Lectin cytochemistry of cell types in human and canine pituitary

Summary Labeled lectins specific for different sugars were employed to identify different cell types in pituitaries from six human autopsies and seven dogs. To determine the lectins bound by each cell type, fixed-paraffin embedded sections serial to those stained with lectins were immunostained for specific hormones and the serial pairs were examined in a comparison microscope. In human pituitaries corticotrophs stained selectively with lectins having affinity for -l-fucose and the core region of complex type N-glycosyl-proteins. Some corticotrophs also stained for the presence of terminal -galactose. Thyrotrophs stained selectively with a periodate oxidation-borohydride reduction-concanavalin A sequence. Some mammotrophs evidenced content of glycoconjugate with terminal -galactose. Dendritic cells stained selectively for abundant glycogen with the periodate-reduction-concanavalin A sequence and a lectin from Griffonia simplicifolia. Adenohypophyseal cells of dog pituitary differed in showing absence of terminal -galactose in corticotrophs, presence of terminal -galactose in thyrotrophs, presence of glycoconjugate with N-glycosidically bound oligosaccharide in thyrotrophs and gonadotrophs and presence of terminal -galactose with a different lectin affinity in mammotrophs. The main contributions of lectin histochemistry applied to the pituitary include providing an additional histologic method for identification of some cell types, and localizing glycosylated prohormone or other biochemically unrecognized non-hormone glycoconjugates whose function in pituitary cells remains to be explained.This research was supported by NIH Grants AM-10956 and HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant #79     

Glycoconjugate localization with lectin and PA-TCH-SP cytochemistry in rat hypophysis

Glycoconjugates were localized by light microscopy with lectin-peroxidase conjugates and by electron microscopy with the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence in immunocytochemically or morphologically identified cell types in rat pituitary. Lectin histochemistry demonstrated sialic acid and glycoconjugates with N-glycosidically linked oligosaccharides in gonadotrophs, thyrotrophs, and corticotrophs. Galactose penultimate to sialic acid was observed mostly in gonadotrophs. The terminal galactose-N-acetylgalactosamine disaccharide was detected in a few gonadotrophs and in a moderate number of mammotrophs. Fucose was localized in only corticotrophs with two fucose-binding lectins and in thyrotrophs with another. Several different monosaccharides were seen in glycoconjugates in melanotrophs and in Herring bodies. Melanotrophs displayed heterogeneous staining with fucose-binding lectins. A small number of nonsecretory cells were also visualized in the pars distalis by virtue of their glycogen content. PA-TCH-SP staining revealed complex carbohydrates in secretory granules and some Golgi cisternae in all types of hormone-producing cells in the pars distalis except for the somatotrophs. Melanotrophs of pars intermedia exhibited stained secretory granules and irregular dense bodies containing a stained meshwork. Corticotrophs of the pars distalis lacked the latter bodies, although they form the same glycoprotein precursor hormone as melanotrophs. Lectin conjugates and the PA-TCH-SP sequence stained some groups of secretion granules in Herring bodies, possibly representing vasopressin-containing granules as well as other cell types in the pars nervosa.     

Lectinhistochemical demonstration of galactose- and N-acetyl-D-galactosamine residues in cells of the rat anterior pituitary

Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. Beta-D-galactose and beta-N-acetyl-D-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.     

Unusual lectin-binding properties of a herpes simplex virus type 1-specific glycoprotein

Lysates from herpes simplex virus type 1-infected cells were subjected to affinity chromatography on soybean and Helix pomatia lectins. One of the virus-specified glycoproteins, probably the herpes simplex virus type 1-specific gC glycoprotein, bound to the lectins and was eluted with N-acetylgalactosamine. The affinity chromatography permitted a high degree of purification of the type-specific glycoprotein with respect to both host cell components and other viral glycoproteins. The lectin affinity pattern of this glycoprotein indicates the presence of a terminal alpha-N-acetylgalactosamine in an oligosaccharide, a finding not reported previously for glycoproteins of enveloped viruses.     

