Experimental Cell Research 98 (1976) 325-337
THE PENETRATION THE SEA URCHIN
OF THE SPERMATOZOON EGG SURFACE
THROUGH
AT FERTILIZATION
Observations from the Outside on Whole Eggs and from the Inside on Isolated Surfaces G. SCHATTEN Department
ofZoology,
University
and D. MAZIA of California.
Berkeley,
CA 94720, USA
SUMMARY The external and cytoplasmic surfaces of the sea urchin egg at fertilization have been examined with the scanning electron microscope (SEM). The outside events were documented by glueing eggs to polylysine coated glass plates, adding sperm and fixing rapidly. To reveal the inner aspects of the surface as the sperm travels through it to reach the egg cytoplasm. the fertilized egg surface was isolated in 0.3 M KCI, 0.35 M glycine, 2 mM MgCI,, 2 mM EGTA, pH 7.5, glued onto a polylysine-coated plate and processed for the SEM. The events of spermatozoon attachment, membrane fusion, sperm entry, rotation and detachment into the egg cytoplasm as well as the associated cortical changes are described. The egg cortex is revealed to be a uniform network of fibrous bundles. The spermatozoon initially attaches to the egg surface by the acrosomal filament. As membrane fusion occurs between the gametes, the plasma membrane of the egg engulfs the sperm, the cortical granules start to discharge and a spreading surface deformation, possibly caused by a cortical contraction, is initiated. The perpendicularly entering spermatozoon is surrounded by a cluster of elongate microvilli which appear to have 235 nm vesicles associated with their bases. The sperm is prevented by the cortex from directly entering the egg cytoplasm and lies upon the egg surface between the plasma membrane and the matrix of cortical fibers. It is subsequently rotated additionally to enter the egg cytoplasm with the posterior end first. A scar is left in the cortex where the spermatozoon penetrated. The egg cortex is shown to consist of XL-200 nm uniformly arranged fibers, and its thickness ranges from 0.2 to 0.5 pm. It is speculated that this structure may be contractile.
In this work the surface events of fertilization of the sea urchin egg are traced from the attachment of the spermatozoon to the outer surface, through the fusion of the membranes of the gametes, to the appearance of the spermatozoon on the inner surface and the attendant cortical reactions. Some newer methods suitable for making the necessary observations with the scanning electron microscope (SEM) and permitting the observation of both the outer and inner faces of the cell surface are used.
A description of fertilization is synthesized from the accumulated evidence from light microscopy, transmission electron microscopy (TEM) and SEM. Wilson & Learning [I] had described the penetration of the spermatozoon into the cytoplasm and its subsequent rotation prior to nuclear diffusion with evidence that left little for classical light microscopy to add. Longo & Anderson [2], using TEM, have presented the ultrastructural aspects of sperm-egg fusion and the cytoplasmic reacExpd Cell Res 98 (1976)
tions associated with the entry of the spermatozoon. Earlier TEM is summarized by Austin [3]. Tegner & Epel [4] have made the basic observations with the SEM, describing the outer surface of the egg, confirming the presence of the vitelline sheet as the outer coat of the egg surface which is the precursor to the fertilization membrane, and relating the cortical reactions to the detachment of unsuccessful sperm. The present work employs the techniques of affixing eggs to a polylysine coated surface [5] which reduces the area available for sperm penetration and permits rapid transfer of the eggs to fixative after the sperm are introduced. It is possible to follow sperm attachment, membrane fusion and sperm entry because the preparations can be fixed more rapidly than eggs in suspension and because fertilization can take place only on the half of the surface which is viewed in the SEM; the other half is glued to the polylysine-coated plate. The observation of the inner face of the unfertilized egg surface was first performed by Vacquier [6] who developed a technique for shearing eggs which are attached to a solid surface in calcium-free sea water; only the attached surface remains and its inner aspect is visible in the SEM. This method is not suitable for examining the entry of the spermatozoon through the egg surface since the upper half of the egg is sacrificed during the isolation. Furthermore, after fertilization the surface of the cell is separated from the fertilization coat; shearing will then detach the egg. For these reasons, the observations of the events of fertilization as seen on the inner aspect of the cell surface were made on cell surface complexes isolated immediately after insemination. The method of isolation used preserves all the features of the cell surface complex known from elecExprlCe//Res
Xl (1976)
tron microscopy of the whole egg. Other methods of isolation exist in the literature [7, 81 and are generally designated as methods of isolating the ‘cortex’. Here we limit the term ‘cortex’ to the structure underlying the plasma membrane. In the method described in detail below, the eggs are inseminated, quickly transferred to the isolation medium and gently cracked to open up the surface; the entire egg surface is maintained. The surface complex, from which the internal contents of the cell are washed away, is glued to a polylysine-coated plate and fixed. It always attaches by the outer surface, with the inner surface facing upward. As will be shown, it is possible to observe the eruption of the spermatozoon through the inner surface. The observation of the egg cortex after the complete cortical granule discharge presented another sort of technical problem since the hardened fertilization coat is resistant to chemical and physical removal. To overcome this, fertilized eggs were placed in mercaptoethylgluconamide to prevent hardening of this coat. The resulting fragile fertilization coat was subsequently stripped off of the egg by passages through fine mesh silk. These ‘naked’ eggs were then glued to polylysine-coated plates and sheared in the presence of the homogenization buffer. These preparations, without the obstruction of the cortical granules, reveal that the inner surface has a uniform matrix of fibers. This confirms the biophysical evidence [9] that a cortex exists beneath the plasma membrane. MATERIALS
AND METHODS
Gametes from the California sea urchin Strongylocentrotus purpuratus were obtained by intracoelomic stimulation with 0.5 M KCI. The eggs, collected in sea water, were dejellied by three passages through bolting silk. Semen was collected ‘dry’ and diluted in sea water just prior to use.
