An Actin-Like Component in Sperm Tails of a Crane Fly (Nephrotoma Suturalis Loew)

J. Cell Sci. II, 491-519 (1972) 491 Printed in Great Britain

AN ACTIN-LIKE COMPONENT IN SPERM TAILS OF A CRANE FLY (NEPHROTOMA SUTURALIS LOEW)

A. FORER* Molecular Biology Institute, Odense University, Niels Bohrs Alle, DK-5000 Odense, Denmark AND O. BEHNKE Anatomy Institute C, Copenhagen University, Raadmandsgade 71, DK-2200 Copenhagen N, Denmark

SUMMARY In negatively stained preparations made from glycerinated crane fly spermatids and sperm, actin-like filaments are seen which bind heavy meromyosin (HMM) to form arrowhead complexes, the reaction with HMM being blocked by ATP and pyrophosphate. In preparations from young spermatids the actin-like filaments are found singly, or in small groups, while in those from mature sperm the actin-like filaments are organized into a structure which we call 'rods'. Both rods and single filaments come from lysed sperm tails. The actin-like filaments in rods bind HMM only when frayed out on a grid. In sections of normal or glycerinated spermatids or sperm, no actin-like filaments are seen, either because they are not preserved through the fixation and embedding procedures, or because they are present in a form which we do not recognize. In sections of glycerinated spermatids incubated with HMM, decorated filaments are seen in non-nuclear regions of spermatids (tails), oriented parallel to the axoneme. These probably correspond to the single filaments identified in the negatively stained preparations and not to the rods, because the actin-like filaments in rods bind HMM only after fraying out on a grid. HMM causes polymerization of filaments: actin-like filaments are not seen in negatively stained preparations of glycerinated cells subsequently incubated in salts solution, yet are seen in such cells after a further incubation in HMM. Thus some of the decorated filaments seen in sections may have been polymerized by the HMM, raising questions about the procedure. After rupture of living cells no single actin-like filaments are seen in negatively stained preparations, raising the question of whether glycerol as well as HMM can cause polymerization of filaments. Rods are seen in such preparations, and thus rods seem to exist as such in living cells. Single actin-like filaments and rods are seen even when cytoplasmic microtubules are not seen (after incubation in colchicine or vinblastine). Thus the filaments do not seem to come directly from cytoplasmic microtubules. The possible presence of actin-like filaments in cilia and in other flagella, the location of actin-like filaments in sperm tails, and the possible role of the actin-like filaments are discussed.

INTRODUCTION F-actin can be identified in preparations negatively stained and observed in an electron microscope: F-actin has a characteristic morphology (Hanson & Lowy, 1963), and binds heavy meromyosin (HMM) to form arrowhead complexes (Huxley, 1963). * Present address: Biology Department, York University; 4700 Keele Street, Downsview 463, Ontario, Canada. 492 A. Forer and O. Behnke Thin (5-6 nm) filaments found in a variety of cells have been identified as 'actin-like' (or 'actinoid') because these filaments become decorated with arrowheads after the cells are glycerinated and HMM is added to the external medium (for example, see Ishikawa, Bischoff & Holtzer, 1969; Pollard & Korn, 1971; Perry, John & Thomas, 1971; Behnke, Kristensen & Nielsen, 1971a, b; Allison, Davies & de Petris, 1971), and because ATP or pyrophosphate prevents the HMM binding (Huxley, 1963; Ishikawa et al. 1969; Behnke et al. 19716). Actin filaments are associated with muscle contraction (Huxley, 1969). Since actin-like filaments seem to be associated with amoeboid movements (Pollard & Ito, 1970; Pollard & Korn, 1971; Nachmias, Huxley & Kessler, 1970; Wooley, 1970) and are found in many different kinds of cells, from protozoa (above) to those of man (Behnke et al. 1971a, b), and could be responsible for the motility in these cells (in conjunction with corresponding myosin-like filaments), it seemed likely that all motile systems contain actin-like and myosin-like proteins which interact to cause motility. We therefore looked in sperm tails for actin-like filaments (i.e. those giving arrowhead-complexes with HMM). In this report we describe the occurrence, localization and some properties of actin-like filaments found in sperm tails of crane flies.

METHODS Animals and testes Crane flies {Nephrotoma suturalis Loew) were reared in the laboratory (Forer, 1964). All the cells in any one crane fly testis are spermiogenic and are in roughly the same stage of development at any given time. We studied the following stages of maturation of the sperm: (1) 'young spermatids', in testes which contained both spermatocytes and spermatids, about 8-9 days after the fourth larval instar; (2) 'middle larva spermatids', in testes which contained only spermatids, about 1 day after meiosis; (3) 'old larva spermatids', in testes dissected from larvae about 1 day before pupation and about 2 days older than the 'middle spermatid' larvae; (4) 'pupa spermatids', in testes dissected from pupae, about 1-2 days after pupation; and (5) 'mature sperm', in testes dissected from virgin adult males. Only mature sperm are motile. Dissections were performed under oil, as described elsewhere (Forer, 1965), and testes remained under oil until transferred to one of the experimental solutions.

Solutions The compositions of the various solutions are given here: the exact sequence in which the solutions were used will be described in the text, as will temperatures. For all glycerinations, the glycerol solution was at room temperature. 'Glycerol I standard salts' (G/SS) is a 1:1 mixture of pure glycerol plus standard salts solution. ''Glycerol'/high salts' (G/HS) is a 1:1 mixture of pure glycerol plus high salts solution. Standard salts solution (SS) contained (final concentrations) 0-05 M KC1, 0005 M MgCl2, and 0006 M Sorensen's phosphate buffer (KH2PO4/Na2HPO4), and had a final pH of 70. High Salts solution (HS) contained (final concentrations) 0-5 M KC1, 0-005 M MgCL, and 0006 M Sorensen's phosphate buffer, at a final pH of 7-0. 'ATP/standard salts' (ATP/SS) contained 0-008 M disodium adenosine-s'-triphosphate (Sigma) in SS, at a final pH of 67. 'Pyrophosphatejstandard salts' contained 001 M sodium pyrophosphate in SS, at a final pH of 6-8. Ringer's solution was essentially Ephrussi-Beadle insect Ringer solution (Ephrussi & Beadle, 1936) with buffer, and contained (final concentrations) 013 M NaCl, 0005 M KC1, 0003 M CaClj, and 001 M Na,HPO

Fixation and embedding Testes were fixed with 2 % glutaraldehyde solution which was at the same temperature as the testes (either 4 °C or room temperature). The glutaraldehyde was either 2 % in the same solution as were the testes immediately prior to fixation, or 2% in o-i M cacodylate buffer, pH 68. The testes were placed in glutaraldehyde for between 2 and 36 h, rinsed in SS, post- fixed for 2 h in 1 % osmium tetroxide (in SS), rinsed in H2O, and left overnight in 1 % uranyl acetate (in H2O). They were then dehydrated through a graded series of ethanols, followed by propylene oxide, and embedded in either Araldite or Epon. Sections were stained with 1 % uranyl acetate (in H2O), followed by lead citrate (Reynolds, 1963), and were observed using a Philips EM 300 electron microscope.

