The role of metalloproteases in fertilisation in the ascidian Ciona robusta

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.

Figure 1

GM6001 inhibited fertilisation of C. robusta eggs only in the presence of VC. (a,b) Eggs were inseminated in the presence (left) and in the absence of inhibitors and were then exposed to inhibitors (right). Fertilised eggs are at the 2- or 4-cell stages. (c) The dose dependency of the inhibitory effect of GM6001 on the ratio of cleaving eggs. (d,e) Eggs with the VC intact or deprived were inseminated in the presence or absence of GM6001 (N = 3, markers indicate independent data points, bars indicate means).

Figure 2

Digestion of the VC by the sperm was inhibited by GM6001. (a) Isolated VC and intact sperm of C. robusta were incubated together in the absence or presence of GM6001. Changes in the intensity caused by sperm were observed in the five labelled bands (1–5), all of which were cancelled by the addition of GM6001. These bands in lanes 3 and 4 were analysed by mass spectrometry. The full-length gel image is available in Fig. S6. (b) The top hit proteins detected from the bands and their protein scores (Mascot ver 2.4.1).

Figure 3

The domain compositions of C. robusta Tast gene products predicted by NCBI Conserved Domain Search. Detected conserved motifs with the E-values less than 1E-04 are shown.

Figure 4

The loci of Tast genes in the genome of C. robusta (KH2012). Exons are indicated with rectangles. Introns are shown with bent lines. The predicted ORFs are highlighted green. Arrows and labels indicate the target sites and the names of the TALEN pairs (Fig. S4).

Figure 5

Depletion of metalloproteases caused developmental anomaly at the metamorphic stage. (a) Examples of 6 day juveniles injected with C1.533-1 TALEN pairs (right) and their wild type siblings (left). (b) Development of wild type fertilised eggs in the absence or presence of 25 μM GM6001. Embryos seemed normal until the hatching was slightly delayed in embryos exposed to GM6001. Larvae in both conditions went through tail contraction; however, only GM6001-free larvae achieved metamorphosis.