Squamous cell carcinoma in rudd Scardinius erythrophthalmus: histopathology, ultrastructure, and transmission

The histopathology and ultrastructure of a skin neoplasm in rudd Scardinius erythrophthalmus (L.) are described and the neoplasm is diagnosed as squamous cell carcinoma. In the early stage, tumour cells appear singly or in clusters close to the epidermis; then tumour cells are seen in the dermis as epithelial pearls/nests, cords or sheets with central keratin; and with further development of the neoplasm, tumour cells appear as large sheets and masses, enclosing extensive keratinous formations and foci of necrotic cells, infiltrating deeper into the dermis and penetrating the muscle layers. Increasing vascularity and inflammation are associated with all stages of progression. In some cases metastases are observed in the viscera. Electron microscopic examination shows that the neoplastic cells are joined by desmosomes; the cytoplasm contains abundant mitochondria and rough endoplasmic reticulum. The neoplasm was successfully transmitted experimentally to healthy rudd when tumour cells were inoculated subcutaneously. Eight of 19 surviving test fish developed tumour growth at the site of tumour cell injection and/or in a corresponding site on the opposite of the body. One of these 8 fish, which developed a neoplasm, also showed a microscopic internal tumour in the viscera. One of the 19 test fish showed a microscopic tumour in the spleen, even though no skin tumour was visible in this fish. No tumours were found in control fish. In contrast, intraperitoneal injection of tumour cells into healthy rudd did not result in transmission of the neoplasm.     

Socializing makes thick-skinned individuals: on the density of epidermal alarm substance cells in cyprinid fish, the crucian carp (Carassius carassius)

In cyprinid fish, density of epidermal club cells (i.e. alarm substance cells) has been found to vary between lakes with different predator fauna. Because predators can be labelled with chemical cues from prey, we questioned if club cell density could be controlled indirectly by predators releasing prey cues. In particular, we suspected a possible feedback mechanism between chemical alarm signals and their cellular source. We raised crucian carp singly and in groups of four. For both rearing types, fish were exposed to skin extracts of either conspecifics or brown trout (without club cells), and provided either low or high food rations. Independent of rearing type, condition factor and club cell density increased with food ration size, but no change was found in club cell density following exposure to conspecific alarm signals. However, the density of club cells was found significantly higher for fish raised in groups than for fish raised alone. We conclude that an increased condition factor results in more club cells, but crucian carp may also possess an awareness of conspecific presence, given by higher club cell densities when raised in groups. This increase in club cell density may be induced by unknown chemical factors released by conspecifics.     

Epidemic trichodinosis associated with severe epidermal hyperplasia in largemouth bass, Micropterus salmoides, from North Carolina, USA

An epidemic of trichodinosis associated with severe epidermal hyperplasia occurred in adult largemouth bass (Micropterus salmoides) from the Chowan River drainage, North Carolina (USA) in late winter to early spring 2002. Initial reports by anglers of fish with a "jelly-like slime coat" on the skin prompted an electrofishing survey in which about 10% of sampled largemouth bass had a very thick, bluish-white "mucoid layer" on the body and fins. Moderate to heavy infestations of the ciliate Trichodina were detected in wet mounts of skin from five of five fish having the mucoid layer; these fish also had significant gill infestations. An additional two fish with only mild reddening and four asymptomatic fish (no skin lesions) had mild skin infestations but no gill infestations. Two asymptomatic fish had no skin parasites. Four fish with the mucoid layer were necropsied and had extremely severe epidermal hyperplasia on the body and fins. The hyperplasic epidermis had relatively few mucus cells and typically was about 5-10 times thicker than healthy epidermis. The upper four fifths of the epidermis consisted of finely vacuolated, highly flattened, somewhat disorganized epithelial cells. No other significant clinical or histopathologic abnormalities were detected. No systemic infection by pathogenic bacteria was noted. The environmental cause of the epidemic is uncertain but the lesions suggest that some chronic stressor was involved.     

Werner''s syndrome: proliferation in vitro of clones of cells bearing chromosome translocations.

