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Studies on the acrosome
Experimental
Cell Research 19, 13-28 (1960)
STUDIES VI.
FINE
13
ON THE ACROSOME
STRUCTURE
OF THE JEAN
Ochanomizu
STARFISH
ACROSOME
C. DAN
University,
Tokyo, Japan
Received March 15, 1959
IN a good many species of invertebrate animals [9], and at least two vertebrate groups,1 it has been found that the acrosomal region of the spermatozoon undergoes a change which is interpreted as a triggered response to the presence of the unfertilized egg of its own species. Typically, this change involves the disappearance of at least part of the material making up the acrosome, and the appearance of a structure which for want of a more precise term has been called a “filament”. Depending on the species, this structure may be very long (35-90 ,u in the holothurian, Thyone briareus [6]), or short and stumpy as in some echinoids. Among many problems which must be solved before a satisfactory accounting for this phenomenon can be made is the question of how the filaments originate: whether they are built up to their final dimensions during spermatogenesis and stored compactly in the acrosome to be released explosively under appropriate conditions, or whether their rapid formation from precursor substances is part of the triggered response that occurs when the spermatozoon arrives in the vicinity of an egg. The first of these possibilities characterizes the mechanism found in coelenterate nematocysts; the second is characteristic of ciliate trichocysts [16]. There is of course no reason to believe that the .mechanism of acrosome discharge or the mode of filament formation must conform to either of these examples. They are brought into the discussion not as models, but rather as furnishing assurance that cell organelles of a size roughly comparable to that of spermatozoan acrosomes are capable both of ejecting preformed tubular filaments and of forming such structures very rapidly from non-fibrous precursor materials. Phase contrast observation has failed to reveal a consistent correlation r A filament extending from the acrosome of cyclostome spermatozoa was figured by Ballowitz [4] and Retzius [15]; this observation has recently been confirmed by electron microscopy [3] and cinematography [ll]. In the frog Biscogtossus, Favard [lo] reports the appearance of a neutral red-staining droplet, 2-3 p in diameter, near the tip of the acrosome; he interprets this as a reaction of the acrdsome similar to those occurring among invertebrates. Kille [li], on the other hand, finds that the spermatozoa of these animals form a long acrosome filament. Experimental
Cell Research
19
Jean C. Dan between the size of the intact acrosome and the length of the filament which it produces. The small size and high refringence of these organelles, moreover, preclude resolution of their exact dimensions and internal structure with light microscopy, and make resort to thin sectioning necessary to secure suc,h information. A study by Afzelius [l, 2,3] has already shown that the sea urchin acrosome in its intact state contains no inclusion which even approximates the tibrillar structure and dimensions of the acrosome filament found in sections of reacted spermatozoa. This result points squarely to the second of the two mechanisms suggested above. The filaments of sea urchin spermatozoa, however, happen to be among the least conspicuous acrosome filaments so far known. In order to widen the basis for drawing a general conclusion concerning the mode of origin of such filaments, and to study a form in which a longer filament results from the acrosome reaction, starfish spermatozoa were fixed in the intact state and after treatment to induce the acrosome reaction, and sectioned for electron microscopy. MATERIAL
AND
METHODS
The spermatozoa of Aster& forbesii were used in this study. For fixing unreacted spermatozoa, “dry sperm” was diluted with sea water to make a 1 per cent suspension; to 1 ml of this suspension was added an equal amount of 2 per cent 0~0, in sea water (pH 8) at room temperature. After 30 minutes of osmium fixation the suspension was post-fixed for 20 hours in 7 per cent formalin-sea water, washed in sea water with light centrifugation and dehydrated with alcohol. Samples were embedded in butyl- plus 15 per cent methyl-methacrylate. To induce the acrosome reaction in these spermatozoa, freshly shed dry sperm was diluted to a 2 per cent suspension with 0.001 M versene in sea water. To this was added an equal amount of egg water (prepared by removing the supernatant from a 50 per cent egg suspension after 30 minutes’ standing). The resulting 1 per cent sperm suspension was fixed one minute after addition of egg water and embedded as described above. The material was sectioned with a Porter-Blum or Sjostrand Microtome and observed with Phillips and Akashi electron microscopes, as indicated in the descriptions of the individual figures. RESULTS’
Intact acrosomes .,The acrosome of the starfish spermatozoon (Fig. 1 and Text-fig. 1A) is set in a characteristic depression in the nucleus, comprising a bowl-shaped region about 0.8 ,u in greatest diameter and 0.5 p in depth, ~1 In the following description, only general terms are used to designate the various regions and substancesof the acrosome,in spite of the resulting awkwardness,sinceit is felt that the assigning of definitive namesshould be basedon a more complete knowledge of their respective functions. Experimentut Cell Research 19
Fine structure of starfish acrosome
15
the base of which opens into a sac-shaped sub-depression about 0.15 ,u in diameter and 0.3 ,u in depth. This whole concavity is lined with the strongly osmiophilic nuclear membrane (Text-fig. 1 A, l).l
Text-fig. Numbers
L-Diagrammatic sketches of Asterias forbesii acrosomes. indicate substances and structures described in text.
A, unreacted.
B, reacted.
The anterior face of the nearly spherical sperm head is radially symmetrical, consisting of an encircling rim of nucleus enclosing the acrosome surface. Although the cytoplasmic membrane (A, 2) is not distinguishable with certainty in the sections presented here, observation of living spermatozoa under experimental conditions indicates that the whole sperm head is covered by a single membrane which can be caused to expand so that it forms a larger sphere within which the nucleus, acrosome and middle piece lie as three separate bodies (see [8], Fig. 2b). Under this closely investing cytoplasmic membrane the topography of the exposed acrosome surface is highly constant, having in the center a raised circular crater (A, 3) surrounded by a peripheral ring of a different substance (A, 4), which is slightly depressed below the adjacent rim of nucleus. In sections through the long axis of the spermatozoon, the acrosome is seen to contain in its center a spherical or somewhat top-shaped mass of homogeneously electron-dense material (A, 5), between 0.2 and 0.3 p in diameter, which appears in all sections to be lacking a bounding membrane. This is completely surrounded (Figs. 1 and 2) by a thick layer of much less dense material (Text-fig. 1 A, 6) which, while not precisely homogeneous, does not show any definite organization, although in some cases there seems to be a suggestion of radial arrangement. This layer is of quite uniform width, 1 Reference
will subsequently
be given to Text-Fig.
