Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17ß on mRNA expression.

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish.

Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.

Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.

Ovarian development and related changes in steroid hormones in female wild common carp (Cyprinus carpio carpio), from the south-eastern Caspian Sea.

Wild common carp (Cyprinus carpio carpio) is a native valuable but threatened species from the south-eastern Caspian Sea in which the endocrine control of its reproduction has not been studied. The objectives of this research were to study the reproductive strategy and changes in steroid hormones during ovarian development. From October 2009 to June 2010, 65 adult females were caught from the Golestan coast (Iran) and the ovarian histology, and gonadosomatic and hepatosomatic indices (GSI and HSI) were studied. Also, the plasma profiles of steroid hormones including testosterone (T), 17ß-estradiol (E2) and 17α-, 20ß-dihydroxyprogesterone (DHP) were measured by radioimmunoassay. The GSI increased gradually during sampling times and reached maximum value at the peak of reproduction season, but the HSI decreased during spawning season. All stages of ovarian development, except the stage of Balbiani bodies, were recorded macro- and microscopically. Spent fish were caught at six of nine sampling times. The peaks of spawning were at late winter and early spring. The results of this study showed that the majority of wild carp collected during the sampling period displayed asynchronous oocyte development. Plasma T showed no significant differences during sampling times or at different stages of ovarian development. The level of E2 decreased gradually during sampling times reached minimum value at the spawning season, and highest value was recorded at tertiary vitellogenesis stage. The plasma levels of DHP during late winter and early spring were significantly higher than those of other sampling periods and its maximum level associated with oocyte maturation stage.

Follistatin is an early player in rainbow trout ovarian differentiation and is both colocalized with aromatase and regulated by the wnt pathway.

In mammals, follistatin (FST) plays an important role in early ovarian differentiation, acting downstream of the Wnt pathway. In teleost fish, fst is implicated in folliculogenesis and oocyte maturation, and an early and specific expression during ovarian differentiation has been described in rainbow trout, Oncorhynchus mykiss. By in situ hybridization, we demonstrated that during rainbow trout gonadal differentiation, fst shares a similar expression pattern with cyp19a1a, the gene encoding ovarian aromatase, a key steroidogenic enzyme needed for ovarian differentiation in fish. Expression of fst and cyp19a1a was first detected in a few scattered cells in the embryonic ovary several days before hatching. Then, after histological differentiation, fst and cyp19a1a expression was localized in clusters of cells lining the future ovarian lamellae. As FST expression is known to be induced by the Wnt/ß-catenin pathway in mammals, the Wnt pathway was inhibited in vivo with the IWR-1 molecule, and we analyzed by qPCR the effects of this treatment on fst expression. We found that IWR-1 decreased fst expression in female gonads, consistent with a regulation of fst expression by the Wnt pathway in rainbow trout. Furthermore, expression of cyp19a1a was also downregulated, suggesting an implication of the Wnt pathway in ovarian differentiation.

Hydride generation using a metallic atomizer after microwave-assisted extraction for inorganic arsenic speciation in biological samples.

The present speciation method reports the determination of inorganic arsenic forms, using metallic furnace hydride generation atomic absorption spectrometry. The inorganic As speciation is carried out using mild conditions for hydride formation, such as slightly acid pH media (4.50) and low tetrahydridoborate(1-) concentration (0.1% (w/v)). Limits of detection and quantification of 2.0 and 6.6 µg L(-1) of iAs(III) are obtained using optimized conditions. Additionally, microwave-assisted extraction using water as solvent is carried out to provide the appropriate environment for As species extraction as well as impeding inter-conversion between species. With these analytical strategies, As was accurately determined (at 99.9% confidence level) in water and plankton samples.

Sex differentiation in an all-female (XX) rainbow trout population with a genetically governed masculinization phenotype.

Sex determination is known to be male heterogametic in the rainbow trout, Oncorhynchus mykiss; however, scattered observations that deviate from this rather strict genetic control have been reported. Here, we provide a detailed morphological and histological characterization of the gonadal differentiation and development (from 43 days postfertilization to 11 months of age) in an all-female (XX) population with a genetically governed masculinization phenotype. In comparison with control males and females, the gonadal differentiation in these animals was characterized by many perturbations, including significantly fewer germ cells. This decrease in germ cells was confirmed by the significantly decreased expression of 2 germ cell maker genes (vasa and sycp3) in the masculinized XX populations as compared with the control females and control males. Although only a proportion of the total adult population was partially or fully masculinized, this early differentiating phenotype affected nearly all the sampled animals. This suggests that the adult masculinization phenotype is the consequence of an early functional imbalance in ovarian differentiation in the entire population. We hypothesize that the lower number of germ cells that we observed in this population could be one cause of their masculinization.

