| Eriocheir sinensis is an economically important aquaculture species in China. While male reproductive biology research in this species has received considerable focus in the areas of spermatogenesis, spermatophore formation, sperm ultrastructure, sperm metabolism, and testicular EST sequence analysis, accessory sex gland research remains a largely unaddressed discipline despite its confirmed reproductive significance in mammals via nutritional contributions for newly developed spermatozoa and yet unidentified factors capable of inducing a cascade of spermatozoa membrane alterations, and its influence on sperm vitality, physiological state, motility and capacitation, as well as fertilization capacity. However,the function and contents of accessory sex gland from Eriocheir sinensis and the specific has never been known.The acrosin enzyme, which is a trypsin-like protease, is essential for fertilization, dissolving the zona pellucida and permitting sperm penetration of the egg; however, it can be found in the sperm head in an inactive form in vivo. Thus, the level of acrosin enzyme activity may have a direct impact on successful fertilization. Sperm acrosome reaction defects are keys examples of sperm dysfunction, and in vivo and in vitro experiments show that fertilization is prevented if the acrosome enzyme is inhibited . Conversely, the level of the enzyme activity is positively correlated with sperm quality, and directly impacts the outcome of fertilization. Therefore, sperm acrosin enzyme activity has become an important indicator and evaluation tool of semen quality, and as such is one of the more sensitive markers of unexplained clinical male infertility.Further, our laboratory has demonstrated for the first time that the accessory sex gland of Eriocheir sinenses plays an important role during male reproduction and in spermatophore breakdown via SSH library constructs (unpublished data). This aims of this study are to investigate the potential role of the accessory sex gland in spermatophore breakdown and to determine if the accessory sex gland influences sperm acrosome enzyme activity (via quantification of acrosin activity). Results will also shed light on potential mechanisms of action during reproduction in Decapod Crustacea males.Proteome defined as expressed by the genome of all protein, which means all the proteins in cells and its activities, more clearly the expression of cell or tissue, or organism at the specific time and space to express all proteins. As a new discipline, proteomics studys the proteome, which studys the composition of the proteins and the law of the control activities at the overall level. It could provide us with the following information: whether and when gene products are translated from the gene sequences, the relative concentration of gene products, the degree of post-translational modification.These three points are not accurately predicted from the nucleic acid sequence, separately, proteome analysis can not dependent on DNA sequence information, and also can contribute to a better understanding of the genome in turn. It is an effective complement to the differential display, microarray expression sequence tag (EST) analysis, directly or indirectly subtractive hybridization, linkage analysis of chromosomes and nucleic acid sequencing, and so on. Early studies on Eriocheir Sinensis in our laboratory, we found that accessory sex glands can make spermatophore broken down and release the sperm. However, the function of accessory sex glands from Eriocheir Sinensis is also limited. To the end, this study of the secretions of accessory sex glands used two-dimensional electrophoresis and MALDI-TOF mass spectrometry in order to understand the role of accessory sex glands in the development and clarify the mechanism of reproductive of Eriocheir Sinensis.1, The Effect of Sperm Isolation Method on Sperm Survival Rate and Acrosomal Enzyme ActivityIn this study, we digested spermatophore with accessory sex glands,trypsin and mechanical homogenate respectively to obtain sperm and tested sperm survival and acrosomal enzyme activity of this sperm. The results showed that protein digestion method of gland the sperm activity higher was (4.290±0.095)μIU/10~6; The homogenate mechanical of sperm activity the lowest, was (1.423±0.109)μIU/10~6; Three methods of acrosin activities were significant difference (P<0.05) .2, Effects of Reproductive Tissue Homogenates on Sperm Acrosome Enzyme ActivityFree sperm isolated by trypsin digestion were exposed to different reproductive tissue homogenates and the acrosomal enzyme activity was measured. As predicted, the lowest enzyme activity was observed in the negative control. Enzyme activity after seminal vesicle homogenate treatment was also low and did not differ significantly from the negative control. While treatment with spermatheca homogenate and accessory sex gland homogenate remarkably and significantly increased enzyme activity compared to the negative control (P<0.05), they did not differ from one another. Further, combined treatment of spermatheca and accessory sex gland homogenates resulted in an additive effect on enzyme activity, although enzyme activity was still lower than observed in sperm collected from recently mated females.3, Identification of proteins of accessory sex gland from Eriocheir sinensis by two-dimensional electrophoresis and MALDI-TOF mass spectrometryIn order to study the function of accessory sex gland from Eriocheir sinensis secretions, the secreted proteins of early and peak periods were separated by using two-dimensional electrophoresis and mass spectrometry. The secretions of accessory sex gland were through 17 cm 2-dimensional SDS-PAGE, Coomassie blue staining, and the peptide mapping analysed with PDQuest software. From the positions of these protein spots in the gel, most of spots focusd in the region of pI 4-7, and the main proteins is small molecular weight. 13 differentially expressed proteins were analyzed by mass spectrometry after digestion. 9 proteins were preliminary identificated. These proteins involved in sperm capacitation; acrosome reaction and sperm-egg binding; the protection of sperm, including the antioxidant capacity and sperm damage; sperm motility; transcription and regulation, and so on. The characteristics of these proteins confirmed that accessory sex gland secretions combined with sperm plays an important role in the sperm physiological status. The proteomics analysis of accessory sex gland build the foundation on learning more about the function of accessory sex gland secretions. |
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