Microtubule Organization and the Distribution of Γ-Tubulin in Spermatogenesis of a Beetle, Tenebrio Molitor (Tenebrionidae, Coleoptera, Insecta)

Home , Acrosome, Micronucleus

Journal of Cell Science 108, 3855-3865 (1995) 3855 Printed in Great Britain © The Company of Biologists Limited 1995 JCS3135

Microtubule organization and the distribution of γ-tubulin in spermatogenesis of a beetle, Tenebrio molitor (Tenebrionidae, Coleoptera, Insecta)

Klaus Werner Wolf1,* and Harish C. Joshi2 1Institut für Biologie, Medizinische Universität Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Deutschland 2Department of Anatomy and Cell Biology, Emory University, School of Medicine, Atlanta, Georgia 30322, USA *Author for correspondence

SUMMARY

The present study focuses on the restructuring of the prominent cytoplasmic MT system of primary spermato- microtubule (MT) cytoskeleton and microtubule-organiz- cytes in prophase, microtubule nucleation apparently ing centres (MTOCs) throughout spermatogenesis of a occurs in the absence of immunologically detectable γ- darkling beetle, Tenebrio molitor (Tenebrionidae, tubulin. At the poles of the meiotic spindles, MTs are Coleoptera, Insecta). To this end, serial ultrathin sections directly inserted into γ-tubulin-containing material and this through male germ cells were studied using transmission connection is considered responsible for their nucleation. electron microscopy. Additionaly, spindles and young sper- The interzone spindle MTs of telophase cells contain γ- matids were isolated from testes under MT-stabilizing con- tubulin and this may confer stability to them. Finally, ditions and doubly labeled with antibodies against β- and manchette MTs of spermatids originate in the vicinity of γ-tubulin. The latter is a tubulin isoform detected in the acrosome precursor but are not inserted into this body. MTOCs of a wide variety of species. The observations The acrosome precursor is surrounded by a membrane and suggest that microtubules may be nucleated from sites with is clearly detected by the antibody against γ-tubulin. and without high γ-tubulin content and that these sites do not necessarily possess canonical centrosomes. In a Key words: acrosome, centrosome, meiosis, spermatid

INTRODUCTION spindles which bring about chromosome segregation during two sucessive divisions. The cells arising from the second All eukaryotic cells possess skeletal elements in the form of meiotic division develop into spermatids. These are slender microtubles (MTs). Most MTs have one end close to an element elongated cells containing a haploid genome. Spermatids which is believed to organize the microtubular cytoskeleton. ﬁnally transform into spermatozoa ready for fertilization. The The expressions centrosome (e.g. Mazia, 1984) or microtubule- basic cytological features of canonical spermatogenesis are organizing-centre (MTOCs) (Pickett-Heaps, 1969) are widely well known. The present study is aimed at the restructuring of used in this context. The last few years have seen the discovery the microtubular cytoskeleton during spermatogenesis of the of a series of components contained within MTOCs (for a darkling beetle Tenebrio molitor (Tenebrionidae, Coleoptera, review, see Kalt and Schliwa, 1993). A highly conserved Insecta). This organism has been selected on technical tubulin isoform, γ-tubulin, has been found to be enriched within grounds: rearing is easy and its size renders it a handy system MTOCs from a wide variety of species and cell types (for a for cytological work. In addition, conventional bipolar spindles review, see Joshi, 1994). The consistent presence of γ-tubulin are formed in male meiosis of the beetle (Wolf and Hellwage, in MTOCs renders it a prime candidate for a role in MT 1995) and sperm structure is regular (Baccetti et al., 1973). nucleation. In order to gain further insight into the function of Thus, T. molitor has the potential of serving as a model γ-tubulin, it appears worthwhile to study its distribution in organism for Coleoptera, the largest insect order with more relation to the changes in the microtubular cytoskeleton in a than 350,000 species described so far. A ﬁne structure analysis complex developmental process such as spermatogenesis. A of MTOCs in spermatogenesis of T. molitor supplements the polyclonal antibody, generated against a conserved portion of immunological work. γ-tubulin (Joshi et al., 1992), renders this feasible. The antibody used in this study was made against a peptide that was carefully chosen to be unique to γ-tubulin (Table 1). MATERIALS AND METHODS During spermatogenesis, profound alterations of the cells in terms of size, shape and function occur. Primary spermatocytes The experimental animal in prophase are characterized by the pairing of homologous Male pupae from a laboratory strain of Tenebrio molitor, reared on chromosomes. Cells of the ensuing stages possess meiotic rolled oats, were used in this study. 3856 K. W. Wolf and H. C. Joshi

