| Oogenesis and vitellogenesis constitute the basic process of ovarian development,and vitellogenesis fundamentally means the rapid accumulation of yolk protein in oocytes.Vitellogein?Vg?is a key gene involved in the vitellogenesis process.After being synthesized,Vg can be processed with nutrients like carbohydrates and lipids into vitellin?Vn?,which is one of the most essential protein of the yolk.The expression and synthesis of Vg,therefore,are critical enough for the yolk accumulation of oocytes to influence the ovarian development.The Chinese mitten crab,Eriocheir sinensis,is an important economic species in China with the characteristic of explosive yolk accumulation during its adult stage,making it an appropriate model for vitellogenesis study.For most egg-laying vertebrates,steroid hormones play an important role in the endocrine regulation of vitellogenesis,and their specific effects and mechanisms on the transcription level of Vg are relatively clear.But in E.sinensis,the specific effect of steroid hormones on the vitellogenesis remains obscure due to the lack of some important steroid receptors,making the its mechanism more complicated as well.In this paper,the endogenous characteristics of Vg expression and distribution in E.sinensis were defined firstly.Then the characteristics of endogenous estrogen and its potential correlation with Vg/Vn protein were predicted and inferred.The regulatory effects of estrogen and ecdysone on the expression of Vg-mRNA were also explored.After confirming the effects of these two steroid hormones on vitellogenesis,the potential genomic pathways regulating Vg expression by steroid hormones were finally explored.The major findings are listed as follows:1. The expression and localization characteristics of Vg and its receptor during ovarian development of E.sinensisThe result of in situ hybridization showed that Vg-mRNA was mainly distributed in the resorptive cells?R cells?and Fibrillar cells?F cells?in the hepatopancreas.In the ovary,Vg-mRNA signals mainly appear in follicular cells,cytoplasm of endogenously vitellogenic oocytes,and nucleus of exogenousy vitellogenic oocytes.The result of RT-q PCR showed that the expression level of Vg-mRNA rose continuously in hepatopancreas and ovary during ovarian development stage ?–?,with the Vg-mRNA expression levels in both tissues increasing dramatically at stage ?.The Vg-mRNA expression level in ovary decreased significantly at stage V.For the vitellogenin receptor?Vg R?,its expression level maintained a high level during stage ? to ?,with no significant difference found among the Vg R-mRNA expression levels at stage ?,?,and ?.The result of immunofluorescence showed that the positive signals of Vg/Vn could be detected mainly in F cells and R cells during the all the ovarian development stages.For the ovary,Vg/Vn positive signals were mainly detected in cytoplasm of endogenously vitellogenic oocytes,exogenously vitellogenic oocytes,nearly mature oocytes,and mature oocytes.According to these results,the vitellogenesis of E.sinensis had a significant characteristic of endogenous and exogenous stages.The main contributor of vitellogenesis at endogenous stage was ovary,while the main contributor of exogenous vitellogenesis was hepatopancreas.2. The expression and localization characteristics of Vg and its receptor during ovarian development of E.sinensisThis study measured the 17?-estradiol?E2?content in hemolymph,hepatopancreas,and ovary during ovarian development of E.sinensis by ELISA.For the hemolymph,E2 contents of stage ? and ? were significantly lower than that of other stages.The lowest E2 content(31.91 pg m L-1)in hemolymph was achieved at stage ?;For hepatopancreas,the lowest E2 content(91.49 pg m L-1)was also detected at stage ?,but E2 content increased significantly and maintained stable during stage ? to ?.The highest E2 content(180.74 pg g-1)was found at stage ?,while E2 content at stage V decreased to 146.25 pg g-1.The E2 content in ovary also appeared at stage ?,and then increased significantly and kept stable during the follow stages.The E2contents in three tissues all showed a similar changing trend of first increasing and then declining.The result of E2 localization showed that E2in ovary mainly distributed in follicle cells and cytoplasm of oocytes.E2in hepatopancreas mainly distributed in F cells and nucleus of R cells.E2in brain ganglion distributed in nervalcells,and E2in thoracic ganglion distributed in nerval cells and