Response of PAS-positive cells of the pituitary pars intermedia in the teleost Carassius auratus to acid water

Summary The PAS-positive or PIPAS cells in the pars intermedia of goldfish are activated after reduction of the pH of the ambient freshwater from 7.5 to 3.5. The cells increase in number and exhibit a five-fold increase in cell volume. Granular endoplasmic reticulum occupies most of the cytoplasm. Goldfish PIPAS cells (also termed calciumsensitive cells) are thought to have a hypercalcemic function. Therefore, their activation in acid water may be caused by the severe drop in plasma calcium concentration following exposure of the fish to low water pH. However, activation of the PIPAS cells in response to acidification of the water is not prevented when the calcium concentration of the water is increased to levels that result in hypercalcemia instead of hypocalcemia. Activation of the PIPAS cells occurs also in fish exposed to acidified freshwater enriched with NaCl to an osmolarity similar to that of the blood. This prevents the reduction in plasma osmolarity and Na⁺ and Cl^- concentrations that follow exposure of goldfish to acidified normal freshwater. Our observations do not support the hypothesis that the PIPAS cells in goldfish produce a hypercalcemic hormone, or indeed any hormone involved in calcium metabolism or osmoregulation. The cells may be implicated in acid-base regulation (a characteristic of many types of fish when exposed to acidified water) but the evidence is indirect.     

Gene expression and intracellular localization of somatolactin in the pituitary of rainbow trout

The gene expression and intracellular localization of somatolactin (SL), a putative pituitary hormone structurally related to both growth hormone and prolactin, were investigated in the pituitary of rainbow trout, Oncorhynchus mykiss. Using an in situ hybridization technique, we demonstrated the gene expression of the SL molecule in cells bordering the neurohypophysial tissue in the pars intermedia. These cells were identified immunocytochemically as SL-cells on the adjacent section. Electron-microscopic immunocytochemistry by means of the protein A-gold technique, also revealed that the SL-immunoreactivity was located mostly on the secretory granules in SL-cells. Our findings clearly indicate that SL is biosynthesized and stored in the granules in these cells.     

Immunohistochemical localization of bioactive peptides and amines associated with the chromaffin tissue of five species of fish

Biogenic peptides and amines associated with the chromaffin tissue in Atlantic cod (Gadus morhua), rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), spiny dogfish (Squalus acanthias) and Atlantic hagfish (Myxine glutinosa) were identified utilizing immunohistochemical techniques. Within the posterior cardinal vein (PCV) in cod, trout and eel, a subpopulation of chromaffin cells displayed immunoreactivity to tyrosine hydroxylase (TH) and dopamine--hydroxylase (DH) but not to phenylethanolamine-N-methyltransferase (PNMT). TH-like immunorectivity was observed within cells in hagfish hearts. Nerve fibres displaying vasoactive intestinal peptide (VIP) immunoreactivity and pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity innervated cod, trout and ell chromaffin cells. In eel, neuropeptide Y (NPY)-like and peptide YY (PYY)-like immunoreactivity was located within cells in the PCV, including chromaffin cells. Serotonin-like immunoreactivity was observed within eel and cod chromaffin cells and in hagfish hearts. In the dogfish axillary bodies, nerves displaying TH-like, VIP-like, PACAP-like, substance P-like and galanin-like immunoreactivity were observed. These results are compared with those of other vertebrates, and potential roles for these substances in the control of catecholamine release are suggested.     

Electron microscopic-immunocytochemical localization of adrenocorticotropin and melanocyte stimulating hormone in the pars intermedia cells of rats and mice

