Studies on a widely-recognized snail model species (Lymnaea stagnalis) provide further evidence that vertebrate steroids do not have a hormonal role in the reproduction of mollusks.

Experiments were carried out to determine whether, as with other mollusks that have been studied, the snail, Lymnaea stagnalis, can absorb, esterify and store vertebrate steroids that are present in the water. We also carried out experiments to determine whether neural tissues of the snail could be immunohistochemically stained with an antibody to human aromatase (a key enzyme that catalyzes the conversion of testosterone [T] to 17ß-estradiol [E2]); and, if so, to determine the significance of such staining. Previous studies on other mollusks have reported such staining and have proposed this as decisive evidence that mollusks have the same steroid synthesis pathway as vertebrates. We found that snails absorb, esterify and retain esterified T, E2, progesterone and ethinyl-estradiol (albeit with an absorption rate about four times slower, on a weight basis, than the mussel, Mytilus edulis). We also found that not only anti-human aromatase, but also anti-human nuclear progesterone receptor (nPR) and anti-human gonadotropin-releasing hormone antibodies immunohistochemically stained snail neural cells. However, further experiments, involving gel electrophoretic separation, followed by immunostaining, of proteins extracted from the neural tissue, found at least two positively-stained bands for each antibody, none of which had masses matching the human proteins to which the antibodies had been raised. The anti-aromatase antibody even stained the 140 kDA ladder protein used as a molecular weight marker on the gels. Mass spectrometric analysis of the bands did not find any peptide sequences that corresponded to the human proteins. Our findings confirm that the presence of vertebrate-like sex steroids in molluscan tissues is not necessarily evidence of endogenous origin. The results also show that immunohistochemical studies using antibodies against human proteins are grossly non-specific and likely to have little or no value in studying steroid synthesis or activity in mollusks. Our conclusions are consistent with the fact that genes for aromatase and nPR have not been found in the genome of the snail or of any other mollusk. Our overarching conclusion, from this and our previous studies, is that the endocrinology of mollusks is not the same as that of humans or any other vertebrates and that continuing to carry out physiological and ecotoxicological studies on mollusks on the basis of this false assumption, is an unconscionable waste of resources.

The Uptake of Ethinyl-Estradiol and Cortisol From Water by Mussels (Mytilus spp.).

Previous toxicokinetic studies have shown that mussels (Mytilus spp.) can readily absorb the three main mammalian sex steroids, estradiol (E2), testosterone (T) and progesterone (P) from water. They also have a strong ability to store E2 and the 5α-reduced metabolites of T and P in the form of fatty acid esters. These esters were shown to have half-lives that were measured in weeks (i.e. they were not subject to fast depuration). The present study looked at the toxicokinetic profile of two other common steroids that are found in water, the potent synthetic oestrogen, (ethinyl-estradiol) (EE2; one of the two components of 'the pill'), and cortisol, a natural stress steroid in vertebrates. In the first three hours of uptake, tritiated EE2 was found to be taken up at a similar rate to tritiated E2. However, the levels in the water plateaued sooner than E2. The ability of the animals to both esterify and sulphate EE2 was found to be much lower than E2, but nevertheless did still take place. After 24 h of exposure, the majority of radiolabelled EE2 in the animals was present in the form of free steroid, contrary to E2, which was esterified. This metabolism was reflected in a much lower half-life (of only 15 h for EE2 in the mussels as opposed to 8 days for E2 and >10 days for T and P). Intriguingly, hardly any cortisol (in fact none at all in one of the experiments) was absorbed by the mussels. The implications of this finding in both toxicokinetic profiling and evolutionary significance (why cortisol might have evolved as a stress steroid in bony fishes) are discussed.

A critical evaluation of some of the recent so-called 'evidence' for the involvement of vertebrate-type sex steroids in the reproduction of mollusks.

Many studies on the control of reproduction in mollusks have focused on hormones (and proteins associated with the production and signaling of those hormones) which were originally discovered in humans, in the belief that if they are also present in mollusks, they must have the same role. However, although human sex steroids can be found in mollusks, they are so readily absorbed that their presence is not necessarily evidence of endogenous synthesis. A homolog of the vertebrate nuclear estrogen receptor has been found in mollusks, but it does not bind to estrogens or indeed to any steroid at all. Antibodies against human aromatase show positive immunostaining in mollusks, yet the aromatase gene has not been found in the genome of any invertebrates (let alone mollusks). This review will deal with these and other examples of contradictory evidence for a role of human hormones in invertebrate reproduction.

Insights into the development of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from UK estuaries.

