GSK3β Is Involved In The Spermatogenesis Of Chinese Mitten Crab Eriocheir Sinensis

Glycogen synthase kinase3β (GSK3β) is a Serine/Threonine kinase involved in metabolic reactions, and the Wnt, Akt, Hedgehogand BMP/Smadl signaling pathways. It regulates cell metabolism, the cell cycle and cell fate through the phosphorylation of a diverse array of substrates. Its disorders lead to testicular degeneration, testicular cord disruption and many other reproductive diseases that often lead to infertility. In mammals, GSK3P play an important role in the regulation of spermatogenesis. To explore GSK3β’s functions during spermatogenesis in Eriocheir sinensis, we cloned the full-length GSK3β cDNA sequence from the testis of E. sinensis (EsGSK3β). The 1161-nucleotide open reading frame encodes a 386-amino-acid protein with a predicted molecular weight of 43 kDa. Additionally, we examined the expression pattern of EsGSK3β in different tissues and testis developmental stages using real time PCR and western blotting. EsGSK3 β is a typical Serine/Threonine kinase, it has a functional domain (S_TKc). In this functional domain has ATP binding site, axin binding site, active site, activation loop, and polypeptide substrate binding site. Multiple sequences alignments show that there has been very similar amino acid sequence of GSK3P protein from different species. Polygenetic tree displays that EsGSK3β protein was initially clustered with arthropods, including F. arisanus, and then with invertebrate C. gigas, and finally with the vertebrates.The qRT-PCR results showed that the EsGSK3β mRNA was expressed in all tissues investigated, including the gill, stomach, intestine, accessory gonad, testis, ovary and heart. The expression was high in the accessory gonad tissue and the ovary. EsGSK3β was expressed in the remaining tissues in the following descending order: stomach, heart, testis, intestine, gill. The EsGSK3β protein expression in various tissues was examined using western blotting. qRT-PCR analysis showed that the expression level of EsGSK3β was detected in every stage of testis development, with relatively high expression in July and August, and the lowest from September to November. In December and January, expression was significantly high, with a maximum in December. Western blotting showed that the expression of the EsGSK3β protein was high from July to August, and declined slightly during September. The expression level showed no significant difference in the subsequent four months.Localization analysis of EsGSK3β RNA and protein by in situ hybridization and immunofluorescence, respectively, showed that molecules translocated from the cytoplasm and nucleus to the regions of the acrosomal tubule and apical cap during spermatogenesis. Therefore, EsGSK3β might play an important role during E. sinensis spermatogenesis.