Ultrastructural changes in the pars intermedia of the goldfish kept in calcium-free environments

The PAS-positive-calcium-sensitive (Ca-s) cells of the pars intermedia (PI) were studied in goldfish kept in fresh water (FW), deionized water (DW), 1/3 sea water (SW) and 1/3 Ca-free SW. Ultrastructural studies show that Ca-s cells of control goldfish kept in FW have a low activity with elongated or deeply indented nuclei. This activity is slightly reduced after 19 days in 1/3 SW. A considerable stimulation of most Ca-s cells is noted in goldfish kept in DW for 20 or 40 days. The stimulation is similar in 1/3 Ca-free SW, but it affects sometimes a smaller percentage of cells and may be less marked in peripheral areas of the PI. Exocytotic figures are more numerous in Ca-s cells of goldfish in 1/3 Ca-free SW than in DW. A basal lamina is rarely present and direct contacts between PI cells and nervous tissue are frequent, although a single synaptic contact with a type B fiber was observed. MSH cells are not affected in goldfish kept in DW. They are stimulated in 1/3 Ca-free SW: the physiological significance of this response remains unclear. Few agranular (Agr) cells are scattered in the PI. Evident changes are not observed in the different environments. The present ultrastructural data support the hypothesis that the Ca-s cells of the PI secrete a factor involved in calcium regulation in some teleosts.     

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.     

Response of prolactin cells to environmental calcium in the eel (Anguilla anguilla L.)

Prolactin (PRL) cell activity was investigated in eels kept in fresh water (FW), deionized water (DW) supplemented or not with Ca (2 mM), in Ca-enriched FW (10 mM), in normal (Ca 3.4 mM) or Ca-free 1/3 sea water (SW), and in SW (Ca 10.2 mM) or Ca-free SW (Ca 0.15 mM). Light-microscopic studies, including measurement of the nuclear area and cell height, showed that PRL cell activity, reduced in DW, is not affected by Ca supplementation. Activity is reduced in Ca-enriched FW, in 1/3 SW and in SW, conditions inducing an increase in the plasma sodium level. The lack of calcium in saline environments partly suppresses the nuclear atrophy occurring in SW. There is no significant correlation between external or total plasma calcium concentration and PRL cell activity. In artificial Ca-free SW, eels show a rapid increase in plasma osmolarity and sodium levels; there is a significant negative correlation between these two plasma values and the nuclear area or cell height of PRL cells. As in some other teleosts, plasma osmolarity and plasma sodium seem to play a more important role than external or internal calcium in controlling PRL secretion. This correlation is not apparent in eels kept in SW, having unstimulated PRL cells but active calcium-sensitive (Ca-s) cells in the pars intermedia.     

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

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

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.     

Specific effect of calcium ions on the calcium-sensitive cells of the pars intermedia in the goldfish

Summary Cytological changes in the calcium-sensitive (Ca-s) cells (formerly termed PAS-positive cells) of the pars intermedia were investigated in the goldfish after adaptation to deionized water (DW), with or without addition of sodium, potassium and magnesium. These ions were added as chloride salts at concentrations similar to those present in fresh water (FW). The marked stimulation of the Ca-s cells is not inhibited in DW supplemented with Na⁺ (0.35 mM/1), K⁺ (0.05 mM/1), and Mg²⁺ (0.2mM/1) for a period of 24 days. The inhibition of the response to DW with calcium chloride (2 mM/1) is reproduced with calcium formiate (2 mM/1). These data show that chloride ions are not responsible for the regression of the Ca-s cells observed in goldfish kept in DW supplemented with calcium chloride. The effect of calcium ions on the Ca-s cells appears to be specific. These results support the hypothesis that the Ca-s cells synthesize a factor (hypercalcin?) involved in calcium regulation, and that its release is influenced by the calcium content of the environment. The role of the pars intermedia in calcium metabolism is strengthened by the present results. Biochemical data suggest the presence of a hypercalcemic factor in the pituitary of fish (Parsons et al. 1978) and are in agreement with the present cytological findings.     

