Interpretation of plasma cortisol levels in wild-caught fish is confounded by the stress of capture. Measurement of cortisol metabolites in fish bile could provide a method for assessing the stress level of wild fish because the time-lag for metabolism, conjugation and excretion into bile avoids the effects of sampling stress. To determine which biliary metabolite(s) to target, four Atlantic cod, Gadus morhua L., were injected with radioactive cortisol. After 22 h, the bile was collected and found to contain 30% of the injected activity. Cortisol metabolites were extracted from diluted bile samples using solid phase extraction and the radioactive metabolites separated by several different chromatographic procedures. The metabolites were predominantly present as sulfates (95%) with the remainder being glucuronidated. Chromatography split the sulfates into at least seven peaks, and acid solvolysis (which removes sulfate groups from steroids) generated four major radioactive steroids. These were identified, using microchemical reactions and re-crystallization to constant specific activity, as: 11β,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), 3α,11β,17,21-tetrahydroxy-5β-pregnan-20-one (tetrahydrocortisol; THF), 3α,17,21-trihydroxy-5β-pregnane-11,20-dione (tetrahydrocortisone; THE) and 3α,17,20β,21-tetrahydroxy-5β-pregnan-11-one (β-cortolone). The last of these was the most abundant, and thus a likely target for a biliary stress assay. Studies were also carried out to determine the best method for extraction and solvolysis of sulfates. Solid phase extraction (i.e. using octadecylsilane) was found to be too unreliable for routine use. Even though the extraction efficiency could be improved by acidifying the bile, this caused premature solvolysis of sulfated steroids. Acid solvolysis of unextracted bile worked best (c. 90% converted to free steroids) on volumes that were 1 μL or lower. Aryl sulfatase digestion of unextracted bile did not work well (only 20% of radioactivity was converted to free steroids).

Progestins (progestogens, C21 steroids) have been shown to regulate key physiological activities for reproduction in both sexes in all classes of vertebrates except for Agnathans. Progesterone (P) and 15α-hydroxyprogesterone (15α-P) have been detected in sea lamprey (Petromyzon marinus) plasma, but the expression patterns and functions of putative progestin receptor genes have not yet been investigated. The first objective of this study was to determine the differences in mRNA expression levels of nuclear progestin receptor (nPR) and the membrane receptor adaptor protein 'progesterone receptor membrane component 1' (pgrmc1) in putative target tissues in males at different life stages, with and without lamprey GnRH-I and -III treatment. The second objective was to demonstrate the function of progestins by implanting prespermiating males (PSM) with time-release pellets of P and measuring the latency to the onset of spermiation and plasma concentrations of sex pheromones and steroids. The third objective was to measure the binding affinity of P in the nuclear and membrane fractions of the target tissues. Expression levels of nPR and pgrmc1 differed between life stages and tissues, and in some cases were differentially responsive to lamprey GnRH-I and -III. Increases in nPR and pgrmc1 gene expressions were correlated to the late stages of sexual maturation in males. The highest expression levels of these genes were found in the liver and gill of spermiating males. These organs are, respectively, the site of production and release of the sex pheromone 3 keto-petromyzonol sulfate (3kPZS). The hypothesis that pheromone production may be under hormonal control was tested in vivo by implanting PSM with time-release pellets of P. Concentrations of 3kPZS in plasma after 1week were 50-fold higher than in controls or in males that had been implanted with androstenedione, supporting the hypothesis that P is responsible for regulating the production of the sex pheromone. P treatment also accelerated the onset of spermiation. Saturation and Scatchard analyses of the target tissues showed that both nuclear and membrane fractions bound P with high affinity and low capacity (KD 0.53pmol/g testis and 0.22 pmol/g testis, and Bmax 1.8 and 5.7 nM, respectively), similar to the characteristics of nPR and mPR in other fish. The fact that a high proportion of P was also converted in vivo to 15α-P means that it is not yet possible to determine which of these two steroids is the natural ligand in the sea lamprey.

The presence of the vertebrate steroids, testosterone (T) and 17β-estradiol in mollusks is often cited as evidence that they are involved in the control of their reproduction. In this paper, we show that a likely source of T in at least one species, the common mussel (Mytilus spp.), is from uptake from water. When mussels were exposed to waterborne tritiated T ([3H]-T) in a closed container, the radioactivity decreased rapidly and exponentially until, by 24h, approximately 35% remained in the water. The rate of uptake of radiolabel could not be saturated by concentrations as high as 16.5μgL-1 (mean measured) of non-radiolabeled T, showing that the animals have a very high capacity for uptake of T. At least 30% of the applied radioactivity could be extracted from the tissues of the animals with organic solvents and most of this (26% of the total applied radioactivity) was in the fatty acid ester fraction. Following alkaline hydrolysis, reverse phase HPLC and TLC, this fraction was shown to consist predominantly of 5α-dihydrotestosterone and 5α-androstane-3β,17β-diol, while T was a minor component. These steroids were definitively identified in the fatty acid ester fraction by mass spectrometry. Overall, less than 5% of the [3H]-T applied to the system remained untransformed at the end of exposure. After ten days of depuration there was no reduction in the total amount of radioactivity in the tissues, nor any changes in the ratio of the metabolites in the ester fraction. These findings show that any association between T presence and reproductive status or sex is confounded by their significant capacity for uptake, and that T undergoes extensive metabolism in mussels in vivo and therefore may not be representative of the androgenic burden of the animals. Consequently, measurements of T in mussel tissue offer little utility as an indicator of reproductive status or sex.

