DNA microarrays detect 4-nonylphenol-induced alterations in gene expression during zebrafish early development.

Technological advances in the biological sciences have led to a growing realization of the inherent complexity of the toxic actions of man-made chemicals and industrial compounds. An organism's response to toxic exposure is often a complex summation of the individual responses of various different cell types, tissues, and organs within an individual. Furthermore, within a population, various factors including gender, age, fitness, exposure history, genetic variation, and developmental stage significantly affect how each individual will react following exposure. Because of this complexity, characterizing the responses of organisms to environmental toxin exposure is an area of research well suited to the utilization of the gene-expression profiling capability of DNA microarrays. Microarrays are capable of screening large numbers of genes for response to environmental exposure, with the resulting genesets comprising de facto biomarkers for such exposures. In many cases, the genesets described contain response transcripts anticipated from known mechanistic pathways, but in other cases, equally indicative biomarkers may be found that are unexpected. We investigated the response of zebrafish embryos exposed in vitro to the environmental contaminant 4-nonylphenol (4NP). Nonylphenol is one of several alkylphenol ethoxylate compounds widely used in agricultural and industrial processes that have become ubiquitous environmental contaminants. By combining data from differing levels of exposure, we have identified a group of genes that appear indicative of embryo exposure to 4NP at concentrations ranging from high near-lethal levels to lower, more environmentally relevant levels. These biomarker sets can be further expanded and adapted for use in environmental monitoring as well as in mechanistic studies of complex toxicological mechanisms during both early and adult developmental stages.

Proteomics in zebrafish exposed to endocrine disrupting chemicals.

Embryonic zebrafish were examined for changes in protein expression following exposure to sublethal concentrations of 17beta-estradiol (E2) and the estrogen mimic 4-nonylphenol (4-NP). Protein Expression Signatures were derived from embryo homogenates by two-dimensional electrophoresis and digital imaging. In both experiments approximately 30% of the proteins sampled were specific to either E2 or 4-NP and about 33% were common to the control, 4-NP and E2. However, of the proteins induced by either E2 or 4-NP, 28% were common to both chemicals at 1 ppm but only 7% were common to both at 0.1 ppm. While there are many proteins that respond specifically to each chemical, relatively few are common to the two chemicals suggesting that the response pathways of the two chemicals are distinct.

An interlaboratory study on the use of steroid hormones in examining endocrine disruption.

In recent years, there has been an increased use of the measurement of sex steroid hormone levels in the blood of animals exposed to chemicals as an indicator of reproductive impairment or an alteration in endocrine function. Although levels of hormones are often compared among animals and laboratories, there has been no study to examine the between-laboratory variability in actual steroid measurements. Therefore, we initiated a study with white sucker collected from a site receiving pulp mill effluent, previously documented as having reduced steroid levels, to address this issue. Samples of plasma and media from in vitro gonadal incubations were delivered to eight outside laboratories with the ability to measure steroid hormones. These laboratories ranged from well-established fish endocrine laboratories to wildlife toxicology laboratories, which have recently implemented the methods to measure steroid hormones. In this study, we have considered both the absolute measure of steroid content between laboratories as well as the ability to discriminate between reference and exposed populations as important criteria when evaluating the utility of these measures. Of the eight outside laboratories conducting the analyses, six detected identical site differences in circulating levels of testosterone and 17beta-estradiol to those documented by our Burlington laboratory (ON, Canada). However, the absolute value of the steroid hormones measured in the plasma varied significantly (plasma testosterone 0.6-23.1 ng/ml, 17beta-estradiol 77.6-1782.7 pg/ml) with coefficients of variation of 70.4% and 60.3% respectively. Similar results were demonstrated for the measurement of steroid hormones in media following in vitro gonadal incubation. Although there was a fair amount of variability in the absolute measure of steroid hormone levels, we would predict a far greater coherence of interlaboratory results through the sharing of reagents and the use of a common methodology between laboratories. These results are very promising, providing evidence for the inclusion of steroid hormones in monitoring endocrine disruption in wildlife species.

Reproductive Cycling in Female Fundulus heteroclitus.

