Spermiogenesis in the Fern Marsilea. Microtubules, Nuclear Shaping, and Cytomorphogenesis

J. CellSci. 23> 57-83 (-977) 57 Printed in Great Britain

SPERMIOGENESIS IN THE FERN MARSILEA. MICROTUBULES, NUCLEAR SHAPING, AND CYTOMORPHOGENESIS

DIANA GOLD MYLES AND PETER K. HEPLER Department of Biological Sciences, Stanford University, Stanford, California 94305 U.S.A.

SUMMARY Spermiogenesis in Marsilea is a complex series of events, including the formation of a helically wound organelle coil. The coil begins to appear in early development when a multilayered structure (MLS) is formed de novo in association with a mitochondrion. The outermost layer of the MLS is a ribbon of microtubules. As the microtubules grow out from the MLS, they grow along the nuclear envelope. The nucleus elongates parallel to the longitudinal axis of the microtubule ribbon until it makes 4-5 gyres. The coil mitochondrion associated with the MLS grows until it completes approximately 9 gyres, following the path of the nucleus in its posterior gyres. The microtubule ribbon is associated with both of these organelles along their exterior edge and separates them from the developing flagellar band. This band arises from dense material that is associated with the basal bodies from, early development, increases in volume, and finally condenses into a solid band, interconnecting the 100—120 basal bodies of the sperm. The flagella are distributed along the entire organelle coil. Besides the microtubules of the ribbon, a set of cytoplasmic tubules is seen during spermiogenesis. These microtubules radiate in bundles from a region of dense nodules located near the MLS. Late in development there is a gross rearrangement of the cytoplasm. This rearrangement begins as the outgrowth of a ring of cytoplasm from the posterior end of the sperm, and continues to grow down around the organelle coil in the anterior end, and fuses to form a bridge, enclosing the anterior end and all the flagella. This bridge fills with cytoplasm that migrates from the posterior region, including many mitochondria and ribosomes, and is finally shed by the sperm when it is released from the microspore and becomes motile. The possible involvement of chromatin condensation and/or microtubules in nuclear morphogenesis is considered. Because much of the shaping of the nucleus occurs before bulk chromatin condensation, the possibility that it is the cause of shaping is ruled out. We postulate that microtubules are involved, not in providing the mechanical force, but as a guide along which shape formation occurs. Furthermore, microtubules of the ribbon were observed to have a close structural relationship to the condensing chromatin.

INTRODUCTION During spermiogenesis profound structural changes occur in the spermatic! cell to produce a sperm. In Marsilea these changes include considerable reshaping of existing organelles, as well as the de novo formation of new ones, to produce a helical organelle coil composed of nucleus, mitochondrion, microtubule ribbon, multilayered structure (MLS), and a flagellar band with over 100 flagella. The structure of the mature sperm has been described in detail elsewhere (Rice & Laetsch, 1967; Myles 58 D. G. Myles and P. K. Hepler & Bell, 1975). The study of spermiogenesis in Marsilea is greatly facilitated by its rapid and synchronized development, since an entire population of single-celled microspores incubated at 20 °C will develop mature sperm in 11 h. During this investigation we have been particularly interested in nuclear differentiation. Nuclear differentiation can be divided into 2 parts: shaping and chromatin condensation. Many previous studies of spermiogenesis have considered these 2 processes to be either directly or indirectly related, and often nuclear-associated microtubules have been implicated in either one or both phenomena. Suggestions of causal relationships include the ideas that: (1) chromatin condensation causes nuclear shaping, either alone or with microtubules as mechanical support (e.g. Fawcett, Anderson & Phillips, 1971); (2) microtubules induce chromatin condensation that in turn is responsible for nuclear shaping (e.g. Stanley et al. 1972); and (3) microtubules are acting directly on nuclear shaping (e.g. Kessel, 1966; Mclntosh & Porter, 1967). In this case they could also be separately involved in chromatin condensation. We have concluded from this study that neither of the first 2 alter- natives is possible in Marsilea, since most of the chromatin condensation occurs after nuclear shaping is largely completed. Our results lead us to support the third alternative as being most likely, with the added consideration that another system, existing near to or as a component of the nuclear envelope, may be involved in both nuclear shaping and chromatin condensation. We also discuss the other important morphogenetic events during spermiogenesis, including shaping of the coil mitochondrion and positioning of the flagella, in relation to the possible role of microtubules in these processes.

MATERIALS AND METHODS Sporocarps were grown to maturity on plants, then harvested and stored dry until use. To produce spermatids, dry sporocarps were ground 3-4 s in a coffee grinder, and shaken through a series of graded sieves to separate the microspores from the megaspores and sporocarp debris. The microspores were then hydrated 1-2 h at 4 °C, given a temperature shock of 30 °C, and cultured at 20 °C in a salt solution with 3 % sucrose (Laetsch, 1967). Under these conditions, the population of spores is close to synchronous in its development. At 20 °C there are 9 mitoses in the first 45 h to produce an endosporic gametophyte with 32 spermatid cells. The spermatids develop into mature sperm by 11 h. Spores were fixed at o-5-h intervals during this development. Fixation was in 2 % glutaraldehyde in 0-05 M phosphate buffer at room temperature for 2-5 h. Spores were rinsed several times with the same buffer and then broken open in a French pressure cell. Cracked spores and free cells were postfixed in 2 % OsO4, collected on a Millipore filter, and embedded in a thin film of warm agar. Dehydration was in 2-methoxyethanol, followed by ethanol and propylene oxide. The material was flat-embedded between 2 fluoro- carbon-coated glass slides in an Araldite-Epon mixture. Sections were made on a Reichert OMU2 with a diamond knife, post-stained with 2 % aqueous uranyl acetate and lead citrate and examined in a Hitachi HU11E electron microscope. For further details see Hepler (1976). Spermiogenesis in Marsilea 59

