Clone Characterization And Involvement In The Process Of Acrosome Reaction Of P38 In The Chinese Mitten Crab, Eriocheir Sinensis

Mitogen-activated protein kinases (MAPK) cascades is one of the important ways of eukaryotes signal transmission network. P38 MAPK signal pathway belongs to MAPK signal pathways which cover other three members. P38 MAPKs comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. It has been confirmed in mammal that p38 protein play an important role in cell cycle, differentiation, apoptosis, blastocyst development, inflammatory response, growth and migration of tumor cells.This study is based on a p38 cDNA fragment from testis and accessory gland transcriptome analysis in Eriocheir sinensis, using the RACE technique to clone 5’ and 3’fragments and overlap the sequences to obtain the full length cDNA, for the first time the full length cDNA sequence of E. sinensis p38 gene (E.s-p38 gene) were cloned. The sequence includes a 186 bp 5’Untranslated Regions, a 1098bp Open Reading Frame (ORF), a 693bp 3’Untranslated Regions. The ORF encoded a p38 protein of 365 amino acids from E.sinensis (E.s-p38 protein). We analyzed the functional domain, secondary structure and three-dimensional structure of E.s-p38 protein through bioinformatics. E.s-p38 protein is a typical serine/threonine kinase, it has a functional domain of p38 MAPK (STKc_p38). In this functional domain has ATP binding sites, substrate binding sites, KIM docking sites, and lipid binding sites. Multiple sequences alignments show that there has been very similar amino acid sequence of p38 protein from different species, especially, A-loop and T-G-Y motif. Polygenetic tree displays that E.s-p38 protein clusters with shrimps and crabs together first, then gets other Arthropoda together orderly. It is in accordance with the results of traditional taxonomy.Tissue expressions of E.s-p38 gene were determined by RT-PCR and quantitative real-time PCR analysis. E.s-p38 gene expressed in various tissues, but it expressed higher in the muscle, hepatopancreas, intestine, ovary and testis. E.s-p38 gene expression had no significant difference in Spermatocyte stage, spermatid stage and earlier sperm stage of testis, it expressed higher in the middle and later sperm stage evidently. Such expression pattern was positively correlated with the development of gonads and reproduction seasons in E. sinensis, so the experimental results suggested that E.s-p38 gene may involve in testis development and spermatogenesis of E. sinensis.From the perspective of protein research, E.s-p38 protein expression was determined in testes at different developmental stages by Western blot. The results showed that total E.s-p38 protein expression level gradually increased during the process of spermatogenesis, while the phosphorylated E.s-p38 was much higher in the spermatid than the spermatocyte and sperm stages. A23187 calcium ionophore was used to induce the acrosome reaction of mature sperm in Chinese mitten crabs in vitro. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction induced by pre-incubation with A23187 in vitro, providing a basis for subsequent experiments. Using immunofluorescence techniques to locate E.s-p38 protein on the sperms in different stages of AR and track its distribution and migration during the whole process. Through the experiment, we found that the activated E.s-p38 protein translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis.In summary, this paper for the first time confirmed the presence of p38 in Chinese mitten crab and discussed the important function p38 may possess in regulating the acrosome reaction process of mature sperm in Chinese mitten crab from two angles:gene and protein. The research results achieved in the study will provide important references for future exploring the role of MAPK signal pathway played in the regulation of male reproduction of Chinese mitten crab.