Immunocytochemical identification of growth hormone (GH) cells in the pituitary of three anuran species using an antiserum against purified bullfrog GH.

An antiserum was prepared against the recently purified bullfrog (bf) growth hormone (GH); it was applied to sections of brain and pituitary of three urodele (Ambystoma, Pleurodeles and Cynops) and three anuran (Xenopus, Bufo vulgaris and B. japonicus) species. No immunostaining was obtained in the urodele pituitary, being consistent with the results of immunoblot analysis of the pituitary homogenate. In the three anuran species, strong immunoreactivity was observed in GH cells that were concentrated in the posterodorsal region of the pars distalis. No GH-like immunoreactivity was detectable in the brain of any of the species. A comparison using adjacent sections stained with anti-bf prolactin (PRL) confirmed the anteroventral localization of PRL cells. Colocalization of GH and PRL was not apparent. These data suggest that the molecular structure of amphibian GHs is considerably different between anurans and urodeles. The antiserum used in the present work shows a high species specificity, recognizing only anuran GHs. In contrast anti-bfPRL labeled PRL cells in all the amphibian species studied in the present work, suggesting that PRLs possess common amino acid sequences recognized by the anti-bfPRL.

Immunocytochemical localization of a galanin-like peptidergic system in the brain of two urodele and two anuran species (Amphibia).

Galanin-like immunoreactivity was localized in the brain of Urodela (Ambystoma, Pleurodeles) and Anura (Bufo, Xenopus) by immunocytochemistry with anti-porcine galanin antiserum. In the four species, immunoreactive perikarya were observed in the telencephalon (striatum, amygdala), diencephalon preoptic area mainly along the anterodorsal wall of the preoptic recessus, suprachiasmatic nucleus, lateral hypothalamus, ventral and dorsal infundibular nuclei, paraventricular organ, and rhombencephalon (nucleus of the solitary tract). Galaninergic fibres extended in similar regions and in the medial septum, ventral telencephalon, ventral hypothalamus, median eminence, and various mesencephalic and rhombencephalic regions. Contacts with the cerebrospinal fluid cavity occurred along the preoptic recessus (Ambystoma) and the ventral infundibular wall (all species). Fibres were scarce in the neurohypophysis. The distal and intermediate lobes of the pituitary were virtually devoid of immunoreactivity. The galaninergic system appeared more developed in adult amphibia than in young animals, suggesting the stimulating influence of sex steroids on the expression of galanin as previously described in Anguilla. The extensive distribution of the galanin-like immunoreactive neurons in amphibian brains suggests that this peptide may act as a neuromodulatur and/or neurotransmitter.

Corticotropin-like immunoreactivity in the brain of intact, hypophysectomized, cortisol- and metopirone-treated eels. Comparison with changes in pituitary corticotropes and brain corticotropin-releasing factor.

An ACTH-like peptidergic system was demonstrated in the brain of three teleost species by immunocytochemistry. In order to investigate the origin of brain ACTH and factors modulating its synthesis, similar techniques were applied to the brain of eels (1) submitted to hypophysectomy in order to suppress pituitary ACTH and plasma cortisol, (2) injected with cortisol to inhibit pituitary ACTH synthesis and release, and (3) injected with metopirone to block cortisol synthesis and stimulate ACTH synthesis and release. Hypophysectomized eels showed a normal distribution of immunoreactive perikarya in the ventral hypothalamus and fibers in the brain, suggesting that brain ACTH does not arise from the pituitary. In cortisol-treated eels immunostaining was markedly reduced in brain perikarya and pituitary corticotropes, suggesting a reduced synthesis. In metopirone-injected eels, one third of the animals showed an increased immunostaining in perikarya and a dense network of immunoreactive fibers, suggesting that ACTH synthesis was increased. Brain ACTH was not affected in other animals. Pituitary corticotropes were rapidly degranulated. Responses of ACTH in the brain and pituitary occur independently when cortisol synthesis is inhibited. These responses are compared to those of the corticotropin-releasing factor system in the same eels.

Responses of brain and pituitary immunoreactive corticotropin-releasing factor in surgically interrenalectomized eels: immunocytochemical study.