OBSERVATIONS The mature spermatozoid of Marsilea is shaped like a pear with the narrow anterior end containing a complex coil of organelles spiralled 9-10 times around the edge of the cell. This coil includes the nucleus (found only in the 4-5 posterior gyres), a single long mitochondrion, a ribbon of microtubules, a multilayered structure (in

Plaitid

Nucleus

Mlcrotubule ribbon Multilayered structure Mitochondrion

Fig. 1. A spermatic! during early development. The nucleus is beginning to coil around the anterior edge of the cell. A ribbon of microtubules runs along the exterior edge of the nucleus and of the coil mitochondrion. Between the microtubule ribbon and mitochondrion is the multilayered structure. Other mitochondria are clustered near the nuclear envelope. Plastids are restricted to the posterior region of the cell. The basal bodies have been omitted to reveal the other organelles of the coil. This drawing is based on light- and electron-microscopic observations. the anterior gyre only), and a flagellar band interconnecting the approximately 100-120 flagella spread along the entire length of the coil (Rice & Laetsch, 1967; Myles & Bell, 1975). Formation of the organelle coil is a complex process involving reshaping of the pre-existing nucleus and mitochondria and de novo formation and growth of the microtubule ribbon, multilayered structure, and flagellar band. Differentiation of the spermatid begins immediately after the last spermatogenous division. Early spermatid cells are associated in tetrads. The broad face of the cell, slightly curved and directed away from the other cells of its tetrad (Fig. 4), is the D. G. Myles and P. K. Hepler

*•---**£/"*• m/s Spermiogenesis in Marsilea 61 edge that will become the anterior end of the sperm. A polarity in the cell becomes delineated early in development when the nucleus comes to lie in the anterior region of the cell, and plastids are confined to the region of the cell that later becomes the posterior end of the sperm (Figs, i, 4).

Early development (5-6 h) Nucleus. At 5 h the nucleus is approximately spherical, with a small depression on one side (Fig. 2). The nucleoplasm is mostly uniform, with occasional dense areas that may be the remains of micronucleoli (Fig. 2). During early development the nucleus elongates (Figs. 1, 4), and then the anterior end begins to coil around the cell and becomes narrowed (Fig. 1). Blepharoplast and basal bodies. At 5 h a blepharoplast is located in the indentation of the nuclear envelope, on the outward-facing or presumptive anterior side of the cell. The blepharoplast arises de novo earlier in development, acts as a spindle pole body during cell division (Hepler, 1976), and is now enlarging and transforming into basal bodies (Figs. 2, 3). Triplet and doublet microtubules of the forming basal bodies can be seen in Fig. 3. At this stage the basal bodies are still in a cluster, but as development proceeds, they become distributed along the length of the microtubule ribbon near the nuclear envelope (Figs. 4, 5). Microtubules and multilayered structure (MLS). Also in this nuclear indentation is a multilayered structure (MLS), closely associated with a mitochondrion on one side and the blepharoplast material on the other (Figs. 2, 3). The appearance of the MLS changes, depending on the angle at which it is cut. The outer layer, in contact with the blepharoplast, is the easiest to define, consisting of a ribbon of microtubules in a closely associated single row (Figs. 1, 2, 5). The next layer is best distinguished when the microtubules are cut obliquely and consists of a layer of fine partitions (Fig. 3). The inner layer, closest to the mitochondrion, appears as a dense plaque when the microtubules are cut in cross-section (Fig. 5). As the nucleus elongates, the microtubule layer grows out from behind the MLS and extends along the surface of the nuclear envelope in a direction parallel to nuclear elongation (Figs. 1, 4, 5). The microtubules are in a single row running parallel to one another (Fig. 1). Besides the microtubules of the ribbon, there is an additional, less-ordered set of microtubules in the cell. They are inserted in the dense, amorphous material derived from the blepharoplast and associated with the basal bodies and are particularly frequent near the MLS (Fig. 5). They become more prominent and are better defined as a set during the middle stages of development.

Fig. 2. Section through a spermatic) at 5 h, showing an almost spherical nucleus («). In an indentation of the nuclear envelope is a breaking-up blepharoplast (b). x 17000. Fig. 3. The blepharoplast region at a higher magnification. Forming basal bodies are cut in cross-section, and the newly formed multilayered structure (mis) is shown with its associated mitochondrion (m). x 34000. 5 CEL 23 62 D. G. Myks and P. K. Hepler

bb V

" 1L Spermiogenesis in Marsilea 63 Mitochondrion. The mitochondrion of the coil first becomes defined by its early association with the multilayered structure (Figs. 2, 3). Throughout spermiogenesis it maintains this association with the MLS and extends its length backwards from it as the MLS migrates in an anterior direction. General. Throughout early development dictyosomes are active and both helical polysomes and rough endoplasmic reticulum are common (Figs. 2-5).

