Evolutionary development of three gonadotropin-releasing hormone (GnRH) systems in vertebrates.

Gonadotropin-releasing hormone (GnRH) is the neuropeptide that links the brain to the reproductive system. Most vertebrate species express two forms of GnRH, which differ in amino acid sequence, localization, distribution, and embryological origin. The GnRH system in the ventral forebrain produces a species-specific GnRH form and projects toward the gonadotropic cell in the pituitary. The GnRH neurons of this system originate from the olfactory placode and migrate into the brain during early development. The other GnRH system is localized in a nucleus in the midbrain, where large cells express chicken-GnRH-II, of which the function is still unclear. In modern teleosts, a third GnRH system is present in the terminal nerve, which contains salmon GnRH. The three GnRH systems appear at different times during fish evolution. Besides the two accepted lineages in GnRH evolution (of conserved chicken GnRH-II in the midbrain and of mammalian GnRH or species-specific GnRH in the hypophysiotropic system), we propose a third lineage: of salmon GnRH in the terminal nerve.

Development of three distinct GnRH neuron populations expressing two different GnRH forms in the brain of the African catfish (Clarias gariepinus).

The early development of both the catfish gonadotropin-releasing hormone (cfGnRH)- and the chicken GnRH-II (cGnRH-II) system was investigated in African catfish by immunocytochemistry by using antibodies against the GnRH-associated peptide (GAP) of the respective preprohormones. Weakly cfGnRH-immunoreactive (ir) neurons and fibers were present at 2 weeks after hatching (ph) but only in the ventral telencephalon and pituitary. Two weeks later, cfGnRH fibers and neurons were also observed in more rostral and in more caudal brain areas, mainly in the preoptic area and hypothalamus. Based on differences in temporal, spatial, and morphologic appearance, two distinct cfGnRH populations were identified in the ventral forebrain: a population innervating the pituitary (ventral forebrain system) and a so-called terminal nerve (TN) population. DiI tracing studies revealed that the TN population has no neuronal connections with the pituitary. The cGnRH-II system is present from 2 weeks ph onward in the midbrain tegmentum and only their size and staining intensity increased during development. Based on the comparison of GnRH systems amongst vertebrates, we hypothesize that during fish evolution, three different GnRH systems evolved, each expressing their own molecular form: the cGnRH-II system in the midbrain, a hypophysiotropic GnRH system in the hypothalamus with a species-specific GnRH form, and a salmon GnRH-expressing TN population. This hypothesis is supported by phylogenetic analysis of known GnRH precursor amino acid sequences. We hypothesize, because the African catfish is a less advanced teleost species, that it contains the cfGnRH form both in the ventral forebrain system and in the TN population.

Gonadal steroids and the maturation of the species-specific gonadotropin-releasing hormone system in brain and pituitary of the male African catfish (Clarias gariepinus).

The effect of testosterone (T), 11-ketotestosterone (KT) and estradiol (E(2)) on the development of the catfish gonadotropin-releasing hormone system (cfGnRH) of male African catfish (Clarias gariepinus), at the onset of puberty [between 10 and 12 weeks post hatching (ph)] was investigated. The cfGnRH neurons, located in the ventral forebrain, were visualized by immunofluorescence and their numbers were determined and the amounts of cfGnRH-associated peptide (cfGAP) in the pituitary were measured by RIA. Steroid treatments did not significantly alter the numbers of immunoreactive GnRH neurons. However, T and E(2) caused an increase in the amount of GnRH, demonstrated by the intensity of the immunostaining of GnRH neurons and fibers in the brain and the amount of cfGAP in the pituitary. Treatment with KT, the main circulating androgen in adult male catfish, neither changed the number of cfGnRH neurons, nor elevated the cfGnRH content in the pituitary. In previous experiments with younger, prepubertal fish (2-6 weeks ph), T caused an elevation of the number of cfGnRH neurons to the same level as present in pubertal fish of 12-14 weeks. We conclude that the onset of puberty in the male African catfish coincides with the completion of the steroid-dependent structural maturation of the cfGnRH system in the brain. T and/or E(2), however, are still able to exert a positive influence on the amounts of cfGnRH during the later stages of pubertal development, thus still playing a role in the control of the cfGnRH system.