The egg surface during fertilization The images of the outer surface events were obtained by glueing eggs to polylysine coated plates, adding dilute sperm and transferring the plates to ftxative rapidly. The preparations were fixed with 5% elutaraldehvde in 80% sea water (PH 8.2) for 30 min. The observations of the inner’ events were performed by fertilizing and homogenizing eggs in suspension and then rapidly glueing and fixing the isolated surfaces on polylysine-coated plates. This could be done in 45 sec. The homogenization medium imitates known features of the internal environment of the sea urchin egg; the cationic ratios according to Rothschild & Barnes [IO1 and the use of glvcine for osmotic balance, according io Kavanau [III it contains, however, the calcium ion chelator ethyleneglycol-bis-@aminoethyl ether) NJ’-tetraacetic acid (EGTA) to prevent cortical granule discharge and membrane healing. Thirty seconds after insemination the eggs were washed twice with IO vol of 0.3 M KCI, 0.35 M glycine, 2 mM MgCI,, 2 mM EGTA, pH 7.5 with Na,CO,, hereafter called homogenization buffer. A IO% egg suspension was gently homogenized by one passage of a Teflon pestle; only a single crack in the egg surface is necessary. Egg surfaces were collected by centrifugation (700 g for I5 set), glued onto polylysine-coated plates and rinsed in homogenization buffer. This rinse washes the rest of the adhering cytoplasm from these splayed open inner surfaces. Fixation was performed in 0.5 M KCI, 2 mM M&I,, 2 mM EGTA,. I % acrolein; 2.5 % glutaraldehyde~ pH 7.5 for I5 min. The surface complex of the fertilized egg is more difficult to isolate in suspension and the method of shearing attached eggs was employed. To ensure that the shearing plane in these eggs will be the surface complex, the fertilization coat was stripped off prior to glueing them to the polylysine coated substrate. To do this, fertilized eggs, at the first sign of membrane elevation, were transferred to 0. I % mercaptoethylgluconamide, IO mM ethylenediamine tetraacetic acid (EDTA) in sea water (pH 8.2). The high and very thin fertilization coat was stripped off of the eggs by three passages through bolting silk. These ‘naked’ eggs tend to clump and are fragile; they must be poured rather than pipetted. Ten minutes after fertilization they were affixed to polylysine-coated plates, washed twice in homogenization buffer and then their tops were sheared off by a squirt of this buffer. Fixation was performed as described for the previous sample. All specimens were dehvdrated in ethanol. transferred in graded series to Freon I I3 and critically point dried in Freon 13. The olatinum:50 00. Fig. 13. The bulging on the inner surface car I be discerned to be an entering spermatozoon. Th ie membraneless spermatozoon lies between the pl asnna membrane and the cortex and is separated fri om the egg cytoplasm by the network of cortical fibers .X 6000. Fig. 14. The intimate association between the cortical fibers and the spermatozoon can be obse rve d as the rotation of the spermatozoon through the co lrtex occurs. X7500.
The egg surface during fertilization
Fig. 15. The spermatozoon (black arrow) has completed its rotation and is about to be detached into the egg cytoplasm. The scar (white arrow) in the cortex where it penetrated can be observed just above the spermatozoon. The outer surface can be seen on the right and measurements of the thickness of the cortex can be performed on these cut surfaces. x3 000.