Summary Electron microscopic localization of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) in light, dark and ACTH cells in the pars intermedia (PI) of rats and mice is attempted by using antisera to _p 1–24, _p 17–39 ACTH and _b MSH with the immunoglobulin-peroxidase bridge technique. All of the PI parenchymatous cells (light, dark and ACTH cells), except the marginal cuboidal and the ependymal like cells, in rats and mice show very good localization of ACTH and MSH staining. In the light and dark cells, stain of varying intensity is seen on the secretory granules, vesicles and also in many places on the surface of the rough endoplasmic reticulum. There is no staining on the mitochondria, in the nuclei or in the granules inside and around the cisternae of the Golgi complex. Dark stained dense core granules become larger and larger as they appear farther and farther away from the Golgi complex. On the other hand, in the ACTH cells of the PI, ACTH antisera show stronger stained granules in the Golgi complex including the cisternae, similar to the pars distalis (PD) ACTH cells. From these observations it is concluded that the corticotropin in light and dark cells, is not packaged or condensed in the Golgi complex like that in the ACTH cells. MSH synthesis in light and dark cells also seems to be similar to that of ACTH synthesis. It is likely that the granules accumulate ACTH and MSH secretions after they are liberated from the Golgi cisternae, and thus become bigger and bigger in size. In case of ACTH cells of PI and PD, corticotropin may be packaged in Golgi cisternae and the size of the granule does not change much. This shows that there are distinct immunocytochemical differences between the light, dark and ACTH cells of the PI. At the moment, it is difficult to say whether ACTH and MSH are present in the same granule or not.The present and previous studies show that the ACTH and MSH secretion in the PI of rats and mice depends on the hypothalamic neural control.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledge the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré. He wishes to acknowledge Mr. Gatson Lambert for his photography.     

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.     

Responses of the PAS-positive pars intermedia cells in the cichlid fish Sarotherodon mossambicus to ambient calcium and background adaptation

Summary The function of the PAS-positive pars intermedia cells in fish has been connected with control of background adaptation and of plasma calcium levels. Since background reflectivity and illumination influence calcium metabolism in S. mossambicus, we have tested the hypothesis that the effects of background reflectivity on the PAS-positive cells are mediated by changes in plasma calcium. However, total plasma calcium does not correlate with the activity of the PAS-positive cells as estimated by morphometrical criteria. Transfer of S. mossambicus to a white background leads to a drop in plasma calcium, and a marked reduction of the activity of the PAS-positive cells. Adaptation to low-calcium freshwater, on a neutral background, has the same effect on plasma calcium but has no effect on the PAS-positive cells. The characteristic structural features of highly active PAS-positive cells in fish from a black background are not due to the slight hypocalcemia that occurs in these fish, since addition of calcium to the water in concentrations that fully prevent the drop in plasma calcium does not suppress the PAS-positive cells. These findings make it very unlikely that these cells produce a hypercalcemie factor in S. mossambicus.     

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel

Summary The structure of the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia was investigated in eels kept in deionized water (DW) or fresh water (FW) supplemented with Ca²⁺ or Mg²⁺. Ca²⁺ (2mM) reduces considerably the response to DW; plasma osmolarity, Na⁺ and Ca²⁺ levels are not significantly affected. In eels adapted to DW for 21 or 28 days, showing highly stimulated Ca-s cells, an addition of CaCl₂ for 2 days inhibits the release of granules, but does not immediately block their synthesis and the mitotic activity. The nuclear area is reduced, osmolarity and plasma sodium increase, but the rise in calcium is not always significant. Magnesium, at a 10-fold greater concentration than in FW (2 mM), slightly inhibits the release of secretory granules without reducing other indicators of stimulation. In Ca-enriched FW, the Ca-s cells appear inactive. These data show that the PAS-positive cells in the pars intermedia of the eel are calcium-sensitive, similar to those of the goldfish; their role in calcium regulation is briefly discussed.     