Negative staining Testes were placed in a drop of solution (described in the text), the testes sheaths were removed, and the contents of each testis sucked into a i-ml syringe fitted with a 26 G gauge hypodermic needle, and the cells ruptured by repeatedly and forcibly expelling and sucking the drop through the syringe. Drops were then placed on carbon-Formvar coated grids and after about 1 min, blotted off. HMM was added at this point for some grids, and after 1 min was blotted off. The grids (either with or without HMM) were then rinsed with a few drops of 1 % formalin (in SS), fixed for 1 min with 1 % formalin, and negatively stained with 1 % uranyl acetate (Huxley, 1963).

RESULTS Actin-like filaments in negatively stained preparations Specificity of HMM-binding. Actin-like filaments were identified in negatively stained preparations (and in sections) by their ability to form arrowhead complexes with HMM. While there is no evidence indicating that the reaction is not specific, several workers have discussed the question of specificity and have shown that, as observed in negatively stained preparations, HMM does not bind to collagen fibrils or 10-nm diameter filaments (Ishikawa et al. 1969; Hitchcock, 1971), and, as observed in sections, HMM does not bind to 'tonofilaments', to collagen, or to intact microtubules (Ishikawa et al. 1969; Behnke et al. 1971a, b). Nor does HMM bind to the protofilaments of fraying microtubules of blood platelets (Behnke et al. 1971a, b). HMM does not bind to actin which has been briefly fixed with 1 % formalin (on the grid) prior to adding HMM (O. Behnke & A. Forer, unpublished) nor to 10-nm filaments found after lysis of BHK cells (R. D. Goldman, unpublished; O. Behnke & K. Nilausen, unpublished). Thus, since none of these other types of fibrous material seems to react with HMM, the reaction seems specific. 494 A. Forer and O. Behnke Actin-like filaments in glycerinated spermatids and sperm. Testes with young sperm- tids were placed in glycerol/standard salts solution (G/SS) at room temperature: after 2-7 days negatively stained preparations were made by rupture of the cells either in G/SS, or in standard salts solution (SS) (after the testes were briefly rinsed in one change of SS). In both cases filaments were seen which looked like actin and which reacted with HMM to form arrowhead complexes. Without HMM, the actin-like filaments were about 6 nm wide and appeared beaded, with a staggered arrangement of subunits (Figs, i, 2). The filaments were sometimes single (Figs. 1, 2) and sometimes grouped in what appeared to be loose bundles of more or less parallel filaments; they were often several microns long, and while they were sometimes found near the axoneme microtubules, they were also often completely separate from the axoneme. Arrowheads were seen on the filaments after reaction on the grid with HMM (Fig. 3). On any given filament these arrowheads were all pointed in the same direction, and were regularly spaced along the filaments about 36 nm apart. This spacing agrees well with that seen in negatively stained preparations of other actin-like filaments (reviewed in Weihing & Korn, 1971). When ATP/SS or pyrophosphate/SS were added on the grid (after HMM and before formalin), or when the HMM was made up in pyrophosphate/SS no arrowhead complexes were seen, further sub- stantiating the actin-like nature of the filaments. The same procedure was followed for testes containing spermatids at various stages of maturity. In mature sperm the actin-like filaments are organized into structures we have termed 'rods': as the young spermatids became more and more mature there were fewer and fewer single actin-like filaments and more and more rods. In mature sperm the only actin was in rods. The rods generally are about 80 nm wide, they vary in length from 0-2 to several microns, and they contain closely packed filaments arranged parallel to the length of the rods (Fig. 4). The rods are distinct morphologically from 9 + 2 and cytoplasmic microtubules (described in Behnke & Forer, 1967): they are thicker than microtubules, the subunits are arranged in a staggered manner rather than presenting the linear array of globules seen in microtubules, and, unlike microtubules, single filaments are not always parallel to each other. (Protofilaments of microtubules do diverge when microtubules break down on a grid, but otherwise are parallel to each other. In rods, however, one or two filaments are often at an angle to the rest of the filaments.) Furthermore, some rods are wider than 80 nm and branch into 2 or more small rods, 80 nm wide (Fig. 5). Individual filaments in rods react with HMM to form arrowhead complexes, but only the outermost filaments react, and reaction occurs only when the individual filaments fray and separate somewhat from the main body of the rods (Figs. 7, 8). Thus, while it is certain that some of the filaments in rods are actin-like it is not certain that all are. The individual filaments in a rod all have arrowheads pointing in one direction, and while the arrowheads on different filaments may point in the same direction, often they point in opposite directions (Figs. 7, 8): the individual actin-like filaments in any one rod are thus often of opposite polarity. As with the single actin-like Actin-like component in sperm tails 495 filaments, arrow-head formation is inhibited by ATP and pyrophosphate (applied as described above for single actin-like filaments). It has not been possible to detect a consistent arrangement of rods in relation to other structures (in the sense that the 9 + 2 tubules are often arranged parallel to each other in negatively stained preparations). Rods, cytoplasmic microtubules, and axonemal microtubules are often present in the same negatively stained preparation. Microtubules do not seem to react with HMM, and despite long and extensive searching, using various methods of preparation, there is no evidence that rods or single actin-like filaments can transform into microtubules, nor is there any evidence of continuity between microtubules and rods or between microtubules and single actin-like filaments. Since the only part of the sperm that lyses in these preparations is that without the nucleus, the rods and other actin-like filaments come from sperm tails. Direct observation confirms this: rods are often seen leaving an otherwise unbroken axoneme. We have seen nothing which resembles the myosin-like filaments in negatively stained preparations from other glycerinated cells (Behnke et al. 19716; Shoenberg, 1969). Both actin-like filaments and microtubules are stable for extended periods in glycerol/SS: after initial room temperature glycerination and storage for 3 days at room temperature, testes were transferred (in glycerol/SS) to 4 °C, and stored for 2-3 months, after which time negative staining in SS revealed an abundance of actin- like filaments (rods and single filaments), and sections of such cells contained the usual microtubules (described below).

