The role of metalloproteases in fertilisation in the ascidian Ciona robusta

In the ascidian Ciona robusta (formerly C. intestinalis type A), the mechanism underlying sperm penetration through the egg investment remains unknown. We previously reported that proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats are abundant in the sperm surface protein-enriched fraction of C. robusta. Here we investigated the involvement of those proteins in fertilisation. We refined the sequences of astacin metalloproteases, confirmed that five of them are present in the sperm, and labelled them as tunicate astacin and thrombospondin type 1 repeat-containing (Tast) proteins. Fertilisation of C. robusta eggs was potently inhibited by a metalloprotease inhibitor GM6001. The eggs cleaved normally when they were vitelline coat-free or the inhibitor was added after insemination. Furthermore, vitelline coat proteins were degraded after incubation with intact sperm. These results suggest that sperm metalloproteases are indispensable for fertilisati...