Effects Of Estradiol On Ovarian Development And Gene Expression Of Estrogen Related Receptor Of Swimming Crab, Portunus Trituberculatus

The swimming crab, Portunus trituberculatus, is an important marine-culture crab in China, and poor ovarian development is one of the important hinder for the sustainable development farming industry of this species. Estradiol（E₂） is one of the important sex steroid hormones for crustaceans, which plays an important role during the process of ovarian development. Although E₂ is involved into the modulation of ovarian development of P.trituberculatus, the mechanism of E₂ remains unclear. In the study, the immunofluorescence assay was used to identify the immune positive distribution and variation of E₂ in the ovary, hepatopancreas, thoracic ganglia, brain ganglion and the mandibular organ during the ovarian development of P.trituberculatus; the further experiments were conducted to investigate the effects of E₂ on ovarian development and gene expression of estrogen-related receptor（ERR） and vitellogen（Vg） of P.trituberculatus by the vitro and vivo assays; finally, the methodology of ERR-RNA interference was established and the potential function of ERR gene was preliminarily studied by ERR-RNAi in vitro assay during the ovarian development of P.trituberculatus. The results are shown as follows:1. Immunolocalization of E₂ in the P.trituberculatus during ovarian developmentThe Immunohistochemistry results showed that:（1）During the ovarian development, follicle cells were stained with strong positive E₂ at each ovarian stage. In germinal cells, positive E₂ was found in the cytoplasm during the late ovarian development stages（II-V）, which showed a trend from rise to decline;（2）In the hepatopancreas（I-IV）, positive E₂ was mainly distributed in the F cell and R nucleus, and the biggest positive in stage IV;（3）In neural tissue（I-IV）, positive E₂ was mainly distributed in the nerve cells and nerve medulla of thoracic ganglion and the cell nucleus of cerebral ganglion nerve cells, which has maintained a strong positive reaction;（4）In the mandibular organ（I-IV）, strong positive E₂ always distributed in the nucleus of mandibular gland cells and the cytoplasm around the mandibular gland cells nucleus; These results suggest that of the ovary, hepatopancreas, thoracic ganglion, cerebral ganglion, and mandibular gland may were the target organs of E₂ action. There maybe complex interactions between different target organs to regulate ovarian development of P.trituberculatus.2. Effects of E₂ on ovarian development and related gene expression of P.trituberculatusBy the vitro and vivo assays, the further experiments were conducted to investigate the effects of E₂ on ovarian development and gene expression of estrogen related receptor（ERR） and vitellogen（Vg）, the results showed that:（1）The exogenous E₂ injection had no significant effects on GSI and HSI of stage III female P.trituberculatus;（2）The different concentrations of exogenous E₂ had significant effects on gene expression of ERR-mRNA and Vg-mRNA, mainly for the different concentrations of exogenous E₂ can promote Vg-mRNA expression increased significantly in ovary and hepatopancrea, whereas the expression of ERR-mRNA decreased significantly;（3）In the nerve tissue and mandibular organ, different concentrations of exogenous E₂ could reduce the expression level of ERR, it can be speculated that E₂ regulated ovarian development by regulating two gene antagonistic expression of ERR and Vg;（4）In vitro incubation, the stage III ovary was used, the results showed that E₂（10-8mol/L） can promote the expression of ERR-mRNA significantly3. The establishment of ERR RNAi systems and the preliminary study of ERR RNAi functionBy reverse transcription in vitro, three different interference segments of ERR dsRNA were synthesized. The stage III ovary were treatmented for interference 1 h, 2 h, 4 h, 8 h in 26°C constant temperature incubator, which found that there was a segment sustained interference and the concentration of interference fragment was 2μg /ml. The stage III ovary were treatmented by four different culture conditions:（1）2μg GFP dsRNA（A）;（2）2μg ERR dsRNA（B）;（3）mixture 2μg ERR dsRNA and 10-8mol/L E₂（C）;（4）10-8mol/L E₂（D）, the results showed that, compared with the group A, the expression of ERR mRNA decreased significantly in group B and C, but the ERR mRNA expression was significantly increased in group D, which indicated that the selected ERR-dsRNA could decrease the expression levels of ERR-mRNA significantly, but 10-8mol/L E₂ could promote the expression levels of ERR-mRNA. These results indicated ERR maybe play the important role during the ovarian development of P. trituberculatus, and E₂ may regulate the vitellogenesis and ovarian development by the ERR signal pathway. The mechanism and ERR pathway should be investigated for female P. trituberculatus in the future.