptic area.     

Forebrain gonadotropin-releasing hormone neuronal development: insights from transgenic medaka and the relevance to X-linked Kallmann syndrome

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.     

Differential brain distribution of gonadotropin-releasing hormone receptors in the goldfish

The present study describes the differential distributions in the brain of the two goldfish gonadotropin-releasing hormone (GnRH) receptors, using both immunohistochemistry and in situ hybridization approaches. The goldfish GnRH GfA and GfB receptors are variant forms of the same receptor subtype, although with distinct differences in ligand binding characteristics, and differential distributions in the pituitary and body tissues [Proc. Natl. Acad. Sci. USA 96 (1999) 2526]. The goldfish GnRH GfA receptor was found to be widespread throughout the brain, with neurons showing immunoreactivity in the olfactory bulbs, telencephalon, preoptic region, ventro-basal hypothalamus, thalamus, midbrain, motor neurons of the fifth, seventh, and tenth cranial nerves, reticular formation, cerebellum, and motor zone of the vagal lobes. The tracts in the posterior commissure, optic tectum, and motor zone of the vagal lobes also demonstrated immunoreactivity. While the brain was not systematically surveyed for in situ hybridization, hybridization was found in similar locations in the telencephalon, preoptic region, ventro-basal hypothalamus, cerebellum, and optic tectum. Hybridization was additionally found in the medial hypothalamus. The goldfish GnRH GfB receptor was found to have a more restricted distribution in the brain, with neurons showing immunoreactivity in the telencephalon, preoptic region, and ventro-basal hypothalamus. In situ hybridization demonstrated a somewhat wider distribution of expression of the receptor, with hybridization occurring in the preoptic region, ventro-basal and medial hypothalamus, as well as in the thalamus, epithalamus, and optic tectum. The widespread distribution of GnRH GfA receptor, and in particular its localization in the midbrain tegmentum in the region of the GnRH-II neurons, suggests that this receptor may be involved in the behavioral actions of GnRH peptides in the goldfish.     

Immunocytochemical studies on the pituitary gland of Anguilla anguilla L., in relation to early growth stages and diet-induced sex differentiation

Mammalian and teleost antisera against pituitary hormones were used to identify and localize pituitary cell types in the European eel (Anguilla anguilla L.). The investigation was conducted on unpigmented glass eels of 5.6-6.2 of total body length (L(T)) caught in river mouths, then on yellow eels reared from the pigmented glass eel (or elver) stage up to 12-14 cm of L(T), in an eel farm in warm freshwater. Treated elvers were fed with commercial paste food supplemented with mature carp ovaries, containing oestradiol, that induced an early ovarian differentiation and a higher growth rate. The antisera detected seven types of immunoreactive (ir) cells, six of which were already found in glass eel adenohypophysis, suggesting differentiation of these cell types during the leptocephalus stage. In 12-14 cm treated yellow eels with small ovaries, a seventh type (ir-GtH) was detected in the proximal pars distalis; in the same animals the ir-TSH cells increased in number and size. From unpigmented glass eels to 12-14 cm yellow eels, the whole pituitary volume of controls increased nearly four times, while that of treated ones increased nearly six times. The larger volume of pituitary in treated eels was mainly due to volume increase of proximal pars distalis and rostral pars distalis. The %GH, that is the potential index of GH production, was significantly higher in treated yellow eels with gonads differentiating into ovaries than in controls; no difference was detected in %PRL between treated and control eels. The above results strongly suggest that in eels the feminizing effects of oestrogen is first exerted on the pituitary, probably through the hypothalamus, and later on the gonads.     

Differential distribution of gonadotropin-releasing hormone-immunoreactive neurons in the stingray brain: functional and evolutionary considerations