Glycoconjugate profiles of adrenocorticotropic and melanotropic cells in the pituitary of adult sea lampreys (Petromyzon marinus): a lectin histochemical study

In the lamprey, adrenocorticotropin (ACTH) and melanotropins (MSHs) are produced from two distinct precursors, proopiocortin (POC) and proopiomelanotropin (POM). Both POC and POM have been suggested to be glycoproteins. The present study aimed to demonstrate glycoconjugates in ACTH and MSH cells in the pituitary of adult sea lampreys (Petromyzon marinus) by means of a lectin histochemistry. A total of 19 kinds of lectins were tested. ACTH cells were distributed in both the rostral pars distalis and the proximal pars distalis, and were stained positively with N-acetylglucosamine binding lectins (i.e., succinylated wheat germ agglutinin), N-acetylgalactosamine binding lectins (i.e., soybean agglutinin), D-mannose binding lectins (i.e., Lens culinaris agglutinin), and D-galactose binding lectins (i.e., Erythrina cristagall lectin). MSH cells were distributed in the pars intermedia, and were stained with N-acetylgalactosamine binding lectins (i.e., Dolichos biflorus agglutinin), D-mannose binding lectin (Pisum sativum agglutinin) and D-galactose binding lectins (i.e., peanut agglutinin). These results suggested that ACTH and MSH cells produce different types of glycoconjugates which may be attributed to the difference in glycoconjugate moieties between the precursor proteins, POC and POM.     

Histochemical evaluation of secretory glycoproteins in human salivary glands with lectin-horseradish peroxidase conjugates

Paraffin sections of submandibular, sublingual, minor salivary, and parotid glands from ten human autopsy cases were stained with a battery of ten lectins conjugated to horseradish peroxidase. Variable affinity for one or another lectin between mucous cells in a gland evidenced cellular heterogeneity in mucin production. Mucous cells of a given type of gland varied among individuals, but for a single individual appeared markedly but not completely similar from one type of salivary gland to another. The individual variation related, in part, to the ABO blood group and secretor status of the individual. For mucous cells in secretors of blood group A and B all antigens stained strongly for the presence of terminal alpha-N-acetylgalactosamine or alpha-galactose, respectively. Mucous cells in AB secretors contained both antigens, whereas those of O (H) secretors lacked both. Mucous cells of three presumed nonsecretors, two of whom were immature infants and possibly too young to produce ABO antigen, failed to stain. Mucous cells in glands from the presumed nonsecretors, however, revealed a staining pattern consistent with the presence of Lea antigen. Mucous cells of nonsecretors stained with Lotus tetragonolobus agglutinin but not with Ulex europeus I agglutinin, whereas mucous cells of ABO secretors stained with both lectins. This difference in lectin binding indicated that sites reactive only with Lotus tetragonolobus agglutinin contain 1----4 linked fucosyl residues and sites stained by both lectins contain fucose linked 1----2 to the oligosaccharide. Staining of mucous cells of nonsecretors with Pisum sativum agglutinin indicate that either the lectin binds to internal N-acetylglucosamine of Lea substance or the mucous cells contain an N-glycosidic glycoprotein of the type thought to bind this lectin. Serous cells stained less strongly than mucous cells and differed in lectin affinities from one type of gland to another in an individual. Staining of serous cells of a given gland varied markedly among different subjects. This individual variability did not relate to blood group as terminal sugars demonstrative of A or B blood group antigens were not detected in any serous cells. Serous cells in the submandibular glands from the two immature infants were unreactive with all lectin conjugates. Secretions in parotid and submandibular serous cells generally contained a higher content of fucose than those in sublingual serous cells, which contained higher levels of a terminal galactose-sialic acid dimer. Some but not other cells of striated and interlobular ducts of submandibular glands of one subject stained for alpha-N-acetylgalactosamine.     

