Immunocytochemical Localization of Secretory Phospholipase A₂-like Protein in the Pituitary Gland and Surrounding Tissue of the Bullfrog, Rana catesbeiana

Abstract

S U M M A R YPreviously, we obtained a protein that has considerable amino acid sequence homology with secretory phospholipase A 2 (PLA 2 ) 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 PLA 2 -like protein were observed predominately in these secretory granules. These findings support the view that this PLA 2 -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. D uring 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 ␤ -subunits of bullfrog luteinizing hormone and follicle-stimulating hormone and reversed-phase high-performance liquid chromatography (Sakai et al. 1992). We finally succeeded in separating this protein from the main TSH fraction by affinity column chromatography for the bullfrog ␣ -subunit of pituitary glycoprotein hormones and by hydroxyapatite column chromatography. Partial amino acid sequence (N-terminal 45 amino acids) analysis of this protein revealed considerable amino acid sequence homology with secretory phospholipase A 2 (PLA 2 ) (Sakai 1992) and was identical to that of otoconin-22, the major protein of aragonitic otoconia in the saccule of the inner ear of Xenopus , which is considered to play a crucial role in depositing calcium carbonate . In general, it is well known that the membranous labyrinth of the inner ear consists of two main parts, i.e., the utriculus and sacculus, which make stones from calcium carbonate and are involved in the sense of balance. In most vertebrates the endolymphatic sac, which arises

Developmental Expression of Otoconin-22 in the Bullfrog Endolymphatic Sac and Inner Ear

“…In stage 25 (early stage) embryos with a head length of 3 and 5 mm, the endolymphatic sac displayed follicular structures consisting of a single epithelium, which reacted strongly with the DIG-labeled cRNA probe and anti-otoconin-22 serum (Figures 2d and 2e). A positive reaction was observed in the apical cytoplasm of the epithelial cells, which is consistent with the results obtained in the adult (Yaoi et al 2001).…”

“…The lumen of these sacs contains many tiny crystals consisting of calcium carbonate, which are formed by otoconin-22 protein. In a previous study, we found that otoconin-22 protein is present in the endolymphatic sac surrounding the pituitary gland and the paravertebral lime sac in the bullfrog (Yaoi et al 2001). Later, we cloned and sequenced a cDNA encoding bullfrog otoconin-22 and found that calcitonin regulates the expression of otoconin-22 mRNA in the endolymphatic sac, thereby stimulating the formation of calcium crystals in the lumen of the endolymphatic sac (Yaoi et al 2003b).…”

Cloning and Expression of Vacuolar Proton-Pumping ATPase Subunits in the Follicular Epithelium of the Bullfrog Endolymphatic Sac

“…Antisera were raised in rabbits immunized with the ST-170 or ST-173 peptides coupled to keyhole limpet hemocyanin (KLH) with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Pierce, Rockford, Ill.) as described previously (Tanaka and Kurosumi, 1992). The antibody against bullfrog otoconin-22 protein, prepared and characterized as described by Yaoi et al (2001), was a guinea pig antiserum against a synthetic peptide corresponding to N-terminal amino acids 1-13 (ST-135: TPAQFDEMIKVTT) of bullfrog otoconin-22.…”

“…The lumen of these sacs contains many tiny crystals composed of calcium carbonate. Yaoi et al (2001) determined that otoconin-22 protein is present in the ELS surrounding the pituitary gland and the PVLS in the bullfrog. Otoconin-22 is believed to be important in creating conditions that favor nucleation and, subsequently, in controlling the crystal growth of the calcium carbonate lattice in otoconia (mineral particles embedded in the otolithic membrane).…”

Otoconin-22 and Calcitonin: A Novel Modality of Regulating Calcium Storages in Lower Vertebrates?

“…Therefore, mechanisms for localized concentrations of the reactants are required, a role generally played by acidic glycoproteins in other carbonate-based calcification systems. Each type of otoconia is characterized by a distinct set of proteins collectively named otoconins (2)(3)(4). Interestingly, the major proteins of otoconia are related to the secretory phospholipase A2 (2)(3)(4).…”