A Correlation Between Pituitary and Integumentary Structure in the Nine-Spined Stickleback,Pungitius pungitius L.

Benjamin, M. 1980. A correlation between pituitary and integumentary structure in the nine-spined stickleback, Pungitius pungitius L. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(2): 105–109. Naturally occurring, large pituitary cysts, that almost excluded prolactin cells from the pituitary of Pungitius pungitius L., offered a unique opportunity to study a target organ for prolactin (the integument) in fish that were “partially hypophysectomised” without any accompanying surgical trauma. In fish with large pituitary cysts, the epidermis from the lateral body wall was thinner and its mucocytes less abundant, than in fish with normal pituitaries and thus many prolactin cells. In support of the hypothesis that prolactin produced the observed integumentary changes, was the similarity of other pituitary cells in animals with and without cysts. Evidently, prolactin can influence the integument of Pungitius, even though it does not seem essential for freshwater survival.     

Comparative ultrastructural studies on the prolactin cells in the pituitary of nine-spined sticklebacks,Pungitius pungitius L., with and without adenohypophyseal cysts

Summary The ultrastructure of the prolactin cells in nine-spined sticklebacks (Pungitius pungitius L.) with and without large pituitary cysts was compared. Prolactin cells from animals with cysts were less active as regards hormone synthesis and release than those from animals without cysts. Rough endoplasmic reticulum, mitochondria, Golgi apparatus and exocytotic figures were all more prominent in animals without cysts. Lysosomes and mature secretory granules were more plentiful in animals with cysts. The correlation between inactive prolactin cells and pituitary cysts argues against the hypothesis that cysts store prolactin released by holocrine secretion. It is more likely that the origin of cysts from degenerating cells signals extreme inactivity in the rostral pars distalis.     

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.     

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     

Effects of changes in environmental calcium on prolactin secretion in Japanese eel,Anguilla japonica

Effects of changes in environmental Ca²⁺ on the secretion of prolactin, a possible hypercalcemic hormone, were examined both in vivo and in vitro in the Japanese ecl, Anguilla japonica. Transfer of seawater- or freshwater-adapted fish to fresh water, fresh water containing 10 mmol Ca²⁺ · 1^-1 sea water, Ca²⁺-free sea water, or deionized water was accompanied by significant changes in plasma Ca²⁺ levels after 7 days, except for the fish transferred from fresh water to fresh water and from sea water to sea water. Changes in external Ca²⁺ concentrations did not affect plasma prolactin levels, although plasma prolactin levels as well as pituitary prolactin contents were significantly greater in fish in a hypotonic environment than those in a hypertonic environment, regardless of the external Ca²⁺ concentration. Hypercalcemia, induced by removal of the corpuscles of Stannius, did not alter plasma prolactin levles. Incubation of the pituitary in the medium with different Ca²⁺ concentrations (up to 2.9 mmol·l^-1) did not affect the basal release of prolactin, except at an extremely low Ca²⁺ concentration (less than 0.1 mmol·l^-1) where prolactin release was inhibited. Addition of Ca²⁺ ionophore (A23187) to the medium led to a marked and significant increase in prolactin release, indicating that an increase in intracellular Ca²⁺ stimulates prolactin release. However, the effect was not specific to prolactin cells; a similar increase was seen in growth hormone release. These results indicate that changes in environmental Ca²⁺ concentration may not be the primary factor influencing prolactin secretion in the eel; changes in environmental osmolality or Na⁺ levels seem to be more critical for the regulation of prolactin secretion.Abbreviations CSX stanniectomy - DMSO dimethylsulphoxide - DW deionized water - FW fresh water - GH growth hormone - PRL prolactin - SW sea water     

Environmental influences on prolactin cell development in the cyprinodont fish,Cynolebias whitei

Summary A combined immunocytochemical and morphometric study on the development of the prolactin (PRL) cells of the annual cyprinodont Cynolebias whitei, transferred as newly hatched larvae to water with different salinities and/or Ca²⁺-concentrations, was carried out. The percentage of the pituitary volume occupied by PRL cells and the affinity of PRL cells for immunocytochemical staining were used as criteria for their activity. Exposure of the larvae for one day to salt water (260 mOsm/kg) led to a significant reduction in the pituitary volume occupied by PRL cells, indicating an osmoregulatory function of PRL shortly after hatching. In fish reared in diluted artificial seawater (70 and 260 mOsm/kg) or Na⁺-enriched fresh water the development of PRL cells was significantly retarded, but such an effect was not observed in fish placed in Ca²⁺-enriched fresh water. These experiments show that in C. whitei the development and activity of PRL cells are influenced by changes in environmental osmolarity and not by changes in ambient Ca²⁺-concentration.     