The Nme gene family in fish.

The Nme gene family, also known as Nm23 or NDPK, is a very ancient gene family that can be found in all kingdoms of life. In the late eighties, a gene of the Nme family, NME1, was identified as the first metastatic suppressor gene, resulting in a major interest for this family. Due to the complexity of the family, the need for a unified and evolutionary-supported gene nomenclature was recently stressed by the scientific community. Based on a complete evolutionary history study of the gene family in metazoans and vertebrates, a unified nomenclature was recently proposed and accepted by gene nomenclature consortia. In addition to its well-documented role in tumor metastasis, members of the Nme family are also involved in a wide variety of cellular and physiological processes. Available data in non-mammalian species remain, however, scarce with the noticeable exception of Drosophila in which a major role in development was reported. In fish, very few studies have specifically investigated the role of nme genes. Several transcriptomic and proteomic studies have, however, revealed the expression of nme genes in various fish organs and tissues, in mature oocytes, and during embryonic development. Altogether, interest for the Nme gene family in fish is growing and new functions/roles in fish biology are expected to be discovered in the forthcoming years. Here, we briefly review the current knowledge of the Nme family in fish.

Unusual cases of adult intersexuality in the European eel Anguilla anguilla.

Two cases of intersexuality are reported for the first time in European eel, Anguilla anguilla (at the beginning of the silver eel stage), within 140 fish caught as glass eels in the south-west of France and reared in tanks at 17°C. Cysts containing spermatozoa were observed in ovaries with pre-vitellogenic oocytes. This feature is very uncommon, especially owing to the fact that male cells do not normally reach this stage in captivity, and an environmentally controlled transdifferentation process may not be excluded. Besides, the expression of the gonadal aromatase gene (cyp19a1a) was found to be higher in these 2 intersexual fish compared to normal females, although these results must be considered with caution since only 2 intersexual fish were available. A possible feminizing effect of this 'abnormal' upregulation of aromatase is discussed.

Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis.

In contrast to the classical model describing the synthesis of androgens and estrogens as restricted to somatic cells, a previous study demonstrated that Xenopus laevis oocytes participate in androgen synthesis. The objective of our study was to determine whether Xenopus oocytes are also involved in estrogen synthesis. More precisely, we analyzed aromatase expression by in situ hybridization and RT-QPCR and measured aromatase activity. Aromatase, the enzyme responsible for estrogen synthesis, appears to be expressed and active not only in the follicular cells but also in the vitellogenic oocytes. During late oogenesis, aromatase oocyte expression and activity decreased concomitantly with the trend observed in surrounding follicular layers. In order to investigate the role of estradiol-17ß (E(2)), we studied its effect on oocyte meiotic resumption. It appears that, as in Rana pipiens, E(2) inhibited the follicle-enclosed maturation of Xenopus oocytes, likely through inhibition of LH-induced maturation-inducing steroid synthesis. In addition, E(2) exerted a slight enhancing action on denuded oocyte maturation whose biological significance remains unclear. Together, our results demonstrate that Xenopus oocyte significantly participates in ovarian E(2) synthesis and this may be a common feature of vitellogenic vertebrates.

Disruption of the secretion and action of 17,20ß-dihydroxy-4-pregnen-3-one in response to a rise in temperature in the Arctic charr, Salvelinus alpinus. Consequences on oocyte maturation and ovulation.

