Expression Characteristics Of H1,Its Variants And Molecular Chaperones During Spermatogenesis In Eriocheir Sinensis

Eriocheir sinensis,as one of the most important economic crabs in China,belongs to Arthropod,Crustacea,Decapoda,Grapsidae.The spermatozoon nucleus of male E.Eriocheir are deconcentrated,whose chromatin is different from the dense structure of the general animal,and unknown about formation mechanism.Histones are the important parts of nucleosome,which have effects on post-translational modification,histone variants on the level of protein and DNA binding and the level of DNA transcription,thereby further regulating chromatin or chromosome structure to exert desired biology function.At the same time,molecular chaperones have an auxiliary effect on nucleosomes in which histones and DNA form the the correct conformation in vivo.In order to explore the cause of decondensed nucleus formation during spermatogenesis in E.sinensis,histone H1 with low conservatism was regarded as the entry point to explore the expression characteristics and dynamic distributions of its variants histone H1.1 etc.,molecular chaperones Nucleophosmin1 pseudogene9(NPM1P9)and Nucleosome assembly protein 1 like 1(NAP1L1),in addition the interaction between histone H1 and NPM1P9 in order to conduct a comprehensive exploration at the levels of molecule and cell.By molecular cloning,the expression systems and corresponding proteins of H1,H1.1 and NPM1P9 were constructed and expressed,respectively.Monoclonal antibody H1(McAb-H1)and Monoclonal antibody H1.1(McAb-H1.1)were prepared by antibody preparation technology,and used them to explore the expression situations and the dynamic distribution characteristics of histone H1 and H1.1 during spermgenesis by western blot and immunofluorescence.The relative expression situations of H1 and H1.1 were quantified in heart,muscle,hepatopancreas and testis by quantitative real-time PCR.Amino acid compositions and protein structures of histone H1 and H1.1 were analysed by bioinformatics.The interaction between histone H1 and molecular chaperone NPM1P9 was explored by Co-immunoprecipitation.The results showed that the histone H1,H1.1 and NPM1P9 were successfully expressed by their vectors in vitro after transformation,induction and purification.McAb-H1 and McAb-H1.1 were obtained by mouse immunity and cell fusion,whose titers both reached over 1:204800.Histone H1 and phosphorylated histone H1.1 were found in testis,whose splicing variants H1-delta-like and H1-delta might exist in spermatozoon.In testis,the expression level of H1.1 was higher than H1 presented significant difference(P <0.05).The expression of H1.1 in testis was significantly higher than that in heart,muscle and hepatopancreas presented extremely difference(P <0.01).During spermatogenesis,NPM1P9 and NAP1L1 transferred from cytoplasm to nucleus,NPM1P9 variants probably existed in spermatozoon.There was an interaction between histone H1 and its molecular chaperone NPM1P9.The study established the foundation for further explaining the packing of chromatin and the formation of decondensed nucleus during spermatogenesis.It has important theoretical values and provides reliable basics for quality evaluation of spermatozoon among other Decapodas.