ARTICLE |
Correspondence to: Shigeyasu Tanaka, Dept. of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan. E-mail: sbstana@ipc.shizuoka.ac.jp
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Summary |
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Previously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A2 (PLA2) from a bullfrog pituitary fraction obtained during the purification of thyrotropin (TSH). Subsequently, partial amino acid sequence (N-terminal 45 amino acid residues) analysis revealed this protein to be identical to the N-terminal amino acid sequence of otoconin-22, the major protein of aragonitic otoconia in the Xenopus saccule. In this study we developed an antibody against the N-terminal peptide of the bullfrog protein and applied it for immunocytochemical study of the pituitary and its surrounding tissue. Western blotting analysis showed that this antibody recognizes a 20.4-kD protein that has a molecular mass close to that of otoconin-22. Immunohistochemical reaction with the antibody was not found in any anterior pituitary cells but was intense in the monolayer epithelial cells of the endolymphatic sac surrounding the pituitary gland, which is a major storage site of calcium carbonate in amphibians. An electron microscopic study revealed that the cuboidal cells in the endolymphatic sac contained large, polymorphic secretory granules in their apical cytoplasm. Immunogold particles indicating the presence of a PLA2-like protein were observed predominately in these secretory granules. These findings support the view that this PLA2-like protein obtained during purification of TSH was derived from the endolymphatic sac adhering to the pituitary and that this protein is a bullfrog otoconin. (J Histochem Cytochem 49:631637, 2001)
Key Words: phospholipase A2-like protein, otoconin, secretory granule, immunocytochemistry, endolymphatic sac, anterior pituitary gland, bullfrog
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Introduction |
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During purification of bullfrog pituitary thyrotropin (TSH) from a pituitary glycoprotein fraction, we noted a protein that did not separate from the main TSH fraction through several steps of chromatography, including anion-exchange column chromatography, affinity column chromatography for b-subunits of bullfrog luteinizing hormone and follicle-stimulating hormone and reversed-phase high-performance liquid chromatography (
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Materials and Methods |
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Animals
Adult male and female bullfrogs (Rana catesbeiana) were purchased from Ouchi (Misato, Japan). They were kept under normal laboratory conditions for at least 1 week and were fed crickets. Their pituitary glands and surrounding tissues and their paravertebral lime sacs were removed under anesthesia with MS 222 (Nacalai Tesque; Kyoto, Japan), and then processed for light or electron microscopic examinations and for biochemical analysis.
Antisera
To develop an antibody against the bullfrog PLA2-like protein (bPLP), we used a synthetic peptide corresponding to bPLP's N-terminal amino acids 113 (ST-135: TPAQFDEMIKVTT), with an amino-terminal cysteine residue added to assist in coupling the peptide to keyhole limpet hemocyanin (KLH) (Pierce; Rockford, IL). Coupling was done with m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce) according to the method of
A rabbit was injected with the KLH-coupled peptide every 2 weeks for 2 months by the multiple-site method described previously (
Western Blotting Analysis
To separate the proteins from the endolymphatic sac and from the calcium carbonate crystals in the endolymphatic sac, we removed the calcium carbonate from the sac and the crystal by treating them with 10% ethylenediamine-N,N,N,N'-tetraacetic acid (EDTA) in water at 4C for 3 days according to the method of
To see whether the immunoreactive protein(s) was glycosylated, the extracts from the endolymphatic sacs and calcium carbonate crystals in the endolymphatic sacs were treated for 1 hr at 37C with peptide-N-glycosidase F (Daiichi Pure Chemicals; Tokyo, Japan) before SDS-PAGE and Western blotting.
Light Microscopic Immunocytochemistry
The anterior pituitary gland and its surrounding endolymphatic sac and the paravertebral lime sac were fixed by immersion in BouinHollande solution for 2 days. After the tissues had been treated with 10% EDTA in water at 4C for 3 days to demineralize the calcium carbonate crystals, they were dehydrated and embedded in Paraplast. Sections were cut at 4 µm and mounted on gelatin-coated slides.
Immunohistochemistry was performed by the indirect enzymeantibody method. Deparaffinized sections were incubated sequentially at room temperature (RT) with the following reagents: 5% normal goat serum for 2 hr, rabbit anti-bPLP serum (1:8000) for 16 hr, peroxidase-conjugated donkey anti-rabbit IgG (1:200; Jackson Immunoresearch, West Grove, PA) for 1.5 hr. They were then rinsed three times with PBS, stained with 15 mg of 3,3'-diaminobenzidine tetrahydrochloride (Dojin Laboratory; Kumamoto, Japan) and 0.005% H2O2 in 100 ml of 0.05M Tris-HCl (pH 7.6) to detect peroxidase activity, rinsed with distilled water, stained with Mayer's hematoxylin, dehydrated by passage through a graded ethanol series, and mounted in Entellan (Merck; Darmstadt, Germany). The specificity of the immunostaining was also examined by a preabsorption test. The diluted antiserum was mixed with the antigen peptide at a final concentration of 1 or 10 µg/ml and pre-absorbed for 12 hr at 4C before being used in the specificity test.
