Electron Microscopic Observation on Sperm Penetration and Pronuclear Formation in the Fish Egg T. IWAMATSU AND T. OHTA Department of Biology, Aichi Uniuersity of Education, Igaya-cho, Kariya-shr, Aichi 448, Japan

ABSTRACT Unfertilized eggs of the medaka, Oryzias latipes were inseminated after being mechanically freed from the chorion. In the first step of penetration, as soon as i t reached the vitelline surface, the whole spermatozoon was quickly enclosed by ooplasmic protrusions (fertilization cone) of the vitelline surface. In the second step, the egg plasma membrane fused with various regions of t h e plasma membrane of t h e enclosed spermatozoon; finally t h e sperm flagellum was also incorporated into the ooplasm. Initial disappearance of the nuclear envelope of the sperm with vesiculation a t the apical region of the head is followed by dispersal of the sperm chromatin. The nuclear envelope is then reformed by fusion of elongated or flattened vesicles along the margin of dispersing nuclear chromatin. The mature male pronucleus has a large nucleolus within a wrinkled envelope. It seems that the fertilization process in this fish involves some features of that occurring in the marine invertebrate and the mammalian eggs. Process of sperm penetration in the egg of invertebrates (cf. Colwin and Colwin, '67b; Dan, '67) and of mammals (cf. Piko, '69; Yanagimachi and Noda, '70c; Bedford, '68, '72) has been observed in detail with the electron microscope. So far, similar electron microscopic observations have not been described in the fish egg, though teleost fertilization has been investigated by light microscopy (Ginsburg, '63). Since it has no acrosome it is of particular interest as to determine how the teleost spermatozoon penetrates into the egg cytoplasm. It is very difficult to find a spermatozoon undergoing fertilization in ultra-thin sections of the large eggs of fish and frogs, but the occurrence of polyspermy does increase the chance of finding fertilizing spermatozoa. So far, fine-structural studies of the sperm penetration and pronuclear formation are largely based on polyspermic eggs, even in small eggs of marine invertebrates and mammals with the exception of studies carried out after natural mating in rabbits (Bedford, '72). Polyspermic eggs of Oryzias latipes have shown abnormalities a t the step of blastodisc formation and in their later development (Sakai, '61; Iwamatsu, '72). In these cases, however, very early developmental events in these polysperJ. EXP. ZOOL. (1978)205: 157-180.

mic eggs including the mode of sperm penetration do not seem basically different from that in monospermic eggs. The present paper describes the process of sperm penetration up to the point of pronuclear formation, in polyspermic eggs inseminated a f t e r mechanical removal of t h e chorion. MATERIALS A N D METHODS

Adult females of t h e medaka, Oryzias latipes which had spawned every morning under environmentally-controlled reproductive conditions were isolated from the males before ovulation time ( 7 : O O A.M.). The ovary with mature eggs ovulated in the ovarian lumen was removed from the body cavity by laparotomy, after pithing of the brain, and put into salt solution (NaC1 6.5 mg/ml, KC1 0.4 mg/ml, CaCl,. 2H,O 0.15 mg/ml, MgC1,. 7Hz0 0.15 mglml, adjusted to pH 7.4 with M/2 NaHCO,). The mature eggs were denuded by carefully squeezing the vitellus itself out of the chorion through a large pore made a t the vegetal pole with iridectomy scissors. They were then inseminated by immersion in a sperm suspension (22"-23"C) prepared by mincing the testis from an adult male. At various times after insemination, the

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eggs were transferred for 18 hours into 4% glutaraldehyde containing 4.25%sucrose, buffered with 100 mM phosphate buffer (pH 7.4, 4°C). After rinsing in buffers, they were postfixed with 1%osmium tetroxide for 1.5 hours (4"C), dehydrated with graded concentrations of ethanol and finally with acetone, and embedded in Epon 812 (Luft, '61). Ultra-thin sections cut on a JUM-7 ultramicrotome, were stained with uranyl acetate and lead citrate and examined with a JEM-100B electron microscope. OBSERVATIONS