Middle development (6-5-8 h) Nucleus. Between 6-5 and 8 h the nucleus elongates to form between one and two gyres (Fig. 6). The posterior region of the nucleus is shaped into a slightly narrowed end, but remains relatively fixed in its position in the cell. The major extension of the nucleus is in an anterior direction, along the microtubule ribbon, and in this region it becomes markedly attenuated so that the nucleus assumes an asymmetrical spindle shape, lying in a coil in the cell (Fig. 6). Also, during this stage, a fine line of dense material becomes visible along the nuclear envelope in the region associated with the microtubule ribbon (Fig. 12). It is assumed that this material is, at least partially, chromatin. Microtubules and multilayered structure (MLS). The multilayered structure keeps its position at the anterior tip of the organelle coil, making approximately 0-5 to 1 gyre (Fig. 6). Microtubules of the ribbon continue to grow out from the MLS in a posterior direction, but there is no evidence that the microtubule ribbon extends in front of the MLS. The greatest number of microtubules are encountered a short distance behind the tip of the coil, with the largest count most frequently appearing in the second cross-section of the coil (approximately 40 microtubules) (Figs. 6, 11). The number of microtubules then decreases to about 30 in successive gyres. At this stage, the second set of microtubules becomes prominent. These microtubules are focused on a dense, amorphous material found in the region of the MLS (Figs. 8, 13, 14) that may be the amorphous material of the blepharoplast (Figs. 3, 5). In Fig. 14 some microtubules appear to end in dense nodules. From a point near the tip of the coil, they radiate out in all directions and are associated in bundles (Fig. 8). Mitochondrion. Many mitochondria are seen surrounding the nucleus as it begins to elongate (Figs. 6, 7). Often these mitochondria present profiles indicative of

Fig. 4. Longitudinal section through a spermatid cell at 5-5 h when the nucleus (n) is already starting to elongate. Basal bodies (bb) are distributed along one edge of the nucleus. At this stage, the cell has a marked polarity with the nucleus occupying the future anterior end of the sperm and plastids (p) restricted to the posterior region, x 10000. Inset: Nomarski phase-contrast micrograph at about the same stage in development, x 1500. Fig. 5. A higher magnification showing a portion of the nucleus (it), microtubule ribbon (mtr), multilayered structure (mb) and its associated mitochondrion (TO). Basal bodies (bb) lie outside the ribbon and are embedded in a dense material. Radiating from this dense material is a set of cytoplasmic microtubules (mt). x 43 500. 5-2 64 D. G. Myles and P. K. Hepler

Plastid

Microtubule ribbon

Nucleus Multllayered structure

Mitochondrion

Fig. 6. A spermatid during the middle stage of development. The nucleus is coiled slighdy more than one complete gyre. A coil mitochondrion extends a short distance in front of the nucleus and is growing backwards toward the posterior end of the nucleus. The microtubule ribbon is continuous along the length of the coil except for a short distance in the posterior end of the nucleus. The multilayered structure makes about one gyre at this stage. Mitochondria cluster around the nucleus and plastids are restricted to the posterior region of the cell. The flagellar band and flagella have been omitted to reveal the other organelles of the coil. This drawing is based on light- and electron-microscopic observations. fusion; it is probable that the coil mitochondrion is growing by the eventual fusion of these mitochondria to it (Figs. 7, 10). As the coil mitochondrion continues to extend its length in a posterior direction away from the MLS (Fig. 6), it remains closely associated with the growing microtubule ribbon, but does not extend into the posterior-most region of the coil, where both the microtubule ribbon and nucleus can be found (Figs. 6, 12). Flagellar band. While the nucleus, microtubule ribbon and mitochondrion are

Fig. 7. Section through 65-h spermatid that cuts longitudinally through part of the nucleus that has already formed one complete gyre. This section shows mitochondria collecting along the nuclear envelope, x 11750. Fig. 8. The cytoplasmic set of microtubules at 65 h. * indicates dense nodules at the central focus of the radiating microtubules. x 26000. Spermiogenesis in Marsilea

v v • ?."*.' ^•d».;.*^-k,r r *.>fi.i-- D. G. Myles and P. K. Hepler Spermiogenesis in Marsilea 67 elongating and making a spiral within the cell, a new organelle, the flagellar band, begins to form along the exterior edge of the coil. It first appears as a dense, amorphous material that is distributed with the basal bodies along the organelle coil (Figs. 9, 11). Because of its appearance and location, we think that this material might be derived from the amorphous regions of the transforming blepharoplast (Fig. 3). During these stages of flagellar band formation, the flagella have not yet reached their final position in the cell and are still oriented at odd angles to each other (Figs. 9, 10). General. By 8 h the overall shape of the cell is not markedly changed, but it is beginning to form a small anterior nose (Fig. 6). Dictyosomes continue to be active, and helical polysomes and rough endoplasmic reticulum are still abundant (Figs. 8, 10).