The ovaries of female Fundulus heteroclitus living in the northeastern Florida saltmarsh recrudesce in January and the fish initially spawn heavily during the subsequent full moons (a lunar pattern); they later spawn with less intensity during both the new and full moons (a semilunar pattern), and then regress in late September. In the laboratory, fish spawning against a vertical screen showed only semilunar periodicities, as observed for seven spawning groups under constant conditions (temperature 26 +/- 1°C; photoperiod 14 h light to 10 h dark; excess food). Regardless of collection times (January, April, August, or September), these seven groups exhibited similar patterns of semilunar spawning for five to eight consecutive cycles whose periods (14.4 to 16.0 days) and phases (-1.7 to +8.4 days) were variable compared with concurrent full/new moon and spring tide cycles. These semilunar cycles, which occurred over the entire year in the laboratory, were thus free-running without entrainment and represent endogenous circasemilunar rhythms. In addition to annual and lunar/semilunar cycles, a tidal spawning cycle was also observed in the habitat. Fish apparently select the higher of the two semidiurnal tides for spawning, regardless of the daily light-dark cycle. This tidal cycle has not yet been tested in the laboratory.

Responses of fish populations and communities to pulp mill effluents: a holistic assessment.

Fish populations residing in a river receiving bleached kraft mill effluents (BKME) and in an uncontaminated river were investigated to evaluate causal relationships between exposure to BKME and various indicators of fish health. The Index of Biotic Integrity demonstrated that species richness and composition were much lower in the contaminated river with an obvious imbalance in the trophic structure of the fish community. Biomolecular and biochemical responses such as DNA damage and elevated activity of detoxification enzymes indicated that fish in the contaminated river had been exposed to toxicants. The status of various condition indices such as the liver and visceral somatic indices suggested metabolic and nutritional imbalances in sunfish as a result of exposure to pulp mill effluents. Fish populations in the contaminated river also demonstrated an abnormal size distribution and age structure. Female redbreast sunfish from the BKME-impacted river contained a large number of atretic oocytes and had lower serum levels of estradiol than fish from the reference site. These observations plus data from previous developmental toxicity studies suggest that the primary mechanism by which BKME may affect fish populations in this river is through reproductive dysfunction and recruitment failure. Decreased recruitment may have caused a reduction in population size and resulted in decreased competition and increased resource availability. With increased food and habitat availability, more energy may have been available for growth and lipid storage of survivors in the contaminated river.

Analytical and experimental studies on the relationship between Na+, K+, and water uptake during volume increases associated with Fundulus oocyte maturation in vitro.

Oocytes of marine and estuarine teleosts often undergo pronounced volume increases during the maturation phase of development that precedes ovulation and fertilization. To examine the physiological correlates of these volume increases, prematuration follicles of the saltmarsh teleost, Fundulus heteroclitus, were cultured in vitro with a maturation-inducing steroid (17 alpha-hydroxy-20 beta-dihydroprogesterone). Mean follicle volume rose significantly (75%) during a 40-h incubation period. Similar to the situation previously found in vivo, uptake of water by the maturing follicle was responsible for this volume increase in vitro, with the water content increasing from 62% to 78% of the total follicle mass. The follicle contents of two probable osmotic effectors--Na+ and K(+)--also rose, the increase in K+ being twice that of Na+. The influx of K+ even exceeded water uptake, resulting in a net increase in the concentration of this cation. It thus appears that the influx of these cations, in particular K+, is a major cause of the uptake of osmotically obligated water and subsequent volume increase experienced by maturing F. heteroclitus follicles. In a search for operant mechanisms, it was found that follicle hydration, but not maturation, was strictly dependent on external K+ in a concentration-dependent manner. The mechanism by which K+ accumulates in the follicle was insensitive to ouabain, so that a typical Na+, K(+)-ATPase mechanism does not appear to be involved. The ability of external K+ to promote follicle hydration was gradually lost during the maturation process as the oocyte dissociated from the surrounding granulosa cells in preparation for ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroidogenesis in Fundulus heteroclitus. II. Production of 17 alpha-hydroxy-20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by various components of the ovarian follicle.

Fundulus heteroclitus prematurational follicles (1.3-1.4 mm) were dissected into various components and cultured in vitro to examine the type of cells involved in the synthesis of steroids upon F. heteroclitus pituitary extract (FPE) stimulation or addition of exogenous precursors (25-hydroxycholesterol or pregnenolone). Culture media and follicular tissue extracts were assayed for 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha-OH.20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) content using specific radioimmunoassays. Complete removal of the follicle wall (denuded oocytes) eliminated steroid accumulation induced by FPE treatment. Removal of the theca/epithelium layer (defolliculated oocytes) did not affect the steroidogenic response (17 alpha-OH,20 beta-DHP, T, E2 production) of the follicles to FPE or exogeneously added precursors (25-hydroxycholesterol or pregnenolone). Isolated theca/epithelium layers secreted only T. Isolated follicular preparations that did not contain the oocyte (theca/epithelium layers or follicle cells) secreted higher levels of steroids to the culture media than did intact follicles. We conclude from these results that (1) the follicle cells (granulosa cells) are the primary source of the various steroids produced by the F. heteroclitus ovarian follicle in response to FPE stimulation: (2) the synthesis of 17 alpha-OH,20 beta-DHP and E2 does not require the involvement of two cell types as shown in other teleosts; (3) the theca/epithelium layer is able to produce T but lacks the aromatase activity necessary for E2 synthesis; and (4) steroids synthesized in the follicle wall are both secreted to the medium and accumulated in the oocyte.

