Cloning And Expression Pattern Of JNK And JIP4 In Spermatogenesis And Acrosome Reaction In The Chinese Mitten Crab, Eriocheir Sinensis

The c-Jun N-terminal protein kinase mitogen-activated protein kinases (JNK MAPKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in controlling cell growth, differentiation and apoptosis in mammalian testis and germ cells. As a scaffold protein of the JNK pathway, JIP4 is found in some mammalian spermatozoa and participates in certain male reproductive processes. However, few have reported on roles of JNK and JIP4 in the Chinese mitten crab Eriocheir sinensis.Based on transcriptome sequences from the testis and accessory gland of E. sinensis, here we successfully cloned full-length cDNAs of JNK and JIP4 (designated Es-JNK and Es-JIP4, respectively). The Es-JNK ORF has 1266bp, encoding an Es-JNK protein of 421 amino acids; The Es-JIP4 ORF has 3936bp, encoding 1311 amino acids. Bioinformatics analysis showed that Es-JNK protein has a conserved JNK MAPK function domain, with a special dual-phosphorylation site named the T-X-Y motif. The Es-JNK protein amino acid sequence were showed high similarity with other species; The Es-JIP4 protein has a conserved JNK-SapK_ap_N domain, however, the Es-JIP4 protein amino acid sequence does not have high similarity with other species. In the phylogenetic tree analysis, JNK and JIP4 from E. sinensis clustered with its homolog from crustaceans first, then with those from insects, and finally vertebrates.Western blotting showed relatively high expression of Es-JNK and Es-JIP4 proteins in the testis. Expression levels of total Es-JNK, phosphorylated Es-JNK (Es-p-JNK) and Es-JIP4 proteins decreased gradually during spermatogenesis. Es-JNK and Es-JIP4 mRNAs were mainly localized in the nucleus from the spermatogonium stage to secondary spermatocyte stage and were widely distributed in the nuclear cup (NC), apical cap (AC), acrosomal tubule (AT) and acrosomal vesicle (AV) of spermatids. However, Es-JNK, Es-p-JNK and Es-JIP4 proteins were mainly dispersed in the cytoplasm and intercellular substance of spermatogonium and primary spermatocytes, in the nucleus of secondary spermatocytes, and in the AC, AT, AV and NC of spermatids. During the acrosome reaction (AR) induced by the calcium ionophore A23187, Es-JNK, Es-p-JNK and Es-JIP4 proteins were found mainly expressed in the AC and AV by immunofluorescence microscopy. The results implicate functions for Es-JNK and Es-JIP4 in spermatogenesis and the AR in E. Sinensis.