Each of several cultures of Werner's syndrome (WS) fibroblasts and lymphoblasts examined was found to be composed of one or several clones of cells with mutated chromosome complements. Two "sister" fibroblasts cell lines (FCLs) that were derived from a mixture of explants cut from the same WS skin biopsy were found to have completely different rearranged chromosome complements. Daily observation of the skin explants from which these two sister FCLs were derived revealed not only that no more than a few fibroblasts ever migrated from a given explant but also that fibroblasts migrated from only a few of the explants. Two of three lymphoblastoid cell lines (LCLs), each probably developed as an independent clone from a different cell from the same WS blood sample, were mosaic, comprised of cells having both normal and rearranged chromosome complements. The third LCL studied, although nonmosaic, had a rearranged chromosome complement, but one that was completely different from those in the other two lines. Based on the observations described, hypotheses have been formulated to explain both the preponderance in long-term WS cultures of clones with mutated chromosome complements and the abbreviated lifespan characteristic of WS fibroblast cultures.     

Apoptosis in skin pigment cells of the medaka, Oryzias latipes (Teleostei), during long-term chromatic adaptation: the role of sympathetic innervation

Many teleost fish can adapt their body color to a background color by changing the morphology and density of their skin pigment cells. Melanophore density in fish skin decreases during long-term adaptation to a white background. Although cell death, especially apoptosis, is thought to be involved in these morphological changes, there are no data clearly supporting this mechanism. Using medaka fish, Oryzias latipes, we observed that, on a white background, melanophore size was reduced first and this was followed by a decrease in melanophore density caused by gradual cell death. The process of cell death included loss of cell activity, cell fragmentation, phagocytosis of the fragments, and clearance via the epidermis. Apoptosis was assessed by the appearance of phosphatidylserine on the cell surface of melanophores that had lost motile activity, and DNA fragments involved in cell fragmentation were detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) assay. However, when chemically denervated fish were used, although melanophore size was reduced as expected, cell death was suppressed even on a white background. In skin tissue culture, apoptosis in melanophores was stimulated significantly by norepinephrine, but not by melanin-concentrating hormone. These results indicate that melanophore density decreases by apoptosis, and suggest that sympathetic innervation has an important role in the regulation of apoptosis in melanophores. In analogous fashion, leucophores showed a significant decrease in density with an increase of cell death on a black background. We suggest that apoptosis regulates the balance of pigment cells in the skin of medaka fish to adapt their body color to a particular background.     

Changes in marine turbot (Scophthalmus maximus) epidermis and skin mucus composition during development from bilateral larvae to juvenile flat fish

Alike other flat fish, marine turbot has the particularity that changes from larvae with bilateral symmetry to adult with asymmetry, in terms of the position of the eyes. As expected, the skin configuration of this species is also affected by the development and transformation suffered by fish during metamorphosis. In this context, changes in the epidermis of marine turbot were studied using conventional staining and histochemical techniques using six lectins (UEA-I, PNA, RCA-I, WGA, Con A and SBA). During development from larvae to juvenile (3–300 days post-hatching), the epidermis increased in both thickness and the number of cell layers. In fact, the simple cuboidal epithelium observed in larvae at day 3 already became stratified at days 10–12, which sequentially increase in thickness with fish development. Turbot epidermis is composed basically of four cell types: epithelial and mucous or secretory cells that are present through the development, and pigmented cells and a type that the authors described as club-like cells that appear during and post-metamorphosis. The Alcian blue-periodic acid Schiff (AB-PAS) histochemical method revealed the presence of neutral glycoconjugates in mucous and club-like cells at post-metamorphic stages of fish. Accordingly, lectin analysis showed mucous cells containing glycoproteins rich in fucose (UEA-I labelling) and glycoconjugates rich in the sequence galactose-N-acetyl galactosamine (PNA and RCA-I labelling) when this cell type appears. Interestingly, melanophores were observed in the dorsal epidermis of post-metamorphic juveniles. This type of cell contains a black-to-brown pigment that provides the skin the typical colour of this fish species. Changes in mucous coat composition were observed during fish development, which was attributed to different roles of the glycoconjugates.     

Ultrastructural features of mitochondria-rich cells in stenohaline freshwater and seawater fishes