22-761801
333
16. The scar in the cortex persists after the spermatozoon has detached into the egg cytoplasm.
Fig.
X5000. Fig. 17.
Ten minutes after fertilization the cortex appears as an overlapping network of fibers 0.24.5 wrn thick. x 1000.
Exprl Cdl Res 98 (1976)
334
Schutten und Mazicl
rtilization
coat
plasma membrane from spermatozoon
fertilization
lasmo
235 nm particle
Exptl Cell Res 98 (1976)
membrane
coat
The egg surface during fertilization
the two membranes, as is permitted by current information about the fluidity of membranes [13]. The events may also, however, involve absolute decreases or increases in the area of the fusing membranes, the egg membrane expanding at the site of fusion and the sperm membrane decreasing in area. For this most common phenomenon, observed every time a cell changes shape, there is no accepted theory. Cortical granule discharge is initiated at the time when membrane fusion between the gametes occurs (text-fig. 1E). The initial area of cortical granule discharge may be as great as 10 pm. The perpendicularly penetrating spermatozoon is surrounded by a cluster of about twenty elongate microvilli (fig. 6). The first cortical event reveals twenty to twenty-five particles arranged in a circle 2.6 pm in diameter (fig. 11); the basal diameter of the clustered microvilli is 2.5 pm. Since the size, diameter and time of appearance correspond, it is postulated that the particles are associated with the bases of the microvilli. Longo & Anderson [2] described rod-containing vesicles of the same dimensions associated with the basal part of the fertilization cone; the 235 nm particles may correspond to the vesicles seen in the TEM. Text-fig. I. (A) The spermatozoon initially contacts the egg with its acrosome (and see Discussion); (B) the egg membrane bulges at the site of membrane fusion; (C) the sperm-egg membrane appears as a column around the sperm head and the egg membrane, indicated by the presence of microvilli, is uplifted; (D) the microvilli cluster and elongate around the spermatozoon as the plasma membrane appears slack and convoluted; (E) as the cortical granules discharge and the vitelline sheet separates from the plasma membrane, the spermatozoon rotates to lie between the membrane and the cortex; (F) the membrane-less spermatozoon establishes contact with the cortical fibers as it lies on the egg surface; (G) the rotation of the spermatozoon through the cortex is initiated at its anterior end as the vitelline sheet elevates to form the fertilization coat; (H) finally the spermatozoon is detached from the cortex into the egg cytoplasm after having transcribed a 180” rotation on the surface. The scar in the cortex persists after the spermatozoon has detached.
335
Membrane fusion also seems to initiate a surface deformation which spreads in magnitude and diameter as the spermatozoon penetrates (cf figs 4-7). Evidence for an apparent surface contraction is inferred from the spreading surface deformation and the clustering of the microvilli. It is speculated that the indentations in the tips of the papillae of the vitelline sheet (fig. 7) occurred when the underlying microvilli withdrew their support from the papillae. Furthermore in view of the thickened and distorted appearance of the cortical fibers at this stage (fig. 1l), it is tempting to implicate the cortex in this alleged contraction. As the deformation spreads, the vitelline sheet appears to drape loosely upon the egg surface as if the contour of the vitelline sheet and the plasma membrane no longer correspond; it is at this stage that the vitelline sheet is probably detached from the plasma membrane. It is speculated that this spreading surface deformation may be involved in temporarily endowing the egg with some resistance to supernumerary sperm. As penetration continues, the spermatozoon rotates to lie upon the inner surface of the egg (figs 13, 14, text-fig. lE, F). After membrane fusion the spermatozoon and egg are bounded by a common plasma membrane and there does not appear to be any support holding the spermatozoon in the perpendicular orientation in which it attached and fused. The tendency to minimize surface area might be the motive force in causing the sperm to lie between the plasma membrane and the cortex (compare text-fig. 1D, E). It is at this stage that the spermatozoon disappears from the outside surface and that a bulging appears on the inner aspect (fig. 12). Since the cortex has been measured to be maximally 0.5 pm thick, the sperm would not be expected to Exptl Cell Res 98 (I 976)
336
Schutten trncl Mtrzirr
be able to ‘hide’ in the surface. The entire spermatozoon, less its plasma membrane, enters the egg and seems to be forced onto the cortex. The rotation of the spermatozoon continues as it enters the egg cytoplasm (fig. 15, text-fig. 1G). This rotation occurs on the cortex and seems to be initiated at the anterior end of the spermatozoon. The cortical fibers probably perform this movement. The rotation of the spermatozoon has been known since 1895 [1] and it is now demonstrated that it occurs on the surface in two stages. First the spermatozoon rotates to lie between the plasma membrane and tne cortex (text-fig. lE), and then it is rotated to enter the egg cytoplasm, posterior end first (text-fig. 1G, H). The scar produced in the cortex is most likely the result of the spermatozoon’s passage through this structure (fig. 16, text-fig. 1H). The reappearance of the twenty smaller vesicles is noteworthy. The scar appears to heal by 10 minutes after fertilizaion, leaving a complete cortex enveloping the inner surface of the egg (fig. 17).
