Electron microscope study of spermiogenesis in Locusta migratoria (Insect Orthoptera)

JOURNAL OF ULTRASTRUCTURERESEARCH 50, 322-346

(1975)

Electron Microscope Study of Spermiogenesis in Locusta migratoria (Insect Orthoptera) ANNETTE SZOLLOSI 1

Laboratoire d'Histophysiologie des Insectes, Universit~ PARIS VI et Laboratoire de Microscopie ~lectronique appliqu~e & la Biologie, CNRS, Paris, France Received May 9, 1974 In a study of spermiogenesis ofLocusta migratoria, the differentiation process has been divided into ten developmental stages. A clearly recognizable character is chosen as the main feature to define each stage, so that in the forthcoming investigations involving experimental treatment, the chosen feature may serve as a guide line. All cell organelles are described in each of the stages. During the first three stages, the mitochondrial nebenkern is formed. It divides into two derivatives while the proaerosome transforms into a conical-shaped structure. From stages 5 to 9 the chromatin rearrangment leads to a homogeneous compact and slender nucleus, while the residual cytoplasm is sloughed off and while the axoneme reaches its definitive configuration. The spermatodesms, formed during stage 9, are transferred into the seminal vesicles during stage 10. Then all microtubules, other than the axonemal ones, disappear. The extracellular fibrous coat of the sperm cell is formed in an early stage. It differentiates very progessively and reaches its high degree of organization only during the last stage.

Spermiogenesis in Acridids has been the subject of several investigations in light microscopy which were reviewed in 1958 by Gatenby and Tahmisian in a study including the first electron microscope observations (17). During recent years, in contrast, most studies have dealt with points of detail such as transformations of the nucleus or evolution of some particular cytoplasmic organelle. Evidently, such studies leave many questions unanswered but, above all, they do not permit a thorough understanding of the correlations between states of development of the different organelles. To interpret some experimental data obtained in Schistocerca gregaria and Locusta migratoria, it was felt there was a need for a new study o f the entire process. It has been shown that spermiogenesis in Acridids is extremely sensitive to slight changes of temperature. In Schistocerca gregaria, males are fertile when raised at 30°C during the day and 20°C during the night; they are sterile if the day tempera1Previous works of the author have been published under the name A. Cantacuz~ne.

ture is lowered, only by 2 ° to 28°C (7, 25, 26). The same appears to be true also in Locusta migratoria which, under similar conditions, are totally sterile (34). The study of these sterile males shows that the first observed alterations are subtle changes in shape and orientation of individual organelles and modifications of their relative rate of transformations. A detailed knowledge of the normal differentiation process was therefore needed to interpret such changes. The present study describes the normal events in spermiogenesis of Locusta migratoria. It proposes a scheme of development in which the process of differentiation is divided into 10 stages. The first four stages are defined on the basis of the mitochondrial morphology while the nuclear transformation is used as the main feature to define the later stages. Some aspects of organellogenesis, not previously reported in other Acridids, are described and discussed. MATERIAL AND METHODS The males of Locusta migratoria migratorioides used in this study were 2-3 wk old adults. The whole testis was immersed in the fixative and individual 322

Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA follicles were immediately dissected and cut into small pieces. In most cases, the following procedure was used: the tissues were fixed for 1 hr at room temperature in 2.5-5% glutaraldehyde in 0.15 M caeodylate buffer, pH 7.4. After a short rinse, the segments were postosmicated for 45 min in a 2% solution of osmium textroxide in the same buffer and dehydrated by a graded series of ethanol. They were left overnight in a propylene oxide-epoxy mixture and embedded in Araldite-Epon. Thick sections were stained with Azur II-Methylene blue for orientation while thin sections were contrasted with uranyl acetate and lead citrate. Observations were made in a Philips EM-300 electron microscope. Occasionally, other procedures were used in order to get specific information. A fixative with high molarity (up to 900 mOsmole) was found suitable for studying the late stages when the cap of the spermatodesm has already formed. Sucrose was added to the fixative to increase its osmolarity. Extractions by pepsine and pronase were performed either on material embedded in GMA (22) or following embedding in Alraldite-Epon (24). For detection of polysaceharids two methods were used: TCI~-silver proteinate (36) and phosphotungstic acid (29). RESULTS

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are in connection with their sister-cells by large c y t o p l a s m i c bridges with a typical dense m a t e r i a l lining the p l a s m a m e m b r a n e along the " r i n g " canal (Fig. 2). In the s p e r m a t o c y t e stage, two centrioles are lying at right angle to each other in the vicinity of the nucleus and one of t h e m is facing a vesicle, initiating flagellum formation (Figs. 3 and 4). In contrast, during late a n a p h a s e of the second meiotic division (Fig. 2), and in the young s p e r m a t i d , we never observed more t h a n one centriole which plays the role of a basal body and is always located at the p e r i p h e r y of the cell. T h e short flagellum elongates within a c y t o p l a s m i c a m p u l l a , which is itself ineluded in a vesicle limited by the p l a s m a m e m b r a n e . A halo of dense m a t e r i a l surrounds the centriole while m i c r o t u b u l e s project in all directions from it. At this stage, they are not observed in other p a r t s of the cell (Fig. 5).

Stage 1

Stage 2

T h e m o s t characteristic features observed in a young s p e r m a t i d are the cluster of m i t o c h o n d r i a which forms at one pole of the cell (Fig. 1) and the d e v e l o p m e n t of a short flagellum at the cell periphery. T h e s h a p e of the s p e r m a t i d is slightly triangular with spherical nucleus m e a s u r i n g app r o x i m a t i v e l y 5 # m in d i a m e t e r . T h e nuclear content is finely fibrillar with several h e t e r o c h r o m a t i c regions dispersed at random. In the s p e r m a t i d s which possess the X c h r o m o s o m e , the X - h e t e r o c h r o m a t i c m a s s is easily recognizable by its h e m i spherical shape and its peripherial position. S m a l l cisternae of e n d o p l a s m i c retieu l u m are s c a t t e r e d t h r o u g h o u t the cytop l a s m ; m o s t of t h e m are a l m o s t s m o o t h with just a few ribosomes a t t a c h e d . In contrast, in a limited area of the cell, some rough cisternae are found in parallel alignm e n t similar to t h a t observed in p r i m a r y s p e r m a t o c y t e s . M o s t of the c y t o p l a s m i c free ribosomes form polysomes. Few myelin figures a n d o c c a s i o n a l m u l t i v e s i c u l a r bodies are observed. T h e young s p e r m a t i d s

