Immunohistochemically detected ontogeny of prolactin and growth hormone cells in the African catfish Clarias gariepinus

The chronological appearance of selected endocrine cells in the pituitary of African catfish Clarias gariepinus (Burchell, 1822) was studied morphologically, histologically and immunohistochemically by using antisera raised against catfish growth hormone (cgGH) and recombinant tilapia prolactin I (tPRL). cgGH- and tPRL-like immunoreactive cells were visible from day 1 post fertilisation (hatching) throughout the juvenile and the adult stage. From 1 to 90 days after hatching, the larval pituitary is oval in shape with a distinctly shaped rostral pars distalis, proximal pars distalis and pars intermedia. From day 120 onwards allometric growth of the rostral and proximal pars distalis extended the prolactin and growth hormone cells anteriorily and posteriorily, respectively. Size and activity of the prolactin and growth hormone cells, measured by the ratio of cell surface to nuclear surface remained constant until day 40 and showed a growth spurt thereafter. Growth hormone content, measured with a catfish-specific radio-immunoassay from hatching until 60 h post hatching, increased exponentially between 30 and 60 h.     

Immunocytochemical studies on the pituitary pars distalis of the Japanese long-fingered bat,Miniopterus schreibersii fuliginosus

Summary Immunocytochemical studies were performed to describe the characteristics of cell types and their distribution in the pars distalis of Japanese long-fingered bat, Miniopterus schreibersii fuliginosus, collected at various stages of the reproductive cycle. Six distinct cell types have been identified in the pars distalis by the unlabeled immunoperoxidase technique and by the ABC method. Growth hormone (GH) and prolactin (PRL) cells were immunostained with antisera against chicken GH and ovine PRL. The GH-immunoreactive cells were round or oval orangeophilic cells distributed throughout the pars distalis with prominent aggregation in the posterolateral region. The PRL cells were pleomorphic carminophilic cells that occurred in small groups within the central and dorsocaudal regions of the pars distalis. They were sparsely distributed in the central region of the pars distalis in the hibernating bats, but increased significantly in the pregnant and lactating bats. The adrenocorticotropic (ACTH) cells were large round or polygonal amphophilic cells in the rostroventral and ventrolateral regions of the pars distalis. The thyrotropic (TSH) cells were small rounded or polygonal and distributed mainly in the ventrolateral region of the pars distalis. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) cells were identified immunocytochemically with antisera against the specific beta subunits of ovine LH and rat FSH. There were two populations of LH and FSH cells, one aggregated in the zona tuberalis and the other scattered singly throughout the rest of the pars distalis. The aggregated cells were immunoreactive with both antisera directed to LH and FSH, while scattered cells were reactive solely with antiserum to either LH or FSH and exhibited seasonal variations. In females, the proportional volume of the pars distalis occupied by LH cells was significantly reduced during pregnancy and lactation. No evidence of involution was observed in pars distalis cells except for PRL cells in males or females during hibernation.     

Peptides with NH2-terminal tryptophan in adrenocorticotrophic hormone and melanocyte-stimulating hormone granules of adenohypophysis.

Fluorescence microscopy has demonstrated formaldehyde-ozone-induced fluorescence in the pars intermedia cells (melanocyte-stimulating hormone cells) and in certain cells of the pars distalis of the mammalian pituitary. From histochemical and chemical evidence the fluorescence is believed to reflect the presence of peptides with NH2-terminal tryptophan. In the pars distalis of hamster, cat and pig pituitary, the cells that exhibit formaldehyde-ozone-induced fluorescence have now been identified as adrenocorticotrophic hormone (ACTH) cells by immunohistochemistry. Granules from pig pituitaries were purified by passage through a succession of Millipore filters followed by centrifugation on a continuous sucrose gradient. Two granular fractions were identified by electron microscopy and found to contain high concentrations of peptides with NH2-terminal tryptophan as well as high ACTH bioactivity. These fractions, when pelleted and analyzed histochemically, displayed formaldehyde-ozone-induced fluorescence and ACTH-like immunoreactivity.     