Location of actin-like filaments in spermatids Fixed and sectioned spermatids were studied in order to discover where in the spermatids the actin-like filaments are located. Sections of non-glycerinated spermatids. Testes at various stages of development were dissected and placed directly into glutaraldehyde, and processed for sectioning. In the following the relevant main features of this development are briefly summarized. Young spermatids contain one or more sets of 9 + 2 tubules, as well as mitochondria and cytoplasmic microtubules: the latter are always separated from each other by at least 10 nm, usually more, and though sometimes found in groups of 2 or 3 (Behnke & Forer, 1967) are usually more or less randomly distributed in the cytoplasm (Behnke & Forer, 1967). There are generally 20-50 cytoplasmic microtubules per spermatid (Behnke & Forer, 1967). Each cytoplasmic microtubule is usually surrounded by a less-dense 'clear zone', extending roughly 10 nm outside the microtubule wall. Besides microtubules, groups of 10 or 12 darkly staining 'dots', about 5 nm in diameter, are sometimes seen in cross-sections of spermatids, and, although these have not been identified in longitudinal sections, we previously called them ' tonofilaments' (see fig. 3 of Behnke & Forer, 1967). It must be emphasized that 'tonofilaments' are not regularly seen, and when seen there are at most only 10-20 per cell. As the spermatids mature, accessory tubules appear near each of the 9 doublets (cf. Kaye, 1964), while the cytoplasmic microtubules are grouped more and more around 32 CEL II 496 A. Forer and O. Behnke the mitochondria (see Behnke & Forer, 1967); concomitantly the cells get thinner. In old larva spermatids and especially in pupa spermatids the 9 + 2 and accessory tubules are electron-dense (Fig. 9), with substructure as described in other sperm (e.g. Kaye, 1964). Associated with the mitochondrion is a dense body, with some substructure. A single row of microtubules remains around the outside edge of this body (Fig. 9), and in favourable sections thin electron-dense lines can be seen which seem to connect these microtubules to the dense body. Pupa spermatids also contain dense tubules near each accessory tubule, and, further, the 'secondary filaments' associated with each spoke of the 9 + 2 (Gibbons & Grimstone, i960) are more prominent than in larva spermatids (Fig. 9). In mature sperm the microtubules associated with the peri- mitochondrial body are not seen, while the 9 + 2 tubules, accessory tubules, and tubules associated with the accessory tubules remain electron-dense, with substructure discernible but not well studied by us. Some tonofilaments are seen occasionally at all stages of spermatid development, from young spermatids to pupa spermatids, but not many are seen per cell, and they are not regularly seen. Because muscle actin appears in sections as dense filaments about 6 nm in diameter (Huxley, i960), and because actin-like filaments in other cells also appear as 5-nm filaments in sections (Pollard & Ito, 1970; Behnke et al. 1971b; Kristensen, Nielsen & Rostgaard, 1971), we expected to find such filaments in spermatids. As described above, nothing regularly seen in non-treated cells looks like the expected filaments. Perhaps they are present in another form, or are masked by some other material, in which case they might be made visible after glycerination. Sections of glycerinated spermatids. Testes at various stages of development were glycerinated for 3 days in G/SS, fixed with glutaraldehyde, and processed for sectioning. In all stages there was some variation from cell to cell in the degree of damage and in the appearance of the cells. In all cells the mitochondria and endoplasmic reticulum were rather swollen, or could not be identified at all. In young spermatids the 9 + 2 tubules look the same as in non-glycerinated cells, as does each individual cytoplasmic microtubule; the latter are not evenly distributed, but are present in groups of 6-12. The tubules in each group are quite close to each other and often touch (Figs. 12, 13); instead of a clear zone there is amorphous material associated with most cytoplasmic microtubules (Fig. 13). In pupa spermatids the 9 + 2 and accessory tubules look the same as in non- glycerinated cells, as do the peri-mitochondrial bodies and their associated microtubules (Fig. 10). In no stage are 6-nm filaments seen. Thus we are in a predicament: the negative- staining experiments show that actin-like filaments are present in glycerinated spermatids of all stages of development, yet no such filaments can be regularly identified in sectioned material, either in non-glycerinated or glycerinated cells. This must mean either that the filaments are present in a form which we do not recognize as actin- like in the sections, or that the filaments are not preserved during the procedure used to prepare cells for sectioning. Thus, prior to fixation we added HMM to the cells to see if this would reveal the location of the actin-like filaments. Actin-like component in sperm tails 497 HMM-treated spermatids. Testes with spermatids at various stages of development were glycerinated for 3 days in G/SS, transferred to and stored in HMM/SS for 24-36 h at 4 °C, fixed with glutaraldehyde, and processed for sectioning. In sectioned material, actin-like filaments are seen in larva and pupa spermatids (Figs. 11, 14-17) which look like those described in other cells after reaction with HMM: they appear 'fluffy' (or 'fuzzy'), over 10 nm thick (though this is difficult to measure with precision), and sometimes arrowheads can be seen spaced regularly along them. In young spermatids the actin-like filaments are sometimes found singly but are generally in loosely packed bundles, 0-1-0-2 fan wide, in the non-nuclear region of the spermatid, oriented parallel to the 9 + 2 tubules (Figs. 14-17), confirming the association of filaments with the axoneme region of spermatids. Sometimes the bundles are close to the axoneme but sometimes they are near the cell periphery. Almost all sections, longitudinal as well as oblique, contain identifiable actin-like filaments; thus many more actin-like filaments are present than can be accounted for by tonofilaments. Often several loosely packed bundles of actin-like filaments are present in individual spermatids (Fig. 14). In young spermatids treated in this way cytoplasmic microtubules are sometimes seen and sometimes not. When seen, the microtubules had less associated amorphous material than those in cells fixed prior to the HMM step. In pupa spermatids the actin-like filaments are also seen in loosely packed bundles, in the non-nuclear region of the spermatid, oriented parallel to the axoneme (Fig. 11). Negatively stained preparations were made of spermatids after incubation for 24- 36 h in HMM/SS (these testes were processed in parallel with the fixed-and-sectioned testes and acted as controls for the fixation procedure). The spermatids were ruptured in HMM/SS and then negatively stained. Decorated single filaments are seen in negatively stained preparations from both young spermatids and pupa spermatids. Rods are seen in negatively stained preparations from pupa spermatids, but only some of the rods have filaments with arrowheads, suggesting that HMM does not bind to intact rods in cells but only to those filaments which have frayed out on the grid. In order to see the actin-like filaments in sections without HMM being bound to the filaments, testes were incubated for 36 h in HMM/SS, transferred to room temperature ATP/SS for 5 min, and then fixed with glutaraldehyde and processed for sectioning. No filaments are seen in spermatids treated in this way. We conclude from the results that ATP indeed causes HMM to be released from the filaments, but that either the release causes the filaments to revert to their previous undetected (or unpreserved) form, or ATP causes filament breakdown as well as release of HMM. Because standard salts solution might cause the actin-like filaments to change from an 'undetected' packing arrangement to single filaments, allowing the HMM to bind to the single filaments, we tested the effect of standard salts solution on the actin- like filaments.