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel

Summary Freshwater eels were adapted to calcium-free sea water (SW) or 1/3 Ca-free SW. Survival was generally poor in Ca-free SW, although three eels were still in good condition after 19–30 days; survival in 1/3 Ca-free SW was excellent. Osmotic disturbances (increase of plasma osmolarity and sodium levels), which initially occur in Ca-free SW, were no longer detectable after 19–30 days, or in eels in 1/3 Ca-free SW after one month. Plasma calcium sharply decreases initially; it is less depressed after 19–30 days and in 1/3 Ca-free SW. Alterations in the mucus production may be involved in the osmotic changes. Under these conditions no clear stimulation of the calcium-sensitive (Ca-s) cells of the pars intermedia was registered, but in Ca-free SW (1/3 or full strength) the inhibitory effect normally observed in SW does not occur. In a hyperosmotic environment, other ion(s), possibly magnesium, may reduce the response of the Ca-s cells to a lack of environmental calcium.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus contains two structurally different endocrine cell types. The predominant cell type is assumed to synthesize MSH and related peptides. The second cell type is PAS positive; its function and products are unknown. In this second cell type changes occur in relation to background colour and illumination. Thus, PAS positive cells of fish adapted to a white background are less numerous and metabolically less active than those of fish adapted to a black background, and are most active in fish kept in total darkness. In blinded fish, whether in light or in darkness, the activity of the PAS positive cells is similar to that of the black background-adapted animals. The significance of these responses in relation to the control of background adaptation is discussed.     

Distribution of PACAP (pituitary adenylate cyclase-activating polypeptide)-like and helospectin-like peptides in the teleost gut

Pituitary adenylate cyclase-activating polypeptide (PACAP) and helospectin are two vasoactive intestinal polypeptide (VIP)-related neuropeptides that have recently been demonstrated in the mammalian gut; the aim of this study was to reveal their occurrence and localisation in the gastrointestinal tract, swimbladder, urinary bladder and the vagal innervation of the gut of teleosts, using immunohistochemical methods on whole-mounts and sections of these tissues from the Atlantic cod, Gadus morhua and the rainbow trout, Oncorhynchus mykiss. Both PACAP-like and helospectin-like peptides were present in the gut wall of the two species. Immunoreactive nerve fibres were found in all layers but were most frequent in the myenteric plexus and along the circular muscle fibres. Immunoreactivity was also demonstrated in nerves innervating the swimbladder wall, the urinary bladder and blood vessels to the gut. Immunoreactive nerve cell bodies were found in the myenteric plexus of the gut and in the muscularis mucosae of the swimbladder. In the vagus nerve, non-immunoreactive nerve cells were surrounded by PACAP-immunoreactive fibres. Double staining revealed the coexistence of PACAP-like and helospectin-like peptides with VIP in all visualized nerve fibres and in some endocrine cells. It is concluded that PACAP-like and helospectin-like peptides coexist with VIP in nerves innervating the gut of two teleost species. The distribution suggests that both PACAP and helospectin, like VIP, are involved in the control of gut motility and secretion.     

Immunocytochemical identification of prolactin cells in the pituitary gland of tilapia larvae (Oreochromis mossambicus: Teleostei)

Summary Using an antiserum to highly purified chum salmon prolactin, prolactin cells were identified in the putative rostral pars distalis of newly hatched tilapia larvae (Oreochromis mossambicus) by the immunogold method for the electron microscope. In the putative rostral pars distalis, some cells had another kind of secretory granule which was much less numerous, much smaller in size, and without immunoreactivity to salmon prolactin antiserum. Controls incubated with salmon prolactin-preabsorbed antiserum or normal serum showed no immunoreactive cells, confirming the specificity of the antiserum. The possible role of prolactin in the osmoregulation of tilapia larvae is discussed.     

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

Summary 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 andMugil, 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 andMyoxocephalus, 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.     

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout,Oncorhynchus mykiss,during ovarian development

Summary Biosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH I- and II-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH -subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, and I messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH -and II-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH -mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although I-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.     

Response of calcium-sensitive cells in the pars intermedia of the goldfish adapted to diluted sea water with different calcium and magnesium contents

Summary Goldfish kept in diluted Ca-free sea water (SW) (23 or 30 %) or in Ca-Mg-deficient SW (23 %) have a limited survival, release large amounts of mucus, and show spasmodic seizures. Plasma calcium decreases. The PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia show a low activity in diluted SW and in 23 % Mg-free SW. In diluted Ca-free SW, Ca-s cells are stimulated, but cell hypertrophy is not uniform and often restricted to an area adjacent to the proximal pars distalis. Nuclear hypertrophy is significant in the reactive area, although less pronounced or even absent at the periphery of the lobe. Mitotic activity occurs in the Ca-s cells of goldfish gradually adapted to diluted Ca-free SW and Ca-Mg-deficient SW, and sacrificed after 19 and 28 days, respectively. A general stimulation of the Ca-s cells, which remains less intense than that in goldfish kept in deionized water (DW), appears unable to ensure the survival of the goldfish in an isosmotic Ca-free environment containing Mg²⁺ (0.1, 12 or 16.8 mM). These data are compared with those obtained in the eel kept in Ca-free SW.     

