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.     

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.     

Molecular cloning of three cDNAs encoding different GnRHs in the brain of barfin flounder

To examine the reproductive endocrinology of a large pleuronectiform fish, barfin flounder, Verasper moseri, a promising candidate for aquaculture and resource enhancement in northern Japan due to its high commercial value, three gonadotropin-releasing hormones (GnRHs) in the brain was identified by isolation of their cDNAs. This species had three molecular forms of GnRH; salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and seabream GnRH (sbGnRH). Each GnRH cDNA encoded a signal peptide (SP), GnRH, and a GnRH-associated peptide (GAP), which was connected to GnRH by a Gly-Lys-Arg sequence. The sGnRH cDNA encoded an SP composed of 23 amino acids and a GAP composed of 54 amino acids. The cGnRH-II cDNA encoded an SP of 23 amino acids and a GAP of 49 amino acids. The sbGnRH cDNA encoded an SP of 26 amino acids and a GAP of 57 amino acids. In situ hybridization showed that the genes for sGnRH, cGnRH-II, and sbGnRH are expressed in the ventromedial olfactory bulbs and the terminal nerve ganglion, the midbrain tegmentum, and the preoptic area, respectively. These results indicate that sbGnRH neurons in the preoptic area are involved in gonadotropin secretion in barfin flounder.     

The gonadotropin-releasing hormone neuronal system of the male Djungarian hamster: distribution from the olfactory tubercle to the medial basal hypothalamus

The neuroanatomical distribution and morphology of neurons that produce gonadotropin-releasing hormone (GnRH) in the brain of the postpubertal male Djungarian hamster was studied using light microscopic immunocytochemistry. Analysis of every section from the rostral olfactory tubercle to the medial basal hypothalamus indicate 356 +/- 37 immunoreactive GnRH perikarya per brain (mean +/- SE; n = 4 brains). Over 90% of GnRH cell bodies were found in 6 brain regions; the largest number of somata were located in the medial preoptic area followed by the diagonal band, lateral hypothalamus, lateral preoptic area, lateral septum and anterior hypothalamus. Morphologically, two predominant types of GnRH neurons were identified: unipolar GnRH cells with an ovoid soma and only a single distinct process (about 40% of all GnRH neurons), and bipolar cells with a fusiform-shaped perikaryon. Overall and in most brain regions, the ratio of unipolar to bipolar GnRH perikarya was 2:3 or greater. A significant proportion of GnRH neurons had an unusually "thick" process(es) that exited the soma and tapered gradually. GnRH fibers were evident in most sections, forming dense plexuses in the arcuate nucleus-median eminence, the periventricular region of the third ventricle and organum vasculosum of the lamina terminalis. These findings indicate that the Djungarian hamster is similar to other rodent species, especially the white-footed mouse, in the neuroanatomical distribution of GnRH neurons. The present report provides a working atlas for the rostral ventral forebrain of the postpubertal Djungarian hamster.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Projections of arcuate nucleus and rostral periventricular kisspeptin neurons in the adult female mouse brain

The important role of kisspeptin neurons in the regulation of GnRH neuron activity is now well accepted. However, the ways in which kisspeptin neurons located in the arcuate nucleus (ARN) and rostral periventricular area of the third ventricle (RP3V) control GnRH neurons are poorly understood. The present study used anterograde and retrograde tracing techniques to establish the neuronal projection patterns of kisspeptin cell populations in the female mouse brain. Anterograde tracing studies revealed that kisspeptin neurons in the ARN innervated a wide number of hypothalamic and associated limbic region nuclei, whereas RP3V kisspeptin neurons projected to a smaller number of mostly medially located hypothalamic nuclei. Retrograde tracing confirmed a major projection of RP3V kisspeptin neurons to the ARN and showed that kisspeptin neurons located in the rostral half of the ARN projected to the rostral preoptic area. Peripheral administration of Fluorogold was found to label the majority of GnRH neurons but no kisspeptin neurons. Together, these studies highlight the complexity of the brain kisspeptin neuronal system and indicate that both ARN and RP3V kisspeptin neurons participate in a variety of limbic functions. In relation to the GnRH neuronal network, these investigations demonstrate that, alongside the RP3V kisspeptin cells, rostral ARN kisspeptin neurons may also project to GnRH neuron cell bodies. However, no kisspeptin neurons innervate GnRH nerve terminals in the external layer of the median eminence. These studies provide a neuroanatomical framework for the further elucidation of the functions of the ARN and RP3V kisspeptin neuron populations.     

