Secretion of melanophore-stimulating hormone (MSH) in long-term cultures of pituitary neurointermediate lobes

Summary Neurointermediate lobes from pituitaries of the frog, Rana berlandieri forreri (Rana pipiens, sensu lato), were maintained in organ culture in media with and without serum for up to six months. The cultured tissues were examined periodically by light microscopy and transmission electron microscopy and by bioassay of the melanophore-stimulating hormone (MSH) secreted and present in the culture media. Light-microscopic observations revealed a high degree of preservation of the pars intermedia at four weeks with isolated areas of some glands maintaining histological integrity for the entire six months. Similarly, at the ultrastructural level the cells appeared morphologically intact and to be actively synthesizing and secreting hormone. Bioassays showed the glands to be continuously secreting MSH; however, larger yields of hormone were obtained in media lacking serum. No significant ultrastructural differences between cells grown in the presence or absence of serum were detected. The difference in concentration of MSH between the two groups therefore apparently results from enzymatic degradation of the hormone by the serum. Organ culture of the vertebrate neurointermediate lobe may provide a unique method for the production of large quantities of MSH and for the study of other melanotropic and opiate peptides as they may be synthesized and secreted by the pars intermedia.     

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.     

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.     

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.     

Demonstration of stellate cells of the pars intermedia of the pituitary gland using a new silver impregnation technique

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 micron sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.     

Demonstration of Stellate Cells of the Pars Intermedia of the Pituitary Gland Using a New Silver Impregnation Technique

The pars intermedia of the pituitary gland in the rat and other species contains, in addition to secretory cells, stellate cells first characterized by using electron microscopy. The distribution and relationships of these cells is difficult to assess at the ultrastructural level. We have developed an ammoniacal silver nitrate method for stellate cells of the pars intermedia. Staining is carried out in 15 µl;m sections of buffered formalin fixed, paraffin embedded pituitaries. This method shows that in the rat pars intermedia, stellate cells showing numerous cytoplasmic projections are abundant in, and evenly distributed throughout, pars intermedia lobules.     

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.     

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⁺.     

CHANGES IN THE MELANOPHOROTROPIC FUNCTION OF THE PITUITARY GLAND ACCOMPANYING BLINDNESS IN XENOPUS LAEVIS DAUDIN

A number of cases of blindness appeared among a group of cultured Xenopus laevis. The blind toads lacked eyeballs and optic nerves and were consistently dark in color. Similar anatomical changes in the head and pigmentation were produced experimentally by removing the eyeballs of tadpoles or young adult toads. The dark pigmentation of blind Xenopus: was shown to be due to the continuous release of MSH from the pars intermedia of the pituitary, since hypophysectomy led to the complete blanching of the dermal and epidermal melanophores. MSH activity in the pars intermedia of blind toads was extremely high in comparison with that of normal animals. No special difference with respect to the general growth and behaviour was found between normal and blind Xenopus, which suggests that the pituitary functions other than that of the pars intermedia are not affected by the state of the optic nerves. These observations suggest that the retrogressive degeneration of the optic nerves exerts a profound effect on the secretory activity of the pars intermedia via the hypothalamic controlling center.     

The effect of change of background colour on the ultrastructure of the pars intermedia of the pituitary of the eel (Anguilla anguilla)

Summary Colour change in the eel resulted in marked ultrastructural changes in the pre-dominating (Type II) secretory cells of the pars intermedia of the pituitary. The effects on these cells of transferring eels from white to black backgrounds for periods of up to 56 days were: a) hypertrophy of the rough endoplasmic reticulum, which increased from 12 to 35% of the cytoplasmic volume; b) loss of secretory granules which decreased from 38 to 5% of the cytoplasmic volume; c) development of a system of fine (25–35 nm) tubules located especially at the secretory poles of the cells but also found in the region of the Golgi apparatus. The tubules were seen to connect with the plasma membrane, with the limiting membrane of the secretory granules, and in one instance to connect a granule with the plasma membrane. After glutaraldehyde fixation at pH 5, electron dense material similar to that found in the secretory granules was observed in the lumen of many of the tubules.The changes that occurred in black background eels are taken to indicate that the Type II cells of the pars intermedia are responsible for MSH secretion, particularly since these changes were reversed by returning eels to white backgrounds. The cytoplasmic tubules found in Type II cells may indicate a process for MSH release which does not involve granule extrusion, but rather direct transport of material from the Golgi apparatus to the cell membrane.The electron microscope facilities used in this investigation were funded by the Medical Research Council.     

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.     

Life span changes in the presence of alpha-melanocyte-stimulating-hormone-containing cells in the human pituitary.

Since recent circumstantial evidence has suggested possible functions of alpha-MSH in intrauterine growth and labour, the presence of this hormone in the human pituitary was determined by means of the indirect immunofluorescence procedure during development and adulthood. Cross reaction of the antibodies with other peptides was measured after which they were purified by solid phase absorption. Experiments on the rat pituitary showed that staining of alpha-MSH- and ACTH-containing cells could be obtained well until 48 h after death. In the pars distalis the ability of ACTH-containing cells to take up stain increased during the period of post-mortem storage. In the youngest human fetus studied (15 weeks) only alpha-MSH-containing cells were found in the pars intermedia and no ACTH-containing cells were observed. In the other fetal pituitaries a distinct pars intermedia containing more alpha-MSH cells than ACTH cells was found. In the pars distalis of the fetuses more ACTH- than alpha-MSH-containing cells were observed. From birth to 19 years, progressively fewer alpha-MSH containing cells could be detected in the 'zona intermedia' and pars distalis, while in adults only a few such cells were found in either area. Irrespective of age, sex, cause of death or therapy, alpha-MSH-containing cells were found in all pituitaries throughout life. The number of ACTH containing cells gradually increased in the zona intermedia and pars distalis and reached a high adult level in the latter structure. In the pituitaries of seven anencephalics, no alpha-MSH-containing cells were present. The presence of alpha-MSH in the fetal pars intermedia, the change in the ratio of the alpha-MSH/ACTH cells during the course of development, and the absence of alpha-MSH in anencephaly all support the possibility that human fetal pituitary alpha-MSH is involved in both intrauterine growth and fetal adrenal function and thus also in parturition.     

