Functions Of The Vasa3 Gene In Schistosoma Japonicum As Assessed By RNA Interference

Objective: Vasa gene is firstly found in Drosophila. It is a necessary factor of the embryonic formation and the development of germ cell. In zebrafish, the Vasa protein,as the product of maternal gene,is mainly stored in the cytoplasm of oocytes,and is a key factor in the formation of germ cells. Since the Vasa gene is closely related to the growth and development of the reproductive organs of the majority of species. We hypothesized that the protein, encoded by the Vasa gene of Schistosoma japonicum,also plays a role in the development of reproductive organs.Methods: The Vasa3 gene was expressed at 18, 24, 36 and 42 days at different developmental stages by whole mount in situ hybridization. The function of SjVasa3 gene was determined by RNAi. SjVasa3 siRNA1, siRNA2 and siRNA3 were designed by software in three different targeting regions according to the principle of siRNA screening. The most effective siRNA was determined by qPCR. Then the siRNA was transfected into the control medium Schistosoma japonicum adults. After 10 days, The SjVasa3 gene transcript level and protein level reduction was determined by qPCR and Western Blot. The ovaries, vitellarium, testes, sperm and intrauterine eggs were analyzed using confocal laser scanning microscopy (CLSM). In addition, eggs in the culture medium were collected and then counted by bright-field microscopy on the 4th,7th and 10th day, respectively.Results: In this study, the SjVasa3 gene were located at different developmental stages by Whole mount in situ hybridization. There was no signal in the female and the male worms of 18 days after infection. However, strong positive signals were detected in the posterior ovary of adult female at days 24, 36 and 42 worms, and showed similar density. SjVasa3 transcript levels were analyzed by qPCR to determine the RNAi effects. the results showed that the SjVasa3 transcript level was decreased by approximately 80% at the 10th day compared with untreated and scrambled group. The SjVasa3 protein level was decreased by approximately 60% compared with the untreated and scrambled group. The SjVasa3 siRNA1 treated worms were stained with hydrochloric carmine and morphologically analyzed using confocal laser scanning microscopy. In SjVasa3 siRNAl treated worms, this morphology changed considerably.The testicular lobes were seriously shrunk. Significant cracks were observed in each testicular lobe. A lack of elongated mature sperm was observed in most of the anterior sperm vesicle,which instead contained round undifferentiated spermatocytes. The number of immature as well as the large mature oocytes was distributed all over the ovary and no longer concentrated to the posterior part. They lost their close arrangement and appeared apoptosis. No significant changes were found on the 4th and 7th day.However, the number of eggs in the medium and intrauterine eggs were decreased by approximately 60% and 68%, respectively. after SjVasai siRNA1 treatment on 10th day compared to untreated and scrambled siRNA treated worms. Meanwhile, On the 7th and 10th day, the worms pairing stability were decreased by approximately 30% and 47%,respectively.Conclusion: The expression of Vasa3 gene in Schistosoma japonicum was reduced by RNAi technique. The morphology of female ovary, vitellarium and male testis was changed obviously. The female fecundity was also significantly affected. It was proved that Vasa3 gene plays an important role in the development of reproductive organs and the formation of eggs.