the membrane implied some structure that held them in place. Thus far [ 16, 171a cortical structure has not been found in TEM studies. Vacquier [6] has described interconnections between the cortical granules in an SEM study of the inner aspect of the surface of sea urchin eggs. In the present work, it has been possible to inspect the cortex directly, ‘head-on’, as is shown in fig. 15. Such cross-sectional images reveal a cortex about 0.5 pm thick, with only the cortical granules well resolved. Additional information about a layer underlying the plasma membrane is given in images such as that in fig. 14, where an eruption of the sperm nucleus and axoneme through the under surface of the membrane makes clearer the presence of a fibrous layer over that surface. In this fixed material, one does not see a thick fibrous layer over that surface, but a thinner one, immediately associated with the inner surface of the membrane. This might account for the rigidity of the surface which has been deduced from its mechanical properties [9]. The vivid evidence of active movement of the surface at the time of fertilization, of which fig. 7 is one example, makes atExistence of the egg cortex tractive the idea of a contractile surface Many descriptions of the surface of the sea layer and invites speculation on the role of urchin egg postulate the presence of a cor- such contraction in the surface changes tex, defined as a relatively thick and rigid which prevent polyspermy. There is evilayer underlying the plasma membrane. dence for the presence of a myosin-like proChambers in 1917 [14] inferred the exist- tein in the work of Mabuchi [ 181on isolated ence of such a gelated layer, about l-2 pm cortices of sea urchin eggs. More generally, there is growing evidence of microfilaments thick, on the basis of micromanipulation studies. Various biophysical experiments and of actin (e.g. [19]) on the under surface (reviewed by Hiramoto [9]) have deduced of cells of various kinds and there are moproperties of the cortex from studies of the dels relating to contractile layer to the deformation of the egg. Mitchison [ 151con- membrane (e.g. [20]). cluded that the cortex in the living egg was about 2 pm thick. The existence of a layer This research was supported by USPHS grant GM 13882 to D. Mazia. The SEM was purchased by the of secretory vesicles commonly called ‘car- Etec tron Microscope Laboratory at this institution tical granules’ in a layer immediately under with NSF grant GM 38-359. Exptl Cd Kes 98 (1976)
The egg surface during fertilization REFERENCES 1. Wilson, E B & Learning, E, An atlas of fertilization and karyokinesis of the ovum. Macmillan & Co, New York (1895). 2. Longo, F & Anderson, E, J cell biol39 (1968) 339. 3. Au&, C R, Ultrastructure of fertilization. Holt, Rinehart &Winston. New York (1968). 4. Tegner, M & Epel, D, Science 179(1973) 685. 5. Mazia, D, Schatten, G & Sale, W, J cell biol 66 (1975) 198. 6. Vacquier, V D, Dev biol 43 (1975) 62. 7. Sakai, H, J biophys biochem cytol8 (1960) 603. 8. Kane, R E & Stephens, R E, J cell biol 41 (1969) 133. 9. Hiramoto, Y, Biorheology 6 (1970) 201. IO. Rothschild, L & Barnes, H, J exp biol 30 (1953) 534.
337
I I. Kavanau, J L, J exp zoo1 122(1953) 285. 12. Tilney, L, Hatano, S, Ishikawa, H & Mooseker, M, J cell biol 59 (1973) 109. 13. Singer, S J, Ann rev biochem 43 (1974) 805. 14. Chambers, R, Am j physiol 43 (1917) I. 15. Mitchison, J M, Quart j microscop sci 97 (1956) 109. 16. Mercer, E H & Wolpert, L, Exp cell res 27 (1962) I. 17. Harris, P, Exp cell res 52 (1968) 677. 18. Mabuchi, J, J cell biol 59 (1973) 542. 19. Clarke, M, Schatten, G, Mazia, D & Spudich, J, Proc natl acad sci US 72 (1975) 1758. 20. Yahara, I & Edelman, G M, Exp cell res 91 (1975) 125. Received August 12, 1975
ExprlCe/IRrs98
(1976)
THE PENETRATION THE SEA URCHIN
OF THE SPERMATOZOON EGG SURFACE
THROUGH
AT FERTILIZATION
Observations from the Outside on Whole Eggs and from the Inside on Isolated Surfaces G. SCHATTEN Department
ofZoology,
University
and D. MAZIA of California.
