Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg

Transcription

1 Develop. Growth & Differ., 30 (6), (1988) Sperm Incorporation Independent of Fertilization Cone Formation in the Danio Egg (Brachy danio / fertiliza tion / fertilizat ion cone / fish Ringer s solution) JOSEPH S. WOLENSKI AND NATHAN H. HART Department of Biological Sciences, Nelson Biological Labs-Busch Campus, Rutgers Universiy, P. 0. Box 1059, Piscataway, N. J. U.S.A The responses of the egg to insemination in a modified Fish Ringer s solution (FRS) were examined in eggs of the zebrafish (Brachydanio rerio) primarily by scanning electron microscopy. FRS is a physiological saline which temporarily inhibits parthenogenetic activation of the egg for 5-8 min. Spermatozoa were collected in a small volume of water and pipetted over eggs in FRS. Eggs inseminated in FRS typically incorporated the fertilizing sperm within 3-4 min. Inseminated cells showed an absence of a fertilization cone and no cortical granule exocytosis. The deep conical depression in the egg surface beneath the micropyle remained unaltered. Control eggs inseminated in tank water developed a large fertilization cone during sperm incorporation. Occasionally, eggs inseminated in water were observed to incorporate the entire sperm head prior to egg activation. Our results corroborate earlier findings showing that in the zebrafish, cortical granule exocytosis, fertilization cone formation and elevation of the sperm entry site are not triggered by the fertilizing sperm in experimental conditions (18, 19). Furthermore, sperm incorporation requires neither egg activation nor formation of a fertilization cone in this fish. INTRODUCTION The fertilization cone is an egg cytoplasmic projection that has generally been regarded as the vehicle responsible for sperm incorporation (4, 12, 15, 16, 17). Studies on several animal species, particularly echinoderms, have shown that the fertilization cone forms at any site on the egg surface which fuses with a sperm (12, 15, 16, 17). In inseminated eggs of teleost fish, by contrast, the fertilization cone develops at a predetermined site beneath the micropyle and gradually engulfs the bound spermotozoan (7, 8, 9, 10, 11, 13). Several electron microscope studies have provided a detailed morphological analysis of sperm incorporation into the teleost egg cortex (6, 8, 9, 14). Unfortunately, the precise role of the fertilization cone in the process of sperm incorporation remains unclear. A difficulty in analysis of the functional relationship between fertilization cone formation and sperm incorporation in some species is an apparent lack of synchronization between the onset of fertilization cone growth and the movements of the fertilizing sperm cell into the egg cytoplasm. In Oryzias (7) and Rhodeus (14), for example, the entire sperm head is incorporated into the egg cortex prior to the development of a true fertilization cone. By contrast, the fertilization cone develops either prior to or concomitant with sperm incorporation in eggs of Cyprinus (9, ll), Plecoglossus (lo), Oncorhynchus (8) and Bruchydunio (18). We have previously shown in Bruchydunio that fertilization cone formation is initiated 619