A diurnal rhythm of pituitary prolactin activity with diurnal effects of mammalian and teleostean prolactin on total body lipid deposition and liver lipid metabolism in teleost fishes*

Previous investigations have shown that mammalian prolactin has a pronounced effect on lipid deposition in teleost fishes (e.g. Meier et al., 1971; de Vlaming & Sage, 1972). Prolactin can be either lipogenic or lipolytic depending on when during the day it is injected. Studies were initiated to determine whether the diurnal effects of prolactin on lipid metabolism in Fundulus similis vary with season or photoperiod acclimation. Data presented here indicate that the diurnal rhythm of response to prolactin persists in the pre-spawning and post-spawning seasons if F. similis is maintained on a long photoperiod. When animals are acclimated to a short photoperiod, treatment with mammalian prolactin either early or late in the photoperiod causes lipid depletion. Homogenates of the portion of the pituitary containing only prolactin cells cause lipid depletion in F. similis when injected early during a long photoperiod, but stimulate lipogenesis if injected late in the photoperiod; these results imply that fish prolactin may be involved in lipid metabolism. Furthermore, prolactin activity in the pituitary of F. similis varies diurnally. In vitro studies of lipid metabolism in Notemigonous crysoleucas and Ictalurus natalis suggest that the liver may be one of the target organs for prolactin.     

The Response of Prolactin,ACTH, and Growth Hormone Cells in the Pituitary Gland of the Three-Spined Stickleback,: Gasterosteus aculeatus L. form leiurus,to Increased Environmental Salinities

Benjamin, M. 1980. The response of prolactin, ACTH, and growth hormone cells in the pituitary gland of the three-spined stickleback, Gasterosteus aculeatus L. form leiurus, to increased environmental salinities. (Department of Anatomy, University College, Cardiff, Wales, U.K.) — Acta zool. (Stockh.) 61(1): 1–7. The time-sequence of response of the prolactin, ACTH and growth hormone cells in the pituitary gland of the leiurus form of the three-spined stickleback, Gasterosteus aculeatus L., to a transfer from freshwater to seawater, was studied by light microscopy. The appearance of the pituitary was compared with that of animals caught in brackish or sea water. The prolactin cells respond only slowly to seawater by cytological changes interpreted as signs of decreased secretory activity. It is thus suggested that prolactin is unlikely to be important for osmoregulation in this stickleback. The growth hormone cells are more active in all seawater-adapted animals, while the ACTH cells are less active—although only in laboratory experiments. The differing responses of the pituitaries of the leiurus and trachurus forms of G. aculeatus and Pungitius pungitius to high salinities are compared. Even in species whose pituitaries are virtually identical, the response to high salinites may differ widely.     

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.     

In vivo evidence for catecholaminergic inhibition of prolactin secretion in the teleostPoecilia latipinna

Summary Injections of L-dopa in freshwater (FW) fish reduced the size of the prolactin (PRL) cell nuclei, suggesting inhibition of PRL synthesis. A single injection of 6-hydroxydopamine (6-OHDA) in one-third seawater (1/3SW) fish reduced pituitary PRL content and increased PRL cell nuclear size at 6 h and 12 h, indicating stimulation of both synthesis and release of PRL. Two daily injections of 6-OHDA in 1/3SW fish led to PRL cell nuclear enlargement and elevated pituitary PRL content at 48 h after the second injection, indicating strong stimulation of PRL synthesis. Consideration of other parameters (plasma and body sodium levels, plasma osmotic pressure) suggests that the PRL cell responses to 6-OHDA were not mediated by internal osmotic changes. Pretreatment with 6-OHDA also appeared to accelerate the PRL cell activation induced by the transfer of fish from 1/3SW to FW.L-dopa opposed the enhancement of PRL release induced by a single injection of 6-OHDA. Injections of 3,4-dimethoxyphenylethylamine (DMPEA), a specific dopamine antagonist, caused short-term depletion of pituitary PRL, indicating enhanced PRL release.These results suggest that PRL secretion is subject to catecholaminergic inhibition, probably by dopamine. Considering these findings together with previous in vitro results (Wigham et al., 1975), it appears that the PRL cells are innervated by inhibitory catecholaminergic nerve fibres.Abbreviations DMPEA 3,4-dimethoxyphenylethylamine - L-dopa L-dihydroxyphenylalanine - FW freshwater - 6-OHDA 6-hydroxydopamine - PRL prolactin - 1/3SW one-third seawater     

