Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Immunohistochemically detected ontogeny of prolactin and growth hormone cells in the African catfish Clarias gariepinus

The chronological appearance of selected endocrine cells in the pituitary of African catfish Clarias gariepinus (Burchell, 1822) was studied morphologically, histologically and immunohistochemically by using antisera raised against catfish growth hormone (cgGH) and recombinant tilapia prolactin I (tPRL). cgGH- and tPRL-like immunoreactive cells were visible from day 1 post fertilisation (hatching) throughout the juvenile and the adult stage. From 1 to 90 days after hatching, the larval pituitary is oval in shape with a distinctly shaped rostral pars distalis, proximal pars distalis and pars intermedia. From day 120 onwards allometric growth of the rostral and proximal pars distalis extended the prolactin and growth hormone cells anteriorily and posteriorily, respectively. Size and activity of the prolactin and growth hormone cells, measured by the ratio of cell surface to nuclear surface remained constant until day 40 and showed a growth spurt thereafter. Growth hormone content, measured with a catfish-specific radio-immunoassay from hatching until 60 h post hatching, increased exponentially between 30 and 60 h.     

An immunocytochemical study of the pituitary gland of the white seabream (Diplodus sargus)

The adenohypophysis of the white seabream (Diplodus sargus) was studied using histochemical and immunocytochemical techniques. The adenohypophysis was composed of rostral pars distalis, proximal pars distalis and pars intermedia. Prolactin (anti-chum salmon prolactin positive) and adrenocorticotropic (anti-human ACTH positive) cells were found in the rostral pars distalis. Prolactin cells were organized into follicles, while ACTH cells were arranged in cords around neurohypophyseal tissue branches that penetrated the rostral pars distalis. In the proximal pars distalis, somatotropic (anti-chum salmon and anti-gilthead seabream growth hormone positive), gonadotropic (anti-chum salmon -gonadotrophin II and anti-carp -gonadotrophin II positive, but anti-chum salmon -gonadotrophin I negative) and thyrotropic (anti-human -thyrotropin positive) cells were observed. Growth hormone cells were restricted to the dorsal and ventral part of the proximal pars distalis. They were clustered or surrounded the neurohypophyseal branches. Only one type of gonadotrophin cell was identified and they were clustered or isolated in the proximal pars distalis. Scattered groups of thyrotropin cells were located throughout the proximal pars distalis. In the pars intermedia somatolactin (anti-chum salmon and anti-gilthead seabream somatolactin positive) and melanotropic (anti--melanotropic hormone positive) cells were localized. In addition, gonadotrophin cells surrounded the pars intermedia or distributed evenly between somatolactin and melanotropic hormone cells. Somatolactin cells were periodic acid-Schiff negative and surrounded the neurohypophyseal branches intermingled with melanotropic cells. These cells were also immunoreactive to anti-human ACTH antiserum.     

草鱼催乳素抗血清的制备与鉴定

应用从草鱼垂体中分离纯化的催乳素免疫兔子制备了特异抗血清。用常规酶联免疫吸附测定法检测表明该抗血清与马哈鱼生长激素及促性腺激素完全没有交叉反应，而与草鱼生长湟级向弱交叉反应。免疫细胞化学染色表明该抗血清主要与草鱼垂体前叶催乳素细胞发生结合反应，与垂体间叶细胞有微弱染色反应，但与神经垂体琢垂体后叶细胞则完全没有免疫结合反应。     

Fine structure of the adenohypophysis in immature sockeye salmon,Oncorhynchus nerka

Summary The ultrastructure of the secretory cells of the adenohypophysis of juvenile sockeye salmon was investigated. Pituitary glands were collected from immature fish transferred experimentally to sea water and subsequently returned to fresh water. The rostral pars distalis contained three cell types: ACTH cells, prolactin cells, and non-secretory cells. The prolactin and non-secretory cells were joined together in the form of follicles by desmosomes and they both had cilia and microvilli projecting into the follicle lumen. Various follicular structures such as lumen, multivesicular structures, and peripheral basement membrane are discussed as possible sites of prolactin cell granule release. The columnar ACTH cells were found at the junction of the rostral pars distalis and the neurohypophysis. The cytoplasmic granules in these cells were characteristically separated from their limiting membrane by a clear space. Multivesicular structures were also found in association with this cell type. The caudal pars distalis also contained three cell types: one acidophil (putative somatotrop) and two basophils (putative thyrotrops and gonadotrops), all of which were similar to those described in adult fish. The pars intermedia contained only one cell type. They appeared to be active cells and were characterized by containing membrane-bounded granules similar to those found in the ACTH cells. Changes in ambient salinity had no apparent effect on any cell type described.The work was supported by a grant in aid of research from the National Research Council of Canada. We wish to thank Mr. R. Lindsay, Mr. C. Cooper, and Mr. G. Longworth for their technical assistance. We would also like to thank Mr. S. Killick of the International Pacific Salmon Fisheries Commission for his assistance in the collection of fish and Dr. H. Cook for his helpful discussion of the project. This paper is No. 058 in the University of Guelph Migration Series.     

