ARTICLE |
Correspondence to: Tsuyoshi Watanabe, Dept. of Anatomy II, Asahikawa Medical College, Midorigaoka-higashi 2-1-1-1, 078-8510 Asahikawa, Japan. E-mail: tyshwata@asahikawa-med.ac.jp
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Summary |
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Secretogranin III (SgIII) is one of the acidic secretory proteins, designated as granins, which are specifically expressed in neuronal and endocrine cells. To clarify its precise distribution in the anterior lobe of the rat pituitary gland, we raised a polyclonal antiserum against rat SgIII for immunocytochemical analyses. By immunohistochemistry using semithin sections, positive signals for SgIII were detected intensely in mammotropes and thyrotropes, moderately in gonadotropes and corticotropes, but not in somatotropes. The distribution pattern of SgIII in the pituitary gland was similar to that of chromogranin B (CgB), also of the granin protein family, suggesting that the expressions of these two granins are regulated by common mechanisms. The localization of SgIII in endocrine cells was confirmed by immunoelectron microscopy. In particular, secretory granules of mammotropes and thyrotropes were densely and preferentially co-labeled for SgIII and CgB in their periphery. Moreover, positive signals for SgIII were occasionally found in cells containing both prolactin and TSH in secretory granules. These lines of evidence suggest that SgIII and CgB are closely associated with the secretory granule membrane and that this membrane association might contribute to gathering and anchoring of other soluble constituents to the secretory granule membrane. (J Histochem Cytochem 51:227238, 2003)
Key Words: secretogranins, chromogranins, anterior pituitary, mammotropes, thyrotropes, immunocytochemistry, rats, secretory granules
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Introduction |
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A FAMILY of acidic secretory proteins, known as granins, is widely expressed in endocrine and neuronal cells, where these proteins are sequestered in secretory granules with peptide hormones and/or bioactive amines (for reviews see
In addition to these major members of the family, at least two other acidic secretory proteins, 7B2 and secretogranin III (SgIII), are also regarded as granins. 7B2 was originally identified as a pituitary-specific protein (
In contrast to 7B2, biochemical properties and the tissue distribution of SgIII have not been fully elucidated. SgIII was first identified as a product of a neuron-specific gene 1B1075 of mouse and, on the basis of its biochemical properties, it was later renamed SgIII (
Five types of endocrine cells are present in the anterior lobes of mammalian pituitary glands: corticotropes, gonadotropes, thyrotropes, mammotropes, and somatotropes. Each endocrine cell possesses characteristic secretory granules and secretes distinct peptide hormones in response to appropriate stimuli. Further understanding of the precise localization of SgIII in these endocrine cells of the anterior pituitary would provide us with important information on its putative function in endocrine cells. Moreover, we recently identified SgIII as a physiological binding protein for CgA by yeast two-hybrid screens, and demonstrated that the deletion of a SgIII-binding domain of CgA results in missorting of CgA into a constitutive secretory pathway in endocrine-derived AtT-20 cells (
The present report describes the immunocytochemical localization of SgIII and other granins in the anterior lobe of the male rat pituitary gland. For this purpose we raised an antiserum against a recombinant protein containing a partial amino acid sequence of rat SgIII. The results showed that SgIII was distributed in anterior pituitary endocrine cells, except for somatotropes, and particularly in mammotropes and thyrotropes. Interestingly, its localization in these endocrine cells was limited to the peripheral region of secretory granules together with CgB, suggesting a role in the organization of secretory granules. We also discuss the relationship between granin expression and the cell lineage of endocrine cells in the anterior lobe of the pituitary gland, by comparing the localization of SgIII with those of other granins.
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Materials and Methods |
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Antibodies
A rabbit polyclonal anti-SgIII antiserum (SgIII-C#6) was raised against a GST-fusion protein containing the C-terminal sequence of rat SgIII (residues 373491). The antigen emulsified with an equal volume of TiterMax (CytRx; Norcross, GA) was injected SC to rabbits six times at 2-week intervals. The rabbits were bled 7 days after the sixth injection, and specificities of the antisera were checked with immunoblotting analyses (see Results).
