OSANAI KENZI

OSANAI, KENZI

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INDUCTION OF ACROSOME REACTION WITH THE ISOLATED CHORION IN POLYCHAETE SPERMATOZOA

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INDUCTION OF ACROSOME REACTION WITH THEISOLATED CHORION IN POLYCHAETE SPERMATOZOA著者 OSANAI KENZIjournal orpublication title東北大学浅虫臨海實驗所報告volume 17number 3page range 159-164year 1983-03-30URL http://hdl.handle.net/10097/00131456BuLL. MAR. BroL. STN. AsAl\IUSHI, TOHoKu UNrv., 17(3). 159-164, 1983 INDUCTION OF ACROSOME REACTION WITH THE ISOLATED CHORION IN POLYCHAETE SPERMATOZOA'> KENZI 0sANAr2> Marine Biological Station, Tohoku University, A'samushi, Aomori, 039-34, Japan Sperm-chorion interaction was examined in the polychaete, Tylorrhynchus heterochaetus. In the presence of calcium ions the spermatozoa underwent acrosome reaction on the isolated chorion and adhered to it using the acrosomal process. In calcium-free media they attached to the isolated chorion without acrosome reaction. This finding suggests that a sperm-chorion binding (sperm attachment) precedes acrosome reaction in fertilization process. The polychaete, Tylorrhynchus · heterochoetus, is one of organisms suitable for analyzing sperm-egg interaction in fertilization process (OSANAI 1978). The spermatozoa undergo acrosome reaction on the outer surface of the chorion. The lobular acrosomal process penetrates the outer thin layer of the chorion and adheres to the surface of the thick inner layer (SATO and 0SANAI, 1981, 1982). Spermatozoa adhere to the chorion isolated from the unfertilized egg (OSANAI, 1976). This suggests that the chorion triggers acrosome reaction, aud leads me to examine whether the acrosome reaction is induced by the isolated chorion. MATERIAL AND METHOD Gametes. Swarming worms of the brackish-water polychaete, Tylorrhynchus heterochaetus (QUATREFAGES) were collected in Natori, Miyagi Prefecture, and stored in cold 30% sea water. Gametes were discharged from the body cavity by cutting the body wall. Unfertilized eggs were preserved in 30% sea water and sperm in ordinary sea water (cf. OsANAI, 1978). Physiological media. Natural sea water filtered with a paper filter (Toyo-roshi No. 2) and HERBST's artificial sea water modified by MoTOMURA (OSANAI, 1975) were used as physiological solution. Divalent cation-free sea water was prepared by substituting NaCl for Ca012 and Na2S04 for MgS04• To remove bivalent cation contamination ethyleneglycol-bis-(/)-aminoethylether) N,N'-tera-acetic acid (EGTA) was added to Ca, Mg-free sea water. Isolation of egg chorions. Unfertilized eggs were suspended in 30% artificial sea water (3 parts of artificial sea water plus 7 parts of distilled water) and 1) Contribution from the Marine Biological Station, Tohoku University, No. 480 2) :!<F'l illli'll 159 160 K. OSANAI then gently homogenized with a Teflon homogenizer. The homogenate was centrifuged at 2000 rpm for 3 minutes. After removing the supernatant, sedimented chorions were resuspended in fresh 30% artificial sea water and then centrifuged again. After repeating the same procedure several times a semi-transparent chorion fraction was obtained. The isolated chorions were suspended in a small amount of distilled water or 30% artificial sea water and stored in a refrigerator until use. Bioassay. Isolated chorions and diluted sperm were mixed in experimental media and then the mixture was centrifuged with a hand centrifuge for removing free spermatozoa. After washlng two or three times with the centrifuge method, spermatozoa remaining on the sedimented chorions were observed with a phase-contrast microscope. RESULTS Acrosome reaction on isolated chorions The Tylorrhynchus spermatozoon has a cylindrical head with a round vertex. A cap-like acrosomal vesicle is located in front of the nucleus (SATO and 0SANAI, 1982). The