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Acrosome formation in the pseudoscorpion Diplotemnus sp.
JOURNAL OF ULTRASTRUCTURE AND MOLECULAR STRUCTURE RESEARCH
98, 105-118 (1988)
Acrosome Formation in the Pseudoscorpion Diplotemnussp. GERHARD WERNER* AND S. R. BAWA~ *Medical Biology, Medical Faculty of the Saar University, D-6650 Homburg/Saar, Federal Republic of Germany, and #Department of Biophysics, PatUab University, Chandigarh-160014, India
Received Ju/y 29, 1987 Acrosome morphogenesis in the Pseudoscorpion, Diplotemnus sp., is unusually complex in many respects. The acrosome exhibits polarized organization from the very beginning: An electron-dense granule close to the cell membrane becomes a prospective anterior part and the electron-lucent half of the vesicle is on the side opposite the posterior part. The latter ultimately is transformed into a helical band. First the apical part appears cone-like with characteristic acuteangled helical ridge to which the ceil membrane adheres. In this manner a considerable segment of the cell membrane, which in late spermatids forms a temporary large vacuole, is invaginated. Simultaneously with the elongation of the anterior part, the spherical posterior part of the acrosome together with the surrounding Golgi apparatus shifts deeper into the cytoplasm and contacts the nucleus. The Golgi apparatus contributes additional material only to the posterior part. Anterior as well as posterior parts develop further in quite different ways. The anterior part grows into that region of the acrosome which extends in front of the nucleus, whereas the posterior part first appears as a sheath around the nucleus and then is transformed into a multicoiled helical band around the nucleus. During the unusually long and complicated development of the acrosome the Goigi apparatus changes its appearance several times and discloses characteristic organization attributable to various steps in acrosome morphogenesis. © 1988AcademicPress.Inc. Preliminary studies o f B a w a et al. (1971) In A r a c h n i d a s p e r m i o g e n e s i s is m a r k e d on D i p l o t e m n u s sp. g a v e i n d i c a t i o n s o f b y m a n y characteristic features. Especially r e m a r k a b l e are the e n c y s t e d s p e r m a t o z o a s o m e f e a t u r e s hitherto not reported. In this w h i c h exist in several orders, including the c o m m u n i c a t i o n w e p r e s e n t details w h i c h P s e u d o s c o r p i o n s . O u r k n o w l e d g e o f sper- s u p p l e m e n t p r e v i o u s o b s e r v a t i o n s and also miogenesis in P s e u d o s c o r p i o n s is b a s e d on a t t e m p t to c o r r e c t s o m e o f the i n a c c u r a light m i c r o s c o p y studies b y S o k o l o w (1926) cies. Diplotemnus s p e r m m o r p h o l o g y is rather on Obisium m u s c o r u m and Chelanops cyrneus and N e s t e r (1932) on Chelanops u n u s u a l and v e r y c o m p l e x . We i n t e n d , corticis and electron m i c r o s c o p y investiga- therefore, to handle s e p a r a t e l y the differtions by Boissin and Manier (1966, I967), entiation o f the various c o m p o n e n t s o f the Tuzet et al., (t966) on Hysterochelifer rner- e n c y s t e d s p e r m a t o z o a , In this p a p e r w e idianus, Boissin (1974) on Garypus beau- d i s c u s s the m o r p h o g e n e s i s o f the a c r o s o m e voisi, and L e g g (1973) w h o d e s c r i b e d ma- and the c o n c o m i t a n t c h a n g e s o f the Golgi ture s p e r m a t o z o a in 18 species. Recently, a p p a r a t u s . F o r o t h e r s t r u c t u r e s m e n t i o n e d Dallai and Callaini (1983) reported on the in this c o n n e c t i o n , such as the large central d e v e l o p m e n t o f the giant a c r o s o m e s o f v a c u o l e , the cell wall, or nuclear or mitoGeogarypus and Chthonius but their w o r k chondrial c h a n g e s , the r e a d e r is referred to w a s not s t i p p l e m e n t e d with the requisite f o r t h c o m i n g publications. electron micrographs. S o m e o f the n e w e r MATERIALS AND METHODS findings, h o w e v e r , are c o n t r a d i c t o r y and i n c o m p l e t e . L e g g ' s (1973) s t a t e m e n t that Adult Diplotemnus sp., found hidden in the crevices " a noticeable feature is the a b s e n c e o f an of the bark of Ficus indica, were collected in the vio b v i o u s a c r o s o m e " (p. 437) s e e m s incor- cinity of Chandigarh, India. A pair of pinhead sized testes were excised and examined under oil immersion rect. 105 0889- ! 60_5/88 $3.00 Copyright © 1988 by Acadcmi,. Press, inc. AIJ rights of reproduction in any form reserved.
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ACROSOME OF PSEUDOSCORPION phase contrast optics (x 100). For electron microscopy, testes were fixed for 2 br in 0.1 M cacodylatebuffered 2% glutaraldebyde at room temperature, and postfixed for 2 hr in cacodylate-buffered 2% osmium tetrox,;.de. Subsequently material was dehydrated in ascending concentrations of acetone and embedded in Araldite. Ultrathin sections were cut with diamond knives (Diatome) using an Ultracut (Reichert). Sections mounted on uncoated copper grids were double stained with uranyi acetate and lead citrate after Reynolds and examined with Zeiss EM 109 and Jeol 1200 EX transmission electron microscopes at 80 kV.
RES U LTS
In late spermatocytes the Golgi apparatus comprises many dictyosomes, located in a characteristic polarized fashion (Fig. I). From the endoplasmic system associated with dictyosomes one can detect the budding off of the coated vesicles. During this phase of development the first cistern of the cis face of most of the dictyos o m e s a p p e a r s d i s t e n d e d . Invariably among the dictyosomes vesicles having electron-dense contents are dispersed. These vesicles do not seem to contribute to the formation of the acrosome. At some later time, the dictyosomes aggregate to one place near the cell periphery; by now most of them have lost their individuality (Fig. 2). Small vesicles with floccular contents show up at the lrans face. A large vesicle with similar material approaches the cell membrane. Dilated tubules appear specifically in that region of the Golgi apparatus in which the acrosomal vesicle is developing. As soon as the acrosomal vesicle contacts the cell membrane, an electrondense granule makes its appearance (Figs.
