Immunoreactivity of the corpuscles of Stannius of the garpike,Lepisosteus osseus,to antisera against salmon and trout stanniocalcins

Stanniocalcin-immunoreactive cells were localized in the corpuscles of Stannius of a holostean fish, the garpike (Lepisosteus osseus), using antisera against salmon and trout stanniocalcins and the peroxidase-antiperoxidase and protein A-gold immunohistochemical methods. The stanniocalcin-immunoreactive cells were periodic acid-Schiff-positive, and antibody staining was abolished if the antiserum was preabsorbed with corpuscle homogenate. Immunocytochemistry revealed two reactive cell types in the glandular parenchyma, and immunoreactivity was confined to the secretory granules. Staining of the granules was also abolished when the antisera were blocked with crude corpuscle homogenate. When corpuscle extracts from garpike were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot analysis, a single dense band was evident with a molecular weight of 68 kDa under non-reducing conditions, whereas three bands were observed (29, 31, and 34 kDa) under reducing conditions. Staining of all bands disappeared following preabsorption of the antiserum with salmon stanniocalcin, trout stanniocalcin, or garpike corpuscle extract. The results are compared with stanniocalcins from another extant holostean, the bowfin (Amia calva), and from more modern bony fishes, the teleosts.     

The corpuscles of Stannius in arawana (Osteoglossum bicirrhosum), an ancient teleost

The corpuscles of Stannius of arawana (Osteoglossum bicirrhosum), an ancient teleost, were examined by routine light and electron microscopy and following their immunoreactivity to salmon and trout stanniocalcin antisera. Periodic acid-Schiff positive cells of the corpuscles of Stannius had a follicular arrangement and demonstrated a strong immunohistochemical reaction with both stanniocalcin antisera. Fine structural analysis of the paired, posteriorly located, and perirenal ovoid glands revealed two morphologically distinct cell types the basal laminae of which were ramified by nerve terminals. Immunocytochemistry demonstrated that osmiophilic secretory granules in both cell types were immunoreactive to the stanniocalcin antisera. When extracts of arawana corpuscles of Stannius were subjected to sodium dodecyl sulphate electrophoresis and Western blot analysis a diffuse molecular weight band was evident ( approximately 68 kDa) in the non-reduced condition. In all cases, immunoreactivity was abolished by preabsorption of the antisera with salmon stanniocalcin or with a crude extract of arawana corpuscles of Stannius. The corpuscles of Stannius of arawana are similar to those in more recent teleosts with respect to cell structure and their anatomical distribution but their stanniocalcin is more similar in molecular weight to that present in at least one other non-teleost actinopterygian (the gar) which has an ancient lineage.     

Corpuscles of Stannius and stanniocalcin-like immunoreactivity in the white sucker (Catostomus commersoni): evidence for a new cell-type

The distribution of stanniocalcin immunoreactivity was examined in the corpuscles of Stannius of the white sucker (Catostomus commersoni) by using a chum salmon stanniocalcin antiserum, Western blotting, and light and electron microscopy. The white sucker possesses at least two stanniocalcin-immunoreactive corpuscles in the most posterior portions of the kidneys. Immunocytochemistry and ultrastructure revealed two cell-types in the corpuscle parenchyma, only one of which was immunoreactive. The nonimmunoreactive cells contained dense-cored vesicles and long processes that extended between the immunoreactive cells and terminated at perivascular spaces. When corpuscle extracts were subjected to electrophoresis and Western blotting, three nonreduced stanniocalcin-like immunoreactive bands (approximately 56, 61, and 64 kDa) were observed. However, in the presence of a reductant, a diffuse band migrating in the range of 28 to 32 kDa was noted. The results of this study on the white sucker demonstrate the presence of a dimeric stanniocalcin-like molecule and present evidence of a previously uncharacterized cell-type in the corpuscles of Stannius.     