2 620 J. s. 'NOLENSKI AND N. H. HART shortly after binding of the fertilizing sperm to the egg plasma membrane (18). Other experiments have shown that the fertilization cone will form in its natural spawning medium independently of sperm binding and incorporation (18). This suggests that the fertilization cone is a natural response to egg activation and may not be required for sperm incorporation. To test this possibility, eggs of the zebra danio were inseminated in Fish Ringer's solution (FRS) and examined at selected time intervals postinsemination using primarily scanning electron microscopy. Eggs stripped from the female fish directly into FRS normally show little evidence of activation for 5-8 min (18). Our results indicate that FRS-immersed eggs incorporate the fertilizing sperm, but fail to develop a characteristic fertilization cone. MATERIALS AND METHODS Adult fish were maintained in aquaria at 26'C on a 12-hr light and 12-hr dark photoperiod. Gametes were obtained from male and female fish according to techniques described by Hart and Messina (5). Experimental eggs were fertilized at room temperature in Fish Ringer's solution (FRS) modified after Ginsburg (3). The FRS per liter consisted of 7.5 g NaCI, 0.5 g MgS04, 0.5 g Na2HP04, 0.25 g KCL, 0.2 g NaHC03, and 0.3 g CaC1,. Approximately unactivated eggs were collected in a watchglass (25 mm) containing 2-3 ml of FRS. An aliquot of 8-10 eggs was quickly pipetted into water and examined under the light microscope to determine if they were mature and capable of activation. Criteria of activation included cortical granule breakdown and elevation of the chorion. Sperm were then collected from a single male in 0.1 ml of water and pipetted over the FRS-immersed eggs. The gamete suspension was then gently swirled. Aliquots of eggs were then removed at selected time intervals and fixed in 2% glutaraldehyde (4 C) in 0.1 M cacodylate buffer (ph 7.4) for scanning electron microscopy (SEM). The remaining eggs were removed from the FRS at 5 min postinsemination, rinsed in 50 ml of water, and observed under the light microscope for evidence of first cleavage furrow formation. SEM observations of experimental conditions were made on 126 eggs collected from four different female fish. Control experiments included eggs that were collected in ml of FRS and inseminated with sperm collected in 0.1 ml of water. The gamete suspension was then gently agitated for 10 sec and immediately diluted with an additional 5-10 ml of water. During the next minute of development, the eggs were rinsed twice with 5 ml of fresh water. Observations were also carried out on eggs collected in water and immediately mixed with water-collected sperm. All control eggs were then fixed at selected time intervals after insemination for study with either SEM or transmission electron microscopy (TEM). Preparation of cells for SEM and TEM was carried out according to previously published methods (18, 19). RESULTS The general organization of the sperm entry site and the morphology of sperm incorporation have been previously described in the danio egg (18, 19). Therefore, we will only describe briefly the site of sperm entry and the events that follow insemination of the zebrafish egg in tap water. 1. Observations on unactivated eggs The funnel-shaped micropylar apparatus of the chorion was located in the center of the - Fig. 1 TEM through the sperm entry site of an unactivated egg. The micropyle of the chorion has a single aperture which is centered around the sperm entry site microvilli (arrows). An electron dense homogeneous band (arrow heads) is located beneath the cell membrane at the base of the conical depression. X5,400. Fig. 2 SEM of the conical depression in an unactivated naked egg (chorion is removed). The sperm entry site (arrow) is visible as a tuft of microvilli at the base of the depression. pb, polar body. X 1,800.

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4 622 J. S. WOLENSKI AND N. H. HART animal pole and closely approximated the conical depression in the egg surface (Fig. 1). The conical depression was typically um deep, and um across at its upper margin (Figs. 1,2). The cluster of microvilli forming the sperm entry site was located at the base of the egg surface depression (Figs. I, 2). A thin band of electron-dense, homogeneous material was visible subjacent to the plasma membrane lining the lower half of the conical depression (Fig. 1). The first polar body was located approximately 40 um from the sperm entry site (Fig. 2). 2. Observations on fertilized control eggs Eggs collected in water activated parthenogenetically and developed a fertilization cone beneath the elevated micropyle. Our experience indicated that the sperm suspension must be added to water-collected eggs within 30 sec in order for successful fertilization to occur. By contrast, eggs collected in FRS could be maintained for 5-8 min at room temperature before showing evidence of activation. When FRS-collected eggs were inseminated and immediately rinsed with excess water, they developed in similar fashion to eggs collected and inseminated in water. Activation of control, inseminated eggs was first detected between sec after the mixing of gametes with the onset of the cortical reaction and initiation of elevation of the chorion. By sec postinsemination, a distinct cytoplasmic swelling or fertilization cone was formed at the base of the conical depression (Fig. 3). The head of the fertilizing sperm was located at the apex of the fertilization cone (Figs. 3, 4). By this time, fusion had occurred between the plasma membranes of the interacting gametes and the sperm nucleus appeared to be in contact with the homogeneous, electron-dense band subjacent to the egg plasmalemma (Fig. 4). The homogeneous band beneath the plasma membrane limiting the fertilization cone appeared to be substantially thicker than the same band beneath the sperm entry site of the unactivated egg; it measured about nm in thickness (compare Fig. 4 to Fig. 1). Continued growth of the fertilization cone was accompanied by a gradual, upward movement of the conical depression. By 3-4 min postinsemination, the fertilization cone and the conical depression were no longer visible as landmarks in the animal pole. Images from SEM and TEM confirmed that the head and midpiece of the sperm were fully incorporated into the egg cortex by this time. During our studies of eggs collected in FRS and inseminated in water, approximately 5% of the eggs were observed in which activation was not detected until sec postinsemination. Sections prepared through the sperm entry site of one of these eggs fixed at 75 sec postinsemination showed the cortical granules to be intact and the micropyle closely associated with the oolemma (Fig. 5). The same sections showed clearly that the fertilizing sperm was completely incorporated into the egg cytoplasm (Fig. 6). However, very little cytoplasmic upheave1 was detected at the base of the conical depression (Fig. 6). 3. Observations on eggs inseminated in FRS with water-collected sperm Eggs collected in 3 ml of FRS and inseminated with 0.1 ml of water-collected sperm showed no evidence of activation during the 5 min observation period. Sperm were motile in this medium since SEM views of fertilized eggs showed incorporation of the sperm head within 3-4min of preparing the gamete suspension. As in control cells, binding of the