Immunohistochemical Localization of Growth Hormone and Prolactin in the Pituitary Gland of Acipenser güldenstaedti Brandt (Chondrostei)

Abstract Prolactin (LTH) and growth hormone (GH) containing cells in A. güldenstaedti have been localized by means of anti-ovine prolactin and anti-bovine growth hormone respectively, coupled indirectly to peroxidase, and localized histochemically with hydrogen peroxide as substrate and 3,3′-diaminobenzidine as capturing agent. The distribution of the anti-prolactin positive cells has been demonstrated and correlated histologically with the acidophilic cells both in the rostral and proximal pars distalis. This cell type is elongated and arranged in follicles in the rostral pars distalis; in the proximal pars distalis they are smaller and oval, without any special orientation. Neither of the other cell types which are scattered among these acidophils contain prolactin. The anti-bovine growth hormone positive cells are evenly distributed in the proximal pars distalis above the hypophysial cleft, and some are also found in the pars intermedia. The anti-GH positive cells have been correlated histologically with the amphiphilic cells in the proximal pars distalis. These cells are arranged in cell cords in close contact with the secondary capillary plexus, near its origin from the primary capillary plexus covering the median eminence.     

Immunocytochemical identification and ontogeny of adenohypophyseal cells in a cave fish,Phreatichthys andruzzii (Cypriniformes: Cyprinidae)

The morphogenesis of the pituitary gland and the chronological appearance of adenohypophyseal cells were investigated for the first time in the Somalian cave fish Phreatichthys andruzzii by immunocytochemistry. The adult adenohypophysis contained: a rostral pars distalis, with prolactin (PRL) cells arranged in follicles and adrenocorticotropic (ACTH) cells, a proximal pars distalis with somatotropic (GH), β‐thyrotropic (TSH), β‐gonadotropic type I (FSH) and type II (LH) cells and a pars intermedia with α‐somatolactin (SL), α‐melanotropic (MSH) and β‐endorphin (END) cells. All regions were deeply penetrated by neurohypophyseal branches. At hatching (24 h post‐fertilization) the pituitary was an oval cell mass, close to the ventral margin of diencephalon. The first immunoreactive cells appeared as follows: PRL at 0·5 days after hatching (dah), GH and SL at 1·5 dah, END at 2 dah, TSH, ACTH and MSH at 2·5 dah, FSH at 28 dah and LH at 90 dah. The neurohypophysis appeared at 5 dah and branched extensively inside the adenohypophysis at 130 dah, but there was no boundary between rostral pars distalis and proximal pars distalis at this stage. The potential indices of prolactin and growth hormone production increased until 28 and 60 dah, respectively. The potential index of growth hormone production correlated positively with total length. Activity of PRL and GH cells, measured as ratio of cell area to nucleus area, was significantly higher in juveniles than in larvae.     

Ultrastructure of the adenohypophysis in the teleost Poecilia latipinna

Summary In the sailfin molly, Poecilia latipinna, seven morphological endocrine celltypes could be distinguished with the electron microscope. Each of these was identified with one of the seven cell-types distinguished with the light microscope, to most of which endocrine functions have previously been allocated. Corticotrophs and prolactin cells form the rostral pars distalis, and the proximal pars distalis consists of an outer layer of gonadotrophs and an inner zone containing growth hormone cells and thyrotrophs. The pars intermedia contains two cell-types, of uncertain function. Stellate cells (interstitial cells) occur throughout the adenohypophysis, but are most numerous and prominent in the rostral pars distalis. The inner proximal pars distalis contains a cell-type not previously distinguished in this species with the light microscope, the Z-cell, which could be aminergic.The ultrastructural features of each cell-type are described in detail, and discussed in comparisons with the homologous cells described in other teleosts. There is good agreement for different teleosts in the ultrastructural details of each cell type.We thank Mr. L. Ethridge, Mr. M. P. Hancock, Mr. D. Hollingworth and Mr. W. Thomson for technical assistance, and Mr. D. Taylor of the Nuffield Institute of the Zoological Society of London for permission to use the Nuffield Institute electron microscope. We are grateful to Dr. Harry Grier, who collected and embedded glands from P. latipinna in its natural fresh-water habitat in Florida, U.S.A. T. Batten is in receipt of an S.R.C. Research Studentship.     