Chum salmon (Oncorhynchus keta) stanniocalcin inhibitis in vitro intestinal calcium uptake in Atlantic cod (Gadus morhua)

Summary Chum salmon (Oncorhynchus keta) stanniocalcin was purified, partially identified and tested for bioactivity in an assay on the intestinal calcium uptake in a marine teleost (Gadus morhua). Basic ethanol extraction, ion exchange chromatography, gel filtration and reverse-phase high-performance liquid chromatography resulted in the isolation of a homogenous glycoprotein that appears as a 46-kDa product under non-reducing conditions and as a 23-kDa product under reducing conditions after sodium dodecylsulphate-polyacrylamide gel electrophoresis. The glycoprotein is likely to be a homodimer composed of two subunits of 23 kDa each. Further characterization indicates homology to Australian eel, sockeye salmon, coho salmon and rainbow trout stanniocalcin, and the glycoprotein is thus concluded to be stanniocalcin. Stanniocalcin-like immunoreactivity was demonstrated in the corpuscles of Stannius of the Atlantic cod, with a specific antiserum raised against purified chum salmon stanniocalcin. The physiological importance and the biological activity of chum salmon stanniocalcin was tested by evaluating its effect on intestinal calcium uptake by the Atlantic cod in vitro. The intestine was perfused, both vascularly and through the intestinal lumen, and the calcium mucosa-to-serosa flux was measured using ⁴⁵Ca²⁺ as a tracer. Stanniocalcin decreased the intestinal calcium uptake in a dose-related manner by 13.5% and 22.4% at doses of 2.2 and 10.9 nM stanniocalcin, respectively. The results establish the intestine as a target organ for stanniocalcin in marine teleosts.Abbreviations BIS balanced intestinal solution - CS corpuscles of Stannius - dpm disintegrations per minute - FW freshwater - J _in ^Ca influx of calcium across the intestinal mucosa - MW molecular weight - NRS normal rabbit serum - PBS phosphate buffered saline - PBST phosphate buffered saline containing 0.05% Tween-20 - PITC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC phenyl isothiocyanate - rp-HPLC reverse phase - SW seawater - STC stanniocalcin - TFA Trifluoroacetic acid - Tris Tris(hydroxymethyl) aminomethan - V volume per fraction     

Fine Structure of the Adrenocortical Homolog and the Corpuscles of Stannius of Amia calva L.

A comparison is made between the fine structure of yellow corpuscles and white corpuscles located within the kidneys of the holostean fish, Amia calva L. The yellow corpuscles are composed of epithelial cells possessing all the features of steroid-producing tissues, namely an abundance of vacuoles, tubular smooth endoplasmic reticulum, and mitochondria with tubular cristae. The Golgi apparatus is also a conspicuous component of their cytoplasm. These cells are homologous to adrenocortical cells of higher vertebrates and they have cytoplasmic projections which extend into the lumina of surrounding sinusoids. The white corpuscles possess epithelial cells of variable appearance but all cells contain secretory granules and an extensive rough endoplasmic reticulum. The secretory granules appear to originate at the Golgi apparatus and occasionally are observed intact in the intercellular space. However the method of release of these granules was not clearly defined. These corpuscles are similar to the corpuscles of Stannius which have been described in modern bony fish. The presence of multivesicular bodies and smooth endoplasmic reticulum in some cells may reflect the origin of the corpuscles of Stannius from the tubular nephron. A. calva appears to be a suitable organism for comparative studies into the function of the adrenocortical homolog and corpuscles of Stannius in “primitive” fish.     

The kidney,adrenocortical homolog,and corpuscles of Stannius in the cockscomb pricklebackAnoplarchus purpurescens

The morphology of the kidney, adrenocortical homolog, and the corpuscles of Stannius was examined in the cockscomb prickleback,Anoplarchus purpurescens, a marine teleost which inhabits the intertidal zone. The paired kidneys of this fish are fused throughout most of their length, there is essentially a single posterior cardinal vein on the right side, they possess renal corpuscles, and there is no distal segment of the tubule. The tubule is specialized, in descending order, into ciliated neck and two proximal segments before entering the system of collecting tubules and ducts. The cells of the latter system are specialized for mucous secretion, as are cells of the main excretory ducts, the paired archinephric ducts. Tubulogenesis occurs in the kidneys in close apposition to the archinephric ducts. The presumptive adrenocortical homolog is located around the posterior cardinal veins in the head kidney while paired corpuscles of Stannius are confined to the posterior end of the kidney. All of the above features are consistent with those found in the kidneys of many other marine teleosts.     

