Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzi: Effects of protease inhibitors

Hoshi, Motonori; Numakunai, Takaharu; Sawada, Hitoshi

doi:10.1016/0012-1606(81)90322-5

Cited by 92 publications

(46 citation statements)

References 17 publications

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“…The solitary ascidian H. roretzi type C (15), which is cultivated in Onagawa Bay, Japan, was purchased from fisherman and stored at Asamushi Marine Biological Station, Tohoku University (Aomori, Japan). The spawning of H. roretzi was induced by controlling the seawater temperature and light conditions (16,17). The mature and immature oocytes were collected as described (12-14, 16, 17).…”

Section: Methodsmentioning

confidence: 99%

“…The thus-treated sperm was mixed with egg suspension (Ϸ200 eggs) in buffered seawater, and the mixture was incubated at 13°C. At 1 and 2 h after insemination, the fertilization ratio was examined on the bases of the expansion of perivitelline space and first cleavage, respectively, as described (16,17).…”

Section: Isolation Of Vitelline Coats and Hrvc70 From Mature And Immamentioning

confidence: 99%

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

Sawada

Tanaka

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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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.

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Section: Methodsmentioning

confidence: 99%

Section: Isolation Of Vitelline Coats and Hrvc70 From Mature And Immamentioning

confidence: 99%

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

Sawada

Tanaka

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Fertilization experiments with this broadcast spawning animal are much easier than those with mammals. We have reported previously that the sperm trypsin-like and chymotrypsin-like proteases are indispensable for sperm penetration through the vitelline coat in H. roretzi by examining the effects of various protease inhibitors on the fertilization of intact as well as naked eggs (6,7). We have purified two trypsin-like proteases (which we designated as ascidian acrosin and spermosin) from H. roretzi sperm, and we showed that these two proteases play key roles in ascidian fertilization (8 -10).…”

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“…The eggs were incubated previously with each oligopeptide, CUB1 or CUB2, in seawater for 30 min at 13°C and were then inseminated. The fertilization ratio was determined as described previously (6,7).…”

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confidence: 99%

cDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

Kodama

Baba

Yokosawa

et al. 2001

Journal of Biological Chemistry

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cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His 76 , Asp 132 , and Ser 227 , which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys 56 -His 57 ) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.In fertilization, sperm must bind to and penetrate through the extracellular glycoprotein matrix surrounding the egg, which is called the zona pellucida in mammals and the vitelline coat in marine invertebrates. After this process, membrane fusion occurs between the sperm and egg. Upon primary binding of the sperm to the vitelline coat, it undergoes an acrosome reaction, which is an exocytosis of the acrosomal vesicle located on the tip of the sperm head. A lytic agent called lysin is exposed on the surface of the sperm head and partially released into the surrounding seawater during the acrosome reaction. In mammals, it has been believed that a trypsin-like enzyme called acrosin (EC 3.4.21.10) is a zona lysin (1, 2). However, recent studies using acrosin-gene knockout mice have revealed that acrosin is not essential for mouse fertilization, although a significant delay (about 30 min) in sperm penetration through the zona pellucida was observed (3, 4). From these results it is currently thought that sperm proteases other than acrosin may participate in sperm penetration through the mammalian egg coat and that acrosin may be involved in the dispersal of the acrosomal matrix (5).Ascidians (Urochordata) occupy a phylogenetic position between vertebrates and "true" invertebrates. Although all ascidians are hermaphrodites, several ascidians including Halocynthia roretzi are strictly self-sterile. The vitelline-coat lysin system is thought...

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“…Halocynthia roretzi (21). They are indispensable for the sperm to penetrate through the egg envelope (22)(23)(24).…”

Section: Sectionmentioning

confidence: 99%

Cytological Characterization of Self Incompatibility in Gametes of the Ascidian, Ciona intestinalis

1987

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We have determined how many elements are involved in the regulation of self-fertilization in the solitary ascidian, Ciona intestinalis that is an incompletely self-sterile species. Animals collected in the field were repeatedly induced to spawn in order to examine their selfing ratios. About 20% of them were self-fertile, although the ratios fluctuated considerably among respective spawnings. Naturally or acid-induced self-fertile gametes required much longer time for selfing than that for crossing. Egg-suspending seawater (egg water) as such activated sperm motility, but it lowered conspicuously the self-fertilization ratio. Self-sterile spermatozoa could scarcely bind to the vitelline coat (VC) of glycerinated autologous eggs, or in case they bound well to it the sperm flagella ceased to beat within five min of the 'insemination'. The staining of sectioned gametes with DAPI, a fluorescent dye of DNA, showed that in selfing the spermatozoa could hardly penetrate the VC even though they bound well to it. The results of this study show that the block of self-fertilization can be classified into four elements from a phenomenal viewpoint, such as egg water, low affinity of sperm-VC binding, inactivation of bound sperm and difficulty in sperm penetration through the VC.

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Evidence for participation of sperm proteinases in fertilization of the solitary ascidian, Halocynthia roretzi: Effects of protease inhibitors

Cited by 92 publications

References 17 publications

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

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

cDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

Cytological Characterization of Self Incompatibility in Gametes of the Ascidian, Ciona intestinalis

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