Expression And Distribution Characteristics Of Aquaporins In Spermatogenic Cells Of Eriocheir Sinensis

Aquaporins(AQPs)are integral membrane water channels that allow water and small solutes transport across cell membranes.There are evidences that AQPs play vital roles in osmotic regulation and spermatogenesis in mammals and fish.The Chinese mitten crab(Eriocheir sinensis)is one of the important economic crabs in China,also known as an important representative of crustaceans.But,the expression and distribution characteristics of AQPs in spermatogenesis of this species are unknown.Besides,when their gonads are fully developed,reproductive migration from freshwater to saltwater becomes necessary.Therefore,it is worth exploring whether the AQP types of mature sperm are different from those of spermatogenic cells in the testis.In this study,the expression and distribution characteristics of AQPs in spermatogenic cells and mature sperm of E.sinensis were studied by molecular biology,cell biology and other techniques.The results would lay a foundation for revealing the role of AQPs in the reproduction and development of this species,and also provide basic data for the study of AQPs in crustaceans.The full-length sequences of open reading frame of AQP1 and AQP3,777 bp and 909 bp respectively,were cloned from the testis of E.sinensis,and the bioinformatics analysis showed that both of them had two conserved NPA motifs.The evolutionary relationship of AQP1 and AQP3 were respectively consistent with the evolutionary status of the species,and the conservation of the AQPs was relatively high.Real-time fluorescence quantitative PCR was used to analyze the expression levels of AQP1 and AQP3 in the testicles among juvenile crab,immature,precocious and mature crabs.The results showed that there was a significant difference between adult crab and other periods in AQP1(P<0.01),and it respectively was 2.49 times,4.22 times and 2.58 times of the expression in the testis of adult crabs.Juvenile crab and immature was 1.99 times and 4.75 times of the expression in the testis of adult crabs,which showed that there was a significant difference between adult crab and the two times in AQP3(P<0.01).But there was no different between adult crab and precocious(P>0.05).It can be seen that the expression of AQP1 and AQP3 in the testes of E.sinensis presents a stage specificity.Analysis of the expression level of AQP1 in seminal vesicles showed that the relative expression level of AQP1 in seminal vesicles of early maturity crabs was 0.31 times that of adult crabs,with a significant difference(P<0.05).The distribution characteristics of AQP1,AQP3 and AQP4 in spermatogenic of E.sinensis were studied by immunofluorescence technology.The results showed that AQP1,AQP3 and AQP4 were localized on cell membrane of the spermatogonium,spermatocytes,spermatids of E.sinensis.However,in mature sperm,only AQP1 was localized on the acrosomal cap.To sum up,both AQP1 and AQP3 have two conserved NPA motifs,and it was different that the expression and distribution of different AQPs in the testes and mature sperm of E.sinensis.Only AQP1 was found in mature sperm.These results lay a foundation for further study in the reproduction and osmotic regulation of E.sinensis.