Antifreeze protein in winter flounder, Pleuronectes americanus, gill epithelial cells isolated and grown in culture

Winsor, Stephen B. (2000) Antifreeze protein in winter flounder, Pleuronectes americanus, gill epithelial cells isolated and grown in culture. Masters thesis, Memorial University of Newfoundland.

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Abstract

Antifreeze proteins (AFPs) have been found in the blood of many teleost species and have the ability to bind to ice crystals and inhibit their growth. Type I AFP was later discovered in several body tissues of the winter flounder (Pleuronectes americanus) including the skin, scales and gills. In order to further our understanding of these proteins that are not produced in the liver, epithelial cells of the winter flounder gill were isolated and maintained in culture to look for the presence of skin type I AFP. The present study is the first that describes the isolation and culture of gill epithelial cells from winter flounder. The isolation procedure used was based in part on the gill cell isolation method developed by Pärt et al [Pärt, P., Norrgren, L., Bergström, E. & Sjöberg, P.(1993) J. Exp. Biol 175, 219 - 232]. The presence of type I AFP in the cells was determined using immuno-histochemistry. The results indicate that epithelial cells stained positive for winter flounder type I AFP antisera against liver, skin or a recombinant form of type I AFP whereas cells stained with sea raven type II AFP antiserum showed no reaction. The distribution pattern of AFP seen in these cells suggests the AFP is located intracellularly. The AFP produced within these cells is believed to be the skin type I AFP and is thought to react with the liver and recombinant antisera due to the close similarity between these AFPs. In short term culture, individual cells were of three predominate shapes. Round cells were 8.3 ± 2.6 μm in diameter, crescent cells were 19.8 ± 5.2 µm x 10.4 ± 2.9 μm and elongated cells were 21.5 ± 5.0 µm x 6.8 ± 1.7 µm. Scanning electron microscopy (SEM) showed individual cells with an elevated nuclear region with a low and somewhat ruffled outer cytoplasmic area. Dishes that contained a relatively high number of isolated gill cells supported the formation of a confluent monolayer of cells. Scanning electron micrographs of confluent cells 8 days in culture show a highly flattened apical surface with no distinguishing characteristics. The cells attached to culture dishes and were capable of forming confluent monolayers of cells similar to pavement cells seen in other gill epithelial cultures. However, SEM revealed that they do not contain microridges typically seen in pavement cells isolated from the sea bass and rainbow trout. The use of the fluorescent stain DASPEI showed these cells to be mitochondria-rich, a characteristic of gill epithelial chloride cells. Preparation of cultures such as these provide a means of examining the mechanisms involved in skin type I AFP production, regulation and also how these proteins function in gill epithelia.

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