This stage is characterized by the formation of the m i t o c h o n d r i a l nebenkern. T h e end to end fusion of m i t o c h o n d r i a forms this organelle, a spherical body of 3-4 ttm in diameter. T h e intertwined mitochondrial strands are s e p a r a t e d by a c o n s t a n t c y t o p l a s m i c space of a b o u t 400/~ (Fig. 6). T h e basal body and the nucleus have come in contact and the flagellum begins to elongate. S o m e t i m e s the nucleus itself is close to the p l a s m a m e m b r a n e and then, the flagellum projects outside from the cell (Fig. 7). In m o s t cases, however, it grows within a cylindrical vesicle which is p a r t l y enclosed in the c y t o p l a s m . Therefore, in transverse sections, it a p p e a r s as being s u r r o u n d e d by two p l a s m a m e m b r a n e s (Fig. 8). T h e a x o n e m e is of the 9 + 2 type, with e m p t y central tubules. R e m n a n t s of the parallel rough cisternae are still observed in the slightly elongated p a r t of the cell (Fig. 10) b u t the vesicular E R is m a i n l y of the s m o o t h type. D i c t y s o m e s are found n e a r b y the nucleus, c o m p o s e d of three to five strongly curved lamellae limiting a

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ANNETTE S Z(~LL(~SI

FIGS. 1-5. Spermatids of stage 1.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

central area (Fig. 11) or a central vesicle. Several small multivesicular bodies are found, sometimes scattered in the cytoplasm but more frequently associated with the nuclear envelope. In a section, the number of the internal vesicles present in these bodies is most of the time close to 10, but up to 20 were seen occasionally (Fig. 9). In the nucleus several inclusion bodies are associated with heterochromatin. Some are electron-dense, others have a fibrillogranular texture, some show short and coiled ribbon-like structures (Fig. 12). On the plasma membrane of the young spermatid a fibrillar external coat begins to develop.

Stage 3 The main character of this stage is the formation of the centriolar adjunct and of the proacrosomal granule (Fig. 13). The cell is pear-shaped and from this stage on, it begins clearly to elongate. The nucleus is slightly ovoid, the X-chromosome as well as the other heterochromatic masses move from the nuclear envelope towards the center of the nucleus where the fibrillar euchromatin is rather homogenous and pale. A narrow dense chromatin condensation lines the nuclear envelope. Convoluted ribbons irregularly intertwined and denser than the surrounding chromatin form a "coiled body" often associated with another fibrillo-granular spherical dense body. It can be well observed following osmium fixation (Fig. 14).

3')5

The basal body, associated with the nuclear envelope in the region of the nebenkern, has been followed in its migration by the deeply invaginated plasma membrane which is lined at its innermost edge by a dense material encircling the centriole. This structure corresponds to the "ring centriole" as it was formerly known (Fig. 15). During this and the following stage, the "ring" slips caudally along the flagellum and subsequently the nebenkern comes closer to the axoneme. The centriolar adjunct appears as a granular organelle surrounding the basal body which, at this stage, has retained the typical configuration of a centriole (Fig. 16). In the nebenkern, mitochondrial strands reorganize concentrically in an onionlike configuration while the outer part becomes progressively electron-lucent (Fig. 18). It corresponds to the "chromophobic" nebenkern of the light microscopists. In the vicinity of both, the nucleus and the nebenkern, a new organelle appears (Figs. 13, 17, and 19); it is a spherical to ovoid dense body of about 0.5 #m in diameter, limited by what appears to be a double membrane. This granule is the proacrosome and approaches the nucleus until its limiting envelope lies parallel and in contact with it. It does not seem to be closely associated with any of the numerous cup-shaped Golgi complexes scattered in the neighbouring cytoplasm. Clear vesicles lined internally by a fibrillar material are regularly found around the organelle (Fig. 13).

FIG. 1. Spermatids of the late stage 1. The nucleus N is sperical and the mitochondria are already almost organized in a nebenkern NB. x 7 200. Fro. 2. Connections between two spermatids. The two cells communicate by a large cytoplasmic bridge whose walls are reinforced, forming a "ring" (arrows). Anaphase chromosomes are visible in the cell below, while in the upper one, the basal body forms a short flagellum, projecting into a vesicle. × 11 000. FIG. 3. Centriole of a spermatocyte. The basal body is apposed to the nuclear envelope and often elaborates a short flagellum (F1). x 29 000. FIG. 4. Two centrioles of a spermatocyte. They lie near the nucleus and are at right angles to each other. × 23 000. FIG. 5. Flagellum formation in the spermatid. The flagellum begins to elongate within a cytoplasmic ampulla which is enclosed in a vesicle. The basal body is located at the periphery of the cell and is surrounded by microtubules mt, Pm plasma membrane. × 36 000.

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ANNETTE SZ()LLOSI

Fins. 6-12. Spermatids of stage 2.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

Stage 4 In this stage, the nebenkern divides into two derivatives, lying on the opposite sides of the flagellum (Fig. 20). They are long ovoid organelles in which cytoplasmic islands remain trapped. The centriolar adjunct is hemispherical, with a diameter approximating 1.5 t~m (Fig. 21). The granules composing it are 250-300 ~ in diameter and are regularly arranged. Towards the nucleus, the granules form a distinct row and a space of 250 • separates the two organelles. Chromatin begins to condense along the internal face of the nuclear envelope, in the region of apposition. Some microtubules surround the centriolar adjunct. The spherical proacrosomal granule is now attached to the anterior pole of the nucleus, opposite to the centriolar adjunct. In the anterior cytoplasmic region, there are no Golgi complexes but two laterally placed cisternae of endoplasmic reticulum usually accompanies the granule. Following osmium fixation, the content of this organelle appears denser on the side facing the plasma membrane (Fig. 22). Its limiting membrane still seems to be double. A line of electron-dense material forms between the acrosome and the nucleus while some granular substance is laid down at the anterior end of the vesicle, underneath the plasma membrane. During stage 4, the acrosome elongates (Fig. 23) and by the

327

end of the stage, it consists of a dense cone surrounded by an external coat. The latter originates from the anterior cytoplasmic deposit and its tip enlarges and forms an electron-translucent ampulla (Fig. 24). The spermatids are lying in an extracellular substance containing granules and very thin fibrils. Some patches of this material begin to condense around the acrosome. The two membranes of the nuclear envelope are closely apposed at the anterior pole of the nucleus but are still distinct near the centriole where there are many pores. Few microtubules appear in the cytoplasm surrounding the nucleus (Fig. 25). In the axoneme, the subfiber B of each doublet forms an external arm (Figs. 26 and 27) which will give rise during the next stage to the external accessory tubule; endoplasmic reticulum extends between the mitochondrial derivatives and the axoneme. A multitubular body of unknown origin forms in the peripheral cytoplasm (Fig. 26). This body is made of a bundle of slightly curved tubules lying in close contact and parallel to each other. Their number is variable in different spermatids and their length has not been determined. Their diameter is 450 •, that is about the double than the axonemal tubules; some of them have a central dot. With the exception of the acrosome region, the plasma membrane is covered by an external fibrous coat with perpendicular