1, A and B only as A or B in brackets. Experimental
Cell Research 19
Jean C. Dan
16
about 0.15 ,u, and follows the shape of the central granule. Its anterior surface is capped (Figs. 3,4) by a thin, dense layer (A, 7) possessing sufficient rigidity to maintain the form of the crater-shaped protuberance (A, 3) which lies exactly over the central granule. The lower margin of this layer is difficult to determine; in most sections it seems to fade out a short distance below the surface, while in some it is apparently continuous with a similar layer (A, 8) which will be described below. Encircling the sides of the mass of non-electron-absorbing substance there is a band, about 0.15 ,u wide and 0.1 ,u thick, of homogeneous material (A, 4), that in most sections is nearly as electron-dense as the central granule (Figs. 1, 2). This band is also strikingly uniform in width at any given level (Fig. 2), and no membrane can be detected between its inner side and the less dense material it surrounds. It also seems to be in immediate contact with the nuclear membrane on its outer side, and its anterior edge extends to the exposed face of the acrosome, where it spreads to some extent under the cytoplasmic membrane (Figs. 1, 2, 3), forming the outer side of the depression which surrounds the central crater-shaped protuberance. Enclosing the posterior half of the mass of least dense material there is a cup-shaped layer (A, 8) which is uniformly thin at the sides and has a thickened plate at its base where it bridges the opening into the sub-depression. Its anterior rim extends a short distance inside the posterior edge of the thick band; the section shown in Fig. 2 lies just at the limit of this layer but not exactly perpendicular to the sperm axis, so that a cross-section of the layer appears at the deeper (lower) side in the micrograph but not at the shallower side. The contents of the sub-depression which is surrounded by nuclear membrane and located approximately in the center of the sperm head present a rather complicated and surprisingly consistent picture. Directly in contact with the outside of the layer (A, 8) described above is a small mass of substance (A, 9) similar in density and general appearance to the central granule (A, 5) in some sections, and to the material of the thick band (A, 4) in others. This fills the anterior part of an inner sac bounded by a strongly osmiophilic membrane (A, 10) apparently identical with that surrounding the nucleus, from which it is separated by a layer of substance (A, 11) about Fig. l.-Longitudinal section through Asferias forbesii acrosome. Section cuts center of acrosome but misses insertion of sperm tail. Regions of acrosome identified in text figure and described in text. M, midpiece N, nucleus (Akashi EM TRS-50). Fig. 2.-Transverse section through greatest diameter of acrosome, nearly parallel to anterior surface of sperm head, but slightly deeper at lower side of micrograph. (Akashi EM TRS-50.) Experimental
Cell Research 19
Fine structure of starfish acrosome
17
Jean C. Dan
18
200 A thick and homogeneously dense like the material (A, 9) inside the membrane. This inner membrane, however, does not form a complete secondary sac. Several sections (Figs. 1, 5) show it joining the nuclear membrane at one side (A, 12); the frequency of occurrence of such sections in the available micrographs is four, compared with three sections showing a symmetrical structure as in Fig. 3. The posterior part of the inner sac often contains one or more vesicles (A, 13) somewhat similar to those found in Strongylocentrotus droebachiensis acrosomes by Afzelius [l]. These have an osmiophilic bounding membrane surrounding non-homogeneous contents, and sometimes appear quite empty (Figs. 1, 3, 5). Reacted acrosomes.-The head of the starfish spermatozoon fixed after exposure to egg-water (Figs. 6, 7, 8, 9, 10; Text-fig. 1 B) presents a strikingly different appearance under the electron microscope from the picture just described. The whole complex of acrosomal substances and structures is missing, the nucleus has tilled in the space previously occupied by the acrosome (B, 14), thus reducing the size and changing the shape of the sperm head, and a tilamentous projection extends forward from its anterior surface. In unreacted spermatozoa the cytoplasmic membrane is extremely delicate and lies so closely against the strongly osmiophilic nu.clear membrane that its presence is difficult to detect except where it bridges the junction between midpiece and nucleus. In reacted cells, on the other hand, a fairly thick layer lies outside the nuclear membrane, often without a clearly defined outer margin. On first inspection this gives an impression of poor fixation or damage during preparation, but since unreacted cells in the same sample show no such general effect, it is probable that some rather drastic change has occurred in the relation of the cell membrane to the rest of the cell. This will be discussed below. Examination of many sections shows that there is usually a cone-shaped depression in the nucleus (B, 15) immediately under the filament, lined with nuclear membrane (B, 16). This depression may be shallow (Fig. 6) or deep Fig. 3.-Longitudinal section through acrosome slightly to side of center, showing complete inner membrane of sub-depression (see description of Text-fig. 1, A-12 in text). (Phillips EM 100a.) Fig. 4.-Longitudinal sections of starfish acrosomes. Upper section cuts through center of outer surface but grazes subdepression; lower section parallel to long axis of spermatozoon somewhat to side of center. (Phillips EM 100a.) Fig. J.-Part of section through abnormal depression. (Akashi EM TRS-50.) Experimental
Cell Research 19
double spermatozoon,
enlarged to show detail of sub-
Fine sfrucfure of starfish acrosome
19
Jean C. Dan (Fig. 8) or apparently lacking (Fig. 7), although the possibility exists that the plane of section has missed the depression in such cases. Encircling this depression there is a wide ring of homogeneous, relatively dense material (B, 17) lying as a slightly convex plate on the anterior surface of the nucleus so that it forms a broad base for the fibrous strand of the filament (B, 19), which passes through the center of this ring. A delicate but rather strongly osmiophilic membrane covers this region (B, 18) and is continuous anteriorly with the outermost layer of the filament, and posteriorly with the nuclear membrane. Examination of a number of sections shows that this membrane covering the outside of the base of the filament has characteristics closely resembling those of the nuclear membrane lying beneath the filament. In most sections the cell membrane appears not to extend over this basal plate. The filament itself, surrounded by the thin osmiophilic membrane, shows longitudinal striations throughout its interior; in some places separate fibrils having a diameter of the order of 100 hi, can be seen (Figs. 6, 9, 10). This filamentous material has a low electron density and does not form a single solid strand, in cross-section showing apparently empty spaces in irregular arrangement. The filament typically narrows immediately beyond its basal plate to a uniform diameter of about 0.1 ,U (Figs. 6, 7). Some sections show a somewhat wider basal region, which is usually associated with the presence of nonfibrous material (Figs. 8, lo), suggesting incomplete conversion of the filament precursor substance into the fibrous form. DISCUSSION
Even with phase contrast microscopy it is apparent that a profound change has occurred in starfish spermatozoa which have reacted to the presence of egg jelly substance, either surrounding the egg or in sea water solution. In addition to the appearance of the filament, there is a conspicuous rounding-up of the mid-piece, indicating a reduction in tension of the membrane \\hich in the unreacted state held the mid-piece tightly against the posterior Fig. (i.-Acrosome filament of Asterius forbesii spermatozoon, structure throughout filament shaft. (Phillips EM 100a.)
showing
longitudinal
fibrous
Fig. 7.-I.ongitudinal section through reacted spermatozoon, probably somewhat off center. Kate oval shape of nucleus, disrupted appearance of cytoplasmic membrane at left side, relation of membranes at periphery of basal plate. (Phillips EM lOOa.) Fig. X.--Section through under base of filament. Exprimer~lul
reacted Asterius spermatozoon (Phillips EM 100a.)
