Immunocytochemical demonstration of close relationships between neuropeptidergic nerve fibers and hormone-producing cell types in the adenohypophysis of the sea bass (Dicentrarchus labrax)

Light microscopic double immunocytochemical stainings, performed on sea bass hypothalamo-hypophysial sections, revealed the projection of different neuropeptide-immunoreactive neurons innervating the hormone-producing cell populations in the pituitary gland. In the rostral pars distalis (PD) the ACTH cells were found in close proximity to fibers immunoreactive for somatostatin (SRIF), growth hormone-releasing hormone (GRF), corticotropin-releasing hormone (CRF), vasotocin (VT), isotocin (IT), substance P (SP), neurotensin, and galanin (GAL), while the PRL cell zone seemed only innervated by nerve fibers immunopositive for GAL. In the proximal PD, fibers immunoreactive for SRIF, GRF, VT, IT, cholecystokinin, SP, neuropeptide Y, and GAL formed a close relationship with the growth hormone cells. The gonadotrophs were observed near nerve fibers immunostained for gonadotropin-releasing hormone, IT, and less obviously GRF and VT, while fibers positive for GRF, CRF, VT, IT, SP, and GAL penetrated between and formed a close association with the thyrotrophs. In the pars intermedia the MSH cells and the PAS-positive (PAS+) cells seemed both innervated by separate nerve fibers immunoreactive for GRF, CRF, melanin concentrating hormone, VT, IT, and SP. All these results suggest a functional role of the neuropeptides in the adenohypophysis of the sea bass, possibly in the synthesis and/or release of hypophysial hormones from the different cell types.     

Purification and characterization of follicle-stimulating hormone from pituitary glands of sea bass (Dicentrarchus labrax)

Follicle-stimulating hormone (FSH) was purified from pituitaries of sea bass (Dicentrarchus labrax), and its biochemical and biological properties were studied. Sea bass FSH (sbsFSH) was purified by ethanol extraction-precipitation (40–85%), followed by anion-exchange chromatography on a LKB Ultropac TSK-DEAE column using a linear gradient of ammonium bicarbonate (50–1000 mM) and reverse phase chromatography on a RESOURCE 15RPC column with a linear gradient of acetonitrile (0–50%), using a FPLC system. The molecular mass of the purified sbsFSH, estimated by mass spectrometry, was of 28.5 kDa for the dimer, 12.6 kDa for the glycoprotein α (GPα) and 13.6 kDa for FSHβ subunits. After separation by SDS–PAGE under reducing condition, the intact sbsFSH was dissociated in the respective subunits (GPα and FSHβ). Subunit identity was confirmed by immunological detection and N-terminal amino acid sequencing. Deglycosylation treatment with N-glycosidase F, decreased the molecular mass of both subunits. Intact sbsFSH activated the sea bass FSH receptor stably expressed in the cell line HEK 293, in a dose dependent manner. Purified sbsFSH showed gonadotropic activity, by stimulating the release of estradiol-17β (E2) from sea bass ovary and testosterone (T) and 11-ketotestosterone (11KT) from testicular tissue cultured in vitro, in a dose and time dependent manner. These results showed that the purified sbsFSH is a heterodimeric hormone, composed of two distinct glycoprotein subunits (GPα and FSHβ), and has biological activity judged by its ability to stimulate its receptor in a specific manner and to promote steroid release from gonadal tissue fragments.     

Thyrotropin-releasing hormone-immunoreactive system in the brain and pituitary gland of the sea bass (Dicentrarchus labrax, Teleostei)

