Florence Le Gac

PRESENCE OF SPECIFIC GROWTH HORMONE BINDING SITES IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) TISSUES : CHARACTERIZATION OF THE HEPATIC RECEPTOR

The present work outlines the presence of specific binding for chinook salmon growth hormone (sGH) in different tissue preparations of rainbow trout. Optimal incubation conditions (pH, Tris, MgCl2) were determined. Specific binding was very sensitive to salt concentration during incubation. The specific binding reached a plateau after 15 and 25 hr of incubation at 12 and 4 degrees. At 20 degrees, specific and nonspecific binding were not stable. Specific binding dissociation was slower than association and was only partial. The binding was saturable (Bmax = 187 +/- 167 pmol), of high affinity (Ka = 2.4 +/- 0.8 10(9) M-1), and very specific for GH, properties which are in agreement with the characteristics of hormonal receptors. Sea bream and mammalian GH appeared 2- and 30-fold, respectively, less potent than cold sGH2 for displacing 125I-sGH2. Tissue preparations from ovary, testis, fat, skin, cartilage, gill, blood pellet, brain, spleen, kidney, and muscle showed significant saturable binding.

Insulin-like growth factor (IGF-I) mRNA and IGF-I receptor in trout testis and in isolated spermatogenic and Sertoli cells.

Few data exist concerning the occurrence and potential role of an insulin‐like growth factor (IGF) system in fish gonads. Using Northern and slot blot hybridization with a specific salmon IGF‐I cDNA, we confirmed that IGF‐I transcription occurs in trout testis. Testicular IGF‐I mRNA abundance may be increased by long‐term GH treatment in juvenile fish, while shorter treatment with growth hormone (GH) or a gonadotropin (GTH‐II) in maturing males had no statistically significant effect. Radiolabelled recombinant human IGF‐I binds with high affinity to crude trout testis preparation, to cultured isolated testicular cells, and to a membrane fraction of these cells (Ka = 0.2 to 0.7 × 1010 M1; Bmax = 10 to 20 fmol/107 cells, and 68 fmol/mg protein of membrane). The binding site was identified as type 1 IGF receptor by its binding specificity (IGF‐I > IGF‐II ⋙ insulin) and the molecular size of its α‐subunit labelled with 125HGF‐I (Mr125 ‐ 140 kDa). 125HGF‐II also bound to the type 1 receptor whereas IGF‐II/mannose 6 phosphate receptors could not be detected.

Generation of a large scale repertoire of Expressed Sequence Tags (ESTs) from normalised rainbow trout cDNA libraries

BackgroundWithin the framework of a genomics project on livestock species (AGENAE), we initiated a high-throughput DNA sequencing program of Expressed Sequence Tags (ESTs) in rainbow trout, Oncorhynchus mykiss.ResultsWe constructed three cDNA libraries including one highly complex pooled-tissue library. These libraries were normalized and subtracted to reduce clone redundancy. ESTs sequences were produced, and 96 472 ESTs corresponding to high quality sequence reads were released on the international database, currently representing 42.5% of the overall sequence knowledge in this species. All these EST sequences and other publicly available ESTs in rainbow trout have been included on a publicly available Website (SIGENAE) and have been clustered into a total of 52 930 clusters of putative transcripts groups, including 24 616 singletons. 57.1% of these 52 930 clusters are represented by at least one Agenae EST and 14 343 clusters (27.1%) are only composed by Agenae ESTs. Sequence analysis also reveals that normalization and especially subtraction were effective in decreasing redundancy, and that the pooled-tissue library was representative of the initial tissue complexity.ConclusionDue to present work on the construction of rainbow trout normalized cDNA libraries and their extensive sequencing, along with other large scale sequencing programs, rainbow trout is now one of the major fish models in term of EST sequences available in a public database, just after Zebrafish, Danio rerio. This information is now used for the selection of a non redundant set of clones for producing DNA micro-arrays in order to examine global gene expression.

Expression profiling of rainbow trout testis development identifies evolutionary conserved genes involved in spermatogenesis

BackgroundSpermatogenesis is a late developmental process that involves a coordinated expression program in germ cells and a permanent communication between the testicular somatic cells and the germ-line. Current knowledge regarding molecular factors driving male germ cell proliferation and differentiation in vertebrates is still limited and mainly based on existing data from rodents and human. Fish with a marked reproductive cycle and a germ cell development in synchronous cysts have proven to be choice models to study precise stages of the spermatogenetic development and the germ cell-somatic cell communication network. In this study we used 9K cDNA microarrays to investigate the expression profiles underlying testis maturation during the male reproductive cycle of the trout, Oncorhynchus mykiss.ResultsUsing total testis samples at various developmental stages and isolated spermatogonia, spermatocytes and spermatids, 3379 differentially expressed trout cDNAs were identified and their gene activation or repression patterns throughout the reproductive cycle were reported. We also performed a tissue-profiling analysis and highlighted many genes for which expression signals were restricted to the testes or gonads from both sexes. The search for orthologous genes in genome-sequenced fish species and the use of their mammalian orthologs allowed us to provide accurate annotations for trout cDNAs. The analysis of the GeneOntology terms therefore validated and broadened our interpretation of expression clusters by highlighting enriched functions that are consistent with known sequential events during male gametogenesis. Furthermore, we compared expression profiles of trout and mouse orthologs and identified a complement of genes for which expression during spermatogenesis was maintained throughout evolution.ConclusionA comprehensive study of gene expression and associated functions during testis maturation and germ cell differentiation in the rainbow trout is presented. The study identifies new pathways involved during spermatogonia self-renewal or rapid proliferation, meiosis and gamete differentiation, in fish and potentially in all vertebrates. It also provides the necessary basis to further investigate the hormonal and molecular networks that trigger puberty and annual testicular recrudescence in seasonally breeding species.