Electron Microscopic Immunocytochemistry
The pituitary and its surrounding endolymphatic sac were fixed with a mixture of 0.5% glutaraldehyde, 4% paraformaldehyde, and 0.2% picric acid in 0.1 M cacodylate buffer, pH 7.4, at 4C for 2 hr and then decalcified by the procedure described above. The tissues were postfixed for 2 hr at 4C in 1% osmium tetroxide and then reduced with 1.5% potassium ferrocyanide in the same buffer. They were dehydrated through a graded ethanol series, infiltrated with propylene oxide, and embedded in an Epon/Araldite mixture. Ultrathin sections were cut with a Reichert Ultracut-E microtome (ReichertJung; Vienna, Austria) equipped with a diamond knife, mounted on nickel grids, and treated according to the following procedure: (a) etching in 3% H2O2 for 5 min at RT; (b) rinsing well with water; (c) bleaching with saturated sodium metaperiodate solution for 20 min (
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Results |
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Gross anatomy revealed that the caudal part of the bullfrog pituitary gland was covered by a sac containing a large quantity of calcium carbonate. This sac was connected to the membranous labyrinth in the inner ear and also to the endolymphatic sac (referred to as the paravertebral lime sac) which extends along the entire length of the spinal canal (Fig 1). In the tissue sections, the endolymphatic sac attached to the caudal region of the pars distalis was observed as a tissue with a follicular structure. This follicular structure consisted of a monolayer of cuboidal epithelial cells, with the basal side surrounded by a thin layer of connective tissue and blood vessels. When sections, including the pituitary gland and its surrounding endolymphatic sac, were immunostained with antiserum against bPLP, no immunoreaction was observed in any of the cells in any part of the pituitary gland (pars distalis, pars intermedia, and pars nervosa). However, the endolymphatic sac was immunopositive for this antiserum (Fig 2a). The apical side of the cytoplasm in the epithelial cells of the sac displayed an intense positive reaction with the antiserum (Fig 2b). The inclusion materials in the lumen of the follicular structures were also positively immunostained. These immunostainings were completely eliminated by preabsorption of the antiserum with 1 µg/ml of the antigen (Fig 2c). Similarly, when we examined the paravertebral lime sac, a positive reaction for bPLP was seen in the apical region of the simple cuboidal epithelial cells (Fig 2d).
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To test the specificity of the antiserum toward the bullfrog endolymphatic sac, the calcium carbonate crystals in the sac, and the anterior pituitary, we conducted Western blotting analysis of their extracts. In the extract of endolymphatic sac and calcium carbonate crystals in the endolymphatic sac, the antiserum detected a major band at 20.4 kD and a minor smear band between 24.6 and 30 kD with the sample from the endolymphatic sac, and two bands at 20.4 and 21.7 kD, and several diffuse bands between 24.6 and 30 kD with that of the calcium carbonate crystals. No band was detectable in the anterior pituitary gland extract (Fig 3A). The bands above were not detected when anti-bPLP was preabsorbed with the peptide used as immunogen (Fig 3B). To confirm that the immunoreactive bands are glycosylated, we performed a digestion experiment using pepide-N-glycosidase F. After digestion, most of the stained bands were converted to a band of 16.4 kD, suggesting that the bands of apparent higher molecular mass represented glycosylated forms of the 16.4-kD PLA2-like protein (Fig 3C).
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Electron microscopy revealed that the cuboidal cells in the endolymphatic sac contained two kinds of secretory granules in their apical cytoplasm: one was large, polymorphic, and electron-lucent; the other type was dense and was fewer in number. The cuboidal cells in the sac had many mitochondria in its apical cytoplasm and well-developed microvillus-like structures at its apex. The surface of the follicular epithelium was covered by an amorphous thin layer, which appeared to have been derived from secretory materials (Fig 4a). These ultrastructural characteristics are very similar to those of the endolymphatic sac of tree frogs reported by
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Discussion |
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In this experiment bPLP, which was isolated as a minor fraction during purification of bullfrog TSH, was studied immunocytochemically. Immunoreactivity was found in the epithelial cells of the endolymphatic sac surrounding the pituitary gland but not in any anterior pituitary cells, supporting our view that the bPLP was mainly derived from the endolymphatic sac adhering to the pituitary.
Because the amphibian endolymphatic sac connects with the membranous labyrinth, the tiny crystals of calcium carbonate that are found in the endolymphatic sac are believed to be homologous to the otoconia in the utriculus and the sacculus. Our scanning electron microscopical examination indicated that the crystals in the bullfrog endolymphatic sac were of a prismatic shape (unpublished data), as reported for several species of amphibians (
In this study, the Western blotting analysis and immunocytochemical staining showed that the anterior pituitary gland does not have PLA2-immunoreactive bands, strongly suggesting that the anterior pituitary gland does not contain such materials. Taken together, the present findings indicate that the PLP fraction obtained during TSH purification was most probably derived from the endolymphatic sac contained in the starting material for TSH purification.
There are some arguments about the precise cellular sites at which otoconia are formed.
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (ST).
Received for publication October 26, 2000; accepted December 20, 2000.
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