Prolactin regulation in the coho salmon,Oncorhynchus kisutch

Summary Parr and smolt sea water acclimated coho salmon,Oncorhynchus kisutch were subjected to gradual and direct transfers to fresh water. Plasma osmotic pressure, Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ were similar in freshwater (FW) fish and seawater (SW) transferred controls for the 24 h following transfer. In spite of the similarity in osmotic pressure and ion levels, plasma cortisol concentrations were significantly increased immediately following salinity change while both pituitary and plasma prolactin decreased indicating enhanced secretion by the pituitary and clearance from the blood. In vitro experiments showed greater incorporation of tritiated leucine into prolactin (PRL) cells immediately after transfer to FW while prolactin injections into intact fish lowered activity in rostral pars distalis (RPD) cells as measured by the same technique, providing evidence of hormonal feedback. These experiments show that the increased synthesis and release of PRL that occurs in coho following movement into FW is not obviously correlated with plasma osmotic pressure, Na⁺ or Ca⁺⁺ concentrations as has been observed in other species of teleosts.Abbreviations FW freshwater - SW seawater - PRL prolactin - RPD rostral pars distalis     

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.     

The Study of Prolactin in the Pituitary Gland of the Atlantic Eel (Anguilla anguilla) and the Atlantic Salmon (Salmo salar) by Immunofluorescence Technique

The prolactin containing cells in the Atlantic eel and the Atlantic salmon have been localized by fluorescein-labelled antibodies to ovine prolactin. Two fluorescence-methods have been used: the direct and the indirect. Erythrosinophilic cells in the follicles of the rostral pars distalis were stained by the fluorescent antibodies in both eel and salmon. Eels from brackish water were transferred to fresh or salt water and differences in the contents of prolactin in the hypophyses were tested. Attempts were made at a semi-quantitative estimation of prolactin, using a fluorescent antibody technique. Eels, which had been kept for three days in fresh water contained a smaller quantity of prolactin in their hypophyses, than the eels which were kept in salt water.     

Prolactin-producing Cells in Clupea harengus membras,Polypterus palmas and Calamoichthys calabaricus Studied by Immuno-histochemical Methods

The prolactin-producing cells in Clupea harengus membras, Polypterus palmas and Calamoichthys calabaricus have been localized by fluores-ceinlabelled antibodies to ovine prolactin. Pituitary sections were cut in a cryostat and both direct and indirect protein tracing methods have been used. As an additional method the paraffin embedding technique was carried out with Polypterus palmas. The prolactin-producing cells in Clupea harengus membras were localized to the follicle epithelium in the rostral pars distalis. In Polypterus palmas and Calamoichthys calabaricus the prolactin producing cells were scattered all over the pars distalis. A possible relationship between the erythrosinophilic prolactin cells and the epithelium cells in the buccohypophyseal canal is discussed.     

The origin of pituitary cysts in the rostral pars distalis of the nine-spined stickleback,Pungitius pungitius L.

Summary Pituitary cysts in the nine-spined stickleback, Pungitius pungitius L., develop next to blood vessels passing through the prolactin zone of the rostral pars distalis to the connective tissue capsule at its periphery. Cysts were most frequent when pituitaries were large compared with body lengths. However, the incidence of cysts could not be closely related to body length alone. As the rostral pars distalis is more highly vascularised in fish with large pituitaries, and as there was no evidence of accumulating secretion around these blood vessels, it is suggested that cysts develop when vascular demands (or supplies) become excessive. Associated with the greater vascularity of the rostral pars distalis in large pituitaries was a remarkable development of non-granulated cells. Indeed cysts may originate as enlarged intercellular spaces between such cells, as the latter often surround blood vessels. The non-granulated cells are also important in enlarging cyst cavities (by phagocytosing prolactin-cell debris) and perhaps for maintaining their structural integrity. It is suggested that the macrophages within cyst cavities are derived from the non-granulated cells.     

The effect of water acidification on prolactin cells and pars intermedia PAS-positive cells in the teleost fish Oreochromis (formerly Sarotherodon) mossambicus and Carassius auratus

Summary Although exposure to acid water (pH 3.5) induces severe and prolonged reduction in plasma osmolarity and total plasma calcium concentration in tilapia (Oreochromis mossambicus) and goldfish (Carassius auratus), the responses of the hypophyseal cells are clearly different. In tilapia, the size of the rostral pars distalis of the pituitary gland is enlarged as a result of the increase in size and number of prolactin cells. The pars intermedia PAS-positive (PIPAS) cells are not noticeably changed. Conversely, in goldfish, prolactin cells are unaffected, whereas the number of enlarged PIPAS cells increases markedly. Stimulation of prolactin secretion may be responsible for the partial restoration of plasma osmolarity and calcium levels observed in tilapia after two weeks exposure to acid water. Prolactin cells apparently play a role in the adaptation to acid stress by counteracting osmoregulatory disturbances. Goldfish show no restoration of plasma osmolarity during the course of the experiment. Plasma calcium levels tend to increase. Although prolactin may have an osmoregulatory function in goldfish under steady state conditions, goldfish prolactin cells do not seem to participate in the physiological adaptation to environmental changes that disturb water and ion homeostasis. The function of PIPAS cells in tilapia remains unclear and is apparently unconnected with ion regulation. The observations on these cells in goldfish are consistent with the hypercalcemic activity suggested for them.     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard Anolis carolinensis

Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its beta-subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine beta-LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.     