Immunohistochemical Localization of Growth Hormone and Prolactin in the Pituitary Gland of Acipenser güldenstaedti Brandt (Chondrostei)

Abstract Prolactin (LTH) and growth hormone (GH) containing cells in A. güldenstaedti have been localized by means of anti-ovine prolactin and anti-bovine growth hormone respectively, coupled indirectly to peroxidase, and localized histochemically with hydrogen peroxide as substrate and 3,3′-diaminobenzidine as capturing agent. The distribution of the anti-prolactin positive cells has been demonstrated and correlated histologically with the acidophilic cells both in the rostral and proximal pars distalis. This cell type is elongated and arranged in follicles in the rostral pars distalis; in the proximal pars distalis they are smaller and oval, without any special orientation. Neither of the other cell types which are scattered among these acidophils contain prolactin. The anti-bovine growth hormone positive cells are evenly distributed in the proximal pars distalis above the hypophysial cleft, and some are also found in the pars intermedia. The anti-GH positive cells have been correlated histologically with the amphiphilic cells in the proximal pars distalis. These cells are arranged in cell cords in close contact with the secondary capillary plexus, near its origin from the primary capillary plexus covering the median eminence.     

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken

 The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large α-subunit and small β-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis. Accepted: 26 August 1997     

CXCL14-like immunoreactivity in growth hormone-containing cells of urodele pituitaries

Immunohistochemical techniques were employed to investigate the distribution of a chemokine, namely, CXCL14-like immunoreactivity in the axolotl (Ambystoma mexicanum) and Japanese black salamander (Hynobius nigrescens) pituitaries. CXCL14-immunoreactive cells concentrated at an area of the pars distalis adjacent to the pars intermedia. We found that these cells correspond to the cells immunoreactive to an antibody against rat growth hormone (GH). Immunoelectron microscopy indicated that the CXCL14-like substance and GH coexisted on the secretory granules in the axolotl pituitary. Western blot analysis of axolotl pituitary extracts revealed the anti-human CXCL14 antibody labeled an approximately 16.6-kDa band that was not labeled by the anti-GH antibody. The CXCL14-like substance in the pars distalis may participate in GH functions in these species.     

Orexin-B-like immunoreactivity localizes in both luteinizing-hormone-containing cells and melanin-concentrating hormone-containing fibers in the red-bellied piranha (Pygocentrus nattereri) pituitary

We examined orexin-like immunoreactivity in the pituitary of the red-bellied piranha (Pygocentrus nattereri). Orexin-B-immunoreactive (IR) cells corresponded to luteinizing hormone (LH)-containing cells in the pars distalis, and orexin-B-IR fibers corresponded to melanin-concentrating hormone (MCH)-containing fibers in the pars nervosa. In the pars distalis, orexin-B-IR puncta that were also immunoreactive for MCH were observed around the orexin-B-IR cells. In the ventral hypothalamus, orexin-B-IR and MCH-IR neurons were found in the nucleus lateralis tuberis. Immunoelectron-microscopic analysis revealed that the orexin-B-like substance co-localized with LH in secretory granules and with MCH in MCH-containing neurons. Some of the MCH secreted in the pituitary might participate in the modulation of LH secretion from the gonadotrophs, together with orexin-B, leading to food intake by the stimulation of growth hormone secretion from the somatotrophs.     

Expression of mRNA for growth hormone-releasing hormone and splice variants of GHRH receptors in human malignant bone tumors

Splice variants (SV) of receptors for growth hormone-releasing hormone (GHRH) have been found in several human cancer cell lines. GHRH antagonists inhibit growth of various human cancers, including osteosarcomas and Ewing's sarcoma, xenografted into nude mice or cultured in vitro and their antiproliferative action could be mediated, in part, through these SV of GHRH receptors. In this study, we found mRNA for the SV(1) isoform of GHRH receptors in human osteosarcoma line MNNG/HOS and SK-ES-1 Ewing's sarcoma line. We also detected mRNA for GHRH, which is apparently translated into the GHRH peptide and secreted by the cells, as shown by the presence of GHRH-like immunoreactivity in the conditioned media of cell cultures. In proliferation studies in vitro, the growth of SK-ES-1 and MNNG/HOS cells was dose-dependently inhibited by GHRH antagonist JV-1-38 and an antiserum against human GHRH. Our study indicates the presence of an autocrine stimulatory loop based on GHRH and SV(1) of GHRH receptors in human sarcomas. The direct antiproliferative effects of GHRH antagonists on malignant bone tumors appear to be exerted through the SV(1) of GHRH receptors on tumoral cells.     