Fundulus heteroclitus Gonadotropin(s) 2. Year-round husbandry of animals with active pituitaries and responsive follicles.

Mummichogs (Fundulus heteroclitus) were exposed to various regimens of temperature and photoperiod in order to develop a routine husbandry procedure so as to have throughout the year a population of reproductively healthy fish with a good quantity and quality of prematuration follicles (1.2-1.5 mm) suitable for carrying out homologous bioassays ofF. heteroclitus gonadotropin. During the fall and winter months, wild fish in the field all had regressed ovaries (gonadosomatic index <1). On the other hand, laboratory-maintained fish, with or without cold temperature (15°C) and short photoperiod (10 h light/day) pretreatment, generally had sexually mature ovaries (GSI>10) when maintained on a warm temperature (25°C) and long photoperiod (14 h light) protocol. Ovarian follicles retrieved from laboratory fish were responsive toF. heteroclitus pituitary extract stimulation, and underwent germinal vesicle breakdown normallyin vitro. Hence these ovarian follicles served well as a bioassay forF. heteroclitus gonadotropin even outside of the normal breeding season. The pituitary glands retrieved from laboratory fish in winter also retained high gonadotropic potencies, in terms of maturational and steroidogenic activities. Our results thus demonstrated that active gametogenesis in laboratory-maintained fish can be extended five months beyond the end of the normal breeding season. Apparently,F. heteroclitus in Florida is potentially a continuous breeder when under favorable conditions, but has a reproductive quiescent period imposed upon it by some environmental stressor(s). Although the design of the present experiments did not determine the relative importance of nutritional factors, temperature, and photoperiod on the annual reproductive cycle ofF. heteroclitus, there are indications that diatary factors may play a much more dominant role in the reproductive cycle than previously recognized.

Oocyte maturation in the mummichog (Fundulus heteroclitus): effects of steroids on germinal vesicle breakdown of intact follicles in vitro.

The effects of several steroids on the maturation of follicle-enclosed oocytes of the mummichog Fundulus heteroclitus in vitro were examined. At a relatively high concentration (1.0 microgram/ml), a number of different steroids, including pregnenolone, 17 alpha-hydroxypregnenolone, corticosterone, cortisol, 11-deoxycorticosterone, 11-deoxycortisol, androstenedione, testosterone, progesterone, 17 alpha-hydroxyprogesterone, 20 beta-dihydroprogesterone, and 17 alpha-hydroxy-20 beta-dihydroprogesterone, were able to induce germinal vesicle breakdown (GVBD) in prematuration oocytes. Cholesterol, 17 beta-estradiol, 11-ketotestosterone, and 11 beta-hydroxytestosterone were totally ineffective. In general, 11-oxysteroids tended to be less effective than their 11-deoxysteroid counterparts. Two 20 beta-dihydroprogestins--17 alpha-hydroxy-20 beta-dihydroprogesterone and 20 beta-dihydroprogesterone--were the most potent maturation-inducing steroids, initiating 50% GVBD at 1 ng/ml in follicles obtained from ovaries containing mature or maturing follicles in vivo, or at 2.5-4.0 ng/ml in follicles from ovaries lacking mature or maturing oocytes in vivo. These results are consistent with several previous studies involving salmonids and various other teleosts, and suggest a possible physiological role for a 20 beta-dihydroprogestin in the resumption of meiotic maturation in F. heteroclitus.

Changes in teleost yolk proteins during oocyte maturation: correlation of yolk proteolysis with oocyte hydration.

Yolk proteins of prematuration occytes and postmaturation eggs were compared by SDS gel electrophoresis in several teleosts, including freshwater species that produce demersal eggs, estuarine and marine species with demersal eggs, and marine species with pelagic eggs. In certain teleosts distinct changes in yolk protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins at this time; proteolysis is most pronounced in marine fishes with pelagic eggs. In many teleosts the oocyte swells by hydration during maturation; this hydration is also most pronounced in marine fishes with pelagic eggs. The extent of yolk proteolysis is well correlated with the extent of oocyte hydration during maturation.