Plasma levels of 17,20ß-dihydroxy-4-pregnen-3-one (17,20ßP), and the timing of ovulation were investigated in female Arctic charr (Salvelinus alpinus) reared at 5°C and at 10°C during the pre-spawning period. The effects of switching from 5 to 10°C, and from 10 to 5°C were also investigated. 17,20ßP plasma levels were higher at 5°C than at 10°C. A switch from 10 to 5°C stimulated 17,20ßP secretion, whereas a switch from 5 to 10°C had the opposite effect. Ovulation occurred spontaneously in the females kept at 5°C, and in those switched from 10 to 5°C. In contrast, ovulation was inhibited in females reared at 10°C, and in those switched from 5 to 10°C. Oocyte maturation at 5°C and at 10°C in the presence of LH or of 17,20ßP was also investigated in vitro using donor females reared at 5 or 10°C. Both LH and 17,20ßP stimulated oocyte maturation more effectively in oocytes incubated at 5°C than at 10°C. At both incubation temperatures, the rearing temperature of the donor females had a significant impact on their responsiveness to LH stimulation, but had no effect on their responsiveness to 17,20ßP stimulation. In addition to the inhibition of LH secretion, which had already been reported, the results reported here show that in Arctic charr raising the temperature above the physiological range reduces both follicular responsiveness to LH stimulation and the sensitivity of oocytes to 17,20ßP stimulation.

Seasonality of reproduction and production in farm fishes, birds and mammals.

A very large majority of farm animals express seasonal variations in their production traits, thus inducing seasonal availability of fresh derived animal products (meat, milk, cheese and eggs). This pattern is in part the consequence of the farmer's objective to market his products in the most economically favourable period. It may also be imposed by the season-dependent access to feed resources, as in ruminants, or by the specific requirements derived from adaptation to environmental conditions such as water temperature in fish. But seasonal variations in animal products are also the consequence of constraints resulting from the occurrence of a more or less marked seasonal reproductive season in most farm animal species including fish, poultry and mammals. Like their wild counterparts, at mid and high latitudes, most farm animals normally give birth at the end of winter-early spring, the most favourable period for the progeny to survive and thus promote the next generation. As a consequence, most species show seasonal variations in their ovulation frequency (mammals and fish: presence or absence of ovulation; birds: variations or suppression of laying rates), spermatogenic activity (from moderate to complete absence of sperm production), gamete quality (variations in fertilisation rates and embryo survival), and also sexual behaviour. Among species of interest for animal production, fishes and birds are generally considered as more directly sensitive to external factors (mainly temperature in fish, photoperiod in birds). In all species, it is therefore advisable that artificial photoperiodic treatments consisting of extra-light during natural short days (in chickens, turkeys, guinea fowl, sheep and goats) or melatonin during long days (in goats, sheep) be extensively used to either adjust the breeding season to animal producer needs and/or to completely overcome seasonal variations of sperm production in artificial insemination centres (mammals) and breeder flock operations (poultry, fish farming). Pure light treatments (without melatonin), especially when applied in open barns, could be considered as non invasive ones which fully respect animal welfare.

Gonadal changes and blood sex steroids levels during natural sex inversion in the protogynous Mediterranean red porgy, Pagrus pagrus (Teleostei: Sparidae).

Changes in gonadal structure and serum levels of sex steroids were investigated during natural sex inversion from female to male in reared populations of the protogynous Mediterranean red porgy, Pagrus pagrus. Four developmental phases were identified by histological observation: female, early transitional (ETr), late transitional (LTr), and male phases. At female phase, a few nests of spermatogonia were observed at the posterior-ventral part of the gonad mainly in females out of the breeding season. At ETr phase, spermatogonial proliferation occurred while perinucleolar oocytes showed signs of degeneration. At LTr phase, seminiferous lobules were formed and spermatogonial proliferation expanded along the ovary which degenerated. All types of male germ cells could be found. At male phase, functional testis underwent active spermatogenesis while small ovarian remnants associated to fat tissue could be detected. Both 17beta-estradiol (E2) and estrone (E1) blood levels were significantly lower in fish at transitional and male phases in comparison to breeding females, while levels of 11-ketotestosterone (11-KT) and testosterone (T) gradually increased in the transitional and male phases. In conclusion, the protogynous P. pagrus possess a delimited type bisexual gonad with a medio-dorsal ovarian area and a latero-ventral testicular zone. Sex inversion starts mainly after the female breeding season with an active spermatogonial proliferation. The testis tissues develop while ovarian tissues regress to disappear completely in the functional male. This process is accompanied by a sharp decrease of estrogens levels and a progressive increase of androgens levels. The physiological significance of such endocrine changes is discussed.

Androgen and estrogen treatments alter steady state messengers RNA (mRNA) levels of testicular steroidogenic enzymes in the rainbow trout, Oncorhynchus mykiss.

