Sex differences in hepatic lipids of Toxaphene-exposed juvenile yellowtail flounder (Pleuronectes ferrugineus Storer).

Histochemical and biochemical effects of Toxaphene on liver were investigated in laboratory-bred female and male juvenile yellowtail flounder (Pleuronectes ferrugineus). Fish were fed uncontaminated food, or food contaminated with hexane (the solvent for Toxaphene) or with one of two concentrations of Toxaphene (0.02 or 0.2 microg/g fish/day) for 2 weeks. Males were more advanced in sexual maturity than females, although all were from the same year-class (0(+)). Liver tissue examined histochemically (Sudan black B, oil red O) revealed that Toxaphene affected storage of total and neutral lipids according to sex and dose. The sexes differed in the amount of total and neutral lipids. Neutral lipid droplets were considerably larger in the liver of females. Lipids were extracted and analyzed using the latroscan TLC/FID system. Triacylglycerols comprised the majority of lipids. Animals exposed to the lowest concentration of Toxaphene stored low amounts of total and neutral lipids and high amounts of polar lipids, while animals exposed to a 10 times higher concentration showed the reverse. Sterols were highest in animals exposed to the highest dose. Thus Toxaphene can alter the lipid composition in the liver of yellowtail flounder, which may have consequences for physiological processes involving the liver, such as lipid metabolism and reproduction.

Effect of toxaphene on isolated hepatocytes of the yellowtail flounder, Pleuronectes ferrugineus storer.

The histochemical and enzyme cytochemical effects of Toxaphene were investigated using isolated hepatocytes in suspension culture from laboratory-bred juvenile, female yellowtail flounder (Pleuronectes ferrugineus). Hepatocytes were kept in suspension culture for 4 days and exposed for 3 days to a control medium, to a medium with hexane (the solvent of Toxaphene), or to a medium with Toxaphene in two different concentrations (1 and 10 mocrog/ml). Subsequently, the cultivated cells were examined histochemically (Sudan black B, oil red O, Schmorl's reaction) and enzyme cytochemically (acid phosphatase, NADPH-ferrohemoprotein reductase). Toxaphene decreased the viability of the isolated cells significantly, as compared to the control suspensions. Toxaphene also increased the storage of total and neutral lipids (as demonstrated by Sudan black B and oil red O, respectively) in a dose-dependent manner. In addition, Toxaphene increased the enzymatic activity of acid phosphatase, and increased the storage of lipofuscin pigment (as demonstrated by the Schmorl's reaction) within the hepatocytes, suggesting an increase in the number and/or size of the lysosomes. Hexane did not have a significant toxic effect on the isolated hepatocytes. It is concluded that Toxaphene is potentially toxic to fish in a marine environment and that this in vitro system may provide a model for assessing the direct effect of various toxicants on fish hepatocytes.

Immunocytochemical localization of bovine thyrotropin and thyroid auto-antibodies in porcine thyrocytes.

Interactions of receptor-bound bovine thyrotropin (bTSH) and immunoglobulins G from sera of patients with Graves' (G-IgG) or Hashimoto's (H-IgG) disease with porcine thyrocytes were studied by immunocytochemistry. Porcine thyroid fragments were fixed and prepared for immunoreaction or enzymatically dissociated with collagenase and dispase II. The dispersed cells were cultured in primary monolayer in a hormone-free medium or in a medium with bTSH (150 micrograms/ml) for 7 days. After immunostaining the thyrocytes in fragments and monolayers were stained with periodic acid Schiff (PAS) or with PAS and haemalum. Cultivation of the isolated thyrocytes in bTSH-enriched medium leads to a monolayer with globular aggregates, i.e., reconstructed three-dimensional follicles. Follicular cells in these monolayers and in fragments give a weak to moderate immunoreaction to anti-bTSH and a strong reaction to G-IgG and H-IgG (vs. control IgG). Precipitate is found particularly in the perinuclear area and to a lesser degree throughout the cytoplasm. Cells cultured in the absence of bTSH show minimal immunoreaction to anti-bTSH, but moderate reaction to G-IgG and H-IgG. Preincubation with bTSH leads to a strong reduction of immunoreaction to G-IgG but does not affect reaction to H-IgG. Morphological results indicate that G-IgG and H-IgG interact with the same cellular sites as bTSH. Hashimoto's disease antibodies bind to a determinant on the TSH receptor separate from the one on which TSH and Graves' IgG bind.