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3, 26a). Thereafter, less electron-dense material accumulates at the opposite side of the attached granule (Fig. 4). Thus the polarity of the acrosome is established with the electron-dense granule lying toward the prospective anterior part. Sometimes the acrosomal vesicle seems to drift toward the nucleus, carrying along with it the cell membrane (Fig. 5). Such inpockets of the cell membrane are always identifiable in the later phases of acrosomal development inasmuch as these contain electron-dense granules interspersed with small vesicles of variable size. Figure 7 perhaps represents a later stage of this unusual situation. In general, however, the acrosomal vesicle elongates in situ and becomes conical, its tip remaining associated with the cell surface (Figs. 6, 26b). The membrane of the conical part reveals stepwise configuration with sharp corners to which the cell membrane adheres and thus is invaginated when the cone elongates. The dilated tubules together with the whole of the Golgi apparatus continue to be associated only with the base of the acrosome, which retains its round vesicular shape. This vesicle enlarges, gets filled with fibrillar material, and then sits on the nucleus with another dense granule at the place of contact (Fig. 7). The developing acrosome is now clearly differentiated into two parts (Fig. 8). The anterior part is delimited by the electron-dense material disposed along the cone. The posterior part is bound by a smooth membrane and material is further contributed only to this vesicular part by the elaborate Golgi apparatus. It appears that the stiffening of
Fro. i. Late spermatoeyte. Areas of dictyosomes with distended first cisterna of the cis face, groups of vesicles with dense contents among them (arrowheads). × 7000. FIG. 2. Beginning of acrosome formation. Goigi apparatus near the cell periphery, vesicle (A) with floccular contents moves to the cell membrane. Dilated tubules (T) in association with the acrosomal vesicle. × 14 250. FIC~. 3. Acrosomal vesicle (A) containing a dense granule in contact with the cell membrane. Dilated tubules (T) near the acrosomal vesicle. × 14 250. FIo. 4. Less dense material accumulates at the site of the attached granule within the acrosomal vesicle (arrowhead). × 14 250. FIo. 5. The acrosomal vesicle along with the cell membrane shifts toward the nucleus. The inpockets contain small dense granules and vesicles (arrowhead). × 14 500. F]o. 6. Acrosomal vesicle in contact with the cell membrane (arrowheads), beginning of cone formation of the anterior part. Golgi apparatus (GA) surrounds the posterior part. × 9000.
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the cone membrane is due to the accumulation of electron-dense material also on its outer face. The acrosome further elongates with the stepwise electron-dense contents becoming more pronounced and its edges always remaining in contact with the inpocketed cell membrane (Fig. 9). The anterior portion of the acrosome is comprised of coarse electron-dense material, whereas the materi~.! on the inner face of the vesicle is deposited in a helical manner. In favorable sections, it is possible to recognize regions of the acrosome with the deposited electron-dense material alternating with regions devoid of such a material (Fig. 10). The posterior portion of the acrosome, however, remains spherical. The Golgi apparatus continues to contribute material only to this part of the acrosome. As soon as chromatin condenses, the spherical posterior portion of the acrosome exhibits "whorl-like" fine fibriilar contents. At this stage the electron-dense granule not only increases in size but appears duplex, its less electron-dense component flattening on the membrane (Fig. 11). It is pertinent to mention that subacrospinal material is conspicuous by its absence at this stage. The anterior portion of the acrosome lengthens considerably, as is evident in the phase-contrast micrographs (Fig. 25), and it is rather rare to have it complete in one section, though its contact
with the cell membrane is retained (Figs. 12, 26c). While the nucleus elongates it becomes ensheathed by the posterior portion of the acrosome (Figs. 13-15, 26c). The latter, no longer is spherical, instead shows pointed contours. The elongated posterior portion of the acrosome initially forming a complete envelope around the nucleus becomes increasingly helicalized (Figs. 14, 15) and then interrupted in the groove, resulting in the winding of the helical band in many turns around the nucleus (Figs. 18, 19). Its fibrillar contents become much more electron dense (Fig. 14). The Golgi apparatus is associated only with this posterior part. The dilated tubules disappear; instead flattened narrower cisternae appear on the t r a n s face (Fig. 26d). The ends of these cisternae often are in close contact with the edges of the developing helical band. The acrosomal membrane, especially at the corners, appears bordered by short filaments at this time. When the chromatin is transformed into distinct lamellae and as these lamellae further condense the Golgi apparatus enters into another active phase (Figs. 16, 26e). During this period randomly orientated cisternae located in the medullary region of the Golgi apparatus appear stuffed with electron-dense material. At many places one finds the interposition of elongated flat cisternae between the aforementioned cisternae (Figs. 17, 26f).
FIG. 7. Differentiation of anterior part (left) and posterior part (right) of the acrosomal vesicle (A): anterior part with the electron-dense material now distributed along the membrane and in contact with the inpocket of the cell membrane (arrowhead); posterior part with smooth membrane surrounded by the Golgi apparatus. Granule at the place of contact with the nucleus (N). × 7500, FxG. 8. Elongation of the anterior part (left) of the acrosome (A), with acute corners always in contact with invaginated cell membrane (arrowheads). Posterior part (right) spherical with smooth membrane surrounded by the Golgi apparatus. Dilated tubules (T) nearby. × 8000. FIG. 9. Longitudinal axis of the anterior part (left) of the acrosome (A) tangential to the nucleus (N). Inpockets of the cell membrane in contact with the acute corners (arrowheads). Coarse material becomes condensed. Posterior part in Contact with the nucleus remains unchanged. × 7500. FIG. 10. Higher magnification of the anterior part of the acrosome. Electron-dense coat of the membrane. The coarse material is deposited on the inside of the membrane in a helical band as indicated by the lines. Invaginated membrane in contact with the corners (arrowhead's). × 22 100. FIG. 11. Beginning of chromatin condensation within the nucleus (N). Anterior part of the acrosome (A) filled with coarse material. Posterior part now shows fine fibrillae. Dense granule with less electron-dense base at the place of contact with the nucleus, x 7000. FIG. 12. Anterior part of the acrosome elongates further while its posterior part is still spherical. Contacts of the cell membrane with the anterior part at the acute corners (arrowheads). x 8000.