The immunohistochemical distribution of thyrotropin-releasing hormone-like immunoreactivity (TRH-ir) in the brain and pituitary of the sea bass (Dicentrarchus labrax) was examined on cryostat sections of tissues perfuse fixed in a formaldehyde-glutaraldehyde mixture. TRH-ir fibres were found in many areas of the brain: dorsal and ventral telencephalon, preoptic and tuberal hypothalamus, thalamus, midbrain tegmentum, optic tectum, and medulla oblongata. In the hypothalamus the densest area of innervation was the nucleus anterioris tuberis and medial nucleus recessus lateralis, where small TRH-ir cell bodies were also found. In the pituitary gland, TRH-ir fibres were numerous in the posterior neurohypophysis, and these appeared to form varicosities between groups of melanocorticotropic cells of the pars intermedia. No clear relationship was seen between TRH-ir fibres and the thyrotropic cells, or any other cell type of the pars distalis. In the brain stem a notable feature was the prominent innervation of groups of motoneurons by beaded TRH-ir fibres. These observations suggest that in teleost fishes the role of TRH may be related to pars intermedia function, rather than the thyrotropin- or prolactin-releasing function established in tetrapods. In addition the tripeptide may act as a central neurotransmitter involved in sensory and autonomic motor integration.     

Chronological appearance of the different hypophysial hormones in the pituitary of sea bass larvae (Dicentrarchus labrax) during their early development: an immunocytochemical demonstration

Antisera raised against chum salmon prolactin (PRL), rainbow trout growth hormone (GH), mammalian adrenocorticotropic hormone (ACTH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) were used to study the chronological appearance of immunoreactivity for PRL, GH, ACTH, TSH, LH, and melanocyte-stimulating hormone (MSH) in the pituitary of sea bass larvae (Dicentrarchus labrax) during the first 26 days after hatching. The anti-ACTH gives positive immunostaining in the ACTH cells as well as in the MSH cells; however, the two cell types can easily be distinguished by their different localization in the pituitary: ACTH in the rostral pars distalis, MSH in the pars intermedia. The first day after hatching cells immunoreactive for TSH, GH and ACTH could already be noticed, ACTH reacted strong in the pars intermedia but very weak in the rostral pars distalis. Cells immunopositive for PRL became visible between Days 9 and 15. With anti-LH, no positive reaction could be obtained during the first 26 days after hatching.     

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) I. Immunocytochemical characterization of glucagon- and PP-related peptides

PP-, PYY-, and glucagon-immunoreactive cells were immunocytochemically identified in the pancreatic islets of Dicentrarchus labrax (sea bass). PYY cells also reacted with anti-PP serum. The specificity control showed that preabsorption of PP antiserum by PYY peptide abolished the immunostaining, while the reaction did not change when the PYY antiserum was preabsorbed by PP. These results suggested the existence of a PP/PYY molecule in the sea bass islets. The islet distribution of PP/PYY-immunoreactive cells differed markedly. Thus, in the principal islet and some intermediate islets few PP/PYY-immunoreactive cells are present (type I islets), whereas in the smaller and some intermediate ones they are numerous (type II islets). Adjacent sections stained by peroxidase-antiperoxidase (PAP) technique and individual sections stained by immunofluorescence double staining showed the coexistence of glucagon and PP/PYY-like immunoreactivities. Both islet types contained cells with PP/PYY coexisting with glucagon peptide, while cells showing solely glucagon immunoreactivity were found in type I islets only.     

Cloning of a proopiomelanocortin cDNA from the pituitary gland of the sea bass (Dicentrarchus labrax) and assessment of mRNA expression in different tissues by means of real-time PCR

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.     

Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) II. Immunocytochemical study of insulin and somatostatin peptides.

Insulin (INS)- and somatostatin (SST)-immunoreactive cells were demonstrated by light immunocytochemistry in the endocrine pancreas of sea bass (Dicentrarchus labrax). INS-immunoreactive cells were identified using bovine/porcine, bonito, and salmon (s) INS antisera; the immunostaining was abolished when each antiserum was preabsorbed with its respective peptide but not with unrelated peptides. These cells also reacted with mammal (m) SST-28 (4-14) antiserum. The immunoreaction did not change when this antiserum was preabsorbed by bovine INS. INS-immunoreactive cells were located in the central part of the endocrine areas of the principal, intermediate, and small islets. Two SST-immunoreactive cell types (D1 and D2) were revealed. D1 cells, immunoreactive to SST 14 (562) and sSST-25 antisera, were located next to the glucagon-immunoreactive cells in the peripheral part of the endocrine areas. D2 cells, immunoreactive to SST-14 (562), SST-14 (566), and mSST-28 (4-14) antisera, were found in apposition to the INS-immunoreactive cells. The specificity controls showed that D1 cells expressed sSST-25-like peptides, while D2 cells might contain SST-14 and/or mSST-28-like peptides. The close topographic association between the different SST-immunoreactive cells and both glucagon- and insulin-immunoreactive cells might indicate the existence of a specific paracrine regulation of each endocrine cell type in the sea bass endocrine pancreas.     

