Immunolocalization of Na+/K+-ATPase, carbonic anhydrase II, and vacuolar H+-ATPase in the gills of freshwater adult lampreys, Geotria australis

As adults, anadromous lampreys migrate from seawater into freshwater rivers, where they require branchial ion (NaCl) absorption for osmoregulation. In teleosts and elasmobranchs, pharmological, immunohistochemical, and molecular data support roles for Na⁺/K⁺-ATPase (NPPase), carbonic anhydrase II (CAII), and vacuolar H⁺-ATPase (V-ATPase) in two different models of branchial ion absorption. To our knowledge, these transport-related proteins have not been studied in adult freshwater lampreys, and therefore it is not known if they are expressed, or have similar functions, in lampreys. The purpose of this study was to localize NPPase, CAII, and V-ATPase in the gills of adult freshwater lampreys and determine if any of these transport-related proteins are expressed in the same cells. Heterologous antibodies were used to localize the three proteins in gill tissue from pouched lamprey (Geotria australis). Immunoreactivity (IR) for all three proteins occurred between, and at the base of, lamellae in cells that match previous descriptions of mitochondrion-rich-cells (MRCs). NPPase-IR was always on the basolateral side of cells that did not stain for CAII or V-ATPase. In contrast, CAII-IR was always on the apical side of cells that also contained diffuse V-ATPase-IR. Therefore, we have identified two types of MRC in adult freshwater lamprey gills based on immunohistochemical staining for three transport proteins. A model of ion transport, based on our results, is proposed for adult freshwater lampreys. 

Egg-laying-hormone immunoreactivity in the neural ganglia and ovary of Haliotis asinina Linnaeus

Immunoreactivity against the abalone egg-laying hormone (aELH) was detected in the fine granules of type 1 and 2 neurosecretory (NS) cells, neurites in the neuropil, and blood sinuses in the connective tissue sheath of the cerebral, pleuropedal, and visceral ganglia of the tropical abalone, Haliotis asinina Linnaeus. The number of positive NS cells, and the intensity of staining in the ganglia, varied and might be related to the stage of ovarian cycle. At any stage, positive cells were most numerous in the pleuropedal, and least numerous in the visceral ganglion. In addition, several cells of the statocyst and associated nerves also exhibited the immunoreactivity. In the ovary, the most intense reactivity was detected in the follicular and granular cells adjacent to mature oocytes, in the trabeculae and the ovarian capsule. The cytoplasm of mature oocytes was also moderately stained. The results indicate that the cerebral, pleuropedal, and visceral ganglia are the main sites of aELH-producing cells. The ovary may also produce aELH locally.

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 μm of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.