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Often, narrow electron-dense processes extend from the helical band (Figs. 18, 26f). During late spermiogenesis, the Golgi apparatus wanes off at the time when the nucleus appears h o m o g e n e o u s l y electron dense (Figs. 19, 20). The helical band is reorganized and becomes slimmer, apparently due to the pinching off of the slender processes. The helical band now mainly is comprised of tubular components (Fig. 19). Masses of granular material with electronlucent internum are seen interspersed among the acrosomal elements (Figs. 19, 21). Further elongation of the nucleus is accompanied by a simultaneous lengthening of the posterior part of the acrosome, resulting in increased helicalization (Fig. 26g). The anterior part of the acrosome is confluent with the posterior part and at the junction of the two is seen an incomplete ring of cytoplasmic material (Fig. 22). Ahead of this ring appears a large globular element with granular material of variable densities which differentiates further (Figs. 23a, 23b). Components of this material surround /he whole of the anterior part of the acrosome except where the invaginated cell membrane is still in contact with the acrosome. Only during late spermiogenesis does subacrosomal material emanate from an invagination of the nucleus and extend into a channel of the anterior part of the acrosome (Fig. 22). An anterior centriole, as m e n t i o n e d by Boissin and Manier (1967), was not found at this place in Diplotetanus. Once the reconstruction of the acrosome is completed the unwanted acrospinal elements disappear. Note that by
I 11
now various organelles in the spermatid are disposed of in a circular fashion around a large temporary vacuole (Figs. 23a, 23b, 25c, 25d). Therefore in many a section both anterior and posterior parts of the acrosome can be found in close apposition to each other. L a t e r on, the s p e r m a t i d shrinks, rounds off, and becomes encysted (Fig. 24). The vacuole tends to become smaller and the various organelles become tightly packed. The final stages of encystment of the spermatozoon will be discussed in a subsequent paper. DISCUSSION
The acrosome formation in Diplotemnus turns out to be a very complicated process, deviating from the usual pattern in many details. From the outset the Golgi apparatus is orientated with its trans face toward the cell periphery. When the acrosomal vesicle appears, although inconspicuous, it abuts the cell membrane. The formation of the acrosome in close apposition with the cell membrane has not yet been reported in Arachnids although it has been observed in Annelids (Anderson and Ellis, 1968). In Diplotemnus the polarity of the anterior and posterior parts of the acrosome is established fairly early in spermiogenesis. So far, these early stages of its morphogenesis have not been reported. Sokolow (1926) and Nester (1932) no doubt described early stages of acrosome formation in the Pseudoscorpions but due to limited resolution of light optics they could not resolve its intricate details. Boissin and
FIG. 13. Beginning of nuclear elongation (N) and its wrapping by the posterior part of the acrosome (A) which develops sharp pointed contours. Golgi apparatus associated with the posterior part. x 7000. FIG. 14. Transformation of the posterior part of the acrosome (A) with condensation of its fibrillar contents. Appearance of long flattened cisternae of the t r a n s face of the Golgi apparatus often ending near the sharp corners (arrowheads). x 12 500. FIG. 15. Elongating nucleus (N) wrapped by the posterior part of the acrosome with increasing helicalization. The arrowhead indicates the junction between the anterior and posterior parts of the acrosome, x 7500. FIG. 16. Longitudinal section through posterior part of the acrosome and nucleus with lamellar chromatin (N) surrounded by the Golgi apparatus. The innermost cisterna of the t r a n s face begins to accumulate electrondense material. The formerly complete wrapping is transformed into the helical band due to interruption in the groove (arrowheads). x 13 500.
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Manier (1967), employing electron microscopy, on the other hand, have described the beginning of acrosome formation only in late spermatids, corresponding to the stage depicted in Fig. 13. From the beginning, both parts of the acrosome develop in different ways but remain interconnected. Sokolow (I926) and Nester (1932) gave correct descriptions of the elongating acrosome but both of them regarded the two parts as distinctly separate structures: "acrosom" and "kopfbliischen" and "acrosome" and "ovoid body," respectively. Generally, the elongating anterior part of the acrosome pushes the posterior part with the associated Golgi apparatus closer to the nucleus. In exceptional instances we find a small vesicle with associated cell membrane traversing the cytopl~iSm to be placed on the nucleus later. In any case it is important to mention that the anterior part of the acrosome remains in contact with the cell membrane. During progressive helicalization of the elongating acrosome, invagination of the adhering cell membrane is further accentuated (Figs. 26a-26d). Possibly such an unusual differentiation process may be due to the fact that these cell membranes later fuse to form a large central vacuole, the details of which will be presented in a forthcoming paper. To the best of our knowledge, to date, such a process has not been observed in any other species. As soon as the condensation of the chromtin ensues the nucleus begins to elongate in the longitudinal axis thereby indenting the spherical posterior part of the acrosome; the latter subsequently en-
113
sheaths the nucleus. At the apical end of the nucleus it passes into its anterior part without any interruption. Simultaneously the smooth membrane of the posterior part forms acute angles and becomes wound in a helix around the nucleus with an increasing number of coils. This sheath, which is complete in the beginning, is transformed later into an interrupted helical band by a very strange process: The sheath is cut through the grooves following the coils of the helix and the thinly extended ends are bent outside and apparently removed. In contrast, Boissin and Manier (1967) imagine the formation of the "spiral" band quite differently. According to them the acrosomal vesicle glides along the head and forms around it a keel-like band which curls itself up as a helix. We also disagree with their contention that the helical band is a product of the Golgi apparatus but only that part of the acrosome which lies in front of the nucleus is characterized as "'complex acrosomien." A helical band as part of the acrosome was, however, described by Osaki (1969) in Heptathela kirnurai, a liphistid spider. Boissin and Manier (1967) in a schematic diagram describe a " c e n t r o s o m e anterieur" lying in a channel at the anterior tip of the nucleus. On the other hand, in Di. plotemnus sp. we find in this place the subacrosomal fiber, and the two coaxially disposed centrioles are located rather in a channel at the posterior end of the nucleus. As the helical band always remains continuous with the anterior part it is an integral part of the acrosome and not a separate entity. The anterior part extending from the
FIn. 17. Cross section through helical band and nucleus with condensed chromatin (N). Advanced stage of accumulation of electron-dense material originating from the Golgi apparatus. × 14 250. FIG. 18. Longitudinal section through the posterior part of the nucleus (N) with adhering nuclear envelope (NE), centriole (C), and flagellum (F). Nucleus and helical band surrounded by the Golgi apparatus. The helical band with slender extended processes (arrowheads). x 13 000. FIn. 19. Oblique section through nucleus (N). Reorganization of the helical band: slender processes are cut off (arrowheads), the contents become tubular, x 23 500. FIn. 20. Cross section through nucleus (N) and helical band. Final stage of its reorganization. The Golgi apparatus has disappeared. The dense material is globular. × 17 500.
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tip of the nucleus is also helicoid with a long pitch. Peculiar to this part is its association with different cytoplasmic structures. In correspondence with the highly complicated structure of the acrosome one observes an unusually prolonged activity of the Golgi apparatus. It begins in the young spermatid and continues thereafter in acrosome reconstruction until the nucleus is completely condensed. Usually the Golgi apparatus parts company with the developing acrosome when the nucleus begins to condense. During spermiogenesis in Diplotetanus sp. one can witness several structural peculiarities of the Golgi apparatus which may be correlated to different functions (compare also Fig. 26): (1) Before acrosome formation (still in late spermatocytes and in very young spermatids)--each dictyosome is characterized by distended first cistern-forming small vesicles of unknown significance. (2) First phase of acrosome formation up
to the stage when fine fibrillae appear in the spherical posterior partmlarge cup-shaped Golgi apparatus associated with dilated tubular system (which seems not to be part of the Golgi apparatus). (3) Elongation and helicalization of the posterior part of the acrosome until chromatin appears lamellar--long, narrow cisternae at the trans face. (4) Transformation of the posterior part into a helical band--last activity phase of the Golgi apparatus prior to its disappearance. The Golgi apparatus forms large quantities of secretory product which apparently help shape up the helical band, Thus, in Diplotemnus, the Golgi apparatus performs different functions in the long history of acrosome morphogenesis. The Golgi apparatus during such a complicated procedure seems to reconstruct and partly degrade its own product. One could say that the Golgi apparatus makes its own raw material to suit its size and shape.
