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.

DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss.

DMRT1 has been suggested to be the first conserved gene involved in sex differentiation found from invertebrates to human. To gain insight on its implication for fish gonadal differentiation, we cloned a DMRT1 homologue in the rainbow trout, Oncorhynchus mykiss (rtDMRT1), and showed that this gene is expressed during testicular differentiation, but not during ovarian differentiation. After 10 days of steroid treatment, expression was shown to be decreased in estrogen-treated male differentiating gonads but not to be restored in androgen-treated differentiating female gonads. This clearly reinforces the hypothesis of an important implication for DMRT1 in testicular differentiation in all vertebrates. In the adults a single 1.5 kb transcript was detected by Northern blot analysis in the testis, and its expression was found to be sustained throughout spermatogenesis and declined at the end of spermatogenesis (stage VI). Along with this expression in the testis we also detected by reverse transcriptase-polymerase chain reaction a slight expression in the ovary. We also obtained new DM-domain homologous sequences in fish, and their analysis suggest that at least four different genes bearing 'DM-domain' (DMRT genes) exist in fish just as in all vertebrate genomes.

17beta-estradiol treatment decreases steroidogenic enzyme messenger ribonucleic acid levels in the rainbow trout testis.

In fish, estrogens are well known for their involvement in ovarian differentiation and have been shown to be very potent feminizing agents when administrated in vivo during early development. However, the mechanism of action of exogenous estrogens is poorly understood. We report here on the feminizing effects of estrogen treatment on the testicular levels of some steroidogenic enzyme messenger RNAs [mRNAs; cholesterol side-chain cleavage (P450scc), 17-hydroxylase/lyase (P450c17), 3beta-hydroxysteroid dehydrogenase (3betaHSD), 11beta-hydroxylase (P45011beta), and aromatase (P450aro)] in the rainbow trout, Oncorhynchus mykiss. Treatment was carried out by dietary administration of 17beta-estradiol (E(2); dosage of 20 mg/kg diet) to a genetically all male population. Steroidogenesis in the differentiating testis was demonstrated to be strongly altered by E(2), as this treatment resulted in considerable decrease in P450c17, 3betaHSD, and P45011beta mRNAs after only 10 days of treatment. In contrast, P450scc and P450aro mRNA levels were unaffected by E(2), with P450scc mRNA levels remaining unaltered and P450aro not stimulated by this feminizing estrogen treatment. To better characterize this E(2) effect, the same treatment was applied on postdifferentiating males, and roughly the same expression pattern was detected with a considerable decrease in testicular P450c17, 3betaHSD, and P45011beta mRNAs and a significant, but reduced, decrease in P450scc mRNA. In the interrenal, these steroidogenic enzyme mRNAs were not significantly affected by this E(2) treatment, except for a slight, but significant, decrease in P450scc mRNA. These results clearly demonstrate that estrogens have profound effects on testicular steroidogenesis and that they are acting specifically on the testis by decreasing mRNA steady state levels of many steroidogenic enzyme genes. The decrease in P45011beta mRNA, and thus inhibition of the synthesis of testicular 11-oxygenated androgens, may be an important step required for the active feminization of these genetic males.

Evidence for two distinct functional glucocorticoid receptors in teleost fish.

Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tIssues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K(d)=5.05+/-0.45 nM; GR2 K(d)=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-)(6 )M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor. Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.

Endocrine and environmental aspects of sex differentiation in gonochoristic fish.

This paper reviews current knowledge concerning the endocrine and environmental regulation of gonadal sex differentiation in gonochoristic fish. In gonochoristic fish, although potentially active around this period, the hypothalamo-pituitary axis is probably not involved in triggering sex differentiation. Although steroids and steroidogenic enzymes are probably not the initial triggers of sex differentiation, new data, including molecular approaches, have confirmed that they are key physiological steps in the regulation of this process. Environmental factors can strongly influence sex differentiation in gonochoristic fish. The most important environmental determinant of sex would appear to be temperature. Interactions between environmental factors and genotype have been suggested for gonochoristic fish.

Expression of cytochrome P450(11beta) (11beta-hydroxylase) gene during gonadal sex differentiation and spermatogenesis in rainbow trout, Oncorhynchus mykiss.

Androgens and especially 11-oxygenated androgens are known to be potent masculinizing steroids in fish. As a first step to study their physiological implication in gonadal sex differentiation in fish, we cloned a testicular cytochrome P450(11beta) (11beta-hydroxylase) cDNA in the rainbow trout, Oncorhynchus mykiss. We isolated a 1882 bp P450(11beta) cDNA (rt11betaH2, AF217273) which contains an open reading frame encoding a 552 putative amino acids protein. This sequence was highly homologous (98% in nucleotides and 96.5% in amino acids) to another rainbow trout P450(11beta) sequence (AF179894) and also to a Japanese eel P450(11beta) (68% in amino acids). Northern blot analysis detected a single transcript of 2 kb which was highly expressed in the testis (stage II) and to a lesser degree in the anterior kidney (containing the interrenal tissue). No signal was detected in the posterior kidney, brain, liver, skin, intestine and heart. In the testis this transcript was highly expressed at the beginning of spermatogenesis (stages I and II), followed by a decrease during late spermatogenesis (stages III to V). By semi-quantitative reverse transcription polymerase chain reaction, P450(11beta) expression during gonadal differentiation was estimated to be at least 100 times higher in male than in female gonads. This difference was first detected at 55 days post-fertilization (dpf), i.e. 3 weeks before the first sign of histological sex differentiation, and was sustained long after differentiation (127 dpf). Specific P450(11beta) gene expression was also demonstrated before testis differentiation (around 50 dpf) using virtual Northern blot, with no expression detected in female differentiating gonads. From these results, and also based on the already known actions of 11-oxygenated androgens in testicular differentiation in fish, it is now suggested that P450(11beta) gene expression is a key factor for the testicular differentiation in rainbow trout.