Gonadotropin-releasing hormone fibers innervate the pituitary of the male African catfish (Clarias gariepinus) during puberty.

The development of the catfish gonadotropin-releasing hormone (cfGnRH) fiber network in the pituitary of male African catfish (Clarias gariepinus) was investigated in relation to puberty. Double immunolabeling studied by confocal laser scanning microscopy revealed a concomitant development of gonadotropes and of pituitary cfGnRH innervation during the first wave of spermatogenesis. Catfish GnRH-immunoreactive fibers in the proximal pars distalis (PPD) of the pituitary were initially observed at the age of 10 weeks (onset of spermatogonial proliferation) and gradually reached the adult pattern at the age of 20 weeks (spermatozoa present in the testis). The content of cfGnRH-associated peptide (cfGAP, part of the prohormone) in the pituitary similarly increased during puberty. At the electron microscopical level, fibers containing cfGAP-ir granules came into close proximity of the gonadotropes at 18 weeks of age. In vitro studies indicated a progressively increasing basal and cfGnRH-stimulated luteinizing hormone (LH) secretion during pubertal development. The LH secretion patterns were similar in response to exogenous cfGnRH (0.1 microM) or to endogenous cfGnRH, the release of which was induced by forskolin (1 microM). Castration experiments demonstrated that the innervation of the pituitary with cfGnRH fibers continued after surgery, accompanied by an increase in the cfGAP levels. However, gonadotrope development was retarded, suggesting a differential regulation of the two maturational processes. Since testosterone stimulates both processes, other testicular factors may also be involved. Puberty-associated changes in LH release patterns appear to reflect changes in the GnRH sensitivity and in the pool of releasable LH, while availability of cfGnRH does not appear to be a limiting factor.

Testosterone accelerates the development of the catfish GnRH system in the brain of immature African catfish (Clarias gariepinus).

The effects of two endogenous steroids on the maturation of the catfish GnRH and the chicken GnRH-II system in the African catfish were investigated. Immature fish (2 weeks of age, which is before sexual differentiation; thus male and female genotypes present) were fed with food pellets containing either testosterone (T), 11beta-hydroxyandrostenedione (OHA) or no steroid (control). After 2 and 4 weeks of treatment, the effects on the two GnRH systems were investigated immunocytochemically, using specific antibodies against the respective GnRH-associated peptides. By means of fluorescence microscopy the number of GnRH perikarya and the cell surfaces were determined. Confocal laser scanning microscopy was applied to verify spatial distribution and staining intensity. After 2 weeks of treatment no difference in any of the parameters between the groups was observed. However, 4 weeks T treatment resulted in significantly more cfGnRH-ir perikarya in the brain compared to the OHA and control groups. In addition, in the T group the number of immunoreactive fibers was markedly higher and the staining of the perikarya and axons was more intense. The distribution of cfGnRH-ir neurons over the ventral forebrain differed between the two age groups: in 4-week-old fish, the largest concentration of neurons was localized in the ventral telencephalon, while 2 weeks later the number of neurons in the supraoptic area had markedly increased, suggesting that the cfGnRH system is still undergoing developmental changes during this period. In 6-week-old fish the average volume of the cfGnRH perikarya (expressed as surface size in the microscopical sections) in both the OHA and the T group was significantly bigger than that in the control group. The cGnRH-II-ir neurons in the midbrain tegmentum showed strong immunoreactivity in all groups, both treated and nontreated. In contrast to the cfGnRH neurons, the staining intensity and the number of cGnRH-II neurons did not change after steroid treatment. The results of this study show that T is able to accelerate the development of the cfGnRH system, whereas OHA has only minimal effects; the cGnRH-II system develops independent from these steroids.