Cell liesearch 19
with uuusually
deep depression in nucleus
Fine structure of starfish acrosome
Experimental
21
Cell Researc :h 19
Jean C. Dan
22
side of the nucleus. The heads of spermatozoa being drawn through the jelly layer often have an elongated shape (see Fig. 22, g, h in [6]) which is in marked contrast to the turgid roundness of the unreacted cells. This difference shows up clearly in sections (cf. Figs. 7, 8 and 9 with Fig. l), with respect to both the over-all shape of the nucleus and mid-piece and the state of the cytoplasmic membrane. Moreover, the thickened and often disrupted appearance of the cell membrane is definitely correlated with the reacted state; unreacted spermatozoa in the treated suspension show no change in the cell membrane in spite of having been exposed to egg-water. The considerable reduction in volume of the sperm head resulting from the loss of the whole acrosomal mass is enough in itself to account for a marked slackening in the tension of the surrounding membrane. In addition, the cell membrane in the acrosome region must be affected in some way by the formation of the filament. A local rupturing of the membrane is the simplest possibility, but this would not necessarily affect the membrane tension over the whole cell unless the cytoplasmic membrane were free to slip over the nuclear membrane, which does not seem likely. Many of the sections of reacted spermatozoa show this thickened surface layer rather clearly stopping at or near the periphery of the plate forming the base of the filament. Evidence is also available from other forms that fertilizing or egg-watertreated spermatozoa show a tendency to come apart. In both Crassostrea echinata and Urechis unicinctus, the sperm tail drops off when the head enters the egg in about half the cases (Dan, unpublished). In Figs. 17, 20 and 22 of [5], showing Hydroides hexagonus spermatozoa partly within the vitelline membrane, the cell membranes of the spermatozoa are almost completely separated from the nuclear membranes and mid-pieces. Moreover, sea urchin spermatozoa treated with egg-water show separation of the cell membrane from the enclosed structures [2], as well as a shifting in the position of the mid-piece which Tyler [17] has interpreted as a preliminary to the complete separation of the sperm components within the egg cytoplasm. An attempt to visualize, by comparing the morphology of spermatozoa before and after reaction, the process which brings about the observed changes must begin with the fact that the acrosome is no longer embedded in the nucleus. This immediately raises the question as to whether the nucleus Figs. 9 and IO.-Reacted Asterias spermatozoa. nature of basal plate. (Akashi EM TRS-50.)
Note fibrous structure
of filament
and non-fibrous
Fig. Il.-Replica of reacted Asterias amurensis spermatozoon fixed with osmium vapor and dried on glass surface. Rigidity of basal plate of filament is shown by its resistance to flattening. (From Dan [S], permission Biol. Butt.) Experimentot
Cell Research 19
Fine structure of starfish acrosome
23
Jean C. Dan actively undergoes some convulsive form-change which forces out the acrosome contents, or whether the initiative lies with the acrosome. It seems almost a foregone conclusion that the latter must be the case, since it is hard to imagine a cytoplasmic trigger medhatiism which would set off nuclear activity, and even harder to conceive how a nucleus cou’ld generate the force necessary for an explosive change of shape. It will be assumed, then, that the triggered explosion occurs wholly within the acrosome, making its component parts interact to form a filament outside the cell, while the mass of relatively fluid nucle&asm ,flls in the space formerly occupied by the acrosome. The nucleus thus acquires a simple spherical or ovoid shape between the filament attached anteriorly and the posterior mid-piece and flagellum. All the sections of reacted spermatozoa give the impression of a soft, easily deformable state in contrast to the turgid regularity of the unreacted cells. While the mature spermatozoon, as it is shed, is a cell highly specialized for the purpose of finding and uniting with an egg, it can be considered to undergo a final specializing and stripping-down process when the acrosome reacts to the presence of an egg by transforming its precursor material into a filament.1 At least in the starfish spermatozoon, although the mitochondrial mid-piece and the flagellum are stili in place, their functions may be considered ended, once the spermatozoon has established contact with the egg surface. The final implement for introducing the sperm nucleus into the egg must be this filament, which activates the egg and is then drawn into its cytoplasm, dragging the nucleus after it. In order to carry out this function, a firm attachment is necessary between filament and nucleus. All sections of reacted spermatozoa show that such an attachment is secured by the basal plate of the filament which fits like a cap over the anterior surface of the nucleus. In an electron micrograph of a replica taken from reacted Asterins nmurensis spermatozoa dried on a glass slide after osmium vapor fixation (Fig. 1 I), this plate is seen to be extremely rigid in comparison with the adjacent nucleus. Careful examination of micrographs fails to reveal any fibrillar organization in this structure. It is rather certain that the precursor of this plate is the band labelled [4] in Text-fig. 1A. In the unreacted acrosome this band stands perpendicular to the surface of the sperm head, on which it later comes to Iie as a nearly flat plate. It seems most probable that this changed orientation is brought about by an outward spreading of the anterior edge so that it becomes the periphery of the plate, while the inner surface of the band is turned anteriorly. ____-’ Favard [lo] proposes that such a reaction of the acrosome be considered the maturation process-“la maturation propre du spermatixoi’de” (p, 391).
the final
step in
Fine structure of starfish acrosome
25
That such a drastic change can take place indicates that the rigidity seen in the basal plate of the filament is acquired secondarily, during or after the acrosome reaction. It is conceivable that the precursor substance making up this band in the unreacted acrosome undergoes some progressive change in state which induces a hardening together with the change in its form, so that it exerts an expelling force on the central mass of acrosome contents. It seems rather more likely, however, that the expulsion of the rest of the acrosome as it changes into filament leaves a space into which the material of the band is pushed as the nuclear contents round up. The nature of the attachment between the basal plate of the filament and the anterior part of the nucleus presents an interesting problem. In all the reacted spermatozoa, the thin membrane covering this region and extending out around the filament is sharply osmiophilic, closely resembling the nuclear membrane which lies beneath this structure. Moreover, all the available micrographs give the impression that these two membranes unite at the periphery of the plate to make up the membrane covering the rest of the nucleus (vd. Figs. 6, 7, 8, 9, 10). Granted that the nuclear membrane is two-layered, it is still not easy to imagine how the material constituting the basal plate of the filament could intervene between the two layers, even given the possibilities for drastic local rearrangement that would be afforded by an explosive acrosome reaction. At present the simplest explanation would be one postulating that an especially close connection is established between acrosome and nuclear surface during spermatogenesis. If it can be established that the acrosome vesicle displaces the outer layer of the nuclear membrane so that the later differentiation of the acrosome takes place in direct contact with the inner layer of the nuclear membrane, the major part of the problem will have been solved. With respect to the origin of the membrane covering the filament, the two obvious possibilities are that it may be some sort of precipitation membrane laid down on the surface of the Iibrillar core as this extends into the sea water at the time of its formation, or that it may be formed by a tremendous stretching of the acrosome surface membrane. Fig. 6 of reference [8] indicates that it can be separated from the underlying fibrillar core, and that the tip is closed; these characteristics support but do not prove the latter of the alternatives suggested above. By constructing a plasticene scale model of the intact acrosome and then rolling it out into a rod of the appropriate diameter, it was found that the Experimental
