Long term hypoxia suppresses reproductive capacity in the estuarine fish, Fundulus grandis.

Human nutrient input has significantly altered dissolved oxygen (DO) cycles in coastal waters such that summertime hypoxia (DO <2 mg/L) and anoxia of bottom water are common worldwide. Prolonged hypoxia usually reduces metabolic rate in fish and potentially reduces reproduction, particularly in a spring and summer spawning species such as the Gulf killifish, Fundulus grandis. To evaluate the effects of long term hypoxia on reproduction, Gulf killifish were subjected to either normoxia (6.68+/-2.1 mg/L DO) or hypoxia (1.34+/-0.45 mg/L DO) for one month. Fecundity, growth, gonadosomatic index (GSI), circulating sex steroids (testosterone, T; 11-ketotestosterone, 11KT; and estradiol-17beta, E2), and egg yolk protein (vitellogenin, VTG) were measured. Hypoxia significantly reduced growth and reproduction. E2 was 50% lower in females and 11KT was 50% lower in males, although the precursor hormone T was unchanged in either sex after hypoxic exposure. Hypoxia-exposed females produced significantly fewer eggs and initiated spawning later than control fish. Plasma VTG concentration was unchanged, suggesting that hypoxia may delay VTG uptake by oocytes. Long term laboratory exposure clearly suppressed reproductive capacity in Gulf killifish. Wild populations experience cyclic hypoxia which could have equivalent effects if daily hypoxic periods are long and frequent - a potential consequence of anthropogenic nutrient enrichment in marsh systems.

Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption.

Vitellogenin (VTG) synthesis in male oviparous vertebrates is used as an indicator of environmental estrogen exposure, but the relationship between elevated VTG levels and the effects of environmental estrogens on reproductive success are poorly understood. To examine whether altered VTG expression predicts reproductive impairment, we exposed medaka (Oryzias latipes) for 2 or 8 weeks posthatch to 0, 0.5, 1.0, 2.5, and 7.5 ppb of the environmental estrogen o,p'-DDT. Fish were sampled 2, 4, and 8 weeks after hatch to examine VTG expression and gonad development. After exposure, fish were transferred to clean water, grown to sexual maturity, and placed in mating pairs. We collected eggs for 7 days and scored them for fecundity (number of eggs), fertility (percent fertilized), and hatching success (percent hatched). DDT had no effect on VTG expression after a 2-week exposure, whereas all doses induced VTG after 8 weeks. At both exposure durations, the highest doses of DDT caused a female-skewed sex ratio in adults. Gonadal feminization appeared to be progressive: some ovotestes were observed after 2- or 4-week exposure to the two highest doses, but the proportion of ovaries increased after 8 weeks. Both 2- and 8-week exposures significantly reduced fertility and hatching success at all doses, with lower doses having a greater effect after longer exposure. Fertility and hatching success were more sensitive to estrogenic disruption than were gonad differentiation and vitellogenin expression. We suggest that VTG expression may be interpreted as a warning of reproductive consequences, but absence of expression cannot be interpreted as absence of consequences.

Gender benders at the beach: endocrine disruption in marine and estuarine organisms.

Several consensus definitions of the term endocrine disruptor have appeared recently, but all definitions include the important, though frequently implicit, stipulation that the animal is not distressed or in obvious discomfort. Instead, a superficially healthy animal is experiencing alterations in hormone synthesis, transport, receptor interaction, metabolism, excretion, or feedback regulation. In addition, hormone disruption may occur during sex differentiation, and its effects may not be manifested until after sexual maturation. Many cases of chemically induced reproductive impairment have been reported for both freshwater and marine species. However, reproductive impairment may not necessarily result from hormone disruption and should be considered suggestive, but not conclusive, evidence of endocrine disruption. A suite of in vivo and in vitro assays will more adequately assess whether a compound is truly endocrine disrupting. This review will cover basic endocrinology of marine and estuarine invertebrates and vertebrates, methods for detecting endocrine disruption, and examples of endocrine disruption in various species.

Sex steroids relative to alternative mating behaviors in the simultaneous hermaphrodite Serranus subligarius (Perciformes: Serranidae).