Effects of 4-Nonylphenol on Sex Differentiation and Puberty in Mosquitofish (Gambusia holbrooki)

Three days post-parturition mosquitofish were exposed to different concentrations of 4-NP following a semi-static protocol. Exposure lasted up to the development of male anal fin in male individuals of the control group. Exposure to 50 μg/L 4-NP resulted in 100% females considering secondary sexual characters, while external sex-ratio did not statistically differed from unity in control group. In group exposed to 0.5 and 5.0 μg/L sex-ratio did not differ from unity but incompletely developed gonopodium was observed in several individuals. Individuals exposed to 50 μg/L 4-NP exhibited female or undeveloped gonads, while gonadal sex-ratio did not statistically differ from unity in control group. Percentage of undeveloped gonads increased with 4-NP concentration. Additional observations demonstrated hepatic histopathology in fish exposed to the highest concentration and growth reduction dependent on 4-NP concentration. In a complementary experiment, extensive metabolism of [3H]4-n-NP was characterized following in vivo exposure of juvenile mosquitofish suggesting that metabolism could modulate 4-NP toxicity. This study suggests susceptibility of early life stages of mosquitofish to endocrine modulators with regard to development of reproductive capabilities.

In vivo and in vitro effects of prochloraz and nonylphenol ethoxylates on trout spermatogenesis.

We investigated the effects of in vivo exposure to non-lethal concentrations of two chemicals commonly discharged into the aquatic environment, prochloraz and nonylphenol diethoxylate (NP2EO - Igepal(R) 210), on the development of spermatogenesis in trout. The in vitro effects on basal and insulin-like growth factor-1 (IGF-I) stimulated DNA synthesis by early germ cells were also studied. In vivo, rainbow trout were exposed for 2 or 3 weeks to waterborne prochloraz (21 and 175 nmol/l) and/or NP2EO (68-970 nmol/l) renewed continuously, or periodically. Only the highest concentrations of NP2EO (225-970 nmol/l) induced a significant increase in blood plasma vitellogenin in juvenile or maturing male trout. When prepubertal fish were exposed for 15 days to prochloraz, the spermatogenetic process was significantly inhibited as shown by the stage of gonadal development reached 3 weeks after exposure. This effect was, to a great extent, reversible within 9 weeks post-exposure. When fish in the initial stage of spermatogenesis were exposed for 21-27 days to 580 nmol/l NP2EO, a 20-40% reduction of the gonadosomatic index was observed 4.5 weeks post-exposure, and the spermatogenetic process was partly inhibited. In vitro, testicular cells obtained at different stages of spermatogenesis were cultured for 4.5 days in the presence or not of the tested molecules and with IGF-I or not. 3H-thymidine (3H-Tdr) incorporation was measured according to Loir (Mol. Reprod. Dev. 53 (1999) 424) and 125I-IGF-I specific binding was determined according to Le Gac et al. (Mol. Reprod. Dev. 44 (1996) 35). Irrespective of the spermatogenetic stage, basal 3H-Tdr incorporation was decreased by prochloraz concentrations > or =10 micromol/l. The presence of IGF-I (10-100 ng/ml) stimulated 3H-Tdr incorporation; this response to IGF-I began to decrease at 25-50 micromol/l prochloraz. In parallel, a dose-dependent increase of IGF-I specific binding was induced by prochloraz 1-100 micromol/l. Similarly, basal and IGF-I-stimulated 3H-Tdr incorporation was decreased by nonylphenol polyethoxylate (NpnEO; starting at 10 micromol/l), NP2EO and NP (30 micromol/l); a dose-dependent increase of IGF-I specific binding was also induced by NP and NPnEO. While 1-100 nmol/l 17beta-estradiol had no effect in our in vitro system, Triton(R) X-100 acted as NPnEO on 3H-Tdr incorporation. Beside their known endocrine disrupting effects on sex steroid production or action, these lipophilic molecules could act on germ cells by disrupting cell membrane receptivity to peptide hormones like growth factors.