32-2 498 A. Forer and O. Behnke

Effect of salts solutions on actin-like filaments in spermatids Testes containing larvae or pupae spermatids were glycerinated for 3 days in G/SS, transferred to SS in which they were stored for 24 h at 4 °C, and then either used for negative staining or fixed and processed for sectioning. In negatively stained preparations single actin-like filaments are not seen in any stage, nor are bundles of actin-like filaments seen. Rods are seen in negatively stained preparations from pupa spermatids, and filaments in these rods bind HMM when this is added on the grid. Thus, single actin-like filaments seem to be depolymerized in standard salts solution, while rods seem to be stable (an alternative possibility is discussed below). In sections neither actin-like filaments nor cytoplasmic microtubules are seen in young spermatids, and no filaments are seen in pupa spermatids. Except for the absence of cytoplasmic microtubules from young spermatids and middle larva spermatids, these cells looked just like those fixed in glycerol/SS. From analogy with actin from skeletal muscle (Huxley, 1963), one would expect actin-like filaments to be more stable in a solution of high KCl concentration than in a solution of low KCl concentration (such as is found in SS). The experiment was therefore repeated but testes were incubated in high salts solution (HS): in negatively stained preparations single actin-like filaments are not seen, but rods are. In sections no filaments are seen. Thus, single actin-like filaments seem to be depolymerized in HS, while rods seem to be stable. The 2 central tubules of the 9 + 2 complex are not seen in sections of larva spermatids after incubation in HS: apparently this solution selectively dissolves them while not affecting the 9 doublets. These tubules are not dissolved in pupa spermatids, indicating that they change properties as spermatids mature. Summarizing the results so far, actin-like filaments are present in glycerinated spermatids, as seen in negatively stained preparations; they are not seen in sections unless the spermatids are first transferred to HMM/SS for 24 h. As based on negatively stained preparations, filaments are not present in spermatids after 24 h in SS; therefore HMM binding to the actin-like filaments apparently protects them against depolymerization.