Interrenal (IR) cell islets, mainly located in the anterior and posterior cardinal veins, can be surgically removed in the eel. Immunocytochemical studies showed that sham operation slightly affected the distribution of immunoreactive corticotropin-releasing factor (ir-CRF) in the preoptic nucleus (PON) and neurohypophysis (NH). Surgically interrenalectomized (IRX) eels showed a significant increase in the cross-sectional area of the magno- and parvocellular perikarya of the PON and a noticeable decrease in their immunostaining after 8-9 days. In the pituitary, the immunostaining of the rostral nerve fibers facing the corticotropic (ACTH) cells was greatly reduced whereas ir-CRF in the caudal NH was not clearly affected. Immunostaining of the ACTH cells was strongly reduced and that of melanocorticotrops slightly decreased. These data suggest that the suppression of cortisol synthesis is responsible for the stimulation of the CRF ir-perikarya and the CRF release along the rostral pars distalis, inducing ACTH cell degranulation. However, the stressed condition of these eels may accentuate the response to IRX. The neurointermediate lobe, although slightly affected, does not seem to be directly controlled by the plasma cortisol level. These results are compared to those obtained after a pharmacological IRX produced by metopirone.

Galanin-like immunoreactivity is increased in the brain of estradiol- and methyltestosterone-treated eels.

A galanin-like peptidergic system was demonstrated in the brain of Anguilla. A group of immunoreactive perikarya was located in the nucleus preopticus periventricularis close to the recessus preopticus. Galaninergic fibers occurred in various brain areas. Galanin identified in mammalian pituitary cells was undetectable in fish adenohypophysial cells. Estradiol increased the immunostaining of the rostral perikarya and brain fibers in both male and female European eels kept in fresh water and in female American eels in sea water. Methyltestosterone, an aromatizable androgen, increased galanin immunoreactivity in rostral perikarya and brain fibers of male European eels and female American eels. The cross-sectional area of these perikarya increased significantly after both treatments whereas cell bodies of the posteroventral hypothalamus were slightly affected. Dihydrotestosterone showed no clear effect. Fibers close to the corticotropes were sometime increased, but galanin synthesis was not induced in pituitary cells. In contrast, estradiol induced galanin synthesis in rat pituitary cells, but had a still controversed effect on hypothalamic galanin. A putative influence of galanin on the pituitary-gonadal axis is discussed as gonadal hormones diversely affect gonadotropes and gonosomatic indices in Anguilla.

Immunocytochemical localization of a galanin-like peptidergic system in the brain and pituitary of some teleost fish.

Immunostaining of brain and pituitary sections of teleost fishes (eels, salmonidae, cyprinidae, gourami, sculpin, mullet) with anti porcine galanin (GAL) revealed the presence of immunoreactive (ir) perikarya and a rich network of fibers. Ir-perikarya were located rostrodorsally to the recessus preopticus, and in the posterior tuberal hypothalamus. Ir-fibers were abundant in basal telencephalon and around diencephalic ventricular recesses but never contacted their lumen. Furthermore, they were observed in basal hypothalamus, brainstem and ventral medulla. Ir-fibers passed along corticotropic (ACTH), gonadotropic cells and somatotropes (GH cells) in eel and trout pars distalis, but rarely ended in caudal neurohypophysis. In goldfish pituitary ir-fibers occurred in neural digitations and among different cell types which however did not contain a GAL-like peptide. The relation GAL fibers/GH cells appeared more evident in species with a high growth rate. The other species showed a similar distribution of brain GAL. In eels and trout, ir-perikarya were not observed in areas containing somatostatin, GH- and ACTH-releasing factor, and ACTH-like perikarya, suggesting that GAL did not coexist with these peptides. The widespread distribution of a GAL-like peptide in teleost brain suggests that it could play a role of neurotransmitter and/or neuromodulator and regulate the secretion of adenohypophysial hormone(s).

Effect of pharmacological adrenalectomy on corticotropin-releasing factor-like and arginine vasotocin immunoreactivities in the brain and pituitary of the eel: immunocytochemical study.