The gsdf gene locus harbors evolutionary conserved and clustered genes preferentially expressed in fish previtellogenic oocytes

The gonadal soma-derived factor (GSDF) belongs to the transforming growth factor-β superfamily and is conserved in teleostean fish species. Gsdf is specifically expressed in the gonads, and gene expression is restricted to the granulosa and Sertoli cells in trout and medaka. The gsdf gene expression is correlated to early testis differentiation in medaka and was shown to stimulate primordial germ cell and spermatogonia proliferation in trout. In the present study, we show that the gsdf gene localizes to a syntenic chromosomal fragment conserved among vertebrates although no gsdf-related gene is detected on the corresponding genomic region in tetrapods. We demonstrate using quantitative RT-PCR that most of the genes localized in the synteny are specifically expressed in medaka gonads. Gsdf is the only gene of the synteny with a much higher expression in the testis compared to the ovary. In contrast, gene expression pattern analysis of the gsdf surrounding genes (nup54, aff1, klhl8, sdad1, and ptpn13) indicates that these genes are preferentially expressed in the female gonads. The tissue distribution of these genes is highly similar in medaka and zebrafish, two teleostean species that have diverged more than 110 million years ago. The cellular localization of these genes was determined in medaka gonads using the whole-mount in situ hybridization technique. We confirm that gsdf gene expression is restricted to Sertoli and granulosa cells in contact with the premeiotic and meiotic cells. The nup54 gene is expressed in spermatocytes and previtellogenic oocytes. Transcripts corresponding to the ovary-specific genes (aff1, klhl8, and sdad1) are detected only in previtellogenic oocytes. No expression was detected in the gonocytes in 10 dpf embryos. In conclusion, we show that the gsdf gene localizes to a syntenic chromosomal fragment harboring evolutionary conserved genes in vertebrates. These genes are preferentially expressed in previtelloogenic oocytes, and thus, they display a different cellular localization compared to that of the gsdf gene indicating that the later gene is not co-regulated. Interestingly, our study identifies new clustered genes that are specifically expressed in previtellogenic oocytes (nup54, aff1, klhl8, sdad1).

Main neuro-endocrine, endocrine and paracrine regulations of fish reproduction, and vulnerability to xenobiotics.

The reproductive function of fish, which is very sensitive to the variations of environmental factors, appears also to be particularly vulnerable to the presence of xenobiotics in the aquatic medium. Many physiological processes can be impaired, from sexual differentiation to female and male gametogenesis, due to disruptions among complex neuro-endrocrine, endocrine or paracrine regulations. This paper describes the main regulation steps that are known or can be suspected to be disrupted by xenobiotics and gives some examples. The large interspecific diversity of reproductive strategies and the complexity of underlying mechanisms are particularly highlighted to draw attention to possible confusions between real endocrine disruptions and natural physiological variations.

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

BackgroundIn rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides 11ketotestosterone (11KT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) 11-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows.MethodsIn this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production.ResultsThe plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10–50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro.ConclusionThese results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction.

Sexuality and gonadal cycle of the common dentex (Dentex dentex) in intensive culture

Abstract The common dentex ( Dentex dentex ) is a highly valued table fish in the Mediterranean region. In culture conditions, this sparid fish has a high specific growth rate and spawns spontaneously. However, the proportion of females that spawn spontaneously and the timing of their spawning are highly unpredictable. In addition, knowledge of the sexual cycles of this species is very limited. The main objective of this study was to provide basic knowledge on the reproductive biology of the common dentex. Study of male and female gametogenesis was performed using four groups of 0 + -, 1 + -, 2 + - and 3 + -year-old fish born in captivity. These groups were sampled either every 1 or 2 months, for either 1 or 2 consecutive years. Histological analysis of the gonads from a total of 448 individuals enabled the definition of five maturity stages for male and five for female fish. Sexual differentiation occurred between 5 and 12 months of age. Among the fish studied, neither bisexual gonads nor any other indication of sexual inversion was found in fish up to 4 years of age, suggesting that common dentex is a gonochoristic fish. In Crete, Greece, spawning took place between the end of March and May. Males and females older than 1 year (1 + ) matured almost simultaneously. All the 2-year-old males produced milt. In our sampling conditions, the highest percentages of females observed undergoing maturation during their second, third and fourth years were 67%, 100% and 100%, respectively. Sexually mature females were detected at the minimum standard length of 21 cm. After the spawning period, and until the following January, all the females were in previtellogenesis and in some males, spermatogenetic activity resumed gradually. In February, under increasing photoperiod, cortical alveoli appeared in growing oocytes and the development of spermatogenesis greatly increased. Between February and April, vitellogenesis occurred in females and the gonadosomatic index (GSI) increased from 0.2–1% to 3–6% in both sexes. The fecundity estimates indicated that common dentex display a spawning strategy similar to many temperate marine teleosts. It is a multiple spawner, exhibiting asynchronous oocyte development (unknown annual fecundity). Batch relative fecundity ranged from 32,000 to 393,000 eggs/kg body weight, and a positive linear relationship between batch fecundity and body size was found.