Modulation of testicular androgen production in adolescent African catfish (Clarias gariepinus).

At 6 months of age the first spermatozoa appear in the testes of the African catfish considered to be adolescent, since the development to adulthood (12 months of age) is accompanied by further morphological and functional differentiation of Leydig cells. There are increasing plasma levels of 11-ketotestosterone (11-KT) and an increasing responsiveness to luteinizing hormone (LH) of testicular androgen secretion in vitro. Whether treatment of adolescent males with key hormones of the brain-pituitary-gonad axis [gonadotropin-releasing hormone (GnRH), LH, and 11-KT] affects the testicular steroidogenic response to a challenge with LH in vitro 7 days later has been investigated. Injection of GnRH (2.5 microg chicken GnRH-II per kilogram of body weight), LH (25 microg/kg), or a high dose of 11-KT (50 microg/kg) down-regulated basal and LH-stimulated testicular androgen secretion to a minimum of 35% of control values. Treatment with LH was, moreover, associated with changes in the ultrastructure of Leydig cell mitochondria which were either swollen and had a less electron-dense matrix or showed an elongated shape. Conversely, a moderate dose of 11-KT (20 microg/kg) enhanced LH-stimulated, but not basal, androgen secretion in vitro to a maximum of 190% of control values. In view of the generally low LH plasma levels and of the steadily increasing 11-KT plasma levels during puberty, 11-KT may be involved in the up-regulation of the testicular steroidogenic capacity observed during development to full maturity.

Pituitary gonadotrophs are strongly activated at the beginning of spermatogenesis in African catfish, Clarias gariepinus.

Pituitary gonadotrophs were studied in male African catfish between 1 and 37 wk of age using antisera against the LH subunits for immunohistological and radioimmunological purposes, and cRNA probes for in situ hybridization. Immunoreactive material was already detectable at the earliest age examined. In juveniles, the signal for the common glycoprotein alpha subunit (GP alpha) was stronger than that for the LH beta subunit. Accordingly, an excess of radioimmunoassayable GP alpha 100 times that of LHbeta was recorded in the pituitary. Using in situ hybridization, the mRNAs were detected 7 (GP alpha) and 13 (LHbeta) wk after hatching. Detection of LHbeta mRNA coincided with a 300-fold increase in the pituitary content of LHbeta and intact LH, whereas GP alpha increased only 15-fold. The number of gonadotrophs per pituitary and the amount of LH per gonadotroph also increased strongly. The strong, initial increase in pituitary LH levels was always associated with the presence of spermatocytes. However, in a limited number of cases (3 out of 12 fish), the pituitary LH content was low despite the presence of spermatocytes. The number of gonadotrophs, the staining intensities (reflecting protein and mRNA), and the pituitary LH content kept increasing, although at a reduced rate, until completion of the first wave of spermatogenesis. In view of the excess of GP alpha over LHbeta, we conclude that expression of the two subunits is regulated in part by different mechanisms, and that expression of LHbeta is rate-limiting for the amount of intact LH. The strong activation of the gonadotrophs shortly after meiosis opens the possibility that a signal of testicular origin stimulates LH expression, in particular its beta subunit. In the absence of a FSH-like gonadotropin in catfish, we propose that LH covers all functions requiring gonadotropic regulation in the African catfish.

Preferential release of newly synthesized, exportable neuropeptides by insect neuroendocrine cells and the effect of ageing of secretory granules.

