Corticosteroid activity, in vitro, in interrenal tissue of Atlantic salmon (Salmo salar) parr. 1. Synthetic profiles.

Fresh interrenal tissue of Atlantic salmon (Salmo salar) parr transformed [3H]pregnenolone and [14C]progesterone, in vitro, to double-labeled cortisol and corticosterone with yields of less than 3% 3H and 5% 14C at 0-5 degrees, 8-10 degrees, and 22-24 degrees over various incubation periods. Incubations at 22-24 degrees gave the highest yields. A time-yield study at 22-24 degrees for 0.5 to 3 hr in late March showed that metabolic patterns did not change with time as metabolic yields increased. Double-labeled metabolites, 11-deoxycortisol, 21-deoxycortisol, and 17 alpha-hydroxyprogesterone, were all detected in hatchery-reared Annapolis River stock in April, but not in November. The 3H:14C ratio of cortisol was significantly lower (P < 0.05) in late April than in early November. Isotopic ratios of corticosterone, but not cortisol, differed significantly (P < 0.05) between immature and precocious parr in November. The isolation of double-labeled corticosterone from all incubations, except immature parr in November, may indicate a metabolic role for the 17-deoxycorticosteroids. No differences in the histology of interrenal tissues were found between Spring (April-May) and Autumn (October-November) parr. Sulfuric acid chromogens on TLC typical of cortisol, corticosterone, and 11-deoxycortisol and other, less polar delta-5-steroids, but not cortisone, in amounts estimated to be > or = 0.1 microgram, were detected in head kidney tissues (0.5-4.5 g) and blood plasma (7-18 ml) of parr in July, late November, and early December.

Corticosteroid activity, in vitro, in interrenal tissue of Atlantic salmon (Salmo salar) parr. 2. Comparative synthetic profiles from Nova Scotian stocks.

Differences observed in autoradiogram profiles and isotope ratios of corticosteroid metabolites (such as cortisol), in vitro, in interrenal activity between and within hatchery reared salmon (Salmo salar) parr stocks were not observed in corresponding wild stocks. Wild fish from nine Nova Scotian rivers (Annapolis, Black, East, Gold, LaHave, Medway. Mushamush, Salmon, and Stewiacke Rivers), sampled during May to December all showed similar corticosteroid profiles. Differences were observed in corticosteroid profiles in December between: (1) hatchery stock held in acidic and circumneutral rivers; and (2) hatchery stock held in cages in a limited river, wild stock from this river, and hatchery stock maintained at the hatchery.

In vitro biosynthesis of 17 alpha,20 alpha,20 beta-dihydroxy-4-pegnen-3-one by the ovaries, testes, and head kidneys of the Atlantic salmon Salmo salar.

Ovaries, testes, and head kidneys of sexually mature Atlantic salmon, Salmo salar, biosynthesized 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) from equimolar amounts of [3H]pregnenolone plus [4-14C]progesterone in vitro. The 3H:14C isotope ratios of steroid metabolites indicated that the biosynthetic pathways to 17 alpha,20 beta-diOHP in the testes differed from those observed in the ovaries and head kidneys. [4-14C]Progesterone appeared to be the principal precursor of 17 alpha,20 beta-diOHP in the testes, whereas both precursors were efficiently biotransformed to 17 alpha,20 beta-diOPH in the ovaries and head kidneys. 17 alpha-Hydroxy-4-pregnen-3-one (17 alpha-OHP) was the immediate precursor to 17 alpha,20 beta-diOHP in all tissues. However, appreciable amounts of 17 alpha,20 beta-diOHP accumulated in vitro in the testes only in the presence of exogenous [14C]progesterone. Incubation of the testes, ovaries, and head kidneys with [14C]pregnenolone resulted in high yields of 17 alpha,20 beta-diOHP in the ovaries and head kidneys but no detectable amounts of the steroid in the testes. The results confirm that progesterone is the favored precursor to 17 alpha,20 beta-diOHP in the testes. The results also suggest that the head kidneys may be an excellent cellular source of 17 alpha,20 beta-diOHP in both male and female. Atlantic salmon and may play an important role in the sexual maturation process in this fish. It is suggested that biosynthetic control mechanism affecting 17 alpha,20 beta-diOHP synthesis and/or spermiation and ovulation may differ in male and female Atlantic salmon.

A simple, rapid, and precise direct radioimmunoassay method for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in salmon plasma.

This report describes a radioimmunoassay method for 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHP) in the blood plasma of Atlantic salmon (Salmo salar). 3H-labeled 17 alpha,20 beta-diOHP was synthesized from 17 alpha-[3H]hydroxy-4-pregnen-3-one (17 alpha-OHP) by enzymatic reduction of the C-20 oxo group and the simultaneous oxidation of coenzyme-reduced nicotinamide adenine dinucleotide. Antiserum to 17 alpha,20 beta-diOHP-3-CMO-BSA exhibited high specificity: all steroids tested against the antiserum gave less than 1% cross-reactivity at 50% displacement. Crude dichloromethane extracts of small volumes (50 microliters or less) of plasma can be assayed directly without prior purification. The working range is 5 to 300 pg. Interassay and intraassay coefficients of variation were less than 10%. Various parallelism tests showed high accuracy and sensitivity. The assay is simple and rapid and at least 75 samples can be easily assayed in duplicate by two analysts in 1 day.