The androgen 11-ketotestosterone (11-KT) plays an important role in reproductive physiology and behaviour in male teleosts. In the three-spined stickleback, Gasterosteus aculeatus, the plasma concentrations of 11-KT are related to the breeding status of the fish. Sticklebacks are relatively small (generally less than 1g) and in order to obtain sufficient plasma for assay of 11-KT, it has been necessary in the past to sacrifice the fish. In this paper, we report on the development of a non-invasive procedure for measuring 11-KT, cortisol and androstenedione (Ad) in the three-spined stickleback. Validation of the procedure included the demonstration that the rate of release of steroids into the water was correlated to their plasma concentrations. Ten males that were kept at a low temperature and short photoperiod were moved to high temperature and long photoperiod to initiate spermatogenesis and breeding. Every two to four days, for a total of 53 days, males were removed and placed in a beaker containing 50-ml water for 30 min. The water was then processed by solid phase extraction for radioimmunoassay. Males were presented with females on days 13/14, 18/19 and 44/45. 11-KT was originally undetectable but built up gradually to reach an average release rate of between 1 and 2.5 ng/g/h between days 16 and 45 and then started to decline (but non-significantly). Ad release reached a plateau of 1 ng/g/h about day 20. However, from days 44/45 to 51, there was a highly significant rise in the rate of release of Ad to 5 ng/g/h. On days 44/45, five of the males mated successfully and five did not. However, there were no significant differences in 11-KT or Ad release rates between the two groups. Cortisol release rates fluctuated with no pattern throughout the study. The results show that it is possible to make measurements on sex and stress steroid production in sticklebacks without recourse to anaesthesia, bleeding or sacrificing the fish. The procedure is potentially a powerful tool for the study of the link between steroids and behaviour in this useful sentinel species.

Previous studies have shown that mussels can pick up 17β-estradiol [E2] and testosterone [T] from water, metabolize them and conjugate them to fatty acids (esterification), leading to their accumulation in tissue. A key requirement for the esterification process is that a steroid must have a 'reactive' hydroxyl group to conjugate to a fatty acid (which in T, and probably E2, is the β-hydroxyl group on carbon 17). Progesterone (P) lacks any hydroxyl groups and theoretically cannot be esterified and hence should not accumulate in mussels in the same way as E2 or T. However, it is already known that mussels have an enzyme that can achieve 5α-reduction of the A ring of T and P and that there is also another reductase that can transform the 3-oxo group of the 5α-reduced A ring of T into a hydroxyl group. We hypothesized that, although intact P cannot be directly esterified, it might nevertheless be transformed into metabolites that can. To test this hypothesis, we investigated the rate and capacity of uptake, metabolism and potential depuration of tritiated P by the common mussel, Mytilus spp. We found that tritiated P was taken up from water at a similar rate to E2 and T (mean clearance rate 49mL-1 animal-1h-1) and that, as found with the other steroids, the rate of uptake could not be saturated by the addition of non-radioactive steroid (even at 7.6μgL-1). We found that up to 66% of the radioactivity that was taken up was present in the ester fraction, suggesting that hydroxylation of the P must indeed have occurred. We then definitively identified two metabolites in the ester fraction: 5α-pregnane-3β,20β-diol and 3β-hydroxy-5α-pregnan-20-one. These same two steroids were also present in the free steroid fraction. Intact P was not detected in either of the fractions. When undergoing depuration (under semi-static conditions), the radioactivity in the ester fractions remained at the same concentration in the animals for at least 10 days. Our findings suggest that the lack of reactive hydroxyl groups on P does not preclude it from being taken up, metabolized and subsequently stored. Many questions remain, not least of which is why, when P seems to be so rapidly metabolized, two previous studies on mussels have reported concentrations of up to 30ngg-1 wet weight of P in their flesh.

Six experiments were carried out to define the optimum conditions for investigating the dynamics of uptake and metabolism of tritiated E2 from water by adult blue mussels, Mytilus spp. Optimum uptake was achieved using 400mL aerated sea water animal-1 and an incubation period of no more than 24h. The pattern of disappearance conformed closest to an inverse hyperbolic curve with the percentage of radiolabel that could be measured in the water reaching an asymptote that was on average 50% of the original. This apparent inability of the animals to absorb all the radiolabel was investigated further. Solvent partition and chromatography revealed that, after 24h, c. 60% of the radiolabel still present in the water was composed of water soluble conjugates, c. 25% was composed of tritiated water and only 15% ran on and around the chromatographic position of E2. The major water soluble constituent was identified by chromatography and mass-spectrometry as 1,3,5(10)-estratriene-3,17β-diol 3-sulfate (estradiol 3-S). The clearance rate of radiolabel was 46.9±1.8mLanimal-1h-1. This was not significantly affected by the addition of as much as 25μgL-1 cold E2 to the water, demonstrating that mussels have a large capacity for E2 uptake. A new procedure involving solvent partition was developed for separating the free, esterified and sulfated forms of E2 present in the flesh of mussels. This involved extracting the soft tissue with organic solvents and then treating a portion of dried extract with a combination of heptane (dissolved fatty acid esters of E2) and 80% ethanol (dissolved free and sulfated E2). The latter fraction was further partitioned between water (sulfate) and diethyl ether (free steroid). This procedure was much cheaper and less time-consuming than chromatography. Approximately 80% of the radioactivity that was taken up by the animals was present in the form of ester. Moreover, E2 was the only steroid identified after saponification of these esters. Of the remaining radioactivity, c. 10% was in the form of unidentified free steroids and c. 10% was estradiol 3-S. In order to determine how rapidly mussels were able to depurate tritiated E2 and its metabolites, two experiments were carried out. Animals from the first experiment purged up to 63% of radioactivity in 20days under flow-through conditions; whereas animals from the second experiment released only 16% of radioactivity in 10days under semi-static conditions. The ratios of the different forms of E2 did not change substantially during the course of depuration.