The Role Of Retinoid X Receptor In Gonadal Differentiation And Development Of Haliotis Diversicolor

The molecular mechanism of gonadal differentiation and development in mollusks has always been the focus of reproductive biology and aquaculture.Although the vertebrate-type steroids are detected in mollusks,the biosynthesis pathway and the origin of these steroids is still unknown and there is no evidence for the presence of hormone-related receptor in the mollusks.So the role of vertebrate-type steroids in mollusks remains controversial.In recent years,a growing body of research of imposex induced by organotin suggests that retinoid X receptor is not only the key factor of imposex in gastropods,but also may play a vital role in the mechanism of mollusks gonadal differentiation and development.In this paper,Real-time quantitative PCR and RNA interference technology were used to analyze the role of retinoid X receptor and cell apoptosis related genes in gonadal differentiation and development of Haliotis diversicolor.The main results were reported as follows:1.The gonad were sampled,fixed in Bouin's fluid,embedded in paraffin and observed under a light microscope every month to examine the histological changes during a year.The gonadal development of Haliotis diversicolor can be divided into five stages according to the color and the volume of gonad,the structure of germ cells,the type of predominant germ cells in the gonad: suspensive stage,proliferation stage,growing stage,maturation stage,breeding stage.The results showed that the small small abalone has only one reproductive cycle in a year and the development of testis and ovary is dyssynchrony.The suspengsive stage of ovary is from January to March,the proliferation stage of ovary is from April to May,the growing stage of ovary is from June to July,the maturation stage of ovary is from August to December.The suspengsive stage of testis is fromFebruary to March,the proliferation stage of testis is from April to May,the growing stage of testis is from June to October,the maturation stage of ovary is from November to next January.2.The best reference genes were selected in different tissues during the reproductive cycle from a set of ten candidates: ?-actin(ACT),18 S ribosomal(18S),28 S ribosomal(28S),elongation factor 1-alpha(ELFA),ribosomal protein L8(RPL8),eukaryotic translation initiation factor 5A(EIF5A),Y-box protein 1(YB1),ornithine decarboxylase antizyme 1(OAZ1),60 S ribosomal protein L3(RPL3),ribosomal protein S9(RPS9).The expression stabilities of the 10 candidate reference genes were evaluated by geNorm,Normfinder and Bestkeeper.The most suitable reference genes were specific to each tissue.In female head and male hepatopancrea,EIF5 A was the most stable reference gene.In ovary,female hepatopancrea and male head,OAZ1,28 S and RPS9 was ranked as the best reference gene,respectively.3.The mRNA expression levels of RXR and retinoid X receptor b(RXRb)were analysized at different gonadal development stages and in different tissues(ovary,female head,female hepatopancrea,male head,male hepatopancrea).In ovary,female head,female hepatopancrea,male head,RXR expression level showed a significant increase at proliferation stage,while in male hepatopancrea,RXR expression level showed a significant decrease at proliferation stage.The significant difference between proliferation and other reproductive stages indicated that the RXR regulated critical period of gonadal development is proliferation stage.4.In this paper,qRT-PCR was used to analyze the mRNA expression level of five apoptosis related genes: Caspase 3(Casp3),Caspase3 like protein(Casp3lp),insulin-like growth factor binding protein 7(IGFBP7),activator protein 1(AP1),defender against cell death 1(DAD1)in gonadal development cycle.In ovary,the expression level of Casp3 and DAD1 showed a peak at suspensive stage,while IGFBP7 and AP1 presented the highest expression level at proliferation stage.As a result,Casp3,DAD1,IGFBP7 and AP1 were involved in the development process of ovary.5.RXR ds-RNAs were in vivo injected into the female hepatopancrea of H.diversicolor and gonads as well as the head were sampled.48 h after injection.There is no significant difference head,while the mRNA level of RXR of RNAi group in ovary was significant decreased compared to that of control group and EGFP group.These results indicate that in vivo injection of RXR ds-RNA could led to knock-down phenotypes in small abalone.