Recent investigations have shown that estrogens have profound inhibitory effects on steroidogenic enzyme gene expressions before and after testicular differentiation in the rainbow trout, Oncorhynchus mykiss. This present study bring new data on juvenile rainbow trout treated with estrogens and androgens. Following a 8 days oral treatment of juvenile male with 17alpha-ethynyl-estradiol (EE2, 20 mg/kg diet) or 11beta-hydroxyandrostenedione (11betaOHDelta4, 10 mg/kg diet), we observed a fast and marked decrease of steady-state mRNA levels for 3betaHSD, P450scc, P450c17, and P450c11 enzymes in the testis. After completion of these treatments, mRNA levels of these enzymes remained low in EE2 treated males whereas in 11betaOHDelta4 treated males they recovered their initial levels in 8 days. This demonstrate that both androgen and estrogen treatments have profound effects on testicular steroidogenesis by decreasing steroid enzymes steady-state mRNA. After in vitro incubation of testicular explants with 17beta-estradiol (E2, 600 ng/ml of medium), we also observed a decrease of mRNA levels for 3betaHSD and P450c11. This suggest that estrogens effects could be triggered, at least to some extend, directly on the testis. We also investigated the hypothesis of a negative feedback of steroids on follicle stimulating hormone (FSH) secretion, but FSH plasmatic levels in treated fish did not showed any significant decrease. This demonstrate that FSH is not implied in this steroids inhibition of steroidogenic enzymes gene expression.

Binding affinities of hepatic nuclear estrogen receptors for phytoestrogens in rainbow trout (Oncorhynchus mykiss) and Siberian sturgeon (Acipenser baeri).

Phytoestrogens are dietary estrogenic contaminants capable of inducing vitellogenin synthesis in rainbow trout and Siberian sturgeon. A competitive-binding assay on their hepatic estrogen receptors (ER) was performed to determine the relative affinity of phytoestrogens compared to estradiol (E(2)). Phytoestrogen concentrations used were 1000 times higher than for E(2), except for genistein and formononetin. For each compound, the competition with 50%-bound labelled E(2) (DC(50)) was considered in order to classify phytoestrogens according to their affinity for ER. The affinities are compared for each species. In rainbow trout, estradiol (DC(50): 7 nM)>formononetin (DC(50): 260 nM)>genistein (DC(50): 570 nM)>equol (DC(50): 5.3 microM)>daidzein (DC(50): 9 microM)>biochanin A (DC(50): 100 microM). In sturgeon, estradiol (DC(50): 5 nM)>genistein (DC(50): 220)>formononetin (DC(50): 1 microM)>equol>(DC(50): 8.3 microM)>daidzein>(DC(50): 80 microM)>biochanin A (DC(50): 100 microM). These results demonstrate that phytoestrogens, mimicking estradiol, can disturb the endocrine system by competing for ER. Also, the higher sensitivity to genistein observed in vivo in Siberian sturgeon (vitellogenin synthesis), compared to rainbow trout, is not due to a higher affinity of genistein for the hepatic ER. Thus, the metabolism of phytoestrogen could be species dependent and affect sensitivity.

Hypothyroidism induces type I iodothyronine deiodinase expression in tilapia liver.

In the current study, the authors examined the effects of experimentally induced hypothyroidism on peripheral thyroid hormone metabolism and growth in two closely related tilapia species: the Nile tilapia (Oreochromis niloticus) and the slower growing black tilapia (Sarotherodon melanotheron). Hypothyroidism, induced by administration of 0.2% methimazole through the food, significantly decreased plasma T(3) and T(4) in both species. This decrease in circulating thyroid hormones was accompanied by an increase in hepatic type II deiodinase (D2) and a decrease in hepatic type III deiodinase (D3). Hepatic type I deiodinase (D1), which is barely expressed in euthyroid tilapia, was significantly upregulated during hypothyroidism. The changes in hepatic D1 and D2 enzyme activity were paralleled by changes in D1 and D2 mRNA levels, indicating pretranslational regulation. Hypothyroidism also resulted in severe growth retardation that was accompanied by an increase in condition factor. Because hyperthyroidism has been shown to decrease the condition factor, these results suggest that thyroid hormones play an essential role in the control of proportional body growth in fish. The authors conclude that (1) hepatic D1 expression is induced by hypothyroidism in tilapia, (2) the changes in hepatic iodothyronine deiodinases during hypothyroidism in tilapia are predominantly regulated at a pretranslational level, and (3) thyroid hormones are involved in the control of proportional body growth in fish.