鱼类斯钙素的研究进展

本文概述了近年来有关硬骨鱼类斯坦尼氏小体（ｃｏｒｐｕｓｃｌｅｓｏｆＳｔａｎｎｉｕｓ,ＣＳ）分泌激素———斯钙素（ｓｔａｎｎｉｏｃａｌｃｉｎ,ＳＴＣ）的研究进展。ＳＴＣ是糖蛋白类激素,为同型二聚体,其表观分子量在天然状态下从４６（大麻哈鱼）～５６（虹鳟）ｋＤａ,还原状态下则为２３～２８ｋＤａ。ＳＴＣ单体的氨基酸序列分析表明,大麻哈鱼、银大麻哈鱼和澳大利亚鳗鲡的氨基酸残基数分别为１７９、２２３和２３１个。研究还表明,ＳＴＣ的分泌受血钙浓度的调节,并且胆碱能神经参与ＳＴＣ的释放。     

The murine stanniocalcin 1 gene is not essential for growth and development

The stanniocalcin 1 (STC1) gene is expressed in a wide variety of tissues, including the kidney, prostate, thyroid, bone, and ovary. STC1 protein is considered to have roles in many physiological processes, including bone development, reproduction, wound healing, angiogenesis, and modulation of inflammatory response. In fish, STC1 is a hormone that is secreted by the corpuscles of Stannius and is involved in calcium and phosphate homeostasis. To determine the role of STC1 in mammals, we generated Stc1-null mice by gene targeting. The number of Stc1-/- mice obtained was in accordance with Mendelian ratios, and both males and females produced offspring normally. No anatomical or histological abnormalities were detected in any tissues. Our results demonstrated that Stc1 function is not essential for growth or reproduction in the mouse.     

Development of the corpuscles of Stannius in the anabantid teleost, Colisa lalia

The development of Stannius corpuscles in the teleost Colisa lalia is described. Certain cells of the mesonephric tubules differentiate and proliferate to form buds which evaginate and finally become separated from the structure of origin. The separated cellular masses undergo further histologic differentiation to attain the adult structures. The corpuscles in C. laha, in contrast to some other teleosts, develop only from mesonephric tubules. A possible explanation for the different types of distribution of Stannius corpuscles in teleosts is suggested, and the possible homology of these structures with part of the adrenal cortex or higher vertebrates is discussed.     

Embryonic origin and development of the corpuscles of Stannius in chum salmon (Oncorhynchus keta)

Summary An immunocytochemical technique was used to follow the embryological origin and development of the corpuscles of Stannius (CS) in the chum salmon, Oncorhynchus keta. Stanniocalcin immunoreactive (ir-) cells can be observed as early as 13 days before hatching. The ir-CS cells appear in clusters of variable size in close association with nephric ducts. In addition, individual ir-cells also occur at this stage amoung epithelial cells of the nephric ducts. these individual cells may give rise to clusters which subsequently increase in size, the largest reaching 100 m in diameter by the time of hatching. During this period, dispersed CS cells become evident and develop into secondary clusters in the vicinity of the primary clusters. These clusters appear to fuse to form larger clusters with a lobular structure. Transfer of the larvae (20 days after hatching) from fresh water to 50% seawater, accelerates the development of the CS tissue, suggesting an important role of the CS in seawater adaptation.     

Hypocalcemic effects of bovine parathyroid hormone (1-34) and Stannius corpuscle homogenates in teleost fish adapted to low-calcium water

Injections of bovine parathyroid hormone (PTH 1-34) and homogenates of corpuscles of Stannius produce hypocalcemia in male killifish and tilapia adapted to calcium-deficient seawater or fresh water, respectively. In fish from water with normal calcium concentrations no effects are noticeable. These results suggest similarity in bioactivity between PTH, the hypercalcemic hormone of terrestrial vertebrates, and the hypocalcemic factor of the corpuscules of Stannius in teleost fish.     