FIG. 6. Nebenkern. Mitochondria fuse end to end, forming strands which are separated one from the other by a space of about 400 ~. × 20 000. FIG. 7. Longitudinal section of the flagellum. In this case, the nucleus has migrated towards the basal body and the cytoplasmic ampulla, surrounding the flagellum F1, projects outside the cell. Nucleus, N. × 6 300. FIG. 8. Cross section of the developing flagellum. In most cases, the cylindrical cytoplasmic ampulla containing the flagellum is located within the cytoplasm of the germ cell; correspondingly, the axoneme is encircled by two plasma membranes, and the space between them is extracellular (ex. s). × 36 000. FIG. 9. Multivesicular body. Up to 10 bodies of this type can be observed around the nucleus. × 36 000. Fro. 10. Cisternae of the rough endoplasmic reticulum. These cisternae in parallel array are similar to those observed in spermatocytes. In the nucleus, the X-heterochromosome can be observed. × 20 000. FIG. 11. Dictyosome. Numerous cup-shaped organelles of this type are randomly scattered in the cytoplasm of the spermatid, × 36 000. FIG. 12. Nucleus. Dense fibrillo-granular and coiled bodies (arrows) interspersed among the fibrillar chromatin are constant components of the spermatid nucleus, D.B., dense body; FGB, fibrillo-granular body. × 29 000.

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ANNETTE SZ6LL6SI

FIGS. 13-19. Spermatids of stage 3.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

329

slightly separated from the rest of the organelle. Stage 5 is the first stage in which a cytoplasmic dense sphere can be observed, Stage 5 always located in the perinuclear region Starting with this stage, the spermatid (Fig. 29) and corresponding to the light is clearly divided into a head containing microscopists' "chromatoid body" (17). essentially the nucleus, and into a tail. The This body is not limited by a membrane; main characteristic is the transformation ribosomes are always observed in its surof the nucleus. Its general shape is ovoid face. The two half-nebenkerns are trans(Fig. 28). The chromatin condenses into fibers have a diameter of 150 A and some formed into two dense and thin mitochonthinner fibrils are interspersed among drial derivatives limited by a double memthem (Figs. 29, 30, and 32). Several dense brane (Fig. 31). They show some internal bodies are observed in the posterior half of cristae but no longer are the cytoplasmic the nucleus. Extracellular substance con- inclusions observable in them. In the axdenses around the acrosome at a constant oneme, the accessory tubules begin to form distance from the cytoplasmic membrane; from the curved arms of subfibers B. The only a few fibers extend between the first completed accessory tubules remain plasma membrane and this extracellular attached to subfibers B of the doublets deposit which represents the first step of which are in the plane of symmetry of the the cap formation, studied later on. spermatid (Fig. 31). The multitubular Around the nucleus thin cisternae of body has migrated into the developing smooth endoplasmic reticulum appear syn- tail (Fig. 33) and cisternae of ER encircle chronously with microtubules oriented the axoneme, forming a sheath around it along the nuclear long axis. These tubules (Fig. 34). lie close to the nuclear envelope in a cytoplasmic area slightly denser than the Stage 6 rest of the cytoplasm and externally limThe spermatids have lanceolate nuclei ited by the cisterna. Within the centriolar in which chromatin fibers are associated adjunct, the size of granules decreases by pairs and form lamellar sheets which except for the juxtanuclear row which is are oriented along the nuclear long axis but orientation to the membrane surface (Fig. 27).

FIG. 13. Nebenkern, centriolar adjunct and proacrosome. The two latter organelles (CA) and (P) appear at this stage and are in close proximity. Clear vesicles are seen nearby and proacrosomal granule (arrow). D, dictyosome; N, nucleus; NB, nebenkern. × 23 000. FIG. 14. Nucleus. Following osmium fixation, the chromatin is poorly stained and intranuclear coiled bodies CB are seen in close contact with dense granular bodies. × 32 000. Fro. 15. Proximal part of the flagellum. The basal body, apposed to the nucleus, is embedded in granules of the centriolar adjunct (CA). It is associated with the plasma membrane (Pm) which is deeply invaginated and is lined at its innermost edge by a dense material. × 39 000. Fro. 16. Cross section of the basal body. The organelle, surrounnded by the centriolar adjunct, has the typical configuration of a centriole. × 110 000. Fin. 17. Proacrosomal granule. Its limiting envelope is double. The external layer is denser than the internal one. The substance of the proacrosome is filamentous. × 130 000. FIG. 18. Nebenkern. Mitochondrial strands have reorganized so that a bilateral symmetry becomes evident. Dictyosome (D), cross section of the flagellum (F1) are also seen. × 19 000. Fro. 19. Proacrosome and dictyosome. No topographical association is seen between the proacrosome (P) and the dictyosome (D). The dense content of the proacrosomal granule has slightly retracted and the double limiting envelope is clearly visible. × 31 000.

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A N N E T T E SZOLL()SI

FIGS. 20-27. Spermatids of stage 4. FIGS. 22-24. Transformations of the acrosome,

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

are not yet regularly organized. Free spaces remain in the chromatin network. The longitudinal lamellae are inserted into a plate of chromatin which has condensed at the posterior end of the nucleus and is located at 200 I from the nuclear envelope (Fig. 35). Dense bodies are still present in this nuclear region (Figs. 35, 36, and 40). In tranverse sections it can be observed that, the lamellae do not form closed tubules and have free extremities sometimes ending by a dense granule (Fig. 37). The nucleus is surrounded by a crown of microtubules and, more externally, by endoplasmic reticulum (Fig. 38). Between the dense cone of the acrosome and the nucleus a rod appears indentating the nucleus as well as the acrosomal cone (Fig. 39). The centriolar adjunct becomes asymmetric: it occupies the space between the posterior end of the nucleus and the mitochondrial derivatives and extends between the derivatives in the plane of symetry of the flagellum. The granules of the centriolar adjunct elongate a n d fuse and the organelle becomes more compact (Fig. 40). The dictyosomes and the chromatoid body have migrated into the tail and an obvious association between this dense

331

body and one of the dictyosome is established. Numerous Golgi vesicles are seen between the Golgi stacks and the chromatold body (Figs. 41 and 42). The mitochondrial derivatives reorganize and periodic clear spaces appear along their length. A row of microtubules develops around them and, immediately external to it, collapsed cisternae of endoplasmic reticulum constitute a sheath around the entire axonemal complex (axoneme + derivatives). In the peripheral cytoplasm, vesicles of ER fuse to form large cisternae with fairly dense content. Small ER vesicles, helicoidal and rosettelike polysomes are still found freely in the cytoplasm. Deformations of the tail indicate the beginning of the sloughing off of cytoplasmic lobes containing large cisternae of ER and free ribosomes (Fig. 43).