Growth hormone regulation of growth hormone-releasing hormone gene expression

Slot-blot hybridization technique was used to evaluate growth hormone-releasing hormone (GHRH) mRNA levels in the hypothalamus of long-term (14 days) hypophysectomized (HPX) rats treated or not with 125 micrograms hGH/rat, twice daily IP, since the first day postsurgery. In addition, mRNA levels were determined in the hypothalamus of short-term (4 days) GH-treated (250 micrograms hGH/rat, twice daily IP) intact rats. GHRH mRNA levels were increased in HPX rats, and GH treatment partially counteracted this rise. Short-term administration of GH decreased GHRH mRNA levels in intact rats. These results, evaluated together with previous findings showing decreased hypothalamic GHRH-like immunoreactivity in both HPX rats and intact rats given GH (6, 7, 9), indicate that GH exerts a negative feedback action on the synthesis and release of GHRH.     

Quantitative aspects of growth hormone cell maturation in the normal and little mutant mouse

Growth hormone (GH) cells were analyzed by means of ultrastructural morphometry in the pars distalis of pituitary glands from male adult and immature normal (C57BL) and homozygous little (lit/lit) mutant mice. Thin sections were exposed to anti-GH serum and processed immunocytochemically with the colloidal-gold technique. In the pars distalis of adult lit/lit mice, the mean volume density of GH cells/total tissue was 24% of the normal value, granules/GH cells was 58% of normal, and granules/total tissue was only 12% of normal. Deficits in all of these parameters likewise occurred in immature glands, though to a lesser extent than in the adults. The results indicate that the GH deficiency in this mutant reflects quantitative deficits in both the secretory granule content of GH cells, as well as the GH cell content of the gland, with the latter being the more severely affected.     

Immunocytochemistry of somatotrophs,gonadotrophs, prolactin and adrenocorticotropin cells in larval sea bream (Sparus auratus) pituitaries

Summary The chronological appearance of endocrine cells in the pituitary of sea-bream (Sparus auratus) larvae was studied using antisera against salmon prolactin, trout growth hormone, salmon gonadotropin and N-terminal human adrenocorticotropin. The larval pituitary (1–12 days after hatching) was oval in shape and was composed of a dense mass of cells with few neurohypophysial fibres. By 60 days after hatching it began to resemble the adult and was divisible into a distinct rostral pars distalis containing prolactin and adrenocorticotropin cells; a proximal pars distalis containing somatotrophs and gonadotrophs and a pars intermedia. Cells immunoreactive with antisera against growth hormone were observed immediately after hatching (2 days post-fertilization). Weakly staining prolactin cells were observed 2 days later in the region corresponding to the rostral pars distalis. Cells immunoreactive with anti-gonadotropin and anti-adrenocorticotropin sera were observed in the pituitary 6 and 8 days after hatching, respectively. All the cell-types studied were immunoreactive from the time they were first identified until the final samples 90 days after hatching.     

Immunocytochemical effects of thyroxine stimulation on the adenohypophysis of dwarf (dw) mutant mice

The effects of dietary thyroxine on the immunoreactivity of cells in the pars distalis of the adenohypophysis in dwarf (dw/dw) mice were determined by ultrastructural immunocytochemistry. In nontreated dwarfs only adrenocorticotropic hormone (ACTH) cells and luteinizing hormone (LH) cells showed positive reactions to their respective antibodies, whereas no cells showed immunoreactivity to antibodies to growth hormone (GH), thyroid-stimulating hormone (TSH), or prolactin (Prl). In dwarfs supplemented postnatally with dietary thyroxine for 9 wks, the treatment failed to produced immunoreactive GH, TSH or Prl cells. However, LH cells became more prominent and fully developed, with denser concentrations of immunoreactive particles overlying the secretory granules than occurred in nontreated dwarfs. In thyroxine-treated dwarfs, ACTH cells were similar in ultrastructural features and immunoreactivity to those in nontreated dwarfs.     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA)     

Age-dependent changes of ultrastructure and thyroid-stimulating hormone immunoreactivity in the pars tuberalis of the rat