The release of newly synthesized neuropeptides was studied in an in vitro system using the adipokinetic hormone (AKH)-producing cells of an insect (Locusta migratoria) as a model system. Tritiated phenylalanine incorporated into three hormonal neuropeptides, AKH I, II and III, was used to distinguish newly synthesized hormones from older, preexisting ones. After pulse-chase labeling experiments of varying duration, the secretion of AKHs by the AKH cells was stimulated. Both hormones released into the incubation medium after stimulation and non-released hormones extracted from the tissue were separated by reversed-phase high performance liquid chromatography. Their radioactivity was measured by scintillation counting of the column eluate. The ratio between the specific radioactivities of the released and the non-released neuropeptides was always greater than 1.0, which indicates that the newly synthesized peptides are preferentially released. The percentages of newly synthesized (radioactive) AKHs which are released, increased until 8 1/4 h and decreased thereafter. The results indicate that after the packaging of the prohormones into secretory granules and their processing to bioactive AKHs, some further maturation of the secretory granules is required before they can release their content. After an 8 1/4 h incubation, secretory granules with radioactive AHKs enter a non-releasable pool consisting of older secretory granules.

Testicular responsiveness to gonadotropic hormonein vitro and Leydig and Sertoli cell ultrastructure during pubertal development of male African catfish (Clarias gariepinus).

The gonadotropin (GTH)-stimulated testicular androgen secretionin vitro and the ultrastructure of Leydig and Sertoli cells was studied during the pubertal development in male African catfish. Testicular weight increased from less than 1 mg in the ninth week of age to nearly 600 mg in the 28th week. Immature testes (stage I: spermatogonia) were highly sensitive to GTH and secreted very high amounts of androgens per mg of tissue. The secretion per mg tissue decreased gradually in stages II (spermatogonia and spermatocytes) and III (spermatogonia, spermatocytes, and spermatids), but precipitously in stage IV (all germ cell stages, including spermatozoa). However, due to the testicular weight gain, the total androgen output per pair of testes increased slightly in stage III and strongly in stage IV. The sensitivity to GTH decreased with the appearance of haploid germ cells in stage III. Leydig cells but not Sertoli cells showed the ultrastructural characteristics of steroid producing cells. Leydig cell morphology did not change in stages I-III, while in stage IV, more smooth endoplasmic reticulum was present. The ultrastructural characteristics of Sertoli cells did not change prominently. Thus, spermatogonial multiplication and spermatocyte formation takes place when the testicular steroidogenic system is highly active and responsive to GTH; whereas the differentiation of haploid germ cells is accompanied by a reduced responsiveness to GTH and by the secretion of several-fold lower androgen amounts per mg of tissue.

Expression and distribution of two gonadotropin-releasing hormones in the catfish brain.

The expression of prepro-catfish GnRH mRNA and prepro-chicken GnRH-II mRNA was investigated by means of in situ hybridization. The differential distribution of cells expressing the respective mRNAs was compared with the distribution of cells immunoreactive for (1) catfish (cf) GnRH and chicken (c) GnRH-II and (2) both GnRH-associated peptides (GAPs). It was found that the prepro-cfGnRH mRNA expressing cells were located in the ventral forebrain, with a similar distribution of the cfGnRH- and cfGAP-immunoreactive perikarya. The prepro-cGnRH-II mRNA expressing cells were exclusively located in the midbrain tegmentum, at the same position as a group of large cGnRH-II- and CIIGAP-immunoreactive perikarya. It was concluded that the peptidergic neurons in the ventral forebrain contain cfGnRH, whereas cGnRH-II perikarya are restricted to the midbrain. The proximal pars distalis of the pituitary, containing the gonadotropin cells, is innervated by fibers immunoreactive for both cfGnRH and cfGAP and originating from the cfGnRH neurons in the ventral forebrain. We could, however, not detect fibers innervating the pituitary that were immunoreactive for cIIGAP.

The adipokinetic cells in the corpus cardiacum of Locusta migratoria preferentially release young secretory granules.