Rabbit polyclonal anti-CgA (CgA-C#101), anti-CgB (CgB-C#4), and anti-SgII (SgII-C#23) antisera were raised against synthetic peptides containing C-terminal sequences of rat CgA (amino acid residues 430442; AIEAELEKVA-HQL), CgB (residues 602618; YDSEEQMGPHQEAEDEK), and SgII (residues 568583; YLNQEQAEQGREHLAK), respectively. These peptides were conjugated to keyhole limpet hemocyanin (Sigma Chemical; St Louis, MO), emulsified with TiterMax, and injected SC to rabbits as described above. The specificities of the antisera obtained were also checked with immunoblotting analyses (see Results).
To identify the various endocrine cell types in the anterior pituitary, the following antibodies and antisera were used: anti-ACTH [rabbit polyclonal (code AB902), Chemicon, Temecula, CA]; anti-human LHß [mouse monoclonal (code O374), Immunotech, Marseille, France]; anti-TSHß [rabbit polyclonal; generated by Dr. Wakabayashi (Gunma University, Japan)]; anti-prolactin [mouse monoclonal (code 12205), QED Bioscience, San Diego, CA]; and anti-GH [rabbit polyclonal (code AB940), Chemicon].
Immunoblotting
For immunoblotting analyses, three male Wistar rats were used. The pituitaries and adrenal glands obtained from each rat were individually homogenized with 1 ml of 0.01 M Hepes/NaOH (pH 7.4) containing 0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 10 µg/ml leupeptin (Peptide Institute; Osaka, Japan), 10 µg/ml pepstatin (Peptide Institute), and 0.4 mM p-amidino-phenyl methanesulfonyl fluoride (WAKO Pure Chemicals; Osaka, Japan). Homogenized samples from each rat were individually stored on ice for 1 hr and centrifuged at 10,000 x g for 20 min at 4C to remove debris.
The extracts from each rat were loaded onto 10% SDS-PAGE under reducing conditions, and transferred to nitrocellulose membranes according to
Tissue Preparation
Sixteen male Wistar rats were purchased at 8 weeks of age and then housed for 2 weeks in a well-ventilated room (temperature 23 ± 1C; relative humidity 5565%) with food and water ad libitum. The lighting condition was defined as a 12:12-hr darklight cycle (lights on 07001900 hr).
For light microscopy, eight rats were sacrificed by cervical dislocation under light ether anesthesia. The pituitaries were quickly removed, cut into small pieces, and processed according to
For immunoelectron microscopy, eight rats were anesthetized with pentobarbital (25 mg/kg, IP) and perfused with physiological saline, followed by a solution of 0.1% glutaraldehyde4% p-formaldehyde in 0.1 M phosphate buffer (pH 7.2). After fixation by perfusion, pituitaries were quickly excised, cut into small pieces, and immersed in the same fixative at 4C for 2 hr. After washing thoroughly with 0.1 M phosphate buffer, pH 7.2, containing 7.5% sucrose, samples were dehydrated with 70% ethanol (1 hr, 4C) and infiltrated into pure LR White resin (London Resin; Basingstoke, Hampshire, UK) at 4C for 12 hr. The pituitaries were then placed at the bottom of gelatin capsules filled with fresh LR White resin and polymerized at 55C for 24 hr.
Immunocytochemistry
For light microscopic immunocytochemistry, after removal of the resin by sodium methoxide (
For immunoelectron microscopy, immunogold labeling was performed as described previously (
To semiquantitatively evaluate the subgranular localization of each granin in mammotropes and thyrotropes, cytoplasmic areas of these cells were randomly photographed at a magnification of x12,000 (10 micrographs per each type of endocrine cells) after double immunostaining for granins (SgIII, CgB, or SgII) and hormones (PRL or TSH). The profile area of each secretory granule was divided into two regions: peripheral and central. The peripheral region was defined as the outer 30% of the granule's diameter, and the central region was defined as the rest of the granule matrix in the center. The number of immunogold particles on each region of secretory granules was separately counted, and the percentages of the immunogold particles within peripheral and central regions were calculated. In addition, the distances of the immunogold particles indicative of SgIII, CgB, SgII, and PRL from the limiting membrane of the secretory granule were measured on the profile of mature mammotropes with NIH Image software (written by W. Rasband; National Institutes of Health, Bethesda, MD), and the results were statistically analyzed with KaleidaGraph software.