Cellular localization of somatolactin in the pars intermedia of some teleost fishes

Summary We report here on the cellular localization in the fish pituitary of somatolactin (SL), a putative new pituitary hormone related to growth hormone and prolactin, which has been recently identified in the piscine pituitary gland. Immunocytochemical staining, using anti-cod SL serum, revealed that in the cod pituitary gland, SL is produced by cells in the intermediate lobe, bordering the neural tissue. These cells, staining weakly with periodic-acid-Schiff (PAS), are distinct from the melanocyte stimulating hormone (MSH) cells which, as in all teleosts, are PAS-negative. SL-immunoreactivity was observed in the same location in all other teleost species examined: flounder, rainbow trout, killifish, molly, catfish and eel. In most fish the SL-immunoreactive cells are either strongly or weakly PAS-positive but in rainbow trout are chromophobic, indicating that the SL protein can probably exist in glycosylated and non-glycosylated forms. Thus, in demonstrating the cellular localization of SL, this study provides the first identification of the enigmatic, second cell-type of the fish 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.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

Immunoreactive prolactin in the pituitary gland of cyprinodont fish at the time of hatching

In the developing pituitary gland of embryos of the annual fish Cynolebias whitei and the medaka, Oryzias latipes, prolactin cells have been identified before hatching by means of a light-microscopic immunocytochemical method with antiserum against ovine prolactin. At the time of hatching, changes in the intensity of the immunoperoxidase staining occur. Histological staining by Cleveland and Wolfe's trichrome shows differentiation of cell types in the adenohypophysis only later in ontogeny. Our results indicate that, in teleosts, differentiated prolactin cells are present before hatching and that prolactin may be involved in the endocrine control of the hatching process.     

Ultrastructure of the endocrine cell types in the adenohypophysis of the teleost,Poecilia latipinna — a morphometric model

Summary A morphometric model providing detailed quantitative information on the ultrastructure of the adenohypophysial endocrine cells has been developed for Poecilia latipinna. The model consists of various morphological components quantified in terms of volumes, surfaces or numbers. For prolactin and growth hormone cells, appropriate results are expressed relative to the average volume of that cell type. The difficulties of quantifying EM data on pituitary glands, together with the various sources of error to which the data are clearly open, have been discussed. Some practical applications of quantitative EM to problems in fish pituitary research are outlined.I thank Dr. J.N. Ball for supplying the fish and Mr. L. Ethridge for technical assistance.     

The rostral pars distalis of the pituitary gland of the freshwater and marine alewife (Alosa pseudoharengus)

Summary In the alewife the orohypophyseal duct, a remnant of Rathke's pouch, persists in adults as a tube passing from the rostral pars distalis to the pharyngeal region. Its lumen is not open to the buccal cavity. The prolactin cells are situated around the bifurcations of this duct in the rostral pars distalis. Contents from prolactin cells, such as granules, nuclei, mitochondria and Golgi structures were found in these bifurcations. These contents were indistinguishable from those of intact prolactin cells. Evidence of actual release into the duct was often noted. At the presumptive point of release, the cells lining the lumen separate and the contents, probably of an entire prolactin cell, are extruded. The cilia usually found at the point of extrusion arise from prolactin cells. The prolactin cells of freshwater fish were more heavily granulated than those from a marine environment. Prolactin cells of fish entering freshwater streams were not heavily granulated but showed evidence of increased activity. Granule size was not affected by salinity. The ACTH cells are arranged in bands along branches of the neurohypophysis in the rostral pars distalis. No differences in ACTH cells from fish of different salinities were noted.We would like to thank Mr. D. D. Zumwalt of the John G. Shedd Aquarium in Chicago, Dr. E. D. Warner, Mr. R. L. Flayter, Dr. J. G. Stanley of the University of Wisconsin, Milwaukee, Mr. L. Wells of the U. S. Fish and Wildlife Service, Great Lakes Fisheries Laboratory, Ann Arbor, and Mr. L. N. Flagg of the Department of Sea and Shore Fisheries, Augusta, Maine, for their assistance in obtaining the fish used in this study. Dr. T. N. Tahmisian and Mr. G. T. Chubb of Argonne National Laboratories, and Dr. L. M. Srivastava and Dr. V. Bourne of Simon Fraser University, Canada, kindly made electron microscope facilities available. Finally, we wish to thank Mr. W. Goossens and Mr. D. J. DeJong for valuable assistance. This project was supported by a grant from the National Marine Fisheries Service.     