Differential Effect of Sodium and Magnesium on the Pituitary of Goldfish Adapted to Calcium-Free Environments

Adaptation to deionized water (DW) affects several cell types in the goldfish. The pars intermedia PAS-positive cells are highly stimulated. Their low response or the absence of changes in goldfish kept in 1/3 Ca-free seawater (SW) and in Ca-free SW-adapted eels, respectively, suggest that sodium and/or magnesium are interfering. To test this hypothesis, young goldfish were adapted to DW supplemented or not with sodium (50 and 140 mM) for 8 and 16 days or with magnesium (16.5 and 50 mM) for 16 and 30 days. Cytological and morphometric studies of the pituitary showed that prolactin (PRL) cell activity was reduced by sodium. Thyrotropic (TSH) cells were stimulated. The activity of melanocyte-stimulating (MSH) cells increased in DW + Na+. Stimulation of the pars intermedia PAS+ cells in DW was partly inhibited by adding sodium; the cellular and nuclear areas increased only moderately, the endoplasmic reticulum (ER) was not conspicuous and mitotic activity disappeared. In DW + Mg²⁺ the activity of PRL, TSH and MSH cells tended to be lower after a long-term adaptation. The response of the PAS+ cells was as high as that noted in DW; complete degranulation, enlargement of the ER and important mitotic activity. Differential responses to Na+ and Mg²⁺ are not due to pH differences in the solutions. External sodium is able then to reduce the response of the PAS+ cells in a Ca-free environment, while magnesium is not inhibitory. Other cell types are also affected by high levels of Na+ and Mg²⁺.     

The Calcium-sensitive Cells of the Pars Intermedia in the Eel

In the pituitary, the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia appear less active in seawater (SW)- than in freshwater (FW)-adapted eels. The kinetics of their response during adaptation to SW or readaptation to FW was investigated. Morphometric studies show that transfer to SW induces a rapid nuclear atrophy which accentuates in eels kept for several weeks in SW. Readaptation to FW stimulated the Ca-s cells after 2–10 days; after 1 or 2 months, the cells tend to be similar to those of eels kept in FW. Plasma calcium decreases slightly but significantly in SW eels. The response of the Ca-s cells is not modified by an ovine prolactin treatment inducing hypercalcemia, hypernatremia and stimulation of the corpuscles of Stannius. Minor changes occurring in the MSH cells remain difficult to interpret; the short stimulation during readaptation to FW may be related to a stress effect and/or to release of other peptides present in the MSH cells of fish.     

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.     

Responses of the teleost pituitary (goldfish,eel) to deionized water

Summary The cytological responses of the pituitary gland during adaptation to deionized water (DW) were investigated in the goldfish and the eel. In both teleost species, a stimulation of the prolactin (PRL) cells could not be detected, although the levels of blood electrolytes (Na⁺,Ca²⁺,Cl^–) are reduced in the eel. PRL cells appear less active in DW-adapted eels. A striking stimulation of the PAS-positive cells of the pars intermedia occurs in both species after 3 weeks and, in the eel, is still present after 11 weeks. Cell and nuclear hypertrophy, mitoses and a well-developed endoplasmic reticulum are observed. MSH cells are partially degranulated when pigmentation is affected; a reduced activity of MSH cells is evident after 11 weeks. The amount of neurohypophysial tissue is reduced. In the goldfish and the eel, during adaptation to DW, an unknown factor secreted by the PAS-positive cells of the pars intermedia appears to play a more important role than the secretion of PRL. These two species are able to survive in fresh water without the pituitary. The control of the PAS-positive cells by external sodium or calcium is discussed.     

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.     

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.     

The osmotic response of salmon louse,Lepeophtheirus salmonis (Copepoda: Caligidae), during the transition from sea water to fresh water

Summary The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl^– measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl^– concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.Acclimation to 37% SW is accompanied by changes in body tissue water content and in the content of ninhydrin positive substances and specific amino acids which suggest the presence of cell volume regulation. Glycine is the dominating free amino acid in the cephalothorax tissues but alanine, proline and taurine also occur in high amounts. Lysine is found to increase significantly during FW acclimation of attached parasites. A breakdown of cell volume regulation is suggested to limit the survival of attached salmon louse in fresh water.Abbreviations FW fresh water - NPS ninhydrin positive substances - SW sea water     

Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus)

In order to replace the diffusive loss of water to the surrounding environment, seawater (SW)-acclimated euryhaline fishes have gastrointestinal tracts with higher ion/water flux in concert with greater permeability, and contrast that to freshwater (FW)-acclimated fish. To understand the cellular basis for these differences, we examined cell proliferation and apoptosis in the anterior intestine of mudskipper transferred from one-third SW to FW or to SW for 1 and 7 days, and those kept out of water for 1 day. The intestinal apoptosis (indicated by DNA laddering) increased during seawater acclimation. TUNEL staining detected numerous apoptotic cells over the epithelium of SW-acclimated fish. Cell proliferation ([³H]thymidine incorporation) in the FW fish was greater than those in SW 7 days after transfer. Labeling with a Proliferating cell nuclear antigen (PCNA) antibody indicated that proliferating cells were greater in number and randomly distributed in the epithelium of FW fish, whereas in SW fish they were almost entirely in the troughs of the intestinal folds. There were no changes in cell turnover in fish kept out of water. During acclimation to different salinities, modification of the cell turnover and abundance may play an important role in regulating the permeability (and transport capacity) of the gastrointestinal tract of fish.     

Effect of decreased environmental salinity on growth hormone cells in the gilthead sea bream (Sparus aurata)

The effect of decreased environmental salinity on growth hormone producing cells (GH cells) of the adenohypophysial proximal pars distalis has been studied in the gilthead sea bream ( Sparus aurata L.) adapted to sea water (SW, 980 mosmol kg ⁻¹) and brackish water (BW, 200 mosmol kg ⁻¹). A combined immunocytochemical, morphometric and electron microscopic study was carried out. GH cells offish adapted to BW occupied a greater hypophysial volume (about 21% of the total hypophysial volume in BW, 17% in SW) and had a larger nuclear area (mean 16 μm² in BW, 13 μm² in SW) than GH cells of SW-adapted fish. The immunoreactivity against a salmon GH-antiserum was lower in BW (mean optical density 142 in BW, 159 in SW). Ultrastructural characteristics of GH cells of BW-adapted fish were distended rough endo-plasmatic reticulum and large secretory granules (about 216 nm in diameter for BW, 209 nm in SW). Volumetric, densitometric and ultrastructural evidence suggested that the synthesis and release of GH were activated in S. aurata adapted to hypo-osmotic environment.     

PAS-positive cells of the pars intermedia are calcium-sensitive in the goldfish maintained in a hyposmotic milieu

Summary Cytological changes in the pars intermedia of the goldfish were investigated after adding calcium to deionized water (DW). In fish maintained in DW, the PAS-positive cells are highly stimulated in comparison to cells of fish kept in fresh water (FW). In DW supplemented with calcium at the same concentration as in FW (2 mM/l), the hyperactivity of the PAS-positive cells is prevented. When calcium ions are added 60 h before the animals are sacrificed, the PAS positive cells start to show signs of regression and their granules are stored: the release of the granular material appears to be suppressed by calcium. In the goldfish, the PAS-positive cells, homologous to a similar cell type in the eel, react only very weakly with the PAS technique. The name calcium-sensitive cells appears to be more appropriate in the goldfish for this particular cell type, secreting an unknown factor. This factor, different from the prolactin produced in the rostral pars distalis of the hypophysis, might be an equivalent of a hypercalcin.     

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 glucose on survival, infectivity and linear movement of the cercariae of Echinostoma caproni

The effects of glucose in artificial spring water (ASW) on the survival, infectivity, and linear movement of Echinostoma caproni cercariae were studied. Cercariae maintained at 23 degrees C in 1% glucose in ASW (ASWG) or ASW alone, reached 50% survival at 26 and 23 h, respectively. All cercariae in ASWG and ASW were dead by 50 and 32 h, respectively. Infectivity to juvenile Helisoma trivolvis (Colorado strain) snails was significantly less for cercariae aged 16 h in ASWG compared to cercariae aged 16 h in ASW. Linear movement, i.e. the ability of cercariae to traverse a 1-cm radius, ceased at 16 and 20 h for cercariae maintained in ASWG and ASW, respectively. Glucose added to ASW extended the survival time of E. caproni cercariae but decreased their ability to infect snails or move in a linear direction.     

Ultrastructural identification of catecholaminergic fibers in the goldfish pituitary

Summary The monoaminergic innervation of the goldfish pituitary gland was studied by means of light- and electronmicroscopic radioautography after in vitro administration of ³H-dopamine. The tracer was specifically incorporated and retained by part of the type-B fibers innervating the different lobes of the pituitary. In the rostral pars distalis labeled fibers were most frequently observed in contact with the basement membrane separating the neurohypophysis and the adenohypophysis. In the proximal pars distalis and the pars intermedia, labeled profiles were detected in the neural tissue and in direct contact with the different types of secretory cells.According to the previous data concerning the uptake and retention of tritiated catecholamines in the central nervous system, it is assumed that the labeled fibers are mainly catecholaminergic (principally dopaminergic). This study provides morphological evidence for a neuroendocrine function of catecholamines in the goldfish.