FIG. 21. Longitudinal section through nucleus (N) and helical band. Stage comparable to Fig. 20. Nuclear envelope (NE) surrounds the posterior part of the nucleus. Granular material with electron-lucent internum (arrowheads) among electron-dense Golgi products, x 8000. Fit:;. 22. Oblique section through junctional zone of anterior part of the acrosome (AP) and helical band (H) around the nucleus (N). Different elements of granular material are attached to the anterior part. Incomplete ring (R) and subacrosomal material (arrowhead). × 19 000. Flo. 23. (a) Late spermatid with large central vacuole (V). The capsule of the cyst begins to form underneath the cell membrane (CM). Nucleus (N) with helical band and remnants of the nuclear envelope (NE). Anterior part of the acrosome (AP) in contact with vacuolar membrane, mitochondria (M) and flagellum (F). × 7000. (b) Anterior part of the acrosome (AP) with attached cytoplasmic material of different appearance in contact with the membrane of thelarge central vacuole (V). Ring (R) at the junction between anterior part of the acrosome and the helical band. x 7090. FIG. 24. Encysting spermatid. Cyst wall (CW). Anterior part of the acrosome (AP) with remnants of the former vacuole (V), nucleus (N) with helical band, mitochondria (I',4) and cross sections of the flagellum (F). x 6500. FIO. 25. Phase-contrast micrographs of live spermatids of Diplotemnus sp. (a) Stage corresponding to Figs. 16 and 26e. Condensing chromatin in the longitudinal axis. Dense material in one place near the nuclear envelope (arrowhead). Anterior part (AP) of the acrosome is bent and its posterior helical part is surrounded by Golgi apparatus (GA). A bundle of long filaments emerges from the mitochondria (M) (small arrowheads). (b) Stage corresponding to Fig s. 18 and 26f. Nucleus surrounded by helical band begins to coil. Golgi apparatus (GA), dense material near nuclear envelope (large arrowheads), and bundle of filamentous mitochondria (small arrowheads). (c) Stage corresponding to Figs. 21 and 26g. Increased helicalization of the band near its posterior end. Nuclear er'.'elope (large arrowhead), mitochondriai bundle (small arrowheads), central vacuole (VA). Note the temporary elongation of the cell. (d) Rounding off of the spermatid with the centrally located vacuole (VA), bent anterior part of the acrosome (AP), coiled head with helical band and mitochondrial bundle (arrowheads). (e) Stage corresponding to Fig. 23. Cyst wall (arrowhead), large central vacuole (VA) with head and mitochondrial bundle coiled around it. The granular material (compare Fig. 23b) attached to the anterior part of the acrosome is discernible (white arrowhead). All figures approximately x I000.
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FIn. 26. Schematic drawings of the main stages of acrosome formation and changes of the Golgi apparatus during spermiogenesis in Diplotemnus sp. Drawings are not to scale, nucleus and helical band in the later stages are considerably longer than shown here. (a) Beginning of acrosome formation; acrosomal granule attached to the cell membrane, tubular reticulum nearby. (b) Conical anterior part surrounded by inpockets of the cell membrane which are attached to the acute corner running in a helix along the cone. Spherical posterior part associated with the Golgi apparatus. (c) Growing acrosome together with the Golgi apparatus have reached the nucleus. Inside the spherical part a dense globule appears. (d) Beginning of nuclear condensation and its elongation in the longitudinal axis. Invagination of the posterior part and initiation of its transformation into the helical band. The Golgi apparatus shows long narrow cisternae at its trans face which often come close to the acute corners. (e) The elongating posterior part is cut along the middle between neighboring acute corners thus transforming the formerly uniform sheath of the nucleus into the helical band. Golgi apparatus begins its last activity phase by forming cisternae with electron-dense contents. Membrane invaginations around the anterior part are indicated by profiles attached to the corners. (IF)The extended free ends of the helical band are turned outside and disappear later. Full stage of the last activity phase of the Golgi apparatus. Cisternae of the trans face with electron-dense material and large globules. Membranes around the anterior part are not shown. The apex is bent around various cytoplasmic components. (g) Late spermatid with fully condensed nucleus, remnants of nuclear envelope, and definitive acrosome. Anterior part and helical band are continuous. Subacrosomal material is within a channel formed by invaginations of the nucleus and the acrosome. Goigi apparatus has completely disappeared. The invaginated tubules have fused to form the large central vacuole (not shown here). 117
118
WERNER AND BAWA
Such an unusual process of acrosome formation has no parallel in other animals. It should be pointed out, however, that a postacrosomal activity phase of the Golgi apparatus has been reported in Discogiossus (Sandoz, 1970) and Notonecta (Werner, 1986). In Notonecta obvious lysosomes are formed which devour surplus material, predominantly membrane structures, which is no longer required in the process of sperm maturation. Thus in very elaborate differentiation processes the spermatid seems to develop a lysosomal apparatus of its own and surplus material is used up by the spermatid itself and not simply sloughed off as generally is the case in spermateleosis. In Diplotemnus, there is another feature which needs emphasis. Phillips (1974) has pointed out that microtubules, otherwise usually found as shape-forming elements in spermiogenesis, are missing during nuclear elongation in Scorpions. In Diplotemnus microtubules are nonexistent throughout spermiogenesis (Bawa and Werner, 1983). The absence of such ubiquitous structures is not at all attributed to improper fixation of the tissues, as we have observed large populations of microtubules in the testicular wall cells. Finally, we do not visualize any relationship between the immense size of the acrosome in Diplotemnus and the length of the nucleus and the axoneme as
suggested by Dallai and Callaini (1983) who explain that the acquisition of a giant sized acrosome in Geogarypus results in the concomitant diminution of its flagellum and the nucleus. The authors thank Miss R. Kaur (Chandigarh) for processing, Mrs. E. Maatz for sectioning the material, Mrs. G. Kiefer for the photographic work, and Mrs. E. Frank (all Homburg) for typing the manuscript. One of us (S.R.B.) gratefully acknowledges the support of the Alexander-von-Humboldt-Stiftung. REFERENCES ANDERSON, W. A., AND ELLIS, R. A. (1968) Z. Zellforsch. 85, 398-407. BAWA, S. R., SJOSTRAND, E S., KANWAR, U., AND KANWAR, K. C. (1971)J. Ultrastruct. Res. 37, 251. BAWA, S. R., AND WERNER, G. (1983) its ANDRE, J. (Ed.), The Sperm Cell, Proc. IVth lnternatl. Syrup. Spermatol., pp. 245-248, Nijhoff, The Hague/ Boston/London. BOISSIN, L. (1974). Arch. Zool. E.rp. Gen. 115, 169-184. BOISSlN, L., AND MANIER, J.-E (1967) Btdl. Soc. Zool. France 92, 705-712. DALLAI, R., AND CALLAINI, G. (1983) in ANDRE, J, (Ed.), The Sperm Cell, Proc. IVth Internatl. Syrup. Spermatol., pp 431-435, Nijhoff, The Hague/ Boston/London. LEGG, G. (1973). J. Zool. (London) 170, 429-440. NESTER, H. G. (1932). J. Morphol. 53, 97-131. OSAKI, H. (1969)Acta Arachnol. 22, 1-13. PHILLIPS, D. M. (1974) J. Cell Biol. 62, 911-917. SANDOZ, D. (1970)J. Microsc. 9, 243-262. SOKOLOW, I. (1926) Z. Zellforsch. 3, 615-681. TUZET, O., MANIER, J.-E, AND BOISSIN, L. (1966) C.R. Acad. Sci. Paris 262, 376-378. WERNER, G. (1986)Biol. Cell 57, 169-180.
