Self/nonself recognition in ascidian fertilization: Vitelline coat protein HrVC70 is a candidate allorecognition molecule

Abstract

Ascidians are hermaphrodites releasing sperm and eggs simultaneously, but many species are self-sterile because of a self͞nonself-recognition system in sperm-egg interaction. Here, we show that a 70-kDa vitelline coat protein, HrVC70, consisting of 12 epidermal growth factor-like repeats, plays a key role in self͞nonself recognition during ascidian fertilization. We discovered that the amount of HrVC70 of the self-sterile mature oocytes is markedly higher than that of the self-fertile immature oocytes and that the selfsterile mature oocytes become self-fertile by acid treatment, which is able to release the HrVC70 from isolated vitelline coats. In addition, fertilization is strongly inhibited by the pretreatment of sperm with HrVC70 from a different individual, but not from the same individual, and the number of nonself sperm bound to HrVC70-agarose was significantly higher than that of self-sperm. A sequence analysis of HrVC70 disclosed that several amino acid residues in a restricted region are substituted at an individual level, with no identical sequences among the 10 individuals tested. Furthermore, genomic DNA analysis revealed that the epidermal growth factor-like domains correspond to the exons, and each intron is highly conserved among even-and odd-numbered introns, suggesting that multiple gene duplications or amplification of this region might have taken place during evolution. It was also found that diversity in cDNA sequences is derived from genomic DNA polymorphism probably elicited by crossing over and specific nucleotide substitutions. These results indicate that HrVC70 is a candidate allogeneic recognition molecule in the gamete interaction of the ascidian Halocynthia roretzi.

Allorecognition mechanisms during ascidian fertilization

“…As mentioned above, we showed that the number of nonself-sperm bound to HrVC70-agarose beads is significantly higher than that of self-sperm and that pretreatment of sperm with nonself-HrVC70 more strongly inhibited fertilization than the pretreatment of sperm with selfHrVC70 (Sawada et al, 2004). We also found that HrVC70 is a highly polymorphic protein showing no identical sequence among 10 individuals tested: amino acid substitutions were mainly found in a region between the 3 rd and 4 th Cys residues in each EGF domain, which except for domain-2 is about 6 residues longer than those in other EGF-like repeat-containing proteins such as HrNotch-1, and also in a connecting region between EGF domains (Sawada, 2002;Sawada et al, 2004). From these results, together with the fact that even a single amino acid substitution is sufficient to affect the molecular recognition between EGF-like repeat-containing molecules such as Notch, Delta and Serrate (Artavanis-Tsakonas et al, 1995;Jotel et al, 1996), we proposed that HrVC70 is a promising candidate allorecognition molecule during fertilization of the ascidian H. roretzi.…”

“…We then noticed that the amount of a 70-kDa-VC protein HrVC70, which had been identified as a sperm receptor consisting of 12 EGF-like repeats (Sawada et al, 2002a), markedly increased during oocyte maturation and that this protein is easily and almost specifically extracted from the VC by 1-10 mM HCl (pH 2-3) (Sawada et al, 2004). As mentioned above, we showed that the number of nonself-sperm bound to HrVC70-agarose beads is significantly higher than that of self-sperm and that pretreatment of sperm with nonself-HrVC70 more strongly inhibited fertilization than the pretreatment of sperm with selfHrVC70 (Sawada et al, 2004).…”

“…We then noticed that the amount of a 70-kDa-VC protein HrVC70, which had been identified as a sperm receptor consisting of 12 EGF-like repeats (Sawada et al, 2002a), markedly increased during oocyte maturation and that this protein is easily and almost specifically extracted from the VC by 1-10 mM HCl (pH 2-3) (Sawada et al, 2004). As mentioned above, we showed that the number of nonself-sperm bound to HrVC70-agarose beads is significantly higher than that of self-sperm and that pretreatment of sperm with nonself-HrVC70 more strongly inhibited fertilization than the pretreatment of sperm with selfHrVC70 (Sawada et al, 2004). We also found that HrVC70 is a highly polymorphic protein showing no identical sequence among 10 individuals tested: amino acid substitutions were mainly found in a region between the 3 rd and 4 th Cys residues in each EGF domain, which except for domain-2 is about 6 residues longer than those in other EGF-like repeat-containing proteins such as HrNotch-1, and also in a connecting region between EGF domains (Sawada, 2002;Sawada et al, 2004).…”

“…However, we recently found that a major VC component protein, HrVC70, showed significantly higher affinity to nonself-sperm than to self-sperm, when it was immobilized onto agarose beads (Sawada et al, 2004;see below). It is not known yet whether the SI specificity in H. roretzi is genetically controlled, because its long generation time makes breeding experiments difficult.…”

The ascidian egg envelope in fertilization: structural and molecular features

“…The best characterized candidate molecule for self-nonself recognition is a 70-kDa vitelline-coat protein from Halocynthia roretzi which possesses sperm receptor activity (Sawada et al, 2002a, Sawada et al, 2004. However, several studies presented good evidence that not only the VC but also the FCs are involved in the self-nonself recognition process (Fuke, 1983; DeSantis, 1991;Pinto et al, 1995).…”