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Results |
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Characterization of Antisera Against Rat SgIII and Other Granins
Because the anterior and intermediate lobes of pituitary glands and the medulla of adrenal glands are known major storage sites for granins, the specificities of antisera used in the present study were examined by immunoblotting analyses of extracts of rat pituitary and adrenal glands. As shown in Fig 1A, the antiserum against a GSTSgIII fusion protein, including amino acid residues 373491 of rat SgIII, properly recognized the native form of SgIII (ca. 60 kD) in the extracts of pituitary glands. The immunoreactivity for SgIII was not detectable in the extract of adrenal glands, as reported previously (
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Immunocytochemical Localization of SgIII in the Anterior Lobe of Male Rat Pituitary Glands
As shown in Fig 2, the majority of endocrine cells in the anterior pituitary were immunoreactive for SgIII. To compare the localization pattern of SgIII in the anterior pituitary, immunostaining of other granins was performed. Among these staining patterns, the localization pattern of SgIII in these cells resembled that of CgB (Fig 2A and Fig 2C).
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By immunostaining of adjacent sections with antisera against various pituitary hormones, four types of pituitary endocrine cells, corticotropes, gonadotropes, mammotropes, and thyrotropes, were identified as containing SgIII (Fig 3). The intensities of immunoreactivity for SgIII were relatively high in mammotropes and thyrotropes, moderate in corticotropes and gonadotropes. No significant immunoreactivity for SgIII was observed in somatotropes. The immunocytochemical localizations of four granins in the anterior pituitary are summarized in Table 1.
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Intracellular Localization of SgIII in Pituitary Endocrine Cells
To precisely examine the intracellular localization of SgIII in pituitary endocrine cells, immunoelectron microscopy was applied to the tissues. The data showed that SgIII was localized in the secretory granules of corticotropes, gonadotropes, mammotropes, and thyrotropes, and that its intragranular localizations varied depending on cell type. The immunoreactivity for SgIII was not detected in the rough ER, Golgi complex, and on the plasma membrane of pituitary endocrine cells.
Corticotropes, Gonadotropes, and Somatotropes
In corticotropes, SgIII was co-localized with ACTH (or its precursor, POMC) in some secretory granules, although labeling density was relatively low (Fig 4A).
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Immunogold particles indicative of SgIII were also observed in a limited population of secretory granules within gonadotropes (Fig 4B). The secretory granules immunoreactive for SgIII appeared as large-sized ones in which CgA was specifically accumulated, as we previously reported (
In contrast to these endocrine cells, secretory granules of somatotropes, which contain predominantly GH, were not significantly labeled with the immunogold particles indicative of SgIII, indicating that in a physiological state somatotropes in the rat pituitary gland do not store a detectable level of SgIII (Fig 4C).
Mammotropes
It is widely accepted that there are at least two subtypes of mammotropes, an immature type containing small round secretory granules and a mature type containing large, irregular-shaped granules. Secretory granules of both subtypes of mammotropes, which contain predominantly PRL, were intensely labeled with the immunogold particles indicative of SgIII (Fig 5A). Interestingly, these immunogold particles were specifically concentrated in the peripheral region of the secretory granules, whereas those indicative of PRL were distributed all over the secretory granules. This peripheral localization in the granules was more prominent in mature mammotropes (Fig 5B), but a similar tendency was discerned in the small secretory granules in immature mammotropes (Fig 5E). Immunoreactivities for another two granins, CgB and SgII, were also observed preferentially in the periphery of secretory granules, although the accumulation of these granins in this region was less apparent than that of SgIII (Fig 5C, Fig 5D, Fig 5F, and Fig 5G). These findings were confirmed by counting separately the number of immunogold particles indicative of each granin in the peripheral and central regions of secretory granules (Table 2) and by measuring the distances between each immunogold particle and limiting membrane of secretory granules in mature mammotropes (Fig 6). Secretory granules of mammotropes were not significantly labeled with the immunogold particles indicative of CgA (data not shown).