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that occurs in multiple structural forms among vertebrate species. Bony fishes, amphibians, reptiles, birds, and mammals express different forms of GnRH in the forebrain and endocrine regions of the hypothalamus which regulate the release of reproductive gonadotropins from the pituitary. In contrast, previous studies on bony fishes and tetrapods have localized the chicken GnRH-II (cGnRH-II) nucleus in the midbrain tegmentum and, combined with cladistic analyses, indicate that cGnRH-II is the most conserved form throughout vertebrate evolution. However, in elasmobranch fishes, the neuroanatomical distribution of cGnRH-II and dogfish GnRH (dfGnRH) cells and their relative projections in the brain are unknown. We used high-performance liquid chromatography and radioimmunoassay to test for differential distributions of various GnRH forms in tissues from the terminal nerve (TN) ganglia, preoptic area, and midbrain of the Atlantic stingray, Dasyatis sabina. These experiments identified major peaks that coelute with cGnRH-II and dfGnRH, minor peaks that coelute with lamprey GnRH-III (lGnRH-III), and unknown forms. Immunocytochemistry experiments on brain sections show that dfGnRH-immunoreactive (-ir) cell bodies are localized in the TN ganglia, the caudal ventral telencephalon, and the preoptic area. Axons of these cells project to regions of the hypothalamus and pituitary, diencephalic centers of sensory and behavioral integration, and the midbrain. A large, discrete, bilateral column of cGnRH-II-ir neurons in the midbrain tegmentum has sparse axonal projections to the hypothalamus and regions of the pituitary but numerous projections to sensory processing centers in the, midbrain and hindbrain. Immunocytochemical and chromatographic data are consistent with the presence of lGnRH-III and other GnRH forms in the TN that differ from dfGnRH and cGnRH-II. This is the first study that shows differential distribution of cGnRH-II and dfGnRH in the elasmobranch brain and supports the hypothesis of divergent function of GnRH variants related to gonadotropin control and neuromodulation of sensory function.     

Beta-endorphin-like immunoreactivity in the forebrain and pituitary of the teleost Clarias batrachus (Linn.)

The organization of beta-endorphin-like immunoreactivity in the olfactory system, forebrain, and pituitary of the teleost Clarias batrachus was investigated. Immunoreactivity was prominently seen in the sensory neurons and basal cells in the olfactory epithelium and in some cells in the periphery and center (granule cells) of the olfactory bulb. Immunoreactive fibers in the olfactory nerve enter the olfactory nerve layer of the olfactory bulb and branch profusely to form tufts organized as spherical neuropils in the glomerular layer. While fascicles of immunoreactive fibers were seen in the medial olfactory tracts, the lateral olfactory tracts showed individual immunoreactive fibers. Immunoreactive fibers in the medial olfactory tract extend into the telencephalon and form terminal fields in discrete telencephalic and preoptic areas; some immunoreactive fibers decussate in the anterior commissure, while others pass into the thalamus. While neurons of the nucleus lateralis tuberis revealed weak immunoreactivity, densely staining somata were seen at discrete sites along the wall of the third ventricle. Although a large population of immunoreactive cells was seen in the pars intermedia of the pituitary gland, few were seen in the rostral pars distalis and proximal pars distalis; immunoreactive fibers were seen throughout the pituitary gland.     

Effects of gonadal steroids on the ultrastructure of GnRH neurons in the rhesus monkey: synaptic input and glial apposition

The secretion of the gonadotropins is modulated by the gonadal steroids, but the means by which these effects are mediated are not well understood. The present anatomical study was undertaken to investigate the possibility that the GnRH system responds to alterations in the gonadal steroid environment with reversible changes in synaptic input and glial wrapping such as have been observed in other neuroendocrine systems. The ultrastructure of GnRH neurons was studied in the preoptic area and medial basal hypothalamus of rhesus monkeys in various steroid conditions including five intact cycling, four long-term ovariectomized animals, two long-term ovariectomized animals with steroid replacement (LtOVX+), and two animals replaced with steroid at the time of ovariectomy (StOVX+). Electron micrographic montages of GnRH neuronal profiles were analyzed using computerized morphometrics, and the percentages of the length of perikaryal membrane immediately apposed by glial processes and that with postsynaptic modification were calculated. Ovariectomy resulted in a significant increase in the apposition of glial processes to GnRH perikaryal membranes and a significant decrease in their innervation in both brain regions. There was also a higher incidence of GnRH neurons with immunostaining confined to secretory granules and a decrease in the volume of nucleoli, both of which could be interpreted as indications that GnRH peptide synthesis was reduced in ovariectomized animals. After an ovarian steroid replacement regimen which mimicked two menstrual cycles, the innervation of GnRH neurons was increased and the glial ensheathment was partially reduced. This was true for both the LtOVX+ and StOVX+ steroid-replacement groups. GnRH neurons in the medial basal hypothalamus received more synaptic input than did those in the preoptic area, regardless of the steroid condition of the animal. The degree of glial ensheathment of GnRH neurons in the preoptic area became significantly greater than that in the medial basal hypothalamus after ovariectomy. These observations suggest there may be differences in the role of GnRH neurons in these two brain regions. These immunocytochemical ultrastructural studies provide strong evidence that alterations in the gonadal steroid milieu can produce morphological changes in the GnRH neuron and its immediate environment in the primate.     