Stage 7 The main feature in this stage is the regularisation of the intranuclear sheets which extend all along the nucleus and fuse to form a honeycomb arrangment limiting irregular polygonal spaces free of any visible structure (Fig. 44). In some places, a second external row of microtubules forms along the perinuclear set, both lying in a

FIG. 20. Nebenkern derivatives. The division of the nebenkern gives rise to two symmetrical organelles in which cristae project into a clear matrix. Islets of cytoplasm are enclosed in them. × 15 000. Fro. 21. Proximal part of' the flagellum in longitudinal section. The hemispherical centriolar adjunct (CA) surrounds the basal body and the proximal part of the flagellum. Small vesicles of smooth endoplasmic reticulum (ER) are scattered in the neighbouring cytoplasm. N, nucleus; NB, nebenkern. × 30 000. FIG. 22. The acrosomal granule is attached to the anterior pole of the nucleus (N). Following osmium fixation only, a denser region appears at the anterior face of the organelle. A line of electron-dense material: the interstitial line (il) forms between the acrosome and the nucleus. A substance is also laid down between the plasma membrane (Pro) and the acrosomal granule (A). × 58 000. FIG. 23. The spherical granule has transformed into a conical structure and became more dense. In this figure, the dense interstitial line is not visible, but the outer substance forms a hollow cone around the acrosome. × 43 200. FIG. 24. The anterior part of the outer cone gives rise to a clear ampulla (a). Tbe interstitial line is shown by an arrow. Patches of extracellular substance accumulate around the acrosomal region. An extracellular fibrous coat (F. ex) covers the plasma membrane except over the acrosome. × 49 000. FIG. 25. Perinuclear cytoplasm. Microtubules (arrows) appear between a cisterna of ER and the nucleus N. × 49 000. FIGS. 26-27. Transverse sections of the tail. In the axoneme, subfibers B of each doublet forms an external arm. Two cisternae of ER extend between the mitochondrial derivatives and the axoneme. FIG. 26. Shows a multitubular body. × 29 000. FIG. 27. represents a more posterior section. The fibrils covering the plasma membrane are well visible. × 36 000.

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ANNETTE SZOLLOSI

FIGS. 28-34. Spermatids of stage 5.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

dense cytoplasmic area and surrounded by a collapsed smooth cisternae (Fig. 45). The centriolar adjunct becomes fairly compact and shows irregularly distributed clear spaces (Fig. 46). In the axoneme a dense core forms in the accessory tubules and in the slightly smaller two central ones (Fig. 49). Periodically distributed densities are seen in longitudinal sections between the central tubules and the peripheral doublet (Fig. 50): they probably correspond to radial densities and spokes which are not very clearly visible in transverse sections. In the doublets themselves, a further periodicity is observed (Fig. 50). The disposition of mitochondrial derivatives, microtubules and cisternae varies slightly along the tail but a close association between collapsed cisternae and rows of microtubules is always noticed (Figs. 46, 48, and 49). Towards the caudal extremity of the tail, the mitochondrial derivatives terminate at first, followed by the accessory tubules and, finally, at the tip of the flagellum, the doublets and the central tubules become randomly scattered. The cytoplasmic lobules being sloughed off contain ribosomes, remnants of merebranes and large areas with a flocculent substance (Fig. 47). Dense patches are often observed on the chromatoid body which will later develop into a very elec-

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tron-dense component of the residual spheres.

Stage 8 The parallel alignment of the spermatids against one cell of the cyst wall characterizes this stage: the germ cells begin to form a bundle. Individual caps surround the acrosomes; they are closed to each other but not yet fused (Fig. 51). The spermatid nuclei reach a length of about 50 #m. The sheets of condensed chromatin can be observed for great lengths which correspond probably to the length of the nucleus itself. The clear spaces of the honeycomb structure become tiny. The inner acrosomal cone covers now the tip of the nucleus and extends along it for 4-6 t~m, while its anterior part is only 0.8 tLm in length. Two to three rows of microtubules encircle the nucleus (Figs. 52, 53, 56, and 58). Although compact, the centriolar adjunct still contains some vacuoles (Fig. 54) and it embeds the base of the nucleus (Fig. 55). Whether the basal body is still existing as a morphological entity is not clear. The flagellar tubules terminate anteriorly in a dense material and the central pair extends almost to the nuclear envelope. Several microtubules lie at random in both sides of the axoneme while they form two rows along the mitochondrial deriva-

FIG. 28. Longitudinal section of the spermatid head. A basal condensation of chromatin lines the nuclear envelope in the caudal region of the nucleus. × 9 000. FIa. 29. Chromatoid body. This dense sphere (Ch. B) appears in the perinuclear cytoplasm. × 13 000. Fio. 30. Anterior region of the nucleus. The chromatin condenses in short fibers dispersed without preferential orientation. The extracellular substance forms an external cap (C) around the acrosome (A). x 18 000. FIG. 31. Axoneme. The first accessory tubules are formed from the curved arms of two of the subfibers B (arrows). E R cisternae surround the axoneme, two of which extending between it and the dense slender mitochondrial derivatives. × 36 000. FIG. 32. Perinuclear cytoplasm. Close to the nuclear envelope, the cytoplasm is slightly denser than in other regions, x 29 000. Fro. 33. Multitubular body. The diameter of these tubules is larger than the one of the ordinary tubules (rot) and their wall is thicker. A central dot can be seen in some of them (arrows). × 58 000. FIG. 34. Longitudinal section of the tail. Elongation has taken place but the shape of the tail is irregular, Cisternae of ER, form a sheath (SH) around the axoneme; polysomes are numerous in the peripheral cytoplasm. x 18 000.

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ANNETTE SZ()LLOSI

FIas. 35-43. Spermatids of stage 6.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

tives (Fig. 57). The collapsed cisterna around the axoneme is in contact with the mitochondrial derivatives and two densities develop, forming crescent-shaped wings at their inner edges (Fig. 57). The mitochondrial derivatives reach their almost definitive configuration with one to four longitudinal invaginations. These are not cristae, but infoldings of both of the mitochondrial membranes; they do not extend along the entire length of the derivatives since, in anterior cross sections, three to four are seen while in more caudal sections, there is only one. Some ribosomes are still found in the cytoplasm of the tail.

Stage 9 This is the stage when the spermatodesms are formed. In the extracellular space the periacrosomal material condenses around all the sperm heads of a bundle and forms a more or less flattened cap (Fig. 59). The individuality of each accrosome is nevertheless maintained and the space between the dense material of the cap and the plasma membrane persists (Fig. 61). The bundle of spermatozoa and the cap holding them together form, in their entirety, the spermatodesm. At this stage, the nuclear chromatin is

335

fully condensed. The two rows of perinuclear tubules are still present (Fig. 60) and a dense dot is observed in some of them. The centriolar adjunct has transformed into a solid piece constituted of four concentric lamellae. It surrounds now obviously the base of the nucleus (Fig. 62). In the tail there are still two rows of microtubules encircling the mitochondrial derivatives (Fig. 63). The periaxonemal sheath has totally disappeared and only the two crescent-shaped structures are present above the derivatives. The residual cytoplasm containing ribosomes, chromatold body, multitubular complexe and dictyosomes has been sloughed off and the tail consists only of the axoneme and the mitochondrial derivatives.