Late development (8'5-io-5 h) Nucleus. During the last developmental stage, the final shaping of the nucleus into 4 to 5 gyres occurs (Figs. 15, 16, 23). In its final shape, the nucleus is considerably narrowed towards the anterior end (Figs. 15, 18, 20), and in the posterior gyres the nuclear envelope assumes an irregular conformation (Figs. 23, 24)- The bulk of chromatin condensation takes place during this final stage. At 8-5 h, when the nucleus has already made 3-4 gyres and is close to its final shape, chromatin condensation is limited to a narrow line along that region of the nuclear envelope in contact with the microtubule ribbon, and to a few other scattered patches (Figs. 15-17). As condensation continues, chromatin becomes more concentrated in the region of the nuclear envelope next to the microtubule ribbon (Figs. 20, 22). Near the end of differentiation, fibres can still be recognized in the chromatin (Fig. 24) but eventually the chromatin appears as a solid rod (Myles & Bell, 1975). Microtubule ribbon. The elongation of the microtubule ribbon keeps pace with the growth of the organelle coil so that it remains continuous along all but the posterior-most region of the coil (Fig. 15). As in the previous stage, the microtubules reach their greatest number (approximately 40-50) a short distance behind the tip, and the number decreases progressively in successive gyres until the count may be less than 20 (Figs. 15-18). When the nucleus stops elongating but the coil is still growing, the nucleus and the multilayered structure become separated from each other. At maturity the coil will total approximately 9-10 gyres, with the nucleus

Fig. 9. Cross-section through a 65-h spermatid. Adjacent to the nuclear envelope (ne) is a longitudinally-cut microtubule {mi) of the ribbon. A fine line of what may be condensing chromatin (c) is just inside the nuclear envelope in this region. Basal bodies (bb) are cut in cross-section at successive levels and their proximal to distal structure is seen from left to right, x 37000. Fig. 10. Cross-section through the cell at 7 h. The nucleus (n) goes in and out of the plane of the section, revealing part of one gyre. Flagella (/) are growing out of the cell, along the edge of the elongating organelle coil, x 12500. D rrnA P. K

V\r

?.<•

';•>*

«-*&£

•v»*^ 14 Spermiogenesis in Marsilea 69 found only in the posterior four to five gyres (Fig. 23), and the multilayered structure restricted to the anterior-most gyre (Myles & Bell, 1975). Mitochondrion. The coil mitochondrion continues to grow back along the nucleus until it reaches the posterior-most gyre (Figs. 20, 23). At 8-5 h it has already extended close to the posterior tip of the nucleus (Figs. 15, 16), but it will continue to extend in an anterior direction until it completes 9-10 gyres (Fig. 23). This indicates that either the mitochondrion grows along its length, not strictly in a posterior direction, or that it slips past the nucleus as it grows. When the coil mitochondrion is fully formed, many small mitochondria remain in the cytoplasm and will be shed by the sperm in 2 successive stages, first at the time of sperm release (Fig. 21 D) and later in the mucilage surrounding the megaspore (Myles & Bell, 1975). Flagellar band. During this period, there is an increase in the amount of dense material associated with the basal bodies, especially in the anterior end of the coil (Figs. 16, 18, 25). At 8-5 h, substructure is apparent in the dense material (Fig. 18), but by 10-5 h the condensation of the material makes it difficult to detect (Fig. 25). However, it is known that the substructure is conserved since it has been observed in the mature sperm (Myles, 1975). Accompanying this change in the morphology of the flagellar band, there is a realignment of the basal bodies, which in earlier stages were positioned at an angle to each other, so that they become oriented parallel to one another and are tangential to the flagellar band (Figs. 23, 25). They also show an approximate pairing and are distributed along the entire length of the coil. As the flagella lengthen, they wrap themselves around the anterior cone of the sperm (Fig. 25). General. In the final stages of spermiogenesis, the total number of gyres in the organelle coil increases from less than 4 to between 9 and 10. The development of these gyres produces the anterior cone of the sperm, giving the sperm its characteristic pear shape (Figs. 19, 21 D). AS development concludes, a major reorganization of the cell occurs. A ring of cytoplasm from the posterior region of the cell (Figs. 19, 21 A-B) grows down around the anterior cone of the sperm and fuses with itself, so

Fig. 11. Anterior cross-section through the organelle coil at 7 h, showing the positional relationship of nucleus (n), mitochondrion (m), microtubule ribbon (mtr) and the beginnings of the flagellar band (JV). At this stage, the multilayered structure (mis) and nucleus are still found together in the same cross-section, x 40000. Fig. 12. Posterior cross-section of the organelle coil at 75 h. At this stage, the mitochondrion does not yet extend into the posterior-most part of the coil, which in this region includes only nucleus (n), microtubule ribbon (mtr), and a small amount of flagellar band material. Along that region of the nuclear envelope associated with the microtubules, is a line of condensed material, probably chromatin (c). x 40000. Fig. 13. Cross-section through anterior region of the coil showing the coil mitochondrion (m) and the multilayered structure (mis). Exterior to the microtubule ribbon (mtr) is found a dense material with the cytoplasmic set of microtubules (nit) radiating from it. x 47500. Fig. 14. Detail from a similar region in a 7-h spermatid. In this section, some microtubules appear to end in dense nodules (arrow), x 41000. D. G. Myles and P. K. Hepler

Plastid

Nucleus

Mitochondrion

Microtubule ribbon

Multilayered structure

Fig. 15. A spermatid during late development. The nucleus now makes about 4 gyres, and is attenuated in the anterior end. The nucleus has almost reached its final shape, but the mitochondrion will continue to elongate in an anterior direction until it makes about 9 gyres. The microtubule ribbon increases to its maximum number of microtubules (not all drawn in this illustration) shortly behind the anterior tip and then gradually reduces in number towards the posterior end of the coil. The flagellar band and flagella are omitted to reveal the other organelles of the coil. This drawing is based on light- and electron-microscopic observations. that the anterior cone and all of the flagella are enclosed in an internal, but extracellular space (Figs. 20, 21c). It is not known if this enclosure is complete, but no openings to the outside are observed. This newly formed cytoplasmic bridge fills with ribosomes, mitochondria, endoplasmic reticulum, and other organelles until it reaches a volume nearly equal to that of the sperm (Fig. 20). When the sperm are released from the microspore, they will shed this body of cytoplasm from their anterior end (Fig. 21 D), but they will carry with them the other organelles, including plastids and many mitochondria, which remain in their posterior vesicle until the sperm approach the egg (Myles & Bell, 1975). The plastids at this stage appear to have increased in size and decreased in number (Figs. 15, 16, 20). Rough endoplasmic reticulum and helical polysomes are still observed in the cytoplasm (Fig. 23). St>ermiosenesis in Marsilea 71 4