Berkeley,
CA 94720, USA
SUMMARY The external and cytoplasmic surfaces of the sea urchin egg at fertilization have been examined with the scanning electron microscope (SEM). The outside events were documented by glueing eggs to polylysine coated glass plates, adding sperm and fixing rapidly. To reveal the inner aspects of the surface as the sperm travels through it to reach the egg cytoplasm. the fertilized egg surface was isolated in 0.3 M KCI, 0.35 M glycine, 2 mM MgCI,, 2 mM EGTA, pH 7.5, glued onto a polylysine-coated plate and processed for the SEM. The events of spermatozoon attachment, membrane fusion, sperm entry, rotation and detachment into the egg cytoplasm as well as the associated cortical changes are described. The egg cortex is revealed to be a uniform network of fibrous bundles. The spermatozoon initially attaches to the egg surface by the acrosomal filament. As membrane fusion occurs between the gametes, the plasma membrane of the egg engulfs the sperm, the cortical granules start to discharge and a spreading surface deformation, possibly caused by a cortical contraction, is initiated. The perpendicularly entering spermatozoon is surrounded by a cluster of elongate microvilli which appear to have 235 nm vesicles associated with their bases. The sperm is prevented by the cortex from directly entering the egg cytoplasm and lies upon the egg surface between the plasma membrane and the matrix of cortical fibers. It is subsequently rotated additionally to enter the egg cytoplasm with the posterior end first. A scar is left in the cortex where the spermatozoon penetrated. The egg cortex is shown to consist of XL-200 nm uniformly arranged fibers, and its thickness ranges from 0.2 to 0.5 pm. It is speculated that this structure may be contractile.
In this work the surface events of fertilization of the sea urchin egg are traced from the attachment of the spermatozoon to the outer surface, through the fusion of the membranes of the gametes, to the appearance of the spermatozoon on the inner surface and the attendant cortical reactions. Some newer methods suitable for making the necessary observations with the scanning electron microscope (SEM) and permitting the observation of both the outer and inner faces of the cell surface are used.
A description of fertilization is synthesized from the accumulated evidence from light microscopy, transmission electron microscopy (TEM) and SEM. Wilson & Learning [I] had described the penetration of the spermatozoon into the cytoplasm and its subsequent rotation prior to nuclear diffusion with evidence that left little for classical light microscopy to add. Longo & Anderson [2], using TEM, have presented the ultrastructural aspects of sperm-egg fusion and the cytoplasmic reacExpd Cell Res 98 (1976)
tions associated with the entry of the spermatozoon. Earlier TEM is summarized by Austin [3]. Tegner & Epel [4] have made the basic observations with the SEM, describing the outer surface of the egg, confirming the presence of the vitelline sheet as the outer coat of the egg surface which is the precursor to the fertilization membrane, and relating the cortical reactions to the detachment of unsuccessful sperm. The present work employs the techniques of affixing eggs to a polylysine coated surface [5] which reduces the area available for sperm penetration and permits rapid transfer of the eggs to fixative after the sperm are introduced. It is possible to follow sperm attachment, membrane fusion and sperm entry because the preparations can be fixed more rapidly than eggs in suspension and because fertilization can take place only on the half of the surface which is viewed in the SEM; the other half is glued to the polylysine-coated plate. The observation of the inner face of the unfertilized egg surface was first performed by Vacquier [6] who developed a technique for shearing eggs which are attached to a solid surface in calcium-free sea water; only the attached surface remains and its inner aspect is visible in the SEM. This method is not suitable for examining the entry of the spermatozoon through the egg surface since the upper half of the egg is sacrificed during the isolation. Furthermore, after fertilization the surface of the cell is separated from the fertilization coat; shearing will then detach the egg. For these reasons, the observations of the events of fertilization as seen on the inner aspect of the cell surface were made on cell surface complexes isolated immediately after insemination. The method of isolation used preserves all the features of the cell surface complex known from elecExprlCe//Res
Xl (1976)
tron microscopy of the whole egg. Other methods of isolation exist in the literature [7, 81 and are generally designated as methods of isolating the ‘cortex’. Here we limit the term ‘cortex’ to the structure underlying the plasma membrane. In the method described in detail below, the eggs are inseminated, quickly transferred to the isolation medium and gently cracked to open up the surface; the entire egg surface is maintained. The surface complex, from which the internal contents of the cell are washed away, is glued to a polylysine-coated plate and fixed. It always attaches by the outer surface, with the inner surface facing upward. As will be shown, it is possible to observe the eruption of the spermatozoon through the inner surface. The observation of the egg cortex after the complete cortical granule discharge presented another sort of technical problem since the hardened fertilization coat is resistant to chemical and physical removal. To overcome this, fertilized eggs were placed in mercaptoethylgluconamide to prevent hardening of this coat. The resulting fragile fertilization coat was subsequently stripped off of the egg by passages through fine mesh silk. These ‘naked’ eggs were then glued to polylysine-coated plates and sheared in the presence of the homogenization buffer. These preparations, without the obstruction of the cortical granules, reveal that the inner surface has a uniform matrix of fibers. This confirms the biophysical evidence [9] that a cortex exists beneath the plasma membrane. MATERIALS
AND METHODS
Gametes from the California sea urchin Strongylocentrotus purpuratus were obtained by intracoelomic stimulation with 0.5 M KCI. The eggs, collected in sea water, were dejellied by three passages through bolting silk. Semen was collected ‘dry’ and diluted in sea water just prior to use.
