Rune Male

Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol

Alkylphenol ethoxylate degradation products such as nonylphenol and octylphenol are shown to have estrogenic effects. Nonylphenol induces synthesis of vitellogenin (a precursor of egg yolk proteins) and zona radiata proteins (eggshell proteins) in juvenile and/or male fish. Little is known about the molecular mechanisms of estrogenicity of environmental chemicals such as nonylphenol. To study the mechanisms of estrogenic effects of 4-nonylphenol (NP), we examined its in vivo effects on the expression of the estrogen receptor (ER), vitellogenin (Vtg) and zona radiata protein (Zrp) genes in juvenile Atlantic salmon liver. We show that the ER mRNA synthesis is induced by NP in a dose-dependent manner in juvenile Atlantic salmon liver. The induction of the ER mRNA synthesis is followed by the induction of Zrp and Vtg mRNA synthesis. The ER transcripts reach peak levels earlier than the Zrp and Vtg mRNA and proteins, which is in agreement with the physiological effects of estradiol during zonagenesis and vitellogenesis. Various studies have also shown that NP competitively inhibits the binding of 17 beta-estradiol (E2) to ER. Our results further suggest that NP directly mimics E2 in inducing the ER, Zrp and Vtg genes in salmon liver.

The aryl hydrocarbon receptor-mediated disruption of vitellogenin synthesis in the fish liver: Cross-talk between AHR- and ERα-signalling pathways

BackgroundIn the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERα). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERα genes, in primary cultured salmon hepatocytes, have been investigated.ResultsThe expression of the genes encoding VTG and ERα were strongly induced by 17β-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor α-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERα mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERα genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERα genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERα mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERα. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERα or that activated AHR blocked the ability of ERα to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA.ConclusionIn fish hepatocytes E2 induces ERα and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERα. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERα- and AHR-signalling pathways is unidirectional.

Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar)

Alkylphenols such as 4-nonylphenol (NP) are one of the wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of eggshell zona radiata proteins (Zrp) and vitellogenin (Vtg) mRNA and protein synthesis in the liver are widely used biomarkers for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the pituitary. To evaluate pituitary effects and develop new potential biomarkers for xenoestrogens, the influences of NP and 17beta-estradiol (E2) on the mRNA levels of pituitary gonadotropic hormone (GTH) beta subunits [leutinizing hormone beta (LH beta or GTH II beta) and follicle stimulating hormone beta (FSH beta or GTH I beta)], prolactin (PRL), growth hormone (GH) and the pituitary specific transcription factor (Pit-1) were investigated in individual male and female juvenile Atlantic salmon (Salmo salar), 3 days after a single intraperitoneal (i.p.) injection. In one experiment, fish were injected with NP (125 mg/kg body weight (BW)) or E2 (5 mg/kg BW) and a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to analyze LH beta and FSH beta mRNA levels. In the second experiment, fish were injected with three doses of NP (10, 50, 125 mg/kg BW) or a single dose of E2 (5 mg/kg BW) and Northern blot analysis was used to quantify LH beta, FSH beta, PRL, GH and Pit-1 mRNAs. Both NP (50 and 125 mg/kg BW) and E2 significantly induced LH beta mRNA levels (P<0.01), but only in females. The highest dose of NP (125 mg/kg BW) significantly induced Pit-1 mRNA in males (P<0.01). NP did not have significant effects on any of the other pituitary transcripts. NP induced LH beta mRNA synthesis in females by up to 6-fold and the changes appeared to correlate with the increases in hepatic Vtg and Zrp mRNA levels. The results show that LH beta mRNA assay in female juvenile salmonids may be used as a marker for pituitary effects of xenoestrogens. The data also suggest that NP may have the potential to perturb the regulation of LH beta gene expression by mimicking E2.

Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish.

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.

Pharmacological characterization, localization and quantification of expression of gonadotropin receptors in Atlantic salmon (Salmo salar L.) ovaries

The gonadotropins Fsh and Lh interact with their receptors (Fshr and Lhr, respectively) in a highly specific manner in mammals with little overlap in biological activities. In fish, the biological activities seem less clearly separated considering, for example, the steroidogenic potency of both Fsh and Lh. Important determinants of the biological activity are the specificity of hormone-receptor interaction and the cellular site of receptor expression. Here, we report the pharmacological characterization of Atlantic salmon Fshr and Lhr, identify receptor-expressing cells in the ovary, and validate receptor mRNA quantification systems. For the pharmacological studies, we used highly purified coho salmon gonadotropins and found that the Fshr preferentially responded to Fsh, but was also activated by approximately 6-fold higher levels of Lh. The Lhr was specific for Lh and did not respond to Fsh. Photoperiod manipulation was used to generate ovarian tissue samples with largely differing stages of maturation. Specific real-time, quantitative (rtq) PCR assays revealed up to 40-fold (fshr) and up to 350-fold (lhr) changes in ovarian expression levels, which correlated well with the differences in ovarian weight, histology, and circulating oestrogen levels recorded in January and June, respectively. Vitellogenic ovaries were used to localise receptor-expressing cells by in situ hybridization. Granulosa cells of small and large vitellogenic follicles were positive for both receptors. Also theca cells of small and large vitellogenic follicles expressed fshr mRNA, while only in large vitellogenic follicles theca cells were (weakly) positive for lhr mRNA. While only ovulatory Lh levels seem high enough to cross-activate the Fshr, expression by both receptors by granulosa and theca cells suggests that homologous ligand receptor interaction will prevail.