Fluorochrome-coupled lectins reveal distinct cellular domains in human epidermis

The distribution of saccharide moieties in human interfollicular epidermis was studied with fluorochrome-coupled lectins. In frozen sections Concanavalin A (Con A), Lens culinaris agglutinin (LCA), Ricinus communis agglutinin I (RCAI), and wheat germ agglutinin (WGA) stained intensively both dermis and viable epidermal cell layers, whereas peanut agglutinin (PNA) bound only to living epidermal cell layers. Ulex europaeus agglutinin I (UEAI) bound to dermal endothelial cells and upper cell layers of the epidermis but left the basal cell layer unstained. Dolichos biflorus agglutinin (DBA) bound only to basal epidermal cells, whereas both soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) showed strong binding to the spinous and granular cell layers. On routinely processed paraffin sections, a distinctly different staining pattern was seen with many lectins, and to reveal the binding of some lectins a pretreatment with protease was required. All keratin-positive cells in human epidermal cell suspensions, obtained with the suction blister method, bound PNA, whereas only a fraction of the keratinocytes bound either DBA or UEAI. Such a difference in lectin binding pattern was also seen in epidermal cell cultures both immediately after attachment and in organized cell colonies. This suggests that in addition to basal cells, more differentiated epidermal cells from the spinous cell layer are also able to adhere and spread in culture conditions. Gel electrophoretic analysis of the lectin-binding glycoproteins in detergent extracts of metabolically labeled primary keratinocyte cultures revealed that the lectins recognized both distinct and shared glycoproteins. A much different lectin binding pattern was seen in embryonic human skin: fetal epidermis did not show any binding of DBA, whereas UEAI showed diffuse binding to all cell layers but gave a bright staining of dermal endothelial cells. This was in contrast to staining results obtained with a monoclonal cytokeratin antibody, which showed the presence of a distinct basal cell layer in fetal epidermis also. The results indicate that expression of saccharide moieties in human epidermal keratinocytes is related to the stage of cellular differentiation, different cell layers expressing different terminal saccharide moieties. The results also suggest that the emergence of a mature cell surface glycoconjugate pattern in human epidermis is preceded by the acquisition of cell layer-specific, differential keratin expression.     

Topographical distribution of the gonadotrophs,mammotrophs, somatotrophs and thyrotrophs in the pituitary gland of the baboon (Papio cynocephalus)

The identification and regionalization of four pituitary parenchymal cell types, gonadotrophs, mammotrophs, somatotrophs and thyrotrophs, were studied in the baboon (Papio cynocephalus) hypophysis using immunocytochemistry. The gonadotrophs were homogeneously distributed throughout the entire pars distalis. Both mammotrophs and somatotrophs predominate at the superior and inferior poles of the organ. The medial and anteromedial regions are populated by mammotrophs and thyrotrophs, while the lateral and posterior portions of the pars distalis contain large numbers of somatotrophs.     

Lectinhistochemical demonstration of galactose- and N-acetyl-d-galactosamine residues in cells of the rat anterior pituitary

Summary Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. -d-galactose and -N-acetyl-d-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Characterization of the glycoconjugates of boar testis and epididymis

Lectin histochemistry was used to perform in situ characterization of the glycoconjugates present in boar testis and epididymis. Thirteen horseradish peroxidase- or digoxigenin-labelled lectins were used in samples obtained from healthy fertile boars. The acrosomes of the spermatids were stained intensely by lectins with affinity for galactose and N-acetyl-galactosamine residues, these being soybean, peanut and Ricinus communis agglutinins. Sertoli cells were stained selectively by Maackia ammurensis agglutinin. The lamina propria of seminiferous tubules showed the most intense staining with fucose-binding lectins. The Golgi area and the apical part of the principal cells of the epididymis were stained intensely with many lectins and their distribution was similar in the three zones of the epididymis. On the basis of lectin affinity, both testis and epididymis appear to have N- and O-linked glycoconjugates. Spermatozoa from different epididymal regions showed different expression of terminal galactose and N-acetyl-galactosamine. Sialic acid (specifically alpha2,3 neuraminic-5 acid) was probably incorporated into spermatozoa along the extratesticular ducts. These findings indicate that the development and maturation of boar spermatozoa are accompanied by changes in glycoconjugates. As some lectins stain cellular or extracellular compartments specifically, these lectins could be useful markers in histopathological evaluation of diseases of boar testis and epididymis.     

Human pituitary corticotrophs: their amphophilic stainability and immunohistochemical characterization

Immunohistochemical characterization of the human pituitary beta(R) cells was investigated through the findings of the immunoreactivities with anti-porcine ACTH, -rat TSH, -rat FSH sera. Immunostained corticotrophs are oval or round in shape and localized in the anteromedial wedge. It is shown on the adjacent sections that they correspond to the beta(R) cells with amphophilic stainability with PAS-iron hematoxylin. In this wedge, amphophilic cells are preponderant, but PAS-positive thyrotrophs and gonadotrophs are not numerous. Amphophilic stainability varies in degree from cell to cell: One cell contains numerous medium-size of secretory granules weakly stained with iron hematoxylin and strongly with PAS in the PAS-positive cytoplasm, and the other cell is filled with big secretory granules intensively stained with iron hematoxylin and weakly with PAS. The immunostained TSH, LH and FSH cells are different from the beta(R) corticotrophs, because anti-ACTH serum never reacts to the TSH, LH and FSH cells in the two adjacent sections. LH and FSH reactivities are observed in the single cells. It is concluded that human corticotrophs are amphophilic beta(R) cells filled with secretory granules, and that they have quite a different appearance from the rat chromophobic stellate corticotrophs with a row arrangement of secretory granules along the plasma membrane.     