The egg surface during fertilization The images of the outer surface events were obtained by glueing eggs to polylysine coated plates, adding dilute sperm and transferring the plates to ftxative rapidly. The preparations were fixed with 5% elutaraldehvde in 80% sea water (PH 8.2) for 30 min. The observations of the inner’ events were performed by fertilizing and homogenizing eggs in suspension and then rapidly glueing and fixing the isolated surfaces on polylysine-coated plates. This could be done in 45 sec. The homogenization medium imitates known features of the internal environment of the sea urchin egg; the cationic ratios according to Rothschild & Barnes [IO1 and the use of glvcine for osmotic balance, according io Kavanau [III it contains, however, the calcium ion chelator ethyleneglycol-bis-@aminoethyl ether) NJ’-tetraacetic acid (EGTA) to prevent cortical granule discharge and membrane healing. Thirty seconds after insemination the eggs were washed twice with IO vol of 0.3 M KCI, 0.35 M glycine, 2 mM MgCI,, 2 mM EGTA, pH 7.5 with Na,CO,, hereafter called homogenization buffer. A IO% egg suspension was gently homogenized by one passage of a Teflon pestle; only a single crack in the egg surface is necessary. Egg surfaces were collected by centrifugation (700 g for I5 set), glued onto polylysine-coated plates and rinsed in homogenization buffer. This rinse washes the rest of the adhering cytoplasm from these splayed open inner surfaces. Fixation was performed in 0.5 M KCI, 2 mM M&I,, 2 mM EGTA,. I % acrolein; 2.5 % glutaraldehyde~ pH 7.5 for I5 min. The surface complex of the fertilized egg is more difficult to isolate in suspension and the method of shearing attached eggs was employed. To ensure that the shearing plane in these eggs will be the surface complex, the fertilization coat was stripped off prior to glueing them to the polylysine coated substrate. To do this, fertilized eggs, at the first sign of membrane elevation, were transferred to 0. I % mercaptoethylgluconamide, IO mM ethylenediamine tetraacetic acid (EDTA) in sea water (pH 8.2). The high and very thin fertilization coat was stripped off of the eggs by three passages through bolting silk. These ‘naked’ eggs tend to clump and are fragile; they must be poured rather than pipetted. Ten minutes after fertilization they were affixed to polylysine-coated plates, washed twice in homogenization buffer and then their tops were sheared off by a squirt of this buffer. Fixation was performed as described for the previous sample. All specimens were dehvdrated in ethanol. transferred in graded series to Freon I I3 and critically point dried in Freon 13. The olatinum:50 00. Fig. 13. The bulging on the inner surface car I be discerned to be an entering spermatozoon. Th ie membraneless spermatozoon lies between the pl asnna membrane and the cortex and is separated fri om the egg cytoplasm by the network of cortical fibers .X 6000. Fig. 14. The intimate association between the cortical fibers and the spermatozoon can be obse rve d as the rotation of the spermatozoon through the co lrtex occurs. X7500.
The egg surface during fertilization
Fig. 15. The spermatozoon (black arrow) has completed its rotation and is about to be detached into the egg cytoplasm. The scar (white arrow) in the cortex where it penetrated can be observed just above the spermatozoon. The outer surface can be seen on the right and measurements of the thickness of the cortex can be performed on these cut surfaces. x3 000.
