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Studies on the Function of the Accessory Sex Gland Protein in Eriocheir Sinensis

Author: MaoZuo
Tutor: WangQun
School: East China Normal University
Course: Zoology
Keywords: Chinese mitten crab Vice gonadal Sperm acrosin Two-dimensional electrophoresis Mass Spectrometry Peptide mass fingerprint analysis
CLC: S917.4
Type: Master's thesis
Year: 2009
Downloads: 67
Quote: 0
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Abstract


Chinese mitten crab (Eriocheir sinensis) is one of China's important economic freshwater crabs, on male reproductive biology research is currently focused in spermatogenesis, spermatophore formation sperm ultrastructure (Spermultrastructure), sperm metabolism (Sperm metabolism), as well as the testis EST sequence analysis, and vice gonads research is relatively weak. Vice gonads is an important part of the the Chinese mitten male reproductive system, mammalian studies confirmed that Vice gonads has an important influence on male reproductive not only provide nutrition for sperm, certain factors can make the sperm surface series changes affect sperm motility, physiological state, sports and his fertilizing capacity. But for a specific function and role of the Chinese mitten crab, vice gonadal not yet understood its contents of specific ingredients, there has never been a study. The sperm acrosin exist in body of spermacrosome, a trypsin (Trypsin), the top body exists to original acrosin inactive precursor form. Acrosin as a key enzyme in the process of fertilization, the sperm penetrate the egg zona pellucida from role of dissolved transparent tape, provided the conditions for the sperm-egg binding, and thus its activity level can be directly affect the pregnancy. Acrosome reaction defect is one of the important reasons for sperm dysfunction, in vitro tests showed that the process of fertilization is prevented when the the acrosome enzyme is inhibited, the the acrosin activity level of a positive correlation with the quality of the sperm directly affect the with the results of the fertilization. Therefore, sperm acrosin activity has become an important indicator of the quality of semen evaluation, clinical unexplained male infertility patients with a more sensitive biological indicator of Recently, our laboratory by build the Chinese mitten Vice gonadal SSH library and found an important role in the organs of the male reproductive, and found for the first time the important role of the Deputy gonadal protein spermatophore rupture. In this paper, on the vice gonadal spermatophores disruption and acrosin activity carried out related research, in order to provide a theoretical basis for further study on the mechanism of the of Decapod crustaceans male reproductive. Proteome (Proteome) is defined as \proteins. As an emerging discipline, proteomics proteome research object, study the protein composition of the regulation on the overall level of activity patterns. Can provide us with the following information: whether and when the gene product is translated, the relative concentration of the gene product, the degree of post-translational modifications predicted from the gene sequence. These three points are not separate from the nucleic acid sequence accurately predict proteome analysis does not depend on DNA sequence information carried, and in turn contribute to further understanding of the genome. It is an effective complement to differential display and the microarray expression sequence tag (EST) analysis, directly or indirectly subtractive hybridization, chromosome linkage analysis and nucleic acid sequencing, genome research methods. Our previous study found that the gonads of the Chinese mitten crab, vice, vice-gonadal secretion protein allows it to The spermatophore rupture, thus releasing the sperm spermatophore. However, there is limited understanding of the the crab vice gonadal function. To this end, the use of two-dimensional electrophoresis and mass spectrometry Vice gonadal secretions, in order to understand the role of Vice-gonadal protein during development, helps to clarify the Chinese mitten crab reproductive mechanism. A different free sperm to get sperm viability and acrosin activity of this study were to use Vice gonadal protein digestion, trypsin digestion, mechanical homogenization of three different methods to obtain the mitten crab sperm and sperm survival and acrosin activity detection, found that sperm acrosin activity maximum Vice gonadal protein digestion (4.290 ± 0.095) μIU/10 ~ 6; the sperm enzyme mechanical homogenization minimum, as (1.423 ± 0.109) μIu/10, 6; obtained by the three methods, the presence of enzyme activity was significant difference (P LT; 0.05). The related tissue protein acrosin activity affect the study of the Chinese mitten crab spermatophores trypsin digestion can be obtained free of sperm in unmated mature male crab seminal vesicle were satisfied seminal vesicle homogenate seminal vesicle wall homogenate, Vice gonadal protein homogenate satisfied seminal vesicles and vice gonad homogenate mixture processing sperm measured sperm acrosin activity. In addition, mating after 10 hours satisfied seminal vesicle free sperm determination of sperm acrosin vitality. The results showed that: after mating satisfied seminal vesicle sperm acrosin activity highest (21.729 ± 0.138) μIU/10 ~ 6; the trypsin digestion sperm prior to mating, Vice gonadal protein and satisfied seminal vesicle mixed evenly after different treatment slurry treatment sperm enzyme activity has improved greatly, seminal vesicle homogenate treated the sperm enzyme with untreated no significant differences. Third, the the Chinese mitten Vice gonadal protein two-dimensional electrophoresis and mass spectrometric analysis of differentially expressed proteins in order to study the function of Vice gonadal secretions, this study using two-dimensional electrophoresis and mass spectrometry proteomics separation Chinese mitten crab Vice gonadal development of early and late a secreted protein. The vice secretion of gonadal protein by 17cm2-dimensional SDS-PAGE, a Coomassie Blue stained polypeptides Spectrum Analytical PDQuestsoftware. From the position of the distribution of the protein spots in the gel, mostly distributed in the range of the isoelectric point pI4-7, the smaller the molecular weight of the main protein. There are significant differences in expression levels which 13 protein spots were in-gel digestion, mass spectrometry, initially identified nine proteins, these proteins are involved in sperm capacitation; acrosome reaction and sperm-egg binding; sperm protection including anti-sperm oxidation ability, sperm damage; sperm motility; transcriptional regulation, and. These characteristics of the protein after the description of the sub-gonadal secretions combined with sperm, while maintaining sperm physiological state to play an important role. Deputy gonadal tissue proteomics analysis method thus constructed, laid a foundation for further understanding of the function of the sub-gonadal secretions.

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