•K * V1 \ *

Fig. 16. Longitudinal section through an 8s-h spermatid. The organelle coil now makes more than 4 gyres, and successive cross-sections through it are numbered from the anterior end. The nucleus is attenuated in the anterior region. The cell is beginning to acquire its final pear shape, x 11500. Inset: Nomarski phase-contrast micrograph at about the same stage in development, x 1500.

DISCUSSION Spermiogenesis is a complex process requiring extensive cellular reorganization and organelle morphogenesis. The resultant sperm cell has a markedly different shape from the spermatid cell and has eliminated a large part of its cytoplasm. The remaining organelles become specifically relocated within the cell and some of them become extensively modified (i.e. the nucleus and mitochondrion), while new organelles (i.e. microtubule ribbon, multilayered structure, and flagellar band) are being formed de nemo. One of the most-discussed and intriguing features of spermiogenesis is the morphogenesis of the nucleus. In both plant and animal sperm, precisely shaped nuclei with condensed chromatin are frequently formed. It is not known what D. G. Myles and P. K. Hepler

\ 1 Spermiogenesis in Marsilea 73 determines the final shape or what provides the force for shaping. Most of the discussion about what is responsible for nuclear shaping centres on 2 ideas: either (1) the final nuclear shape results from the condensation of chromatin within the nucleus, or (2) microtubules are controlling the nuclear shape from outside the nucleus. Evidence that chromatin condensation is determining nuclear shape comes from an example where condensing chromatin takes the final form of the nucleus, and then the nuclear envelope conforms to that shape (Stanley, 1969, and quoted in Fawcett et al. 1971). In addition, cases where microtubules do not appear to be in the right place at the right time (Fawcett et al. 1971) or are entirely missing during spermiogenesis (Lyke & Robson, 1975; Phillips, 1974; van Deurs, 1975) are used in arguments against the hypothesis that microtubules are controlling shaping and therefore indirectly to implicate chromatin condensation as being the responsible force. Sometimes in cases where microtubules are present they have been considered to have a secondary role in shaping, either by imposing external restraints on the shaping by chromatin condensation (Rattner, 1972; Rattner & Brinkley, 1972) or by supporting the developing nucleus (Grier, 1975). In other cases the possibility has been considered that microtubules are acting to induce chromatin condensation which in turn determines the nuclear shape (Stanley et al. 1972; Lora Lamia Donin & Lanzavecchia, 1974). Arguments that microtubules are responsible for shaping nuclei during spermiogenesis follow 2 lines. The first line of evidence is the documentation of their close association with the nucleus during shaping (e.g. Kessel, 1966; Mclntosh & Porter, 1967; Ferraguti & Lanzavecchia, 1971; Duckett, 1973; Lai & Bell, 1975; Szollosi, 1975). An impressive example of this relationship comes from the study of rooster spermiogenesis where microtubules are found in a double helix around the nucleus (Mclntosh & Porter, 1967). These authors postulated that nuclear shaping was the result of microtubules sliding past one another to squeeze the nucleus externally into its elongated form. At maturity these microtubules are replaced with a second set, postulated to support the nucleus and give it a slight bend. Another example of microtubules closely associated with the nucleus during spermiogenesis occurs in Drosophila. In this case microtubules were never seen surrounding the developing nucleus, as they do in rooster spermatids, and therefore

Fig. 17. Higher magnification of a section adjacent to Fig. 16, showing organelle coil cross-sections 7 and 0. In cross-section 0, the mitochondrion has ended and is not seen associated with the nucleus (n). Chromatin (c) is beginning to condense especially along that region of the nuclear envelope associated with the microtubule ribbon (mtr), but scattered patches of condensation occur elsewhere (arrows), x 64500. Fig. 18. Higher magnification of Fig. 16 showing organelle coil cross-sections 2, 4 and 6. Coil mitochondrion (m), microtubule ribbon (mtr), and the nucleus (n) are cut in cross-section. At this stage of development, the dense material forming the flagellar band (fb) has increased in volume. However, it is not yet fully condensed and some substructure can be seen along its outer edge (arrow), x 45 500. 74 D. G. Myles and P. K. Hepler