Metopirone, a blocker of the 11 beta-hydroxylase, was used to suppress cortisol synthesis in the eel, producing a pharmacological adrenalectomy. After 1 and 2 days, corticotropin-releasing factor (CRF) immunoreactivity was reduced in the preoptic nucleus (PON) and was increased in the rostral neurohypophysis (NH) where CRF fibers ended in close proximity to ACTH cells. After 4 days, immunoreactive (ir) CRF increased in the PON or was similar to that of controls, and was reduced in the rostral NH. It was not affected in the caudal NH among intermediate lobe (IL) ramifications. Arginine vasotocin (AVT) immunostaining was often slightly increased in the PON, but changes were not apparent in the pituitary and AVT-ir fibers remained very scarce in the rostral NH. Metopirone significantly increased the cross-sectional area of CRF- and AVT-ir perikarya in the parvocellular region and the number of these perikarya as well as the ratio of CRF to AVT cell bodies in the totality of the PON. Colocalization of CRF and AVT, barely detected in control eels, occurred in 9.6% of CRF perikarya after 1-2 days, but in only 2.4% after 4 days. ACTH cells were rapidly and markedly stimulated. These immunocytochemical studies suggest that CRF synthesis, axonal transport, and release are increased in metopirone-treated eels, acting on the pituitary to stimulate ACTH release and interrenal cell activity. The participation of AVT in ACTH cell stimulation is less clear. These data are compared to those reported in mammals.

Corticotropin-like immunoreactivity in the brain and pituitary of three teleost species (goldfish, trout and eel).

Immunostaining of brain and pituitary sections of three teleost species (goldfish, trout and eel) with antisera to porcine and human ACTH 1-39 revealed the presence of an ACTH (adrenocorticotropic hormone)-like peptide in the ventral hypothalamus. Perikarya were localized in the rostral, median and posterior portions of the nucleus lateralis tuberis (NLT); some were in contact with the cerebrospinal fluid. A dense network of immunoreactive (ir) fibers occurred in the peri-infundibular region and extended into the periventicular tissue, around the lateral and posterior recesses. Rostrally directed ir-fibers reached the telencephalon either ventrally or mediodorsally; some were observed in the olfactory lobe. In the mesencephalon, ir-fibers penetrated into the optic tectum of the goldfish. In the pituitary, both antisera intensely labeled rostral ACTH cells. Small groups of labeled cells were scattered in the rostral pars distalis and the proximal pars distalis. A gradient of activity was evident among ACTH cells: those located along the rostral neurohypophysis containing corticotropin-releasing factor nerve terminals were larger and often more marked than those farther away from the neural tissue. ACTH-like peptide in the brain may act as a neuromodulator, mainly in the NLT and the preoptic nucleus, and around the nuclei of the ventricular recesses containing serotonin and catecholamines.

Localization of growth hormone-releasing factor-like immunoreactivity in the hypothalamo-hypophysial system of some teleost species.

An antiserum to growth hormone-releasing factor (GRF) 1-44 was applied on brain and pituitary sections of nine teleost species. Immunoreactive (ir) perikarya were demonstrated in parvo- and magnocellular portions of the preoptic nucleus (PON) and occasionally in the nucleus lateralis tuberis. The two tracts originating in the PON ran ventro-laterally toward the optic chiasm and then caudally in the basal hypothalamus. In the pars distalis (PD) of the eel, carp, goldfish and salmonids, GRF-ir fibers did not enter the rostral PD and few fibers passed close to somatotropes. In Myoxocephalus and Mugil, a variable number of ir-fibers passed close to cells of the rostral and proximal PD. In the neurointermediate lobe, GRF-ir fibers were located exclusively in the neural tissue of the eel and trout. In goldfish, carp and Myoxocephalus, GRF-ir fibers entered the intermediate lobe. This antiserum also labeled corticotrops and, to a lesser extent, melanotrops in the pituitary of cyprinids. A variable number of perikarya contained both GRF and vasotocin in the PON of the eel. In all teleost species studied so far, the distribution patterns of GRF are different, and the function of the various adenohypophysial cell types appears to be differently modulated, according to the variable distribution of GRF in the pituitary.

Quantitative changes of CRF-like immunoreactivity in eels treated with reserpine and cortisol.