The influence of flight activity on the release of secretory granules from the adipokinetic cells in the corpus cardiacum of Locusta migratoria was studied. Two labeling methods, an enzymatical and a radioactive one, were used to label young, newly synthesized secretory granules and so distinguish them from older, preexisting granules. Both methods demonstrated that the ratio between the numbers of labeled and unlabeled secretory granules was lower in flight-stimulated adipokinetic cells than in unstimulated cells. This ratio was lower in both the cell bodies and the cell processes of flight-stimulated cells. After flight there was no detectable change in the total number of secretory granules, which indicates that the synthesis of new secretory granules is not inhibited by flight activity. Rather, the tendency of flight-stimulated cells to have more trans-Golgi networks labeled with wheat-germ agglutinin-conjugated horseradish peroxidase suggests that the synthesis of new secretory granules was enhanced by flight. The results led to the conclusion that young secretory granules were preferentially released over older secretory granules.

Pubertal development of male African catfish (Clarias gariepinus). Pituitary ultrastructure and responsiveness to gonadotropin-releasing hormone.

Pubertal development was studied in male African catfish by immunocytochemical examination of pituitary gonadotrophs and by monitoring the responsiveness of gonadotropin (GTH) secretion to salmon GnRH analogue (sGnRHa) in vitro. Experiments were carried out with fish from 9 to 28 wk of age. Fish were assigned to four groups, according to the stage of spermatogenesis: I, spermatogonia alone; II, spermatogonia and spermatocytes; III, spermatogonia, spermatocytes, and spermatids; IV, all germ cell stages, including spermatozoa. Basal and sGnRHa-stimulated secretion of the LH-like GTH II increased 3- to 4-fold from stage I to II and from stage II to III, whereas a 15-fold increase was recorded from stage III to IV. The ED50 values of sGnRHa varied between 0.08 and 0.49 nM, stages II and III being less sensitive. The highest dosage of sGnRHa (100 nM) led to a reduction of GTH secretion. In the first three stages, the pituitary secreted large amounts of free alpha-subunit while free GTH II beta-subunit was not detected at any stage of development. Antisera against GTH II and its alpha- and beta-subunits were used for immunocytochemical studies. In stages I and II, two subtypes of gonadotrophs, which differed in the size and labeling intensity of their secretory granules, were present. Both types of granules were immunopositive for the two subunits of GTH II. In stages III and IV, only gonadotrophs of the subtype with the larger granules were found. Globules and irregular, membrane-bound masses (IMs), probably arising through fusion of secretory granules, appeared in the gonadotrophs in stage III and became more prominent in stage IV. Globules and IMs were immunopositive for the beta-subunit but negative for the alpha-subunit. We conclude that the two subtypes of gonadotrophs represent different developmental stages of GTH II-producing cells, as they shared immunolabeling for the alpha- and the beta-subunits of GTH II. The scarcity of GTH II beta-subunit may be rate-limiting for the amount of intact GTH II available for secretion, particularly at early stages of development. In contrast, at more advanced stages when the readily releasable pool of GTH II has greatly increased, the amount of GTH II also appears to be controlled by modification or elimination of the alpha-subunit from globules and IMs.

Two gonadotropin-releasing hormones in the African catfish, Clarias gariepinus: localization, pituitary receptor binding, and gonadotropin release activity.