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting European flounder (Platichthys flesus), particularly from estuaries historically impacted by pollution. This study demonstrated that the HFI prevalence range was 6-77% at several UK estuaries, with Spearman rank correlation analysis showing a correlation between HFI prevalence and sediment concentrations of ∑PBDEs and ∑HBCDs. The data showed that males exhibit higher HFI prevalence than females, with severity being more pronounced in estuaries exhibiting higher prevalence. HFI were not age associated indicating a subacute condition. Electron microscopy confirmed that HFI were modified proliferating rough endoplasmic reticulum (RER), whilst immunohistochemistry provided evidence of VTG production in HFI of male P. flesus. Despite positive labelling of aberrant VTG production, we could not provide additional evidence of xenoestrogen exposure. Gene transcripts (VTG/CHR) and plasma VTG concentrations (>1 µg ml-1), were only considered elevated in four male fish showing no correlation with HFI severity. Further analysis revealed that reproductively mature female P. flesus i.e. >3-year-old, did not exhibit HFI, whereas males of all ages were affected. This, combined with previous reports that estradiol (E2) can impair mixed function oxygenase activity, supports a hypothesis that harmful chemical metabolites (following phase 1 metabolism of their parent compounds) are potentially responsible for HFIs observed in male and ≤ 3-year-old female fish. Consequently, HFI and xenoestrogenic induced VTG production could be independent of each other resulting from different concurrent toxicopathic mechanisms, although laboratory exposures will likely be the only way to determine the true aetiology of HFI.

Immunoassays are not immune to errors: Examples from two studies of steroid output from freshwater trout farms.

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L-1) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L-1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed.

Is there any value in measuring vertebrate steroids in invertebrates?

This brief review questions the belief that just because it is possible to measure vertebrate steroids (such as estradiol-17ß, testosterone and progesterone) in the tissues of invertebrates, this necessarily means that they are endogenously derived or are hormones. There is a surprisingly large number of studies, mainly on mollusks, showing that they can readily absorb vertebrate steroids from the environment. They are also able to conjugate these steroids to fatty acids with great efficiency, and subsequently retain them for very long periods (with half-lives measured in weeks rather than days). This, plus the fact that key enzymes that are required for the biosynthesis of vertebrate steroids (e.g. aromatase) do not appear to be present in invertebrates, calls into doubt the claims in many studies on invertebrates that steroid concentrations are functionally linked to reproductive cycles or that invertebrates can be used as biomarker for vertebrate-type endocrine disrupters.

Uptake and metabolism of water-borne progesterone by the mussel, Mytilus spp. (Mollusca).

Previous studies have shown that mussels can pick up 17ß-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 ß-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-3ß,20ß-diol and 3ß-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.

Data on the uptake and metabolism of testosterone by the common mussel, Mytilus spp.

This article provides data in support of the research article entitled "Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp." (T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, 2017) [1]. The uptake of tritiated testosterone (T) from water by mussels is presented. The two main radioactive peaks formed from T and present in the fatty acid ester fraction of mussel tissues were shown to have the same elution positions on a thin layer chromatography plate as 17ß-hydroxy-5α-androstan-3-one (DHT) and 5α-androstan-3ß,17ß-diol (3ß,17ß-A5α). Reverse phase high performance liquid chromatography of the non-esterified (80% ethanol) fraction of the mussel tissue extracts also presented radioactive peaks at the elution positions of DHT and 3ß,17ß-A5α. There was no evidence for sulfated T in this fraction. It was shown that aeration led to significant losses of radiolabeled testosterone from the water column.

Rapid uptake, biotransformation, esterification and lack of depuration of testosterone and its metabolites by the common mussel, Mytilus spp.

The presence of the vertebrate steroids, testosterone (T) and 17ß-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-3ß,17ß-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.

Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17ß from water.

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-3,17ß-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.

Data on the uptake and metabolism of the vertebrate steroid estradiol-17ß from water by the common mussel, Mytilus spp.