5 SPERM ENTRY INTO THE UNACTIVATED EGG 623 Figs Control eggs were collected in FRS and inseminated with sperm in water. The gamete mixture was then swirled for 10 sec and immediately diluted with an additional 10 ml of water. Fig. 3 Control egg showing characteristicgrowth of a large fertilization cone (fc) beneath the sperm head (s). Note the growth of the cone from within the surrounding depression. Fixed 1 min postinsemination. X 6,000. Fig. 4 TEM of a typical control egg fixed at 1 min postinsemination. The fertilization cone (fc) measures about 4-5,urn in height and 12,urn across at its base. Note the homogeneous band (arrow heads) beneath the cell membrane. s, sperm head. x 5,600. Fig. 5 Light micrograph of a control egg which has completely incorporated the sperm head (arrow) prior to activation. Although this egg was fixed at 90 sec postinsemination, the conical depression is unchanged and there is no evidence of a fertilization cone. Note that the chorion (c) closely approximates the oolemma and the cortical granules (arrow heads) are not exocytosed. x 1,200. Fig. 6 TEM of the same egg as in Fig. 5. The sperm nucleus (s) is beneath the egg cell membrane. The sperm entry site cytoplasm has elevated approximately 750 nm and formed a small bleb around the nucleus. X 15,000.

6 624 J. S. WOLENSKI AND N. H. HART fertilizing sperm in some cells occurred within 15 sec of the mixing of gametes. By 60 sec postinsemination in these eggs, the microvilli of the sperm entry site were no longer distinct and had presumedly fused with the sperm plasma membrane (Fig. 7). Compared to control eggs, there was no evidence of fertilization cone formation and no detectable movement of the sperm into the cytoplasm (compare Fig. 7 to Figs. 3,4). Between 1-3 rnin postinsemination, the sperm head and midpiece of experimental cells were partially incorporated into the egg cytoplasm (Fig. 8). The sperm head in these preparation appeared slightly swollen, and its plasma membrane was thrown into several short, stubby ridges (Fig. 8). Between 3-5 rnin postinsemination, the entire sperm head and midpiece became fully incorporated into the egg cytoplasm; the site of sperm entry was marked by a portion of the sperm flagellum (Fig. 9). At this time, the conical depression appeared unchanged from the unactivated state, and was still visible as a prominent invagination in the egg surface (Fig. 10). There was no evidence of cortical granule exocytosis or formation of a fertilization cone (Figs. 9, 10). Eggs fertilized and maintained in FRS failed to undergo cleavage. Indeed, such cells frequently showed leakage of the cytoplasm through the micropylar canal after minutes in the FRS. When fertilized eggs were rinsed in 20 ml of water after 5 min in FRS and examined under the light microscope, they showed cortical granule exocytosis, formation of a cytoplasmic cap, and cleavage furrow formation. The first cleavage furrow typically formed min postinsemination or some 5-10 rnin after the control eggs. DISCUSSION The site of entry of the fertilizing sperm into the egg undergoes dramatic changes in organization upon insemination. Typically, a prominent cytoplasmic projection or fertilization cone forms beneath the region of fusion between male and female gametes (9,11, 17, 18). Studies with invertebrate eggs treated with cytochalasins show inhibition of both fertilization cone growth and sperm incorporation (4, 12, 15, 16). This suggests that fertilization cone growth is necessary for normal entry of the sperm into the egg cytoplasm. The teleost egg offers a unique opportunity to examine the functional relationship between the fertilization cone and movement of the sperm into the egg. Immersion of zebra danio eggs in natural spawing medium (water) triggers several morphological changes at the surface associated with activation, including cortical granule exocytosis, chorion elevation and fertilization cone formation (18, 19). By contrast to observations on Oryzius (7) and Figs Experimental eggs were collected in 3 ml of FRS and fertilized with sperm in 0.1 ml water. Eggs were maintained in FRS and gently agitated until fixation. All are SEM images of the sperm entry region in naked eggs. Fig. 7 A sperm cell (s) fused to the sperm entry site microvilli (arrow) of an egg fixed 60 sec postinsemination. x 10,000. Fig. 8 The site of sperm entry in an egg fixed 3 rnin postinsemination. The sperm head is partially incorporated into the egg cortex and is covered with short microvilli-like projections (arrows). No fertilization cone is visible. f, sperm flagellum. x 10,000. Fig. 9 The sperm entry site in an egg fixed 4 min postinsemination. The sperm head, no longer visible, is completely incorporated into the egg cortex. Note that the site of incorporation is flattened and populated with microvilli. No fertilization cone is visible. f, sperm flagellum. ~5,000. Fig. 10 A low magnification view of the animal half of the same egg as in Fig. 9. Note that the conical depression (arrow) is still visible and that there is no evidence of cortical granule exocytosis. The sperm flagellum is visible extending from the base of the conical depression. X 1,200.