In order to elucidate the functional significance of accessory cells in freshwater fishes, such as the rainbow trout, which displays a poor adaptability to seawater life, a search for such cells was performed in two stenohaline freshwater fishes: the loach and the gudgeon. Accessory cells were never encountered in these species; but, in contrast, two types of chloride cells were observed consistently that strikingly resembled the alpha- and beta-cells previously described in the guppy, a freshwater-adapted euryhaline fish. The alpha-cell, a pale and elongated chloride cell, was located at the base of the secondary lamellae in close contact with the arterioarterial pillar capillary. Darker, ovoid chloride cells resembling the beta-cell were found exclusively in the interlamellar region of the primary epithelium facing the central venous sinous. The latter cells frequently formed multicellular complexes linked together by deep, narrow, apical junctions. In another experiment, a stenohaline seawater fish, the turbot, was adapted to diluted 5% saltwater and to fresh water. In seawater, the gill epithelium contained only one type of chloride cell, always associated with accessory cells. Due to numerous cytoplasmic interdigitations between the accessory cells and the apical portion of the chloride cell, there was a noticeable increase in the length of the shallow apical junction, sealing off the intercellular space between the two cell types. In 5% saltwater, there was a decrease in the number of these interdigitations and a concomitant decrease in the length of the shallow apical junction. In fresh water, chloride cells were partially or completely separated from the outside medium by modified accessory cells. It is thus concluded that accessory cells are found exclusively in fish living in seawater or preadapted to seawater and that they probably are involved in the formation and modulation of paracellular pathways for ionic excretion. In contrast, the respective roles of the two types of chloride cells observed in freshwater fishes are still to be determined.     

Immunohistochemical detection of ACTH and MSH cells in the hypophysis of the hermaphroditic teleost, Diplodus sargus

Hypophyseal ACTH and MSH cells were immunohistochemically characterised in the teleost fish, Diplodus sargus, using anti-ACTH (1-24) and anti alpha-MSH polyclonal antisera. ACTH cells were found both in the pars distalis and in the pars intermedia. In the former region, they appeared small, round-shaped and clustered; in the latter, they were either small or large and elongated. Moreover, a few ACTH-immunoreactive cells resembling microglia were present in the neurohypophysis. Conversely, MSH cells were found only in the pars intermedia, and were similar to the larger ACTH cells of the same region. In the pars intermedia, co-localisation of ACTH and MSH immunoreactivity in the same cell was revealed by double immunostaining, though the two hormones were also observed in distinct cell types. The distribution of ACTH cells appeared quite uniform, without any marked difference between the specimens tested. Conversely, MSH cell amount varied according to the stage of the sexual cycle of this teleost fish, which is characterised by protandrous hermaphroditism. In fact, a lower amount of MSH cells were observed in females, whereas no significant difference was found between immature and male specimens.     

Rapid morphological oscillation of mitochondrion-rich cell in estuarine mudskipper following salinity changes

Morphological changes in the chloride cells or mitochondrion-rich (MR) cells in the skin under the pectoral fin of the estuarine mudskipper (Periophthalmus modestus) were examined in relation to intertidal salinity oscillation in river mouth. MR cells were distinguished between those in contact with the water (cells labeled with both mitochondrial probe DASPEI and Concanavalin-A, an apical surface marker of MR cells) and those that are not (DASPEI-positive only). After transfer of the fish from seawater to freshwater, no difference in the total MR cell density was observed, but the subpopulation of MR cells that are Concanavalin-A-positive decreased dramatically within 30 min. After 6 hr in freshwater, the fish were returned to seawater; the number of Con-A-positive MR cells increased to the initial levels rapidly. Thus, in seawater, mudskippers seem to open the apical crypts of the MR cells to secrete salt; in freshwater, they close the crypt of the MR cells tentatively, and tolerate hypotonicity until the rising tide. This unique response of chloride cells may also be seen in gills of other estuarine species.     

Parabiosis and transplantation models show no evidence of circulating dermal fibroblast progenitors in bleomycin-induced skin fibrosis

To test the hypothesis of an extra-dermal origin of dermal fibroblasts, parabiosis, and transplantation models were developed utilizing a collagen promoter green fluorescent protein (GFP) reporter transgene expressed in dermal fibroblasts. Parabiotic pairs were treated with bleomycin to induce the skin fibrosis that was evaluated for a dense deposition of collagen and inflammatory cell infiltrates in the thickened dermis in comparison with parabiotic pairs treated with saline. Although, in all cases, repeated injection of bleomycin for 4 weeks induced skin fibrosis, only a few GFP positive cells were detected in skin samples from some of the treated non-transgenic mice. Unexpectedly, similar results were observed in saline treated controls. Furthermore, bone marrow chimeras were created in which non-transgenic recipient mice received injections of bone marrow cell preparations isolated from pOBCol3.6GFP transgenic mice. After bone marrow chimerism had been successfully established, fibrotic lesions in the skin were induced by local bleomycin injections. Donor GFP expressing cells were observed in the skin from all recipient mice. However, no difference in the presence of GFP expressing cells was observed between non-treated mice or mice treated with bleomycin or saline. A large number of GFP expressing cells were observed in the lung preparations from all chimeric mice. Mac-3 antibody immunostaining confirmed a macrophage phenotype for these GFP expressing cells suggesting the expression of the pOBCol3.6GFP transgene in a non-collagen producing cell. Based on these observations, we found no evidence of circulating dermal fibroblast progenitors that participate in the development of bleomycin-induced skin fibrosis.     

