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Smnmary--We describe here an/n vitro technique to assess the estrogenic activity of chemicals. This technique is based on rainbow trout hepatocytes incubated in a basic medium free of any additional growth factors or estrogenic chemicals and uses the production of vitellogenin (VTG) as a marker for the estrogenic potency of the compounds tested. The system allows at least some of the metabolic transformations which are undertaken by the liver cells/n vivo and could therefore be used for xenobiotic compounds which exhibit estrogenic activities after liver metabolic transformation. A dose-response curve was always consistently obtained using estradiol-17fl (E2), with a mid point at around 100 nME 2 and a maximum response at around 1000nM. Established estrogens such as 17al ethynylestradiol (EE2) or diethylstilboestrol (DES) were also tested. EE 2 appeared to be equipotent with E2 and DES slightly less potent. E2 conjugates were, perhaps surprisingly, also very potent. Estradiol-3-sulfate was equipotent with E2 and estradiol-17fl-glucuronide approx. 10% as potent. Other steroids such as androgens and progesterone, though active in the bioassay, were 3 orders of magnitude less potent than E 2. Of the various steroids tested, only cortisol, at concentrations up to 50 #M, was completely inactive. Six different phytoestrogens were tested in the assay. All were weakly estrogenic, possessing approximately one thousanth the potency of E 2 (they were as potent as the androgens and progesterone). All six phytoestrogens, as well as the androgens and progesterone, were tested in the presence of tamoxifen. In all cases tamoxifen reduced the production of VTG significantly, demonstrating that the estrogenic action of all of these compounds was most likely mediated by the E 2 receptor. The potencies determined here may not reflect the situation/n vivo but can provide complementary results about the activity of chemicals which need an hepatic metabolization to be estrogenic. Hepatocyte cultures would profitably be developed in other species to sustain these results.

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.

SummaryWe have previously described the cloning, sequencing and in vitro expression of a full-length rainbow trout estrogen receptor cDNA (rtER cDNA). This full cDNA randomly labelled was used to study the estrogen induction of hepatic rtER mRNA in correlation with vitellogenin (Vg) mRNA in different physiolo~cal situations.In this paper, we show that in the liver two mRNA species are under hormonal control and their level increases about g-fold after estrogen stimulation.These two mRNAs are expressed and induced in the liver as early as the hatching stage in correlation with the expression of Vg mRNA. A long-term analysis of rtER mRNA after estradiol (E2) injection shows a transient induction of the nuclear ER and its mRNA which recover to the basal level after 2 weeks. Nevertheless, a memory effect was observed on the expression of the Vg gene which does not appear to be directly related to the estrogen receptor level.