Endorphin in rat pituitary glands: its distribution within the gland, and age related changes in gland content in male and female rats

Endorphins were extracted with glacial acetic acid:acetone from freshly microdissected single lobes of rat pituitary and assayed for inhibition of ³H-etorphine stereospecific binding to rat brain opiate receptors. Highest tissue concentrations of endorphin were found in pars intermedia and pars distalis, with minimal activity observed in pars nervosa. In addition, mature female rats exhibited a significantly higher anterior lobe endorphin content than males, reflecting the larger gland weight, although no significant differences were found between neurointermediate lobe endorphin content of either sex. Pituitary endorphin content increased substantially with age, rising rapidly between the fifth and tenth week after birth. This elevation was still apparent at twenty-five weeks. These studies emphasize the importance of employing rats of closely similar age and weight in any experiments designed to examine pituitary endorphin function, and suggest that ontological development of pituitary endorphin may proceed in a manner similar to that of other pituitary hormones (e.g., MSH) derived from the same precursor peptide.     

Ultrastructure of the male rat hypophysis chronically grafted under the kidney capsule

Summary Male rats were divided in two experimental groups. In group I two partes distales of the hypophysis were grafted under the kidney capsule and in group II two complete hypophyses were transplanted. Animals were killed 5 to 22 months after the operation. The grafted tissue was excised and processed for light and electron microscopy.The transplanted pars distalis tissue showed a well developed vascularisation in contrast to the pars intermedia which appeared poorly vascularised. Six different cell types were observed in grafted pars distalis. They correspond to the different types of cells found in the rat pars distalis in situ. The predominant cell type in the graft displayed all the morphological characteristics of stimulated prolactotrophs. Pars intermedia cells appeared hypertrophied resembling the MSH cells under stimulation. Two types of syncytial formations were frequently seen. One of them appeared to originate from prolactotrophs and the other from MSH cells. Bodian impregnated fibres and structures resembling either growth cones of axons or typical nerve endings were observed in the pars intermedia of long-term grafted hypophyses. Pituicytes remained as isolated clusters of cells. Canaliculi lined by two or more pituicytes were observed. Saccular formations resembling the hypophyseal cleft appeared in all grafts studied. The present findings suggest that in the male rat the chronically grafted pituitary gland is capable of synthesising most or all the hormones which are known to be produced by the gland in situ. Furthermore, prolactin and MSH seem to be the predominant secretion of the transplanted pituitary.Supported by the National Research Council of Argentina. Grant 7315/74Members of the Research Career of Conicet, Argentina     

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.     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis

Summary Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH₂-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no α-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

Observations on the functional cytochemistry of pars intermedia of the rat hypophysis.

Light and electron microscopic histochemical reactions were studied in the cells of pars intermedia of the rat. The possible correlations between enzymatic reactions and endocrine functions of these cells were discussed. By combined formaldehyde and chloral vapour treatment the cells of the pars intermedia exhibited a strong yellow fluorescence suggesting the presence of a peptide or peptides with NH2-terminal tryptophan. Masked metachromasia after acid hydrolysis was probably due to these peptides. Only a weak or no alpha-glycerophosphate dehydrogenase and nonspecific esterase activity was observed in the cells of pars intermedia compared to the cells of pars distalis suggesting low production rate of hormone synthesis. Specific and non-specific cholinesterases were demonstrated light and electron microscopically constantly in the cells bordering the lobules. These cells probably represent a certain type of glial cells. In the other cells the enzymatic activities varied markedly in intensity and distribution showing different ultrastructural localizations. Thus cholinesterase activities in the cells of pars intermedia reflect possibly different functional stages of the cells in their hormone production, storage and secretion processes.     

The influence of environmental lighting on the amphibian pars intermedia: Receptors and possible pathways

The well-known "background adaptation" response of the anuran amphibians constitutes a clearly defined example of an effect of environmental illumination on pituitary secretion. Morphological studies strongly suggest that the secretion of MSH, the hormonal agent of background adaptation, is controlled by neurons which terminate in the intermediate lobe of the pituitary (pars intermedia). We have investigated the possible role of this innervation in the photic control of the pars intermedia ofRana pipiens by recording the electrical activity of single, intermediate lobe neurons with gold-plated, platinum-tipped micro-electrodes. These studies have shown that changes in illumination affect the activity of some neurons in the intermediate lobe, ant that the photoreceptors which mediate this response are clearly the lateral eyes. Light responsive neurons in the pars intermedia do not exhibit any detectable response to illumination of the pineal region. The latency of the response to light is consistent with the existence of a neural circuit linking the eyes with the pars intermedia, and studies of retinal projections in amphibians suggest several possible routes for a retino-pars intermedia pathway. The unique availability of the input and output stages of this retino-hypophyseal pathway may make it a useful model for the study of photo-neuroendocrine mechanisms.     

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.     

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.