Immunohistochemical identification of cells in the pars distalis of the pituitary of the lizard anolis carolinensis

Summary Immunocharacteristics of the pars distalis cells of the pituitary of the male lizard A. carolinensis are determined by employing the immunoperoxidase technique with antisera to mammalian pituitary hormones. On the basis of their immunoreactivity, 5 different cell types with characteristic anatomical distribution are recognized. ACTH cells are found in the rostral half of the pars distalis, and PRL cells in the rostral two thirds of the pars distalis. GH and TSH cells are located in the caudal half of the pars distalis. GTH cells are distributed throughout the gland. When consecutive sections are stained with antiserum to ovine FSH or its -subunit and to ovine LH, the same cells show immunoreactivity to all the three antisera. None of the GTH cells show positive immunoreactivity to ovine -LH antiserum. The results suggest the existence of one gonadotropic cell type in the pituitary of this lizard.Supported by U.S. Council for International Exchange of Scholars (to D.R.N.) and PHS Grant NS09914     

Immunocytochemical devidence for hypophysial projections of the cells of the nucleus lateralis tuberis pars lateralis in the rainbow trout (Salmo gairdneri)

In the rainbow trout the pars lateralis is the most prominent part of the nucleus lateralis tuberis (NLT). To demonstrate a morphological relationship between this lateral part of the NLT and the pituitary, immunocytochemistry was applied as a staining method. Experiments were carried out on glutaraldehyde-picric acid-acetic acid-fixed brain sections of mature male and female rainbow trout using the peroxidase-anti-peroxidase immune technique with an antiserum against 27-S-methylglucagon as the first antibody. Most of the cells in the NLT/pars lateralis reacted with the antiserum. Axons from these cells enter the pituitary, extending exclusively in the numerous neurohypophysial digitations in the pars intermedia. No immunoreactive neurohypophysial protrusions were found in those parts of the adenohypophysis where the gonadotropic cells are located, indicating that the lateral part of the NLT is not directly involved in the control of gonadotropin secretion. In addition to cells of the NLT/pars lateralis only prolactin cells in the rostral pars distalis of the adenohypophysis reacted with the antiserum used.     

Histology of the pituitary gland of the grey mullet, Mugil cephalus L.

The present communication deals with a histological study of the pituitary gland of the teleost fish Mugil cephalus , found in the estuarine waters of Cochin area. Six different cell types were identified in the pituitary gland on the basis of their grouping, distribution and staining properties. The prolactin and the TSH cells (thyroid stimulating hormone producing cells or thyrotrops) were identified in the rostral pars distalis and the ACTH cells (adrenocorticotropic hormone producing cells or corticotrops) in the interphase between the neurohypophysis and the rostral pars distalis. The STH cells (somatotropic hormone producing cells or somatotrops) and the gonadotropic cells were distinguished in the proximal pars distalis and the MSH cells (melanin stimulating hormone producing cells or melanotrops) in the pars intermedia.     

Prolactin Cells: Study of the Pituitary Gland of the Roach (Leuciscus rutilus) by Immuno-histochemical Methods

Fluorescein-labelled antibodies against ovine prolactin have been used to localize the prolactin-producing cells in the pituitary gland of the roach, Leuciscus rutilus. Two methods of protein tracing have been used: the direct and the indirect. In the direct method gammaglobulin fractions of rabbit-antisera were conjugated with fluorescein-isothiocyanate. Frozen sections of the pituitary gland were treated with conjugate. Cells with a content of prolactin gave off green fluorescence. Conjugate made from normal rabbit-serum failed to give this result. Blocking-experiments were also used to control the conjugate fluorescence. In the indirect method sheep-anti-rabbit-gammaglobulin was labelled with fluorescein. First the sections of the hypophysis were treated with antiserum from rabbits. After careful rinsing the sections were exposed to the dyemarked sheep-anti-rabbit-gammaglobulin. For orientation in the pituitary gland histological staining was carried out in parallel to that by fluorescence. The investigation has shown that the erythrosinophilic cells in the rostral pars distalis contained prolactin. Since no other cells in the gland were fluorescent, it can be concluded that the eta-cells are the prolactin cells.     

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.