No abstract

We have recently shown that water-soluble materials from the egg extracellular cortical rods (wsCRs) exert the ability to induce the sperm acrosome reaction in Penaeus monodon. In this study, we further demonstrated that the thrombospondin protein family (TSP) existed in wsCRs, and that their mRNA transcripts were detected in developing oocytes as early as stage I. Full sequence analysis revealed that our pmTSP sequence was considerably different from the recently reported pmTSP in the 5' nonconserved region and in many TSP signature domains, hence, the name pmTSP-II was given to our variant. The transcripts of pmTSP-II were detected only in early developing oocytes (stage-I and -II) while TSP-like proteins were detected in all developing oocytes, particularly at the outer rim of cortical rods situated in the extracellular crypts of the mature, stage-IV oocytes. In addition, wsCRs contained anti-TSP-reactive proteins, suggesting that TSP-like proteins are dissolved in and are part of the egg water during spawning. The functional importance of TSP-like proteins was evident by the interference of a wsCR-induced acrosome reaction response with anti-TSP in a concentration-dependent manner. In summary, we found that pmTSP-II transcripts were present in the developing oocytes and pmTSP-II protein accumulated in cortical rods, which are partly secreted and thus solubilized to produce dissolved TSP-like proteins that participate in induction of the sperm acrosome reaction-a novel reproductive role for TSP protein family.

Glycoconjugates in egg extracellular matrices are known to serve several functions in reproductive processes. Here, the presence of N‐linked mannose (Man) glycoconjugates on shrimp thrombospondin ( pmTSP‐II) and their physiological functions were investigated in the black tiger shrimp Penaeus monodon. A molecular analysis of pmTSP‐II demonstrated anchorage sites for N‐linked glycans in both the chitin‐binding and TSP3 domains. The presence of Man residues was verified by concanavalin A lectin histochemistry on the purified fraction of pmTSP‐II (250 kDa with protease inhibitor). The function of the Man glycoconjugates was evident by the Con A interference with the pmTSP‐II‐induced acrosome reaction (AR) as well as by the ability to recover the induction of the AR by the inclusion of Mans in the treatment mixture. In addition, the recombinant proteins of the three signature pmTSP‐II domains expressed in E. coli (lacking glycosylation) and mannosidase‐treated pmTSP‐II showed a minimal ability to initiate the AR response. Together, these results provide evidence of the pivotal role that Man‐linked pmTSP‐II plays in modulating the shrimp sperm AR, a novel role for a TSP family protein in shrimp reproductive biology.