Hypothalamus-hypophysis and testicular GnRH control of gonadal activity in the frog, Rana esculenta: seasonal GnRH profiles and annual variations of in vitro androgen output by pituitary-stimulated testes

The binding of a gonadotrophin-releasing hormone (GnRH) long acting analog (GnRHA), D-Ser (But)6,Pro9-NEt GnRH (HOE 766), to pituitary and testicular extracts and the presence of GnRH-like material in testes and hypothalamuses were measured in the frog, Rana esculenta. Also, the cellular localization of immunoreactive GnRH was investigated in testes by immunohistochemical staining. Furthermore, lyophilized preparations of pituitary crude homogenates from animals caught monthly were tested in vitro for their ability to stimulate androgen production by December testes. Satisfactory results on specific 125I-GnRH binding were difficult to obtain in view of its low binding capacity. Moreover, binding in testicular homogenates was of the same order of magnitude (about 2%) as that found in pituitaries. In a cospecific radioimmunoassay for GnRH nonapeptide, both hypothalamic and testicular extracts gave displacement parallel to the standard curve. Immunoreactive GnRH did not significantly fluctuate in hypothalamuses, while it peaked in testes during December and July. Immunoreactive GnRH was evidenced in June and September testes employing immunohistochemical staining. In particular, the interstitial cells and the Sertoli cells were faintly stained. Testes of December animals stimulated by February pituitaries produced larger quantities of androgens as compared with testes stimulated with hypophyseal preparations from the remaining periods of the year. In conclusion, the present results are consistent with the idea that seasonal changes of the hypothalamus-hypophyseal activity play an important role in regulating the hormonal response in vertebrate testes. Moreover, we report that, in addition to rats, GnRH-like material is present in frog testes and for the first time it has been shown that such putative intratesticular material undergoes seasonal fluctuations in a vertebrate.     

Origin and development of GnRH neurons.

Gonadotropin-releasing hormone (GnRH) is an essential decapeptide, with both endocrine and neuromodulatory functions in vertebrates. GnRH-containing cells of the forebrain were thought to originate in the olfactory placode and migrate to their central nervous system destinations, and those of the midbrain to arise locally from the neural tube. Here, the embryonic origins of GnRH cells are re-examined in light of recent data suggesting that forebrain GnRH cells arise from the anterior pituitary placode and cranial neural crest, from where they migrate to their final destinations. The emerging picture suggests that GnRH cells do not originate from the olfactory placodes, but arise from multiple embryonic origins, and transiently associate with the developing olfactory system as they migrate to ventral forebrain locations.     

Different modes of gonadotropin-releasing hormone (GnRH) release from multiple GnRH systems as revealed by radioimmunoassay using brain slices of a teleost, the dwarf gourami (Colisa lalia)

It has become a general notion that there are multiple GnRH systems in the vertebrate brains. To measure GnRH release activities from different GnRH systems, we conducted a static incubation of brain-pituitary slices under various conditions, and GnRH released into the incubation medium was measured by RIA. The slices were divided into two parts, one containing GnRH neurons in the preoptic area and axon terminals in the pituitary (POA-GnRH slices), and the other containing the cell bodies and fibers of terminal nerve-GnRH neurons and midbrain tegmentum-GnRH neurons (TN-TEG-GnRH slices). We demonstrated that GnRH release was evoked by high [K(+)](o) depolarizing stimuli (in both POA-GnRH and TN-TEG-GnRH slices) via Ca(2+) influx through voltage-gated Ca(2+) channels. The most prominent result was the presence of conspicuous sexual difference in the amount of GnRH release in the POA-GnRH slices. The GnRH release from TN-TEG-GnRH slices also showed a small sexual difference, which was by far more inconspicuous than that of POA-GnRH slices. Immunohistochemical analysis using an antiserum specific to the seabream GnRH (sbGnRH; suggested to be specific to POA-GnRH neurons) revealed the presence of a much larger number of POA-GnRH neurons in males than in females. This clear morphological sexual difference is suggested to underlie that of GnRH release in the POA-GnRH slices.     