The state of the actin-like material in spermatids To understand the function of the actin-like material in spermatids, it is necessary to know both the location of the actin-like material in the cells and how it is organized (i.e., as filaments, monomers, or filaments packed into a superstructure, etc.). In sections, actin-like filaments have been identified only after HMM was added. Let us accept for the sake of argument that actin-like filaments are organized into a superstructure that we do not recognize. The following experiment was done to test the possibility that standard salts solution (SS) causes the superstructure to revert to single filaments, and that HMM binds to the single filaments and prevents their further solubilization. Glycerination in the presence of HMM. Testes were glycerinated for 3 days in G/SS Actin-like component in sperm tails 499 which contained HMM, and then either used for negative staining or fixed and processed for sectioning. For negative staining the spermatids were rinsed and ruptured either in the incubation medium or in SS. In the negatively stained preparations from larva and pupa spermatids there are decorated single actin-like filaments. There are rods in preparations from pupa spermatids: in preparations from cells ruptured in SS the actin-like filaments of the rods seen do not have arrowheads, whereas in preparations from cells ruptured in HMM-containing solution the actin-like filaments in some of the rods do have arrowheads. These results show firstly that the testes need not be first transferred to SS in order to achieve HMM binding. Secondly, the lack of arrowheads on the filaments in the rods negatively stained without HMM in the medium substantiates the conclusion that rods do not bind HMM unless the component actin-like filaments first fray out on a grid, and thus shows that in sectioned material rods will not be detected by HMM binding. Perhaps non-frayed rods do not bind HMM because there is a non-actin component in the rods which sterically hinders binding to the actin-like filaments. In sections of both larva and pupa spermatids there are bundles of actin-like filaments in the sperm tail, oriented parallel to the axonemes, which look like those described previously. Thus, since HMM binds to filaments when added in G/SS and the filaments are visible without prior transfer to SS, a 'breakdown' of a higher level of organization induced by standard salts does not seem to be a valid explanation for visibility of filaments after adding HMM. Polymerization of actin-like filaments by HMM. Though admittedly it is difficult to be quantitative in negative-staining experiments, there seem to be many more actin- like filaments in sections of spermatids than in negatively stained preparations. Since rabbit skeletal muscle G-actin can be converted to F-actin by addition of HMM (Yagi, Mase, Sakakibara & Asai, 1965; Kikuchi, Noda & Maruyama, 1969; Tawada & Oosawa, 1969; Cooke & Morales, 1971; J. Emmersen, O. Behnke & A. Forer, in preparation), it is possible that the same change occurs in spermatids. For the sake of argument, consider the possibility that some of the actin-like material in glycerinated spermatids is in monomeric form, that HMM causes this to polymerize to actin- like filaments, and that we detect the HMM-induced filaments in sections and in negatively stained preparations. This possibility was tested as follows. As described above, single actin-like filaments are not present in spermatids after 24 h incubation in SS; if such testes are subsequently incubated with HMM, any actin-like filaments seen must have been formed by the HMM. Larva and pupa testes were glycerinated for 3 days in G/SS, transferred to SS and stored at 4 °C for 24 h; some testes were then fixed and processed for sectioning, whilst others were used for negative staining, and yet others were transferred to HMM/SS and stored for 24 h at 4 °C. After this time the latter testes either were fixed and processed for sectioning, or were used for negative staining (for which the spermatids were ruptured in SS). In negatively stained preparations from both larva and pupa spermatids, single actin-like filaments are not seen in the control preparations incubated in SS, while 500 A. Forer and O. Behnke decorated filaments are seen in preparations made after HMM incubation. In sections fixed after the incubation in SS no actin-like filaments are seen, while in testes fixed after incubation with HMM bundles of actin-like filaments are seen, oriented parallel to the 9 + 2 tubules, though there are certainly many fewer bundles than in the usual preparations. Thus, we conclude that HMM has caused polymerization of some actin-like filaments. If the same procedure is followed but high salts solution (HS) is used after glycerination instead of SS then some bundles of actin-like filaments are seen in sections after incubation in HMM/SS, but fewer than when standard salts solution is used. Since HMM can cause polymerization of actin-like filaments, and since we are concerned about the state of the actin-like material in the spermatids, the question arises whether glycerol, too, might cause polymerization of actin-like filaments. We thus did the following experiments. Negatively stained preparations from living cells. Negatively stained preparations were made from living spermatids of all ages. Testes were transferred to Ringer solution, and cells were then ruptured in (i) Ringer, (2) SS, or (3) HS. Single actin- like filaments were not seen in any preparations, while rods were seen in negatively stained preparations of pupa spermatids in all 3 experiments (Fig. 6), and, like those in preparations from glycerinated spermatids, were seen leaving otherwise unbroken axonemes. Filaments in these rods bound HMM on grids. Thus rods containing actin-like filaments exist as such in living pupa spermatids. Since single actin-like filaments are not seen, it is possible that actin-like material is present in monomeric form in living cells and that glycerol causes polymerization. Alternatively, the single filaments might be unstable under the conditions used, and while they may exist as filaments in living cells, we can only say that we have not seen them in negatively stained preparations. Summarizing the results to this point: as seen in negatively stained preparations actin-like filaments are present in glycerinated spermatids, but they cannot be identified in sections. If HMM is added to the cells decorated filaments are seen in the sections; however, since HMM can cause polymerization of actin-like filaments, some of the filaments seen in sections after HMM may not have been present before HMM. Single actin-like filaments are not seen when living cells are lysed, which raises the question of whether glycerol as well as HMM can cause polymerization of actin-like filaments. Rods containing actin-like filaments are present in negatively stained preparations of lysed cells, both living and glycerinated. Rods do not bind HMM unless the component filaments are fraying out on a grid, so they cannot be identified in sections as HMM-binding material. Table 1 summarizes our experimental results. The last 2 rows in Table 1 concern 'sheath cells': this is a single cell layer, the testis 'sheath', which is removed for negative staining but which is included in sections. These cells contain 5-6 nm diameter filaments in microvilli and apical cytoplasm which are seen in sections of non-treated testes. Since these sheath cell filaments are seen in both normal and glycerinated cells, and are like the more conventional actin- like filaments seen in other cells, they served as a control on our techniques. Do actin-like filaments derive from microtubules? Despite seeing no evidence in Actin-like component in sperm tails 501 negatively stained preparations that microtubules are continuous with or convert directly into actin-like filaments, the following experiments were done to test whether the presence of actin-like filaments requires the previous presence of intact cytoplasmic microtubules. Testes containing young spermatids or middle larva spermatids were placed in colchicine solution; after 60 min some were fixed with glutaraldehyde and processed for sectioning, whilst the rest were glycerinated, either with G/SS containing HMM, or with G/SS alone. Those glycerinated with HMM were fixed after 3 days of glycerination, and then processed for sectioning. Those glycerinated without HMM either were fixed after 3 days of glycerination and processed for sectioning, or after 3 days of glycerination were used for negatively stained preparations, for which the cells were ruptured in SS. As expected, cytoplasmic microtubules are not seen in sections of spermatids after any of the fixations described above (Behnke & Forer, 1967). Filaments are not seen in spermatids fixed immediately after colchicine (Behnke & Forer, 1967), nor in testes fixed after glycerination without HMM. In sections bundles of actin-like filaments are seen in spermatids glycerinated with HMM, and in negatively stained preparations single filaments are seen that bind HMM on the grid. Thus cytoplasmic microtubules need not be present as such in order for the glycerination procedure to make actin-like filaments detectable. Since colchicine usually does not affect the peri-mitochondrial microtubules in late larva spermatids, another 'microtubule poison', vinblastine, was used, as follows. Pupa testes were placed in vinblastine solution for 1 h. Some were then fixed with glutaraldehyde and processed for sectioning, whilst others were treated by one of the two following procedures. (1) Testes were glycerinated with G/SS for 3 days, incubated in HMM/SS for 24 h at 4 °C, and then fixed and processed for sectioning. Bundles of actin-like filaments are seen in sections, and the microtubules near the peri-mitochondrial body are not seen, but neither are vinblastine crystals. This demonstrated that intact microtubules are not required in order to see actin-like filaments, but in order to eliminate the possibility that microtubule monomers contribute to the actin-like filaments, we wanted to demonstrate both vinblastine crystals and actin-like filaments in the same cells. Because the crystals might have been dissolved in the G/SS (Nagayama & Dales, 1970), vinblastine was added to it. (2) Testes were placed in vinblastine solution for 1 h and then were glycerinated with G/SS containing vinblastine at a final concentration of io~3 M. After glycerination for 3 days some testes were fixed and processed for sectioning, whilst others were used for negative staining, and yet others were incubated in HMM/SS, as previously, and then fixed and processed for sectioning. Typical vinblastine 'bedspring' crystals (Marantz & Shelanski, 1970) are seen in sections of spermatids fixed after glycerination or fixed after HMM, and the microtubules near the peri-mitochondrial body are not seen. In sections, decorated filaments are seen after HMM, and in negatively stained preparations 'bedsprings', single actin-like filaments, and rods are seen. Thus, presuming that microtubule proteins are bound quantitatively in vinblastine crystals (Wilson, Bryan, Ruby & Table. I. Summary of expe~imentson crane j?y testes

In~tlaltreatment of Into G/SS tcatrs z days, room temp.--, I I I I 1 1 C 1 4 Subsequent treatment HMM/SS, ATP/SS, SS, 24 h, HMM/SS, SS, 24 h, HS, 24 h, HMM/SS, HS, for rinse

24 h, 4 OC --+ 5 min 4'C -----f 24 h, 4 "C room temp. 4 OC --+ 48 h, 4 'C and rupture 1 I 1 1 1 I 1 of spermatids

Larva testis Negative Rupture in Single - No filaments Some single - No filaments - - spermntids stam G/SS or SS; decornted filaments, with single actln- filaments arrow-hends like filaments +groups of filaments a Fix and No filaments Filament No filaments Aro fikunenb Fewer No filaments No filaments, A few - o% section bundles, or cytoplasmic filaments. hTo or cytoplasmic cytoplasmic filaments. 3 parallel to m~crotubules cytoplasm~c microtubules microtubules, near 9 + axoneme m~crotubules or central microtubules L of 9+2 -0 Pupa testis Negative Some single Some single - Rods, no single Rods. Some - Rods. No - Rods. No spermatids stam filaments, filaments, w~th filaments slngle filaments filaments single a-5 some in loose arrowheads. with filaments 3 bundles; Rods, with arrowheads ~1, mostly rods. and without (Rupture In arrowheads GISS or SS.) FIXand No filaments Filament No filaments No filaments Fewer - Central No filaments - section bundles, filaments microtubules parallel to of 9+2. No axoneme filaments

Larva sheath Section Filaments Filaments bind F~laments,no Filaments Fdaments bind - - F~lamentsbmd - cells HMM HMM HMM HMM binding

Pupa sheath Section F~laments Filaments bind Filaments, no Filaments F~lamentsbind - Some - - cells HhlM HMM H MIM filaments binding Table I (cont.)