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8 626 J. S. WOLENSKI AND N. H. HART Cyprinus (11), the formation of the fertilization cone does not require sperm binding and its growth is not dependent upon the rate of gamete membrane fusion. When the danio egg is inseminated in water, the fertilizing sperm binds to a cluster of microvilli on the egg plasma membrane and is gradually internalized at the apex of a developing fertilization cone. Temporally, therefore, cone growth and sperm incorporation under natural conditions are coupled events. Results of the present study show that incorporation of the fertilizing sperm can occcur independently of egg activation and formation of the fertilization cone. When danio eggs are immersed in physiological saline (FRS) and inseminated, there is little evidence of either fertilization cone formation or cortical granule breakdown. This confirms prior experiments showing that the fertilizing sperm is not required for danio egg activation (18, 19). However, these same eggs consistently show incorporation of the sperm head and midpiece, although movement into the cytoplasm appears to be temporally delayed by 1-2 minutes when compared to control cells. Sperm incorporation in the absence of activation has previously been reported in salmonid fish eggs inseminated in saline (3). By contrast, Oryzias eggs fertilized in saline will activate, form a fertilization cone, and develop to the blastula stage (7). It was not possible to trace the development of FRS-inseminated danio eggs because they begin to lyse after minutes. However, if these eggs are removed from FRS at 5 minutes and rinsed in water, their development through the first cleavage appears similar to control cells. Sperm incorporation into the danio egg is undoubtedly related to complex and rapid changes in the cytoskeleton at the site of sperm entry. There is some evidence for the presence of an actin-based cytoskeleton in this region of the teleost egg (8, 18, 19). Filaments measuring about 8 nm in diameter have been identified at the sperm entry site of danio eggs (18, 19). They are visible in the microvilli to which the fertilizing sperm binds and in an electron-dense band of material subjacent to the plasma membrane in this part of the cell. Actin filaments have also been observed at the sperm entry site of chum salmon eggs (8). Also, treatment of eggs with cytochalasins either before or after insemination blocks sperm incorporation in a concentration-dependent manner (19). The mechanism of how actin filaments mediate the movement of the sperm into the cytoplasm remains to be elucidated. However, it is likely that alterations in the cytoskeleton leading to sperm incorporation are highly localized. Little movement of the cortical cytoplasm appears required for sperm entry. This is also suggested in studies of Fundu~~ (1,2), Oncorhynchus ), and Rhodeus (14) eggs in which the initial stages of sperm incorporation appear to take place with little upheaval of the cortical cytoplasm. Furthermore, the fertilizing sperm is capable of triggering these reorganizational changes quite independently of egg activation. One important function of the fertilizing sperm, therefore, may be to directly initiate the changes in the egg cortex that result in its internalization. The entry of the fertilizing sperm into the danio egg in the absence of the formation of a fertilization cone raises an interesting question. What is the role(s) of the fertilization cone in the development of the teleost egg? In the zebrafish, the cone may act as a catalyst and expedite the movement of the sperm into the cytoplasm. As shown in this study, eggs which are inseminated in water develop a fertilization cone and generally incorporate sperm more rapidly than eggs inseminated in FRS and lacking a fertilization cone. The possibility is also