Isolation and identification of testosterone from the serum and testes of the American lobster (Homarus americanus).

Testosterone was isolated and quantified from male lobster serum and testes by solvent extraction, sequential thin-layer chromatography, and high-performance liquid chromatography. The identity of the isolated steroid was established by its isopolarity and isomorphicity with authentic radiolabeled testosterone and its acetate derivative. The concentrations of testosterone were determined by high-performance liquid chromatography, by a double-isotope derivative assay, and by radioimmunoassay. The concentrations of testosterone as determined by the three methods were the same and were 0.3 ng/ml and 14.3 ng/g in lobster serum and testes, respectively.

Bioconversion of steroids by the testes of the American lobster, Homarus americanus, in vitro.

Lobster testes have been demonstrated to contain steroid 20-ketone reductase by their capacity to convert [14C]progesterone to 20 alpha-dihydroprogesterone (20 alpha-DHP). The major product was isopolar and identical with 20 alpha-DHP during thin-layer chromatography, high-pressure liquid chromatography, mass spectrometry, and acetate derivative formation. The vas deferens from the lobster was also capable of the same conversions but to a lesser extent. Lobster testes converted [14C]pregnenolone to a major product identified as 20-dihydropregnenolone (20-DHPe) by mass spectrometry after purification by thin-layer chromatography and high-pressure liquid chromatography. The presence of delta 5,3 beta-ol dehydrogenase and delta 5,delta 4-isomerase in the lobster were also indicated. [14C]Cholesterol was not transformed to steroid hormones by lobster testes under the same experimental conditions.

The blood sex hormone levels in sexually mature male atlantic salmon (Salmo salar) in the Westfield River (pH 4.7) and the Medway River (pH 5.6), Nova Scotia.

Blood samples were taken from fourteen sexually mature (ripe) male Atlantic salmon (Salmo salar), eight from the acidic Westfield river (pH 4.7) and six from the less acidic (pH 5.6) Medway river to determine if there is any difference in sex hormone production in the fish in the two rivers. The plasma levels of the two principal male sex hormones, testosterone and 11-ketotestosterone, which normally peak at functional sexual maturity, were significantly lower in salmon from the more acidic Westfield river compared to those in salmon from the Medway river. Since these fish were of the same stock, and were in the same state of sexual maturation, it is suggested that the more acidic Westfield River has affected the production and/or utilization of sex hormones in this species.

Testicular abnormalities in cod (Gadus morhua) fed Aroclor 1254.

Cod were fed herring containing Aroclor 1254, a polychlorinated biphenyl (PCB), at diet levels of 0, 1, 5, 10, 25, and 50 microgram/g for a period of 5 1/2 months. Histological examinations of the gonads of surviving male fish revealed various testicular abnormalities in 9 of 17 PCB-fed fish but in none of four experimental control and four stock control fish. The abnormalities were observed in testes that were either at functional maturity or in a stage of rapid spermatogenic proliferation but not in testes that were sexually immature or regressed. The testicular abnormalities included disorganization of lobules and spermatogenic elements, inhibition of spermatogenesis, fibrosis of lobule walls, fatty necrosis, and, in one case, total disintegration of the elements in many lobules. There was a significant uptake of PCB by testicular and liver tissues of fish that were fed the higher levels (greater than 1 microgram/g) of Aroclor 1254.

A study of the effects of methyl mercury, cadmium, arsenic, selenium, and a PCB, (Aroclor 1254) on adrenal and testicular steroidogeneses in vitro, by the gray seal Halichoerus grypus.

An in vitro study on the effects of the contaminants polychlorinated biphenyl (Aroclor 1254) (PCB), methyl mercury (MeHg), arsenic (As), cadmium (Cd), and selenium (Se) on the biosynthesis of steroid hormones in the gray seal (Halichoerus grypus) indicated altered steroid biosynthesis. Biotransformed delta4-androstene-3, 17-dione (delta 4A), dehydroepiandrosterone, 11-ketotestosterone (11-KT), and testosterone (T) were detected in all seal testicular incubates. Yields of 11-KT were greatly increased in the presence of Aroclor 1254. All contaminants except As and Se stimulated the in vitro biosyntheses of T, with the greatest increase in production of T being in the Cd-treated tissue. Cortisol (F), corticosterone (B), aldosterone (ALDO) but no cortisone (E), were biosynthesized by the seal adrenal tissue. Corticosterone (B) was the principal transformation product in all incubations with less B produced by the treated adrenals than by the control. The lowest yeild of B was achieved by the Se-treated adrenal. The yeild of ALDO was also lower in all contaminant treated incubations, with Se and Cd giving the greatest inhibition. More F was biosynthesized by all the treated adrenals than by the control. The greatest increase of production of F(6-fold) from progesterone was by the As-treated adrenal.