| Estrogen, one kind of sex steroid hormones, widely exists in humans and other higher animals, and has very important physiological functions on skeletogeny, lipometabolism, the development of reproductive systems, and the formation of central nervous systems. Estrogen works mainly through estrogen receptors(ERs). Therefore, the study of ERs on vitellogenesis can help us understanding well the estrogen signal pathways.In the present study, the full length cDNA sequences of three ERs subtypes(ERα, ERβ1 and ERβ2), and three vitellogenins(Vtgs) subtypes(Vtg-A, Vtg-B and Vtg-C) cDNA fragments were cloned in Scatophagus argus. Expression of ERs in 11 tissues, and that in liver of S. argus at different ovary development stages were determined by semiquantitative reverse transcription PCR(SqRT-PCR). Effects of estradiol(E2) and estrogen receptor antagonists(ICI182780, MPP and Cyclofenil) on ERs and Vtgs mRNA expressions in male liver were examined using quantitative real-time PCR(qPCR). In addition, levels of VTG were measured by enzyme-linked immunosorbent assay(ELISA). The main results were as following: 1. Molecular cloning and characterization of ERs in S. argus.Three subtypes of ERs were cloned in S. argus. The full length cDNA of was 1876 bp with 1770 bp ORF which encoded 589 amino acids. The complete ERβ1 cDNA was 1811 bp with 1683 bp ORF which encoded 560 amino acids, and that of ERβ2 was 2111 bp with 1974 bp ORF which encoded 657 amino acids. Amino acid alignment analyses showed that ERs in S. argus is the member of nuclear receptor superfamily, which contain all the typical characters of this families. 2. Temporal and spatial expression of ERs in S. argus.In the detected 11 tissues(brain, pituitary, testis, ovary, liver, kidney, spleen, intestines, heart, gill and muscle) in S. argus, the ER mainly expressed in gonad, spleen, heart, brain and pituitary; The ERβ1 distributed in tissues except gill in male, and gill and spleen in female; The ERβ2 expressed not only in gonad, spleen, intestines, brain, pituitary and heart in both male and female, but also in kidney in male, as well as liver, muscle and gill in female.Both ERα and ERβ1 expressed weakly in the liver in S. argus at the previtellogenesis stage, but increased in that at the vitellogenesis stage. However, no difference was found in liver ERβ2 expression between these two stages. Results suggested that and ERβ1 might involve in the vitellogenesis induced by estrogen. 3. Effects of estradiol(E2) on Estrogen receptors(ERs) and vitellogenin(VTG)expression in S. argusThree Vtgs cDNA fragements, 235 bp for Vtg-A, 1089 bp for Vtg-B, and 1087 bp for Vtg-C were cloned respectively from the liver of S. argus.The expression of ERα, ERβ1 and Vtgs mRNA, as well as the content of VTG increased significantly(P<0.05) at 24 hours incubated with E2(dose from 1 to 10μM), which indicated that both ERα and ERβ1 participated in the vitellogenesis in S. argus. 4. Effects of estrogen receptor antagonists on the expression of ERs and VTGs in S. argusEstrogen receptor broad-spectrum antagonist ICI182780(0.01, 0.1 and 1μM) significantly inhibited the expression of ERs and Vtgs, as well as the content of VTG in a dose-dependent manner, which suggested that ERα and ERβ1 co-regulated the vitellogenesis and promoted the yolk protein accumulation in teleost.Estrogen receptor alpha antagonist MPP suppressed the expression of ERα, Vtg-B and Vtg-C, as well as the content of VTG, while promoted the expression of ERβ1 and Vtg-A mRNA; However, estrogen receptor beta antagonist Cyclofenil inhibited that of ERβ1, ERβ2, Vtg-A and Vtg-C, as well as the content of VTG while accelerated that of ERα and Vtg-B mRNA. Results indicated that ERα mainly regulated the expression of Vtg-B while Vtg-A. Results suggested that the ERα-mediated signal pathway begin a process to increase the ERα and Vtg-B expression when ERβ1-mediated signal pathway was blocked, while ERβ1-mediated signal pathway increased ERβ1 and Vtg-A expression when ERα-mediated signal pathway was blocked. |
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