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.

Expression of sGnRH mRNA in gonads during rainbow trout gametogenesis.

The salmon gonadotropin-releasing hormone (sGnRH) is the major form of GnRH decapeptide expressed in the salmonid brain and it acts as a gonadotropin releaser. In rainbow trout, sGnRH-1 and sGnRH-2 mRNA forms were found in brain and gonads. We analyzed the expression of both forms in trout gonads at different stages of gametogenesis. Northern blot demonstrated that sGnRH-2 mRNA was the major sGnRH form in testis and ovary. In testis but not in ovary, brain or pituitary, alternatively spliced sGnRH-2 transcripts which coded for prepro-sGnRH with a truncated GnRH-associated peptide due to a premature stop codon in retained intron 2 were detected. In testis, sGnRH mRNA was highly expressed before the onset of spermatogenesis, it disappeared at stage II and then increased progressively up to stage VI. In ovary, the expression of sGnRH was high in immature pre-vitellogenic fish and progressively decreased throughout vitellogenesis. At ovulation it reached its maximum and came down again after stripping. The decrease of sGnRH mRNA expression during the period of active spermatogonial proliferation in testis and increase during meiosis occurrence in testis and ovary suggest an anti-proliferative and meiosis-stimulating effect of sGnRH during rainbow trout gametogenesis.

[Erysipelas in the young population of a military hospital]. / Erysipèles dans la population jeune d'un hôpital militaire.

INTRODUCTION: Erysipelas usually affects either elderly patients or patients with predisposing factors. We studied erysipelas in a young healthy military population. SUBJECTS AND METHODS: A retrospective study of patients, less than 30 years old, admitted to the Hôpital d'Instruction des Armées Robert Picqué between 1991 and 1997 was performed. RESULTS: Eighty-one patients were studied, 80 were men, mean age was 21.4 years. Localization of erysipelas was: face: 2; leg: 74. Facilitating factors were: portal of entry in 100 p. 100; venous insufficiency in 1 case; alcohol abuse in 1 case. Anti-inflammatory agents had been given at the beginning of treatment in 32.1 p. 100 cases. 7.1 p. 100 patients had a recurrence.. Complications were: abscess in 8 cases and bursitis in 1 case. No facilitating factor was detected. The course was not related with bacterial findings. No thrombo-embolic complication was observed. DISCUSSION: In a young healthy population, erysipelas is not a rare infection. Face is an exceptional localization of erysipelas in relation to older population. The main risk factor for developing this infection is local factor found in 100 p. 100 cases but general factors as repeated hikes, care access, long standing could also be incriminated. Use of anti-inflammatory agents is frequent but role of anti-inflammatory agents in developing complications is not clear. Complications are rare and benign except for frequence of recurrences while the good health.

[Human papillomavirus 6/11 in conjunctival papilloma]. / Papillome conjonctival à virus des papillomes humains 6/11.

INTRODUCTION: Human papilloma virus is highly prevalent, but rarely localized in the conjunctiva. CASE REPORT: A 19-year-old man with no past history of skin or mucosal disease consulted for tumefaction of the conjunctiva. Exeresis revealed a viral papilloma. In situ hybridization was positive for human papilloma virus 6/11. DISCUSSION: Conjunctival lesions of human papilloma virus are often located in the caroncula as in our patient. The papillomatous aspect of the tumour may suggest squamous cell carcinoma (sometimes induced by human papillomavirus 16/18). Contamination may be manual or occur at birth via the maternal genital mucosa. Treatment usually is based on wide exeresis with cryo-application although spontaneous regression is possible.

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.

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.

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.

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.

Gonadal in vitro androstenedione metabolism and changes in some plasma and gonadal steroid hormones during sex inversion of the protandrous sea bass, Lates calcarifer.

Steroidogenesis in the gonad of the protandrous sea bass, Lates calcarifer, was examined in vitro in spermiating testis, previtellogenic ovary, and transitional gonads. Gonadal tissues were incubated with tritiated androstenedione. Metabolites were analyzed by thin-layer chromatography, high-performance liquid chromatography, microchemical reactions, and crystallization to constant specific activity. 17 beta-Hydroxysteroid dehydrogenase, 5 beta-reductase, and 3 alpha-hydroxysteroid dehydrogenase activities were found in all of the sex types. On the other hand, 11 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxylase activities were found only when testicular tissue was present, i.e., in testis and early transitional gonad. A low aromatase activity leading to estrone synthesis was detected in the previtellogenic ovary. In late transitional gonads, a major metabolite (metabolite X) was suggestively identified as a 3-ester of 17 beta-estradiol according to its chemical and immunological characteristics. Levels of 17 beta-estradiol (E2), the metabolite X, testosterone (T), and 11-ketotestosterone (11KT) were also measured by radioimmunoassay in plasma, before (January and February) and during (March and April) the sex inversion process. Plasma E2 was virtually undetectable (means below 25 pg/ml), although higher levels of metabolite X were found in transitional fish (485 +/- 432 pg/ml in March). Throughout this period, plasma levels of T and 11KT and the androgens/estrogens ratio were significantly higher in males than in transitional fish, where these levels decreased during the sex inversion period. The level of in vitro synthesis of metabolite X was high in transitional gonads, but their concentrations were very low (0.07 +/- 0.09 ng of equivalent E2/g in transitional gonads against 0.22 +/- 0.37 ng of equivalent E2/g in testes and 2.16 +/- 2.7 ng of equivalent E2/g in ovaries).