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.     

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 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.     

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.     

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.     

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.     

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.     

Immunocytohistochemical characterization of pituitary cells of the bluefin tuna, Thunnus thynnus L

In this paper we report the first complete mapping of the pituitary in a tuna species. The various different adenohypophysis cell types of the bluefin tuna, Thunnus thynnus L. have been identified and located using different antisera against mammalian and piscine hormones and various histochemical techniques: PAS, Alcian Blue pH 2.5 and lectins -ConA and WGA(Neutral and Acidic Glycoproteins); Bromophenol Blue (Proteins) and Tioglycollate-Ferric-Ferricianide-FeIII (-S-S- groups). Prolactin (PRL) and adrenocorticotrophic (ACTH) cells were located in the rostral pars distalis (RPD) of the pituitary, while the proximal pars distalis (PPD) displayed gonadotrophic (GTH), thyrotrophic (TSH), somatotrophic (GH) and also a few PRL cells. Moreover, somatolactin (SL) and melanotrophic (MSH) cells were identified inside the pars intermedia (PI). Interestingly, some SL-immunoreactive fibers were also detected in the neurohypophysis. Some GTH cells were also located on the exterior surface of the PI. Glycoproteins containing mannose (Man) and/or glucose (Glc); N-acetyl-glucosamine (GlcNAc) and/or sialic acid sugar residues, as well as -S-S- groups, were observed in GTH, TSH and SL cells. The Bromophenol Blue technique stained amphiphilic SL, acidophilic GH cells and weakly ACTH cells. GH and ACTH cells were unreactive to PAS, Alcian Blue, Tioglycollate-Ferric-Ferricianide-FeIII and lectin (Con A and WGA) techniques. Finally, PAS reaction was positive in amphiphilic SL cells, which were PbH unreactive, while MSH and ACTH cells were stained with PbH technique.     

Pituitary cytology of radiothyroidectomised teleost Heteropneustes fossilis (Bloch.).

The pituitary of Heteropneustes fossilis is divisible into rostral (RPD) and proximal (PPD) pars distalis, and pars intermedia (PI). Neurohypophysis extensively ramifies in the PI. The RPD is formed of acidophilic prolactin cells and lead hematoxylin (H. Pb) positive ACTH cells. The latter is arranged around the neurohypophysial extensions. The PPD is comprised of thyrotrophs (TSH) and gonadotrophs which are stainable with PAS, AF, and aniline blue. Acidophilic somatotrophs (STH) and chromophobes are scattered throughout the PPD. In the pars intermedia PAS positive and H. Pb positive cells are present. The latter is believed to secrete MSH whereas the function of the former is yet to be confirmed. In the partly and completely thyroidectomised H. fossilis the TSH cells exhibited marked hypertrophy. They are located in the middle of the PPD flanked by the gonadotrophs on either side. Hypertrophy of the ACTH cells were also evident. But the other cell types did not show any marked changes as compared to the controls.     

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.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.     

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.     

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 MSH producing cells in the pars intermedia of Sarotherodon mossambicus have been shown to be involved in background adaptation processes. Reflected light received by the eyes affects the activity of these cells. In the present study the hypothesis has been tested that also the pineal organ, as a second photoreceptor, is involved in regulation of the metabolic activity of the MSH cells. The pineal organ appears to contain photoreceptor cells and is considered to be capable of transferring information about light conditions to the animal. Removal of the pineal organ of fish kept on a black background has no effect on activity of MSH cells, whereas the activity of these cells in fish kept in darkness is increased. Thus it seems that the pineal organ exercises its influence on MSH cells only in darkness and that this influence results in a reduced activity of these cells. It is therefore concluded that the metabolic activity of MSH cells is inhibited not only by reflected light received by the eyes, but also by the action of the pineal organ as a result of the absence of illumination.No structural signs of secretory activity can be observed in the pineal, which might indicate synthesis or release of substances like melatonin. However, administration of melatonin reduces the activity of MSH cells. Neither pinealectomy nor treatment with melatonin has any influence on the second cell type of the pars intermedia, the PAS positive cells.     