Two GnRH peptides have recently been identified in brain extracts of the African catfish, chicken-II GnRH ([His5,Trp7,Tyr8]GnRH, cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH, cfGnRH). Using three experimental approaches, we investigated whether both peptides are involved in the regulation of pituitary gonadotropin secretion. First, the presence of cfGnRH and cGnRH-II in the pituitary was studied by biochemical and immunocytochemical techniques, as GnRH reaches the pituitary via axonal transport in teleost fish. Pituitary extracts contained cfGnRH- and cGnRH-II-immunoreactive material, showing the same HPLC retention times as the respective synthetic GnRH peptides; cfGnRH was present in 37-fold higher amounts than cGnRH-II. Using single and double labeling immunocytochemical techniques, both peptides were localized in the same peptidergic nerve fibers and often within the same secretory granules in the vicinity of the gonadotropes. Second, the two peptides were tested for their capacity to induce an increased secretion of the LH-like gonadotropin-II (GTH-II). In vivo studies showed that both GnRHs released GTH-II, but 100-fold higher cfGnRH than cGnRH-II doses were necessary to induce similar increases in circulating GTH-II levels. In vitro experiments using pituitary tissue fragments in a perifusion system also revealed a clearly higher GTH-II-releasing capacity of cGnRH-II compared to that of cfGnRH. Third, the peptides were tested for their ability to displace [125I]salmon GnRH analog ([D-Arg6,Trp7,Leu8,Pro9-NEt] GnRH, sGnRHa), a high affinity GnRH receptor ligand, from catfish pituitary membrane preparations. Chicken GnRH-II competed with [125I]sGnRHa for pituitary GnRH-binding sites, whereas cfGnRH did so only slightly. The present data show that cGnRH-II is the more potent GTH-II secretagogue, although a role for cfGnRH in the regulation of GTH-II secretion cannot be excluded. The high biological activity of cGnRH-II may be related to the regulation of GTH-II secretion surges, such as those associated with spawning, whereas cfGnRH may be involved in regulating moderate changes in GTH-II plasma levels. The peptides' potency differences appear to be related to their different binding affinities for the pituitary GnRH receptor.

Gonadotropin-releasing hormones, including a novel form, in snook Centropomus undecimalis, in comparison with forms in black sea bass Centropristis striata.

The molecular forms of gonadotropin-releasing hormone (GnRH) in brain-pituitary extracts were determined for snook Centropomus undecimalis and black sea bass Centropristis striata. The extracts were analyzed in both isocratic and gradient high performance liquid chromatography (HPLC) programs. Eluted fractions were tested in radioimmunoassays with 4 different antisera made against 3 distinct GnRH peptides. Results show that snook contain 3 forms of GnRH, all of which are present in males and females irrespective of the stage of the reproductive cycle. Larger quantities of these GnRH peptides are present in snook in the nonreproductive phase than in snook in the reproductive phase. One form of snook GnRH is immunologically and chromatographically similar to salmon GnRH, and a second form is similar to chicken GnRH-II. However, the third snook GnRH appears to be distinct from the 7 known forms of the vertebrate hormone. In contrast, sea bass contain only the salmon GnRH-like and chicken GnRH-II-like forms of GnRH and, hence, appear to match the more usual pattern of GnRH peptides in teleosts. We speculate that one of the GnRH genes was duplicated and then altered in a fish ancestral to snook but not sea bass, even though both species of fish are in the recently evolved Perciformes order.

GTH-cells in the pituitary of the African catfish, Clarias gariepinus, during gonadal maturation: an immuno-electron microscopical study.

In an ultrastructural immunocytochemical study we investigated the development of the gonadotropic cells in the pituitary of two to six months old male African catfish in relation to testicular development. In this period, pituitary and testicular tissue samples were collected on five occasions (groups I-V). Blood samples could only be taken from the fish in groups III-V. The testicular development was divided in three stages i.e., immature (only spermatogonia, group I), early (spermatogonia and spermatocytes, groups II and III) and advanced (all germ cell stages including spermatozoa, groups IV and V) spermatogenesis. 11-Ketotestosterone blood levels were low, except for the last group. Antisera were raised against the complete catfish α,ßGTH-II, as well as to the separate α- and ß-subunits of catfish GTH-II. In the proximal pars distalis of immature fish, undifferentiated cells, somatotrops, putative thyrotrops (pTSH) and putative gonadotrops (pGTH) were found. In the two latter, secretory granules were labeled with anti-αGTH, but not with anti-ßGTH-II. pTSH- and pGTH-cells were distinguished on the basis of the size of their secretory granules. During early spermatogenesis, two classes of putative gonadotrops could be distinguished. One type had the same immunocytochemical and ultrastructural characteristics as in immature fish; the secretory granules in the second cell type, which was more abundant, were also immunopositive for anti-ßGTH-II. The mean volume of the secretory granules in these GTH-II cells was three times larger than that in the early appearing pGTH-cells. In addition, the later appearing GTH-II cells contained large inclusions, known as globules. These structures labeled with anti-αßGTH-II and with anti-ßGTH-II, but not with anti-αGTH. It is assumed that the globules are involved in a differential storage and/or breakdown of the GTH-II subunits. During advanced spermatogenesis the two gonadotropic cell types could still be distinguished, but the early appearing pGTH-cell type was scarce. The present observations permit the conclusion that the early appearing cells may be GTH-I cells. However, definitive proof about their identity depends on the availability of antibodies or cDNA probes specific for GTH-I.

Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity.

A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt, Triturus alpestris (Urodela).

Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA- and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, originating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.

Application of cryosubstitution in neurohormone- and neurotransmitter-immunocytochemistry.

Pituitaries of the African catfish, Clarias gariepinus, were prefixed in aldehyde fixatives, frozen in liquid propane and submitted to a cryosubstitution procedure. Ultrathin sections of the Lowicryl HM20-embedded tissue were treated with primary antisera raised in rabbits to gonadotropin releasing hormone (GnRH), vasopressin or gamma amino butyric acid (GABA) respectively. Binding of the primary antisera was visualized with goat anti-rabbit (GAR) labeled with gold. The general morphology of the tissue components in the cryosubstituted pituitaries matches with that obtained after routine embedding procedures. In addition, a strong labeling intensity of the neuropeptides/neurotransmitters investigated in the present study was demonstrated. Due to these qualities cryosubstitution provides optimal conditions for studying co-localization of neurosecretory products, using double-immunostaining procedures. In the pars distalis of the catfish pituitary several types of hypothalamus-derived nerve fibers are present between or synapting on the secretory cells. It is demonstrated that the two known catfish GnRHs are co-localized in the same nerve fiber and within these nerve fibers even co-exist in the same neurosecretory granules. GABA and vasopressin-immunolabeling each occurred in different nerve fibers. The present data demonstrate that cryosubstitution and low temperature-embedding results in an excellent morphological preservation compared to ultracryotomy and a better preserved immunoreactivity of small antigenic molecules in comparison to conventional fixation and embedding techniques.

Distribution of serotonin- and dopamine-immunoreactivity in the brain of the teleost Clarias gariepinus.

The distribution of serotonergic and dopaminergic cell bodies and varicose fibres in the brain of the teleost Clarias gariepinus was studied immunohistochemically using antisera against formaldehyde-conjugated serotonin and dopamine. Many serotonergic and dopaminergic fibres innervated the areas dorsalis telencephali pars medialis and pars lateralis dorsalis, as well as the area ventralis telencephali pars ventralis. In the diencephalon, a large number of serotonergic and some dopaminergic fibres were found in the preoptic nucleus, innervating the cells of this nucleus. In addition, serotonergic and dopaminergic fibres were observed in the pituitary stalk and in all regions of the pituitary gland. Moreover, the diencephalon contained the highest number of serotonin- or dopamine-immunoreactive cell bodies. These cells were confined to the same periventricular nuclei as the nucleus ventromedialis thalami, the nucleus posterior periventricularis, the nucleus lateralis tuberis, the nuclei recessus lateralis and recessus posterioris. Most cells of these nuclei were in contact with the cerebrospinal fluid of the third ventricle. The brainstem contained serotonergic cell bodies in the raphe nuclei and a few serotonergic and dopaminergic fibres. The torus semicircularis was densely innervated by serotonergic fibres and, to a lesser extent, dopaminergic fibres. In the midbrain of Clarias gariepinus, no dopaminergic homologue of the substantia nigra was observed. The results are discussed both in a comparative and a physiological context. In this regard, special attention has been paid to the contribution of hypothalamic monoamines in the regulation of gonadotropin secretion as an essential step in the neuro-endocrine control of reproduction.

Topographical relationships between catecholamine- and neuropeptide-containing fibers in the median eminence of the newt, Triturus alpestris. An ultrastructural immunocytochemical study.

Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.