Cell Research 19
Jean C. Dan
26
amount of available precursor material is roughly adequate to form a filament 25 ,X long, which obviates the necessity for invoking extensive swelling, and points to a change in state of the constituent molecules, such as polymerization or possibly denaturation if the material in question turns out to be protein. In Mytilus edulis it has been shown [ 163 that a lysin which effectively attacks the vitelline membrane of the egg is released from the spermatozoa at the time of acrosome reaction, and the suggestion has been made [7] that the granule at the tip of sea urchin spermatozoa similarly contains a lytic substance. So far, however, no direct evidence has been found to show that echinoderm spermatozoa contain a lysin which attacks echinoderm egg membranes (but see [12]). In the particular case of the starfish, there does not seem to be any place in the series of events making up the fertilization process where a lysin is definitely required. It seems safe to assume that the filament, formed by the acrosome on contact with the outer edge of the jelly layer, is able to pierce this layer with the push derived from the chemical change which causes its formation. The little that is known about the properties of the unfertilized starfish egg surface suggests that it resembles that of the sea urchin egg; electron microscopy shows that the latter is surrounded by a vitelline membrane so delicate that some experienced observers doubt its existence [13]. Presumably a filament with a modicum of rigidity and a diameter of only 0.1 ,u could penetrate such a membrane without having to dissolve it chemically. In view of the fact that pricking with a glass needle is one of the most generally reliable means of activating unfertilized eggs and has been proved effective in echinoderms [ 141, there seems to be no a priori necessity for postulating an activating enzyme in addition to the pricking stimulus delivered by the lilament. At any rate, it is clear that activation of the starfish egg takes place immediately upon penetration of the filament tip, as indicated by cortical granule breakdown, fertilization membrane elevation and cone formation; all these events occur while the sperm head itself is passing through the jelly layer, well away from the egg surface. The next process in which a lysin might conceivably be useful is the passage of the sperm head through the fertilization membrane. However, it is frequently observed that a tip of the fertilization cone extends along the filament and appears outside the fertilization membrane. The advancing sperm head meets and enters the cone, which retreats through the membrane as the sperm head continues to approach the egg surface. This may be interpreted Experimental
Cell Research 19
Fine structure of starfish acrosome
27
as indicating that the hole in the fertilization membrane where the filament pierces it is large enough to permit passage of the sperm head; if a lysin is to be included in the scheme, the fertilization cone tip must be equipped with it as well as the sperm head. Aside from such apparent lack of necessity for lytic activity, the mechanics of lysin transport under the circumstances present some difficulties to the imagination. No indication has been found that a mass of material is carried on the distal end of the filament, as Afzelius [2, 31 has shown to be the case in sea urchins (see also [7], Fig. 14), and since the filament is not tubular, there is no question of a substance being injected through it, even supposing that the tremendous capillary resistance inside a tube 0.1 ,u in diameter could be overcome by some force generated by the reacted spermatozoon. The present study has so far provided no clues at all to the answers of two of the most interesting questions in connection with the structure and reaction of the starfish acrosome: Where is the trigger? and What is the function of the so-called “sub-depression” ? It is hoped that fixation within shorter intervals after initiation of the reaction will provide the solutions to these problems. With respect to the question posed in the introduction, concerning the mode of origin of the filaments in the two groups of echinoderms which have so far been studied in detail, it appears sufficiently certain that any tibrillar elements which may be present are below the limit of resolution of the electron microscopes employed, assuming of course that the method of preparation has removed nothing essential. It is even more certain that neither sea urchin nor starfish acrosomes contain any preformed structures which resemble the definitive filaments, and that in these groups it is appropriate to visualize a rapid transformation of precursor materials into fibrous structures as part of the triggered response of the acrosome. SUMMARY
Electron micrographs of thin-sectioned Asterias forbesii spermatozoa show the acrosome, which is embedded in the nucleus, to be composed of a large (diameter 0.25 ,u) granule of dense material enclosed in a mass of material with low electron density. This is in turn surrounded at its sides by a wide and relatively thick band of a third, rather dense, substance. The anterior surface of the acrosome shows a characteristic profile, with a raised, crater-like structure in the center. Proximally, a double-walled sac containing homogeneous substance and vesicles surrounded by osmiophilic membranes extends into the center of the nucleus. Experimental
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Jean C. Dan
28
Spermatozoa fixed after treatment with egg-water lack this whole complex, and the general appearance of the nucleus and the cytoplasmic membrane indicate a reduction in the volume of the sperm head. From the anterior surface of the nucleus extends a straight rod about 0.1 p in diameter, showing faint longitudinal flbrils within an osmiophilic membrane which is continuous with the outer layer of the nuclear membrane. The base of the filament is set in a slightly convex circular plate which covers the anterior surface of the nucleus and is believed to contain the material of the outermost layer in the intact acrosome. It is concluded that the acrosome filament is not preformed and stored in the intact acrosome, but rather formed very rapidly from precursor substances at the time of the acrosome reaction. This study was performed during the tenure of a Special Research Fellowship granted by the National Cancer Institute, National Institutes of Health, for work in the laboratory of Professor Arthur W. Pollister at Columbia University. The author gratefully acknowledges the kindness of all the Zoology Department members, and particularly the patient instruction of Dr. Jerome Kay and Mr. Robert Ward. The author also wishes to thank Drs. Keith Porter and Montrose Moses, and Mr. Gordon Kaye, of the Rockefeller Institute, for their courtesy in offering use of the Institute’s facilities as well as instruction in thin-sectioning. She further thanks Mr. K. Akashi, of the Akashi Manufacturing Company, for the assistance of his staff in the preparation of some of the material. The Porter-Blum Microtome used in this study was purchased with a grant from the American Philosophical Society. REPERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
AFZELIUS, B. A., Z. Zellforsch.
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42, 134 (1955).
Proc. Stockholm Conf. on Electron Microscopy, p. 167, 1956.
AFZELIUS, B. A. and MURRAY, A., Exptl. Cell Research 12, 325 (1957). BALLOWITZ,E., Arch, mikroskop Anat. u. Enfwickfungsmech. 65, 96 (1904). COLWIN, A. L., COLWIN, L. H. and PHILPOTT, D. E., J. Biophys. Biochem. Cyfol. 3, 489
(1957). COLWIN, L. H. and COLWIN, A. L., Biol. Bull. 110, 243 (1956). DAN, J. C., Biol. Buff. 103, 54 (1952). ~ ibkl. 107, 203 (1954). ~ Intern. Reu. Cyfol. 5, 365 (1956). FAVARD, P., Ann. Sci. nut. zool. et biol. animate lie ser. XVII, 370 (1955). KILLE, R. A., Personal communication (1959). KRAUSS, M., J. Exufi. Zool. 114, 279 (19501. MIT&ON, ‘J. M., *BxpfZ. Cell Research 10, ‘316 (1956). MOSER, F., J. Expfl. Zool. 80, 447 (1939). RETZIUS, G., Bioi. Unfersuch., Neue Fofge XIX, 43 (1921). SEDAR, A. W. and PORTER, K. R., J. Biophys. Biochem. Cyfol. 1, 583 (1955). TYLER, A., Anaf. Record 113, 525 (1952). WADA, S. K., COLLIER, J. R. and DAN, J. C., Bxpif. Cell Research 10, 168 (1956).
