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Previously, we have identified and characterized a male reproduction‐related kazal‐type peptidase inhibitor (MRPINK) gene from the prawn, Macrobrachium rosenbergii. In the present study, MRPINK was discovered to have an inhibitory effect on the gelatinolytic activity of M. rosenbergii sperm and immunofluorescence analysis revealed it bound specifically onto the base of sperm. The proteolytic activity of sperm extracts to vitelline coat components was also detected to be interfered by MRPINK. Furthermore, a novel gelatinase on sperm was found to be specifically inhibited by MRPINK and was named M. rosenbergii sperm gelatinase (MSG). MSG was then isolated and purified by reversed‐phase high performance liquid chromatography combining with gelatinolytic assay. By amino‐terminal amino acid sequence analysis and molecular cloning, the primary structure of MSG was determined. The data presented in this study provided evidence that MRPINK has an inhibitory effect on the gelatinolytic activity as well as proteolytic activity of prawn sperm and specifically blocks the activity of MSG. Mol. Reprod. Dev. 75: 1327–1337, 2008. © 2008 Wiley‐Liss, Inc.

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The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an in vitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.