This study is the first investigation of reproductive endocrinology in a simultaneously hermaphroditic teleost, the belted sandfish (Serranus subligarius). We address two questions: (1) Do steroid hormone levels vary during the spawning season or during the daily spawning cycle of sandfish? (2) Do hormone levels vary relative to an individual's phenotype-size, frequency of spawning and aggressive behaviors, and proportion of testis in the gonad? We analyzed circulating estradiol-17beta (E2), testosterone (T), 11-ketotestosterone (11KT), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20betaS), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) concentrations in a field population. Only E2 levels were significantly higher at the new and full moon, suggesting peak periods of vitellogenesis at these times. Naturally spawning sandfish were sampled every 2 h during the photophase of a 25-h period (12 pm to 1 pm the following day) and gonadosomatic index, degree of oocyte hydration and ovulation, and plasma levels of E2, T, DHP, and 20betaS were analyzed. E2 and T levels did not vary during photophase, suggesting continuous recruitment of oocytes into vitellogenesis. The 20betaS levels peaked around the time of final oocyte maturation. Since frequency of spawning behaviors changes with body size, we captured individuals of various sizes throughout the spawning season and analyzed circulating levels of hormones. 11KT and 20betaS levels increased significantly with body size. In 1992, we quantified frequency of spawning and aggressive behaviors, circulating T and 11KT levels and testicular mass relative to ovotestis mass in focal animals. 11KT levels tended to be positively correlated with frequency of courting male behavior, but were unrelated to the frequency of aggressive behavior or testis mass. Because hormone levels increased with size and frequency of each spawning behavior changes with size, we propose that sex steroids influence growth-related changes in spawning tactics of individuals.

Fathead minnow (Pimephales promelas) vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds.

The egg yolk precursor protein, vitellogenin (VTG), was purified from blood plasma of 17beta-estradiol (E2)-treated male fathead minnows (Pimephales promnelas) by anion-exchange chromatography on DEAE-agarose. A rabbit antiserum was raised against their blood plasma and then adsorbed with plasma from untreated (control) males to render the antiserum specific to VTG. The adsorbed antiserum was used to detect fathead minnow VTG (fVTG) in Western and dot blotting experiments and in an enzyme-linked immunosorbent assay (ELISA). The antiserum recognised fVTG as a approximately 156 kDa protein in plasma from vitellogenic females and E2-injected males but not untreated males. Its identity was confirmed by analysis of: (1) amino acid composition; (2) an internal amino acid sequence; (3) reactivity to the homologous antiserum; and (4) recognition by monoclonal antibodies prepared against the VTG from common carp (Cyprinus carpio) and brown bullhead (Ameiurus nebulosus). Specificity of the homologous antiserum to fVTG was confirmed by Western blotting of serially diluted plasma from vitellogenic females. Utility of the antiserum and purified fVTG for detecting exposure of male fathead minnows to estrogenic compounds was verified using a dot blotting immunoassay of fVTG and detected by chemiluminescence. Adult male fish were exposed to various concentrations of E2 (10(-8), 10(-9) and 10(-10) M) in their rearing water and plasma assayed for the presence of VTG at different time points (2, 7, 14 and 21 days). A competitive, antibody-capture, quantitative ELISA was then developed based on the purified fVTG and its respective antiserum. The ELISA was validated by demonstrating parallel binding slopes of dilution curves prepared with plasma from E2-injected males, vitellogenic females, and aqueous egg extracts as compared with purified fVTG standard. Plasma concentrations of VTG as low as 3 ng ml(-1) were detected in the ELISA, for which inter- and intra-assay coefficients of variation were both less than 5%. Furthermore, plasma from control males was unreactive with the fVTG antiserum. The VTG ELISA could be useful for the detection of estrogenic properties associated with certain compounds and could be easily incorporated into standard laboratory toxicity assays using this species.

Alteration of leopard frog (Rana pipiens) metamorphosis by the herbicide acetochlor.

Based on the geographic correlation between the use of the pre-emergent herbicide acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl) acetamide] and the natural range of Northern leopard frogs (Rana pipiens), we investigated the effects of acetochlor (ACETO) on frog metamorphosis. We specifically examined the interaction of ACETO with thyroid hormone (T3) and corticosterone (CORT), hormones that regulate natural metamorphosis. ACETO, T3, and CORT were administered via immersion. Growth, developmental stage, and onset of metamorphic climax (forelimb emergence, FLE) were measured. We examined three hypotheses: (1) ACETO may alter metamorphosis. Premetamorphic tadpoles with low endogenous T3 were exposed to ACETO +/- 10(-9) M T3 for 7 days. 67% of tadpoles exposed to ACETO + T3 attained FLE, while 0% of T3 treated animals did. (2) ACETO mimics T3 action at the thyroid receptor (TR). Tadpoles were pretreated with T3 for 3 days to induce TR expression, then treated for 7 days with vehicle (DMSO), T3, or ACETO +/- T3. ACETO treatment after T3 priming did not accelerate FLE, suggesting that ACETO does not interact directly with the TR. Cotreatment with ACETO + T3 after T3 priming accelerated FLE relative to tadpoles primed with T3, then treated with T3. Because the ACETO + T3 acceleration of FLE appeared similar to the effect of CORT, we examined a third hypothesis: (3) ACETO may interact with CORT to accelerate FLE. Premetamorphic tadpoles were exposed to various doses of ACETO +/- T3 in the presence or absence of 10(-7) M CORT. CORT inhibited growth and hindlimb development and delayed FLE. ACETO never inhibited growth or hindlimb development, but ACETO did counteract the effects of CORT when T3 was present. ACETO consistently accelerated T3-induced metamorphosis, apparently interacting with T3 via a non-TR-mediated mechanism.