Protein synthesis in tissues cultured from the bovine hoof

Salmon gonadotropin (GTH II) is a heterodimeric glycoprotein hormone ( and II subunits), serving as a maturational GTH, and is produced in a specific gonadotropic cell-type (GTH II-cells) containing small granules and large globules. In trout GTH II-cells, double immunolabeling for the - and II-subunits shows that colocalization of the - and II-immunolabeling is confined to the small granules, indicating storage of functional GTH II. On the other hand, -immunolabeling is absent in the large globules, even though II labeling is abundant throughout the period of seasonal gametogenesis. The -specific antiserum recognizes the intact -subunit as well as the reduced and deglycosylated -subunits by immunoblotting. These results indicate that an accumulation of the II-subunit is specifically generated in the large globules of these cells. In fact, with sexual maturity, the quantity of II-subunits becomes elevated in the trout pituitary due to a marked increase in GTH II-cells containing many large globules. However, the derivation and function of the large globules and the fate of their contained II-subunits remains unknown.     

Co-existence of gonadotrophins (FSH,LH) and thyrotrophin (TSH) in single anterior pituitary cells of the musk shrew,Suncus murinus

According to recent immunocytochemical studies of anterior pituitary cells, it is obvious that the one cell-one hormone theory must be modified. Many pituitary morphologists have demonstrated that there are some cells that contain two hormones. In this study, we demonstrate by means of immuno-electronmicroscopy the co-existence of gonadotrophins (FSH and LH) and thyrotrophin (TSH) in the same anterior pituitary cells of the musk shrew. These cells were remarkably altered in their ultrastructural features by either gonadectomy or thyroidectomy. Double labeling for gonadotrophins and thyrotrophin was present not only in the same cells but also in the same secretory granules. Our ability to demonstrate co-existence of gonadotrophins and thyrotrophin in the same cell may be due to our selection of fixative and embedding media for electron-microscopic immunocytochemistry. Our conclusion that gonadotrophins and thyrotrophin are produced in a single cell type of the anterior pituitary gland in the musk shrew, i.e., thyrogonadotrophs, suggests the need to consider a modification of the classic scheme for classification of anterior pituitary cells.     

Ultrastructural changes in the calcium-sensitive (PAS-positive) cells of the pars intermedia of eels kept in deionized water and in normal and concentrated sea water

Summary The ultrastructure of the calcium-sensitive (Ca-s) (PAS-positive) cells of the pars intermedia was investigated in eels kept in hypo and hyperosmotic environments. Although the cells were moderately active in fresh water (FW), they were highly stimulated in deionized water (DW) and displayed an enlarged Golgi apparatus, a distinct rough endoplasmic reticulum, few secretory granules, some microtubules and an extended area of contact with the basal lamina that separates nervous and glandular tissues. Some mitosing cells were seen. A similar picture was observed in eels kept in sea water (SW) for 45 days, returned to FW and subsequently to DW for 21 days. In SW (30 and 33), and particularly in concentrated SW (50, 60 and 63), the Ca-s cells were inactive. Their granules were significantly smaller than in eels kept in FW, and the area of contact with the basal lamina was greatly reduced. However, signs of granule-release were seen in eels adapted to 50 and 60 SW. Nerve fibers rarely contacted the Ca-s cells and did not synapse with them. The ultrastructural data support the hypothesis that the Ca-s cells of Anguilla, like those of Carassius, are involved in ionic regulation. MSH cells were not greatly affected by the present experiments.     