Immunocytochemical localization of hypocalcin in the endocrine cells of the corpuscles of Stannius in three teleost species (trout,flounder and goldfish)

Summary In order to identify the cell-type responsible for the production of hypocalcin (the recently isolated hypocalcemic hormone of teleost fish), the corpuscles of Stannius (CS) of trout, flounder and goldfish, were immunocytochemically stained with antisera raised against trout hypocalcin. The secretory granules of the type-1 cells of the CS, considered to be the hypocalcin-producing cells, showed intense immunoreactivity in all species examined. However, in trout and flounder, the secretory granules produced by the type-2 cells, which have been suggested to represent a functionally different cell-type, also showed an intense immunoreactivity. In goldfish, no type-2 cells were observed. We tentatively conclude that type-1 and type-2 cells represent structurally different forms of the same functional cell-type.     

Immunocytochemical identification of prolactin cells in the pituitary gland of tilapia larvae (Oreochromis mossambicus: Teleostei)

Summary Using an antiserum to highly purified chum salmon prolactin, prolactin cells were identified in the putative rostral pars distalis of newly hatched tilapia larvae (Oreochromis mossambicus) by the immunogold method for the electron microscope. In the putative rostral pars distalis, some cells had another kind of secretory granule which was much less numerous, much smaller in size, and without immunoreactivity to salmon prolactin antiserum. Controls incubated with salmon prolactin-preabsorbed antiserum or normal serum showed no immunoreactive cells, confirming the specificity of the antiserum. The possible role of prolactin in the osmoregulation of tilapia larvae is discussed.     

Nitrate reductase induction and molecular characterization in rice (Oryza sativa L.)

Summary Barley nitrate reductase cDNA clone bNRp10 was used as a hybridization probe to screen a genomic DNA library of rice (Oryza sativa L.) cultivar M201. Two different lambda clones were isolated, subcloned to plasmids, and partially characterized. The subclone pHBH1 was tentatively identified as encoding a NADH nitrate reductase. Southern and dot blot analysis suggest that, in rice, nitrate reductase is encoded by a small gene family. Regulation of NADH nitrate reductase was investigated in rice cultivars Labelle and M201 representing the subspecies indica and japonica, respectively. In the absence of nitrate, only trace levels of nitrate reductase activity and mRNA were detected in seedling leaves. Upon addition of nitrate to seedling roots, nitrate reductase activity and mRNA increased rapidly in leaves. Nitrate reductase activity continued to increase over a 24 h period, but the mRNA accumulation peaked at about 6 h and then declined. Western blot analysis with a barley NADH nitrate reductase antiserum showed the presence of two bands of approximately 115 and 105 kDa. These protein bands were not detected in extracts of tissue grown in the absence of nitrate.     

Immunological characterization of nitrogenase in the filamentous non-heterocystous cyanobacterium Oscillatoria limosa

The filamentous non-heterocystous cyanobacterium Oscillatoria limosa was subjected to Western blot analyses using two antisera raised against the small subunit (Fe-protein) of the nitrogenase complex. Two polypeptides were recognized in nitrogen-fixing cultures irrespective of the antiserum used while no bands were detectable in nitrate-grown cultures. The apparent molecular weights of the two polypeptides were approximately 40.5 and 39.5 kDa respectively, with the former, probably an inactive form, dominating. In situ immunogold electron microscopy was used to reveal the cellular and subcellular localization on the Fe-protein. All cells of the trichomes of nitrogen-fixing O. limosa showed a dense label. The label was homogeneously distributed throughout the cytoplasm including the thylakoid area. Nitrate-grown cultures contained a very low label.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis This study was supported by the Swedish Natural Science Research Counsil and the M. and M. Wallenberg Fund (to B. Bergman). We are grateful to Dr. S. Nordlund (University of Stockholm, Sweden) for providing us with the antiserum of Rhodospirillum rubrum nitrogenase and to Drs. S. Reich and P. Böger (University of Konstanz, FRG) for the antiserum of Anabaena variabilis. Skilful technical assistence by K. Östlund and E. Danielsson is gratefully acknowledged. We would also like to thank M. Villbrandt (University of Oldenburg, FRG) for providing cultures of Oscillatoria limosa and Dr. P. Lindblad for valuable discussions and suggestions.To whom correspondence should be addressed.     