Nuclear and cytoplasmic lectin receptor sites in rat Py1a osteoblasts

The intracellular distribution of lectin receptor sites was studied in the rat Pyla osteoblasts using immunofluorescence at the confocal microscopy level. This immortalized cell line was found to represent a satisfactory model to study the occurrence and distribution of sugar moieties. Our data showed distinct affinity patterns of lectins recognizing different terminal or internal sugar residues. For some lectins, the binding patterns appeared to be cell cycle-independent, whereas for PNA the cell cycle greatly influenced the nuclear binding. By combining lectin affinity with sialidase degradation and alcoholic saponification the sialic acid acceptor sugars and derivatives were also visualized. In particular, glycoconjugates with sialic acids linked to beta-galactose, and mainly C4 acetylated, were located in the cytoplasm, while glycoconjugates characterized by sialic acids linked to alpha-N-acetylgalactosamine, and devoid of acetyl groups at C4, were almost exclusively found in the nucleus. The comparison of lectin affinities, with and without prior glycosidase digestions, allowed us to gain further insight into the chemical composition of glycoconjugates that act as the lectin receptor sites that appeared to belong to O- and N-linked glycoconjugates. The use of additional enzymatic treatments were useful to better establish the localization of nuclear receptor sites and results were compared with previous studies about endogenous and exogenous lectins in an attempt to reconcile the association of lectins and sugars within the nucleus and their possible involvement in modulation of cell proliferation and/or response to chemical signals. The above digestions also provided information about the cytoplasmic binding patterns.     

An in vitro model for studies of intercellular communication in cultured rat anterior pituitary cells

The formation of intimate associations among different hormone-secreting cells within the rat adenohypophysis may serve as a possible site for physiologic regulation. In this report we describe a high density plating method which enables us to study cell-to-cell interactions within anterior pituitary cell cultures. Trypsin-dispersed pituitary cell suspensions attach rapidly (within 6 hr) and quantitatively (95-97%) to glass or plastic surfaces when plated in medium containing microM calcium concentrations (pH 7.6-7.8). Freshly plated cell suspensions obtained from female pituitary glands contained subpopulations of mammotrophs 49.3%, somatotrophs 30.3%, gonadotrophs 12.6%, corticotrophs 3.4% and thyrotrophs 1.5%. Epithelial cell colonies were formed during a 3-day culture period as the cells flattened and re-established contacts with neighboring cells. Freeze-fracture electron microscopic analysis of these colonies produced morphological evidence for direct intercellular contacts among the hormone-secreting cells. Large areas of tight junctions and small gap junctions were identified on the membranes of the epithelial cells within these colonies. Cells which contained tight junctions usually contained microvilli and morphological signs of active hormone secretion. Small junctional plaques containing tightly packed intramembrane particles were also occasionally found on the membranes of cells which were actively secreting pituitary hormones. The high density plating procedure which is described in this report provides greater opportunity for cell-cell interaction and thus may prove to be a useful model for evaluating the role of intercellular communication within this tissue.     

Aminoglutethimide-stimulated corticotrophs

Thirty-six rats of both sexes in two groups were treated with aminoglutethimide (AG), a steroid synthesis inhibitor, for 1 and 8 weeks respectively. The anterior pituitaries were investigated by light and electron microscopy, the techniques used including immunocytochemical and morphometric methods. Corticotrophs were identified by the avidinbiotin-peroxidase complex technique at the light and electron microscopic levels. AG administration resulted in hyperplasia of ACTH-containing cells. The increase in the volume density of corticotrophs was prevented by simultaneous medication with 3 mg corticosterone in male rats, whereas in female rats a larger dose of corticosterone was required to suppress pituitary corticotroph hyperplasia. Electron microscopy revealed that in AG-treated rats, corticotrophs were larger and contained more secretory granules than those in controls, the mean secretory granule diameter increasing from 100 nm to 165 nm. Under AG stimulation, corticotrophs showed considerable variations in structural features probably reflecting differences in their functional state. It was apparent that only one ACTH-producing cell type existed in the pars distalis of the rat adenohypophysis, although this cell type may undergo substantial morphologic alterations due to changes in endocrine activity. Gonadotrophs and thyrotrophs showed morphologic signs of stimulation and exhibited hyperplasia following AG treatment indicating that the effect of the drug was not restricted to corticotrophs.     