Fine structure of the adenohypophysis in immature sockeye salmon,Oncorhynchus nerka

Summary The ultrastructure of the secretory cells of the adenohypophysis of juvenile sockeye salmon was investigated. Pituitary glands were collected from immature fish transferred experimentally to sea water and subsequently returned to fresh water. The rostral pars distalis contained three cell types: ACTH cells, prolactin cells, and non-secretory cells. The prolactin and non-secretory cells were joined together in the form of follicles by desmosomes and they both had cilia and microvilli projecting into the follicle lumen. Various follicular structures such as lumen, multivesicular structures, and peripheral basement membrane are discussed as possible sites of prolactin cell granule release. The columnar ACTH cells were found at the junction of the rostral pars distalis and the neurohypophysis. The cytoplasmic granules in these cells were characteristically separated from their limiting membrane by a clear space. Multivesicular structures were also found in association with this cell type. The caudal pars distalis also contained three cell types: one acidophil (putative somatotrop) and two basophils (putative thyrotrops and gonadotrops), all of which were similar to those described in adult fish. The pars intermedia contained only one cell type. They appeared to be active cells and were characterized by containing membrane-bounded granules similar to those found in the ACTH cells. Changes in ambient salinity had no apparent effect on any cell type described.The work was supported by a grant in aid of research from the National Research Council of Canada. We wish to thank Mr. R. Lindsay, Mr. C. Cooper, and Mr. G. Longworth for their technical assistance. We would also like to thank Mr. S. Killick of the International Pacific Salmon Fisheries Commission for his assistance in the collection of fish and Dr. H. Cook for his helpful discussion of the project. This paper is No. 058 in the University of Guelph Migration Series.     

Transgenic Medaka Fish Bearing the Mouse Tyrosinase Gene: Expression and Transmission of the Transgene Following Electroporation of the Orange-Colored Variant

Transgenic fish bearing the mouse tyrosinase gene (mg-Tyrs-J) were produced by transfection into fertilized eggs of the homozygous normal orange-colored variant of medaka fish, Oryzias latipes, by means of electroporation. Of 589 eggs transfected, 38 fish (6%) exhibited brownish wild-type skin pigmentation, which was discernible from control siblings. Light microscopy of the skin from the founders thus generated disclosed that 1) melanization occurred and was restricted to melanophores formed presumably from preexisting amelanotic melanophores, 2) there was a wide variation in the degree of melanization observed among melanophores, and 3) no melanin deposition was recognized in xanthophores or leucophores. Immunofluorescence using an antibody raised against mouse tyrosinase disclosed that melanophores at varying stages of maturation were reactive. Thus, it was shown that the transgene in medaka fish expressed its action in a cell type-specific manner. Crossing of transgenic founders with homozygous orange-colored variant fish yielded two groups of offspring expressing either the wild-type or the orange-colored skin pigmentation at an approximate ratio of 1:1. Crossing between founders exhibiting wild-type pigmentation yielded only offspring with melanized skin. Skin melanophores in these offspring formed vertical stripes, which are rare in this species. The hereditary basis of melanized skin was demonstrated in matings of Fl progenies, which resulted in similar degrees of melanization over whole skin melanophores. The sum of these findings implied that the transgene is expressed as a dominant character gene and is transmitted through germ cell lines according to the Mendelian law. PCR analysis combined with nested PCR technique strongly suggested that the transgene was integrated into the medaka genome, even though the copy number deduced from gel banding was largely diminished, possibly as a result of fragmentation or instability within the medaka genome.     

Expression of the polymeric Immunoglobulin Receptor (pIgR) in mucosal tissues of common carp (Cyprinus carpio L.)