Immunocytochemical distribution of FMRFamide-like substance in the brain of the cloudy dogfish,Scyliorhinus torazame

Summary The distribution of the molluscan cardioexcitatory tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH₂) in the brain of the cloudy dogfish, Scyliorhinus torazame, was examined by immunocytochemistry. FMRFamide-like immunoreactivity was demonstrated to occur extensively in various regions of the dogfish brain, except for the corpus cerebelli. Immunoreactive neuronal perikarya were located in the ganglion of the nervus terminalis, the preoptic area, and the hypothalamic periventricular gray matter consisting of the nucleus medius hypothalamicus, the nucleus lateralis tuberis, and the nucleus lobi lateralis. some of the immunoreactive cells in the hypothalamus were identified as cerebrospinal fluid-contacting neurons. The bulk of the immunostained fibers in the nervus terminalis penetrated into the midventral portion of the telencephalon and ran dorsocaudally toward the basal telencephalon and hypothalamus, showing radial projections or ramifications. The labeled fibers were abundant in the midbasal part of the telencephalon and in the hypothalamus, where some fibers were found in loose networks around the cell bodies of the nucleus septi and hypothalamic periventricular nuclei. The fibers demonstrated in the hypothalamus terminated around the vascular wall of the primary capillary plexus of the median eminence or penetrated deeply into the pars intermedia of the hypophysis. These results suggest that, in the dogfish, an FMRFamide-like substance participates in the regulation of adenohypophysial function. This molecule may have a role as a neurotransmitter and/or neuromodulator in the central nervous system.     

Stereological analysis of daily variation in the ultrastructure of the pars intermedia of the pituitary gland of the Djungarian hamster (Phodopus sungorus)

Summary A Stereological analysis has been made of the daily changes occurring in the ultrastructure of the melanotrophs of the pars intermedia of the pituitary gland of the Djungarian or Siberian hamster, Phodopus sungorus, maintained in long day photoperiods. The rough endoplasmic reticulum, Golgi apparatus and lysosomes all declined in fractional volume throughout the photophase reaching minima at mid-scotophase and rising to reach their maxima at about the time of onset of the photophase. The mitochondria reached their peak fractional volume just before the cessation of the photophase but then also declined to a minimum at mid-scotophase. No significant changes were found to occur in the fractional volumes of the nucleus or the secretory granules. These morphological findings are compared with the changes in plasma and pituitary -melanocyte-stimulating hormone levels found in the rat.Supported in part by the Medical Research Council (Project Grant G978-398/C to BW)     

Distribution of neuropeptide Y-like immunoreactivity in the brain and hypophysis of the cloudy dogfish,Scyliorhinus torazame

Summary Using a specific antiserum raised against synthetic neuropeptide Y, we examined the localization of immunoreactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, by the peroxidase-antiperoxidase method. Immunoreactive perikarya were demonstrated in the ganglion of the nervus terminalis, the dorsocaudal portions of the pallium dorsale, the basal telencephalon, and the nucleus lateralis tuberis and the nucleus lobi lateralis in the hypothalamus. Labeled perikarya were also found in the tegmentum mesencephali, the corpus cerebelli, and the medulla oblongata. Some of the immunoreactive neurons in the hypothalamus were of the CSF-contacting type. The bulk of the labeled fibers in the nervus terminalis ran toward the basal telencephalon, showing radial projections and ramifications. Large numbers of these fibers coursed into the nucleus septi caudoventralis and the nucleus interstitialis commissurae anterioris, where they became varicose and occasionally formed fine networks or invested immunonegative perikarya. In the diencephalon, immunoreactive fibers were observed throughout the hypothalamus, e.g., in the pars neurointermedia of the hypophysis, the subependymal layer of the lobus inferior hypothalami, and in the neuropil of the posterior (mammillary) recess organ. Labeled fibers were scattered throughout the rest of the brain stem and were also seen in the granular layer of the cerebellum. These results suggest that, in the dogfish brain, neuropeptide Y or a related substance is involved in a variety of physiological processes in the brain, including the neuroendocrine control of the hypophysis.