20 Spermiogenesis in Marsilea 75 Tokuyasu (1974) has proposed an alternative model for microtubule involvement in Drosophila, not involving mechanical force. He suggests that microtubules could be effective in nuclear shaping by serving as a cytoskeleton, so that, as the nucleus is reduced in volume, it conforms to the microtubule cytoskeleton. In addition, he has suggested the possibility of a more active role, with the microtubules inducing the movement of excess nucleoplasm to the cytoplasm. In Drosophila nuclear elongation occurs mostly before chromatin condensation is much advanced. A second line of evidence that brings compelling support for some type of involvement of microtubules in shaping comes from the study of mutants with an abnormal microtubule population and from disruptions induced by microtubule inhibitors and cold. In colchicine-treated plants of the green alga Nitella, the spermatids lack microtubules and their nuclei are misshapen (Turner, 1970). Misshapen nuclei have also been observed in Drosophila spermatids treated with antimicrotubule agents. Incubation with colcemid produces spermatids essentially without microtubules, and non-elongated nuclei (Wilkinson, Stanley & Bowman, 1974). Vinblastine produces abnormal microtubule populations and abnormally shaped nuclei (Wilkinson, Stanley & Bowman, 1975). Abnormally shaped nuclei were also formed in cold-treated locust spermatids with disrupted microtubule populations (Szollosi, 1976). In addition, mutant Drosophila spermatids were shown to lack the normal cytoplasmic microtubules and had nuclei that did not elongate (Wilkinson et al. 1974). Similarly, Dooher & Bennett (1974) have examined a mouse mutant with spermatids showing both abnormal microtubule populations and distorted nuclear shape. This accumulation of evidence makes it increasingly likely that microtubules are involved in nuclear shaping during spermiogenesis in at least some species. Marsilea is probably one such case. Microtubules are present throughout nuclear elongation and persist in the mature sperm. As in Drosophila, much of the nuclear shaping is completed prior to most of the chromatin condensation, eliminating the possibility that bulk chromatin condensation is responsible for shape formation. However, since the microtubule ribbon is oriented parallel to the axis of nuclear elongation, it cannot be 'squeezing' the nucleus externally as postulated for rooster sperm (Mclntosh & Porter, 1967). Microtubules could be pulling the nucleus into its spiral shape by attaching to successive sites on the nuclear envelope as the microtubule ribbon

Fig. 19. Section, along one edge of developing sperm at 95 h showing the excess cytoplasm beginning to grow down around the anterior end of the cell (arrows). A glancing section through the posterior gyres of the nucleus (n) shows areas of condensing chromatin. The flagellar band (Jb) appears as a dark strip, x 8750. Inset: Nomarski phase-contrast micrograph at about the same stage in development. Arrow indicates anterior organelle coil, x 1 500. Fig. 20. Longitudinal section through a later stage where the sides of the cell have finished growing around the anterior end and fused together. This bridge has filled with cytoplasm including many mitochondria and ribosomes, but the plastids (p) remain in the posterior end. The flagella are now enclosed in an internal, but extracellular space (*). x 8750. Inset: Nomarski phase-contrast micrograph at about the same stage in development. Arrow indicates anterior organelle coil, x 1500. D. G. Myles and P. K. Hepler

Fig. 21. Illustration, based on light- and electron-microscopic observations, showing the cytomorphogenesis that results in the shedding of excess cytoplasm, A, a ring of cytoplasm begins to grow down around the anterior coil of the developing spermatid. B, the ring has almost completely enclosed the anterior end forming a veil of 2 thicknesses of plasmalemma and a thin layer of cytoplasm over the flagella and anterior coil, c, the ring fuses with itself enclosing the flagella and anterior region of the sperm in an internal, but extracellular space; the anterior bridge begins to fill with cytoplasm. D, upon release from the microspore, the sperm pinches off the anterior bridge of excess cytoplasm; the cytoplasm rounds up into a sphere and the sperm swims away. Spermiogenesis in Marsilea 77

Fig. 22. Cross-section through 3 gyres of the organelle coil in a 10-h sperm. The nucleus (n) is close to its final shape and the chromatin (c) is in its final stages of condensation. The microtubule ribbon (mtr) lies external to the nucleus and coil mitochondrion (m) and separates them from the flagellar band (Jb). x 57000.

C EL 23 D. G. Myks and P. K. Hepler

,& Spermiogenesis in Marsilea 79- elongates, but we believe it is more likely that they are acting as a guide for nuclear elongation and are not providing the mechanical force. In addition to nuclear shaping, chromatin condensation in Marsilea may be influenced by microtubules, since numerous observations reveal most of the condensation to be near that region of the nuclear envelope closely associated with the microtubule ribbon. This close association between microtubules and condensing chromatin and an accompanying alteration of the morphology of the nuclear envelope has previously been observed during spermiogenesis (e.g. Ferraguti & Lanzavecchia,. 1971; Lanzavecchia & Lora Lamia Donin, 1972; Stanley et al. 1972; Bergstrom & Arnold, 1974; Fonzo & Esponda, 1975), and it has been suggested that microtubules could be inducing chromatin condensation in these areas. Studies of sperm showing alterations in their microtubule composition indicate that chromatin can condense in these cases, but the condensation may be incomplete or abnormal in its patterns. (Wilkinson et al. 1974, 1975; Szollosi, 1976). In those species where microtubules- have been shown to be lacking during spermiogenesis, chromatin condensation still occurs (Phillips, 1974; Lyke & Robson, 1975; van Deurs, 1975), indicating that microtubules may be participating more as an organizing factor than in a causaL role. In fact, other organelles that become closely associated with the nuclear envelope show a similar relation to modifications of the membrane (Schrankel & Schwalm,. 1974) and even to membrane-associated chromatin condensation (Lora Lamia Donin & Lanzavecchia, 1974). During nuclear morphogenesis in Marsilea, both shaping and chromatin condensation show a close structural relationship with the microtubule ribbon. If microtubules are acting as a guide rather than a force in nuclear shaping,, then it is likely that the force-generating system, possibly actin or an actin-like protein, is situated in or near the nuclear envelope and interacts with the microtubules. to determine the shape. This same system, aligned by the microtubules, could also- be responsible for the ordering of chromatin condensation. In addition to the nucleus and microtubule ribbon other organelles, including the coil mitochondrion, form a spiral helix in the cell. The coil mitochondrion is first distinguishable early in development because of its association with the multilayered structure (MLS). Throughout its growth it remains associated with the multilayered structure at its anterior tip and is aligned along the length of the microtubule ribbon. Its acquisition of a helical shape may also, as is postulated for the nucleus,, involve the use of the microtubule ribbon as a guide. The fusion of small mitochondria to each other and to the original MLS-associated mitochondrion is probably its mode of growth, although the reorganization of a single reticulate mitochondrion has not been conclusively ruled out. The coordination of basal body orientation also appears to be related to the