Immunocytochemical demonstration of pituitary cell types in the teleost Poecilia latipinna, by light and electron microscopy

Using the unlabelled antibody method at the light microscope level, and the immunogold method at the electron microscope level, the distribution of the different adenohypophysial cells was demonstrated in the teleost Poecilia latipinna, by means of antisera to both teleostean and mammalian pituitary hormones and their subunits. Anti-salmon prolactin, but not anti-rat or -ovine prolactin, gave a specific staining of the acidophils of the rostral pars distalis (RPD), while anti-trout growth hormone (GH), but not anti-rat GH, stained similar but always separate cells in the proximal pars distalis (PPD). Antisera to the whole molecules of mammalian glycoprotein hormones stained the entire population of basophils in the PPD, but separate populations of gonadotrophs and thyrotrophs could be discriminated using anti-salmon gonadotrophin and anti-human thyrotrophin beta subunit. Antisera to ACTH (1-24) and (11-24) sequences, as well as beta-endorphin and met-enkephalin, stained the lead haematoxylin-positive cells of the RPD and pars intermedia (PI), whereas anti-alpha-MSH stained only the PI cells. Ultrastructural examination showed that these immunoreactivities were present in the same secretory granules, and were always greater in pale granules rather than electron dense granules. In the RPD, blebs of ACTH-immunoreactive cytoplasm were found to protrude through the gaps in the basement membrane into the neurohypophysis. The second "PAS-positive" cell type of the PI showed a strong cross-reaction with anti-salmon gonadotrophin, suggesting that it may produce a glycoprotein chemically related to the gonadotrophin(s).     

Immunoaffinity purification and partial characterization of sea bass (Dicentrarchus labrax) growth hormone

Growth hormone (GH) was isolated from sea bass (Dicentrarchus labrax) pituitary extract by a simple one-step procedure involving immunoaffinity chromatography. A monoclonal antibody raised against chicken GH and found to immunostain very specifically the GH cells in the pituitary of the sea bass was coupled to CNBr-activated Sepharose 4B. Sea bass pituitary extracts were run on the affinity column, and the eluted material was analyzed on reversed-phase HPLC and found to consist of one single peak. The yield of purified hormone was 2.4 mg/g pituitary. Two monomeric forms (MW = 20,000 and 22,000 Da) of sea bass GH were identified by gel electrophoresis. Gel electrofocusing revealed apparent isoelectric points of 6.15, 6.50, and 6.95. Amino acid composition is consistent with other vertebrate GHs. The immunological relatedness was tested by immunoblotting using antisera raised against GH of different species. Polyclonal antisera raised against the isolated hormone exhibited a specific labeling of the GH cells in sea bass pituitary sections as well as of the immunoblotted purified GH.     

Distribution of cell types and aromatase activity in the sculpin (Myoxocephalus) pituitary

