Abstract
Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.
Highlights
Ascidians, the invertebrate chordates, are hermaphroditic animals releasing gametes nearly simultaneously during the spawning season
As in C. intestinalis, mature oocytes but not immature oocytes are self-sterile in H. roretzi, and the self-sterility is lost by treatment with acidic seawater [9]
Ascidian Sperm Binding Partner for HrVC70 to propose that a putative allorecognition protein might be expressed in immature oocytes as a precursor and that the active form might be generated by a trypsin-like protease, resulting in its attachment to the vitelline coat (VC) during oocyte maturation
Summary
Biological Materials and Preparations of the VC and Lowdensity Detergent-insoluble Membrane (LD-DIM) Fraction— Spawning of H. roretzi, collected near Mutsu Bay in northern Japan, was induced by controlling the seawater temperature and light conditions as described previously [17]. Amino Acid Sequences and Compositions, and LC/MS/MS Analyses—For determination of amino acid sequences of HrUrabins, the LD-DIM fraction was subjected to SDS-PAGE in the presence of thioglycolate, followed by electrophoretic transfer onto a polyvinylidene difluoride membrane. For analyses of amino acid compositions, the 35- and 50-kDa HrUrabin bands in LD-DIM fraction, which were separated by SDS-PAGE, were excised, extracted, and subjected to acid hydrolysis (6 N HCl in in vacuo, 24 h). For bead-binding experiments, isolated HrVC70 from the VC was conjugated to the Protein A-Sepharose beads via the anti-HrVC70 antibody as described previously [13]. Self- or nonself-sperm suspensions in artificial seawater, which had been incubated for 30 min at 13 °C with control or anti-HrUrabin antibodies at a concentration of 2 mg/ml, was added into a small volume of HrVC70 beads.
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