Fig. 23. Longitudinal section of a 105-h sperm, near the time of release. Both the chromatin (c) and the flagellar band (Jb) are very dense. The nuclear envelope (ne) has become irregular in its outline, x 19000. Fig. 24. A glancing section through the organelle coil of a 105-h sperm. Fibres can still be distinguished in the chromatin (arrow), x 37000.

6-2 D. G. Mvles and P. K. Kepler

Fig. 25. An egg's eye view of the sperm, showing the dense flagellar band and the tangential insertion of the flagella winding around the sperm, x 37000. Spermiogenesis in Marsilea 81 microtubule ribbon. Basal bodies become distributed along the microtubule ribbon as it grows in length, along with the dense material that may be derived from the original blepharoplast. There is a gradual increase in the amount of this dense material around the proximal ends of the basal bodies until it finally condenses into a solid band continuous with the basal bodies and interconnecting them (Myles & Bell, 1975). Multilayered structures occur in the spermatid cells of archegoniates including bryophytes (Carothers, 1975), mosses (Lai & Bell, 1975), pteridophytes (Duckett, 1975), and cycads (Norstog, 1975). The association between the microtubule ribbon and the multilayered structure (MLS) may be responsible for the ordered arrangement of microtubules in the ribbon. Possibly the MLS serves as an initiation site or microtubule-organizing centre. However, the location of microtubule growth has not been determined. Microtubule growth at the anterior end of the ribbon would result in either the microtubules protruding in front of the MLS, which is never observed, or sliding past the MLS. On the other hand, if the microtubules are growing at their distal tip, they must slide past the nucleus after it stops elongating, because the distance between the nucleus and the MLS (and therefore the length of the microtubule ribbon in this region) increases during the last stages of development. Evidence for distal tip growth comes from the images of the microtubule ribbon seen after nuclear elongation has stopped. That part of the ribbon anterior to the nucleus demonstrates a hook shape possibly acquired by contact with the nucleus. If the region originally associated with the nucleus is pushed forward by displacement from behind, it would explain the appearance of the hook-shaped ribbon in the area anterior to the nucleus. The extra set of cytoplasmic microtubules seen in the cell is associated with a region near the multilayered structure where they insert on dark nodules of material. These nodules are reminiscent of the material associated with the blepharoplast earlier in development (Hepler, 1976) and may be derived from that material. In earlier stages of development it has been shown that the blepharoblast is active in microtubule initiation (Hepler, 1976). The cytoplasmic microtubules are distinguished from the microtubule ribbon by their much more tenuous association with the multilayered structure and by their lack of a highly ordered association with each other. Also, they are transient, disappearing sometime before the sperm is completely formed. Their location in the cell makes them candidates for functioning as a general cytoskeletal structure. The structure of the mature sperm results from the coordination of numerous morphogenetic phenomena. The microtubule ribbon, probably in conjunction with the cytoplasmic set of microtubules, appears to be an important and possibly controlling factor in this coordination, including nuclear morphogenesis, spiralization of the coil organelles, flagellar distribution, and overall shaping of the cell.

We wish to thank Teppy Williams for her skilful work in both the conceptualization and beautiful execution of the drawings. We also thank Deborah Stairs for assistance with the printing and preparation of the manuscript. This work was supported by a NIH Postdoctoral Fellowship to D.G.M. and NSF Grant no. BMS 74-15245 to P.K.H. 82 D. G. Myles and P. K. Hepkr