Initial treatment of Into G/SS aith HMM, 3 days, testes room temp. In? "W Subsequent treatment - Ruptured in SS - SS for rupture of HS for rupture of - HS, 24 h, 4 "C b spermatlds spermat~ds a Lama testis Negative Single filaments, Single filaments No filaments No filaments - - - 7. spermatids stain with arrowheads with arrowheads (rupture in R) 3 '3 Fix and F~lamentsin - No filaments - - - - section bundles, parallel to 9fz 1 Pupa testis Negatwe Some single Some single Some rods. No hTofilaments. Some rods. No Rods after rupture Rods after rupture spermatids stain decorated filaments. decorated filaments. filaments. Some rods filaments in HS. No ~n HS. No - Rods; some bind Rods wlthout (Rupture in R.) filaments filaments 1 HMRd arrowheads a' .;3 Fix and Fdament bundles, - No filaments - - - - section parallel to axoneme 1 h Larva sheath Sectlon - - Filaments - - - - cells

Pupa sheath Section - - F~laments - - - - cells

-, no observations were made; G, glycerol solution; HS, high salts solution; R, Ringer's solution; SS, standard salts solution. 504 A. Forer and O. Behnke Mazia, 1970), microtubule proteins do not contribute to the actin-like filaments seen.

DISCUSSION Actin-like material is present in sperm tails Actin-like filaments are present in tails of glycerinated Nephrotoma suturalis spermatids at all stages of maturity, the filaments being present in mature, motile sperm only in 'rods' and being present in earlier, non-motile stages of maturity both as rods and as single filaments (or groups of filaments). These filaments are 'actin- like' in that they look like actin, they react with rabbit skeletal muscle HMM to form arrowheads, and ATP or pyrophosphate block the reaction with HMM. Rods are different from single filaments: rods are seen after rupture of living sperm, and thus are present as such in living sperm. Single actin-like filaments, on the other hand, are not seen, and thus this actin-like material may exist as monomer in living sperm; if this is true, the actin-like filaments seen after rupture of glycerinated spermatids were polymerized by the glycerol. As evidence for this indeed being a possibility, rabbit skeletal muscle G-actin polymerized to F-actin when placed in 30 % ethanol (Mihashi & Ooi, 1965), or in 50 % glycerol (J. Emmersen et al. in preparation). Rods are different from single filaments in other respects as well (see Table 1, p. 502): they are stable for 24 h in standard salts solution and in high salts solution, while single filaments are not. Also, the actin-like filaments in the rods seem to bind HMM only after they are frayed out on a grid, while the single filaments bind HMM even in the glycerinated cell. Perhaps these differences arise because the rods contain other components as well as actin, which somehow stabilize the actin. Rabbit skeletal muscle F-actin differs from both single actin-like filaments and rods with respect to stability in salts solutions. Skeletal muscle F-actin is stable in both 0-05 M and 05 M KC1 (Hanson & Huxley, 1957; Kikuchi et al. 1969; Tawada & Oosawa, 1969), which correspond to our standard salts solution (SS) and high salts solution (HS), respectively. Single actin-like filaments from spermatids, on the other hand, seem to be depolymerized in both, for they are not seen in negatively stained preparations of cells lysed after 24 h incubation in either standard salts solution or high salts solution. (One might raise the objection that HS and SS cause extraction of actin-like filaments from spermatids, rather than depolymerization, based on the argument that skeletal muscle actin is extracted during 24 h incubation in 0-5 M KC1 (e.g., Mommaerts, 1958), whilst smooth muscle actin is extracted by low salts as well as high salts (Needham & Shoenberg, 1968). This objection might be valid for incubations with high salts solutions, but seems not to be valid for incubations with standard salts solution: addition of HMM after incubation in SS causes the reappearance of intracellular actin-like filaments, demonstrating that after the incubation in standard salts solution at least some of the actin-like material is present inside the cells. Since the material is present intracellularly and since actin-like filaments are not seen in negatively stained preparations of cells ruptured after incubation in standard salts, the filaments must have been depolymerized by the SS and repolymerized by the HMM.) Actin-like component in sperm tails 505 Rabbit skeletal muscle actin is extracted from muscle during 24 h incubation with 0-5 M KC1 (Mommaerts, 1958). Rods, on the other hand, are still present in spermatids after 24 h incubation in high salts solution. This does not necessarily mean that actin in rods is different from actin in skeletal muscle, for the apparent difference might be explained if the rods contained other components as well as actin. Is actin-like material found in all flagella and cilia, or just in crane fly sperm? A clear answer, of course, would help to assess, for example, whether actin-like material is involved in motility (in which case it might be in all cilia and flagella) or in some other function of sperm (in which case it might be in sperm but not in cilia or flagella). Actin-like material has been looked for in various cilia and flagella; some reports say that no actin-like material is detectable, while other reports say that actin-like material is present. The negative results were obtained using fluorescent antibody techniques (Finck & Holtzer, 1961) and biochemical techniques (Burnasheva, 1958; Burnasheva, Efre- menko, Chumakova & Zueva, 1965). Fluorescent antibodies against chick skeletal muscle actin did not bind in detectable amounts to glycerinated chick sperm or to glycerinated chick ciliated epithelial cells (Finck & Holtzer, 1961). Besides the question of detectability discussed by Finck & Holtzer, one can question this evidence on 2 grounds: first, it appears that antibodies supposedly against actin are in reality not directed against actin but against tropomyosin (Wilson & Finck, 1971). Secondly, the rods in crane fly spermatids seem to bind HMM only on the grid, not in the cells: the same thing which prevents HMM binding in the cells might prevent antibody binding as well. Thus, actin-like material might be present but not detected using those techniques. Actin-like material was not extracted from bull sperm or Tetrahymena cilia using muscle extraction techniques (Burnasheva, 1958; Burnasheva et al. 1965). No actin- like material was found after 24-h extraction with 0-5 M KC1; as discussed above, however, the rods are not extracted from glycerinated crane fly spermatids under these conditions, so perhaps a similar explanation might account for the negative results in bull sperm and Tetrahymena cilia. No actin-like material was found in the water- soluble fraction of acetone powders made from fresh bull spermatozoa or from salt- extracted Tetrahymena cilia. Only 2% of the spermatozoa protein and 10% of the cilia protein are solubilized under these conditions: perhaps rod actin is not soluble. Or perhaps flagella other than crane fly sperm have no actin-like components. (In some preliminary experiments we did not see actin-like filaments in negatively stained preparations of glycerinated sea-urchin sperm (Echinus esculentus) or of isolated Tetrahymena cilia. These cannot be taken as definitive negative results, however.) On the other hand, actin-like material has been reported as being present in bull sperm tails: when sperm tails were treated with high salts plus detergents, a component was extracted which had properties similar to actomyosin, namely, it was soluble in 0-5 M KC1 but precipitated in 0-05 M KCl + o-i mM MgCl2; its viscosity dropped when ATP was added and increased after the ATP was used up; and it had ATPase activity (Young & Nelson, 1968; 1969). A component was separated from the extract which had properties similar to actin, namely, it was depolymerized by KI and 506 A. Forer and O. Behnke repolymerized by KC1 (as measured by viscosity); it had bound nucleotide, and though the data on this point are not absolutely convincing, on mixing with muscle myosin an increase in viscosity was reported, which could be inhibited by ATP. Thus, though here is some indication that actin-like material might be present in bull sperm and absent from isolated Tetrahymena cilia, the present data are incon- clusive. One therefore cannot be certain whether actin-like material is present in cilia and flagella other than sperm tails of crane flies. A corollary to the question of whether actin-like material is present in all cilia and flageUa is whether a myosin-like component is present. Our data on crane fly spermatids are negative: we have not seen filamentous aggregates of myosin in negatively stained preparations, though in our standard salts solution these would be formed from skeletal muscle myosin (Huxley, 1963), smooth muscle myosin (Shoenberg, 1969), and blood platelet myosin (Behnke et al. 1971a, b). Spermatids thus may contain no myosin. On the other hand, perhaps myosin is not seen in ruptured spermatids because spermatid myosin, like Physarum myosin (Adelman & Taylor, 1969), may not aggregate in 0-05 M KC1. Or the rods may consist of both actin-like and myosin-like components, and we cannot identify the myosin-like component in the centre of the rod. A component has been extracted from bull sperm and Tetra- hymena cilia which has solubility properties like myosin and which causes a viscosity increase on mixing with skeletal muscle F-actin, the latter being inhibited by ATP (Burnasheva, 1958; Burnasheva & Raskidnaya, 1968; Young & Nelson, 1968). Thus, while we cannot say whether a myosin-like component is present in crane fly spermatids, there is some evidence for such a component in bull sperm and Tetrahymena cilia.