Electron microscopy Table 1. Peptide used to raise the antibody to γ- Pupal testes were prepared for electron microscopy as reported pre- tubulin and C-terminal peptides of γ-tubulins from yeast viously for Lepidopteran tissue (Wolf, 1994). to man

Anti-tubulin immunofluorescence Peptide EEFATEGTDRKDVFFY-C Human EEFATEGTDRKDVFFY The testes were dissected out in Ringer solution (Wolf, 1994), trans- Mouse EEFATEGTDRKDVFFY ferred into a microtubule-stabilizing buffer (100 mM piperazine-N,N′ Drosophila EDFANDGLDRKDVFFY bis(2-ethane sulfonic acid), pH 6.8, 1 mM MgSO4, 1 mM ethylene Aspergillus EEFATEGGDRKDVFFY glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid, 1% Triton X- Yeast (S. pombe) ESFATEGVDRKDVFFY 100) and minced using tungsten needles. The suspension containing α-tubulin SDKTIGGDDSFNTFF germ cells was spun onto coverslips in a cytocentrifuge (Shandon D L D β GTSDAQLERISVXYNE Cytospin II, 1,500 rpm, 5 minutes). Fixation was carried out with -tubulin 0.25% glutaraldehyde and 2% formaldehyde in microtubule-stabiliz- The antibody to γ-tubulin used in this study was elicited from a C-terminal ing buffer devoid of detergent (30 minutes). For further stabilization peptide conserved among γ-tubulins from yeast to man. The comparison with of the cytoskeletons, the coverslips were subsequently immersed in peptides from α- and β-tubulin shows that cross-reactions are not likely. 100% methanol (−20¡C). In order to block free aldehyde residues, the From Joshi et al. (1992). cells were treated with NaBH4 (0.5 mg/ml in phosphate-buffered saline (PBS) according to the method of Osborn and Weber (1982). Following this and all subsequent incubations, the coverslips were Prometaphase primary spermatocytes are characterized by rinsed three times (5 minutes) in PBS containing 0.02% NaN3 and the presence of an elongated microtubular spindle and chro- 0.1% Triton X-100 and once (5 minutes) in this solution without mosomes scattered throughout the spindle area (Fig. 1d-f). The detergent. remainder of the cytoplasm is virtually devoid of MTs. The Double labeling of γ- and β-tubulin was achieved by using simul- spindle poles are intensely labeled with the antibody against γ- taneously two different antibodies. These were: (1) a mouse mono- tubulin (Fig. 1e). This is also true for metaphase in primary clonal antibody against β-tubulin (Sigma); and (2) a rabbit polyclonal γ spermatocytes, when a regular bipolar spindle forms and the antibody against -tubulin (Joshi et al., 1992). The antibodies were bivalents are aligned in the equatorial plate (Fig. 1g-i). Spindle diluted 1:100 with PBS containing 10 mg/ml bovine serum albumine (BSA) prior to use. structure does not change much in early anaphase, but half Incubation with a biotin-conjugated anti-mouse antibody (Sigma) at bivalents separate from one another (Fig. 1j-l). In primary sper- a dilution of 1:50 in PBS containing 10 mg/ml BSA (1 hour) and finally matocytes in mid telophase I, the spindle elongates and the with rhodamine-coupled avidine (Sigma) diluted 1:50 with PBS con- chromosomes reach the spindle poles. A prominent interzone taining 10 mg/ml BSA (1 hour) was used to visualize the binding of spindle develops between the prospective daughter nuclei. The the antibody against β-tubulin. The specimens were incubated with 1 antibody against γ-tubulin detects two separate fluorescent spots mg/ml polylysine (Serva) in PBS (10 minutes) prior to the last step. The per spindle pole. Additionally, there is label at the poleward antibody against γ-tubulin was rendered visible through a fluorescein- ends of the interzone spindle at the equatorial face of the isothiocyanate (FITC)-conjugated anti-rabbit antibody. In order to µ ′ prospective daughter nuclei (Fig. 1m-o). Primary spermatocytes visualize the chromatin, the specimens were stained with 5 g/ml 4 ,6- in late telophase show two weak fluorescent spots close to the diamidino-2-phenylindole.2 HCl (DAPI) (Serva) in citrate buffer (100 daughter nuclei when labeled with the antibody against γ- mM citric acid, 200 mM Na2HPO4, pH7). The specimens were mounted in PBS AF3 (Citifluor Ltd, London). The preparations were sealed with tubulin. The two spots are further apart from one another than nail varnish. Except for the methanol treatment, all reactions were in the previous stage. The interzone spindle fluoresces faintly carried out at room temperature. The cells were analysed and pho- throughout (Fig. 1p-r). The comparison of spindles in the first tographed using a Laborlux 12 photomicroscope equipped with epiflu- meiosis (Fig. 1a-c to p-r) with spindles stained under identical orescence illumination and a Fluotar ×100 (Leitz, Wetzlar). conditions but with the antibody to γ-tubulin omitted (see Fig. 8a-c) showed that the weak label evenly distributed throughout the microtubular cytoskeleton in primary spermatocytes from RESULTS prophase to early anphase (Fig. 1a-l) represents background. Label in primary spermatocytes in telophase is, however, The first meiotic division clearly above background (Fig. 1m-r). Immunofluorescence The second meiotic division in males of T. molitor is very similar to the first meiosis with respect to the restructuring of Meiotic division in males of T. molitor was studied using the microtubular cytoskeleton and chromosome movements. electron microscopy of ultrathin sections. For details of cell and Therefore, we dispense with showing the entire process in the spindle architecture, the reader is referred to Wolf and Hellwage present context. However, spindles are smaller in the second (1995). Primary spermatocytes in late prophase possess a meiotic division and telophase spermatocytes possess only one prominent microtubular cytoskeleton. A large MT bundle con- β centrosome per spindle pole (compare Fig. 3a-c). Short flagella taining -tubulin can be observed close to the nucleus (Fig. 1a). are present already in primary spermatocytes (see below and The nuclei do not reveal much substructure when stained with compare Wolf and Hellwage, 1995) and we are dealing with a DNA-specific fluorescent dye (Fig. 1c). Using the antibody γ basal bodies at the spindle poles in male meiosis of T. molitor. against -tubulin, two weak fluorescent spots close to the The basal bodies do not replicate during transition from first to nucleus but opposite the prominent MT bundle are detectable. second meiotic division. This is probably the rule in male Their presence marks the positions of the centrosomes (Fig. 1b) insects (compare Friedländer and Wahrman, 1971). and some MTs are visible in their vicinity. However, the bulk of cytoplasmic MTs of late prophase primary spermatocytes is Electron microscopy not organized through the centrosomes. The cellular sites detected by the antibody against γ-tubulin MTOCs in Tenebrio molitor 3857