98, 105-118 (1988)
Acrosome Formation in the Pseudoscorpion Diplotemnussp. GERHARD WERNER* AND S. R. BAWA~ *Medical Biology, Medical Faculty of the Saar University, D-6650 Homburg/Saar, Federal Republic of Germany, and #Department of Biophysics, PatUab University, Chandigarh-160014, India
Received Ju/y 29, 1987 Acrosome morphogenesis in the Pseudoscorpion, Diplotemnus sp., is unusually complex in many respects. The acrosome exhibits polarized organization from the very beginning: An electron-dense granule close to the cell membrane becomes a prospective anterior part and the electron-lucent half of the vesicle is on the side opposite the posterior part. The latter ultimately is transformed into a helical band. First the apical part appears cone-like with characteristic acuteangled helical ridge to which the ceil membrane adheres. In this manner a considerable segment of the cell membrane, which in late spermatids forms a temporary large vacuole, is invaginated. Simultaneously with the elongation of the anterior part, the spherical posterior part of the acrosome together with the surrounding Golgi apparatus shifts deeper into the cytoplasm and contacts the nucleus. The Golgi apparatus contributes additional material only to the posterior part. Anterior as well as posterior parts develop further in quite different ways. The anterior part grows into that region of the acrosome which extends in front of the nucleus, whereas the posterior part first appears as a sheath around the nucleus and then is transformed into a multicoiled helical band around the nucleus. During the unusually long and complicated development of the acrosome the Goigi apparatus changes its appearance several times and discloses characteristic organization attributable to various steps in acrosome morphogenesis. © 1988AcademicPress.Inc. Preliminary studies o f B a w a et al. (1971) In A r a c h n i d a s p e r m i o g e n e s i s is m a r k e d on D i p l o t e m n u s sp. g a v e i n d i c a t i o n s o f b y m a n y characteristic features. Especially r e m a r k a b l e are the e n c y s t e d s p e r m a t o z o a s o m e f e a t u r e s hitherto not reported. In this w h i c h exist in several orders, including the c o m m u n i c a t i o n w e p r e s e n t details w h i c h P s e u d o s c o r p i o n s . O u r k n o w l e d g e o f sper- s u p p l e m e n t p r e v i o u s o b s e r v a t i o n s and also miogenesis in P s e u d o s c o r p i o n s is b a s e d on a t t e m p t to c o r r e c t s o m e o f the i n a c c u r a light m i c r o s c o p y studies b y S o k o l o w (1926) cies. Diplotemnus s p e r m m o r p h o l o g y is rather on Obisium m u s c o r u m and Chelanops cyrneus and N e s t e r (1932) on Chelanops u n u s u a l and v e r y c o m p l e x . We i n t e n d , corticis and electron m i c r o s c o p y investiga- therefore, to handle s e p a r a t e l y the differtions by Boissin and Manier (1966, I967), entiation o f the various c o m p o n e n t s o f the Tuzet et al., (t966) on Hysterochelifer rner- e n c y s t e d s p e r m a t o z o a , In this p a p e r w e idianus, Boissin (1974) on Garypus beau- d i s c u s s the m o r p h o g e n e s i s o f the a c r o s o m e voisi, and L e g g (1973) w h o d e s c r i b e d ma- and the c o n c o m i t a n t c h a n g e s o f the Golgi ture s p e r m a t o z o a in 18 species. Recently, a p p a r a t u s . F o r o t h e r s t r u c t u r e s m e n t i o n e d Dallai and Callaini (1983) reported on the in this c o n n e c t i o n , such as the large central d e v e l o p m e n t o f the giant a c r o s o m e s o f v a c u o l e , the cell wall, or nuclear or mitoGeogarypus and Chthonius but their w o r k chondrial c h a n g e s , the r e a d e r is referred to w a s not s t i p p l e m e n t e d with the requisite f o r t h c o m i n g publications. electron micrographs. S o m e o f the n e w e r MATERIALS AND METHODS findings, h o w e v e r , are c o n t r a d i c t o r y and i n c o m p l e t e . L e g g ' s (1973) s t a t e m e n t that Adult Diplotemnus sp., found hidden in the crevices " a noticeable feature is the a b s e n c e o f an of the bark of Ficus indica, were collected in the vio b v i o u s a c r o s o m e " (p. 437) s e e m s incor- cinity of Chandigarh, India. A pair of pinhead sized testes were excised and examined under oil immersion rect. 105 0889- ! 60_5/88 $3.00 Copyright © 1988 by Acadcmi,. Press, inc. AIJ rights of reproduction in any form reserved.
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ACROSOME OF PSEUDOSCORPION phase contrast optics (x 100). For electron microscopy, testes were fixed for 2 br in 0.1 M cacodylatebuffered 2% glutaraldebyde at room temperature, and postfixed for 2 hr in cacodylate-buffered 2% osmium tetrox,;.de. Subsequently material was dehydrated in ascending concentrations of acetone and embedded in Araldite. Ultrathin sections were cut with diamond knives (Diatome) using an Ultracut (Reichert). Sections mounted on uncoated copper grids were double stained with uranyi acetate and lead citrate after Reynolds and examined with Zeiss EM 109 and Jeol 1200 EX transmission electron microscopes at 80 kV.
RES U LTS
In late spermatocytes the Golgi apparatus comprises many dictyosomes, located in a characteristic polarized fashion (Fig. I). From the endoplasmic system associated with dictyosomes one can detect the budding off of the coated vesicles. During this phase of development the first cistern of the cis face of most of the dictyos o m e s a p p e a r s d i s t e n d e d . Invariably among the dictyosomes vesicles having electron-dense contents are dispersed. These vesicles do not seem to contribute to the formation of the acrosome. At some later time, the dictyosomes aggregate to one place near the cell periphery; by now most of them have lost their individuality (Fig. 2). Small vesicles with floccular contents show up at the lrans face. A large vesicle with similar material approaches the cell membrane. Dilated tubules appear specifically in that region of the Golgi apparatus in which the acrosomal vesicle is developing. As soon as the acrosomal vesicle contacts the cell membrane, an electrondense granule makes its appearance (Figs.