In eggs fixed ten seconds after insemination many spermatozoa were found in the process of attaching to the vitelline surface (figs. 1,2). As soon as the spermatozoon reaches the vitelline surface, i t is caught (or covered) by numerous microvilli or cytoplasmic protrusions (fertilization cone, figs. 3, 4). At this time, the plasma membrane of the spermatozoon is in close contact with that of the egg, but fusion between them can not be recognized. At one minute after insemination the spermatozoon is deeply enclosed within the cortical cytoplasm around which cortical breakdown completes. The vitelline surface forms a cave-like pit which leads to engulfment of the sperm head. The apposed sperm and egg plasma membranes disintegrate partially over various portions of the sperm head (figs. 5, 6). At the same time, the intact flagellum remains free within a cavity which is formed during the engulfment process (figs. 7, 8). By three minutes after insemination the whole spermatozoon lies denuded of its membranes in the egg cytoplasm, with a pit and an electron dense layer remaining a t the vitelline surface. Occasionally, however, in a spermatozoon whose head is completely incorporated, the sperm tail still retains part of the plasma membrane. The nuclear envelope displays various stages of vesiculation along the lateral aspect of the sperm nucleus (figs. 9, 10). Small vesicles are often observed around the swelling sperm nucleus. The nuclear chromatin disperses first in the apical region of the sperm head lying in the egg cytoplasm (fig. 9). Disintegration of the nuclear envelope and dispersion of the chromatin take place more slowly in the caudal region of the sperm head. The decondensed nuclear material progressively disperses, the sperm mitochondria scatter within the egg cytoplasm, and micro-

tubules extend from the centriole region of the swelling sperm head. By five minutes after insemination, complete dispersion of the chromatin of the sperm nucleus has taken place. Smooth-surfaced vesicles accumulate along the margin of the dispersing chromatin (fig. 111,and fuse together to form a double membrane of the male pronucleus (fig. 12). The cytoplasmic vesicles often enclose a minute vesicle-like body within their envelope (fig. 11: arrows). By 15 minutes after insemination, the male pronucleus has become spherical and is surrounded by a nuclear envelope. At this point, the pronucleus still has only small nucleoli. Occasionally, smooth-surfaced vesicles are found incorporated into the nuclear envelope (fig. 13). The sperm flagellum is still detected within the egg in the vicinity of the developing pronucleus. The electron dense layer disappears from t h e cortical cytoplasmic region. By 25 minutes after insemination, the formation of the pronucleus is completed. The pronucleus has a folded nuclear envelope and a fully developed nucleolus (fig. 14). A pit, or the point of entry of the spermatozoon, is still recognizable on the vitelline surface. DISCUSSION

The fish spermatozoon is immediately surrounded and trapped by numerous egg microvilli or cytoplasmic protrusions as soon as it reaches the surface of the egg. This rapid movement of the egg surface against contact of the spermatozoon has been described in echinoderms (Anderson, '68; Longo and Anderson, '68, '70) and in mammals (rat, Piko, '67, '69; Piko and Tyler, '64; hamster, Yanagimachi and Noda, '7Oa,b,c; rabbit, Bedford, '70, '72). The response must be induced in the egg cortex as an immediate result of contact with the spermatozoon. The time required for the activation is less than ten seconds in Oryzias eggs, and thus much shorter than in the mammalian egg. The activated egg cortex plays a role in the incorporation of the spermatozoon which itself is passive during its movement into the egg cytoplasm, as observed in the hamster (Yanagimachi and Noda, ' 7 0 ~ )Uwa . ('67) found that the spermatozoon treated with 0.06% benzalkonium chloride with spermicidal action more than five seconds after insemination is capable of participating in the development of the egg. Judging from this experimental observation and our own in the

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electron microscope, the spermatozoon may be membrane appears to have changed from the shielded or enclosed by the vitelline surface inner acrosomal membrane to the plasma almost instantly a f t e r insemination. The membrane. Spermatozoa of primitive verteresponse of the vitelline surface region to the brates, the lampreys, Lampetra fluviatilia and stimulation of the spermatozoon seems sep- Lampetra planeri (Ballowitz, '04; Afzelius arable from the cortical reaction itself, i.e., and Murray, '57; Kille, '60) and of the fusion of the alveolar envelope with the egg sturgeons Acipenser guldensadti and A. plasma membrane, as suggested by Ginsburg stellatus (Dettlaff and Ginsburg, '63) have ('63). In salmonid eggs the union of the acrosomes and undergo an acrosome reaction, gamete proceeds without egg activation (cor- despite the appearance of micropylar canals in tical reaction) upon insemination either in the egg membrane (chorion). The filament coelomic fluid or in Ringer's solution (Pri- protruded from the acrosome loses its role in volnev, '41; Kusa, '50, '53; Ginsburg, '63). passing through the chorion, because the sperAccording to Ginsburg ('63), this union seems matozoon reaches directly the vitelline surto indicate a partial engulfment by the egg cy- face through the micropylar canal. However, toplasm. A similar result is observed in anaes- it is unknown whether or not the acrosome thetized eggs of Oryzias latipes (Aketa, '66). filament membrane (inner acrosomal memThe spermatozoon probably enters the egg brane) fuses with the egg plasma membrane cytoplasm even if breakdown of the cortical in these fishes. The spermatozoa of teleost alveoli is temporarily prevented (Sakai, salmonid fishesSalmo trutla L. andS. salar L. (Billard and Ginsburg, '73), Oryzias latipes '64a,b; Iwamatsu, '65). In the initial step of engulfment in Oryzias (Sakai, '76) and Oligocottus maculosus eggs, the fusion between egg and spermato- (Stanley, '69) have no acrosomes. The present zoon plasma membranes could not be detected. paper demonstrates that in Oryzias latipes, a Whether or not there is a change of membrane teleost devoid of an acrosome, sperm penetrapotential (Maeno et al., '56; Hori, '58; Ito and tion takes place by the fusion of sperm and egg Maeno, '60) or a rapid movement of the egg plasma membranes by a mode similar to t h a t cortex prior to membrane fusion remains of mammals. The developmental process of the male prouncertain. Partial fusion is observed within at least one minute after insemination. The ear- nucleus proceeds in two steps, disappearance liest disappearance of the cortical alveolus of sperm nuclear envelope followed by formacommences about 20 to 50 seconds after in- tion of nucleoli, as reported in invertebrates semination or pricking with a glass needle in Gea urchin, Franklin, '65; Longo and AnderOryzias eggs (Yamamoto, '61; Uwa, '67; son, '68; Longo, '76; surf clam, Longo and Nakano, '69). Judging from these timed re- Anderson, '70) and in mammals (rat, Szollosi sults, our data seem to indicate that fusion of and Ris, '61; Piko, '64, '67, '69; Piko and Tyler, the sperm plasma membrane and the cortical '64; rabbit, Bedford, '68, '72; Zamboni and alveolar envelope with that of the egg takes Mastroianni, '66; mouse, Stefanini et al., '69; place during or after engulfment of the sper- h a m s t e r , B a r r o s a n d F r a n k l i n , ' 6 8 ; Yanagimachi and Noda, '70b,c). The swelling matozoon in the monospermic egg. The difference between invertebrates and of the sperm nucleus proceeds concurrently mammals in the initial site of membrane with disappearance of the nuclear envelope. fusion of t h e spermatozoon was pointed out by The disappearance of the nuclear envelope Bedford ('70) and Yanagimachi and Noda advances with vesiculation through a process ( ' 7 0 ~ ) Membrane . fusion begins between the of membrane fusion between the inner and inner acrosomal membrane of spermatozoon outer envelopes. The sequence of disappearance of the sperm and the egg plasma membrane in some invertebrates (Colwin and Colwin, '61, '63, '67a,b; nuclear envelope differs among various speFranklin, '65; Niijima and Dan, '65; Pasteels, cies. In Oryzias Zatipes, it progresses from the '65; Tyler, '65), but in mammals this occurs apical to the basal region of t h e sperm head. between the plasma membrane of the post-nu- The nuclear envelope adjacent to the sperm clear cap region and the egg plasma mem- neck region disappears somewhat later as brane (Barros and Franklin, '68; Bedford, '70, compared with the other regions. In the sea '72; Piko and Tyler, '65; Yanagimachi and urchin, a delayed disappearance of the nuclear Noda, '7Oa,b,c). In the process of mammalian envelope at the apex and the base of the sperm evolution, the fusion site with the egg plasma nucleus may be associated with the acrosome

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and centriole (Long0 and Anderson, '68). On the other hand, similar observations on the delayed disappearance of the hamster nuclear envelope in these two regions of the sperm nucleus have been made in vitro by Yanagimachi and Noda ('70~).This delay in dispersion of nuclear material was suggested as associated with either an existence of the acrosome or the centriole to prevent the exposure of the nuclea r envelope to the egg cytoplasm. It seems that disappearance of the sperm nuclear envelope is delayed a t only t h e neck region in t h e teleostean spermatozoon with no acrosome. Formation of the male pronuclear envelope is heralded by the appearance of vesicles along the peripheral region of the dispersing chromatin, and is completed in the fusion of the elongated or flattened vesicles to form a double laminated structure, as studied in sea urchins and mollusks (Pasteels, '63, '65; Longo and Anderson, '68, '69, '70; Longo, '76). In mammals in which none of the sperm nuclea r envelope remains after chromatin dispersion, the vesicles forming the presumptive male pronuclear envelope seems to appear de nouo (Bedford, '70, '72; Yanagimachi and Noda, ' 7 0 ~ ) In . the sea urchin, a majority of the vesicles forming the pronuclear envelope are derived from endoplasmic reticulum and a few come from the sperm nuclear envelope (cf. Longo, '76). In Oryzias latipes, in which the sperm nuclear envelope breaks down by vesiculation, the residues of the nuclear envelope are probably scattered around the dispersing chromatin. Small numbers of such vesicles may be incorporated with other smooth-surfaced vesicles into the structure of the male pronuclear envelope. Thus, this aspect of male pronuclear formation is similar to that in the invertebrate egg. ACKNOWLEDGMENTS

The authors wish to express their deepest appreciation to Doctor J. M. Bedford of the Department of Obstetrics and Gynecology, Cornell University, Medical College, New York City for his kind improvement of the manuscript. LITERATURE CITED Afzelius, B. A,, and A. Murray 1957 The acrosomal reaction of spermatozoa during fertilization or treatment with egg water. Exptl. Cell Res., 12: 325-337. Aketa, K. 1966 A study of t h e block mechanism against polyspermy in t h e medaka, Oryzias latipes. Annot. Zool. Japon., 39: 149-155.

Anderson, E. 1968 Oocyte differentiation in t h e sea urchin, Arbacia punctulata, with particular reference to the origin of cortical granules and their participation in the cortical reaction. J. Cell Biol., 37: 514-539. Ballowitz, E. 1904 Uber die Spermien des Flussneunauges (Petrornyson fluuiatilis L.) und ihre merkwdrdige Kopfborste. Arch. mikr. Anat., 65: 96-120. Barros, C., and L. E. Franklin 1968 Behavior of the gamete membranes during sperm entry into the mammalian egg. J. Cell Biol., 37: C13-Cl8. Bedford, J. M. 1968 Ultrastructural changes in the sperm head during fertilization in the rabbit. Am. J. Anat., 123: 329-358. 1970 Sperm capacitation and fertilization in mammals. Biol. Reprcd. Suppl., 2: 128-158. 1972 An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am. J. Anat., 133: 214-254. Billard, R., and A. S. Ginsburg 1973 La spermiogenesis et le spermatozode dAnguilla anguilla L. etude ultrastructurale. Ann. Biol. anim. Bioch. Biophys., 13: 523-534. Colwin, A. L., and L. H. Colwin 1961 Changes in the spermatozoon during fertilization in Hydroides hexagonus (Annelida). 11. Incorporation with the egg. J. Biophys. Biochem. Cytol., 10: 255-274. 1963 Role of the gamete membranes in fertilization in Saccoglossus kowaleuskii (Enteropneusta). I. The acrosomal region and its changes in early stages of fertilization. J. Cell Biol., 19: 477-500. 1967a Behavior of the spermatozoon during sperm-blastomere fusion and its significance for fertilization (Saccoglossus kowaleuskii: Hemichordata). Z. Zellforsch., 78: 208-220. 1967b Membrane fusion in relation to spermegg association. In: Fertilization. C. B. Metz and A. Monroy, eds. Academic Press, New York, Vol. 1, Chap. VII, 295-367. Dan, J. C. 1967 Acrosome reaction and lysins. In: Fertilization. C. B. Metz and A. Monroy, eds. Academic Press, New York, Vol. 1, Chap. VI, pp. 237-293. Dettlaff, T. A,, and A. S. Ginsburg 1963 Acrosome reaction in sturgeons and the role of Ca-ions in sperm-egg association. Dokl. Acad. Nauk, SSSR, 153: 1461-1464. Franklin, L. 1965 Morphology of gamete membrane fusion and of sperm entry into oocytes of the sea urchin. J. Cell Biol., 25: 81.100. Ginsburg, A. S. 1963 Sperm-egg association and its relationship to t h e activation of t h e egg in salmonid fishes. J. Embryol. exp. Morph., 11: 13-33. Hori, R. 1958 On the membrane potential of the unfertilized egg of the medaka, Oryzias latipes, and changes accompanying activation. Embryologia, 4: 79-91. Itoh, S., and T. Maeno 1960 Resting potential and activation potential of theOryzias egg. I. Response to electrical stimulation. Kumamoto, J. Sci. Ser. B, Sect. 2,5: 100-107. Iwamatsu, T. 1965 Effect of acetone on the cortical changes a t fertilization of t h e egg of t h e medaka, Oryzias latipes. Embryologia, 9: 1-12, 1972 Development of t h e fish egg penetrated by many spermatozoa (in Japanese with English abstract). Zool. Mag., 81: 146-149. Kille, R. A. 1960 Fertilization of the lamprey egg. Exptl. Cell Res., 20: 12-27. Kusa, M. 1950 Physiological analysis of fertilization in t h e egg of t h e salmon, Oncorhynchus keta. I. Why are the eggs not fertilized in isotonic Ringer solution? Annot. Zool. Japon., 24: 22-28. 1953 Physiological analysis of fertilization in the egg of t h e salmon, Oncorhynchus keta. 11. Signifi-

FERTILIZATION IN FISH EGGS cance of the cortical change in the initiation of development of the salmon egg. Annot. Zool. Japon., 26: 73-77. Longo, F. J. 1976 Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs. Develop. Biol., 49: 347-368. Longo, F. J., and E. Anderson 1968 The fine structure of pronuclear development and fusion in the sea urchin, Arbacia punctulata. J. Cell Biol., 39: 339-368. 1969 Cytological aspects of fertilization in the lamellibranch, Mytilus edulis. 11. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes. J. Exp. Zool., 172: 97-120. 1970 An ultrastructural analysis of fertilization in the surf clam, Spisula selidissirna. 11. Development of the male pronucleus and t he association of t he maternally and paternally derived chromosomes. J. Ultrastruct. Res., 33: 515-527. Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414. Maeno, T., H. Morita and M. Kuwabara 1956 Potential measurements on the eggs of Japanese killi-fish, Oryzias latipes. Mem. Fac. Sci. Kyushu Univ., Ser. B, 2: 87-94. Nakano, E. 1969 Fishes. In: Fertilization, C. B. Metz and A. Monroy, eds. Academic Press, New York, Vol. 2, Chap. VII, 259-324. Niijima, L., and J. C. Dan 1965 The acrosome reaction in Mytilus edulis. 11. Stages in the reaction, observed in supernumerary and calcium-treated spermatozoa. J. Cell Biol., 25: 249-259. Pasteels, J. J. 1963 Sur l'origine de la membrane nucleaire du ,pronucleus chez le Mollusque Bivale Barnea candida (Etude a n microscope electronique). Bull. Acad. Roy. Sci. (Belgique), 49: 329-336. 1965 Aspects structuraux de la fkondation vus au microscope electronique. Arch. Biol., 76: 463-509. Piko, L. 1964 Mechanism of sperm penetration in the rat and the Chinese hamster based on fine structural studies. Proc. Vth Int. Congr. Anim. Reprod. Trento, 7: 301-302. 1967 Immunological phenomena in the reproductive process. Int. J. Fertil.. 12: 377-383. 1969 Gamete structure and sperm entry in mammals. In: Fertilization. C. B. Metz and A. Monroy, eds. Academic Press, New York, Vol. 2, Chap. VIII, 325-403. Piko, L., and A. Tyler 1964 Fine structural studies of sperm

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penetration in the rat. Proc. Vth Int. Congr. Anim. Reprod. Trento, 2: 372-377. Privolnev, T. I. 1941 Fertilization of fish in the absence of water. Bull. Inst. Freshw. Fish., Leningrad, 24: 54-59 (in Russian). Sakai, Y. 1961 Method for removal of chorion and fertilization of the naked egg in Oryzias latrpes. Embryologia, 5: 357-368. 1964a Studies on the ooplasmic segregation in the egg of the fish, Oryzias latips. Embryologia, 8: 129-134. 1964b Studies on the ooplasmic segregation in the fish, Oryzias latipes. 11. Ooplasmic segregation of the partially activated egg. Embryologia, 8: 135-145. 1976 Spermiogenesis of t h e teleost, Oryzias latzpes, with special reference to the formation of flagellar membrane. Develop. Grow. Different., 18: 1-13. Stanley, H. P. 1969 An electron microscope study of spermiogenesis in the teleost fish,Oltgocottus maculosus. J. Ultrastruct. Res., 27: 230-243. Stefanini, M., C. Qura and L. Zamboni 1969 Ultrastructure of fertilization in the mouse. 11. Penetration of sperm into the ovum. J. Submicroscop. Cytol., 1: 1-23. Szollosi, D. G., and H. Ris 1961 Observations on sperm penetration in the rat. J. Biophys. Biochem. Cytol., 10: 275-283. Tyler, A. 1965 The biology and chemistry of fertilization. Am. Nat., 99: 309-334. Uwa, H. 1967 An study on relationship between sperm penetration and egg activation in the medaka, Oryzias latipes. J. Fac. Sci. Shinshu Univ., 2: 87-94. Yamamoto, T. 1961 Physiology of fertilization in fish eggs. Intern. Rev. Cytol., 12: 361-405. Yanagimachi, R., and Y. D. Noda 1970a Ultrastructural changes in the hamster sperm head during fertilization. J. Ultrastruct. Res., 31: 465-485. 1970b Physiological changes in the postnuclear cap region of mammalian spermatozoa: A necessary preliminary to the membrane fusion between sperm and egg cells. J. Ultrastruct. Res., 31: 486-493. 1970c Electron microscopic studies of sperm incorporation into the golden hamster egg. Am. J. Anat., 128: 429-462. Zamboni, L., and L. Mastroianni 1966 Electron microscopic studies on rabbit ova. 11. The penetrated tuba1 ovum. J. Ultrastruct. Res., 14: 118-132.

PLATE I EXPLANATION OF FIGURES

1 , 2 A spermatozoon on the vitelline surface. A sperm head has been caught by microvilli of t h e egg ten seconds after insemination. Note no fusion between sperm and egg plasma membranes (fig. 2). Figure 1 x 28,000; figure 2 x 56,000.

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PLATE 1

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PLATE 2 EXPLANATION OF FIGURES

3, 4 A spermatozoon surrounded by microvilli and a cytoplasmic protrusion (fertilization cone, FC; see insert.) of the egg ten seconds after insemination. The sperm tail (ST)is also trapped by microvilli (MV). Note no fusion between egg and sperm plasma membranes (fig. 4). Figure 3 x 16,800; figure 4 x 56,000; insert X 250.

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PLATE 2

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PLATE 3 EXPLANATION OF FIGURES

5 , 6 Spermatozoa enclosed by cytoplasmic protrusions of the eggs one minute after insemination, indicating the partial fusion (arrows) of the plasma membrane with that of the egg. Figure 5 X 28,000; figure 6 x 28,000.

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PLATE 3

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PLATE 4 EXPLANATION OF FIGURE

7 Position of a spermatozoon enclosed within a cave-like structure, which is opened from the sperm head (HIt o a pit (arrow) on the vitelline surface. This sperm head is the same one as in figure 5. X 5,600.

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PLATE 5 EXPLANATION OF FIGURE

8 A spermatozoon deeply engulfed inside the electron dense layer (EDL) of cortical cytoplasm of the egg three minutes after insemination. The sperm head (HIand sperm flagellum (STI have been incorporated in the vicinity of the cave-like structure open to the viteliine surface (arrow). VS, vitelline surface. X 3,360.

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PLATE 6 EXPLANATION OF FIGURES

9, 10 An early stage in the dispersal of t h e sperm chromatin three minutes after insemination. Vesiculation (V) of the nuclear envelope and dispersion (arrows) of the chromatin of a spermatozoon in figure 9 begin a t the apical region of the sperm head. The sperm nucleus a t a more advanced stage (fig. 10) of dispersing chromatin has an intact nuclear envelope surrounding t h e centriole (0.Figure 9 X 14,000; figure 10 X 16,800.

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PLATE 6

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PLATE 7 EXPLANATION OF FIGURES

11, 12 The early male pronucleus five minutes after insemination. Long or flattened vesicles frequently containing minute vesicles (arrows) have accumulated along the dispersed chromatin with the centriole (CJ and sperm mitochondria (SMT). I n figure 12, a double membrane is almost formed. MT, microtubule. Figure 11 x 28,000; figure 12 x 28,000.

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PLATE 7

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PLATE 8 EXPLANATION OF FIGURE

13 Male pronucleus fifteen minutes after insemination. The nucleus with a small nucleolus (NO) has a continuous nuclear envelope. x 5,600.

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PLATE 8

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PLATE 9 EXPLANATION OF FlGlJRE

14 Fully grown male pronucleus with a large nucleolus 25 minutes after insemination. The arrow indicates a pit on t h e cortical region which has poor or no electron dense layer. X 5,600

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PLATE 9

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Electron microscopic observation on sperm penetration and pronuclear formation in the fish egg.

Electron Microscopic Observation on Sperm Penetration and Pronuclear Formation in the Fish Egg T. IWAMATSU AND T. OHTA Department of Biology, Aichi Un...
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