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.

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.

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.

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.

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.

The effect of elevated steroids released by reproductive male round gobies, Neogobius melanostomus, on olfactory responses in females.

The round goby, Neogobius melanostomus, is a highly successful invasive species in the Laurentian Great Lakes. Previous behavioral studies implied that females are attracted by pheromones to the nests of reproductive males, and that males release putative steroidal pheromones--unconjugated as well as conjugated forms of 3α-hydroxy-5ß-androstane-11,17-dione (11-O-ETIO)-following stimulation of the hypothalamic--gonadal axis with salmon gonadotropin releasing hormone analog (sGnRHa). In this study, we tested the olfactory system of females in response to extracts containing these released steroids. We compared electrical field potential responses from the olfactory epithelium (electro-olfactogram, EOG) of non-reproductive females to methanol extracts of water that previously held males, collected before and after injection of the males with sGnRHa or saline. The females showed increased EOG responses to the post-injection extracts when males were treated with sGnRHa but not saline. This finding provides further evidence for interactions between male and female N. melanostomus via steroidal reproductive pheromones.

Teleost maturation-inducing hormone, 17,20ß-dihydroxypregn-4-en-3-one, peaks after spawning in Tinca tinca.

During an eight month study of the reproductive cycle in two age groups, and in both sexes, of tench (Tinca tinca L.), it was found that plasma concentrations of the presumptive 'maturation inducing hormone (MIH)' 17,20ß-dihydroxypregn-4-en-3-one (17,20ß-P) did not reach a peak during the spawning season, but as much as two months after spawning had ceased. The cessation of the spawning season was confirmed by histological examination of the gonads and by measurement of 11-ketotestosterone and 17ß-estradiol in the plasma of males and females, respectively. Measurements were also made of the 'alternative MIH' 17,20ß,21-trihydroxypregn-4-en-3-one in the older fish. However, this steroid did not show the same pattern as 17,20ß-P. An assessment was made of the prevalence of primary spermatocytes in the testes of post-spawned fish - to test an alternative hypothesis that 17,20ß-P might be involved in the stimulation of meiosis. However, there was no evidence for any increase in testis differentiation post-spawning. In fact the testes became increasingly undifferentiated as the autumn progressed. The role, if any, of this 'unseasonal' peak of 17,20ß-P production remains to be determined.

Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814).

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.

Reproductive responses in fathead minnow and Japanese medaka following exposure to a synthetic progestin, Norethindrone.

Synthetic progestins, such as Norethindrone (NET), are common ingredients in oral contraceptives and in treatment for post-menopausal problems. Given the widespread use of oral contraceptives and post-menopausal treatments, several reports have targeted and identified progestins in aquatic environments. In fish, progestins play an important role in the stimulation of oocyte final maturation and ovulation in females, stimulation of spermiation and sperm motility in males, and the initiation of meiosis in both sexes. They also have a role as pheromones in some species. Given the pivotal role that progestins play in reproduction, their appreciable daily dose (i.e. microg to mg range in contraceptives and hormone replacement therapies) and continuous use pattern, it is important to understand the potential risk these compounds pose once discharged into the aquatic environment. Since little published data are available on this class of compounds, our research focused on the reproductive effects of NET on the fathead minnow and Japanese medaka. A 28 day static-renewal reproduction study with Japanese medaka indicated that NET produces a significant decrease in fecundity at aqueous concentrations >or=25 ng/L. A 21 day flow-though fathead minnow reproduction study also demonstrated that NET causes a significant decrease in fecundity in the low ng/L range. Fathead minnow morphological changes (i.e. female fin spots) suggest that NET exposure may have a potent androgenic effect on fish; however, plasma 11-Ketotestosterone (11-KT) concentrations were reduced in males at the highest exposure concentration. Collectively, these data indicate that further investigation of reproductive responses associated with synthetic progestins is warranted.

Intercalibration exercise using a stickleback endocrine disrupter screening assay.

The Organisation for Economic Cooperation and Development (OECD) is currently validating a short-term fish screening protocol for endocrine disrupters (estrogens, androgens, and their antagonists and aromatase inhibitors), using three core species: fathead minnow, Japanese medaka, and zebrafish. The main endpoints proposed for the first phase of validation of the screen are vitellogenin (VTG) concentration, gross morphology (secondary sexual characteristics and gonado-somatic index), and gonadal histopathology. A similar protocol is concurrently being developed in the United Kingdom using the three-spined stickleback, with identical endpoints to those for the core species and, in addition, a unique androgen-specific endpoint in the form of spiggin (glue protein) induction. To assess the suitability of this species for inclusion in the OECD protocol alongside the core species, an intercalibration was conducted using 17beta-estradiol (a natural estrogen) and trenbolone (a synthetic androgen), thus mimicking a previous intercalibration with the core species. All three participating laboratories detected statistically significant increases in VTG in males after 14 d exposure to nominal concentrations of 100 ng/L 17beta-estradiol and statistically significant increases in spiggin in females after 14 d exposure to nominal concentrations of 5,000 ng/L trenbolone. The stickleback screen is reliable, possessing both relevant and reproducible endpoints for the detection of potent estrogens and androgens. Further work is underway to assess the relevance and suitability of the screen for weakly acting estrogens, anti-androgens, and aromatase inhibitors.