Screening methods for thyroid hormone disruptors.

The U.S. Congress has passed legislation requiring the EPA to implement screening tests for identifying endocrine-disrupting chemicals. A series of workshops was sponsored by the EPA, the Chemical Manufacturers Association, and the World Wildlife Fund; one workshop focused on screens for chemicals that alter thyroid hormone function and homeostasis. Participants at this meeting identified and examined methods to detect alterations in thyroid hormone synthesis, transport, and catabolism. In addition, some methods to detect chemicals that bind to the thyroid hormone receptors acting as either agonists or antagonists were also identified. Screening methods used in mammals as well as other vertebrate classes were examined. There was a general consensus that all known chemicals which interfere with thyroid hormone function and homeostasis act by either inhibiting synthesis, altering serum transport proteins, or by increasing catabolism of thyroid hormones. There are no direct data to support the assertion that certain environmental chemicals bind and activate the thyroid hormone receptors; further research is indicated. In light of this, screening methods should reflect known mechanisms of action. Most methods examined, albeit useful for mechanistic studies, were thought to be too specific and therefore would not be applicable for broad-based screening. Determination of serum thyroid hormone concentrations following chemical exposure in rodents was thought to be a reasonable initial screen. Concurrent histologic evaluation of the thyroid would strengthen this screen. Similar methods in teleosts may be useful as screens, but would require indicators of tissue production of thyroid hormones. The use of tadpole metamorphosis as a screen may also be useful; however, this method requires validation and standardization prior to use as a broad-based screen.

Potential mechanisms of thyroid disruption in humans: interaction of organochlorine compounds with thyroid receptor, transthyretin, and thyroid-binding globulin.

Organochlorine compounds, particularly polychlorinated biphenyls (PCBs), alter serum thyroid hormone levels in humans. Hydroxylated organochlorines have relatively high affinities for the serum transport protein transthyretin, but the ability of these compounds to interact with the human thyroid receptor is unknown. Using a baculovirus expression system in insect cells (Sf9 cells), we produced recombinant human thyroid receptor ss (hTRss). In competitive binding experiments, the recombinant receptor had the expected relative affinity for thyroid hormones and their analogs. In competitive inhibition experiments with PCBs, hydroxylated PCBs (OH-PCBs), DDT and its metabolites, and several organochlorine herbicides, only the OH-PCBs competed for binding. The affinity of hTRss for OH-PCBs was 10,000-fold lower (Ki = 20-50 microM) than its affinity for thyroid hormone (3,3',5-triiodothyronine, T3; Ki = 10 nM). Because their relative affinity for the receptor was low, we tested the ability of OH-PCBs to interact with the serum transport proteins--transthyretin and thyroid-binding globulin (TBG). With the exception of one compound, the OH-PCBs had the same affinity (Ki = 10-80 nM) for transthyretin as thyroid hormone (thyroxine; T4). Only two of the OH-PCBs bound TBG (Ki = 3-7 microM), but with a 100-fold lower affinity than T4. Hydroxylated PCBs have relatively low affinities for the human thyroid receptor in vitro, but they have a thyroid hormonelike affinity for the serum transport protein transthyretin. Based on these results, OH-PCBs in vivo are more likely to compete for binding to serum transport proteins than for binding to the thyroid receptor.

Environmental signaling: a biological context for endocrine disruption.

Endogenous and exogenous chemical signals have evolved as a means for organisms to respond to physical or biological stimuli in the environment. Sensitivity to these signals can make organisms vulnerable to inadvertent signals from xenobiotics. In this review we discuss how various chemicals can interact with steroid-like signaling pathways, especially estrogen. Numerous compounds have estrogenic activity, including steroids, phytoestrogens, and synthetic chemicals. We compare bioavailability, metabolism, interaction with receptors, and interaction with cell-signaling pathways among these three structurally diverse groups in order to understand how these chemicals influence physiological responses. Based on their mechanisms of action, chemical steroid mimics could plausibly be associated with recent adverse health trends in humans and animals.

Purification, amino acid sequence, synthesis, and receptor selectivity of alligator gastrin.

Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.