Immunocytochemical techniques were applied to brain and pituitary sections of European eels after experimental manipulation of the pituitary-interrenal activity. A corticotropin-releasing factor (CRF) antiserum allowed the identification of a CRF-like peptide in the preoptic nucleus (PON) and rostral and caudal neurohypophysis (NH). CRF-immunoreactivity (ir) was not affected in solvent-injected eels compared to noninjected eels. Reserpine induced a stimulation of the pituitary interrenal axis, decreased ir-CRF in the rostral NH, but did not affect hypothalamic ir-CRF. Cortisol reduced the immunostaining of hypothalamic CRF-ir perikarya and perikarya cross-sectional area. In the rostral NH, CRF-ir fibers decreased in number and almost disappeared in long-term treated eels. The immunostaining of ACTH cells with ACTH antiserum was greatly reduced. These data suggest that cortisol induces a marked reduction in the activity of the CRF-corticotrop axis. The intensity of the ir-CRF staining observed in the caudal NH, close to the intermediate lobe (IL) was not significantly affected in reserpine-treated eels, and only slightly reduced in long-term cortisol-treated eels. The intensity of ir-CRF staining in the caudal NH did not correlate with melanocorticotropic activity or plasma cortisol level. These data suggest that immunoreactive CRF fibers in the rostral and caudal NH are differently regulated.

Effect of hypophysectomy on the hypothalamic corticotropin-releasing factor (CRF) neuronal system in the eel: immunocytochemical study.

Male and female eels were hypophysectomized by a buccal approach and examined either 2 and 7 weeks (males) or 4 and 5 months (females) later. Intact and sham-operated eels served as controls. Fish were killed by decapitation, and the brains fixed in sublimated Bouin-Hollande solution and embedded in paraffin wax. Frontal and sagittal sections were immunostained by the peroxidase-antiperoxidase method, using antisera against cortiotropin-releasing factor (CRF) or arginine vasotocin (AVT). Immunoreactive (ir)-CRF was detected in parvo- and magnocellular perikarya of the preoptic nucleus. Sham hypophysectomy did not affect ir-CRF in cell bodies and fibres. After hypophysectomy, mainly after 4 or 5 months, there was an apparent increase in the number of ir-CRF cells. The intensity of immunostaining in the cell bodies was variable but not significantly different from the controls, but the two ventral axonal tracts were much thicker and contained more immunoreactive material than in sham-operated eels, while ir-CRF accumulated close to the sectioned pituitary stalk. The existence of negative feedback by cortisol on the CRF neurons is discussed.

Cytological responses of the pituitary (rostral pars distalis) and immunoreactive corticotropin-releasing factor (CRF) in the goldfish treated with dopamine antagonists.

The in vivo effects of three dopamine (DA) antagonists on the cytology of the rostral pars distalis (RPD) were investigated in young goldfish (Carassius auratus L.). Pimozide, sulpiride, and domperidone were injected for 5 (low dose, Experiment I) and 7 days (higher dose, Experiment II). Cytological and immunocytochemical techniques using antisera to (1-24) ACTH and (1-39) ACTH, human beta-thyrotropin (TSH beta), and synthetic (1-41) CRF were applied to pituitary and brain sections. Cytometrical studies showed that the three drugs induced similar quantitative changes in the cells of the RPD. Prolactin (PRL)-secreting cell hypertrophy was significant in Experiment II, whereas the nuclear enlargement was significant in both experiments. The numbers of cytoplasmic granules were similar in control and treated goldfish. Thyrotropic (TSH) cells and their nuclei were significantly enlarged in both experiments; their content in immunoreactive TSH was not clearly modified. Corticotropic (ACTH) cells showed significant nuclear and cellular hypertrophy, and labeled granules were often concentrated along the cell membrane. The amount of immunoreactive CRF present in the rostral neurohypophysial ramifications was reduced in the majority of treated fish. Solvent-injected controls showed no significant changes in the RPD. These results suggest that DA inhibits PRL cell activity in goldfish. TSH and ACTH cells appear stimulated by DA-receptor blockers, although differential effects on synthesis and release cannot be evaluated in in vivo experiments. A release of corticotropin-releasing factor may be involved in the ACTH cell stimulation. These data are compared with those obtained in other vertebrates.

Coexistence of corticotropin-releasing factor-like immunoreactivity and vasotocin in perikarya of the preoptic nucleus in the eel.