The mucosal immune system seems to be an important defence mechanism for fish but the binding of IgM in mucosal organs is poorly described in fish. In this study the gene encoding the polymeric Immunoglobulin Receptor (pIgR) in carp has been isolated and sequenced from a liver cDNA-library and aligned with other species. The pIgR of carp consists of 2 Ig domains, a transmembrane and an intracellular region, together 327 amino acids. In situ hybridisations with sense and anti-sense DIG-labelled pIgR RNA probes were performed on liver, gut and skin of common carp (Cyprinus carpio L.) and in these organs only anti-sense probes were found to hybridise. In liver the majority of hepatocytes was stained around the nucleus. In gut and skin, staining could be detected around the nucleus of the epithelial cells, but in gut also a subpopulation of lymphoid cells was stained in epithelium and lamina propria. The specific in situ hybridisation of the epithelia and hepatocytes coincides with the in situ binding of FITC-labelled carp IgM to the same cells. RT-PCR results indicate the expression of the pIgR gene in all lymphoid organs of carp, but not in muscle. Macrophages/neutrophils enriched by adherence or sorted B cells (MACS) did not show expression of the pIgR gene and are excluded as the pIgR expressing lymphoid cells in the intestine. The relevance of pIgR staining and gene expression in mucosal organs is discussed.     

Damage to the gills,skin and other tissues by lysenin and the coelomic fluid of the earthworm Eisenia foetida in two teleosts,Tanichthys albonubes and Oreochromis mossambicus

Lysenin is a 33-kDa protein found in the coelomic fluid (CF) of the earthworm Eisenia foetida. Purified lysenin binds specifically to sphingomyelin (SM). In the present studies, we found that the white cloud mountain minnow Tanichthys albonubes and the Mozambique tilapia Oreochromis mossambicus died in solutions of lysenin (at concentrations above 2.5 microg/ml) and CF (0.6%, v/v) within 2 h. The gills of both species of fish were damaged similarly by lysenin and by CF. Most gill lamellae became irregularly bent or curled, with swelling of the epithelial cells of the lamellae. Red blood cells in the lamellar vascular sinuses, in the central venous sinuses, and in the blood vessels of the entire body became swollen and lysed, choking the sinuses. Epithelial cells in the skin were also damaged. When fish of both species were treated with lysenin or CF that had been incubated with SM-liposomes, they did not die. Their behavior remained normal and there was no damage to any cells or tissues. These findings suggest that SM might be involved in the lethal effects of lysenin and CF. It is likely that purified lysenin and lysenin in CF bound to SM in the cell membranes of the tissues mentioned above, damaging the cells. The presence of SM in the gills and skin was confirmed, supporting this hypothesis. The damage to gills and hemolysis might have resulted in lethal respiratory problems. Damage to the skin might disturb the exchange of ions through the skin, hastening death. Damage by lysenin and CF to epithelial cells of the cornea and the wall of the oral cavity was also recognized, but there was no such damage to the intestine.     

Mucous cell responses in gill and skin of brown trout Salmo trutta fario in acidic, aluminium-containing stream water

Morphometric examination was carried out on the gills and skin of wild and caged hatchery brown trout Salmo trutta fario in an acidic (pH 4.9 to 5.4; Al 203 to 250 microg l(-1)) and in a non-acidic (pH 6.7 to 7.0; Al 27 to 67 microg l(-1)) stream in the Vosges Mountains (NE France) to assess the sublethal effects of acidic water on the mucous cell response. The caged fish were randomly collected after 2, 4, 7 and 11 d and the wild fish were obtained by electrofishing. After 2 d, a reduction of both mucous cell (MC) number and size was observed in the gills of fish held in the acidic stream, suggesting a massive mucus discharge. Hyperplasia and hypertrophy of cells immediately followed this mucus secretion. In the same fish population, skin examination showed a slight and delayed decrease of MC number but a significant increase of cell size. The number of mucous cells of gills and skin was similar in both wild trout populations, whereas a significant MC hypertrophy was observed in the wild fish of the acidic stream. The present field experiment indicates that caged fish could be useful as early indicators of acidification. In addition, the examination of wild populations suggested the occurrence of adaptive mechanisms, information that might be of importance in the context of river recovery programs.     

Ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs) are acquired from their diet by medaka fish (Oryzias latipes) but not by SKH-1 hairless mice