Reproduction in the Mexican leaf frog, Pachymedusa dacnicolor. V. Immunohistochemical localization of gonadotropin-releasing hormone in the brain.

The presence and distribution of immunoreactive gonadotropin-releasing hormone (GnRH) in brains of adult male and female Pachymedusa dacnicolor has been studied immunohistochemically using antisera against mammalian, chicken-II, and salmon GnRHs. The distribution map of the immunoreactive-GnRH elements in the brain of P. dacnicolor is extremely simple, being limited to the anterior preoptic area-infundibulum-median eminence circuit. No sex- or reproductive status-related difference in either the distribution pattern or intensity of immunoreaction was revealed in this study. This is also the first immunohistochemical evidence of the presence of different structural forms of GnRH in the brain of an amphibian.     

Development and differentiation of gonadotropin hormone-releasing hormone neuronal systems and testes in the Japanese eel (Anguilla japonica).

In this study we investigated the relationship between the development of the olfactory, preoptic, and midbrain gonadotropin-releasing hormone (GnRH) neuronal systems and testicular differentiation in eels (Anguilla japonica) from embryonic stages through adulthood (5.4-50 cm body length). GnRH-synthesizing neuronal populations were first observed in the youngest fish ( approximately 5.0 cm) at the rostrobasal and caudalmost olfactory bulbs immunoreactive to a "promiscuous" (nonspecific) GnRH antiserum (635.5), and in the preoptic area and midbrain tegmentum immunoreactive to chicken GnRH II antiserum. The eel brains lacked salmon and seabream GnRH immunoreactivity. The evidence from our study suggests that the olfactory, preoptic, and midbrain GnRH populations have origins independent from those of proliferative periventricular zones within the brain. However, the olfactory GnRH neurons could have migrated out of the olfactory placodes during ages earlier than those observed in this study. Although all three GnRH neuronal populations contribute to pituitary innervation to some degree, the preoptic GnRH innervation was pronounced in the pituitary when primordial germ cells (animals approximately 5.0 cm) differentiated into male germ cells (animals 14-16 cm) and, therefore, an association can be assumed between preoptic GnRH expression and testicular differentiation in the Japanese eel.     

Gonadotropin-releasing hormone neuronal development during the sensitive period of temperature sex determination in the pejerrey fish,Odontesthes bonariensis

The development of gonadotropin-releasing hormone (GnRH) neurons was studied in relation to the sensitive period of thermolabile sex determination in the pejerrey Odontesthes bonariensis, an atherinid fish from South America. Fish were raised from hatching at three different temperatures: 17 degrees C (100% females), 24 degrees C (70% females), and 29 degrees C (100% males). Three groups of immunoreactive GnRH (ir-GnRH) neurons were identified at the terminal nerve ganglion (TNG), the midbrain tegmentum (MT), and the preoptic area (POA). Immunoreactive GnRH (ir-GnRH) neurons were identified in the TNG at hatching (day 0) and in the MT at day 3 at all the experimental temperatures. In the POA ir-GnRH neurons were identified in the nucleus preopticus periventricularis simultaneously with the first appearance of ir-GnRH fibers in the pituitary on days 11, 14, and 17 for larvae kept at 29, 24, and 17 degrees C, respectively. The number of ir-GnRH neurons in the TNG did not show any statistical difference between temperatures. The number of ir-GnRH neurons in the MT increased in number during the experiment for larvae kept at 17 and 24 degrees C but decreased between days 17 and 31 in larvae kept at 29 degrees C. The number of ir-GnRH neurons in the POA increased during development with a peak at day 28 for all temperatures studied and the magnitude of this peak showed a correlation with incubation temperature. These results reinforce the notion that the hypothalamus-pituitary-gonadal axis is active during sex determination in pejerrey suggesting a possible role of the central nervous system and GnRH in this process. It is also suggested that GnRH neurons located in the preoptic area might be the physiological transducers of temperature during the temperature sensitive period in this species.     