nerve medulla.The correlation between E2 and Vg/Vn is revealed by colocalization and correlation analysis.For hemolymph,at endogenously vitellogenic stage,E2 and Vg/Vn content showed a positively linear-dependent relationship?R2>0.8?.At exogenously vitellogenic stage,the correlation between E2 and Vg/Vn content conformed to the power function curve?R2>0.8?.For hepatopancreas,the E2 and Vg/Vn content showed a negative correlation?R2>0.8?.For ovary,E2 and es Vg/Vn content showed a significant correlation during the vitellogenesis process?0.6<R2<0.8?.Colocalization result showed that E2 and Vg/Vn were colocalized in F cell and R cell.In ovary,E2 was localized in follicle cells,while Vg/Vn was localized in the cytoplasm of endogenously vitellogenic oocytes at early stage of vitellogenesis.There was no direct correlation between their spatial distribution;At exogenously vitellogenic stage,follicle cells were not able to be observed,and E2 and Vg/Vn were only colocalized in cytoplasm of oocytes.These results showed that E2 and Vg/Vn had a certain correlation in content and distribution at early stage of vitellogenesis,but this correlation gradually weakened with the ovarian development.3. The regulation of E2 and 20E on the mRNA expression of Vg and its receptorThis result of in vitro tissue culture showed that in hepatopancreas and ovary of both endogenously and exogenously vitellogenic stage,24 h incubation of 0.01?M E2could improve the Vg-mRNA expression level.The in vivo E2injection experiment also showed that a certain dose of E2could improve the Vg-mRNA expression.20E could regulate the Vg-mRNA expression level in hepatopancreas and ovary both in vitro and in vivo as well,and 0.1?M 20E was a better dose for Vg-mRNA expression inducement in tissue culture experiment.However,compared to effects of E2,the effects of 20E on ovary was not so obvious.24 h incubation in vitro of 0.1?M 20E did not improve the Vg-mRNA expression level significantly.Just as the results in vitro,the injection of 0.2?g g-1 20E could improve the Vg-mRNA expression level,but had no significant effects in ovary.For Vg R,both E2 and 20E could suppress the its expression at endogenously vitellogenic stage.The results above showed that steroid hormone could regulate the Vg-mRNA expression,but might regulate vitellogenesis by affecting other vitellogenesis-related genes such as Vg R.4. The possible mechanism of E2 and 20E regulating the mRNA expression of Vg and its receptorThis study first cloned the full length of estrogen related receptor?ERR?of E.sinensis.es ERR sequence was 2825 bp,encoding 423 amino acids without signaling peptides and transmembrane domains.The identity and similarity of its amino acid sequence with that of Portunus trituberculatus were as high as 97.2%.The results of spatial and temporal expression analysis of ERR revealed that ERR mainly expressed in the muscle,and also expressed in hepatopancreas and ovary with a lower level.As for different ovarian development stages,ERR-mRNA expression level in hepatopancreas was lowest at stage ?,but stable at other stages.In ovary,the ERR-mRNA expression level showed an increasing trend and increased rapidly during stage ?-?.The expression of EcR-mRNA in hepatopancreas increased significantly at stage ?,but there was no significant difference among other four stages;The expression level of EcR-mRNA in ovary was higher at stage ?-?,but significantly decreased at stage V.In vitro RNA interference experiments on ERR and EcR showed that both ERR and EcR knockdown could lead to significant down-regulation of Vg-mRNA expression;ERR and EcR knockdown could also eliminate the improvement of E2 and 20E on Vg-mRNA expression under the inducement of exogenous steroid hormones,suggesting that ERR and EcR might be synergistically involved in the regulation of steroid hormones on vitellogenesis of E.sinensis.The specific binding assay of E2and ERR Recombinant protein showed that E2could not bind to ERR Recombinant protein directly.In this study,a 1717 bp upstream sequence of the Vg 5'UTR was also cloned by DNA walking method,and its transcription start site and core promoter sequence were analyzed.At the same time,three potential 1/2estrogen response element?1/2 ERE?sequence was found on this sequence,suggesting that estrogen may play its function through the interaction between nuclear receptor and transcriptional regulatory elements on the Vg promoter. |
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