REFERENCES BERGSTROM, B. H. & ARNOLD, J. M. (1974). Nonkinetochore association of chromatin and microtubules. A preliminary note. J. Cell Biol. 62, 917-920. CAROTHERS, Z. B. (1975). Comparative studies on spermatogenesis in bryophytes. In The Biology of the Male Gamete (ed. J. B. Duckett & P. A. Racey). Biol. J. Linn. Soc. 7, Suppl. 1, pp. 71-84. London: Academic Press. DOOHER, G. B. & BENNETT, D. (1974). Abnormal microtubular systems in mouse spermatids associated with a mutant gene at the T-locus. J. Embryol. exp. Morph. 32, 749—761. DUCKETT, J. G. (1973). An ultrastructural study of the differentiation of the spermatozoid of Equisetum. J. Cell Sci. 12, 95-129. DUCKETT, J. G. (1975). Spermatogenesis in pteridophytes. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey). Biol. J. Linn. Soc. 7, Suppl. 1, pp. 97-127. London: Academic Press. FAWCETT, D. W., ANDERSON, W. A. & PHILLIPS, D. M. (1971). Morphogenetic factors in- fluencing the shape of the sperm head. Devi Biol. 26, 220-251. FERRAGUTI, M. & LANZAVECCHIA, G. (1971). Morphogenetic effects of microtubules. I. Spermiogenesis in Annelida tubificidae. J. submicrosc. Cytol. 3, 121-137. FONZO, S. & ESPONDA, P. (1975). The relationship between microtubules and chromatin in spermatids of Acrididae. Protoplasma 85, 193-197. GRIER, H. J. (1975). Spermiogenesis in the teleost Gambusia affinis with particular reference to the role played by microtubules. Cell Tiss. Res. 165, 89-102. HEPLER, P. K. (1976). The blepharoplast of Marsilea: its de novo formation and spindle association. J. Cell Sci. 21, 361-390. KESSEL, R. G. (1966). The association between microtubules and nuclei during spermiogenesis in the dragonfly. J. Ultrastruct. Res. 16, 293-304. LAETSCH, W. M. (1967). Ferns. In Methods in Developmental Biology (ed. F. H. Wilt & N. K. Wessells), pp. 319-328. New York: Crowell. LAL, M. & BELL, P. R. (1975). Spermatogenesis in mosses. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), Biol. J. Linn. Soc. 7, Suppl. 1, pp. 85-95. London: Academic Press. LANZAVECCHIA, G. & LORA LAMIA DONIN, C. (1972). Morphogenetic effects of microtubules. II. Spermiogenesis in Lumbricus terrestris. J. submicrosc. Cytol. 4, 247-260. LORA LAMIA DONIN, C. & LANZAVECCHIA, G. (1974). Morphogenetic effects of microtubules. III. Spermiogenesis in Annelida hirudinea. J. submicrosc. Cytol. 6, 245-259. LYKE, E. B. & ROBSON, E. A. (1975). Spermatogenesis in Anthozoa. Differentiation of the spermatid. Cell Tiss. Res. 157, 185-205. MCINTOSH, J. R. & PORTER, K. R. (1967). Microtubules in the spermatids of domestic fowl. J. Cell Biol. 35, 153-173- MYLES, D. G. (1975). Structural changes in the sperm of Marsilea vestita before and after fertilization. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), Biol. J. Linn. Soc. 7, Suppl. 1, pp. 129—134. London: Academic Press. MYLES, D. G. & BELL, P. R. (1975). An ultrastructural study of the spermatozoid of the fern, Marsilea vestita. J. Cell Sci. 17, 633-645. NORSTOG, K. (1975). The motility of cycad spermatozoids in relation to structure and function. In The Biology of the Male Gamete (ed. J. G. Duckett & P. A. Racey), Biol. J. Linn. Soc. 7, Suppl. 1, pp. 135-142. London: Academic Press. PHILLIPS, D. M. (1974). Nuclear shaping in the absence of microtubules in scorpion spermatids. J. Cell Biol. 62, 911-917. RATTNER, J. B. (1972). Nuclear shaping in marsupial spermatids. J. Ultrastruct. Res. 40, 498-512. RATTNER, J. B. & BRINKLEY, B. R. (1972). Ultrastructure of mammalian spermiogenesis. III. Trie organization and morphogenesis of the manchette during rodent spermiogenesis. J. Ultrastruct. Res. 41, 209-218. RICE, H. V. & LAETSCH, W. M. (1967). Observations on the morphology and physiology of Marsilea sperm. Am. J. Bot. 54, 856-866. Spermiogenesis in Marsilea 83 SCHRANKEL, K. R. & SCHWALM, F. E. (1974). Structures associated with the nucleus during chromatin condensation in Coelopa frigida (Diptera) spermiogenesis. Cell Tiss. Res. 153, 45-53- STANLEY, H. P. (1969). An electron microscope study of spermiogenesis in the teleost fish Oligocottus maculosus. J. Ultrastruct. Res. 27, 230-243. STANLEY, H. P., BOWMAN, J. T., ROMKELL, L. J., REED, S. C. & WILKINSON, R. F. (1972). Fine structure of normal spermatid differentiation in Drosophila melanogaster. J. Ultrastruct. Res. 41, 433-466. SZ5LL6SI, A. (1975). Electron microscope study of spermiogenesis in Locusta migratoria (Insect Orthoptera). J. Ultrastruct. Res. 50, 322-346. SZSLLOSI, A. (1976). Influence of infra-optimal breeding temperature on spermiogenesis of the locust Locusta migratoria. II. Abnormalities in differentiation of the nucleus. J. Ultra- struct. Res. 54, 215-223. TOKUYASU, K. T. (1974). Dynamics of spermiogenesis in Drosophila melanogaster. IV. Nuclear transformation. J. Ultrastruct. Res. 48, 284-303. TURNER, F. R. (1970). The effects of colchicine on spermatogenesis in Nitella. J. Cell Biol. 46, 220-234. VAN DEURS, B. (1975). Chromatin condensation and nuclear elongation in the absence of microtubules in Chaetognuth spermatids. J. submicrosc. Cytol. 7, 133—138. WILKINSON, R. F., STANLEY, H. P. & BOWMAN, J. T. (1974). Genetic control of spermiogenesis in Drosophila melanogaster: the effects of abnormal cytoplasmic microtubule populations in mutant MS(3) 10R and its colcemid-induced phenocopy. J. Ultrastruct. Res. 48, 242- 258. WILKINSON, R. F., STANLEY, H. P. & BOWMAN, J.T. (1975). The effect of vinblastine on spermiogenesis in Drosophila melanogaster: evidence for two functional classes of cytoplasmic microtubules. J. Ultrastruct. Res. 53, 354-365. (Received 27 May 1976)