GnRH immunodetection in the brain of the holocephalan fish Chimaera monstrosa L.: correlation to oocyte maturation

Chimera monstrosa (rabbit fish) like other holocephalans is a rare, delicate deep sea fish. Owing to the difficulty of sampling individuals in good shape, there is a paucity of information available on the morphology and physiology of this species especially concerning reproduction. In holocephalans, a hypothalamus-pituitary-gonadal axis has been postulated and a GnRH molecule identical to cGnRH II has been identified. The aim of the present study was to correlate the presence of steroidogenic enzymes in the ovarian follicles with the presence of GnRH in the hypothalamus. Estrogens, the steroids that trigger the accumulation of yolk (vitellogenesis) in the oocytes are synthesized by the somatic cells of the follicle in the vitellogenic stages via a cascade of steroid dehydrogenases involving 3 beta-hydroxysteroid-dehydrogenase (3 beta-HSD; in the inner thecal layer) and aromatase cytochrome (P450; granulosa layer). Our results showed that 3 beta-HSD is present concomitant with the presence of cGnRH II in the preoptic area and in the ventral hypothalamus. Another form of immunoreactive GnRH, mGnRH is also present in the brain of C. monstrosa. It is localized in the ventral telencephalon and in the midbrain caudal diencephalon (boundary between ventral thalamus and tegmentum of the mesencephalon). This form of GnRH is probably correlated with sexual behaviour.     

Gonadotropin-releasing hormone neurons extend complex highly branched dendritic trees outside the blood-brain barrier

GnRH neurons project axons to the median eminence to control pituitary release of gonadotropins and, as such, represent the principal output neurons of the neuronal network controlling fertility. It is well established that the GnRH neurons exhibit a simple bipolar morphology with one or two long dendrites. Using adult GnRH-green fluorescent protein transgenic mice and juxtacellular cell filling, we report here that a subpopulation of GnRH neurons located in the rostral preoptic area exhibit extremely complex branching dendritic trees that fill the organum vasculosum of the lamina terminalis (OVLT). The dendritic nature of these processes was demonstrated at both light and electron microscopic levels by the presence of spines, dendritic ultrastructure, and synapses. Further, electrophysiological recordings showed that GnRH neurons were excited by glutamate as well as kisspeptin puffed onto their dendrites located within the OVLT. Using iv injection of horseradish peroxidase, a molecule unable to penetrate the blood-brain barrier (BBB), we show that GnRH neuron cell bodies and dendrites within 100 μm of the OVLT reside outside the BBB. Approximately 85% of GnRH neurons in this area express c-Fos at the time of the GnRH surge. These observations demonstrate that GnRH neurons extend complex, highly branched dendritic trees beyond the BBB into the OVLT, where they will be able to sense directly molecules circulating in the bloodstream. This indicates a new mechanism for the modulation of GnRH neurons that extends considerably the range of factors that are integrated by these neurons for the control of fertility.     

Characterization and localization of mRNA encoding the salmon-type gonadotrophin-releasing hormone precursor of the masu salmon.

Gonadotrophin-releasing hormone (GnRH) is considered to have an important role in the control of reproduction in salmonid fish, although we do not have any direct evidence. To clarify this problem by molecular techniques, we first determined the nucleotide sequence of the mRNA encoding the precursor of salmon-type GnRH (sGnRH) from the masu salmon, Oncorhynchus masou. The masu salmon sGnRH precursor was composed of a signal peptide, sGnRH and a GnRH-associated peptide (GAP) which was connected to sGnRH by a Gly-Lys-Arg sequence. The amino acid sequence of sGnRH and Gly-Lys-Arg were highly conserved when compared with the corresponding regions of African cichlid sGnRH and mammalian GnRH precursors. However, the GAP region was markedly divergent, with a 66% amino acid similarity to African cichlid GAP and an 8.3-15% similarity to mammalian GAPs. Northern blot analysis indicated the presence of a single mRNA species of about 600 bases in the olfactory bulb and telencephalon and in the diencephalon. The signal was more intense in the former regions. An in-situ hybridization study further revealed that sGnRH neurones were distributed in the olfactory nerve, the ventral part of the olfactory bulb, the ventral part of the telencephalon, the lateral preoptic area and the preoptic nucleus. The sGnRH neurones were thus longitudinally scattered between the olfactory nerve and the lateral preoptic area in the rostroventral part of brain. The intensity of the hybridization signals and the size of hybridization-positive somata were much greater in the olfactory nerve and the rostral olfactory bulb than in the other regions. Preoptic sGnRH neurones were scarcely detected in immature masu salmon, whereas they were more frequently observed in maturing animals. It is possible that the olfactory and the preoptic sGnRH neurones have different physiological roles in salmonid fish.     