The data presented in this article primarily provide support for the research article entitled "Mussels (Mytilus spp.) display an ability for rapid and high capacity uptake of the vertebrate steroid, estradiol-17ß from water" (T.I. Schwarz, I. Katsiadaki, B.H. Maskrey, A.P. Scott, 2016) [1]. Data are presented on the ability of mussels to absorb tritiated estradiol (E2) from water. The data indicate that most of the radioactivity remaining in the water is 1,3,5(10)-estratriene-3,17ß-diol 3-sulfate (E2 3-S) and the radioactivity in the mussel tissue is mainly in the form of fatty acid esters. The latter, following saponification, were identified by ultra-high performance liquid chromatography in conjunction with tandem mass spectrometry (UHPLC-MS/MS) as intact E2. Data are included that indicate that the remaining radioactivity in the tissue is composed of E2 3-S and unidentified free metabolites. Experimental data included also relate to a) the efficiency of extraction of radioactivity from tissue, b) the efficiency of separation of free and esterified E2 using solvents and c) possible factors affecting the recovery of radioactivity. Finally, preliminary data are provided on concentrations of immunoreactive E2 in the free and ester fractions of tissue extracts from mussels caged in the field.

From single chemicals to mixtures--reproductive effects of levonorgestrel and ethinylestradiol on the fathead minnow.

The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants-each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day 'pair-breeding' assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case.

Evidence that progestins play an important role in spermiation and pheromone production in male sea lamprey (Petromyzon marinus).

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.

Identification of cortisol metabolites in the bile of Atlantic cod Gadus morhua L.

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: 11ß,17,21-trihydroxypregn-4-ene-3,20-dione (cortisol), 3α,11ß,17,21-tetrahydroxy-5ß-pregnan-20-one (tetrahydrocortisol; THF), 3α,17,21-trihydroxy-5ß-pregnane-11,20-dione (tetrahydrocortisone; THE) and 3α,17,20ß,21-tetrahydroxy-5ß-pregnan-11-one (ß-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).

Principles of sound ecotoxicology.

We have become progressively more concerned about the quality of some published ecotoxicology research. Others have also expressed concern. It is not uncommon for basic, but extremely important, factors to apparently be ignored. For example, exposure concentrations in laboratory experiments are sometimes not measured, and hence there is no evidence that the test organisms were actually exposed to the test substance, let alone at the stated concentrations. To try to improve the quality of ecotoxicology research, we suggest 12 basic principles that should be considered, not at the point of publication of the results, but during the experimental design. These principles range from carefully considering essential aspects of experimental design through to accurately defining the exposure, as well as unbiased analysis and reporting of the results. Although not all principles will apply to all studies, we offer these principles in the hope that they will improve the quality of the science that is available to regulators. Science is an evidence-based discipline and it is important that we and the regulators can trust the evidence presented to us. Significant resources often have to be devoted to refuting the results of poor research when those resources could be utilized more effectively.

Several synthetic progestins with different potencies adversely affect reproduction of fish.

Synthetic progestins are widely used as a component in both contraceptives and in hormone replacement therapy (HRT), both on their own and in combination with EE2. Their presence in the environment is now established in wastewater effluent and river water and this has led to concerns regarding their potential effects on aquatic organisms living in these waters. We carried out in vivo experiments to determine the potencies of four different synthetic progestins on the reproductive capabilities of the fathead minnow (Pimephales promelas). We then performed a series of in vitro assays to try and determine the reason for the effects seen in the in vivo experiments. In the first experiment, fathead minnow exposed to a single concentration of 100 ng/L of either Levonorgestrel or Gestodene stopped spawning almost completely. The same nominal concentration of Desogestrel and Drospirenone did not affect reproduction (21 d NOECs of 100 ng/L). The second experiment investigated two progestins of different potency: Gestodene at 1, 10, and 100 ng/L and Desogestrel at 100 ng/L, 1 µg/L, and 10 µg/L. Gestodene concentrations as low as 1 ng/L had significant effects on reproduction over 21 d, whereas concentrations of Desogestrel at or above 1 µg/L were required to significantly reduce egg production. The synthetic progestins also masculinized the female fish in a concentration-dependent manner. Results from yeast-based in vitro assays demonstrated that the progestins are all strongly androgenic, thereby explaining the masculinization effects. The results strongly suggest that synthetic progestins merit serious consideration as environmental pollutants.

Do mollusks use vertebrate sex steroids as reproductive hormones? II. Critical review of the evidence that steroids have biological effects.

