Bernadette Ducouret

Differential regulation of two genes implicated in fish reproduction: Vitellogenin and estrogen receptor genes

In rainbow trout as well as in other species, variability of estrogen receptor (ER) gene expression according to the cell type and the physiological state reflects a differential cell and gene sensitivity to estrogen. We previously demonstrated that expression of the rainbow trout estrogen receptor (rtER) and vitellogenin (Vg) genes were induced differently by estrogens in rainbow trout liver. Therefore, these two genes offered a suitable model to study the influence of ER concentration on gene transcriptional activities. In the present study we show that the transcription rate of rtER and Vg genes during an estrogenic treatment are affected differently by variation of cellular ER concentration. We demonstrate that rtER gene exhibits a low threshold response to loaded estrogen receptor, and increasing ER amounts do not affect the transcriptional response of this gene during an estrogenic stimulation. On the contrary, Vg gene expression requires the presence of a higher loaded estrogen receptor level to be induced, and its transcriptional response is directly proportional to the amount of synthesised ER. Mol. Reprod. Dev. 48:317–323, 1997.

Transcriptional Interference Between Glucocorticoid Receptor and Estradiol Receptor Mediates the Inhibitory Effect of Cortisol on Fish Vitellogenesis

Abstract In oviparous species, the synthesis of vitellogenin (Vg) takes place in the liver according to a strictly estrogen-dependent mechanism that first involves an up-regulation of the estrogen receptor (ER) by its own ligand. However, reports from the literature indicate that in trout stress or cortisol may cause a reduction of cytosolic E2-binding sites in the liver and a decrease in plasma Vg levels. To investigate the mechanisms underlying these effects, in vivo and in vitro experiments were designed in rainbow trout (Oncorhynchus mykiss). The results demonstrate that cortisol implanted into maturing females caused a marked decrease of rainbow trout ER (rtER) and rainbow trout Vg (rtVg) mRNA levels in the liver. In vitro experiments on hepatocyte aggregates also showed that dexamethasone (Dex) caused a strong decrease in the basal and E2-stimulated rtER mRNA and to a lesser extent rtVg mRNA. These effects were specific as no other hormones were able to mimic the inhibitory action of Dex. A study of rtER mRNA stability indicated that the effects of glucocorticoids are likely to take place at the transcriptional level. This was further indicated by transfection experiments in CHO-K1 cells, which showed that rainbow trout glucocorticoid receptor (rtGR) strongly inhibited the E2-stimulated transcriptional activity of the rtER promoter. Taken together, these results indicate that the rtGR exerts a transcriptional interference on the expression of the rtER that may explain some of the negative effects of stress or cortisol on vitellogenesis.

Effects of copper on cortisol receptor and metallothionein expression in gills of Oncorhynchus mykiss

Effects of waterborne Cu (2.4 microM) on the expression of glucocorticoid receptor (GR) and metallothionein (MT) in the branchial epithelium of freshwater rainbow trout (Oncorhynchus mykiss) was studied by immunocytochemistry. After 5 days of Cu exposure, the number of GR-immunoreactive (GR-ir) cells in the gill epithelium had decreased, whereas the number of MT-ir cells had increased. Localization of GR in chloride cells was achieved by double staining for Na(+)/K(+)-ATPase; other cell types were identified on the basis of their topology. GRs were present in the chloride cells in both the filaments and lamellae, in respiratory cells in the lamellae, in pavement cells, basal layer cells and undifferentiated cells in the filaments. Co-localization of Na(+)/K(+)-ATPase and MT revealed chat MT was expressed in chloride cells, both in filaments and lamellae. Occasionally, MT immunoreactivity was found in pavement cells and in undifferentiated cells. By double staining for Na(+)/K(+)-ATPase and GR, for Na(+)/K(+)-ATPase and MT and for GR and MT, we can conclude that after 5 days of Cu stress there are chloride cells that express GR and MT, GR or MT alone or neither of the two proteins. This apparent functional heterogeneity of branchial chloride cells may reflect a limited window when chloride cell subpopulations show an adaptive response to Cu.

Peptide insertion in the DNA-binding domain of fish glucocorticoid receptor is encoded by an additional exon and confers particular functional properties.

The trout glucocorticoid receptor (rtGR) contains an additional sequence of nine amino acids located between the two zinc fingers of the DNA-binding domain (DBD) (Endocrinology 136 (1995) 3774). Polymerase chain reaction on trout genomic DNA and sequencing were performed in the DBD region, demonstrating that this peptide is encoded by an additional exon of 27 nucleotides between the two exons encoding the two zinc fingers of other nuclear receptors. This additional sequence in the rtGR confers a better binding affinity of the receptor to a single GRE, as shown by gel shift experiments with GST-DBDrtGR fusion proteins, deleted or not of the nine amino acids (Delta9). This higher affinity is correlated with a higher constitutive transcriptional activity of the receptor on a reporter gene driven by a single GRE, but not with the ligand-induced transcriptional activity. Nevertheless, on a double GRE, the wild type and rtGR-Delta9 are equally active on both constitutive or dexamethasone-induced transcriptional activity. This original DBD structure could have emerged during evolution such as to allow better regulation of glucocorticoid dependent genes in relation to the large spectrum of cortisol physiological functions in fish.

The activity of 3α- and 3β-hydroxysteroid dehydrogenases and 5α-reductase, together with androgen levels in male rat pituitary during sexual maturation

Abstract In all subcellular pituitary fractions, 3α-hydroxysteroid dehydrogenase (3α-ol dehydrogenase) activity is high (1 to 3 pmol/mg/h) with NADH or NADPH as cofactor, and 3β-hydroxysteroid dehydrogenase (3β-ol dehydrogenase) activity much lower. The highest activity of the latter (0.15 pmol/mg/h) is detected in cytosol with NADH as cofactor. During sexual maturation, cytosolic (NADH-dependent) 3α- and 3β-ol dehydrogenase activities remain constant, whereas the 5α-reductase activity is maximum at 37 days. The levels of different pituitary androgens were evaluated by radioimmunoassay. At 28 days, testosterone level is 4 ng/g of tissue, then after 42 days the level remains between 4.5 and 6 ng/g at a level higher than the DHT level. In all cases during the maturation of the rat, the different 5α-reduced androgens are in the same ratio : DHT > 3α-diol > 3β-diol, and the sum of these three 5α-reduced androgens decreases between the 28th and the 90th day.