Stage 10 Once completed, the spermatodesms are liberated from the cyst in which they were formed. The bundles begin to move; they migrate towards the efferent duct through a narrow passage located at the periphery of the follicle, between the follicle wall and the cyst walls (Fig. 64). They reach the spermiduct and, finally, the seminal vesicles. Some morphological transformations takes place during this transfer.

FIGS. 35-36. Posterior region of the nucleus. FIG. 35. Longitudinal section. Sheets of chromatin are associated by pairs (circle) and oriented along the nuclear long axis. They insert into a basal plate of chromatin lining the nuclear envelope, x 41 500. FxG. 36. Oblique section. Five dense bodies lie in the clear spaces among the chromatin sheets. A cisterna of ER completely encircles the nucleus, x 29 000. FIG. 37. Cross section of the nucleus. The chromatin lamellae have free extremities sometimes terminated by a dense granule. A dictyosome is present in the peripheral cytoplasm, x 29 000. FIG. 38. Perinuelear microtubules. Microtubules (rot) extend between the nuclear envelope and the perinuclear cisternae. × 29 000. FIG. 39. Acrosome. A dense rodlet (R) appears between the acrosomal cone (A) and the nucleus (N) indenting both. An individual extracellular cap (C) is formed around the acrosome, x 14 000. F~G. 40. Centriolar adjunct. The granular body is elongated and asymmetric. On the right side of the figure it occupies the space between the nucleus and the mitochondrial derivatives (M) while on the left, it extends much more caudally. The granules are still visible, x 8 100. FIcs. 41-42. Chromatoid body. The organelle (Ch B) has migrated into the tail where it associates with a dictyosome (D). Ribosomes are attached to its surface (arrows). F~G. 41. x 27 000. FIG. 42. x 36 000. Fxa. 43. Posterior region of the tail. Ribosomes and large cisternae of ER accumulate in the peripheral cytoplasm whose deformations indicate the beginning of the sloughing off precess, x 18 000.

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ANNETTE SZOLL()SI

FIGs. 44-50. Spermatids of stage 7.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

The perinuclear microtubules as well as those surrounding the mitochondrial derivatives have disappeared. In longitudinal section of the mitochondrial derivatives, periodically distributed light areas are clearly seen, while one to four infoldings indent the outer side of the organelles. The external fibrils covering the plasma membrane have transformed into a compact, highly organized coat (Fig. 64) about 300 A thick. Tangential sections show a square network while cross sections show that the coat is constituted of two layers (Figs. 68-71). In the acrosomal region, embedded in the cap (Fig. 65), only the more internal layer of the coat is present and the transition between the acrosomal and the more posterior region is sharply defined (Fig. 68). In the posterior tapering end of the tail, the pattern of axonemal microtubules differs from that seen in earlier stages. Observing cross sections more and more caudally, it can be established that the doublets disappear first, followed by the central tubules, and finally by the accessory tubules. The latter are organized in a circle, but at the very tip, they are dispersed at random. The central and accessory tubules react positively with Thiery's

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and Rambourg's methods: they contain polysaccharids (Figs. 72 and 73). DISCUSSION

The synchronous development of isogenic spermatids has been observed for long and in many species. It has been proposed that such a synchronous differentiation could be related to cytoplasmic bridges connecting the cells within a cyst (10, 13). Such connections have actually been found as late as stage 8, in case of Locusta, when spermatids are almost fully differentiated; their disappearance is correlated with the sloughing off of residual cytoplasm. This synchrony and the parallel orientation of cells, occurring in late stages, allow the establishment of a fairly precise developmental sequence. The main features of each stage can be summarized as follows: Stage 1. Aggregation of mitochondria; budding of the flagellum from a basal body Stage 2. Mitochondrial nebenkern formation. Stage 3. Centriolar adjunct and proacrosome formation. Stage 4. Division of the nebenkern into two derivatives.

FIGs. 44-45. Transverse sections of the nucleus. F~G. 44. The chromatin sheets have fused and they form a honeycomb pattern limiting irregular polygonal spaces. Two cross sectional profiles represent more anterior level (arrows). They show two symmetrical nuclear indentations containing the posterior end of the acrosomal cone. × 18 000. Fro. 45. This slightly oblique section shows a complete set of perinuclear microtubules (mt) and a second row in formation. × 36 000. FIG. 46. Centriolar adjunct. The organelle (CA) extends between the two mitochondrial derivatives. × 28 8OO. Fro. 47. Tail of the spermatid. Several ]obules form along the tail. They contain ribosomes and large cisternae which will be rejected during the next stage and will form the residual cytoplasmic spheres. × 36 000. Fins. 48-49. Transverse sections of the tail. Microtubules appear associated with ER cisternae whose membranes have collapsed. Fig. 48 shows three collapsed cisternae; the horizontal one fbrms a straight line with associated microtubules. × 29 000. FIG. 49 represents a slightly later stage: the central and accessory tubules of the axoneme have a dense core. The collapsed cisterna forms a complete sheath around the axoneme and the mitochondria; it is lined by a row of microtubules. × 49 000. FIG. 50. Longitudinal section of the axoneme. The micrograph shows densities extending between the central tubules and the peripheral doublets (long arrows) and a periodicity in the doublets themselves (short arrows). × 90 000.

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Fins. 51-58. Spermatids of stage 8.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA

Stage 5. Chromatin organization into fibers; appearance of the "chromatold body" and, in the axoneme, oI accessory tubules. Stage 6. Organization of lamellar sheets in the nucleus and completion of the acrosome; beginning of cytoplasmic sloughing off. Stage 7. Honeycomb arrangement of the chromatin sheets in the nucleus; formation of a core in accessory and central tubules of the axoneme. Stage 8. Formation of the spermatid bundle; very tiny honeycomb arrangement of the chromatin; formation of concentric rows of microtubules around the nucleus and the mitochondrial derivatives. Stage 9. Formation of the spermatodesm; completion of the cytoplasmic sloughing off; total condensation of the nucleus. Stage 10. Disappearance of all microtubules. In spite of the evident artificial character of any staging, the proposed one provides an adequate guide line for further studies and a useful tool for understanding abhorrealities of spermiogenesis. Besides, in establishing this staging, the morphology of individual organelles was studied and their progressive transformations were followed. New aspects of their development and of their mutual relationships became evident.