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Thyrotropes
In addition to mammotropes, immunolabeling of SgIII was intensely detected in endocrine cells containing small-sized secretory granules that were labeled densely with anti-TSHß antiserum (Fig 7A). The size and appearance of the secretory granules in thyrotropes could hardly be distinguished from those of the immature type in mammotropes. Immunogold particles showing SgIII were localized predominantly in the periphery of secretory granules in thyrotropes, as demonstrated in mammotropes (Table 2). The expression patterns of other granins in thyrotropes were also similar to that in mammotropes; CgB and SgII were expressed in these cells but CgA was not (Fig 7B and Fig 7C).
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Although secretory granules in the greater parts of thyrotropes and mammotropes contain exclusively just one of the hormones, TSH or PRL (Fig 7D and Fig 7E), an intermediate type of endocrine cells containing both TSH and PRL was found among the endocrine cells immunoreactive for SgIII (Fig 7F). This type of endocrine cell, however, was rarely but distinctly found in the anterior pituitary of the adult male rat. This finding is compatible with the fact that mammotropes and thyrotropes originate from a common progenitor of pituitary cells, which is determined by a transcriptional factor Pit-1 during the developmental stages of the gland.
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Discussion |
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After its identification, SgIII's distribution and function were not investigated as extensively as were other granins, probably because mice missing the 1B1075 gene that encodes SgIII exhibit no obvious defects in viability, fertility, or locomotor behavior (
The present study demonstrated that SgIII was intensely expressed in mammotropes and thyrotropes in the anterior lobe of the pituitary gland, and was preferentially co-localized with CgB in the peripheral region within secretory granules. Immunoreactivity for CgA is not detectable in the mammotropes and thyrotropes, suggesting that SgIII in these cells contributes to the sorting and retention of other granins and/or peptide hormones instead of CgA.
The peripheral localization of certain granins within secretory granules has been reported in other endocrine cells. CgA in the pancreatic A-cell is localized more closely to the secretory granule membrane within the secretory granule matrix than is the co-stored hormone glucagon (
Putative specific interaction between granins and the secretory granule membrane have also been demonstrated by biochemical analyses. CgB is tightly associated with the secretory granule membrane of PC12 cells (
It is believed that, to be transported properly to secretory granules, soluble secretory proteins are first aggregated, and the aggregates then may be anchored to certain molecules on the membrane destined for secretory granules (
It is also important to understand whether SgIII and CgB function cooperatively or redundantly in endocrine cells of the anterior pituitary. Considering that no clear-cut phenotype appears in mice deficient solely in SgIII (
The similarity in the expression and subcellular localization of these two granins in mammotropes and thyrotropes reminds us that these two types of endocrine cells originated from a common precursor cell, determined by a pituitary-specific transcriptional factor, Pit-1 (
To date, no study has shown a direct relationship between pituitary-specific transcriptional factors, such as Pit-1, and the regulation of granin expression. Recent analyses on promoter regions of CgA, CgB, and SgII genes have clearly demonstrated that some characteristic elements such as CRE (cAMP response element) and G/C-rich domains contribute to the neuroendocrine-specific and secretagogue-inducible expression of granins in endocrine cells (
In summary, the present immunocytochemical study demonstrated that SgIII in the rat anterior pituitary gland is distributed in all types of endocrine cells except for somatotropes. Among the granin proteins examined, the localization pattern of SgIII was similar to that of CgB at both light and electron microscopic levels. In particular, peripheral localizations of SgIII and CgB within secretory granules of mammotropes and thyrotropes suggested that these two granins might be membrane-associated proteins and play a role in anchoring other soluble constituents of secretory granules.
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Acknowledgments |
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We thank Dr K. Wakabayashi (Gumma University, Japan) for the anti-TSHß antiserum.
Supported in part by a grant-in-aid for Scientific Research (C) from the Japan Society for the Promotion of Science (JSPS) and by a grant from the Akiyama Foundation, Japan.
Received for publication June 25, 2002; accepted October 2, 2002.
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