Dopamine receptor sites in the anterior pituitary.

An immunocytochemical method was developed to visualize dopamine receptor sites on dispersed anterior pituitary cells of the rat. Dopamine receptors were labeled with the antagonist haloperidol. Some cells were incubated with haloperidol and a 100-fold excess of the potent antagonist D-butaclamol to determine nonspecific binding. The labeled sites were stained with an antibody against haloperidol and the peroxidase anti-peroxidase (PAP) technique. PAP complexes which served as markers for dopamine binding sites appeared on the outer plasmalemmal surface of the vast majority of mammotrophs. PAP complexes attached to the inner surface of endocytotic vesicle membrane suggested internalization of receptor-rich portions of the plasmalemma. Some gonadotrophs and somatotrophs were specifically stained to a lesser extent. However, high receptor site density and internalization of PAP complexes were never observed on cell types other than mammotrophs. The presence of dopamine receptors on the plasmalemma of mammotrophs provides strong additional evidence that dopamine acts upon these cells as a prolactin inhibitory hormone.     

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-microns serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.     

Gene expression patterns of pro-opiomelanocortin-processing enzymes PC1 and PC2 during postnatal development of rat corticotrophs.

We examined the expression and localization of the prohormone convertases, PC1 and PC2, in the anterior pituitary cells of developing rats by a double staining procedure using in situ RT-PCR and an immunofluorescence technique. In the adult, both PC1 mRNA and PC2 mRNA were expressed in corticotrophs, gonadotrophs, thyrotrophs, and mammotrophs. These cells, except for corticotrophs, had previously been considered to be ones in which proprotein processing does not take place, but both PC1 and PC2 may be necessary to process other proteins, such as granin family proteins, having proteolytic cleavage sites and located in secretory granules of the above trophs. In addition, no PC1 or PC2 mRNA was expressed in somatotrophs, which is consistent with the fact that somatotrophs do not contain these granins. In addition, 7B2 mRNA was expressed in these PC2-positive trophs, suggesting that there is a functional relationship between PC2 and 7B2 proteins. We found that alpha-MSH was expressed in the corticotrophs of the postnatal rat and that the number of alpha-MSH-immunopositive corticotrophs decreased as development proceeded. Because the changes in the pattern of POMC processing are considered to depend on the relative expression levels of PC1 and PC2, PC1 and PC2 mRNAs were examined in corticotrophs during postnatal development. We found a decrease in the number of PC2 mRNA-positive cells, which coincided with one in the number of alpha-MSH-immunopositive corticotrophs, as postnatal development proceeded. Our present data demonstrate that the alpha-MSH production varies directly in accordance with the expression of PC2. We also discuss the possible significance of alpha-MSH production during the postnatal period.     

Galanin and vasoactive intestinal polypeptide are colocalised with classical pituitary hormones and show plasticity of expression

Summary The identity of galanin- and vasoactive intestinal polypeptide-(VIP) immunoreactive (IR) cells in the rat anterior pituitary was investigated using immunocytochemistry and, since levels of both peptides are stimulated by oestrogen, the effect of oestrogen treatment and gonadectomy on the expression of both peptides was examined. In normal male rats, few galanin-IR and very few VIP-IR cells were found. Colocalisation studies performed on 2-m serial paraffin sections revealed that in these animals galanin IR was present in somatotrophs and thyrotrophs. In normal females in dioestrus many lactotrophs, in addition to somatotrophs and thyrotrophs, expressed galanin, but very few VIP-IR cells were seen. In cryostat sections of normal rat pituitaries, slightly more VIP-IR cells were present. Oestrogen treatment in females produced an increase in frequency of galanin-IR cells, the vast majority of which were lactotrophs, and more VIP-IR cells, identified as lactotrophs, also appeared. VIP was present in a subset of galanin-IR lactotrophs after oestrogen treatment. After ovariectomy female pituitaries resembled those of normal males, with few galanin positive cells none of which were lactotrophs, and hardly any VIP-IR cells. Thus these two peptides are present in specific endocrine cell types of rat anterior pituitary and display plasticity of expression in different cell types under the influence of oestrogen. Their roles in control of pituitary hormone secretion are supported by these findings, and it is possible that both peptides act in a paracrine fashion within the pituitary.