WJiere in the sperm tail is tlie actin-like material? Because of their different properties, we discuss the rods separately from the single actin-like filaments. Rods are present in living spermatids, and though we are unable to identify them in sections, we expect them to be organized into a structure a few hundred Angstroms or more in diameter (though, in fact, we do not know if they would be circular in cross-section). Rods are present in negatively stained preparations after vinblastine caused microtubules to disappear from around the peri-mitochondrial dense body, and thus these microtubules cannot be the structures giving rise to the rods. Structures associated with the 9 + 2 (such as accessory tubules, tubules associated with accessory tubules, prominent secondary fibres) could be the structures giving rise to the rods, especially since there is an increase in presence of rods parallel to the development of these structures. Single filaments are seen in sections of spermatids only after incubation with HMM; they may not even be present as such in living cells, and thus it is difficult to ascertain where the actin-like material is. The following observations, however, suggest that actin-like material giving rise to the single filaments is associated somehow with cytoplasmic microtubules. Cytoplasmic microtubules appear clumped and have associated amorphous materials in sections of glycerinated young spermatids and middle Actin-like component in sperm tails 507 larva spermatids in which no filaments are seen (see Table 1). The microtubules have less associated amorphous material in sections of HMM-treated spermatids, in which many filaments are seen, parallel to the microtubules. To be consistent with the observations after negative staining we must add that actin-like material is present as fiJaments in glycerinated cells but the filamentous form is not preserved by the fixation and embedding procedure, a possibility previously discussed by Pollard & Ito (1970) and Perry et al. (1971). Relevant to this point, Ishikawa et al. (1969) found that decorated filaments were seen near the surface of dividing cells after HMM treatment, but filaments were not seen in cells fixed by usual procedures. We suggest, then, that actin-like material is somehow associated with cytoplasmic microtubules. This is certainly not to say that microtubules are actin, for we have no evidence of a transformation from microtubule to actin-like filament in negatively stained preparations, and biochemically 9 + 2 tubules are different from actin (Stephens & Linck, 1969). Our suggestion then is that the actin-like material is associated with, but not equivalent to microtubules. We might point out that filaments are aligned even in the absence of visible microtubules, and thus the latter are not necessary for alignment.

What is the role of the actin-like material? Until one knows the exact state of the actin-like material in vivo, where exactly it is located, and whether or not myosin is present, discussion of the functional role of actin-like material in sperm tails is necessarily speculative. Nonetheless, we expect actin-like material to be involved in sperm motility (Behnke, Forer & Emmersen, 1971). If any actin-like component functions in crane fly sperm motion it must be the actin-like filaments in the rods, for this is the only form seen in mature motile sperms. Further, as discussed above, rods are present as such in living cells. Consider the 2 points, (a) the presence of actin-like filaments in rods, and (b) actin- like filaments in the same rod often have opposite polarity. One could argue that both things have something to do with sperm motion. When muscles contract the shorten- ing is in the direction of the actin filaments, whereas when cilia and flagellamov e they undulate in complicated 2-dimensional or 3-dimensional waves (Sleigh, 1969). One could speculate that the rod arrangement has something to do with the bending motion. Single actin-like filaments are not found in motile sperm. In spermatids the filaments are oriented longitudinally, and could be involved somehow with force produc- tion for spermatid elongation.

Implications of our results for the HMM-binding technique We have discussed data indicating that HMM causes polymerization of actin-like filaments in spermatids, similar to the skeletal muscle G-actin polymerization to F-actin (Yagi et al. 1965; Kikuchi et al. 1969; Tawada & Oosawa, 1969; Cooke & Morales, 1971). There is also a possibility that unpolymerized actin-like material in spermatids can be polymerized to filaments by 50 % glycerol, similar to the polymer- 508 A. Forer and O. Behnke ization of skeletal muscle G-actin by 30% ethanol (Mihashi & Ooi, 1965), and by 5°% glycerol (J. Emmersen et al. in preparation). Therefore it may be difficult to interpret data from sectioned cells in which HMM-binding occurs: if similar filaments are not seen in sections of non-treated cells, this may be because the filaments are not preserved as such by the fixation and embedding procedures. But on the other hand, either the glycerol or the HMM may have caused polymerization of filaments. Thus, caution is needed in inferring the state of the actin-like material in vivo from its appearance in glycerinated-HMM-reacted cells. We might also point out that the location is uncertain as well: skeletal muscle actomyosin fixed with glutaraldehyde under certain conditions contracts to a gel which occupies only about one third of the original volume (O. Behnke, A. Forer & J. Emmersen, in preparation), indicating that changes in the location of decorated filaments may occur during fixation. Finally, to make explicit a point implicit in our previous discussion: we accept as likely the proposition that actin (and, by implication, myosin as well) in vivo is in an equilibrium between polymerized and unpolymerized forms, and that, depending on the intracellular conditions, the actin may be completely polymerized, or completely unpolymerized, or in an intermediate state, as has been discussed extensively by others (Hatano, Totsuka & Oosawa, 1967; Pollard & Ito, 1970; Behnke et al. 1971a, b; Weiss & Mayr, 1971). Our data on depolymerization and repolymerization of actin- like filaments may even provide some evidence for this point of view.

