Prohormone convertases PC1 and PC2 are endoproteases involved in prohormone cleavage at pairs of basic amino acids. There is a report that prohormone convertase exists in the rat anterior pituitary gonadotrophs, where it had previously been considered that proprotein processing does not take place. In addition to luteinizing hormone and follicle-stimulating hormone, rat pituitary gonadotrophs contain chromogranin A (CgA) and secretogranin II (SgII), two members of the family of granin proteins, which have proteolytic sites in their molecules. In the present study we examined whether there is a close correlation between subcellular localization of prohormone convertases and granin proteins. Ultrathin sections of rat anterior pituitary were immunolabeled with anti-PC1 or -PC2 antisera and then stained with immunogold. Immunogold particles for PC1 were exclusively found in large, lucent secretory granules, whereas those for PC2 were seen in both large, lucent and small, dense granules. The double-immunolabeling also demonstrated colocalization of PC2 and SgII in small, dense granules and of PC1, PC2, and CgA in large, lucent granules. These immunocytochemical results suggest that PC2 may be involved in the proteolytic processing of SgII and that both PC1 and PC2 may be necessary to process CgA.

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Background Biodiversity loss is a serious environmental problem, and human activities might be primarily responsible for the marked decline in animal populations globally. Amphibians, in particular, have significantly decreased in number in recent decades. One example is the endangered Yamato salamander (Hynobius vandenburghi), which is distributed in Central Japan and has a very restricted distribution in Gifu Prefecture. Aims We aimed to discover new populations of H. vandenburghi using a combination of GIS and environmental DNA (eDNA) analysis. Materials & Methods Firstly, we designed two primer sets for amplifying Hynobius species targeting mitochondrial 12S rRNA and cytochrome b genes. Next, we performed aquarium experiments to detect H. vandenburghi DNA in aquarium water. We also conducted sequential eDNA detection surveys in five known habitats in Gifu City, Japan, 17 times from January to August 2016. Following these basic eDNA studies, we used GIS to characterize the vegetation and topography of known habitats of H. vandenburghi. We collected water samples in the potential habitats identified by GIS and analyzed eDNA for the presence of H. vandenburghi using the designed primers. Finally, we conducted physical collection surveys in these potential habitats. Results We successfully developed two Hynobius‐specific primer sets and detected H. vandenburghi eDNA in aquarium water. The eDNA of the target species was detected in almost all (94%–100%) samples from four habitats, whereas only 29% of samples were positive for one habitat. We identified five potential habitats by GIS analysis. The DNA of H. vandenburghi was detected in three of five potential habitats. Finally, we discovered a new population in one of the potential sites. Discussion Our approach combining GIS and eDNA enabled the detection of novel population of an endangered amphibian species. This study was conducted by high school students under the supervision of teachers with the help of university researchers, suggesting the applicability of eDNA studies as a tool of citizen science. Conclusion The combination of GIS and eDNA will allow to detect cryptic populations on which conservation efforts may be focused and to alert people to the need for conservation action.

Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

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