107
3, 26a). Thereafter, less electron-dense material accumulates at the opposite side of the attached granule (Fig. 4). Thus the polarity of the acrosome is established with the electron-dense granule lying toward the prospective anterior part. Sometimes the acrosomal vesicle seems to drift toward the nucleus, carrying along with it the cell membrane (Fig. 5). Such inpockets of the cell membrane are always identifiable in the later phases of acrosomal development inasmuch as these contain electron-dense granules interspersed with small vesicles of variable size. Figure 7 perhaps represents a later stage of this unusual situation. In general, however, the acrosomal vesicle elongates in situ and becomes conical, its tip remaining associated with the cell surface (Figs. 6, 26b). The membrane of the conical part reveals stepwise configuration with sharp corners to which the cell membrane adheres and thus is invaginated when the cone elongates. The dilated tubules together with the whole of the Golgi apparatus continue to be associated only with the base of the acrosome, which retains its round vesicular shape. This vesicle enlarges, gets filled with fibrillar material, and then sits on the nucleus with another dense granule at the place of contact (Fig. 7). The developing acrosome is now clearly differentiated into two parts (Fig. 8). The anterior part is delimited by the electron-dense material disposed along the cone. The posterior part is bound by a smooth membrane and material is further contributed only to this vesicular part by the elaborate Golgi apparatus. It appears that the stiffening of
Fro. i. Late spermatoeyte. Areas of dictyosomes with distended first cisterna of the cis face, groups of vesicles with dense contents among them (arrowheads). × 7000. FIG. 2. Beginning of acrosome formation. Goigi apparatus near the cell periphery, vesicle (A) with floccular contents moves to the cell membrane. Dilated tubules (T) in association with the acrosomal vesicle. × 14 250. FIC~. 3. Acrosomal vesicle (A) containing a dense granule in contact with the cell membrane. Dilated tubules (T) near the acrosomal vesicle. × 14 250. FIo. 4. Less dense material accumulates at the site of the attached granule within the acrosomal vesicle (arrowhead). × 14 250. FIo. 5. The acrosomal vesicle along with the cell membrane shifts toward the nucleus. The inpockets contain small dense granules and vesicles (arrowhead). × 14 500. F]o. 6. Acrosomal vesicle in contact with the cell membrane (arrowheads), beginning of cone formation of the anterior part. Golgi apparatus (GA) surrounds the posterior part. × 9000.
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the cone membrane is due to the accumulation of electron-dense material also on its outer face. The acrosome further elongates with the stepwise electron-dense contents becoming more pronounced and its edges always remaining in contact with the inpocketed cell membrane (Fig. 9). The anterior portion of the acrosome is comprised of coarse electron-dense material, whereas the materi~.! on the inner face of the vesicle is deposited in a helical manner. In favorable sections, it is possible to recognize regions of the acrosome with the deposited electron-dense material alternating with regions devoid of such a material (Fig. 10). The posterior portion of the acrosome, however, remains spherical. The Golgi apparatus continues to contribute material only to this part of the acrosome. As soon as chromatin condenses, the spherical posterior portion of the acrosome exhibits "whorl-like" fine fibriilar contents. At this stage the electron-dense granule not only increases in size but appears duplex, its less electron-dense component flattening on the membrane (Fig. 11). It is pertinent to mention that subacrospinal material is conspicuous by its absence at this stage. The anterior portion of the acrosome lengthens considerably, as is evident in the phase-contrast micrographs (Fig. 25), and it is rather rare to have it complete in one section, though its contact
with the cell membrane is retained (Figs. 12, 26c). While the nucleus elongates it becomes ensheathed by the posterior portion of the acrosome (Figs. 13-15, 26c). The latter, no longer is spherical, instead shows pointed contours. The elongated posterior portion of the acrosome initially forming a complete envelope around the nucleus becomes increasingly helicalized (Figs. 14, 15) and then interrupted in the groove, resulting in the winding of the helical band in many turns around the nucleus (Figs. 18, 19). Its fibrillar contents become much more electron dense (Fig. 14). The Golgi apparatus is associated only with this posterior part. The dilated tubules disappear; instead flattened narrower cisternae appear on the t r a n s face (Fig. 26d). The ends of these cisternae often are in close contact with the edges of the developing helical band. The acrosomal membrane, especially at the corners, appears bordered by short filaments at this time. When the chromatin is transformed into distinct lamellae and as these lamellae further condense the Golgi apparatus enters into another active phase (Figs. 16, 26e). During this period randomly orientated cisternae located in the medullary region of the Golgi apparatus appear stuffed with electron-dense material. At many places one finds the interposition of elongated flat cisternae between the aforementioned cisternae (Figs. 17, 26f).
FIG. 7. Differentiation of anterior part (left) and posterior part (right) of the acrosomal vesicle (A): anterior part with the electron-dense material now distributed along the membrane and in contact with the inpocket of the cell membrane (arrowhead); posterior part with smooth membrane surrounded by the Golgi apparatus. Granule at the place of contact with the nucleus (N). × 7500, FxG. 8. Elongation of the anterior part (left) of the acrosome (A), with acute corners always in contact with invaginated cell membrane (arrowheads). Posterior part (right) spherical with smooth membrane surrounded by the Golgi apparatus. Dilated tubules (T) nearby. × 8000. FIG. 9. Longitudinal axis of the anterior part (left) of the acrosome (A) tangential to the nucleus (N). Inpockets of the cell membrane in contact with the acute corners (arrowheads). Coarse material becomes condensed. Posterior part in Contact with the nucleus remains unchanged. × 7500. FIG. 10. Higher magnification of the anterior part of the acrosome. Electron-dense coat of the membrane. The coarse material is deposited on the inside of the membrane in a helical band as indicated by the lines. Invaginated membrane in contact with the corners (arrowhead's). × 22 100. FIG. 11. Beginning of chromatin condensation within the nucleus (N). Anterior part of the acrosome (A) filled with coarse material. Posterior part now shows fine fibrillae. Dense granule with less electron-dense base at the place of contact with the nucleus, x 7000. FIG. 12. Anterior part of the acrosome elongates further while its posterior part is still spherical. Contacts of the cell membrane with the anterior part at the acute corners (arrowheads). x 8000.
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Often, narrow electron-dense processes extend from the helical band (Figs. 18, 26f). During late spermiogenesis, the Golgi apparatus wanes off at the time when the nucleus appears h o m o g e n e o u s l y electron dense (Figs. 19, 20). The helical band is reorganized and becomes slimmer, apparently due to the pinching off of the slender processes. The helical band now mainly is comprised of tubular components (Fig. 19). Masses of granular material with electronlucent internum are seen interspersed among the acrosomal elements (Figs. 19, 21). Further elongation of the nucleus is accompanied by a simultaneous lengthening of the posterior part of the acrosome, resulting in increased helicalization (Fig. 26g). The anterior part of the acrosome is confluent with the posterior part and at the junction of the two is seen an incomplete ring of cytoplasmic material (Fig. 22). Ahead of this ring appears a large globular element with granular material of variable densities which differentiates further (Figs. 23a, 23b). Components of this material surround /he whole of the anterior part of the acrosome except where the invaginated cell membrane is still in contact with the acrosome. Only during late spermiogenesis does subacrosomal material emanate from an invagination of the nucleus and extend into a channel of the anterior part of the acrosome (Fig. 22). An anterior centriole, as m e n t i o n e d by Boissin and Manier (1967), was not found at this place in Diplotetanus. Once the reconstruction of the acrosome is completed the unwanted acrospinal elements disappear. Note that by
I 11
now various organelles in the spermatid are disposed of in a circular fashion around a large temporary vacuole (Figs. 23a, 23b, 25c, 25d). Therefore in many a section both anterior and posterior parts of the acrosome can be found in close apposition to each other. L a t e r on, the s p e r m a t i d shrinks, rounds off, and becomes encysted (Fig. 24). The vacuole tends to become smaller and the various organelles become tightly packed. The final stages of encystment of the spermatozoon will be discussed in a subsequent paper. DISCUSSION
The acrosome formation in Diplotemnus turns out to be a very complicated process, deviating from the usual pattern in many details. From the outset the Golgi apparatus is orientated with its trans face toward the cell periphery. When the acrosomal vesicle appears, although inconspicuous, it abuts the cell membrane. The formation of the acrosome in close apposition with the cell membrane has not yet been reported in Arachnids although it has been observed in Annelids (Anderson and Ellis, 1968). In Diplotemnus the polarity of the anterior and posterior parts of the acrosome is established fairly early in spermiogenesis. So far, these early stages of its morphogenesis have not been reported. Sokolow (1926) and Nester (1932) no doubt described early stages of acrosome formation in the Pseudoscorpions but due to limited resolution of light optics they could not resolve its intricate details. Boissin and
FIG. 13. Beginning of nuclear elongation (N) and its wrapping by the posterior part of the acrosome (A) which develops sharp pointed contours. Golgi apparatus associated with the posterior part. x 7000. FIG. 14. Transformation of the posterior part of the acrosome (A) with condensation of its fibrillar contents. Appearance of long flattened cisternae of the t r a n s face of the Golgi apparatus often ending near the sharp corners (arrowheads). x 12 500. FIG. 15. Elongating nucleus (N) wrapped by the posterior part of the acrosome with increasing helicalization. The arrowhead indicates the junction between the anterior and posterior parts of the acrosome, x 7500. FIG. 16. Longitudinal section through posterior part of the acrosome and nucleus with lamellar chromatin (N) surrounded by the Golgi apparatus. The innermost cisterna of the t r a n s face begins to accumulate electrondense material. The formerly complete wrapping is transformed into the helical band due to interruption in the groove (arrowheads). x 13 500.