Experimental
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Cell Research 19, 13-28 (1960)
STUDIES VI.
FINE
13
ON THE ACROSOME
STRUCTURE
OF THE JEAN
Ochanomizu
STARFISH
ACROSOME
C. DAN
University,
Tokyo, Japan
Received March 15, 1959
IN a good many species of invertebrate animals [9], and at least two vertebrate groups,1 it has been found that the acrosomal region of the spermatozoon undergoes a change which is interpreted as a triggered response to the presence of the unfertilized egg of its own species. Typically, this change involves the disappearance of at least part of the material making up the acrosome, and the appearance of a structure which for want of a more precise term has been called a “filament”. Depending on the species, this structure may be very long (35-90 ,u in the holothurian, Thyone briareus [6]), or short and stumpy as in some echinoids. Among many problems which must be solved before a satisfactory accounting for this phenomenon can be made is the question of how the filaments originate: whether they are built up to their final dimensions during spermatogenesis and stored compactly in the acrosome to be released explosively under appropriate conditions, or whether their rapid formation from precursor substances is part of the triggered response that occurs when the spermatozoon arrives in the vicinity of an egg. The first of these possibilities characterizes the mechanism found in coelenterate nematocysts; the second is characteristic of ciliate trichocysts [16]. There is of course no reason to believe that the .mechanism of acrosome discharge or the mode of filament formation must conform to either of these examples. They are brought into the discussion not as models, but rather as furnishing assurance that cell organelles of a size roughly comparable to that of spermatozoan acrosomes are capable both of ejecting preformed tubular filaments and of forming such structures very rapidly from non-fibrous precursor materials. Phase contrast observation has failed to reveal a consistent correlation r A filament extending from the acrosome of cyclostome spermatozoa was figured by Ballowitz [4] and Retzius [15]; this observation has recently been confirmed by electron microscopy [3] and cinematography [ll]. In the frog Biscogtossus, Favard [lo] reports the appearance of a neutral red-staining droplet, 2-3 p in diameter, near the tip of the acrosome; he interprets this as a reaction of the acrdsome similar to those occurring among invertebrates. Kille [li], on the other hand, finds that the spermatozoa of these animals form a long acrosome filament. Experimental
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19
Jean C. Dan between the size of the intact acrosome and the length of the filament which it produces. The small size and high refringence of these organelles, moreover, preclude resolution of their exact dimensions and internal structure with light microscopy, and make resort to thin sectioning necessary to secure suc,h information. A study by Afzelius [l, 2,3] has already shown that the sea urchin acrosome in its intact state contains no inclusion which even approximates the tibrillar structure and dimensions of the acrosome filament found in sections of reacted spermatozoa. This result points squarely to the second of the two mechanisms suggested above. The filaments of sea urchin spermatozoa, however, happen to be among the least conspicuous acrosome filaments so far known. In order to widen the basis for drawing a general conclusion concerning the mode of origin of such filaments, and to study a form in which a longer filament results from the acrosome reaction, starfish spermatozoa were fixed in the intact state and after treatment to induce the acrosome reaction, and sectioned for electron microscopy. MATERIAL
AND
METHODS
The spermatozoa of Aster& forbesii were used in this study. For fixing unreacted spermatozoa, “dry sperm” was diluted with sea water to make a 1 per cent suspension; to 1 ml of this suspension was added an equal amount of 2 per cent 0~0, in sea water (pH 8) at room temperature. After 30 minutes of osmium fixation the suspension was post-fixed for 20 hours in 7 per cent formalin-sea water, washed in sea water with light centrifugation and dehydrated with alcohol. Samples were embedded in butyl- plus 15 per cent methyl-methacrylate. To induce the acrosome reaction in these spermatozoa, freshly shed dry sperm was diluted to a 2 per cent suspension with 0.001 M versene in sea water. To this was added an equal amount of egg water (prepared by removing the supernatant from a 50 per cent egg suspension after 30 minutes’ standing). The resulting 1 per cent sperm suspension was fixed one minute after addition of egg water and embedded as described above. The material was sectioned with a Porter-Blum or Sjostrand Microtome and observed with Phillips and Akashi electron microscopes, as indicated in the descriptions of the individual figures. RESULTS’
Intact acrosomes .,The acrosome of the starfish spermatozoon (Fig. 1 and Text-fig. 1A) is set in a characteristic depression in the nucleus, comprising a bowl-shaped region about 0.8 ,u in greatest diameter and 0.5 p in depth, ~1 In the following description, only general terms are used to designate the various regions and substancesof the acrosome,in spite of the resulting awkwardness,sinceit is felt that the assigning of definitive namesshould be basedon a more complete knowledge of their respective functions. Experimentut Cell Research 19
Fine structure of starfish acrosome
15
the base of which opens into a sac-shaped sub-depression about 0.15 ,u in diameter and 0.3 ,u in depth. This whole concavity is lined with the strongly osmiophilic nuclear membrane (Text-fig. 1 A, l).l
Text-fig. Numbers
L-Diagrammatic sketches of Asterias forbesii acrosomes. indicate substances and structures described in text.
A, unreacted.
B, reacted.
The anterior face of the nearly spherical sperm head is radially symmetrical, consisting of an encircling rim of nucleus enclosing the acrosome surface. Although the cytoplasmic membrane (A, 2) is not distinguishable with certainty in the sections presented here, observation of living spermatozoa under experimental conditions indicates that the whole sperm head is covered by a single membrane which can be caused to expand so that it forms a larger sphere within which the nucleus, acrosome and middle piece lie as three separate bodies (see [8], Fig. 2b). Under this closely investing cytoplasmic membrane the topography of the exposed acrosome surface is highly constant, having in the center a raised circular crater (A, 3) surrounded by a peripheral ring of a different substance (A, 4), which is slightly depressed below the adjacent rim of nucleus. In sections through the long axis of the spermatozoon, the acrosome is seen to contain in its center a spherical or somewhat top-shaped mass of homogeneously electron-dense material (A, 5), between 0.2 and 0.3 p in diameter, which appears in all sections to be lacking a bounding membrane. This is completely surrounded (Figs. 1 and 2) by a thick layer of much less dense material (Text-fig. 1 A, 6) which, while not precisely homogeneous, does not show any definite organization, although in some cases there seems to be a suggestion of radial arrangement. This layer is of quite uniform width, 1 Reference
will subsequently
be given to Text-Fig.