To assess whether vertebrates can acquire, from their diet, ultraviolet radiation-absorbing mycosporine-like amino acids (MAAs), medaka fish and hairless mice were maintained for 150 and 130 days, respectively, on diets either including Mastocarpus stellatus (rich in MAAs) or the same diets without this red alga. In medaka, the MAAs palythine and asterina-330, present in trace quantities in the diet with added M. stellatus, were present in significantly greater quantities in the eyes of fish fed this diet than in the eyes of control fish. Only traces of MAAs were present in the skin of medaka fed the diet containing MAAs. Shinorine, the principal MAA in M. stellatus, was not found in any tissues of medaka, which raises questions about the specificity of transport of MAAs. In hairless mice, no dietary MAAs were found in the tissues of the eyes, skin, or liver after maintenance on the experimental diet. Low concentrations of shinorine were present only in the tissues of the small and large intestines. These results indicate that MAAs are acquired from their diet and translocated to superficial tissues by teleost fish, but that mammals may be incapable of such. Thus, dietary supplementation with MAAs may be useful in aquacultured species of fish, but MAAs as ‘dietary sunscreens' may not be an option for mammals, including humans. Nevertheless, our demonstration of the uptake of shinorine by human skin cancer cells in culture raises evolutionary questions regarding the organ specificity of the capacity for the cellular transport of MAAs.     

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.     

Histological process and dynamics of germ cell degeneration in pejerrey Odontesthes bonariensis larvae and juveniles during exposure to warm water

Elevated temperature causes degeneration and disappearance of the germ cells in the males of scrotal mammals. It was recently shown that heat-induced germ cell degeneration occurs also in fish but, unlike in mammals, it occurs not only in males but also in females. The purpose of this study was to clarify the histological process and dynamics of heat-induced germ cell disappearance in pejerrey Odontesthes bonariensis larvae and juveniles. Monosex and mixed-sex fish produced by thermal manipulation of sex (temperature-dependent sex determination) were subjected to 29 degrees C for periods between 1 and 12 weeks, and used to analyze, by histological methods, the changes in gonadal size and the number of normal and degenerating germ cells. Groups exposed to 29 degrees C for 8-12 weeks were subsequently transferred to 24 degrees C to verify if any gonadal damage would be permanent. Germ cell degeneration, histologically characterized by nuclear pyknosis or eosinophilia and cytoplasmic eosinophilia, was observed with increasing frequency at higher temperatures (29>24> 17 degrees C) and more in males than in females. Clear degenerative changes in the germinal epithelium usually began within one week of exposure to 29 degrees C and appeared clearer in females than in males. Complete loss of germ cells was observed only in individuals exposed for periods of 8-12 weeks to 29 degrees C but no treatment produced 100% sterile fish. Germ cells that remained in the gonads after exposure to 29 degrees C retained the capacity to rapidly recolonize germ cell-depleted areas, suggesting that the associated somatic cells in the gonads are little or not affected by this temperature.     

Attachment-inducing capacities of fish tissue extracts on oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae)

When oncomiracidia of Neobenedenia girellae (Monogenea, Capsalidae) were incubated in wells with lyophilized extracts of fish skin epithelia on the bottom, some attached to the well bottom with the haptor unfolded and shed the ciliated epidermal cells. Based on these morphological changes in oncomiracidia, we developed a new assay method to examine the attachment-inducing capacities of various fish extracts for oncomiracidia. Attachment-inducing capacities were found only in extracts of fish skin epithelium and not in other fish extracts. No significant difference in capacities was observed among extracts of skin epithelia of 4 fish species. Wheat germ lectin and concanavalin A suppressed capacities in extracts of skin epithelia of Japanese flounder and yellowtail, respectively. Suppressed capacities were recovered by adding sugars that bound specifically to these lectins. These results indicate that some sugar-related chemical substances that exist specifically in fish epithelium induce the attachment of N. girellae oncomiracidia.     

Probing the skin permeation of fish oil/EPA and ketoprofen-3. Effects on epidermal COX-2 and LOX

This work employed immunocytochemistry (ICC) techniques to study the effect of topically applied fish oil and ketoprofen on cyclooxygenase (COX-2) and lipoxygenase (LOX) within freshly excised porcine ear skin. Maintained in Hanks buffer immediately post excision, full thickness membranes were mounted in Franz diffusion cells and dosed with 1 ml of individual formulations containing ketoprofen, fish oil or both. At different timepoints, the diffused areas were recovered and relative activities of COX-2 and LOX determined. It was found that the fish oil formulation qualitatively inhibited the expression of both COX-2 and LOX enzymes. As expected, ketoprofen had no effect upon LOX expression but a significant decrease on COX-2 expression was observed. The formulation containing both fish oil and ketoprofen proved to be the most effective at inhibiting the expression of both COX-2 and LOX. Considered together with data from earlier papers, a mechanism of EPA permeation enhancement by ketoprofen may be elucidated and also show the ability of such a formulation to inhibit these enzymes and thus indicate the efficacy of such a formulation.