Although aromatase activity is exceptionally high in the teleost pituitary, it is not known which of the secretory cell types are responsible. Pituitary glands from the longhorn sculpin (Myoxocephalus octodecimspinosus) were sectioned transversely into "cephalic" and "caudal" fragments and cultured for 24 hr in medium containing [7-3H]androstenedione. Radiolabeled estrone and estradiol-17 beta production were measured as an estimate of aromatization. In order to determine the distribution pattern of different cell types, the in situ pituitary and dissected fragments were analyzed by standard cytological procedures. Further verification of cell function was obtained by somatostatin (SRIF) and corticotropin-releasing factor (CRF) immunocytochemistry. Estrogen yields obtained from caudal fragments in two separate experiments averaged four times higher per milligram protein than yields from matched cephalic fragments. In addition, female glands synthesized significantly more estrogen than those of males. Due to an anteroflexion of the longitudinal axis and a disposition of the gonadotropic (GTH) cells at the periphery of the gland and surrounding the neurointermediate lobe (NIL), the classical subdivisions of the teleost adenohypophysis were not strictly applicable to the sculpin. The predominance of growth hormone (GH) secreting cells in the caudal fragment suggests their participation in aromatization, a finding which is consistent with a previous study of rodent pituitary; however, a role for gonadotropes and other hypophysial cells in this transformation cannot be ruled out.     

Radioimmunoassay of ovarian steroids in plasmas of ovulating female sea bass (Dicentrarchus labrax)

Radioimmunoassays of the free and conjugated fractions of plasmas from ovulating sea bass (Dicentrarchus labrax) have revealed the presence of several unusual polar steroids. Among them is 3 alpha,17 alpha,20 beta,21-tetrahydroxy-5 beta-pregnane.     

Immunocytochemical localization of LH-RH in the brain and pituitary of the catfish, Clarias batrachus (Linn.)

An elaborate organization of luteinizing hormone-releasing hormone (LH-RH) immunoreactive (ir) cells and fibers was encountered in the olfactory system of Clarias batrachus. In addition to the ir structures in the olfactory nerve, peripheral area of the olfactory bulb, and the medial olfactory tract (MOT), ir cells and fibers were prominently seen in the lamellae of the olfactory organ. Perikarya showing varying degrees of intensity of immunoreaction were observed along the base of the forebrain in the nucleus preopticus basalis lateralis, nucleus preopticus periventricularis, nucleus preopticus, nucleus lateralis tuberis pars posterior, and the pituitary. Some cells were also noticed in the midbrain tegmentum. A well-defined system of ir fibers from the MOT penetrated the telencephalon and curved dorsocaudally into the pars supracommissuralis above the anterior commissure (AC); while some fibers decussate in the AC, others extended posteriorly into the diencephalon. A fairly dense network of beaded ir fibers was seen in the basal forebrain, conspicuous around the organum vasculosum laminae terminalis and caudally traceable as far as the neurohypophysis; some immunostained fibers appear to be directly contacting with the cells of the proximal pars distalis. Fibers were also witnessed in the optic chiasma and in the inner plexiform layer of the retina. Solitary fibers were noticed in certain circumscribed telencephalic areas, caudal hypothalamus, posterior commissure, midbrain tegmentum, cerebellum, and ventral medulla oblongata. The highly organized LH-RH containing system in C. batrachus is indicative of its elaborate role in synchronization of the reproductive processes and the environmental cues.     

Androgen receptor localization in different cell types of the adult rat prostate

To better understand direct and indirect androgen action on rat prostatic growth and function, the various cell populations within the intact adult ventral, dorsal, and lateral prostate lobes were characterized for the presence or absence of androgen receptor (AR). Polyclonal rabbit antibodies raised against amino acids 1-21 of the rat AR (PG-21) were used in combination with a library of monoclonal antibodies directed against cell-specific antigens for positive cellular identification. Luminal epithelial cells were strongly AR positive, with an order of ventral greater than lateral greater than or equal to dorsal. In the lateral lobe, staining intensity was strongest in the peripheral regions, whereas a similar gradient was not apparent in the ventral and dorsal prostate. Basal epithelial cells were AR negative in all regions of the three lobes. Periacinar smooth muscle was strongly positive for AR, and this staining did not vary with the thickness of the muscle layer. Endothelial cells of the vasculature were AR negative, while the perivascular smooth muscle cells were AR positive. The majority of stromal fibroblasts were AR negative, although a number of AR-positive fibroblastic-appearing cells were observed within the ventral and dorsal lobes. Staining with ED2, a specific marker for tissue macrophages, revealed that fixed macrophages were present in significant quantities in the stroma of intact rat prostate lobes. Since these were frequently identified as AR positive, macrophages may partially account for the appearance of AR-positive stromal cells. Thus, the present findings indicate a complex pattern of AR expression among different cell types of the three prostate lobes. Cells types that express AR can potentially be considered as direct targets of androgen action, whereas those lacking AR should be considered as indirect targets or androgen-insensitive cells.     