1, A and B only as A or B in brackets. Experimental
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Jean C. Dan
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about 0.15 ,u, and follows the shape of the central granule. Its anterior surface is capped (Figs. 3,4) by a thin, dense layer (A, 7) possessing sufficient rigidity to maintain the form of the crater-shaped protuberance (A, 3) which lies exactly over the central granule. The lower margin of this layer is difficult to determine; in most sections it seems to fade out a short distance below the surface, while in some it is apparently continuous with a similar layer (A, 8) which will be described below. Encircling the sides of the mass of non-electron-absorbing substance there is a band, about 0.15 ,u wide and 0.1 ,u thick, of homogeneous material (A, 4), that in most sections is nearly as electron-dense as the central granule (Figs. 1, 2). This band is also strikingly uniform in width at any given level (Fig. 2), and no membrane can be detected between its inner side and the less dense material it surrounds. It also seems to be in immediate contact with the nuclear membrane on its outer side, and its anterior edge extends to the exposed face of the acrosome, where it spreads to some extent under the cytoplasmic membrane (Figs. 1, 2, 3), forming the outer side of the depression which surrounds the central crater-shaped protuberance. Enclosing the posterior half of the mass of least dense material there is a cup-shaped layer (A, 8) which is uniformly thin at the sides and has a thickened plate at its base where it bridges the opening into the sub-depression. Its anterior rim extends a short distance inside the posterior edge of the thick band; the section shown in Fig. 2 lies just at the limit of this layer but not exactly perpendicular to the sperm axis, so that a cross-section of the layer appears at the deeper (lower) side in the micrograph but not at the shallower side. The contents of the sub-depression which is surrounded by nuclear membrane and located approximately in the center of the sperm head present a rather complicated and surprisingly consistent picture. Directly in contact with the outside of the layer (A, 8) described above is a small mass of substance (A, 9) similar in density and general appearance to the central granule (A, 5) in some sections, and to the material of the thick band (A, 4) in others. This fills the anterior part of an inner sac bounded by a strongly osmiophilic membrane (A, 10) apparently identical with that surrounding the nucleus, from which it is separated by a layer of substance (A, 11) about Fig. l.-Longitudinal section through Asferias forbesii acrosome. Section cuts center of acrosome but misses insertion of sperm tail. Regions of acrosome identified in text figure and described in text. M, midpiece N, nucleus (Akashi EM TRS-50). Fig. 2.-Transverse section through greatest diameter of acrosome, nearly parallel to anterior surface of sperm head, but slightly deeper at lower side of micrograph. (Akashi EM TRS-50.) Experimental
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Fine structure of starfish acrosome
17
Jean C. Dan
18
200 A thick and homogeneously dense like the material (A, 9) inside the membrane. This inner membrane, however, does not form a complete secondary sac. Several sections (Figs. 1, 5) show it joining the nuclear membrane at one side (A, 12); the frequency of occurrence of such sections in the available micrographs is four, compared with three sections showing a symmetrical structure as in Fig. 3. The posterior part of the inner sac often contains one or more vesicles (A, 13) somewhat similar to those found in Strongylocentrotus droebachiensis acrosomes by Afzelius [l]. These have an osmiophilic bounding membrane surrounding non-homogeneous contents, and sometimes appear quite empty (Figs. 1, 3, 5). Reacted acrosomes.-The head of the starfish spermatozoon fixed after exposure to egg-water (Figs. 6, 7, 8, 9, 10; Text-fig. 1 B) presents a strikingly different appearance under the electron microscope from the picture just described. The whole complex of acrosomal substances and structures is missing, the nucleus has tilled in the space previously occupied by the acrosome (B, 14), thus reducing the size and changing the shape of the sperm head, and a tilamentous projection extends forward from its anterior surface. In unreacted spermatozoa the cytoplasmic membrane is extremely delicate and lies so closely against the strongly osmiophilic nu.clear membrane that its presence is difficult to detect except where it bridges the junction between midpiece and nucleus. In reacted cells, on the other hand, a fairly thick layer lies outside the nuclear membrane, often without a clearly defined outer margin. On first inspection this gives an impression of poor fixation or damage during preparation, but since unreacted cells in the same sample show no such general effect, it is probable that some rather drastic change has occurred in the relation of the cell membrane to the rest of the cell. This will be discussed below. Examination of many sections shows that there is usually a cone-shaped depression in the nucleus (B, 15) immediately under the filament, lined with nuclear membrane (B, 16). This depression may be shallow (Fig. 6) or deep Fig. 3.-Longitudinal section through acrosome slightly to side of center, showing complete inner membrane of sub-depression (see description of Text-fig. 1, A-12 in text). (Phillips EM 100a.) Fig. 4.-Longitudinal sections of starfish acrosomes. Upper section cuts through center of outer surface but grazes subdepression; lower section parallel to long axis of spermatozoon somewhat to side of center. (Phillips EM 100a.) Fig. J.-Part of section through abnormal depression. (Akashi EM TRS-50.) Experimental
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double spermatozoon,
enlarged to show detail of sub-
Fine sfrucfure of starfish acrosome
19
Jean C. Dan (Fig. 8) or apparently lacking (Fig. 7), although the possibility exists that the plane of section has missed the depression in such cases. Encircling this depression there is a wide ring of homogeneous, relatively dense material (B, 17) lying as a slightly convex plate on the anterior surface of the nucleus so that it forms a broad base for the fibrous strand of the filament (B, 19), which passes through the center of this ring. A delicate but rather strongly osmiophilic membrane covers this region (B, 18) and is continuous anteriorly with the outermost layer of the filament, and posteriorly with the nuclear membrane. Examination of a number of sections shows that this membrane covering the outside of the base of the filament has characteristics closely resembling those of the nuclear membrane lying beneath the filament. In most sections the cell membrane appears not to extend over this basal plate. The filament itself, surrounded by the thin osmiophilic membrane, shows longitudinal striations throughout its interior; in some places separate fibrils having a diameter of the order of 100 hi, can be seen (Figs. 6, 9, 10). This filamentous material has a low electron density and does not form a single solid strand, in cross-section showing apparently empty spaces in irregular arrangement. The filament typically narrows immediately beyond its basal plate to a uniform diameter of about 0.1 ,U (Figs. 6, 7). Some sections show a somewhat wider basal region, which is usually associated with the presence of nonfibrous material (Figs. 8, lo), suggesting incomplete conversion of the filament precursor substance into the fibrous form. DISCUSSION
Even with phase contrast microscopy it is apparent that a profound change has occurred in starfish spermatozoa which have reacted to the presence of egg jelly substance, either surrounding the egg or in sea water solution. In addition to the appearance of the filament, there is a conspicuous rounding-up of the mid-piece, indicating a reduction in tension of the membrane \\hich in the unreacted state held the mid-piece tightly against the posterior Fig. (i.-Acrosome filament of Asterius forbesii spermatozoon, structure throughout filament shaft. (Phillips EM 100a.)
showing
longitudinal
fibrous
Fig. 7.-I.ongitudinal section through reacted spermatozoon, probably somewhat off center. Kate oval shape of nucleus, disrupted appearance of cytoplasmic membrane at left side, relation of membranes at periphery of basal plate. (Phillips EM lOOa.) Fig. X.--Section through under base of filament. Exprimer~lul
reacted Asterius spermatozoon (Phillips EM 100a.)