Histological observations on the pituitary gland and the glomerular body of the larvae of the marine teleost,Hexagrammos otakii

Summary In the larvae of the marine teleost, Hexagrammos otakii, the prolactin, ACTH, STH and pars-intermedia cells as well as the neurohypophysis and glomerular body showed no histological change at the time of hatching. These cells or organs do not seem to be specifically involved in the mechanisms of adjustment to the hypertonic osmotic stress at this time. The size of the nuclei of the prolactin cells increased in fresh-water (F. W.) and that of the ACTH cells decreased in 2/3, 1/2 and 1/3 sea-water (S.W.). The amount of chrome-alum haematoxylin-positive material in the neurohypophysis of fish immersed in 1/2 S.W., 1/3 S.W. and F.W. was less than that of the control fish. There was a difference in the glomerular ratio between the fish kept in F. W. and control fish. And the red blood cells in the glomerulus of the larvae kept in 1/2 and 1/3 S.W. apparently increased in number in comparison with the control fish.The author wishes to express his gratitude to Professor Hideshi Kobayashi for his valuable guidance during the course of this study. The author is indebted to Dr. Masaaki Inoue, Kanagawa Prefectural Fisheries Experimental Station, for supplying the eggs of Hexagrammos.     

Gonadotropin-like and adrenocorticotropin-like cells in the pituitary gland of hagfish, Paramyxine atami; immunohistochemistry in combination with lectin histochemistry

The pituitary system of the hagfish remains an enigma. The present study has aimed to detect possible adenohypophysial hormones in the pituitary gland of the brown hagfish, Paramyxine atami, by means of immunohistochemistry in combination with lectin histochemistry. Rabbit antisera raised against ovine luteinizing hormone (LH)β, proopiomelanocortin (POMC)-related peptides, and the growth hormone/prolactin family of tetrapod and fish species were used, and 25 kinds of lectins were tested. Three different types of adenohypophysial cells were revealed in the pituitary of brown hagfish. The first was stained with both anti-ovine LHβ and several D-mannose-binding lectins, such as Lens culinaris agglutinin and Pisum sativum agglutinin. This cell type predominated in the adenohypophysis in adults with developing gonads and thus appeared to be involved in the regulation of gonadal functions. The second was negative for anti-ovine LHβ but was stained with several N-acetylglucosamine-binding lectins, such as wheat germ agglutinin and Lycopersicon esculentum lectin. This cell type exhibited a weak positive reaction with anti-lamprey adrenocorticotropin (ACTH) and thus appeared to be related to POMC-like cells. The second cell type was found in the adenohypophysis regardless of the developmental state of the gonads. The third cell type was negative for both antisera and lectins. Since this cell type was numerous in juveniles and adults without developing gonads, most cells of this type were probably undifferentiated. These findings suggest that GTH and ACTH are major adenohypophysial hormones in the hagfish. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture (to M.N.).     

Distribution of growth hormone-like cells in the pituitary of adult sea lampreys,Petromyzon marinus

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) are members of a pituitary hormone family that are believed to have evolved from a common ancestral gene by duplication and subsequent divergence. Since these hormones are found both in bony fish and cartilaginous fish, their ancestral form(s) should be present in the Agnatha. Thus, although there is no convincing evidence that the lamprey pituitary secretes GH or PRL, GH- and/or PRL-like immunoreactivity was examined in the pituitary of adult sea lampreys (Petromyzon marinus), using antibodies to GHs, PRLs and SL of mammalian and/or fish origins. Our initial attempt with ordinary immunohistochemical procedures failed to detect any positive reactions in the lamprey pituitary. Following the hydrated autoclave pretreatment of the sections, anti-salmon GH, anti-salmon PRL and anti-blue shark GH gave positive reactions in most cells distributed in the dorsal half of the proximal pars distalis. These results suggest that the material immunoreactive to those antibodies is related, to some extent, to GH/PRL, but enhancement of immunoreactivity to reveal this by the hydrated autoclave pretreatment of sections is needed due to low crossreactivity. The similarity of the topographic distributions within the pituitary between lampreys and teleosts suggests that lamprey GH/PRL-like cells are GH cells of the lamprey.