acrosomal vesicle can be clearly recognized with a phase-contrast microscope. When sperm were added to isolated chorions in natural or artificial sea water, numerous spermatozoa adhered to the chorions. All of the adhering spermatozoa underwent acrosome reaction and connected to the chorion with the acrosomal process (Fig. 1a). Thls sperm-chorion connection seems to correspond with the adhesion of the acrosomal process to the inner layer of the chorion, whlch was a Fig. 1. Reaction of spermatozoa on isolated ohorions. \ b ! \ ' \ ---1------"/ ' \ ' \, { ! \ : \, I 'i \ \ ,.: \ ' a: Spermatozoa adhering to the chorion in ordinary sea water. b: Diagram showing sperm attachment and spin movement in Ca-free sea water. ACROSOME REACTION OF POLYCHAE'fE SPERM 161 demonstrated by electron microscopy (SATO and 0SANAI, 1981, 1982). Sperm-chorion attachment prior to acrosome reaction External calcium ions are necessary for acrosome reaction in echinoderm spermatozoa (DAN, 1954; CoLLINS and EPEL, 1977). To examine whether calcium is required for the acrosome reaction of Tylorrhynchus spermatozoa, sperm-isolated chorion interaction under calcium-free condition was observed. Two drops (0.1 ml) of sperm (1/10 in dilution) were suspended in 0.9 ml of_Ca, Mg-free sea water and then 0.1 ml of the suspension was added to isolated chorwns placed in 5 ml of artificial sea water, or Ca-free or Ca, Mg-free sea wate~. The chorions had been previously treated with the following procedure: the rsolated chorions were suspended in Ca, Mg-free sea water containing 0.1 mM EGTA and sedimented with a hand centrifuge. The sediment was resuspended in Ca, Mg-free sea water and re-centrifuged to remove EGTA. The sedimented chorions were placed in experimental media, in which they were mixed with the sperm suspended in Ca, Mg-free sea water (Fig. 2). When the sperm was added to the chorions in ordinary sea water, spermatozoa underwent acrosome reaction and adhered to the chorion (Fig. 3a-c). When the sperm was mixed with the chorions in Ca-free or Ca, Mg-free sea water, however, o, 1 ml o, I ml I--Sperm 1/10 Co,M g-free S\1 I I 0 .9 ml S\1 5 ml 0 .I ml Chorion 1--I t I _, ~ Co-free or 'wJJ Co,Mg-free s 5 ml \1 Co,Mg-free SW Co,Mg-free SW + 0, I mM EGTA Fig. 2. Schematic representation of procedure for observing sperm·chorion interaction in Ca·free sea water. 162 K. OSANAI Fig. 3. Reaction of sperma.tozoa on isolated ch01·ions in artificial sea water (a-c) and Ca, Mg-f ree sea water (d , f ). X 1800. The bar indicates 10 pm. the acrosomal vesicle remained intact. Each spermatozoon attached itself to the chorion with the tip of the head and spun the tail around the attachment point (Fig. 1b). No change in the acrosome of t he attaching spermatozoa was detected with light microscopy (Fig. 3d, f). This observation suggests t hat the outer surface of the sperm head attaches to the chorion prior to acrosome reaction. The sperm-chorion attachment was followed by acrosome reaction in Ca-present ACROSOME REACTION OF POLYCH AETE SPERM 163 condition, but not in Ca-free media. Therefore, it is concluded t hat the presence of external calcium is required for acrosome reaction also in Tylm·rhynchus spermatozoa. DISCUSSION The results described above show that Tylm·rhynchus spermatozoa connect with the chorion by two ways: . (1 ) attachment prior to acrosome reaction and (2) adhesion after acrosome reaction. The former seems to correspond to the binding between the outer surface of the head tip and the outer layer of the chorion, and t he latter that between the acrosomal process and the inner chorion layer (SATO and OsANAI 1982). In sea urchins two ways were reported concerning sperm-vitelline layer binding. The first is the binding between sperm surface glycoproteins and vitelline layer proteins (AKETA, 1975 ; AKETA et al_ , 1978). The