In assessing the evidence as to whether vertebrate sex steroids (e.g. testosterone, estradiol, progesterone) have hormonal actions in mollusks, ca. 85% of research papers report at least one biological effect; and 18 out of 21 review papers (published between 1970 and 2012) express a positive view. However, just under half of the research studies can be rejected on the grounds that they did not actually test steroids, but compounds or mixtures that were only presumed to behave as steroids (or modulators of steroids) on the basis of their effects in vertebrates (e.g. Bisphenol-A, nonylphenol and sewage treatment effluents). Of the remaining 55 papers, some can be criticized for having no statistical analysis; some for using only a single dose of steroid; others for having irregular dose-response curves; 40 out of the 55 for not replicating the treatments; and 50 out of 55 for having no within-study repetition. Furthermore, most studies had very low effect sizes in comparison to fish-based bioassays for steroids (i.e. they had a very weak 'signal-to-noise' ratio). When these facts are combined with the fact that none of the studies were conducted with rigorous randomization or 'blinding' procedures (implying the possibility of 'operator bias') one must conclude that there is no indisputable bioassay evidence that vertebrate sex steroids have endocrinological or reproductive roles in mollusks. The only observation that has been independently validated is the ability of estradiol to trigger rapid (1-5 min) lysosomal membrane breakdown in hemocytes of Mytilus spp. This is a typical 'inflammatory' response, however, and is not proof that estradiol is a hormone - especially when taken in conjunction with the evidence (discussed in a previous review) that mollusks have neither the enzymes necessary to synthesize vertebrate steroids nor nuclear receptors with which to respond to them.

Do mollusks use vertebrate sex steroids as reproductive hormones? Part I: Critical appraisal of the evidence for the presence, biosynthesis and uptake of steroids.

The consensus view is that vertebrate-type steroids are present in mollusks and perform hormonal roles which are similar to those that they play in vertebrates. Although vertebrate steroids can be measured in molluscan tissues, a key question is 'Are they formed endogenously or they are picked up from their environment?'. The present review concludes that there is no convincing evidence for biosynthesis of vertebrate steroids by mollusks. Furthermore, the 'mollusk' genome does not contain the genes for key enzymes that are necessary to transform cholesterol in progressive steps into vertebrate-type steroids; nor does the mollusk genome contain genes for functioning classical nuclear steroid receptors. On the other hand, there is very strong evidence that mollusks are able to absorb vertebrate steroids from the environment; and are able to store some of them (by conjugating them to fatty acids) for weeks to months. It is notable that the three steroids that have been proposed as functional hormones in mollusks (i.e. progesterone, testosterone and 17ß-estradiol) are the same as those of humans. Since humans (and indeed all vertebrates) continuously excrete steroids not just via urine and feces, but via their body surface (and, in fish, via the gills), it is impossible to rule out contamination as the sole reason for the presence of vertebrate steroids in mollusks (even in animals kept under supposedly 'clean laboratory conditions'). Essentially, the presence of vertebrate steroids in mollusks cannot be taken as reliable evidence of either endogenous biosynthesis or of an endocrine role.

Piscine follicle-stimulating hormone triggers progestin production in gilthead seabream primary ovarian follicles.

Ovarian growth (vitellogenesis) in most lower vertebrates is mediated by estradiol-17beta (E2) secreted by the follicles in response to follicle-stimulating hormone (Fsh), whereas oocyte maturation and ovulation are mediated by progestins, such as 17alpha,20beta-dihydroxypregn-4-en-3-one (17,20beta-P), produced in response to luteinizing hormone (Lh). In teleosts, follicular synthesis of 17,20beta-P at the time of maturation is due primarily to up-regulation of the enzymes P450c17-II (Cyp17a2) and 20beta-hydroxysteroid dehydrogenase (Cbr1). Here, we show that follicular cells associated with primary growth (previtellogenic) oocytes of the gilthead seabream also express cyp17a2 and cbr1, in addition to P450c17-I (cyp17a1) and aromatase (cyp19a1), enzymes required for E2 synthesis. Ovaries containing only oogonia and early primary ovarian follicles had a 60-fold higher concentration of 17,20beta-P than ovaries in the succeeding stages and had a higher expression of cbr1 and Fsh receptor (fshra). Stimulation of explants of primary follicles in vitro with recombinant piscine Fsh (rFsh), which specifically activates the seabream Fshra, promoted a rapid accumulation of 17,20beta-P, and synthesis was sustained by an external supply of 17alpha-hydroxyprogesterone. In the presence of Cbr1 inhibitors, rFsh-mediated 17,20beta-P production was reduced, with a concomitant increase in testosterone and E2 synthesis. In primary explants, rFsh up-regulated cyp17a2 and cbr1 transcription and simultaneously down-regulated cyp17a1 and cyp19a1 steady-state mRNA levels within 24 h. In contrast, in explants containing vitellogenic follicles, rFsh had no effect on cyp17a2 and cbr1 expression, but increased that of cyp17a1 and cyp19a1. These data suggest a functional Fshra-activated Cyp17a2/Cbr1 steroidogenic pathway in gilthead seabream primary ovarian follicles triggering the production of 17,20beta-P.