We are grateful to Dr J. Emmersen for his generous gift of HMM, and we acknowledge the competent technical assistance of Silla Petersen and Hanne Thinggaard. This work was sup- ported by grants from the Danish Science Research Council (512-1009/71, to O.B. and 511-703 to A.F.) and the Danish Medical Research Council (512-149/69 and 512-1008/71, to O.B.).

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Electron microscopical observations on actinoid and myosinoid filaments in blood platelets. J. Ultrastruct. Res. 37, 351- 369- BURNASHEVA, S. A. (1958). Properties of spermosin, the contractile protein of spermatozoa. Biokhimiya (English transl.) 23, 523-527. BURNASHEVA, S. A., EFREMENKO, M. V., CHUMAKOVA, L. P. & ZUEVA, L. V. (1965). Isolation of the contractile proteins from the cilia of Tetrahymena pyriformis and investigation of their properties. Biokhimiya (English transl.) 30, 656—662. Actin-Uke component in sperm tails 509 BURNASHEVA, S. A. & RASKIDNAYA, N. V. (1968). Characteristics of the ciliary ATPase of Tetrahymena pyriformis. Dokl. Akad. Nauk SSSR (English transl.) 179, 73-76. COOKE, R. & MORALES, M. F. (1971). Interaction of globular actin with myosin subfragments. J. molec. Biol. 60, 249-261. EPHRUSSI, B. & BEADLE, G. (1936). A technique for transplantation for Drosophila. Am. Nat. 70, 218-225. FINCK, H. & HOLTZER, H. (1961). Attempts to detect myosin in cilia and flagella. Expl Cell Res. 23, 251-257. FORER, A. (1964). Evidence for Two Spindle Fiber Components: A Study of Chromosome Movement in Living Crane Fly (Nephrotoma suturalis) Spermatocytes, Using Polarization Microscopy and an Ultraviolet Microbeam. Ph.D. Thesis, Dartmouth College, New Hampshire (U.S.A.) FoRER, A. (1965). Local reduction of spindle fiber birefringence in living Nephrotoma suturalis (Loew) spermatocytes induced by ultraviolet microbeam irradiation. J. Cell Biol. 25 (No. 1, Pt. 2), 95-H7- GIBBONS, I. R. & GRIMSTONE, A. V. (i960). On flagella structure in certain flagellates. J. biophys. biochem. Cytol. 7, 697-716. HANSON, J. & HUXLEY, H. E. (1957). Quantitative studies on the structure of cross-striated myofibrils. II. Investigations by biochemical techniques. Biochim. biophys. Acta 23, 250—260. HANSON, J. & LOWY, J. (1963). The structure of F-actin and of actin filaments isolated from muscle. J. molec. Biol. 6, 46—60. 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Divalent cation activation of flagellar ATP-phospho- hydrolase from bull sperm. J. cell. Physiol. 74, 315-322. [Received 14 February 1972)

Fig. 1-8 are micrographs of negatively stained preparations. Figs. 9-17 are micrographs of sections. Figs. 1, 2. Single actin-like filaments (arrows) from glycerinated young spermatid testes. x 82000 and x 128000, respectively. Fig. 3. A group of actin-like filaments from glycerinated young spermatids, reacted on the grid with HMM. The filaments are decorated with regularly spaced arrowheads, x 82000. Fig. 4. Actin-like filaments in a 'rod' from glycerinated pupa spermatids. x 128000. Fig. 5. A wide rod (from glycerinated pupa spermatids) splitting into 2 smaller rods, x 128000. Actin-like component in sperm tails 511

•••• -W.JIUW w^xr A. Forer and O. Behnke

Fig. 6. A rod from living pupa spermatids, reacted on the grid with HMM. The rod (r) is somewhat cun'ed and is near the axoneme microtubules (a). Filaments in the rod have formed arrowheads with HMM (indicated by arrows), x 82000. Figs. 7, 8. Rods from glycerinated pupa spermatids, reacted with HMM on the grid, x 92000. Fig. 7. The actin-like filaments are decorated, and 2 adjacent filaments have opposite polarity (this is seen at the bottom of the figure, the polarities being indicated by arrowheads, T and A). Fig. 8. Illustrates that the reaction with HMM apparently takes place only when the individual filaments are freely exposed to the HMM: to the left in the picture no arrowheads are seen; to the right the lateral filaments indicated with arrows are decorated, while the central filaments appear not decorated. Actin-like component in sperm tails 5*3

»»i&'

33-2 514 A. Forer and O. Behnke

Fig. 9. Transverse section of non-treated pupa spermatid, illustrating the axoneme (a), mitochondrion (m), perimitochondrial body (pb) and microtubules (pi) associated with this body, x 86000. Fig. 10. Transverse section of a glycerinated pupa spermatid, illustrating the axoneme (a), perimitochondrial body (pb) and microtubules near the body (pi), x 140000. Fig. 11. Longitudinal section of a glycerinated pupa spermatid fixed after incubation with HMM. Decorated actin-like filaments (/) are seen parallel to the axoneme (a), x 82000. Actin-Uke component in sperm tails

; -11 516 A. Forer and O. Behnke

Figs. 12, 13. Transverse sections of glycerinated young spermatids fixed while in glycerol/standard salts solution. Fig. 12. Axonemes (a) and cytoplasmic microtubules (ct) which are clumped together and are associated with some amorphous dense material. No filaments are seen, x 82 coo. Fig. 13. Transversely sectioned microtubules, illustrating the amorphous material associated with them, x 180000. Fig. 14. Oblique section through an early spermatid which had been glycerinated and incubated in HMM. Besides axonemes (a) and cytoplasmic microtubules (ct), bundles of decorated actin-like filaments are seen (some indicated by arrows), predominantly oriented parallel to the cytoplasmic microtubules. x 50000. Actin-like component in sperm tails 517 518 A. Forer and O. Behnke

Figs. 15-17. Longitudinal sections of glycennated young spermatids incubated with HMM, illustrating at a somewhat higher magnification than Fig. 14 the decorated actin- like filaments (/), cytoplasmic microtubules (ct), axonemes (a), and plasma membrane (p) with associated filaments. Fig. 15 is near the centre of the cell, and Figs. 16, 17 are at the periphery of the same cell, x 82000. Actin-like component in sperm tails 519