339

Flagellum, Basal Body and Centriolar Adjunct Flagellum formation is initiated very early since a short bud of this organelle is already present during late anaphase of the second meiotic division. In contrast to some authors who reported the presence of two centrioles in the young spermatid in various insects (5), only one centriole was found in Locusta in the periphery of the cell. There, it functions as a basal body. Without complete serial sections, it is of course impossible to establish with certainty the absence of a second centriole; however, a great number of sections were searched in vain. On the other hand in three out of the four micrographs submitted by Breland et al. (5) and which demonstrate the presence of two centrioles, the chromatin pattern, the great number of nuclear pores and the localization of the mitochondria suggest that these cells could be spermatocytes rather than spermatids (Figs. 1, 3, and 4). The fourth micrograph shows a spermatid with a very peculiar nucleus, probably a degenerative one. In such a case, the presence of two centrioles could also be abnormal. Several authors (I6, 37) found a single centriole in spermarids but the earliest stage they considered was the stage when the basal body is already apposed to the nuclear envelope and surrounded by the centriolar adjunct

Fro. 51. Bundle formation. The sperm cells align against the nuclear region of one of the cyst cells. × 7 200. FIGS. 52-53. Transverse sections of the nucleus. The size of the clear spaces of the honeycomb is reduced, particularly in the more anterior region (Fig. 52). Under the perinuclear ER, two to three rows of microtubules have developed. × 36 000. FIos. 54-55. Longitudinal sections of the caudal portion of the nucleus. The sheets of chromatin are longitudinally oriented. Although compact, the centriolar adjunct (CA) still contains vacuoles. Fig. 54 is a lateral view showing the anterior part of the mitochondrial derivatives. × 14 000. FIG. 55 shows that the base of the nucleus is embedded by the centriolar adjunct. × 29 000. FrG. 56. Transverse section of the nucleus. Slightly later, three complete rows of microtubules are present around the nucleus. A central dot is present in some of them. × 50 000. Fro. 57. Transverse section of the tail. At the end of the stage, the external fibrous coat of the plasma membrane becomes compact. The mitochondrial derivatives have reached their definitive aspect and show a deep lateral infolding. Above them, the periaxonemal sheath forms two crescent-shaped densities (arrow). × 72 000. FIG. 58. Oblique section of the nucleus. Enlargement from Fig. 51. × 28 000.

340

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SZOLLSSI

Fins. 59-63. S p e r m a t i d s of stage 9. FIa. 59. F o r m a t i o n of t h e s p e r m a t o d e s m . Individual caps of t h e s p e r m cells have fused to form a single cap (C) in which all t h e acrosomes are e m b e d d e d . Cy, cyst cell. × 12 000.

SPERMIOGENESIS OF LOCUSTA MIGRATORIA (stage 3 of the present study). In the latter cases, the presence of a second centriole in a younger s p e r m a t i d cannot be excluded. In Locusta, the granules of the centriolar adjunct a c c u m u l a t e around the basal body which, at least until t h a t stage, is a typical centriole constituted by 9 triplets. This case apparently differs from those described by Phillips (28) who states (p. 260) t h a t the organelle forms around a centriole oriented perpendicularly to the axis of the flagellum. When following experimental t r e a t m e n t s , several centriolar adjuncts form in Locusta, a corresponding n u m b e r of flagellum also form and their base is located in the center of the organelle (35). T h e flagellum forming centriole is ext r e m e l y modified in later stages and no triplets have been seen. At the base of the nucleus, the accessory tubules, doublets and central tubules are e m b e d d e d in a dense matrix. F r o m slightly oblique sections at this level, it appears t h a t the central and accessory tubules almost reach the nuclear envelope. T h e orientation of the doublets is also modified and they become almost radial. Furthermore, there is no basal plate, and thus, no clear separation can be m a d e between a centriolar region and the flagellum: the conclusion must be drawn that there is no real centriole in the m a t u r e sperm. Similar modifications of the basal body have been described in some other insects (28) as well as in m a m m a l s (I4). Flagellum formation is similar to t h a t described by Sorokin (32), the elongating axoneme projecting into a cytoplasmic bud limited by a cuplike vesicle limited by a double m e m b r a n e . T h e accessory tubules

341

start forming as small arms extending from subfibers B (stage 4). The first completed accessory tubules are always located in the axoneme region which is not facing the mitochondrial derivatives, as in Drosophila (33) but no explanation can be given for this fact. By stage 5, all accessory tubules have been formed. A dense core is deposited in the accessory and central tubules during stage 7. In m a t u r e sperm, it was shown to react positively with Thiery's and Rambourg's methods (36, 29) indicating a polysaccharidic content. In longitudinal sections, the stain is often arranged in line of granules. In other Acridids, this material has been identified as glycogenIike (4). The organization of the axoneme is constant all along its length, except for its termination where the typical p a t t e r n is distorted. The dissociation and scattering of axonemal tubules does not proceed, however, as far as in other insect (12, 27). The morphological evolution of the centriolar adjunct has been reported already in a previous paper (6). On GMA sections, the organelle is easily digested by pepsine and pronase in very young spermatid and it is thus d e m o n s t r a t e d t h a t its protein content is quite high as soon as it is formed (unpublished). These new d a t a do not contribute further information as to the eventual RNA content of the organelle.

Acrosome and Cap Formation A description of the mature acrosome of O r t h o p t e r a has been m a d e recently (3) and our observations in Locusta are in general agreement with this report which was based on another Acridid. The proacrosoreal granule and its transformations have

FIG. 60. Nuclear region. Longitudinal microtubules surround the nucleus whose chromatin is fully condensed, x 27 000. Fro. 61. Acrosomal region. The size of the anterior ampulla (a) has decreased. Strands of extracellular material extend between the plasma membrane and the substance of the cap (C). In this figure, the individual cap of the sperm cell has not yet fused with the others, x 27 000. Fla. 62. Centriolar adjunct. The dense organelle is subdivided in several longitudinal lamellae, x 13 000. FIG. 63. Cross section of the sperm tail. The periaxonemal sheath has disappeared but the two dense crescents persist above the mitochondria, x 41 000.

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FIGS. 64-73. Spermatids of stage 10.

S P E R M I O G E N E S I S OF L O C U S T A M I G R A T O R I A

been described in detail elsewhere (34) but questions remain concerning the structure and origin of the organelle. It is first seen in stage 3 and is limited by what appears to be a double membrane. However, a trilaminar structure could not be resolved in either of the two membranes and therefore a doubt remains concerning their nature. If the proacrosome was limited by a single membrane, then it would be a very peculiar one, since the internal dense line would represent a regularly occurring structure mimicking a second membrane. No thorough description of such a structure has yet been given even though Kaye (20) mentions that in the proacrosome of the Cricket the membrane seems to be double in places. Similarly, a double membrane seems to enclose the proacrosomal granule in Sarcophaga (Fig. 4 in Warner (37)) but was not mentioned. This unusual or double membrane of the acrosome could thus be a more general feature in Insect sperm. The origin of the organelle is peculiar in Locusta. The dictyosomes do not aggregate to form a single acroblast as in Gryllids (20) and as in most other animal sperm cells, but small Golgi complexes are randomly scattered in the cytoplasm of the spermatid. Many of them could potentially produce acrosomal material but none have been seen in the immediate vicinity of the proacrosome granule which appears sud-

343

denly at stage 3 between the nebenkern and the nucleus. Possibly, in earlier stages, some dictyosomes specialize in production of acrosomal material while condensation is seen only when the proacrosome is already separated from the dictyosomes. The presence of electron translucent vesicles around the granules has also to be noted since it is a consistent feature. The fibrous coat lining the internal surface of these vesicles is morphologically similar to the external coat, and therefore they represent probably endocytotic vesicles and are not of Golgi origin. Because of the peculiar limiting membrane and disposition of the Locust proacrosome, the question of its origin from the Golgi must be left open. The mature acrosomes of all spermatids of a cyst are progessively embedded in an extracellular substance which binds them together in stage 9 and forms the cap of the spermatodesm. Details regarding the mature acrosome and the spermatodesm formation have been recently described (34).