The pars tuberalis of the rat adenohypophysis was investigated by immunohistochemistry and electron microscopy at different stages of the peri- and postnatal development. A characteristic pattern of changes in thyroid-stimulating hormone immunoreactivity of pars tuberalis-specific secretory cells was observed with an increase in staining intensity after birth, a marked reduction in adulthood and a subsequent increase in senium. Electron microscopy showed age-dependent changes in the number of dictyosomes per cell, in the number of large lysosomes per area of cytoplasm and in the extension of the granular endoplasmic reticulum. The number of secretory granules per area of cytoplasm was maximal perinatally; there was no correlation between granule content and immunoreactivity. Thyrotropes of the pars distalis did not show comparable immunohistochemical or ultrastructural changes.     

A systematic immunohistochemical survey of the distribution patterns of GH, prolactin, somatolactin, β–TSH, β–FSH, β–LH, ACTH, and α–MSH in the adenohypophysis of Oreochromis niloticus, the Nile tilapia

Fish pituitary plays a central role in the control of growth, development, reproduction and adaptation to the environment. Several types of hormone-secreting adenohypophyseal cells have been characterised and localised in diverse teleost species. The results suggest a similar distribution pattern among the species investigated. However, most studies deal with a single hormone or hormone family. Thus, we studied adjacent sections of the pituitary of Oreochromis niloticus, the tilapia, by conventional staining and immunohistochemistry with specific antisera directed against growth hormone (GH), prolactin (PRL), somatolactin (SL), thyrotropin (beta-TSH), follicle-stimulating hormone (beta-FSH), luteinising hormone (beta-LH), adrenocorticotropic hormone (ACTH) and melanocyte-stimulating hormone (alpha-MSH). The pituitary was characterised by a close interdigitating neighbourhood of neurohypophysis (PN) and adenohypophysis. PRL-immunoreactive and ACTH-immunoreactive cells were detected in the rostral pars distalis. GH-immunoreactive cells were present in the proximal pars distalis (PPD). A small region of the PPD contained beta-TSH-immunoreactive cells, and beta-LH-immunoreactive cells covered approximately the remaining parts. Centrally, beta-FSH-immunoreactive cells were detected in the vicinity of the GH-containing cells. Some of these cells also displayed beta-LH immunoreactivity. The pars intermedia was characterised by branches of the PN surrounded by SL-containing and alpha-MSH-immunoreactive cells. The ACTH and alpha-MSH antisera were observed to cross-react with the respective antigens. This cross-reactivity was abolished by pre-absorption. We present a complete map of the distinct localisation sites for the classical pituitary hormones, thereby providing a solid basis for future research on teleost pituitary.     

Bioactivity of growth hormone releasing hormone (1-29) analogues after SC injection in man

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA) This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunoreactivity to gonadotropin-releasing hormone and gonadotropic hormone in the brain and pituitary of the rainbow trout Salmo gairdneri

Summary Immunoreactivity to gonadotropin-releasing hormone (GnRH) and gonadotropic hormone (GTH) was studied at the light-microscopical level in the brain and pituitary of rainbow trout at different stages of the first reproductive cycle using antisera against synthetic mammalian GnRH and salmon GTH. GnRH perikarya were localized exclusively in the preoptic nucleus, both in the pars parvicellularis and the pars magnocellularis. A few somata contacted the cerebrospinal fluid. Not all neurosecretory cells were GnRH-positive, indicating at least a bifunctionality of the preoptic nucleus. We recorded no differences between sexes or stages of gonadal development in the location of GnRH perikarya, whereas gradual changes were found in staining intensity during the reproductive cycle. GnRH fibres ran from the partes parvicellularis and magnocellularis through the hypothalamus and merged into a common tract at the transverse commissure before entering the pituitary. In the pituitary, GnRH was localized in the neural tissue of the neurointermediate lobe and, to a lesser extent, in the neural protrusions penetrating the proximal pars distalis. The bulk of GTH-positive cells was situated in the proximal pars distalis. Some cells were found more rostrally amidst prolactin cells or in the neurointermediate lobe. Only a limited number of GTH cells appeared to be in close contact with GnRH-positive material.     

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.     

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.