Sex steroids and their receptors in lampreys.

The use of steroids and their receptors as ligand-gated transcription factors is thought to be an important step in vertebrate evolution. The lamprey is the earliest-evolving vertebrate to date in which sex steroids and their receptors have been demonstrated to have hormonal roles similar to those found in jawed vertebrates. Sex steroids and their receptors have been examined in several lamprey species, and the majority of studies have focused on the sea lamprey, Petromyzon marinus. While classical steroids appear to be present in lampreys, their function, concentrations, and synthesis have not been determined conclusively. The only classical steroid that is thought to act as a hormone in both males and females is estradiol. Recent research has established that lampreys produce and circulate 15alpha-hydroxylated steroids, and that these steroids respond to upstream stimulation within the hypothalamic-pituitary-gonadal axis. In particular, 15alpha-hydroxyprogesterone is highly sensitive and responds in great magnitude to stimulation, and is likely a hormone. Lampreys also appear to use androstenedione, a precursor to vertebrate androgens, as their main androgen, and a receptor for androstenedione has recently been described. Non-classical steroids are prevalent in many aquatic vertebrates, and the non-classical steroids found in the sea lamprey may represent an evolutionary artifact, or alternatively may be a way to avoid endocrine disruption when ingesting the body fluids of host fish. The lamprey will continue to be an interesting model for examining the evolution of steroid hormones, steroid receptors, and steroid function.

The sea lamprey (Petromyzon marinus) has a receptor for androstenedione.

The use of nuclear steroid receptors as ligand-activated transcription factors is a critical event in vertebrate evolution. It is believed that nuclear steroid receptors arose at or before the vertebrate radiation, except for an androgen receptor (Ar) that evolved only in the gnathostome line. We report an androgen-Ar complex in the male sea lamprey (Petromyzon marinus), an extant jawless vertebrate. The androgen with the highest affinity is not testosterone, but its direct precursor, androstenedione (Ad). To establish that the binding moiety in lamprey testis is a receptor-and not an "androgen-binding protein"-we have shown that it can be extracted from the nucleus as well as the cytosol, that the Ad-receptor complex binds to DNA, and that the receptor is approximately twice the size of an androgen-binding protein extracted from the Atlantic salmon testis. The capacity (and high affinity) of binding of the lamprey Ar is such that much of the Ad present in male lampreys becomes sequestered within the testis (as opposed to circulating in the plasma). Concentrations of Ad (but not of testosterone) in plasma and testis tissue are upregulated by injection of lamprey GnRH. Implantation of male lampreys with exogenous Ad significantly accelerates the development of the testis and growth of at least one secondary male characteristic. It appears that all classes of steroid hormones have contributed to the evolution of the regulatory complexity of steroid receptors found in modern vertebrates.

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility.

The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.).

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.

Dose-response relationship of 15alpha-hydroxylated sex steroids to gonadotropin-releasing hormones and pituitary extract in male sea lampreys (Petromyzon marinus).

The sea lamprey (Petromyzon marinus) is one of the earliest extant vertebrates for which the hypothalamic-pituitary-gonadal (HPG) axis has been shown to control and regulate reproduction in a similar fashion to gnathostome vertebrates. While the two forms of gonadotropin-releasing hormones in the sea lamprey (GnRH I and GnRH III) have been studied extensively, their in vivo effect on synthesis of 15alpha-hydroxytestosterone (15alpha-T) and 15alpha-hydroxyprogesterone (15alpha-P) have only been partially characterized. In the present study, plasma concentrations of 15alpha-T and 15alpha-P were measured in prespermiating sea lampreys that were given a single injection of either GnRH I or GnRH III in doses ranging from 5 to 100 microg/kg, or of pituitary extract (as a source of gonadotropin). Plasma was sampled at 1-6h and 6-48 h post-injection, in separate experiments, in order to characterize the peak and duration of responses. 15alpha-T plasma concentrations increased slightly in response to all three treatments, but not in a dose-dependent manner, and the timing of peak concentrations varied between doses. However, 15alpha-P plasma concentrations showed a greater range of response (between 1 and 100 ng/ml) and were clearly correlated with the injection dose. Plasma concentrations of 15alpha-P also responded to far lower doses of GnRH I and GnRH III than any other steroid previously investigated in lampreys. The plasma concentrations of 15alpha-P peaked at 6h after injection for all three treatments, and levels reached a mean of 53.1 ng/ml. In female lampreys that were injected twice with 50 microg/ml GnRH I or III, 15alpha-T concentrations did not exceed 0.5 ng/ml and 15alpha-P concentrations did not exceed 1 ng/ml. These results lend further support to the hypothesis that 15alpha-P plays an important role in the reproductive endocrinology of male lampreys.