Effect of 17 alpha-methyltestosterone, estradiol-17 beta and synthetic LHRH on production of gonadotropic hormone in pituitaries of rainbow trout (organ culture).

Pituitary glands from 6-month-old sexually immature female rainbow trout, Salmo gairdneri, were kept in organ culture for 48 or 72 h. Certain groups of pituitaries were cultivated for 48 h on either control medium or medium with 17 alpha-methyltestosterone (MT), or with estradiol-17 beta (E2) in concentrations of 8.5 X 10(-7) M. Other groups of pituitaries were cultivated for 72 h on control medium, or for 48 h on either control medium or MT-medium or E2-medium, and subsequently for 24 h on medium with synthetic LHRH in concentrations of 8.5 X 10(-7) M and 8.5 X 10(-10) M. Gonadotropic (GTH) cells are identified by Alcian Blue-Periodic Acid Schiff-Orange G staining and the double-antibody immunoenzyme-cytochemical technique using anti-carp beta GTH as the first antibody. A quantitative histological procedure was used to study the nuclear size of the GTH cells in response to the different hormones. Secretory activity was estimated by measuring the gonadotropin (GTH) content in extracts of pituitaries, plasma, and the culture media every 24 h by radioimmunoassay. Cultivation on MT- or E2-enriched medium results in an increase of the total amount of GTH in the pituitary and medium, an accumulation of GTH in GTH-cells (approximately 20 percentage points) and an increase in their nuclear size, indicating a stimulation of GTH synthesis. However, autonomous GTH-release is not affected by these steroids. Subsequent cultivation of the pituitaries for 24 h with LHRH causes stimulation of GTH synthesis (approximately 20 percentage points). Preincubation with steroids increases the GTH synthesis capacity of LHRH only when used in a concentration of 8.5 X 10(-10) M. Moreover, 8.5 X 10(-7) M LHRH causes a stimulation of GTH-release. Preincubation of the pituitaries with steroids increases the responsiveness of GTH-cells to LHRH. It is concluded that GTH-production in pituitaries of immature female rainbow trout can be directly influenced by gonadal steroids and by a hypophysiotropic substance.

Fine structure and function of isolated gonadotropic cells as revealed from pituitaries of immature rainbow trout, Salmo gairdneri, by means of a new enzymatic dispersion technique.

A procedure has been developed for dissociating pituitary glands of juvenile rainbow trout, Salmo gairdneri, producing a preparation of single dispersed pituitary cells in which morphological and functional integrity is preserved. The pituitaries are dispersed by sequential treatment with 0.1% collagenase, 0.04% ethylene-diamine-tetra-acetic acid (EDTA) and 0.125% dispase. The cell yield is 0.3--0.35 x 10(6) cells per pituitary with a cell viability percentage of 95 +/- 1% and single cell percentage of 87 +/- 4%. The isolated cells are kept in a suspension system and the gonadotropic cells are identified by the double antibody immuno-enzyme-cytochemical technique using anti-carp-beta-gonadotropin as first antibody. Secretory activity is estimated by measuring the gonadotropin content in cells and culture media by radioimmunoassay. Isolated cells show an autonomy of gonadotropin secretion. 17 alpha-Methyltestosterone both in vivo and in vitro stimulates the production of gonadotropin in the cells and seems to inhibit its release from the cells. It is concluded that this in vitro system can be used as a model for studying the control of gonadotropic cells in juvenile rainbow trout.