Evidence for the existence of atrial natriuretic factor-containing neurons in the rat brain

Immunoreactive atrial natriuretic factor- (ANF-)positive nerve fibers and cell bodies were observed in the preoptic area, hypothalamus, mesencephalon, and pons of rats. In colchicine-treated animals a large number of immunoreactive ANF-positive cell bodies were seen in the organum vasculosum of the lamina terminalis, in several hypothalamic nuclei (e.g. periventricular, arcuate, and ventral premammillary nuclei), and in the dorsolateral tegmental nuclei of the pons. Varicose nerve fibers containing ANF were generally observed in the vicinity of the cells. These findings indicate that a widespread network of ANF-containing neurons is present in the brain.     

Unmasking the progesterone receptor in the preoptic area and hypothalamus of the ewe: no colocalization with gonadotropin-releasing neurons

Progesterone powerfully inhibits GnRH secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Visualization of the neural ovine progesterone receptor has proved elusive but, using a high temperature antigen unmasking technique, the progesterone receptor was revealed in the ewe brain. Progesterone receptors were located in the preoptic-hypothalamic continuum, especially in the preoptic area, ventrolateral region of the ventromedial nucleus and the arcuate nucleus. This study also suggests that the inhibitory action of progesterone on GnRH release is not transduced directly through the GnRH neurons as a single GnRH perikaryon of 732 was immunoreactive for the progesterone receptor.     

Primary structure of gonadotropin-releasing hormone from the brain of a holocephalan (ratfish: Hydrolagus colliei).

Gonadotropin-releasing hormone (GnRH) in the ratfish brain has been isolated and purified using reverse-phase high performance liquid chromatography. Amino acid composition and sequence analysis indicate that the primary structure is pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. The presence of the amino terminal pyroglutamic acid has been confirmed by degradation studies with pyroglutamyl aminopeptidase. The amidated carboxy terminus and molecular weight were confirmed using mass spectrometry. Moreover, sequence comparison and coelution studies with one of the synthetic forms of GnRH (chicken GnRH II) indicate that the ratfish and chicken GnRH II molecules are identical. This represents the first sequence data of a GnRH molecule from a cartilaginous fish (class: Chondrichthyes). It is argued that the ratfish GnRH molecule has been retained for over 400 million years of evolution and is expressed in most vertebrate classes.     

Potential for polysialylated form of neural cell adhesion molecule-mediated neuroplasticity within the gonadotropin-releasing hormone neurosecretory system of the ewe

The GnRH neurosecretory system undergoes marked structural and functional changes throughout life. The initial goal of this study was to examine the neuroanatomical relationship between GnRH neurons and a glycoprotein implicated in neuroplasticity, the polysialylated form of neural cell adhesion molecule (PSA-NCAM). Using dual label immunocytochemistry in conjunction with confocal microscopy, we determined that fibers, terminals, and perikarya of GnRH neurons in adult ovariectomized ewes are intimately associated with PSA-NCAM. In the preoptic area, intense PSA-NCAM immunoreactivity was evident around the periphery of GnRH cell bodies. The second goal of this study was to determine whether PSA-NCAM expression associated with GnRH neurons varies in conjunction with seasonal changes in the activity of the GnRH neurosecretory system in ovariectomized ewes treated with constant release implants of estradiol. During the breeding season when reproductive neuroendocrine activity was enhanced, the expression of PSA-NCAM immunoreactivity associated with GnRH neurons was significantly greater than that during anestrus when GnRH secretion was reduced. This difference, which occurred despite an unchanging ovarian steroid milieu, was not observed in preoptic area structures devoid of GnRH immunoreactivity, suggesting that the seasonal change is at least partially specific to the GnRH system. The close association between PSA-NCAM and GnRH neurons and the change in this relationship in conjunction with seasonal alterations in GnRH secretion provide anatomical evidence that this molecule may contribute to seasonal remodeling of the GnRH neurosecretory system of the adult.