22-761801
333
16. The scar in the cortex persists after the spermatozoon has detached into the egg cytoplasm.
Fig.
X5000. Fig. 17.
Ten minutes after fertilization the cortex appears as an overlapping network of fibers 0.24.5 wrn thick. x 1000.
Exprl Cdl Res 98 (1976)
334
Schutten und Mazicl
rtilization
coat
plasma membrane from spermatozoon
fertilization
lasmo
235 nm particle
Exptl Cell Res 98 (1976)
membrane
coat
The egg surface during fertilization
the two membranes, as is permitted by current information about the fluidity of membranes [13]. The events may also, however, involve absolute decreases or increases in the area of the fusing membranes, the egg membrane expanding at the site of fusion and the sperm membrane decreasing in area. For this most common phenomenon, observed every time a cell changes shape, there is no accepted theory. Cortical granule discharge is initiated at the time when membrane fusion between the gametes occurs (text-fig. 1E). The initial area of cortical granule discharge may be as great as 10 pm. The perpendicularly penetrating spermatozoon is surrounded by a cluster of about twenty elongate microvilli (fig. 6). The first cortical event reveals twenty to twenty-five particles arranged in a circle 2.6 pm in diameter (fig. 11); the basal diameter of the clustered microvilli is 2.5 pm. Since the size, diameter and time of appearance correspond, it is postulated that the particles are associated with the bases of the microvilli. Longo & Anderson [2] described rod-containing vesicles of the same dimensions associated with the basal part of the fertilization cone; the 235 nm particles may correspond to the vesicles seen in the TEM. Text-fig. I. (A) The spermatozoon initially contacts the egg with its acrosome (and see Discussion); (B) the egg membrane bulges at the site of membrane fusion; (C) the sperm-egg membrane appears as a column around the sperm head and the egg membrane, indicated by the presence of microvilli, is uplifted; (D) the microvilli cluster and elongate around the spermatozoon as the plasma membrane appears slack and convoluted; (E) as the cortical granules discharge and the vitelline sheet separates from the plasma membrane, the spermatozoon rotates to lie between the membrane and the cortex; (F) the membrane-less spermatozoon establishes contact with the cortical fibers as it lies on the egg surface; (G) the rotation of the spermatozoon through the cortex is initiated at its anterior end as the vitelline sheet elevates to form the fertilization coat; (H) finally the spermatozoon is detached from the cortex into the egg cytoplasm after having transcribed a 180” rotation on the surface. The scar in the cortex persists after the spermatozoon has detached.
335
Membrane fusion also seems to initiate a surface deformation which spreads in magnitude and diameter as the spermatozoon penetrates (cf figs 4-7). Evidence for an apparent surface contraction is inferred from the spreading surface deformation and the clustering of the microvilli. It is speculated that the indentations in the tips of the papillae of the vitelline sheet (fig. 7) occurred when the underlying microvilli withdrew their support from the papillae. Furthermore in view of the thickened and distorted appearance of the cortical fibers at this stage (fig. 1l), it is tempting to implicate the cortex in this alleged contraction. As the deformation spreads, the vitelline sheet appears to drape loosely upon the egg surface as if the contour of the vitelline sheet and the plasma membrane no longer correspond; it is at this stage that the vitelline sheet is probably detached from the plasma membrane. It is speculated that this spreading surface deformation may be involved in temporarily endowing the egg with some resistance to supernumerary sperm. As penetration continues, the spermatozoon rotates to lie upon the inner surface of the egg (figs 13, 14, text-fig. lE, F). After membrane fusion the spermatozoon and egg are bounded by a common plasma membrane and there does not appear to be any support holding the spermatozoon in the perpendicular orientation in which it attached and fused. The tendency to minimize surface area might be the motive force in causing the sperm to lie between the plasma membrane and the cortex (compare text-fig. 1D, E). It is at this stage that the spermatozoon disappears from the outside surface and that a bulging appears on the inner aspect (fig. 12). Since the cortex has been measured to be maximally 0.5 pm thick, the sperm would not be expected to Exptl Cell Res 98 (I 976)
336
Schutten trncl Mtrzirr
be able to ‘hide’ in the surface. The entire spermatozoon, less its plasma membrane, enters the egg and seems to be forced onto the cortex. The rotation of the spermatozoon continues as it enters the egg cytoplasm (fig. 15, text-fig. 1G). This rotation occurs on the cortex and seems to be initiated at the anterior end of the spermatozoon. The cortical fibers probably perform this movement. The rotation of the spermatozoon has been known since 1895 [1] and it is now demonstrated that it occurs on the surface in two stages. First the spermatozoon rotates to lie between the plasma membrane and tne cortex (text-fig. lE), and then it is rotated to enter the egg cytoplasm, posterior end first (text-fig. 1G, H). The scar produced in the cortex is most likely the result of the spermatozoon’s passage through this structure (fig. 16, text-fig. 1H). The reappearance of the twenty smaller vesicles is noteworthy. The scar appears to heal by 10 minutes after fertilizaion, leaving a complete cortex enveloping the inner surface of the egg (fig. 17).