second is the binding between vitelline layer glycoprotein and the acrosomal process by the mediation of proteinous bindin (V ACQUIER and lVIoY, 1978; GLABE and V ACQUIER, 1978). The mediation of an electron-dense mterial derived from the acrosomal vesicle is not found in the adhesion between the acrosomal process and the chorion in Tylorrhynclnts (SATO and 0SANAI, 1981, 1982) . In the process from the first attachment to the confirmed adhesion during normal Tylon·hynchus fertilization, an electron-dense matter is exposed by the opening of the acrosomal vesicle. The matter may mediate the binding between the acrosomal vesicle membrane and the chorion as shown in oyster spermatozoa (BRANDRIFF et al., 1978; OsANAI and KYOZUKA, 1982). The progression from the sperm-chorion attachment to the acrosomal process-chorion adhesion in TylmThynchus requires ext ernal calcium ions. The acrosome F ig. 4. Successive changes of spermatozoa on t he egg chorion. Schematically drawn from electron micrographs by SATO and 0SAN.A.I (1982). a : Sperm-attachment to the outer layer of the chorion. b: Intermediate step from a to c. Electron-dense matter derived from the acrosomal vesicle contacts with the outer chorion layer. c : Adhesion between the acrosomal p rocess and the im1er chorion layer. AP, acrosomal process; AV, acrosomal vesicle; DM, electron-dense matter; IC. inner layer of chorion ; N, sperm nucleus; OL, outer layer of chorion. 164 K. OSANAI reaction of sea urchin spermatozoa is not induced by triggers, such as egg jelly, ionophore A23187 and alkaline, inCa-free sea water (CoLLINS and EPEL, 1977). The acrosome reaction of Tylorrhynchus spermatozoa may be triggered by contact with or attachment to the chorion, but can not proceed in Ca-deficient condition. REFERENCES A.KETA, K., 1975 Physiological studies on the sperm surface component responsible for sperm-egg bonding in sea urchin fertilization. II. Effect of Concanavalin A on the fertilization capacity of sperm. Exptl. Cell Res., 90: 56--62. AKETA, K., S. MIYAZAKI, M. YOSHIDA and H. Tsuzmrr, 1978 A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs. Biochem. Biophys. Res. Comm., 80: 917-922. BRA.NDRIFF, B., G.W. MoY and V.D. VACQUIER, 1978 Isolation of sperm bindin from the oyster (Orassostrea gigas). Gamete Res., 1: 89-99. CoLLINS, F., and D. EPEL, 1977 The role of calcium ions in the acrosome reaction of sea urchin sperm. Regulation of exocytosis. Exptl. Cell Res., 106: 211-222. DAN, J.C., 1954 Studies on the acrosome. III. Effect of calcium deficiency. Bioi. Bull., 107: 335-349. GLABE, C. G., and V.D. V ACQUIER, 1978 Egg surface glycoprotein receptor for sea urchin bindin. Proc. Natl. Acad. Sci., USA, 75: 881-885. 0SANAI, K., 1975 Handling Japanese sea urchin and their embryos. In, The Sea Urchin Embryo, edited by G. Czihak. Springer, Berlin, 26-40. OSANAI, K., 1976 Egg membrane.sperm binding in the Japanese Palolo eggs. Bull. Mar. Bioi. Stn. Asamushi Tohoku Univ., 15: 147-155. 0SANAI, K., 1978 Early development of the Japanese Palolo, Tylorrhynchus heterochaetus. Bull. Mar. Bioi. Stn. Asamushi Tohoku Univ., 16: 59-69. SATO, M., and K. OsANAI, 1981 Sperm entry into Tylorrhynchus eggs after perivitelline space formation caused by pronase treatment. Bull. Mar. Bioi. Stn. Asamushi. Tohoku Univ., 17: 37-46. SATO, M., and K. 0SANAI, 1982 Sperm incorporation at an egg microvillus in the poly. chaete. Dev. Growth Differen., 22: 406. VACQUIER, V.D., and G.W. MoY, 1978 Macromolecules mediating sperm·egg recognition and adhesion during sea urchin fertilization. In, Cell Reproduction, edited by E.E. Dirksen, D.M. Prescott and C.F. Fox. Academic Press, New York, 379-389. 

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INDUCTION OF ACROSOME REACTION WITH THE ISOLATED CHORION IN POLYCHAETE SPERMATOZOA

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