| Sperm proteases play important roles in the mammalian fertilization process. To prevent the detrimental effects of proteases to the reproductive tract, the protease inhibitors precisely control their activities. Researches on proteases and their inhibitors in the male reproductive system of crustaceans are very limited compared with those of mammalian.In our previous studies, the KPI MRPINK from the prawn M. rosenbergii was identified and characterized as having an inhibitory effect on both the gelatinolytic activity and proteolytic activity of prawn sperm. In addition, MRPINK specifically blocked the activity of M. rosenbergii sperm gelatinase (MSG), and then the primary structure of MSG was determined. Therefore, we speculate that MRPINK may play an important role in the fertilization process of this species. Considering the widespread phenomenon of reproductive isolation in animals, the involvement of these male-specific molecules needs to be clarified. Can other male reproductive system-derived KPIs from different crustacean species substitute for MRPINK? Do any KPI molecules show species specificity? On the other hand, the role of MSG involving in the reproduction process remains to be clarified.In the present study, by using RT-PCR and RACE, we successfully cloned two KPI genes from the male reproductive tracts of M. nipponense and Eriocheir sinensis, they were named Man-KPI and Ers-KPI, respectively. The gene expression characterization and their distribution in the male reproductive tracts were studied. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The separate domains were also expressed in Escherichia coli system, the active domains of intact KPI were uncovered. In addition, the inhibition mechanisms of Man-KPI and Ers-KPI were tested. Meanwhile, the inhibitory activities of these two KPIs towards M. rosenbergii sperm were compared with that of MRPINK. On the other hand, by using RNAi technique, we studied the role of MSG in the fertilization process. The results include two parts:1. Both of deduced amino acid sequences of Man-KPI and Ers-KPI contain several typical Kazal domains, the former has three and the latter has four, respectively. Northern blotting showed Man-KPI was expressed in the male reproductive tract only, while Ers-KPI was also expressed in the hemocytes. The results showed that the three-domain Man-KPI has potent inhibitory activity against chymotrypsin and elastase, weak activity against trypsin, and no activity against thrombin. By comparison, the four-domain Ers-KPI was found to strongly inhibit chymotrypsin and elastase, and weakly inhibit thrombin, but not to inhibit trypsin. Only Man-KPI_domain3is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2and3are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2,3and4are responsible for the inhibition of elastase. Furthermore, kinetic analysis revealed that the inhibition mechanisms of Man-KPI against chymotrypsin and elastase and that of Ers-KPI against elastase were competitive in nature, while inhibition of chymotrypsin by Ers-KPI was uncompetitive. Meanwhile, the inhibitory activities of these two KPIs towards M. rosenbergii sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that while MRPINK completely inhibited sperm gelatinolytic activity, Man-KPI and Ers-KPI had little effect.2. MSG was proved to distribute on vas deference and terminal ampullae by western blotting. Immunohistochemical analysis revealed that MSG was expressed in secretory epithelial cells and sperm. Immunofluorescence analysis of individual sperm was performed to determine the localization of MSG on sperm and the result showed that it bound mainly onto the base. To elucidate the functional role of MSG in vivo, we disrupted the MSG gene by RNA interference (RNAi) and evaluated the effect on fertility and several related sperm parameters. RNAi treatment remarkably decreased the sperm gelatinolytic activity, however, fertilization and embryo development was not affected.Our research, thus, indicates that MRPINK can not be substituted by other male reproductive system-derived KPIs from different crustacean species, and KPIs may evolve as species-specific molecules; MSG is not essential for fertilization, but takes part in maintaining the sperm activity during the fertilization process.
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