A corticotropin-releasing factor (CRF)-like system has been identified in eels (Anguilla anguilla, A. rostrata, and A. japonica) with immunocytochemical techniques. Immunoreactive perikarya were located in the preoptic area, in magno- and parvocellular regions of the preoptic nucleus. Fine fibers reached the pituitary and terminated in the neurohypophysis close to the corticotropic cells of the rostral pars distalis and caudally close to the melanocorticotropic cells of the intermediate lobe. Double immunostainings revealed the presence of three subpopulations of perikarya: one population containing only CRF, a second containing only arginine vasotocin (VT), and a third one containing both CRF and VT. This coexistence of CRF and VT was also demonstrated by comparing adjacent sections stained with anti-CRF and anti-VT, respectively. Because antisera used in the present work were carefully absorbed, these data strongly suggest that CRF and VT coexist in some perikarya, at least in the animals studied.

Localization of CRF-like immunoreactivity in the brain and pituitary of teleost fish.

Immunocytochemical techniques were applied to brain and pituitary sections of eleven teleost species. A corticotropin-releasing factor (CRF)-antiserum allowed the identification of a CRF-like system in these species. Perikarya were labeled in the preoptic nucleus. Labeled fibers were traced laterally, then ventrally close to the optic chiasma, forming two symmetrical tracts running through the basal hypothalamus. These ended in the rostral neurohypophysis (NH) close to ACTH cells as shown by double immunostaining. Other fibers, often more variquous, ended in the caudal NH close to melanocorticotropic cells. In Salmo fario, small perikarya also stained in the nucleus lateralis tuberis. The CRF-like system appears distinct from that of somatostatin. In Anguilla, adjacent sections stained with CRF- and vasotocin (AVT)-antisera respectively showed that these two peptides coexist in some perikarya. As few fibers containing only AVT end in the rostral NH, they probably do not control ACTH cells directly. AVT fibers terminate mostly in the caudal NH close to melanocorticotropic cells. Some extra-hypothalamic fibers suggest that CRF may also act as a neurotransmitter. The plurality of hormones showing a CRF-like activity in teleosts is considered.

In vivo effect of dopamine antagonists on melanocyte-stimulating hormone cells of the goldfish (Carassius auratus L.) pituitary.

Young goldfish were injected with three dopaminergic antagonists, pimozide, sulpiride, and domperidone, for 5 (low dose) and 7 days (higher dose). Cytological and immunocytochemical techniques using anti-alpha-melanocyte-stimulating hormone (MSH) serum were applied to the pituitary. MSH cells in the three treated groups showed a decrease in immunoreactive cytoplasmic granules, a significant nuclear hypertrophy, and, after 7 days, a cellular enlargement. The nucleolus and the lamellar endoplasmic reticulum were more developed and some mitotic figures occurred. Erythrophores and occasional melanophores were in a stage of maximal dispersion. These changes were not apparent in the solvent-injected controls. The responses to the three blockers of dopaminergic receptors were similar. These data suggest that MSH release seems to be under a dopaminergic inhibitory control in the goldfish. The other cell type of the pars intermedia (PAS-positive and calcium-sensitive in the goldfish) was not clearly affected by the three drugs.

Immunocytochemical localization and spatial relation to the adenohypophysis of a somatostatin-like and a corticotropin-releasing factor-like peptide in the brain of four amphibian species.

The distribution of somatostatin (SRIF) - and corticotropin-releasing factor (CRF)-like - immunoreactive material was studied in the brain of four amphibian species (Ambystoma mexicanum, Pleurodeles waltlii, Xenopus laevis, Rana ridibunda) by use of immunocytochemistry. A wide network of SRIF-immunoreactive fibers and numerous perikarya were observed in all amphibians examined, with a dense accumulation of nerve endings in the external layer of the median eminence (ELME). In the representatives of the four amphibian species the CRF-like system was more circumscribed. Immunoreactive perikarya were present in the preoptic area, mainly in a ventrobasal position, and in the interpeduncular nucleus. The tract running along the ventral part of the tuber cinereum ends in the ELME facing the rostroventral lobe of the pars distalis that contains corticotrophs. CRF fibers were scarce or absent in the neural lobe. In all species studied in the present work, CRF fibers end in the area of the ELME close to the pituitary lobe containing corticotrophs. This correlation is similar to that reported for the Japanese quail and several teleosts.