Nuclear Trans[ormations Numerous studies exist of the extensive reorganization of the nuclear chromatin in various Orthoptera and the late transformations seem very similar in all species studied (8, 18, 19). They consist mainly in the formation of longitudinally oriented sheets limiting clear areas and in the

Fro. 64. Sperm cells during their transfer towards the efferent duct. Perinuclear microtubules have disappeared (arrow). Follicle cell wall, FW; cyst cell, Cy. × 36 000. FIG. 65. Transverse sections of the cap of the spermatodesm. From center to periphery the sperm ceils are sectioned at different levels: the most anteriorly sectioned sperm cells are in the periphery. × 8 600. Fins. 66-70. Transverse sections of the acrosomal region. In Figs. 66 and 67, only the anterior triangular acrosomal cone is present. Its limiting envelope still appears double. In Fig. 68, the inner rodlet appears and this level shows the transition between the acrosomal plasma membrane and the regular fibrous one (arrows). In Fig. 69, the anterior part of the indented nucleus becomes visible. Fig. 70 shows the nucleus with the lateral posterior part of the acrosomal cone. Substance of the outer cone has totally disappeared at this stage. × 72 000. FIG. 71. Cross section of the flagellum. All microtubules other than in the axoneme have disappeared. × 40 500. Fins. 72-73. Detection of polysaecharids in the axoneme by the Thi~ry's method. FIG. 72. The central and accessory tubules show a positive reaction. On the caudal extremity of the tail, the elements which reach the most posterior level are the accessory tubules (arrows). x 23 000. Fro. 73. In longitudinal section the stain is often arranged in line of granules. × 29 000. Fios. 65-73 represent sperm cells stored in the seminal vesicles.

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ANNETTE SZ6LL~SI

progressive reduction of these areas until the nucleus is fully condensed. Intranu-" clear dense bodies persist until stage 7 when most of the chromatin has already reorganized in sheets; at the moment, their composition and significance is unknown. Clusters of bodies similar to those described in Acrida lata (38) were not found in this study but the "coiled structure" may possibly correspond to the "filamentous elements" delineated in the above paper. The authors' statement concerning the chemical nature of these structures cannot be accepted however, since their conclusions are uniquely based on acid extraction and Unna-Pappenheim reactions carried out on thin and thick Epon sections. These methods have been proved to be unreliable. A coiled structure, morphologically similar to the one described here has been demonstrated in Drosophila where it was suggested to contain highly condensed DNA (20). From stage 5 on, transverse sections of nuclei give an adequate information for staging but, again if one looks in more detail, an antero-posterior gradient of the nuclear transformation becomes evident. In stage 9 in particular, when the heads of spermatid are gathered into a tight bundle, it is obvious that the clear spaces of the honeycomblike chromatin are smaller and fewer in the acrosome region than at the posterior end of the nucleus.

Mitochondrial Derivatives The formation of the nebenkern, its division into two halves, its transformation into two long dense derivatives are similar to the process found in other insects and previously described and summarized by Phillips (28). Until the end of stage 4, a typical mitochondrial profile is retained, with a double limiting membrane and internal cristae. Then a reorganization occurs, and the general electron density increases. Mitochondrial transformations in the spermatozoa of Locusta can be considered as simple in comparison with other

insects (I, 28): the two derivatives are similar, symmetrically displayed and they extend almost to the tip of the long tail. With the technics used in this work, no crystalline material has been detected in these organelles.

Microtubules and Endoplasmic Reticulum During spermiogenesis, ER vesicles with occasionally attached ribosomes are scattered randomly in the cytoplasm. However, some cisternae have a particular fate. They tend to collapse and then, they frequently become associated with microtubules. In stage 5, a cisterna surrounds the ovoid nucleus delimiting a narrow and rather dense cytoplasmic territory within which microtubules develop. At stage 6, the two membranes of this cisterna have collapsed in regions, forming a single dense line. In addition, by this time, the two membranes of the nuclear envelope has also fused. The same is true for the cisterna which surrounds the axoneme and also for those which surround the whole axonemal complex including mitochondrial derivatives, axoneme and centriolar adjunct. Stage by stage the collapse of the ER could be followed and, for this reason, we do not agree with authors who consider this system of narrow membranes as originating from the Golgi (4). In the same way, the cresent-shaped body, associated with the mitochondrial derivatives, appears in contact with ER and not with Golgi material. These collapsed membranes of the flagellum delineate early a peripheral territory whose fate is to be sloughed off with all organelles included. However, in this territory, polysomes are found whose presence could indicate some late protein synthesis until stage 8, when all spermatids are gathered in a bundle. Bridges between spermatids exist also until this late stage and actual passage of newly synthesized proteins would still be possible between the cells. In many places, microtubules develop in rows along the collapsed cisternae which

SPERMIOGENESIS OF LOCUSTA MIGRATORIA disappear when the complete set of microtubules is formed. I t is tempting to see a relation between the two structures because of the synchrony of their appearance and because of their fairly constant topographical association. Maybe, the cisternae delimit a cytoplasmic territory in which microtubules precursors can accumulate, but the functional significance of the collapsing process is not clear. A role of microtubules in cell elongation has often been proposed (23). Although Fawcett et al. (11) developed several arguments to show that these organelles do not play a determinant role in shaping the sperm nucleus, such a role is an attractive hypothesis in the case of the long slender sperm cell of Acridids. Actually, disturbance of the microtubular pattern in experimental conditions (35) is always cotrelated with abnormalities in sperm and nucleus elongation. The possibility remains also that the microtubules could be linked in some fashion to the antero-posterior flow of cytoplasm which accompanies the normal differentiation process.