9 SPERM ENTRY INTO THE UNACTIVATED EGG 627 raised that the cytoplasmic movements involved in cone formation may play a role in bringing about the approximation of male and female pronuclei. Finally, there is evidence in several teleost species that the fertilization cone swells into the micropylar canal following successful sperm binding and functions as a block to polyspermy (9, 10, 11, 13, 14). This does not appear to be the case in eggs of Brachydanio (18) and Oryzias (7). As in eggs of other animal species (20), the reorganization of the teleost egg at the site of gamete union is complex. Further studies employing techniques designed to probe membrane structure and the arrangement of the actin cytoskeleton before and after activation should provide additional insight into the mechanism(s) of fertilization cone growth and the movement of the fertilizing sperm into the cortex. J. S. Wolenski is a predoctoral fellow sponsored by the Charles and Joanna Busch Memorial Fund. This research was supported in part by NIH grant HD to N. H. H and monies from the Leathem Fund of Rutgers University to J. S. W. 1. REFERENCES Brummett, A. R., and J. N. Dumont, heteroclitus. J. Exp. Zool., 210: Brummett, A. R., J. N. Dumont and C. S. Richter, Initial stages of sperm penetration into the egg of Fundufus 2. Later stages of sperm penetration and second polar body and blastodisc formation in the egg of Fundulus heteroclitus. J. Exp. Zool., 234: Ginsburg, A Sperm-egg association and its relationship to the activation of the egg in salmonid fishes. J. Embryol. exp. Morphol., 11: Gould-Somero, M., L. Holland and M. Paul, Cytochalasin B inhibits sperm penetration into eggs of Urechk cuupo (Echiura). Develop. Biol., 58: Hart, N. H., and M. Messina, Artificial insemination of ripe eggs in the zebrafish, Brachydanio rerio. Copeia, 2: Iwamatsu, T., and T. Ohta, Electron microscopic observations on sperm penetration and pronuclear formation in the fish egg. J. Exp. Zool., 205: Iwamatsu, T., and T. Ohta, Scanning electron microscopic observations on sperm penetration in teleostean fish. J. Exp. Zool., 218: Kobayashi, W., and T. S. Yamamoto, Light and electron microscopic observations of sperm entry in the chum salmon egg. J. Exp. Zool., 243: Kudo, S., Sperm penetration and the formation of a fertilization cone in the common carp egg. Develop. Growth and Differ., 22: Kudo, S., Response to sperm penetration of the cortex of eggs of the fish, Pfecogfossus aftivefis. Develop. Growth and Differ., 25: Kudo, S., and A. Sato, Fertilization cone of carp eggs as revealed by scanning electron microscopy. Develop. Growth and Differ., 27: Longo, F. J., Effects of cytochalasin B on sperm-egg interactions. Develop. Biol., 67: Ohta, T., Electron microscopic observations on sperm entry into eggs of the bitterling during cross-fertilization. J. Exp. Zool., 233: Ohta, T., and T. Iwamatsu, Electron microscopic observations on spe;rm entry into eggs of the rose bitterling, Rhodeus oceflurus. J. Exp. Zool., 227: Schatten, H., and G. Schatten, Surface activity at the egg plasma membrane during sperm incorporation and its cytochalasin B sensitivity. Develop. Biol., 78: Schatten, G., and H. Schatten, Effects of motility inhibitors during sea urchin fertilization. Exp. Cell Res., 135: Tilney, L. G., and L. A. Jaffe, Actin, microvilli, and the fertilization cone of sea urchin eggs. J. Cell Biol.. 87:

10 628 J. S. WOLENSKI AND N. H. HART Wolenski, J. S., and N. H. Hart, Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg. J. Exp. Zool., 243: Wolenski, J. S., and N. H. Hart, The effects of cytochalasins B and D on the fertilization of zebrafish (Bmchydanio) eggs. J. Exp. Zool., (In press) Yonemura, S. and I. Mabuchi, Motility and the Cyto., 7: Wave of cortical actin polymerization in the sea urchin egg. Cell (Received April 18, 1988; accepted June 18, 1988)