Cell liesearch 19
with uuusually
deep depression in nucleus
Fine structure of starfish acrosome
Experimental
21
Cell Researc :h 19
Jean C. Dan
22
side of the nucleus. The heads of spermatozoa being drawn through the jelly layer often have an elongated shape (see Fig. 22, g, h in [6]) which is in marked contrast to the turgid roundness of the unreacted cells. This difference shows up clearly in sections (cf. Figs. 7, 8 and 9 with Fig. l), with respect to both the over-all shape of the nucleus and mid-piece and the state of the cytoplasmic membrane. Moreover, the thickened and often disrupted appearance of the cell membrane is definitely correlated with the reacted state; unreacted spermatozoa in the treated suspension show no change in the cell membrane in spite of having been exposed to egg-water. The considerable reduction in volume of the sperm head resulting from the loss of the whole acrosomal mass is enough in itself to account for a marked slackening in the tension of the surrounding membrane. In addition, the cell membrane in the acrosome region must be affected in some way by the formation of the filament. A local rupturing of the membrane is the simplest possibility, but this would not necessarily affect the membrane tension over the whole cell unless the cytoplasmic membrane were free to slip over the nuclear membrane, which does not seem likely. Many of the sections of reacted spermatozoa show this thickened surface layer rather clearly stopping at or near the periphery of the plate forming the base of the filament. Evidence is also available from other forms that fertilizing or egg-watertreated spermatozoa show a tendency to come apart. In both Crassostrea echinata and Urechis unicinctus, the sperm tail drops off when the head enters the egg in about half the cases (Dan, unpublished). In Figs. 17, 20 and 22 of [5], showing Hydroides hexagonus spermatozoa partly within the vitelline membrane, the cell membranes of the spermatozoa are almost completely separated from the nuclear membranes and mid-pieces. Moreover, sea urchin spermatozoa treated with egg-water show separation of the cell membrane from the enclosed structures [2], as well as a shifting in the position of the mid-piece which Tyler [17] has interpreted as a preliminary to the complete separation of the sperm components within the egg cytoplasm. An attempt to visualize, by comparing the morphology of spermatozoa before and after reaction, the process which brings about the observed changes must begin with the fact that the acrosome is no longer embedded in the nucleus. This immediately raises the question as to whether the nucleus Figs. 9 and IO.-Reacted Asterias spermatozoa. nature of basal plate. (Akashi EM TRS-50.)
Note fibrous structure
of filament
and non-fibrous
Fig. Il.-Replica of reacted Asterias amurensis spermatozoon fixed with osmium vapor and dried on glass surface. Rigidity of basal plate of filament is shown by its resistance to flattening. (From Dan [S], permission Biol. Butt.) Experimentot
Cell Research 19
Fine structure of starfish acrosome
23
Jean C. Dan actively undergoes some convulsive form-change which forces out the acrosome contents, or whether the initiative lies with the acrosome. It seems almost a foregone conclusion that the latter must be the case, since it is hard to imagine a cytoplasmic trigger medhatiism which would set off nuclear activity, and even harder to conceive how a nucleus cou’ld generate the force necessary for an explosive change of shape. It will be assumed, then, that the triggered explosion occurs wholly within the acrosome, making its component parts interact to form a filament outside the cell, while the mass of relatively fluid nucle&asm ,flls in the space formerly occupied by the acrosome. The nucleus thus acquires a simple spherical or ovoid shape between the filament attached anteriorly and the posterior mid-piece and flagellum. All the sections of reacted spermatozoa give the impression of a soft, easily deformable state in contrast to the turgid regularity of the unreacted cells. While the mature spermatozoon, as it is shed, is a cell highly specialized for the purpose of finding and uniting with an egg, it can be considered to undergo a final specializing and stripping-down process when the acrosome reacts to the presence of an egg by transforming its precursor material into a filament.1 At least in the starfish spermatozoon, although the mitochondrial mid-piece and the flagellum are stili in place, their functions may be considered ended, once the spermatozoon has established contact with the egg surface. The final implement for introducing the sperm nucleus into the egg must be this filament, which activates the egg and is then drawn into its cytoplasm, dragging the nucleus after it. In order to carry out this function, a firm attachment is necessary between filament and nucleus. All sections of reacted spermatozoa show that such an attachment is secured by the basal plate of the filament which fits like a cap over the anterior surface of the nucleus. In an electron micrograph of a replica taken from reacted Asterins nmurensis spermatozoa dried on a glass slide after osmium vapor fixation (Fig. 1 I), this plate is seen to be extremely rigid in comparison with the adjacent nucleus. Careful examination of micrographs fails to reveal any fibrillar organization in this structure. It is rather certain that the precursor of this plate is the band labelled [4] in Text-fig. 1A. In the unreacted acrosome this band stands perpendicular to the surface of the sperm head, on which it later comes to Iie as a nearly flat plate. It seems most probable that this changed orientation is brought about by an outward spreading of the anterior edge so that it becomes the periphery of the plate, while the inner surface of the band is turned anteriorly. ____-’ Favard [lo] proposes that such a reaction of the acrosome be considered the maturation process-“la maturation propre du spermatixoi’de” (p, 391).
the final
step in
Fine structure of starfish acrosome
25
That such a drastic change can take place indicates that the rigidity seen in the basal plate of the filament is acquired secondarily, during or after the acrosome reaction. It is conceivable that the precursor substance making up this band in the unreacted acrosome undergoes some progressive change in state which induces a hardening together with the change in its form, so that it exerts an expelling force on the central mass of acrosome contents. It seems rather more likely, however, that the expulsion of the rest of the acrosome as it changes into filament leaves a space into which the material of the band is pushed as the nuclear contents round up. The nature of the attachment between the basal plate of the filament and the anterior part of the nucleus presents an interesting problem. In all the reacted spermatozoa, the thin membrane covering this region and extending out around the filament is sharply osmiophilic, closely resembling the nuclear membrane which lies beneath this structure. Moreover, all the available micrographs give the impression that these two membranes unite at the periphery of the plate to make up the membrane covering the rest of the nucleus (vd. Figs. 6, 7, 8, 9, 10). Granted that the nuclear membrane is two-layered, it is still not easy to imagine how the material constituting the basal plate of the filament could intervene between the two layers, even given the possibilities for drastic local rearrangement that would be afforded by an explosive acrosome reaction. At present the simplest explanation would be one postulating that an especially close connection is established between acrosome and nuclear surface during spermatogenesis. If it can be established that the acrosome vesicle displaces the outer layer of the nuclear membrane so that the later differentiation of the acrosome takes place in direct contact with the inner layer of the nuclear membrane, the major part of the problem will have been solved. With respect to the origin of the membrane covering the filament, the two obvious possibilities are that it may be some sort of precipitation membrane laid down on the surface of the Iibrillar core as this extends into the sea water at the time of its formation, or that it may be formed by a tremendous stretching of the acrosome surface membrane. Fig. 6 of reference [8] indicates that it can be separated from the underlying fibrillar core, and that the tip is closed; these characteristics support but do not prove the latter of the alternatives suggested above. By constructing a plasticene scale model of the intact acrosome and then rolling it out into a rod of the appropriate diameter, it was found that the Experimental
