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Abstract
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 fertilisation, probably owing to direct or indirect mediation of vitelline-coat digestion during sperm penetration. TALEN-mediated knockout of Tast genes and the presence of GM6001 impaired larval development at the metamorphic stage, suggesting that Tast gene products play a key role in late development.
Highlights
Egg fertilisation is key to achieving genetic diversity in the generation
To check the role of metalloproteases in fertilisation, C. robusta eggs were inseminated in the presence of the following metalloprotease inhibitors: wide-spectrum metalloprotease inhibitor GM6001, inhibitory activity-free analogue GM6001NC, aminopeptidase-selective inhibitor bestatin, and thermolysin-selective and bacterial metalloprotease-selective inhibitor phosphoramidon (Fig. 1)
Most eggs exposed to inhibitors at the same concentration after insemination underwent cleavage, suggesting that the decreased cleavage ratio was the result of inhibited fertilisation instead of egg cleavage itself
Summary
Egg fertilisation is key to achieving genetic diversity in the generation. In most animals, the eggs are covered with an acellular investment called the egg coat, called the vitelline coat (VC) in some marine invertebrates, including ascidians and zona pellucida in mammals. Sperm are equipped with specialised systems to recognise and penetrate the egg coat This includes a lytic agent which acts against the egg coat, referred to as lysin. We have previously performed proteomic analyses of sperm-surface proteins and those released from the sperm[3] to document the sperm proteins that would mediate gamete interactions in C. robusta. Proteins containing both an astacin metalloprotease domain and thrombospondin type 1 repeats were abundant in the sperm-surface protein-enriched fraction prepared by labeling sperm with a cell-impermeable biotinylating agent followed by affinity purification with avidin. We aimed to elucidate the function of C. robusta metalloproteases in fertilisation
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