Proteolytically Activated, Recombinant Anti-Müllerian Hormone Inhibits Androgen Secretion, Proliferation, and Differentiation of Spermatogonia in Adult Zebrafish Testis Organ Cultures

Anti-Müllerian hormone (Amh) is in mammals known as a TGFβ type of glycoprotein processed to yield a bioactive C-terminal homodimer that directs regression of Müllerian ducts in the male fetus and regulates steroidogenesis and early stages of folliculogenesis. Here, we report on the zebrafish Amh homologue. Zebrafish, as all teleost fish, do not have Müllerian ducts. Antibodies raised against the N- and C-terminal part of Amh were used to study the processing of endogenous and recombinant Amh. The N-terminally directed antibody detected a 27-kDa protein, whereas the C-terminally directed one recognized a 32-kDa protein in testes extracts, both apparently not glycosylated. The C-terminal fragment was present as a monomeric protein, because reducing conditions did not change its apparent molecular mass. Recombinant zebrafish Amh was cleaved with plasmin to N- and C-terminal fragments that after deglycosylation were similar in size to endogenous Amh fragments. Mass spectrometry and N-terminal sequencing revealed a 21-residue N-terminal leader sequence and a plasmin cleavage site after Lys or Arg within Lys-Arg-His at position 263-265, which produce theoretical fragments in accordance with the experimental results. Experiments using adult zebrafish testes tissue cultures showed that plasmin-cleaved, but not uncleaved, Amh inhibited gonadotropin-stimulated androgen production. However, androgens did not modulate amh expression that was, on the other hand, down-regulated by Fsh. Moreover, plasmin-cleaved Amh inhibited androgen-stimulated proliferation as well as differentiation of type A spermatogonia. In conclusion, zebrafish Amh is processed to become bioactive and has independent functions in inhibiting both steroidogenesis and spermatogenesis.

Salmon Eggshell Protein Expression: A Marker for Environmental Estrogens

Abstract: A liver complementary DNA expression library from Atlantic salmon (Salmo salar) pretreated with estradiol-17β (E2) was constructed and screened with antibodies raised against salmon eggshell (zona radiata) proteins. Two clones, SalZr2_19 and SalZr2_23 were sequenced and shown to encode proteins of approximately 50 kDa. SalZr2_23 contains 12 octamer sequence lpqr/kpa/vq repeats also found in SalZr2_19, but only twice. Alignment reveals that the two salmon sequences are similar to piscine zona radiata proteins and mammalian zona pellucida proteins. Several transcripts ranging from 2.3 to 12 kb appeared in liver extracts of E2-treated fish. Juvenile fish treated with E2 or 4-nonylphenol showed strong induction of zona radiata protein. The use of egg envelope transcriptional and translational induction in male or juvenile fish as a biological marker of environmental estrogens is discussed.

Pituitary gonadotropin and ovarian gonadotropin receptor transcript levels: seasonal and photoperiod-induced changes in the reproductive physiology of female Atlantic salmon (Salmo salar).

In female Atlantic salmon kept at normal light conditions, pituitary follicle-stimulating hormone beta (fshb) transcript levels were transiently elevated one year before spawning, re-increased in February, and remained high during spawning in November and in post-ovulatory fish in December. The first increase in plasma 17b-estradiol (E2), testosterone (T) and gonadosomatic index (GSI) was recorded in January; E2 rose up to one month prior to ovulation, while T and GSI kept increasing until ovulation. Pituitary luteinizing hormone beta (lhb) transcript levels peaked at the time of ovulation. Except for transient changes before and after ovulation, ovarian follicle stimulating hormone receptor (fshr) transcript amounts were relatively stable at a high level. By contrast, luteinizing hormone receptor (lhcgr) transcript levels started out low and increased in parallel to GSI and plasma E2 levels. Exposure to continuous light (LL) induced a bimodal response where maturation was accelerated or arrested. The LL-arrested females showed previtellogenic oil droplet stage follicles or primary yolk follicles only, and fshb and E2 plasma levels collapsed while fshr increased. The LL-accelerated females showed elevated lhb transcript levels and slightly elevated E2 levels during early vitellogenesis, and significantly elevated lhcgr E2 and GSI levels in late vitellogenesis. We conclude that Fsh-dependent signaling stimulates recruitment into and the sustained development through vitellogenesis. Up-regulation of lhcgr gene expression during vitellogenesis may reflect an estrogenic effect, while elevated fshr gene expression following ovulation or during LL-induced arrestment may be associated with ovarian tissue remodeling processes.

Properties and mechanism of action of eukaryotic 3-methyladenine-DNA glycosylases.

SUMMARY 3-Methyladenine-DNA glycosylase activities have been identified in all eukaryotic cell systems studied. Some of the results from these studies are reviewed here. The enzymes possess molecular weights between 24×103 and 34×103, they have a broad pH optimum at approximately pH8, require double-stranded DNA and act in the absence of any cofactors. The enzyme can excise several different methylated bases from DNA such as 3-methyladenine, 7-methylguanine and 3-methylguanine. The specific activity of this DNA glycosylase in mouse L-cells was found to be a function of the proliferative state of the cell. In vitro quantification of this DNA repair activity in synchronized mouse L-cells suggests that it is regulated within a defined temporal sequence prior to the onset of DNA replication. Using DNA fragments of defined sequences it was observed that the efficiency of removal of the methylated bases is sequence-dependent.

A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio)

The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses.