Initial stages of sperm-egg fusion in the freshwater teleost Rhodeus ocellatus ocellatus.код для вставкиСкачать
THE ANATOMICAL RECORD 229:195-202 (1991) Initial Stages of Sperm-Egg Fusion in the Freshwater Teleost, Rhodeus ocellatus ocellatus TADAYUKI OHTA Department of Biology, Aichi University of Education, Kariya City, Aichi 448, Japan ABSTRACT The morphology of spermatozoa and the initial stages of spermegg fusion a t fertilization were investigated ultrastructurally in the rose bitterling, Rhodeus ocellatus ocellatus. Each spermatozoon is composed of a spherical head without a n acrosome, two centrioles, a large mitochondrion, and a flagellum. Freeze-fracture of spermatozoa illustrates that specialized arrays of intramembranous particles (IMPS) are present on the protoplasmic facing (PF) surface of the head plasma membrane a t the portion slightly in front of the centrioles. The specialized arrays, whose functions are uncertain, are parallelogram-like in shape. The distribution of the particles is random and less compact in other areas of the head plasma membrane. The number of particles on the PF surface is larger than that on the extracellular facing (EF) surface. The complementary structures of the specialized arrays are also found on a similar portion of the EF surface. An ultrastructural study clearly shows fusion of gamete plasma membranes a t the initial stages of sperm entry into the egg. Membrane fusion is first observed in eggs fixed 10 seconds after insemination in fresh water. The fusion site is the microvillus membrane of a sperm entry site on the egg and the head membrane of the spermatozoon. The plasma membrane fusion of gametes is discussed relative to the distribution of the IMPs and the fusion site. Fertilization is a phenomenon of cell fusion. The primary morphological indication of sperm entry into the egg is the fusion of the involved plasma membranes. Consequently, the fertilizing spermatozoon carries a male nucleus with some cytoplasmic components into the egg. In marine invertebrates and mammals, initial stages of sperm-egg fusion have been investigated by electron microscopy. The results showed that the membrane fusion site of spermatozoa is restricted within specially fixed portions (Colwin and Colwin, 1967; Yanagimachi and Noda, 1970; Bedford and Cooper, 1978). Recently, a distribution of intramembranous particles (IMPs) of the sperm plasma membrane in various animals has been investigated by freeze-fracture (the millipede, Reger and Fitzgerald, 1979; the zebrafish, Kessel et al., 1983; the ascidian, Rosati, 1985; the horseshoe crab, Tilney, 1985; the mammal, see Toyama and Nagano, 1988). From the relationship between the fusion site of the plasma membrane and the distribution of the particles, i t was shown that the membrane fusion site is particle-free (cf. Yanagimachi, 1988). A teleostean fish egg is enveloped by a thick egg membrane (chorion) with a micropyle. As the fertilizing spermatozoon attaches to the egg plasma membrane (of the sperm entry site) directly beneath the micropyle, the fusion site of the egg plasma membrane is destined to become the sperm entry site. Though plasma membrane fusion also occurs in fish gametes, the initial stages of the fusion event have not been researched so far. The first objective of this study was to obtain decisive ultrastructural evidence of the initial stages of gamete c 1991 WILEY-LISS, INC membrane fusion in teleostean fish. The second was to investigate the distribution of the IMPs of fish spermatozoa. MATERIALS AND METHODS Adult male and female rose bitterlings, R. ocellatus ocellatus, were purchased from a fish farmer in Toyohashi City. They were kept in glass containers with freshwater bivalves under conditions of light 14 hours and dark 10 hours a t 20-23°C. Mature eggs were obtained by manually pressing the abdomens of females having a lengthened ovipositor. Mature spermatozoa were obtained from males by a similar method. Unfertilized eggs kept in physiological saline were washed with water (pH adjusted to 7.2 by Nil0 NCl) which was boiled,once and treated with a sperm suspension (5 x 10- concentration). At various time intervals (1,3, 5, 10 seconds) after the start of insemination, the eggs were fixed with modified Karnovsky’s fixative containing 3%sucrose for 4 hours a t 4°C. After being washed with 5% sucrose-0.1 M phosphate buffer (pH 7.2), they were postfixed with 1% OsO4-3g sucrose-0.1 M phosphate buffer for 1.5 hours a t 4°C. For scanning electron microscopy (SEM), some of the eggs were dehydrated in a n alcohol series followed by isoamylacetate and then dried with liquid CO, in a critical point dryer (Hitachi, HCP-2). The dried eggs were coated with gold by ion sputter (JEOL, JFC-1100) *’ Received March 16, 1990; accepted J u n e 26, 1990 196 T. OHTA and examined with a JEOL, JEM-100B electron microscope with attached scanning devices. For superficial observations of spermatozoa, those in physiological saline were put on a thin glass coated with 0.1% polylysine for 15 minutes. They were fixed with modified Karnovsky's fixative containing 3% sucrose then treated similar to the eggs except for the use of goldpalladium instead of gold. For transmission electron microscopy, some of the eggs were dehydrated in a n alcohol-acetone series and were embedded in Quetol 812 (Nisshin EMco., Tokyo). Ultra-thin sections were stained with uranyl acetate and lead citrate, then examined with JEOL, JEM-100B, and JEM-2000FX electron microscopes. Testes fixed for 4 hours with modified Karnovsky's fixative were washed with 0.1 M phosphate buffer (pH 7.2) and kept in 30% glycerin-0.1 M phosphate buffer for about 2 hours. The testes were frozen in degassed liquid nitrogen and fractured at about - 125"C, using a freeze-fracture apparatus (JEOL, JFD-7000). Platinumicarbon followed by carbon was coated onto the fractured surfaces of the testes. Replica membranes were separated from the testies in bleach, rinsed in distilled water, mounted on 150 mesh grids and observed with the electron microscopes. RESULTS Sperm Morphology Each spermatozoon of the rose bitterling is composed of a head, a middle piece, and a tail (Fig. 1).The spermatozoa have no acrosome structure in the head (Fig. 1A). Each has a large mitochondrion and centrioles in the middle piece (Fig. 1).The proximal portion of the flagellum is surrounded by plasma membrane (sleeve, Fig. l A , S). SEM observations showed that the superficial structures of the round shaped heads were smooth without remarkable characters (Fig. lB,C). The distribution of IMPs in the sperm plasma membrane was examined by freeze-fracture. Each portion, the head, middle piece and flagellum, of each fractured spermatozoa was easily differentiated (Figs. 2,3). Specialized arrays, like the parallelogram shapes of IMPs on the protoplasmic facing (PF) surfaces, were seen in a portion of sperm heads on the flagellar side shown in Fig. 1A,B (Fig. 2A-D, arrows). The specialized arrays of IMPs were positioned slightly in front of the centrioles. The extent of their distribution varied depending on individual differences in spermatozoa (Fig. 2A-C). The average size of the IMPs was about 11.7 i 3.5 nm (means of 54 particles 2 standard deviation). The number of the IMPs per 0.25 km2 was about 43 2 8 (means of 130 areas i standard deviation) on the P F surface of Fig. 1. Spermatozoa of R. ocellatus ocellatus. A: An electron micrograph of a spermatozoon. C, centriole (basal body); CF, centriolar fossa; F, flagellum; M, mitochondrion; N, nucleus; S , sleeve. x 27,000. B: A S E M photo of a spermatozoon viewed from the flagellar side (mof A ) . F, flagellum; H, head. x 30,000. C: A S E M photo of a spermatozoon viewed from the mitochondrial side ( m o f A). H, head; MP, middle piece. x 30,000. Fig. 2. Freeze-fracture replicas of the PF surface plasma membranes of spermatozoa. A,B:These photos show the distribution of the IMPS on the sperm membrane viewed from the flagellar side. Specialized arrays (arrows) of the IMPs were found on the sperm head slightly in front of centrioles. A ) x 34,000. B) x 42,500. C: The distribution of IMPs on the apical-side membrane of the sperm head. x 24,000. D Higher magnification ofthe specialized arrays of the IMPs. x 120,000. E: The distribution of the IMPs on the head and middle piece membranes viewed from the mitochondrial side. Note that they are scattered. x 34,000. SPERM-EGG FUSION Fig 2 197 198 T. OHTA Flg. 3. Freeze-fracture replicas of t h e E F surface plasma membranes of spermatozoa. A Parallelogram-like structures (arrows) were found on the complementary portion of t h e PF surface. x 40,000. B: Higher magnification of t h e parallelogram-like structures. x 102.000. C: The distribution of the IMPS on the head and middle piece membranes viewed from the mitochondria1 side. There a r e fewer IMPS on the E F surface than on the PF surface. x 50,000. 199 SI’E IO-EGC; FUSION TABLE 1. Number of eggs with a spermatozoon at various time intervals after insemination Ems Insemination time (seconds) 1 3 5 10 No. of With Without 146 65 93 54 sperm 3 31 68 52 sperm 129 34 25 2 eggs Unclear 14 0 0 0 * the head and about 41 8 on the middle piece. No significant difference in number was recognized among the different areas of the head and middle piece. There were no specialized arrays of the IMPs on the mitochondrial side shown in Fig. 1A,C (Fig. 2E). Complementary structures (Fig. 3B) of the specialized arrays of the IMPs were observed on the extracellular facing (EF) surface in similar positions to the specialized arrays on the PF surface (Fig. 3A,C). Fewer IMPs were observed on the E F surface than the PF (Figs. 2 , 3). Initial Stages of Sperm-Egg Fusion In order to identify the membrane fusion between sperm and egg, inseminated eggs had to be sectioned along the micropyle and then examined by a transmission electron microscope. It was necessary first to investigate the timing of insemination to increase the probability of observing a fusion event. Accordingly, the presence of sperm a t a sperm entry site on the plasma membrane directly beneath the micropyle was examined in eggs fixed at l - 10 seconds after the start of insemination. Naturally, the number of eggs with spermatozoa a t the micropyle increased with the increase of insemination time (Table 1).Spermatozoa were observed at the sperm entry site or the micropyle in 50% of the eggs after 3 seconds and nearly all eggs after 10 seconds. As the chorion of rose bitterling eggs is somewhat thin, it was easy to visualize the sperm entry site superficially. In each egg the micropyle was situated in the chorion of the animal pole where the chorion became most hollow. Some microvilli were found a t the sperm entry site (Fig. 4A). In eggs fixed 10 seconds after the start of insemination, fertilizing spermatozoa had attached to the microvilli of the sperm entry sites by their lateral head portions (Figs. 4B-D, 5A-C). Figures 4 and 5 clearly show that each fertilizing spermatozoon entered into the egg by mutual plasma membrane fusion. The membrane fusion site was formed by the sperm entry site microvilli of the egg and the head portion of the spermatozoon (Figs. 4B-D, 5A-C). Plasma Membrane Fusion Site Between Sperm and Eggs We quickly discovered that plasma membrane fusion occurred in the sperm entry site microvilli of the eggs (Figs. 4, 5). Yet, we found it necessary to obtain more information on the precise site of fusion on the spermatozoa. In eggs fixed 30 seconds after the start of insemination, sperm were observed by the SEM to be attached to the sperm entry sites. Fixed eggs were dechorionated to facilitate our observations. Spermatozoa attached to the microvilli of eggs by various portions of their heads. We obtained the following results: sperm attached to the eggs by the flagellar side (see Fig. 1A,B) of their heads in 69 of 99 eggs, by the mitochondrial side (see Fig. 1A,C) in 8 of 99 eggs and by some point in between in 25 of 99 eggs. DISCUSSION Spermatozoa possess a male nucleus and some cytoplasmic components which differ slightly from species to species. Though the spermatozoa of most animals have a n acrosomal structure in the anterior portion of their heads, those of teleostean fish are known to lack the acrosomal structure (cf. Afzelius, 1978; Yanagimachi, 1988). The spermatozoa of the rose bitterling, a teleostean fish, have also been found to lack the acrosoma1 structure (Ohta and Iwamatsu, 1983).Other features of these spermatozoa include the following: 1) they have a large mitochondrion in their middle piece and a sleeve which encircles the root of their flagella, and 2) their flagella project from one side, not the center, of the head when viewed from the tail. Freeze-fracture electron microscopy has shown that the apical region of acrosomal membrane is almost free of IMPs in marine invertebrates (cf. Rosati, 1985; Tilney, 1985). During the acrosome reaction, the fusion of the acrosomal and plasma membranes occurs at a particle-free site. Mammalian spermatozoa are also known to undergo a similar type of fusion (Friend e t al., 1977). Generally, acrosome-reacted spermatozoa can fuse with the egg. The fusion site on the plasma membrane of these spermatozoa has been shown to be particle-free (cf. Longo, 1987; Yanagimachi, 1988). Similarly, in rat peritoneal mast cells, the area of fusion between vesicles and plasma membranes is known to be devoid of all particles (Lawson et al., 1977). These observations suggest that plasma membrane fusion occurs at particlefree sites. Our present observations on freeze-fractured spermatozoa showed the existence of specialized arrays of IMPs in sperm heads. In contrast to the area of the specialized arrays, the IMP distribution in the other areas of the sperm head was not as compact, and a notable particle-free zone was not present. Therefore, it is difficult to suggest a possible membrane fusion site by the distribution of the IMPs in the rose bitterling; a situation different from other animals. The specialized arrays of the IMPs were observed in the sperm head portion in front of the centrioles. Their pattern was parallelogram-like in shape. Kessel et al. (1983) reported a similar structure in zebrafish spermatozoa. They found that unusual arrays of IMPs appeared as simple hexagons or parallelograms and were localized in an equatorial position in the sperm heads. The functional significance of these characteristic arrays of IMPs is quite unclear a t present. In the rose bitterling, it is known that a sperm-stimulating factor is localized in the chorion near a micropyle and attracts spermatozoa to the egg (Suzuki, 1961). It may be that the arrays of IMPs play the role of receptors to the factor, yet, we have no convincing experimental data to support or refute this. Another possibility is that they play an essential role when a spermatozoon attaches to the egg plasma membrane directly beneath the micropyle. Our present observatons on sperm-egg interactions showed many cases in which the fertilizing spermatozoon fused with the egg a t the membrane side 200 rr. OHTA Fig. 4. Sperm-egg fusion in eggs fixed 10 seconds after insemination. A: A SEM photo of a n unfertilized egg. CH, chorion; SS, sperm entry site. x 8,800. B: A SEM photo in t h e vicinity of t he micropyle of a fertilized egg. A fertilizing spermatozoon seems to attach the mi- crovilli of t h e sperm entry site. x 8,800. C, D A fertilizing spermatozoon clearly fused by its head with the membrane of t h e microvilli. C ) x 12,300. D ) x 38,500. SPERM-EGG FUSION Fig. 5 . Another sperm-egg fusion event in an egg fixed 10 seconds after insemination. A A SEM photo of a fertilizing spermatozoon which seems to have attached the microvilli by the sperm head membrane on the flagellar side. x 10,000 B, C: A fertilizing spermatozoon 20 1 fused with the membrane of the microvilli by the sperm head membrane near the centrioles (arrows). MV, microvilli; ch, chorion. B) x 28,000. C) x 52,500. 202 T. OHTA with the arrays of the IMPS. Therefore, the arrays of the IMPS may play a in egg recognition. To “lve these problems, research using immunocytochemical techniques is in progress. A fertilizing spermatozoonpenetratesinto an egg by gamete plasma membrane fusion. The membrane fusion occurs a t the inner aCrOSOma1 membrane covering the acrosomal process in marine invertebrates (Colwin and Colwin, 1967; cf. Yanagimachi, 1988) and in birds (Okamura and Nishiyamaj It Occurs at the postacrosomal region (Yanagimachi and Noda, 1970) or over the equatorial segment (Bedford and Cooper, 1978) in mammals. fishes, membrane fusion was reported in the medaka (Iwamatsu and Ohta, 19781, the rose bitterling (Ohta and Iwamatsu, 1983; Ohta, 1985), Fundulus heteroclitus (Brummett et al., 19851, the common carp (Kudo and 1985)7and the ze(Wolenski and However, data describing the very early stages of gamete membrane fusion have not been presented so far. The present electron microscopic observations showed the following: A spermatozoon primarily contacted the microvilli of a sperm entry site on the egg. 2 ) The sperm plasma membrane fused with the plasma membrane of the microvilli at the sperm head portion. 3) The membrane fusion had Occurred within lo seconds after insemination. In spermatozoa having an acrosomal structure, the structure is located at the tip Of the head’ Just opposite the site Of the centriolar fossa. However, the ’permatozoa of the rose bitterling have a spherical head and no acrosomal structure. In addition, the single mitochondrion does not completely encircle the flagellum, Because Of these morphological expression of “apical” or “lateral” is not SO easy. At present, it is difficult to determine the real fusion site of the sperm head. marine invertebrates, electron microscop~cobservations have shown clear fusion. From the results of these studies, the time from insemination to membrane fusion is known to be less than 9 seconds in annelida and within 7 seconds in hemichordata (colwin and Colwin, 1967). We found that this event in the rose bitterling required a similar amount of time. Though the membrane fusion event had occurred in eggs fixed 10 after insemination, it seemed to begin earlier. In fact, Longo et al. (1986) reported that membrane fusion occurred as quickly as 5 seconds after the onset of sperm-induced conductance increase in sea urchin eggs. However, the exacttirne between primary ‘Ontact Of the spermatozoon and membrane fusion is uncertain in rose bitterling eggs. ACKNOWLEDGMENTS LITERATURE CITED Afzelius, B.A. 1978 Fine structure of the garfish spermatozoon. J. Ultrastruct. Res., 64:309-314. Bedford, J.M. and G.W. Cooper 1978 Membrane fusion events in the fertilization of vertebrate eggs. In: Membrane Surface Reviews. G. Poste and G.L. Nicolson, eds. Elsevier North-Holland, Amsterdam, vol, 5, pp, 65-125. Brummett, A.R., J.N. Dumont, and C.S. Richter 1985 Later stages of sperm penetration and second polar body and blastodisc formation in the egg of Fundulus heteroclitus. J . Exp. zool., 234: 423439. Colwin, L.H., and A.L. Colwin 1967 Membrane fusion in relation to sperm-egg association. In: Fertilization. C.B. Metz and A. Monroy, eds. Academic Press, New York, vol. 1, pp. 295-367. Friend, D.S., L. Orci, A. Perrelet, and R. Yanagimachi 1977 Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa. J. Cell Biol., 74: 561-577. Iwamatsu, T., and T. Ohta 1978 Electron microscopic observation on sperm penetration and pronuclear formation in the fish egg. J. Exp. Zool., 205: 157-180. Kessel, R.G., H.W. Beams, H.N. Tung, and R. Roberts 1983 Unusual particle arrays in the plasma membrane of zebrafish spermatozoa. J. Ultrastruct. Res., 84: 268-274. Kudo, S., and T. Sat0 1985 Fertilization cone of carp eggs as revealed by scanning electron microscopy. Dev. Growth Differ., 27: 121128. Lawson, D., M.C. Raff, B. Gomperts, C. Fewtrell, and N.B. Gilula 1977 Molecular events during membrane fusion. A study of exocytosis in r a t peritoneal mast cells. J . Cell Biol., 72: 242-259. Longo, F.J. 1987 Fertilization. Chapman and Hall, New York, pp. 1-183. Longo, F.J., J.W. Lynn, D.H. McCulloh, and E.L. Chambers 1986 Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus uariegatus. Dev. Biol., 118: 155-166. Ohta, T. 1985 Electron microscopic observations on sperm entry and pronuclear formation in naked eggs of the rose bitterling in polyspermic fertilization. J. E ~zool., ~ 234: . 273-281. Ohta, T., and T. Iwamatsu 1983 Electron microscopic observations on sperm entry into eggs of the rose bitterling, Rhodeus ocellatus. J . Exp. Zool., 227: 109-119. Okamura, F., and H. Nishiyama 1978 Penetration of spermatozoon into ovum and transformation of the sperm nucleus into the male pronucleus in the domestic fowl, Gallus gallus. Cell Tissue Res., 190: 89-98. Reger, J.F., and M.E. Fitzgerald 1979 The fine structure of membrane complexes in spermatozoa of the millipede, Spirobolus sp., as seen by thin section and freeze-fracture techniques. J. Ultrastruct. Res., 67: 95-108. Rosati, F. 1985 Sperm-egg interaction in ascidians. In: Biology of Fertilization. C.B. Metz and A. Monroy, eds. Academic Press, New York, vol. 2 pp. 361-388. Suzuki, R. 1961 Sperm activation and aggregation during fertilization in some fishes. VI. The origin of the sperm-stimulating factor. Annot. Zool. Japon., 34: 24-29. Tilney, L.G. 1985 The acrosomal reaction. In: Biology of Fertilization. C.B. Metz and A. Monroy eds. Academic Press, New York, vol. 2, pp. 157-213. Toyama, Y., and T. Nagano 1988 Maturation changes of the plasma membrane of r a t spermatozoa observed by surface replica, rapidfreeze and deep-etch, and freeze-fracture methods. Anat. Rec., 220: 43-50. Wolenski, J.S., and N.H. Hart 1988 Sperm incorporation independent of fertilization cone formation in the Danio egg. Dev. Growth Differ., 30: 619-628. Yanagimachi, R. 1988 Sperm-ear fusion. Curr. Top. Membr. Transport., 32: 3-43. Yanagimachi, R., and Y.D. Noda 1970 Electron microscopic studies of sperm incorporation into the golden hamster egg. Am. J . Anat., 128: 429-462. I I The author wishes to express his thanks to Dr. T’ Iwamatsu ofthe Department of Biology, Aichi University of Education for reading the manuscript.