| In order to better understand the role of methyl farnesoate (MF) in the ovary development ofEriocheir sinensis (E. sinensis). We cultured oocytes with mandibular organ (MO) as group1, andoocytes with MO and X-organ-sinuus gland (XO-SG) as group2in vitro. Oocytes cultured withmedium were set as control, and then the oocyte diameters were determined to evaluate the effect ofmandibular organ on ovary development. Eyestalk ablation could induce the activity of Mandibularorgan. The relative expression of the Farnesoic acid methyl transferase (FAMeT) and VTG mRNAwere detected after the eyestalk ablation to discuss the effect of eyestalk ablation on mandibularorgan and ovary development. The E. sinensis were injected with methyl farnesoate whose dose is20μL every day and were sacrificed on the5thday. The Na~+/K~+-ATPase activity of hemolymph andgonad was detected. In addition, oocytes were treated in vitro with10-8mol/L MF in this experiment,and then we monitored the expression of VTG mRNA using real time RT-PCR. PKC activator orinhibitor was applied to treat the oocytes cultured in M199medium, and then we monitored therelative expression of VTG mRNA to study whether the PKC signal transduction pathway wasinvolved in activating the ovary development. The main results were as following.1. The effect of the eyestalk ablation on the oocyte developmentMO excretion could promote oocyte maturation by increasing the oocyte diameter in1st,3rd,6thand14thday after eyestalk ablation, especially in the6thday (P<0.01). XO-SG had a significantinhibition on the oocyte development (P<0.05). The results indicated that the functional material inMO could induce the oocyte maturation and show the strongest biology effect in the6thday aftereyestalk ablation.2. Changes in the relative expression of FAMeT and VTG mRNA after the eyestalk ablationBy means of fluorescent quantitative RT-PCR, the FAMeT level was detected. In mandibularorgan, the expression of FAMeT mRNA increased by265%in the6thand14thday after eyestalkablation, significantly higher than that of the control group (P<0.01), and the expression level in the14thwas lower than in the6thday while no significant changes in the1stand3rdday followed byeyestalk ablation (P>0.05). FAMeT mRNA in MO was regulated by XO-SG which could cause the inhibition of oocyte maturation in vitro. After the eyestalk ablation, the expression of VTG mRNAwas significantly induced, and the expression became stronger with the time passing. Theexpression of VTG mRNA was increased545.62fold in the6thday. The VTG mRNA in the gonadwas downregulated by XO-SG.3. Changes in the Na~+/K~+-ATPase activity by injection of MFMF was injected into the E. sinensis with0.5μg/ml and1μg/ml whose dose is20μL every dayand the E. sinensis were sacrificed on5thday. The Na~+/K~+-ATPase activity in the hemolymph washigher than the control group. The results showed that MF with concentration of0.5μg/mL and1μg/mL had significant stimulation of the Na~+/K~+-ATPase activity in the hemolymph, whichincreased by88%and187%respectively over the control group. In the gonad, the meanNa~+/K~+-ATPase activity in the experimental groups were48.2%and83.3%respectively larger thanthat in contrast group without MF. With increase of hormone concentration, the Na~+/K~+-ATPaseactivity was increased. The increase of Na~+/K~+-ATPase activity could cause a reduction in cellvolume, which created intercellular spaces through which vitellogenin is admitted.4. The effect of MF on the relative expression of VTG mRNAThe experimentation results showed that the MF or PKC inhibitor could increase theexpression of VTG mRNA compared to the control group, while the expression of VTG mRNA wassuppressed by the PKC activator. The VTG expression in the group with5×10-11mol/L MF and0.5μmol/L PMA is lower than the MF group. The result demonstrated that MF could enhance theexpression of VTG mRNA. The inactivation of PKC involved in the progress of VTG expression. |