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Jean C. Dan
26
amount of available precursor material is roughly adequate to form a filament 25 ,X long, which obviates the necessity for invoking extensive swelling, and points to a change in state of the constituent molecules, such as polymerization or possibly denaturation if the material in question turns out to be protein. In Mytilus edulis it has been shown [ 163 that a lysin which effectively attacks the vitelline membrane of the egg is released from the spermatozoa at the time of acrosome reaction, and the suggestion has been made [7] that the granule at the tip of sea urchin spermatozoa similarly contains a lytic substance. So far, however, no direct evidence has been found to show that echinoderm spermatozoa contain a lysin which attacks echinoderm egg membranes (but see [12]). In the particular case of the starfish, there does not seem to be any place in the series of events making up the fertilization process where a lysin is definitely required. It seems safe to assume that the filament, formed by the acrosome on contact with the outer edge of the jelly layer, is able to pierce this layer with the push derived from the chemical change which causes its formation. The little that is known about the properties of the unfertilized starfish egg surface suggests that it resembles that of the sea urchin egg; electron microscopy shows that the latter is surrounded by a vitelline membrane so delicate that some experienced observers doubt its existence [13]. Presumably a filament with a modicum of rigidity and a diameter of only 0.1 ,u could penetrate such a membrane without having to dissolve it chemically. In view of the fact that pricking with a glass needle is one of the most generally reliable means of activating unfertilized eggs and has been proved effective in echinoderms [ 141, there seems to be no a priori necessity for postulating an activating enzyme in addition to the pricking stimulus delivered by the lilament. At any rate, it is clear that activation of the starfish egg takes place immediately upon penetration of the filament tip, as indicated by cortical granule breakdown, fertilization membrane elevation and cone formation; all these events occur while the sperm head itself is passing through the jelly layer, well away from the egg surface. The next process in which a lysin might conceivably be useful is the passage of the sperm head through the fertilization membrane. However, it is frequently observed that a tip of the fertilization cone extends along the filament and appears outside the fertilization membrane. The advancing sperm head meets and enters the cone, which retreats through the membrane as the sperm head continues to approach the egg surface. This may be interpreted Experimental
Cell Research 19
Fine structure of starfish acrosome
27
as indicating that the hole in the fertilization membrane where the filament pierces it is large enough to permit passage of the sperm head; if a lysin is to be included in the scheme, the fertilization cone tip must be equipped with it as well as the sperm head. Aside from such apparent lack of necessity for lytic activity, the mechanics of lysin transport under the circumstances present some difficulties to the imagination. No indication has been found that a mass of material is carried on the distal end of the filament, as Afzelius [2, 31 has shown to be the case in sea urchins (see also [7], Fig. 14), and since the filament is not tubular, there is no question of a substance being injected through it, even supposing that the tremendous capillary resistance inside a tube 0.1 ,u in diameter could be overcome by some force generated by the reacted spermatozoon. The present study has so far provided no clues at all to the answers of two of the most interesting questions in connection with the structure and reaction of the starfish acrosome: Where is the trigger? and What is the function of the so-called “sub-depression” ? It is hoped that fixation within shorter intervals after initiation of the reaction will provide the solutions to these problems. With respect to the question posed in the introduction, concerning the mode of origin of the filaments in the two groups of echinoderms which have so far been studied in detail, it appears sufficiently certain that any tibrillar elements which may be present are below the limit of resolution of the electron microscopes employed, assuming of course that the method of preparation has removed nothing essential. It is even more certain that neither sea urchin nor starfish acrosomes contain any preformed structures which resemble the definitive filaments, and that in these groups it is appropriate to visualize a rapid transformation of precursor materials into fibrous structures as part of the triggered response of the acrosome. SUMMARY
Electron micrographs of thin-sectioned Asterias forbesii spermatozoa show the acrosome, which is embedded in the nucleus, to be composed of a large (diameter 0.25 ,u) granule of dense material enclosed in a mass of material with low electron density. This is in turn surrounded at its sides by a wide and relatively thick band of a third, rather dense, substance. The anterior surface of the acrosome shows a characteristic profile, with a raised, crater-like structure in the center. Proximally, a double-walled sac containing homogeneous substance and vesicles surrounded by osmiophilic membranes extends into the center of the nucleus. Experimental
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Jean C. Dan
28
Spermatozoa fixed after treatment with egg-water lack this whole complex, and the general appearance of the nucleus and the cytoplasmic membrane indicate a reduction in the volume of the sperm head. From the anterior surface of the nucleus extends a straight rod about 0.1 p in diameter, showing faint longitudinal flbrils within an osmiophilic membrane which is continuous with the outer layer of the nuclear membrane. The base of the filament is set in a slightly convex circular plate which covers the anterior surface of the nucleus and is believed to contain the material of the outermost layer in the intact acrosome. It is concluded that the acrosome filament is not preformed and stored in the intact acrosome, but rather formed very rapidly from precursor substances at the time of the acrosome reaction. This study was performed during the tenure of a Special Research Fellowship granted by the National Cancer Institute, National Institutes of Health, for work in the laboratory of Professor Arthur W. Pollister at Columbia University. The author gratefully acknowledges the kindness of all the Zoology Department members, and particularly the patient instruction of Dr. Jerome Kay and Mr. Robert Ward. The author also wishes to thank Drs. Keith Porter and Montrose Moses, and Mr. Gordon Kaye, of the Rockefeller Institute, for their courtesy in offering use of the Institute’s facilities as well as instruction in thin-sectioning. She further thanks Mr. K. Akashi, of the Akashi Manufacturing Company, for the assistance of his staff in the preparation of some of the material. The Porter-Blum Microtome used in this study was purchased with a grant from the American Philosophical Society. REPERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
AFZELIUS, B. A., Z. Zellforsch.
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42, 134 (1955).
Proc. Stockholm Conf. on Electron Microscopy, p. 167, 1956.
AFZELIUS, B. A. and MURRAY, A., Exptl. Cell Research 12, 325 (1957). BALLOWITZ,E., Arch, mikroskop Anat. u. Enfwickfungsmech. 65, 96 (1904). COLWIN, A. L., COLWIN, L. H. and PHILPOTT, D. E., J. Biophys. Biochem. Cyfol. 3, 489
(1957). COLWIN, L. H. and COLWIN, A. L., Biol. Bull. 110, 243 (1956). DAN, J. C., Biol. Buff. 103, 54 (1952). ~ ibkl. 107, 203 (1954). ~ Intern. Reu. Cyfol. 5, 365 (1956). FAVARD, P., Ann. Sci. nut. zool. et biol. animate lie ser. XVII, 370 (1955). KILLE, R. A., Personal communication (1959). KRAUSS, M., J. Exufi. Zool. 114, 279 (19501. MIT&ON, ‘J. M., *BxpfZ. Cell Research 10, ‘316 (1956). MOSER, F., J. Expfl. Zool. 80, 447 (1939). RETZIUS, G., Bioi. Unfersuch., Neue Fofge XIX, 43 (1921). SEDAR, A. W. and PORTER, K. R., J. Biophys. Biochem. Cyfol. 1, 583 (1955). TYLER, A., Anaf. Record 113, 525 (1952). WADA, S. K., COLLIER, J. R. and DAN, J. C., Bxpif. Cell Research 10, 168 (1956).
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