A Catalog of Proteins Expressed in the AG Secreted Fluid during the Mature Phase of the Chinese Mitten Crabs (Eriocheir sinensis)

Abstract

The accessory gland (AG) is an important component of the male reproductive system of arthropods, its secretions enhance fertility, some AG proteins bind to the spermatozoa and affect its function and properties. Here we report the first comprehensive catalog of the AG secreted fluid during the mature phase of the Chinese mitten crab (Eriocheir sinensis). AG proteins were separated by one-dimensional gel electrophoresis and analyzed by reverse phase high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). Altogether, the mass spectra of 1173 peptides were detected (1067 without decoy and contaminants) which allowed for the identification of 486 different proteins annotated upon the NCBI database (http://www.ncbi.nlm.nih.gov/) and our transcritptome dataset. The mass spectrometry proteomics data have been deposited at the ProteomeXchange with identifier PXD000700. An extensive description of the AG proteome will help provide the basis for a better understanding of a number of reproductive mechanisms, including potentially spermatophore breakdown, dynamic functional and morphological changes in sperm cells and sperm acrosin enzyme vitality. Thus, the comprehensive catalog of proteins presented here can serve as a valuable reference for future studies of sperm maturation and regulatory mechanisms involved in crustacean reproduction.

Fig 1. Photograph of the male reproductive system in the E. sinensis.

The male reproductive system of this crab consists of a pair of testes (T), vas deferens (VD), seminal vesicles (SV), accessory glands (AG) and ejaculatory duct (ED).

Fig 2. SDS-PAGE pattern of AG proteins in E. sinensis.

AG proteins (30 μg) were separated using 10 cm 12% SDS-PAGE gels for 1 h at 120 V. The gel was stained with Coomassie Brilliant Blue G250 (Invitrogen) and the protein lane of the stained gel was cut into ten pieces of equal size.

Fig 3. GO annotation of AG proteins in E. sinensis.

The subcellular locations and functions of the identified proteins were determined by their GO component and function terms, respectively, available for download as text-based annotation files from the GO database ftp site: ftp://ftp.geneontology.org/pub/go/

Fig 4. Changes in immunofluorescence staining patterns of antibodies against human Es-serpin in E. sinensis sperm during the acrosome reaction.

Sperm were incubated in eggs water for 40min, and the reaction was terminated by 4% paraformaldehyde respectively at 10min, 20min, 30min, 40min.