No 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...

No abstract

Vitellogenin is a precursor of yolk protein that is necessary for embryonic development. This protein is a large multi-domain protein consisting of a signal peptide, a heavy-chain lipovitellin, a phosvitin, a light-chain lipovitellin, a von Willebrand factor type D domain (vWF-D), and a C-terminal coding region (CT), which are processed to respective domains after uptake into oocytes. It is currently believed that only lipovitellin and phosvitin domains are necessary for nutrient supply to oocytes. Thus, molecular species of vitellogenin lacking these domains are not known. Here, we show that two novel isoforms of vitellogenin, both of which possess vWF-D and CT domains but not a lipovitellin or phosvitin domain, are expressed in the gonad of the ascidian Halocynthia roretzi. In situ hybridization revealed that mRNAs of these proteins are specifically expressed in oocytes and test cells, accessory cells in the perivitelline space of ascidian eggs. Immunohistochemistry showed that these proteins are localized around the surface of test cells in immature oocytes. Immunoelectron microscopy revealed that vitellogenin associates with vesicles located beneath the vitelline coat (VC) before fertilization but that it dissociates from the VC after fertilization. These results, together with our previous results showing that vWF-D and CT domains are capable of binding to the two sperm proteases HrProacrosin and HrSpermosin, led us to propose that novel isoforms of vitellogenin, which are expressed in oocytes and test cells and released to the perivitelline space during oocyte maturation, may participate in gamete interaction upon fertilization.

Ascidians (primitive chordates) are hermaphroditic animals that release spermatozoa and eggs almost simultaneously, but some species, including Halocynthia roretzi, show strict self-sterility. In H. roretzi, a 70-kDa vitelline coat (VC) protein consisting of 12 EGF-like repeats, named HrVC70, appears to be a promising candidate for the self/nonself-recognition (or allorecognition) system during gamete interaction. After spermatozoon recognizes the VC as nonself, sperm 700-kDa extracellular ubiquitin-conjugating enzyme complex appears to ubiquitinate Lys234 of HrVC70, and the ubiquitinated HrVC70 is degraded by the sperm 26S proteasome that is located on the sperm head surface. This novel ubiquitin-proteasome system enables spermatozoa to penetrate through the VC. Sperm trypsin-like proteases, acrosin and spermosin, also participate in fertilization, probably as sperm-side 'movable' binding proteins to the VC.