The Molecular Characteristic And Physiological Function Of TAP In Male Reproductive Tract Of The Prawn, Macrobrachium Rosenbergii

Macrobrachiun rosenbergii is the commercially important organism and widely distributes in the worldwide, mainly in Indian, tropical and subtropical regions of Pacific Ocean. It has high economic value for its fast growth, short reproduction cycle and great size. But the molecular mechanism of fertilization and reproduction remain poorly understand by far. In our present study, we selected the prawn, M.rosenbergii, as the experimental material and focused on the molecules related to sperm maturation and reproduction.Firstly, we used full-length normalization suppression subtraction hybridization (FNSH) to generate a subtracted cDNA library enriched for the male reproductive tract and gained five terminal ampullae-specific transcripts. One of them was found to contain 768 nucleotides with a 264 nucleotides open-reading frame that encodes a putative 88 amino acid precursor protein. The deduced translation product contained a 17 amino acid residue signal peptide and a 71 amino acid residue mature peptide. By blast searches, the gene sequence and the deduced protein had no significant homology with any known gene or protien in GeneBank database. It was a novel gene and named TAP (terminal ampullae peptide).Then, the TAP gene expression characterization was studied. The tissue characterization was demonstrated by semi-quantitative RT-PCR analysis and the results showed that TAP transcript was dominately observed in terminal ampullae of male reproductive tract and the TAP transcript level was steadily increased at different prawn developmental stages. The tissue localization was demonstrated by in situ hybridization (ISH) and the results showed that TAP mRNAs were located in the secretory epithelial cells of terminal ampullae and in androgenic gland cells, and in the spermatogenic cells of testis.Thirdly, the protein tissue distribution was observed. The tissue distribution was demonstrated by Western blotting analysis the results showed that TAP protein was distributed exclusively on terminal ampllae of male reproductive tract and sperm, and the level increased steadily at different prawn developmental stages along with gonad development. The tissue localization was illustrated by immunohistochemical analysis and the results showed that TAP was located in secretory epithelial cells of terminal ampullae and sperm. The immunofluorescence and transmission electron microscope immunohistochemical analyses results further showed the TAP was located on the convex surface of the sperm base and spike.Fourthly, RNAi was performed to study the potential function of TAP gene in sperm maturation and fertilization. The RNAi efficiency was detected by RT-PCR and Western blotting analyses, and the results showed that both the TAP mRNA and protein levels were reduced to less than 10% compared with control samples. The mature male prawns underwent targeted gene disruption shown no differences in morphology and behaviors. No difference between treated and control prawns was observed in the spermatogenesis of testis or spermatophore formation of terminal ampullae at the histological level, and no apparent changes were detected in sperm morphology and amounts. However, the sperm activity has been markedly changed. The sperm proteolytic activity was evaluated by gelatin SDS-PAGE assay and the result showed that the gelatinolytic activity of sperm extracts was obviously reduced in RNAi-induced prawns. Meanwhile, the activity of sperm penetration vitelline coat was evaluated by incubation the sperm extracts with vitelline coat protein in vitro and the result showed that the activity of sperm degradation vitelline coat was severely impaired in RNAi-induced prawn. In other words, TAP performs a pivotal role in regulating the activity of the sperm proteolytic enzymes in terminal ampullae.To study the potential function of TAP gene in fertilization and fertilized eggs development, RNAi-induced male prawns were bred with females of proven fertility. They were found to be fertile. The fertilization ratio and fertilized eggs cell development showed no difference. However, the vitelline coat components of fertilized eggs were great different and the vitelline coat was not degraded in RANi-induced groups. These results indicated that TAP likely performed a pivotal role in regulating the activity of enzymes related to degrade vitelline coat but not influencing the process of sperm entry egg and fertilization.