Immunocytochemical and ultrastructural characterization of the cell types in the adenohypophysis of Sparus aurata L. (teleost)

The structure and immunocytochemistry of the adenohypophysis of sexually mature male specimens of Sparus aurata (gilthead sea bream) were studied. The adenohypophysis was composed of rostral pars distalis (RPD), proximal pars distalis (PPD), and pars intermedia (PI). In the RPD the prolactin cells were organized into follicles which occupied a very reduced area as corresponds to that in saltwater fishes; the corticotropic cells were surrounding the pars nervosa branches and the prolactin follicles. The PPD showed somatotropic, gonadotropic, and thyrotropic cells. The somatotropic cells were isolated, clustered, or surrounding the pars nervosa branches. Only one polymorphic cell type of gonadotropic cells was found in the PPD ventral and dorsal areas and around the PI. The PI was composed mainly of melanotropic cells and a PAS-positive cell layer adjacent to the neurohypophysis. The ultrastructure of the presumptive endocrine cells was reported and their distribution was discussed in relation to those of other teleosts.     

Immunocytochemical localization of angiotensin immunoreactivity in gonadotropes and lactotropes of the rat anterior pituitary gland

Utilizing an immunocytochemical technique wherein two different antigens can be visualized on the same tissue section, angiotensin-like immunoreactivity was found in rat adenohypophyseal cells that showed staining with antisera to hLH, hFSH or human prolactin. Angiotensin-like immunoreactivity was not found in cells that showed staining with antisera to hTSH, C-terminal ACTH or hGH. The nature and possible physiologic significance of the angiotensin-like immunoreactivity is discussed.     

Immunocytochemical study on the localization and distribution of the somatolactin cells in the pituitary gland and the brain of Oreochromis niloticus (Teleostei, cichlidae)

Using specific antibody for chum salmon somatolactin (SL), immunocytochemical studies were employed to determine the distribution of this hormone in the pituitary gland and the brain of Orechromis niloticus. The results indicated that the SL-immunoreactive (ir) cells are found in the pars intermedia (PI) of the pituitary gland. The SL-ir cells showed strong and specific immunoreactivity to anti-chum salmon SL. Moreover, SL-ir cells were found to be widely distributed in most brain regions. Most of the SL-ir cell bodies were scattered along a nearly continuous line extending posteriorly from the olfactory bulb to the medulla oblongata through the nucleus preopticus periventricularis, habenula, and midbrain tegmentum and ventral to the nucleus lateralis tuberis pars posterior through the nucleus preopticus basalis lateralis and organum vasculosum luminae terminalis. Also SL-ir cells were observed in the cerebellum. The synthetic and secretory activity of the SL-ir cells, in the pituitary and the brain, showed an increase during sexual maturation and spawning. The highly organized SL-containing system and the gradual stimulation of SL synthesis and release during sexual maturation and spawning of O. nilotcus suggest that SL may be involved in the control of some steps of reproductive processes.     