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Manier (1967), employing electron microscopy, on the other hand, have described the beginning of acrosome formation only in late spermatids, corresponding to the stage depicted in Fig. 13. From the beginning, both parts of the acrosome develop in different ways but remain interconnected. Sokolow (I926) and Nester (1932) gave correct descriptions of the elongating acrosome but both of them regarded the two parts as distinctly separate structures: "acrosom" and "kopfbliischen" and "acrosome" and "ovoid body," respectively. Generally, the elongating anterior part of the acrosome pushes the posterior part with the associated Golgi apparatus closer to the nucleus. In exceptional instances we find a small vesicle with associated cell membrane traversing the cytopl~iSm to be placed on the nucleus later. In any case it is important to mention that the anterior part of the acrosome remains in contact with the cell membrane. During progressive helicalization of the elongating acrosome, invagination of the adhering cell membrane is further accentuated (Figs. 26a-26d). Possibly such an unusual differentiation process may be due to the fact that these cell membranes later fuse to form a large central vacuole, the details of which will be presented in a forthcoming paper. To the best of our knowledge, to date, such a process has not been observed in any other species. As soon as the condensation of the chromtin ensues the nucleus begins to elongate in the longitudinal axis thereby indenting the spherical posterior part of the acrosome; the latter subsequently en-
113
sheaths the nucleus. At the apical end of the nucleus it passes into its anterior part without any interruption. Simultaneously the smooth membrane of the posterior part forms acute angles and becomes wound in a helix around the nucleus with an increasing number of coils. This sheath, which is complete in the beginning, is transformed later into an interrupted helical band by a very strange process: The sheath is cut through the grooves following the coils of the helix and the thinly extended ends are bent outside and apparently removed. In contrast, Boissin and Manier (1967) imagine the formation of the "spiral" band quite differently. According to them the acrosomal vesicle glides along the head and forms around it a keel-like band which curls itself up as a helix. We also disagree with their contention that the helical band is a product of the Golgi apparatus but only that part of the acrosome which lies in front of the nucleus is characterized as "'complex acrosomien." A helical band as part of the acrosome was, however, described by Osaki (1969) in Heptathela kirnurai, a liphistid spider. Boissin and Manier (1967) in a schematic diagram describe a " c e n t r o s o m e anterieur" lying in a channel at the anterior tip of the nucleus. On the other hand, in Di. plotemnus sp. we find in this place the subacrosomal fiber, and the two coaxially disposed centrioles are located rather in a channel at the posterior end of the nucleus. As the helical band always remains continuous with the anterior part it is an integral part of the acrosome and not a separate entity. The anterior part extending from the
FIn. 17. Cross section through helical band and nucleus with condensed chromatin (N). Advanced stage of accumulation of electron-dense material originating from the Golgi apparatus. × 14 250. FIG. 18. Longitudinal section through the posterior part of the nucleus (N) with adhering nuclear envelope (NE), centriole (C), and flagellum (F). Nucleus and helical band surrounded by the Golgi apparatus. The helical band with slender extended processes (arrowheads). x 13 000. FIn. 19. Oblique section through nucleus (N). Reorganization of the helical band: slender processes are cut off (arrowheads), the contents become tubular, x 23 500. FIn. 20. Cross section through nucleus (N) and helical band. Final stage of its reorganization. The Golgi apparatus has disappeared. The dense material is globular. × 17 500.
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tip of the nucleus is also helicoid with a long pitch. Peculiar to this part is its association with different cytoplasmic structures. In correspondence with the highly complicated structure of the acrosome one observes an unusually prolonged activity of the Golgi apparatus. It begins in the young spermatid and continues thereafter in acrosome reconstruction until the nucleus is completely condensed. Usually the Golgi apparatus parts company with the developing acrosome when the nucleus begins to condense. During spermiogenesis in Diplotetanus sp. one can witness several structural peculiarities of the Golgi apparatus which may be correlated to different functions (compare also Fig. 26): (1) Before acrosome formation (still in late spermatocytes and in very young spermatids)--each dictyosome is characterized by distended first cistern-forming small vesicles of unknown significance. (2) First phase of acrosome formation up
to the stage when fine fibrillae appear in the spherical posterior partmlarge cup-shaped Golgi apparatus associated with dilated tubular system (which seems not to be part of the Golgi apparatus). (3) Elongation and helicalization of the posterior part of the acrosome until chromatin appears lamellar--long, narrow cisternae at the trans face. (4) Transformation of the posterior part into a helical band--last activity phase of the Golgi apparatus prior to its disappearance. The Golgi apparatus forms large quantities of secretory product which apparently help shape up the helical band, Thus, in Diplotemnus, the Golgi apparatus performs different functions in the long history of acrosome morphogenesis. The Golgi apparatus during such a complicated procedure seems to reconstruct and partly degrade its own product. One could say that the Golgi apparatus makes its own raw material to suit its size and shape.