Generation of polyclonal antiserum against the growth hormone secretagogue receptor (GHS-R): evidence that the GHS-R exists in the hypothalamus, pituitary and stomach of rats

Growth hormone (GH) secretagogues (GHSs), which stimulate GH secretion, are synthetic compounds that act through the GHS receptor (GHS-R) which has been recently cloned. We raised an antiserum in a rabbit against a synthetic peptide corresponding to amino acid residues 248-260 of the third intracellular loop of the rat GHS-R. A competitive immunoassay showed that the antiserum had a specific affinity for the target peptide. To confirm the specificity of the antiserum, the GHS-R cDNA was stably expressed in COS-7 cells. In Western blot analysis, the band was detected at 44 kDa in the extracts from COS-7 cells expressing GHS-R (COS-7/tf3-2) but not in those from wild-type COS-7 cells. Furthermore, while COS-7/tf3-2 cells were strongly immunostained for GHS-R, no GHS-R-like immunoreactivity was observed in wild-type COS-7 cells. Immunoreactive bands were also observed at approximately 46 kDa in the extracts from rat hypothalamus, pituitary and stomach by Western blot analysis. These studies are the first to show the existence of GHS-R protein in the stomach. The antiserum for the GHS-R is sensitive and specific, and it would be useful for clarifying the roles of GHS/ghrelin.     

Distribution and Structure of the Adrenocortical Homolog in Polypterus palmas Ayres

The identity, distribution and structure of the adrenocortical homolog (AH) was made in Polypterus palmas Ayres using routine light and electron microscopy and histochemistry for the enzyme δ⁵-3β-hydroxysteroid dehydrogenase (3β-HSD). The AH is confined to yellow corpuscles which are positioned near the posterior cardinal and renal veins in the anterior two-thirds of each kidney. The 46–57 spherial to cigar-shaped corpuscles are placed end to end and are equally distributed in the kidneys. The tortuous cords of epithelial cells of each corpuscle are surrounded by sinusoids and are completely delimited from the haemopoietic and renal tissue of the kidneys. The steroidogenic nature of the cells is demonstrated by their 3β-HSD activity and their ultrastructure, namely lipid droplets, tubular smooth endoplasmic reticulum and mitochondria with tubulovesicular cristae. Giant mitochondria and gap junctions are notable features of AH cells in this fish. The yellow corpuscles of the kidneys in P. palmas represent the AH and this tissue has a distribution which is in accordance with the taxonomic position of Polypteriformes.     

Expression, Purification, and Bioassay of Human Stanniocalcin from Baculovirus-Infected Insect Cells and Recombinant CHO Cells

Stanniocalcin is a calcium- and phosphate-regulating glycoprotein hormone that was first described in fish where it functions in preventing hypercalcemia. Human cDNA clones encoding the homolog of stanniocalcin have been recently isolated. In this study, the full-length cDNA coding for human stanniocalcin (hSTC) was cloned into both baculovirus and CHO expression vectors. Recombinant hSTC was then produced efficiently from both baculovirus-infected insect cells and CHO cells in large-scale bioreactors. Purification protocols were developed and used to purify recombinant hSTC from both sources in four chromatography steps. The hSTCs from both expression systems were secreted as glycosylated proteins and as disulfide-linked homodimers. The results from glycosylation studies indicated that stanniocalcin from both sources contained N-linked oligosaccharides but no O-linked sugars. In anin vivobioassay based on the inhibition of gill calcium transport in fishes, the baculovirus and CHO-expressed protein showed biological activity which is dose dependent. The inhibitory effects of hSTC produced from both systems were essentially equipotent in fishes, despite the differences in glycosylation. Consequently, the precise role of the carbohydrate moiety in recombinant hSTC remains to be determined.     

cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura

A cDNA encoding the general odorant binding protein II (GOBP II) was isolated from the antennae of Spodoptera litura (SlGOBP II, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SlGOBP II was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72. SlGOPB II shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SlGOPB II shared significant identity with the GOBP II from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SlGOBP II was specifically expressed in the antennae. cDNA encoding SlGOBP II was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coli BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPII i.e, 32 kD, which has a 6×His tag at the N-terminus. The recombinant SlGOBP II was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1︰12800, while Western blot analysis showed that the recombinant SlGOBP II was recognized as anti-SlGOBP II an-tiserum.