Localization of growth hormone releasing factor-like immunoreactivity in the hypothalamo-hypophyseal system of the frog (Rana temporaria) and the sea bass (Dicentrarchus labrax)

Using two different antisera, one raised against total human pancreatic growth hormone releasing factor (hpGRF) coupled through a two-step glutaraldehyde method and the other one raised against rat hypothalamic growth hormone releasing factor 1-10 (rGRF1-10), GRF-like immunoreactivity was localized in the hypothalamo-hypophyseal system of the frog (Rana temporaria) and the sea bass (Dicentrarchus labrax). In the frog immunoreactive neurons were found in the nucleus preopticus, pars magnocellularis. The immunopositive fibers were localized in the lateral wall of the preoptic recess, the pars ventralis of the tuber cinereum, the internal and external zone of the median eminence, and the neural lobe. Positive-stained neurons in the sea bass were located in the preoptic nucleus, in the pars magnocellularis as well as in the pars parvocellularis, and in the nucleus lateralis tuberis, pars rostralis. GRF-ir nerve fibers, originating in the hypothalamus, projected to the rostral and proximal pars distalis, the posterior neurohypophysis, and the pars intermedia (PI). Double stainings with anti-GRF and anti-ACTH or anti-trout GH showed some close relationship between GRF immunoreactive nerve fibers and adenohypophyseal cell types. In the PI both the MSH and the PI "PAS" positive cells seemed to be directly innervated by the GRF-ir axons. These results show that a GRF-like system is present in the hypothalamo-hypophyseal system of amphibians and teleosts and that in these lower vertebrates GRF-like material may be secreted directly in the systemic circulation. The function of this GRF, however, is not yet clear.     

Immunocytochemical identification and localization of APUD cells in the gut of seven stomachless teleost fishes

AIM To study the cell types, localization,distribution density and morphology of APUD cells in the intestinal mucosa of stomachless teleost fishes.METHOD By using the peroxidaseantiperoxidase complex ( PAP )immunocytochemical staining technique the identification, localization and morphology of immunoreactive (IR) endocrine cells seattered in the intestinal mucosa of grass carp ( Cyenopharyngodon idellus ), black carp (Mylopharyngodon piceus ) and common carp (Cyprinus carpio ) were investigated with 20 kinds of antisera prepared against mammalian peptide hormones of APUD cells, and likewise by using avidin-biotin-peroxidase complex (ABC)method those of silver carp ( Hypophthalmichthys molitrix ), bighead (Aristichthys nobilis ), silver crucian carp (Carassius gibelio ) and bluntnose black bream ( Megalobrama amblyocephala ) were also studied with 5 different antisera. The replacement of the first antiserum by phosphate buffered saline (PBS) was employed as a control. IR endocrine cells were counted with a square-mesh ocular micrometer from 10 fields selected randomly in every section of each part of the intestine specimen. The average number of IR endocrine cells per mm2 was counted to quantify their distribution density.RESULT Gastrin (GAS)-, Gastric inhibitory peptide (GIP)-, glucagon (GLU)-, glucagon-like immunoreactants ( GLI )-, bovine pancreatic polypeptide (BPP)-, leucine-enkephalin (ENK)-and substance P (SP)-IR endocrine cells were found in the gut of grass carp, black carp and common carp, and somatostatin ( SOM )-IR endocrine cells were only seen in common carp.GAS-, GIP- and GLU-IR endocrine cells were found in the intestinal mucosa of silver carp,bighead, silver crucian carp and bluntnose black bream. Most of IR endocrine cells had the higher distribution density in the foregut and midgut,and were longer in shape. They had a long apical cytoplasmic process extended to the gut lumen and a basal process extended to adjacent cells or basement membrane and touched with it.Sometimes, the basal cytoplasmic process formed an enlarged synapse-like structure in the contiguous part with basement membrane. This phenomenon provided new morphological evidence for neuroendocrine and paracrine secretory function of these enteroendocrine cells.CONCLUTION At least 8 kinds of IR endocrine cells were found in the gut of stomachless teleost species for the first time in China. These IR endocrine cells scattering in the gut mucosa belong to the APUD system. Among them, the hormones secreted by SP-, ENK-, SOM- and GLU-IR endocrine cells belong to the peptides of dual distribution in the brain and gut. This provided new evidence for the concept of brain-gut peptide. According to the cell types,distribution density, morphological characteristics and variety in shape of APUD cells in the gut of stomachless teleost fishes, it is deemed that the digestive tract of fishes is also an endocrine organ of great importance and complexity.