Cytological and ultrastructural changes in the pituitary pars distalis of the goldfish kept in calcium-free environments.

The cytology and ultrastructure of the pars distalis, mainly that of prolactin (PRL) cells, were investigated in goldfish adapted to fresh water (FW) or deionized water (DW) for 20 and 40 days, or gradually adapted to 1/3 artificial sea water (ASW) or 1/3 Ca-free sea water. When compared to PRL cells of goldfish kept in FW, those of goldfish adapted to DW did not show signs of increased activity. The lack of exocytotic activity and the low development of various organelles suggested that cell activity was slightly reduced. In 1/3 ASW, PRL cells were smaller and less active. In 1/3 Ca-free ASW, PRL cells appeared slightly stimulated compared with those of fish in 1/3 ASW. The Golgi area was more developed and a few lamellae of endoplasmic reticulum were observed in some cell islets. However, there was no significant difference between PRL cells of goldfish kept in 1/3 Ca-free ASW and in FW. In 1/3 ASW, which is isosmotic to the blood, thyrotrophs (TSH cells) corticotrophs (ACTH cells) and somatotrophs (STH cells) were not clearly affected. In DW, these cells and their nuclei were significantly enlarged. Their stimulation was also evident in 1/3 Ca-free ASW; values for cellular and nuclear areas were maximal in this environment and significantly higher than those of fish in FW and 1/3 ASW. These data suggest that in addition to the PAS-positive cells of the pars intermedia, highly stimulated in Ca-free environments, other cell types of the pars distalis may be involved in osmoregulation, and that the role of PRL cells is not primordial in the goldfish.

Effects of estradiol and mammalian LHRH on the ultrastructure of the pars distalis of the eel.

Male silver eels were injected with estradiol-17 beta (E2) to induce the development of gonadotropic (GTH) cells. They were subsequently injected with luteinizing hormone-releasing hormone (LHRH). Exocytotic figures and the lysis of some large globules and granules were observed. Morphometric studies showed a significant increase in the percentage of vacuoles after 4 and 6 injections of LHRH and a slight but significant decrease of granules. This response did not, however, occur in all GTH cells which never appeared completely degranulated and did not reach a vesicular stage. Hemi-pituitaries of E2-pretreated eels were incubated with or without LHRH (20 min to 2 h). Although typical exocytoses were not detected, an increased number of small granules near the basal lamina and lytic processes (globules with a raspberry-shaped structure, granules with variable electron density) were observed in the LHRH-incubated hemi-pituitaries compared with those kept in a control medium. The structure of GTH cells and their response to LHRH has been studied only under conditions of artificial stimulation, and their functional similarity to GTH cells of spontaneously maturing eels is discussed. Large female eels had unstimulated GTH cells. Growth hormone (STH), thyrotropic (TSH) and prolactin (PRL) cells were stimulated after E2 and LHRH. As with GTH cells, they regressed slowly after treatment was discontinued.

Effect of 17 alpha-methyltestosterone on the skin and gonads of freshwater male silver eels.

Male silver eels kept in fresh water (FW) were injected with 17 alpha-methyltestosterone (MT). They received 5, 10, 15, or 20 injections (8 micrograms/g every other day) or 6 or 9 injections (4 or 2.5 micrograms/g once a week) and were killed 24 hr after the last injection. Compared to solvent-injected controls, the treated eels appeared darker, the eye diameter was enlarged, the head and the pectoral and caudal fins were black and the melanophore index was maximal. The thickness of the skin (epidermis plus dermis) was significantly increased. Macroscopically, the organs of Syrski (juvenile testes) were slightly larger and less translucent, but the maximal gonosomatic index reached only 1-1.2 in two fishes. Stimulation started in the anterior part of the gonad. Microscopically, all stages of spermatogenesis and spermiogenesis were observed. After 20 injections (high dose, 40 days) or 6 (one case only) and 9 injections (low dose, 57 days), spermatids and spermatozoa were present. These positive results are compared to the negative data previously reported in other countries. The mode of action of the androgen is discussed. It is suggested that it acts for the most part directly on the gonad, but a participation of the pituitary cannot be ruled out, although gonadotrophs appeared only slightly stimulated.