Steroid enzyme gene expressions during natural and androgen-induced gonadal differentiation in the rainbow trout, Oncorhynchus mykiss.

In fish, according to Yamamoto's model, androgens would drive testis differentiation and estrogens ovarian differentiation. In order to study the implication of steroid enzymes in rainbow trout gonadal differentiation, we examined the expression of some steroid enzyme genes during natural differentiation (cholesterol side chain cleavage = P450scc, 17-hydroxylase/lyase = P450c17, 3beta-hydroxysteroid dehydrogenase = 3betaHSD) and androgen-induced differentiation (P450scc, P450c17, 3betaHSD, aromatase = P450aro, and 11beta-hydroxylase = P45011beta). Expressions of P450scc, 3betaHSD, and P450c17 were all detected in male and female gonads at 55 days post-fertilization (dpf), i.e., two weeks before histological differentiation. There were no differences in their expression level respective to the sex. The androgen treatment was carried out by administration of 11beta-hydroxyandrostenedione (11betaOHDelta4) in genetic all-female populations and the resulting sex ratios were found to be 100% male even at a low dosage of 1 mg/kg of food. Following 11betaOHDelta4 treatment, only the expression of P450c17 was found to be sustained when compared with the female untreated control. In contrast, P450scc was clearly up-regulated and 3betaHSD and P450aro down-regulated by the androgen treatment. P45011beta gene expression remained low in gonads of androgen-treated females, as it did in control untreated females. These results together demonstrate that steroidogenesis in rainbow trout is potentially active in pre-differentiating gonads of both sexes, and that one of the masculinizing actions of androgens in the species may be to down-regulate the female-specific gonadal P450aro gene expression. However, in vivo androgen treatment in genetic females does not induce the same pattern of steroid gene expression as in genetic males. These data suggest that exogenous androgens might induce a male differentiation process with P450aro inhibition being one of the steps required. However, this process would not involve endogenously produced 11-oxygenated androgens.

Search for genes involved in the temperature-induced gonadal sex differentiation in the tilapia, Oreochromis niloticus.

In the tilapia, Oreochromis niloticus, sex is determined by genetic factors (XX/XY) but temperature can also influence the gonadal sex differentiation. Elevated temperatures of 35 degrees C can generate functional male phenotypes if applied before and during sexual differentiation. The genes and mechanisms by which temperature acts on the cascade leading to sex differentiation have been investigated. Two strategies have been followed: 1) Search for novel genes by differential display, and 2) Expression studies of candidate genes. Genetically all-female and all-male progenies were reared at 27 degrees C (natural temperature) and at 35 degrees C (masculinizing treatment) and gonads dissected. Using differential display, we isolated a 300 bp cDNA (MM20C) from temperature-masculinized females. Virtual northern analysis revealed a 1.2 kb transcript in 35 degrees C treated females and males, but hardly any expression in natural females (27 degrees C). Semi-quantitative RT-PCR established a several-fold increase in MM20C expression in 35 degrees C masculinized fry. Elevated expression was observed in natural males (27 degrees C) with higher levels detected in those reared at 35 degrees C. Furthermore, we have analyzed as a candidate gene the P450 11beta-hydroxylase, an important androgen steroidogenic enzyme. Low levels of expression were found in natural males. This coincides with low concentrations of 11 ketotestosterone in the gonads before and during gonadal sex differentiation. Higher expression levels of 11beta-hydroxylase were detected in male gonads at 35 degrees C but levels in phenotypic males were similar to those found for natural females. Previous results reported that expression of aromatase is repressed by masculinizing treatments. Our study demonstrated that masculinizing-temperature can also stimulate the expression of other gene(s).

Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus.

