Ascidian Sperm Glycosylphosphatidylinositol-anchored CRISP-like Protein as a Binding Partner for an Allorecognizable Sperm Receptor on the Vitelline Coat

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 detergentinsoluble 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.Ascidians, the invertebrate chordates, are hermaphroditic animals releasing gametes nearly simultaneously during the spawning season. Most ascidians show self-sterility or preference for "non-autologous" (allogeneic) fertilization (1, 2), which appears to be beneficial for the achievement of genetic diversity in the next generation. However, the mechanism of self-sterility during ascidian fertilization is a long-standing enigma. In the early part of the 20th century, Morgan (3-6) studied this problem using the solitary ascidian Ciona intestinalis, and found that a barrier against self-fertilization is abolished by the treatment of eggs with weak acid (citrus juice) or protease (pancreatic extract) (3). Because the vitelline coat (VC) 3 -free naked eggs are self-fertile, he proposed that a self/nonself-recognition molecule must reside on the VC or test cells. Later, it was found that only nonself-sperm tightly and specifically bound to the VC of glycerinated eggs in C. intestinalis (7). From these results, it is currently believed that a certain allorecognition molecule responsible for self-sterility must reside on the VC.We have been studying the mechanism of self-sterility using another solitary ascidian, Halocynthia roretzi, because a large quantity of readily fertilizable gametes can be easily obtained from this species, which is aqua-cultured in Japan for human consumption (8). Although H. roretzi exhibits much more strict self-sterility than C. intestinalis, the mode of self-sterility in...

Allurin: Exploring the Activity of a Frog Sperm Chemoattractant in Mammals

“…As demonstrated by Sawada and coworkers, ascidian sperm display GPI-anchored Crisp proteins that play a role in mediating self-recognition between sperm and egg so as to prevent self-fertilization (Urayama et al 2008 ;Yamaguchi et al 2011 ). In two different species, H. roretzi and C. intestinalis , a GPI-anchored Urabin on the surface of the sperm head binds to a specifi c partner protein in the vitelline coat of the conspecifi c egg (VC70 and VC57 in the two respective species).…”

“…4.1 , truncated members typically contain the PR and Hinge domains along with amino-acid sequences at the C-terminal that vary in length from a few residues to several hundred. Members of this class include allurin, the frog sperm chemoattractant whose characteristics are described in the current chapter, insect venom and hookworm antigen proteins (Burnett et al 2008a ), the protease inhibitor-like proteins P115 and P116 (Gibbs et al 2008 ), Ciona intestinalis (Ci) Urabin and Halocynthia roretzi (Hr) Urabin, recently discovered self-identity ligands on ascidian sperm (Urayama et al 2008 ;Yamaguchi et al 2011 ), and the glioma pathogenesis related-like proteins, whose functions are currently unknown, but which include members that are expressed in a testis-specifi c manner (Gibbs et al 2010b ).…”

Allorecognition mechanisms during ascidian fertilization

“…Furthermore, we identified a novel protein that strongly interacts with HrVC70 by Far western blotting in the sperm LD-DIM (low density-detergent-insoluble membrane, or so-called lipid rafts) fraction (Urayama et al, 2008). This 35-kDa GPI-anchored glycoprotein, referred to as HrUrabin (Halocynthia roretzi unique lipid rafts-derived binding partner), showed a sequence homology to a CRISP (Cys-rich secretory protein) family, some members of which are known to play roles in fertilization (Yudin et al, 2002;Ellerman et al, 2006;Roberts et al, 2006).…”

Phospho-Signaling at Oocyte Maturation and Fertilization: Set Up for Embryogenesis and Beyond Part II. Kinase Regulators and Substrates

“…In sea urchin egg, a protein (p350) was isolated as sperm receptor with the egg plasma membrane-vitelline layer complexes (Giusti et al 1997) and another report have shown that EBR1 gene product serves a speciesspecific sperm-interacting protein on the egg vitelline envelope (Kamei and Glabe 2003). In Ascidians (Halocynthia roretzi), the sperm-egg binding is mediated by the molecular interaction between HrUrabin, a glycosylphosphatidylinositol-anchored CRISP (cysteinerich secretory protein)-like protein on the sperm surface and HrVC70 on the polymorphic vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein (Urayama et al 2008). In Xenopus egg, gp69/64 glycoproteins are two glycoforms in the vitelline envelope and have the same number of N-linked oligosaccharide chains but differ in the extent of O-glycosylation, might serve as sperm receptor (Tian et al 1999).…”