Cloning, Expression Profile And Reproduction Regulation Of Estrogen Receptor In Common Chinese Cuttlefish, Sepiella Japonica

Sepiella japonica is the most important marine cephalopods species along the coast of China. The artificial breeding of S. japonica has made a breakthrough, while a sophisticated precocious puberty under artificial environment has been noted to seriously affect the breeding effect. Then what is the key to solve the problem is to reveal the regulatory mechanism of reproduction and development of the cuttloefish under the condition of breeding. Estrogen receptor is a typical sex steroid hormone receptor, playing an important role in sexual maturity and reproduction of many marine animals. In consideration of the function of ER in reproduction regulation of cuttlefish, clon and analysis of sjER cDNA,sub-cellular localization, potential action sites and characteristic of tissue distributions, the relationship between the E2 content changes and the sjER expression fluctuation during the ovary development and the function in reproduction regulation were analysed in this paper.The main results were as followed:The transcriptome information of the ovarian of S. japonica was obtained using Illumina Hiseq 2000 sequencing. In total, the transcriptome was assembled into 70,039 unigenes, and the average length was 800 bp. 40.7%(28,492) of the unigenes were annotated, among which 38.5%(10,969) were annotated as GO terms. A number of genes were identified according to gene annotation information and gene expression abundance.793 abundant expressed genes(AEGs)(FPKM ? 100) and 38 AEGs were identified. 46,071 unigenes display significantly(P < 0.001) down-regulated expression between the immature and mature ovaries and 1,217 unigenes display significantly up-regulated expression. ER was significantly up-regulated gene in mature ovarian transcriptome, and showed more than 80% identity with that of octopus.The full-length cDNA sequence of sjER was isolated using the rapid amplification of5' and 3' cDNA ends and the sequence was analysised. The results showed that the full-length cDNA sequence of sjER(KT278504) was 1,788 bp, which contained an open reading frame(ORF) of 1,470 bp encoding 489 amino acid(aa) residues, a 5'-untranslated region(UTR) of 280 bp, and a 3'-UTR of 38 bp. As other nuclear receptors, the sjER sequence contains five nuclear receptor characteristic domains: A/B domain(N-terminal,130aa), C domain(DNA-binding domain, 80aa), D domain(the hinge domain 35aa), E domain(the ligand binding domain, 228aa) and F domain(the C-terminal domain, 16aa).The C and E domains of sjER showed more conserved sharing 100% and 96% identity with those of O. vulgaris. The analysis revealed that there were basically three groups of ERs:vertebrate ERs(ER?, ER? and ER?), invertebrate ERs, and the cephalochordate ERs. In molluscs, sjER clustered among the other identified molluscan ERs and showed 89%identity with that of octopus.The characteristic and location of sjER were analyzed, and the results showed that constructing the sjER-EGFP expression vector and stained with DiI and DAPI, the sjER was localized in the nucleus, consistenting with the prediction of NLS and suggesting the nature of a nuclear receptor of sjER functioning through a ligand-dependent activation of specific gene transcription.Real-time quantitative PCR assays were adopted to examine the tissue distribution of sjER mRNA of adult S. japonica. The results showed that more sj ER mRNA expression was generally observed in brain, gonad and liver which were related to reproduction and development than in other tissues of both male and female cuttlefish. During the oogenesis development, the ER expression were up-regulated significantly(P < 0.05) in the oogonium production period, protoplasmic growth period and interstitial growth period, then decreased sharply in the trophoplasmic growth period. The fluctuation of ER expression was in accordance with the changes of E2 content during the reproductive cycle of S.japonica, which may provide further evidence that ER play an important role in gonad development and maturation of cephalopod species.In order to have a look into the function of sjER in reproduction regulation, the HEK293 cells were cotransfected ERE-pGL3 and sjER-Flag and it has been found that the activity of ERE was significantly(P < 0.05) multiplied by sj ER-Flag and significantly(P <0.05) inhibited by Tamoxifen which is the specific inhibitor of ER, suggesting that sjER may promote ERE transcription through the classic nuclear receptor pathway. However the activity of ERE was unchanged(P > 0.05) when treated with E2, suggesting that sjER was a constitutive transcriptional activator such as the other molluscan ERs, rather than depended on the combination of sjER and E2. Treated with E2, there was no Ca2+and cAMP accumulation in HEK293 cells transfected sjER-Flag indicating that sjER may not mediate the membrane signal transduction pathways through interaction with membrane receptors.It was the first report about the characteristic, expression and function of the full-length sequence of sjER, suggesting the function in reproduction regulation, which could provide theoretical basis for artificial breeding and genetic resources conservation.