Distribution of immunoreactivities for adenohypophysial hormones in the pituitary gland of the polypteriform fish, Polypterus endlicheri

Polypteriform fish constitutes the most primitive living descendent of the ancient bony fish. In polypteriform fish, only proopiomelanocortin (POMC) has been identified so far in the adenohypophysis, which is surprising in view of their evolutionary importance. In the present study, distribution of immunoreactive adenohypophysial hormones was examined in juvenile individuals of Polypterus endlicheri. Antisera to tetrapod and fish adenohypophysial hormones were used as immunostaining probes. Adrenocorticotropin (ACTH)-like cells were detected by antisera to salmon POMC N-terminal peptide, porcine ACTH and mammalian alpha-melanotropin (MSH), and were distributed in the rostral pars distalis in close proximity to the hypophysial duct. MSH-like cells were found in the pars intermedia, and were stained by anti-salmon N-Ac-beta-endorphin II as well as anti-mammalian alpha-MSH and anti-salmon POMC-N terminal peptide. Prolactin (PRL)-like cells were detected only after application of anti-sturgeon PRL, and were distributed in the rostral pars distalis, where PRL-positive material was found in columnar mucinous cells lining the diverticuli of the hypophysial duct. Growth hormone (GH)-like cells were stained with antisera to sturgeon GH, human GH, salmon GH and blue shark GH, and were distributed in the proximal pars distalis. Somatolactin (SL)-like cells were stained with anti-salmon SL, and were distributed in the pars intermedia. Two types of glycoprotein hormone-positive cells were detected in the proximal pars distalis. Although both types of cells were stained with several antisera to glycoprotein hormones, such as sturgeon LHbeta and salmon LHbeta, it was difficult to know which types of cells produce LH, FSH, or TSH. Thus, the present study revealed seven types of adenohypophysial hormone-like cells in the Polypterus pituitary gland, which may provide the morphological basis for better understanding on evolution of the pituitary gland and the adenohypophysial hormones in vertebrates.     

Pituitary cytology of the chimaeroid fish Hydrolagus colliei (Lay and Bennett).

The pituitary of Hydrolagus colliei is divided into the adenohypophysis, neurohypophysis and an oral Rachendachhypophyse. The adenohypophysis is further divided into rostral and proximal pars distalis and neurointermediate lobe. The neurohypophysis is restricted to the pars intermedia only. The rostral pars distalis is composed of acidophils, chromophobes, lightly PAS+ cells and amphiphils. The amphiphils were stained with Heidenhain's iron haematoxylin and lead haematoxylin also. The proximal pars distalis is formed of cyanophils where the granules are AF and PAS positive, acidophils, chromophobes and H.Pb+ cells. The pars intermedia has perviascular amphiphils which are H.Pb+, lightly PAS+ cells and chromophobes. Few AF+ cells were also identified. All the component parts of the adenohypophysis have follicular cavities which are probably developed from the hypophysial cavity, which is well seen in the young specimen as a single cavity extending antero-posteriorly throughout the adenohypophysis.     

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.     

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.     

Cyclical mitotic activity in the adeno-hypophysis of Rana temporaria

Summary In the pars distalis, the pars intermedia and the pars tuberalis of the hypophysis of Rana temporaria, an annual mitotic cycle is described.In the pars distalis there exists a gradient of mitotic activity, corresponding to a gradient of distribution of the PAS positive cells.In the pars distalis, the rate of cell degeneration and of cell renewal of the PAS positive cells seems to be faster than of the orange G positive cells.The replacement of degenerated PAS positive cells of the pars distalis seems to occur mainly during the months immediately following on the period of maximal activity of the PAS positive cells.     

Immunohistochemical detection of ACTH and MSH cells in the hypophysis of the hermaphroditic teleost, Diplodus sargus

Hypophyseal ACTH and MSH cells were immunohistochemically characterised in the teleost fish, Diplodus sargus, using anti-ACTH (1-24) and anti alpha-MSH polyclonal antisera. ACTH cells were found both in the pars distalis and in the pars intermedia. In the former region, they appeared small, round-shaped and clustered; in the latter, they were either small or large and elongated. Moreover, a few ACTH-immunoreactive cells resembling microglia were present in the neurohypophysis. Conversely, MSH cells were found only in the pars intermedia, and were similar to the larger ACTH cells of the same region. In the pars intermedia, co-localisation of ACTH and MSH immunoreactivity in the same cell was revealed by double immunostaining, though the two hormones were also observed in distinct cell types. The distribution of ACTH cells appeared quite uniform, without any marked difference between the specimens tested. Conversely, MSH cell amount varied according to the stage of the sexual cycle of this teleost fish, which is characterised by protandrous hermaphroditism. In fact, a lower amount of MSH cells were observed in females, whereas no significant difference was found between immature and male specimens.