Characterization and expression analysis of serpins in the Chinese mitten crab Eriocheir sinensis

• The full-length cDNA of the Esserpin-2 and Esserpin-3 genes were cloned.
• Esserpin-2 and Esserpin-3 share common features with other serpin genes.
• Spatial and temporal distribution of Esserpin-3 expression was observed.
• Esserpin-3 expression increased during the development of the testis.
• Esserpin-3 expression reduced during the acrosome reaction.

Serpins are a family of serine protease inhibitors that regulate physiological functions and are found widely in animals, plants and microorganisms. However, there are few reports on the role of serpins in the Chinese mitten crab, Eriocheir sinensis. We cloned two serpin genes from E. sinensis, named Esserpin-2 and Esserpin-3. Quantitative and semi-quantitative real-time PCR results showed that Esserpin-3 mRNA transcripts were detected in all the examined E. sinensis tissues, but were most highly expressed in immune tissues, including the gills, hepatopancreas and intestines. Lower levels of Esserpin-3 expression were detected in the accessory gland, testis and stomach, while the muscle and heart showed minimal expression. Esserpin-3 was differentially expressed throughout testis developmental stages. Esserpin-3 protein was localized in the tube wall and lumen of the accessory gland. In the testes, Esserpin-3 was detected in spermatogonia, spermatocytes and spermatids. As Esserpin-3 was also detected in the sperm contained within the seminal vesicles, it is suggested to be an intrinsic sperm protein. During the acrosome reaction, Esserpin-3 expression reduced gradually and completely disappeared after the reaction. Together, our results indicate that Esserpin-3 is an intrinsic sperm protein involved in regulation of sperm maturation and the acrosome reaction in E. sinensis.