Chromatoid Body In their work on Orthoptera spermatogenesis, Gatenby and Tahmisian (17) refer to various cytoplasmic organelles: "ordinary chromatoid body .... Y-granules" and "X enigmatic body." The dense sphere described in the present paper corresponds to the "ordinary chromatoid body," it is sometimes associated with a lighter sphere, possibly the X enigmatic body. The relationship of the chromatoid body with other particle aggregates found in earlier stages (spermatogonia and spermatocyte) could not be demonstrated. It appears suddenly as a dense sphere and there is no hint as to its origin. Thus, no analogy can be drawn with the chromatoid body in Mammalian spermatozoa (9, 12). Following a pronase + pepsine treatment of GMA sections, the chromatoid body is extracted, suggesting a proteinaceous content. In another Acridid (39), it was

345

claimed that the chromatoid body had a nuclear origin and that it "contributes to the formation of the centriole adjunct in a visible way." The nuclear origin of the chromatoid body is possible but not convincingly demonstrated by the authors since the "extruded granules" in their Fig. 2 are limited by a membrane and not associated with the nuclear pores. In Locusta, we found multivesicular bodies associated in large number with the nuclear envelope and they possibly correspond to the dense bodies shown in Acrida. In regard to the participation of the chromatoid body in the formation of the centriolar adjunct, our observations are in contradiction with the statement of Yasuzumi et al. (39) for the following reasons: (1) the chromatoid body and the centriolar adjunct are constantly found in two distinct and different cytoplasmic regions, (2) their chemical nature is different, since the centriolar adjunct is easily digested even following short treatment by either pepsine or pronase while extraction of the chromatoid body is obtained after a much longer treatment by these two enzymes in sequence and only in a water-miscible embedding medium, and (3) the dissolution of the chromatoid body cannot be claimed since the organelle migrates toward the tail and is finally found in the residual cytoplasms. There is no decrease in its size during the migration and no obvious loss of its substance occurs. Thus, according to the results of the present study, no relationships between the chromatoid body and the centriolar adjunct can be demonstrated.

Plasma M e m b r a n e and Fibrillar Coat The plasma membrane of the Locust sperm cell has a particularly thick external layer. In addition, it is covered by a specific

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ANNETTE SZOLLOSI

external coat (2, 21, 31). In Locusta, a deli9. EDDY, E. M., Biol. Reprod. 2, 114 (1970). cate fuzzy coat is visible as early as stage 10. FAWCETT,D. W., Proc. Int. Symp. Genetics of the spermatozoon. Edinburgh, 37 (1971). 2 but its thickness progressively in11. FAWCETT,D. W., ANDERSON,W. A., ANDPHmLIPS, creases. During the last stage, and may be D. M., Dev. Biol. 26, 220 (1971). with addition of an amorphous substance, 12. FAWCETT,D. W., EDDY, E. M., AND PHILLIPS, D. it transforms into a highly organized and M., Biol. Reprod. 2, 129 (1970). compact sheath in which two layers can be 13. FAWCETT,D. W., ITO, S., ANDSLAUTTERBACK,D., J. Biophys. Biochem. Cytol. 4, 453 (1959). distinguished. The internal layer is the 14. FAWCETT,D. W., ANDPHILLIPS, D. M., Anat. Rec. only one present in the acrosomal region. 165, 153 (1969). The transition between acrosomal and 15. FOLLIOT, R., C. R. Acad. Sci. Paris 271, 508 more posterior regions is sharply defined (1970). in mature sperm but exists already in early 16. FRIEDL.ANDER,M., ANDWAHRMAN,J., J. Morphol., 134, 383 (1971). stages. Actually, the external layer of the 17. GATENBY,J. B., ANDTAHMISIAN,T. N., La Cellule, fibrous coat was never formed over the de60, 103 (1959). veloping acrosome. 18. GALL, J. G., AND BJORK, L. B., J. Biophys. A very orderly sequence of events takes Biochern. Cytol. 4, 479 (1958). place during spermiogenesis and each or- 19. GIBBONS,J. R., ANDBRADFIELD,J. R., J. Biophys. Biochem. Cytol. 3, 133 (1957). ganelle is in a definite developmental stage 20. KAYE, J. S., J. Cell Biol. 12, 411 (1962). at one given stage. However, it is not 21. KESSEL,R. G., J. Ultrastruct. Res. 18, 677 (1967). known in which way synchronous events 22. LEDUC, E. S., AND BERNHARD,W., J. Ultrastruct. are linked. Does the appearance of one Res., 19, 196 (1967). organelle induce specific transformations 23. MCINTOSH,J. R., ANDPORTER, K. R., J. Cell Biol. 35, 153 (1967). in another? How strictly defined is the 24. MONNERON, A., AND BERNHARD, W., J. Microsc. cytoplasmic region in which they appear? (Paris) 5, 697 (1966). Such questions can partially be answered 25. PAPILLON, M., LAUVERJAT,S., AND CANTACUZENE, by experiments which provoke shifts in this A., J. Insect Physiol. 18, 2005 (1972). highly organized process and which are in 26. PAPILLON, M., AND CANTACUZf~NE-SzI~KELY,A., Bull. biol. Fr. Belg. 107, 116 (1973). progress. The author wishes to thank Mrs. Marcaillou for her technical help and Mr. Morineau for his efficient assistance in printing the micrographs. REFERENCES 1. ANDR~, J., J. Ultrastruct. Res. 6, Suppl. 3 (1962). 2. BACCETTI, B., BIGLIARDI, E., BURRINI, A., DALLAI, R., AND ROSATI, F., VII Congr. Micr. Electr. Grenoble (1970). 3. BACCETTI,B., ROSATI, F. AND SELMI, G., J. Submicrosc. Cytol. 3, 319 (1971). 4. BACCETTI, B., Advanc. Insect Physiol. 9, 315 (1972). 5. BRELAND,O. P., BACKER,K. R., EDDLEMAN,C. D., AND BIESELE, J. J., Ann. Entomol. Soc. A m e r . 61, 1037 (1968). 6. CANTACUZf~NE,A., Comparative Spermatology, p. 553. Academic Press, New York, 1970. 7. CANTACUZENE,A., LAUVERJAT, S., AND PAPILLON, M., J. Insect Physiol. 18, 2077 (1972). 8. DASS, C. M., AND RIS, H., J. Biophys. Biochem. Cytol. 4, 129 (1958).

27. PEROTTI, M. E., J. Submicrosc. Cytol. 1, 171 (1969). 28. PHILLIPS,m. M., J. Cell Biol. 44, 243 (1970). 29. RAMBOURG,A., C. R. Acad. Sci. Paris 265, 1426 (1967). 30. RASSMUSSEN,S. W., Z. Zellforsch. mikrosk. Anat. 140, 125 (1973). 31. ROTH, L. E., J. Biophys. Biochem. Cytol. 3, 816 (1957). 32. SOROKIN,S., J. Cell Biol. 15, 363 (1962). 33. STANLEY,H. G., BOWMAN,J. T., ROMRELL,L. J., REED, S. C., AND WILKINSON,R. F., J. Ultrastruct. Res. 41, 433 (1972). 34. SZSLLgSI,A., Acrida 3, 175 (1974). 35. SZ~JLL{~SI,A., J. Microsc. (Paris) 20, 92 (1974). 36. THII~RY,J. P., g. Microsc. (Paris) 6, 987 (1967). 37. WARNER,F. D., J. Ultrastruct. Res. 35,210 (1971). 38. YASUZUMI, G., SUGIOKA, T., TSUBO, I., AND MATANO, Y., Z. Zellforsch. mikrosk. Anat. 109, 450 (1970a). 39. YASUZUMI, G., SUOIOKA, T., TSUBO, I., AND MATANO, Y., Z. ZeUforsch. mikrosk. Anat. 110 231 (1970b).