Pituitary-testicular function in immature rats after treatment with GnRH antagonist, GnRH antiserum and bromocriptine

The effects of a GnRH antagonist analogue (N-acetyl-Ala1,D-p-Cl-Phe2,D-Trp3,6-GnRH, Ant.) and a GnRH antiserum (A/S) on the development of pituitary-testicular function were studied in immature (23/24-31/32-day-old) rats. In another experiment the Ant. treatment was combined with bromocriptine (BR)-induced hypoprolactinaemia. Ant. and A/S decreased serum and pituitary levels of LH and FSH, and BR those of Prl (P less than 0.01-0.05). Testicular testosterone (T) and progesterone (P) contents were significantly decreased only by Ant. (P less than 0.01). Ant. decreased the weights of the testes, ventral prostates and seminal vesicles, as well as testicular LH, FSH and Prl receptors (R) (P less than 0.01-0.05). BR decreased LH-R but had no effect on Prl-R. Both Ant. and A/S decreased available pituitary GnRH-R (P less than 0.01), but free testicular GnRH-R were reduced only by Ant. BR increased GnRH receptors in the pituitaries. It is concluded that Ant.-induced low gonadotropin levels in immature animals inhibit the developmental increase of testicular weight, gonadotropin and Prl-R, steroidogenesis and androgen action on accessory sex glands. Hypoprolactinaemia had an additive inhibitory effect to the antigonadal effects of Ant. The testis tissue of immature (23/24-day-old) animals already contains GnRH-R. In general, developing animals are clearly very sensitive to the antigonadal actions of Ant. and BR, whereas the effect of GnRH-A/S is less pronounced than in adults.     

Immunoneutralization of circulating inhibin in the hypophysiotropically clamped male rhesus monkey (Macaca mulatta) results in a selective hypersecretion of follicle-stimulating hormone

The purpose of this experiment was to examine directly whether inhibin is involved in the testicular regulation of FSH secretion in the male rhesus monkey. To this end, the pituitary-testicular axis in eight juvenile monkeys was prematurely activated by a chronic iv infusion of GnRH (0.1 microgram/min for 3 min every 3 h). After a minimum of 5 weeks of pulsatile GnRH stimulation, four animals received a brief (30-min) iv infusion of an ovine antiserum to inhibin alpha-subunit (approximately 10 ml/kg BW), and four monkeys received a comparable volume of a control ovine immune serum. The pulsatile GnRH infusion continued without interruption throughout the entire experiment. The FSH response to passive immunization against inhibin was determined by measuring concentrations of this gonadotropin in sequential plasma samples collected immediately before a GnRH infusion and for 3 h thereafter (an inter-GnRH pulse interval) on days 0.5, 1, 2, 4, 8, and 16 after injection of the immune serum. Administration of the antiserum to inhibin alpha-subunit resulted, within 2 days, in a 2- to 3-fold increase in the mean concentration and pulse amplitude of plasma FSH. The hypersecretion of FSH induced by administration of the antiserum to inhibin alpha-subunit was maintained until day 4, and then mean concentrations and mean pulse amplitudes of plasma FSH declined, reaching preantibody control levels by day 16. The time course of the antiserum-induced hypersecretion of FSH was closely correlated to changes in circulating inhibin-binding activity. Most importantly, the hypersecretion of FSH observed during the first 2 days after immunoneutralization of circulating inhibin was indistinguishable from that elicited during the initial 2 days after subsequent bilateral orchidectomy and concomitant testosterone (T) replacement. Administration of a control immune serum did not influence circulating FSH concentrations, and neither the antiserum to inhibin alpha-subunit nor the control immune serum induced changes in pituitary LH secretion and testicular T release. Since the exogenous drive to the pituitary-testicular axis of the animals was clamped in a mode that produced a pattern of pulsatile LH and T secretion comparable to that observed in adult monkeys, the present findings provide evidence for the view that inhibin plays a major role in the testicular regulation of FSH secretion during adulthood by exerting a selective inhibition on the secretion of this gonadotropin directly at the level of the anterior pituitary gland.