In the tilapia Oreochromis niloticus, sex is determined genetically (GSD), by temperature (TSD) or by temperature/genotype interactions. Functional masculinization can be achieved by applying high rearing temperatures during a critical period of sex differentiation. Estrogens play an important role in female differentiation of non-mammalian vertebrates. The involvement of aromatase, was assessed during the natural (genetic all-females and all-males at 27 degrees C) and temperature-induced sex differentiation of tilapia (genetic all-females at 35 degrees C). Gonads were dissected between 486--702 degree x days. Aromatase gene expression was analyzed by virtual northern and semi-quantitative RT-PCR revealing a strong expression during normal ovarian differentiation concomitant with high levels (465 +/- 137 fg/g) of oestradiol-17 beta (E2-17 beta). This was encountered in gonads after the onset of ovarian differentiation (proliferation of both stromal and germ cells prior to ovarian meiosis). Genetic males exhibited lower levels of aromatase gene expression and E2-17 beta quantities (71 +/- 23 fg/ g). Aromatase enzyme activity in fry heads established a sexual dimorphism in the brain, with high activity in females (377.9 pmol/head/hr) and low activity in males (221.53 pmol/head/hr). Temperature induced the masculinization of genetic females to a different degree in each progeny, but in all cases repression of aromatase expression was encountered. Genetic males at 35 degrees C also exhibited a repression of aromatase expression. Aromatase brain activity decreased by nearly three-fold in the temperature-masculinized females with also a reduction observed in genetic males at 35 degrees C. This suggests that aromatase repression is required in the gonad (and perhaps in the brain) in order to drive differentiation towards testis development. Mol. Reprod. Dev. 59:265-276, 2001.

Mercury fluxes in a natural forested Amazonian catchment (Serra do Navio, Amapá State, Brazil).

Mercury (Hg total) fluxes were calculated for rainwater, throughfall and stream water in a small catchment located in the northeastern region of the Brazilian Amazon (Serra do Navio, Amapá State), whose upper part is covered by a natural rainforest and lower part was altered due to deforestation and activities related to manganese mining. The catchment area is 200 km from the nearest gold mining (garimpo). Minimum and maximum Hg concentrations were measured monthly from October 1996 to September 1997 and were 3.5-23.4 ng l(-1) for rainwater, 16.5-82.7 ng l(-1) for throughfall (March-August 1997) and 1.2-6.1 and 4.2-18.8 ng l(-1) for stream water, in natural and disturbed areas, respectively. In the natural area, the inputs were 18.2 microg m 2 year(-1) in rainwater and 72 microg m(-2) year(-1) in throughfall. This enrichment was attributed to dry deposition. The stream output of 2.9 microg m(-2) year(-1) indicates that Hg is being recycled within the forest as other chemical species or is being retained by the soil system, as confirmed by the cumulative Hg burden in the 0-10 cm surface layer, which was 36480 microg m(-2). When the disturbed area of the catchment was included, the stream output was 9.3 microg m(-2), clearly indicating the impact of the deforestation of the lower part of the basin on the release of mercury. The Hg burden in the disturbed area was 7560 microg m(-2) for the 0-10 cm surface layer.

A mineralocorticoid-like receptor in the rainbow trout, Oncorhynchus mykiss: cloning and characterization of its steroid binding domain.

Using reverse transcriptase polymerase chain reaction (PCR) (RT-PCR) with degenerate primers followed by 3' rapid amplification of cDNA ends PCR (3'Race-PCR) we have isolated a new fish steroid receptor cDNA sequence of 1806 bp from rainbow trout (Oncorhynchus mykiss) testis. This sequence has clear homology with various mineralocorticoid receptor cDNA sequences (rat, human, African toad: 68-70% amino acid identity), and encompasses the second part of DNA binding domain (C domain), the whole hinge region (D domain) and the steroid binding domain (E domain) plus 726 bp of 3'untranslated sequence. COS-1 cells transfected with a pCMV5 expression vector containing the whole E domain (pCMV5-rtMR) showed high affinity binding for cortisol (K(a) = 0.53+/-0.03 nM, K(d) = 1.9 nM) in the cytosol, which could not be detected in untransfected cells. Aldosterone displaced (3)H-cortisol binding, though was less effective by than unlabeled cortisol (P<0.05). Competition experiments with other steroids gave the following hierarchy for the displacement of the (3) dexamethasone, whereas 17, 20beta-dihydroxy-4-pregnen-3-one and 17,20beta,21beta-trihydroxy-4 pregnen-3-one (two fish specific progestins) did not show any specific binding. These results strongly suggest that this cDNA sequence encodes a rainbow trout mineralocorticoid-like receptor, and represent the first description of such a receptor in teleost fish where aldosterone, the classic mineralocorticoid, is believed to be absent.