Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i>
We previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian <i>Ciona intestinalis&l...
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2024-11-01
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| author | Hitoshi Sawada Ikuya Hattori Noritaka Hashii Takako Saito |
| author_facet | Hitoshi Sawada Ikuya Hattori Noritaka Hashii Takako Saito |
| author_sort | Hitoshi Sawada |
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| description | We previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian <i>Ciona intestinalis</i> type A (Phlebobranchia). However, whether such a protease is involved in the fertilization of other ascidians is unknown. Here, we investigated the effects of four metalloprotease inhibitors on the fertilization of the ascidian <i>Halocynthia roretzi</i> (Stolidobranchia). Three metalloprotease inhibitors, GM6001, TAPI-0, and TAPI-1, strongly inhibited fertilization at 33 and 11 μM, whereas TAPI-2 weakly inhibited fertilization at 33 μM. In contrast, GM6001NC (negative control) had no effect on fertilization at 100 μM. Furthermore, GM6001 had no inhibitory effect on the fertilization of VC-deprived eggs. The metalloprotease appears to function at the middle or late stage of fertilization. Ten <i>Tast</i> genes were identified in the <i>H. roretzi</i> genome database, among which four genes (<i>HrTast1</i>, <i>HrTast2b</i>, <i>HrTast2c</i>, and <i>HrTast3c</i>) possessed a single transmembrane domain in the N-terminal region. These four genes are transcribed in the testis and ovary, as revealed by RT-PCR. Anti-HrTast2c IgG raised against a peptide corresponding to the Zn-binding consensus sequence weakly inhibited fertilization at 0.5 mg/mL. These results led us to propose that sperm astacin-like metalloproteases may be involved in sperm penetration of the VC during <i>H. roretzi</i> fertilization. |
| format | Article |
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| issn | 2218-273X |
| language | English |
| publishDate | 2024-11-01 |
| publisher | MDPI AG |
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| series | Biomolecules |
| spelling | doaj-art-e1f32f4d3f074ab49a3b72ea666770fa2025-08-20T02:55:44ZengMDPI AGBiomolecules2218-273X2024-11-011412148710.3390/biom14121487Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i>Hitoshi Sawada0Ikuya Hattori1Noritaka Hashii2Takako Saito3Sugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, JapanSugashima Marine Biological Laboratory, Graduate School of Science, Nagoya University, 429-63 Sugashima, Toba 517-0004, JapanDivision of Biological Chemistry and Biologicals, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki 210-9501, JapanDepartment of Applied Life Sciences, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, JapanWe previously reported that five astacin-like metalloproteases with thrombospondin type-1 repeats (Tasts) located on the sperm surface are a promising candidate as the protease involved in sperm penetration of the vitelline coat (VC) during fertilization of the ascidian <i>Ciona intestinalis</i> type A (Phlebobranchia). However, whether such a protease is involved in the fertilization of other ascidians is unknown. Here, we investigated the effects of four metalloprotease inhibitors on the fertilization of the ascidian <i>Halocynthia roretzi</i> (Stolidobranchia). Three metalloprotease inhibitors, GM6001, TAPI-0, and TAPI-1, strongly inhibited fertilization at 33 and 11 μM, whereas TAPI-2 weakly inhibited fertilization at 33 μM. In contrast, GM6001NC (negative control) had no effect on fertilization at 100 μM. Furthermore, GM6001 had no inhibitory effect on the fertilization of VC-deprived eggs. The metalloprotease appears to function at the middle or late stage of fertilization. Ten <i>Tast</i> genes were identified in the <i>H. roretzi</i> genome database, among which four genes (<i>HrTast1</i>, <i>HrTast2b</i>, <i>HrTast2c</i>, and <i>HrTast3c</i>) possessed a single transmembrane domain in the N-terminal region. These four genes are transcribed in the testis and ovary, as revealed by RT-PCR. Anti-HrTast2c IgG raised against a peptide corresponding to the Zn-binding consensus sequence weakly inhibited fertilization at 0.5 mg/mL. These results led us to propose that sperm astacin-like metalloproteases may be involved in sperm penetration of the VC during <i>H. roretzi</i> fertilization.https://www.mdpi.com/2218-273X/14/12/1487ascidianfertilizationmetalloproteaseastacinproteasethrombospondin |
| spellingShingle | Hitoshi Sawada Ikuya Hattori Noritaka Hashii Takako Saito Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> Biomolecules ascidian fertilization metalloprotease astacin protease thrombospondin |
| title | Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> |
| title_full | Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> |
| title_fullStr | Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> |
| title_full_unstemmed | Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> |
| title_short | Involvement of Metalloproteases in the Fertilization of the Ascidian <i>Halocynthia roretzi</i> |
| title_sort | involvement of metalloproteases in the fertilization of the ascidian i halocynthia roretzi i |
| topic | ascidian fertilization metalloprotease astacin protease thrombospondin |
| url | https://www.mdpi.com/2218-273X/14/12/1487 |
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