FIG. 21. Longitudinal section through nucleus (N) and helical band. Stage comparable to Fig. 20. Nuclear envelope (NE) surrounds the posterior part of the nucleus. Granular material with electron-lucent internum (arrowheads) among electron-dense Golgi products, x 8000. Fit:;. 22. Oblique section through junctional zone of anterior part of the acrosome (AP) and helical band (H) around the nucleus (N). Different elements of granular material are attached to the anterior part. Incomplete ring (R) and subacrosomal material (arrowhead). × 19 000. Flo. 23. (a) Late spermatid with large central vacuole (V). The capsule of the cyst begins to form underneath the cell membrane (CM). Nucleus (N) with helical band and remnants of the nuclear envelope (NE). Anterior part of the acrosome (AP) in contact with vacuolar membrane, mitochondria (M) and flagellum (F). × 7000. (b) Anterior part of the acrosome (AP) with attached cytoplasmic material of different appearance in contact with the membrane of thelarge central vacuole (V). Ring (R) at the junction between anterior part of the acrosome and the helical band. x 7090. FIG. 24. Encysting spermatid. Cyst wall (CW). Anterior part of the acrosome (AP) with remnants of the former vacuole (V), nucleus (N) with helical band, mitochondria (I',4) and cross sections of the flagellum (F). x 6500. FIO. 25. Phase-contrast micrographs of live spermatids of Diplotemnus sp. (a) Stage corresponding to Figs. 16 and 26e. Condensing chromatin in the longitudinal axis. Dense material in one place near the nuclear envelope (arrowhead). Anterior part (AP) of the acrosome is bent and its posterior helical part is surrounded by Golgi apparatus (GA). A bundle of long filaments emerges from the mitochondria (M) (small arrowheads). (b) Stage corresponding to Fig s. 18 and 26f. Nucleus surrounded by helical band begins to coil. Golgi apparatus (GA), dense material near nuclear envelope (large arrowheads), and bundle of filamentous mitochondria (small arrowheads). (c) Stage corresponding to Figs. 21 and 26g. Increased helicalization of the band near its posterior end. Nuclear er'.'elope (large arrowhead), mitochondriai bundle (small arrowheads), central vacuole (VA). Note the temporary elongation of the cell. (d) Rounding off of the spermatid with the centrally located vacuole (VA), bent anterior part of the acrosome (AP), coiled head with helical band and mitochondrial bundle (arrowheads). (e) Stage corresponding to Fig. 23. Cyst wall (arrowhead), large central vacuole (VA) with head and mitochondrial bundle coiled around it. The granular material (compare Fig. 23b) attached to the anterior part of the acrosome is discernible (white arrowhead). All figures approximately x I000.
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FIn. 26. Schematic drawings of the main stages of acrosome formation and changes of the Golgi apparatus during spermiogenesis in Diplotemnus sp. Drawings are not to scale, nucleus and helical band in the later stages are considerably longer than shown here. (a) Beginning of acrosome formation; acrosomal granule attached to the cell membrane, tubular reticulum nearby. (b) Conical anterior part surrounded by inpockets of the cell membrane which are attached to the acute corner running in a helix along the cone. Spherical posterior part associated with the Golgi apparatus. (c) Growing acrosome together with the Golgi apparatus have reached the nucleus. Inside the spherical part a dense globule appears. (d) Beginning of nuclear condensation and its elongation in the longitudinal axis. Invagination of the posterior part and initiation of its transformation into the helical band. The Golgi apparatus shows long narrow cisternae at its trans face which often come close to the acute corners. (e) The elongating posterior part is cut along the middle between neighboring acute corners thus transforming the formerly uniform sheath of the nucleus into the helical band. Golgi apparatus begins its last activity phase by forming cisternae with electron-dense contents. Membrane invaginations around the anterior part are indicated by profiles attached to the corners. (IF)The extended free ends of the helical band are turned outside and disappear later. Full stage of the last activity phase of the Golgi apparatus. Cisternae of the trans face with electron-dense material and large globules. Membranes around the anterior part are not shown. The apex is bent around various cytoplasmic components. (g) Late spermatid with fully condensed nucleus, remnants of nuclear envelope, and definitive acrosome. Anterior part and helical band are continuous. Subacrosomal material is within a channel formed by invaginations of the nucleus and the acrosome. Goigi apparatus has completely disappeared. The invaginated tubules have fused to form the large central vacuole (not shown here). 117
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WERNER AND BAWA
Such an unusual process of acrosome formation has no parallel in other animals. It should be pointed out, however, that a postacrosomal activity phase of the Golgi apparatus has been reported in Discogiossus (Sandoz, 1970) and Notonecta (Werner, 1986). In Notonecta obvious lysosomes are formed which devour surplus material, predominantly membrane structures, which is no longer required in the process of sperm maturation. Thus in very elaborate differentiation processes the spermatid seems to develop a lysosomal apparatus of its own and surplus material is used up by the spermatid itself and not simply sloughed off as generally is the case in spermateleosis. In Diplotemnus, there is another feature which needs emphasis. Phillips (1974) has pointed out that microtubules, otherwise usually found as shape-forming elements in spermiogenesis, are missing during nuclear elongation in Scorpions. In Diplotemnus microtubules are nonexistent throughout spermiogenesis (Bawa and Werner, 1983). The absence of such ubiquitous structures is not at all attributed to improper fixation of the tissues, as we have observed large populations of microtubules in the testicular wall cells. Finally, we do not visualize any relationship between the immense size of the acrosome in Diplotemnus and the length of the nucleus and the axoneme as
suggested by Dallai and Callaini (1983) who explain that the acquisition of a giant sized acrosome in Geogarypus results in the concomitant diminution of its flagellum and the nucleus. The authors thank Miss R. Kaur (Chandigarh) for processing, Mrs. E. Maatz for sectioning the material, Mrs. G. Kiefer for the photographic work, and Mrs. E. Frank (all Homburg) for typing the manuscript. One of us (S.R.B.) gratefully acknowledges the support of the Alexander-von-Humboldt-Stiftung. REFERENCES ANDERSON, W. A., AND ELLIS, R. A. (1968) Z. Zellforsch. 85, 398-407. BAWA, S. R., SJOSTRAND, E S., KANWAR, U., AND KANWAR, K. C. (1971)J. Ultrastruct. Res. 37, 251. BAWA, S. R., AND WERNER, G. (1983) its ANDRE, J. (Ed.), The Sperm Cell, Proc. IVth lnternatl. Syrup. Spermatol., pp. 245-248, Nijhoff, The Hague/ Boston/London. BOISSIN, L. (1974). Arch. Zool. E.rp. Gen. 115, 169-184. BOISSlN, L., AND MANIER, J.-E (1967) Btdl. Soc. Zool. France 92, 705-712. DALLAI, R., AND CALLAINI, G. (1983) in ANDRE, J, (Ed.), The Sperm Cell, Proc. IVth Internatl. Syrup. Spermatol., pp 431-435, Nijhoff, The Hague/ Boston/London. LEGG, G. (1973). J. Zool. (London) 170, 429-440. NESTER, H. G. (1932). J. Morphol. 53, 97-131. OSAKI, H. (1969)Acta Arachnol. 22, 1-13. PHILLIPS, D. M. (1974) J. Cell Biol. 62, 911-917. SANDOZ, D. (1970)J. Microsc. 9, 243-262. SOKOLOW, I. (1926) Z. Zellforsch. 3, 615-681. TUZET, O., MANIER, J.-E, AND BOISSIN, L. (1966) C.R. Acad. Sci. Paris 262, 376-378. WERNER, G. (1986)Biol. Cell 57, 169-180.