the membrane implied some structure that held them in place. Thus far [ 16, 171a cortical structure has not been found in TEM studies. Vacquier [6] has described interconnections between the cortical granules in an SEM study of the inner aspect of the surface of sea urchin eggs. In the present work, it has been possible to inspect the cortex directly, ‘head-on’, as is shown in fig. 15. Such cross-sectional images reveal a cortex about 0.5 pm thick, with only the cortical granules well resolved. Additional information about a layer underlying the plasma membrane is given in images such as that in fig. 14, where an eruption of the sperm nucleus and axoneme through the under surface of the membrane makes clearer the presence of a fibrous layer over that surface. In this fixed material, one does not see a thick fibrous layer over that surface, but a thinner one, immediately associated with the inner surface of the membrane. This might account for the rigidity of the surface which has been deduced from its mechanical properties [9]. The vivid evidence of active movement of the surface at the time of fertilization, of which fig. 7 is one example, makes atExistence of the egg cortex tractive the idea of a contractile surface Many descriptions of the surface of the sea layer and invites speculation on the role of urchin egg postulate the presence of a cor- such contraction in the surface changes tex, defined as a relatively thick and rigid which prevent polyspermy. There is evilayer underlying the plasma membrane. dence for the presence of a myosin-like proChambers in 1917 [14] inferred the exist- tein in the work of Mabuchi [ 181on isolated ence of such a gelated layer, about l-2 pm cortices of sea urchin eggs. More generally, there is growing evidence of microfilaments thick, on the basis of micromanipulation studies. Various biophysical experiments and of actin (e.g. [19]) on the under surface (reviewed by Hiramoto [9]) have deduced of cells of various kinds and there are moproperties of the cortex from studies of the dels relating to contractile layer to the deformation of the egg. Mitchison [ 151con- membrane (e.g. [20]). cluded that the cortex in the living egg was about 2 pm thick. The existence of a layer This research was supported by USPHS grant GM 13882 to D. Mazia. The SEM was purchased by the of secretory vesicles commonly called ‘car- Etec tron Microscope Laboratory at this institution tical granules’ in a layer immediately under with NSF grant GM 38-359. Exptl Cd Kes 98 (1976)
The egg surface during fertilization REFERENCES 1. Wilson, E B & Learning, E, An atlas of fertilization and karyokinesis of the ovum. Macmillan & Co, New York (1895). 2. Longo, F & Anderson, E, J cell biol39 (1968) 339. 3. Au&, C R, Ultrastructure of fertilization. Holt, Rinehart &Winston. New York (1968). 4. Tegner, M & Epel, D, Science 179(1973) 685. 5. Mazia, D, Schatten, G & Sale, W, J cell biol 66 (1975) 198. 6. Vacquier, V D, Dev biol 43 (1975) 62. 7. Sakai, H, J biophys biochem cytol8 (1960) 603. 8. Kane, R E & Stephens, R E, J cell biol 41 (1969) 133. 9. Hiramoto, Y, Biorheology 6 (1970) 201. IO. Rothschild, L & Barnes, H, J exp biol 30 (1953) 534.
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I I. Kavanau, J L, J exp zoo1 122(1953) 285. 12. Tilney, L, Hatano, S, Ishikawa, H & Mooseker, M, J cell biol 59 (1973) 109. 13. Singer, S J, Ann rev biochem 43 (1974) 805. 14. Chambers, R, Am j physiol 43 (1917) I. 15. Mitchison, J M, Quart j microscop sci 97 (1956) 109. 16. Mercer, E H & Wolpert, L, Exp cell res 27 (1962) I. 17. Harris, P, Exp cell res 52 (1968) 677. 18. Mabuchi, J, J cell biol 59 (1973) 542. 19. Clarke, M, Schatten, G, Mazia, D & Spudich, J, Proc natl acad sci US 72 (1975) 1758. 20. Yahara, I & Edelman, G M, Exp cell res 91 (1975) 125. Received August 12, 1975
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