ERK Is Involved In The Process Of Acrosome Reaction In Vitro Of The Chinese Mitten Crab, Eriocheir Sinensis

Mitogen-activated protein kinases (MAPK), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation and the acrosome reaction.To investigate ERKs functions in Eriocheir Sinensis reproduction, we successfully cloned the full length ERK from the testis of E. Sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365 amino acids protein with a predicted molecular weight of 42KDa. The bfoinformatics analysis identified an ERK MAPK function domain. In addition, there is a dual-phosphorylation site named the T-E-Y motif unique to ERK MAPK. The phylogenetic tree showed that E. sinensis was clustered together initially with other invertebrates such as Scylla paramamosain, and then gathered with the vertebrates, which corresponded with the traditional taxonomy.Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but Phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. This result show that ES-ERK may participate in the spermatozoa maturation.There is a large number of mature sperm accumulated in the vas deferens and the p-ERK and ERK immunoreactive sites of vas deferens paraffin slides showed that both proteins were located in nucleus and apical cap. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). The morphological changes that occur during AR and this process is divided into four steps:1) apical cap protruding stage; 2) acrosomal vesicle valgus stage; 3) extension of the acrosomal tubule stage; 4) the end of the acrosome reaction. After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.