a b c d e f g h i

j k l m n o p q r

Fig. 1. Meiosis I. (a,d,g,j,m,p) An anti-β-tubulin immunofluorescence image; (b,e,h,k,n,q) an anti-γ-tubulin immunofluorescence image of the same cell; (c,f,i,l,o,r) visualization of the chromatin of the cell using DAPI, a DNA-specific fluorescent dye. (a-c) Late prophase primary spermatocyte. A prominent microtubule bundle (asterisk in a) is detectable next to the nucleus (N). Arrows in b denote the positions of the centrosomes. (d-f) Prometaphase primary spermatocyte. A bipolar spindle is present. The bivalents are scattered throughout the spindle area. (g-i) Metaphase primary spermatocyte. The bivalents are aligned in the equatorial plane. Note the prominent staining of the spindle poles with the antibody against γ-tubulin (h). (j-l) Early anaphase primary spermatocyte. Separation of homologs, situated at the periphery, has started. (m- o) Mid telophase primary spermatocyte. A prominnt interzone spindle is visible between the two prospective daughter nuclei (D). The antibody against γ-tubulin detects two fluorescent spots at each spindle pole (n). Note also the faint staining obtained with this antibody close to the equatorial face of the prospective daughter nuclei. (p-r) Late telophase primary spermatocyte. The width of the interzone spindle is decresing. The antibody against γ-tubulin detects two faint fluorescent spots per spindle pole (arrows in q). The antibody also faintly labels the interzone spindle throughout (q). Bar, 5 µm. were studied in ultrathin sections through germ cells of T. of the spindle equator, MTs show a regular orientation parallel molitor using transmission electron microscopy. In primary to the spindle axis. In contrast, the orientation of numerous spermatocytes in late prophase, two pairs of orthogonally MTs in the apical portions of the interzone spindle close to the arranged centrioles are located in a slender cytoplasmic layer chromatin deviates from the regular path. Many MTs arranged between the nucleus and the plasma membrane. The centrioles obliquely or perpendicularly to the spindle axis are to be seen show the usual pattern of 9 circumferentially arranged micro- there (Fig. 2g). tubular triplets and a central hub in their basal portions. Each centriole pair is surrounded by a thin layer of pericentriolar Spermatid structure material (PCM). Few MTs occur in the vicinity of the centro- Immunofluorescence somes (Fig. 2a-d). As mentioned above, telophase II spindles of T. molitor show In primary spermatocytes in prometaphase, flagellar only one fluorescent spot, the centrosome, per spindle pole outgrowth begins and the centrioles are now referred to as when stained with the antibody against γ-tubulin. The interzone basal bodies. A pair of basal bodies is found per spindle pole. spindle is faintly labeled close to the daughter nuclei. Using A flagellar vesicle forms around the distal end of each basal the antibody against β-tubulin, the interzone spindle appears as body. Most conspicuous, however, is the increase both in the an array of parallel MTs (Fig. 3a-c). As spermatogenesis amount of PCM around the centrioles and the number of MTs proceeds, we see spermatids with spherical nuclei and a long inserted in this mass (Fig. 2e,f). While close to the prospective flagellum attached to the nucleus. At the attachment point, a daughter nuclei, the basal bodies of one pair separate from one weak fluorescent spot appears when the cells are labeled with another in mid telophase primary spermatocytes (Fig. 2g). the antibody against γ-tubulin. This represents a remnant of the Some membraneous sheets are visible around the chromatin centrosomal γ-tubulin situated at the poles of meiotic spindles. mass and this may indicate the onset of nuclear envelope refor- Independent of this label, however, a relatively intense flu- mation. The fine structure of the apical ends of the interzone orescent spot is detectable laterally and close to the nucleus. spindle differs from the central portions. In the neighbourhood Both fluorescent spots were never found very closely neigh- 3858 K. W. Wolf and H. C. Joshi

a b

Fig. 2. Electron micrographs of ultrathin sections through c d e f primary spermatocytes of Tenebrio molitor. (a-d) Late prophase primary spermatocyte. Four consecutive serial sections through a centrosome consisting of two centrioles (arrowheads). One centriole is cross-sectioned whereas the second is longitudinally cut. The location of the entire complex close to the plasma membrane (PM) is obvious. Bar, 200 nm. (e,f) Prometaphase primary spermatocyte. Two consecutive serial sections through the spindle pole. Two basal bodies (arrowheads) with short flagellar stubs are visible. One bears a flagellar vesicle (FV). The entire complex is embedded in electron-dense material. Note the numerous microtubules in the lateral cytoplasm. Bar, 200 nm. (g) Mid telophase primary spermatocyte. A portion of the spindle containing a prospective daughter nucleus (D), separating basal bodies (arrowheads), and some regularly arranged interzone microtubules (asterisk) is visible. Close to the prospective daughter nucleus, numerous membraneous elements and microtubules are to be seen. M, mitochondria. g Bar, 1 µm. bouring. Thus, it is less likely that centrosomal γ-tubulin spermatid nucleus and very close to it. Cytoplasmic MTs migrates directly into the newly formed spot. Instead, the originate there and these give rise to a spermatid-specific MT- newly formed intensely fluorescent spot lateral to the nucleus system, the manchette (Fig. 3g-i). The microtubular manchette appears to recruit its γ-tubulin from the cytoplasm. MTs are is most probably responsible for the compression and not visible in the vicinity of the nucleus at this stage (Fig. 3d- elongation of the nucleus during this period (e.g. Baccetti, f). 1972). A prominent fluorescent spot remains visible with the The connection between the nucleus and the flagellar base antibody against γ-tubulin at the anterior pole of the spermatid is maintained when nuclear elongation begins, but the antibody when the number of manchette MTs originating there increases against γ-tubulin does not detect much material at the site (Fig. 3j-l). At an advanced stage of nuclear stretching, the where these elements are joined. This applies also to the intensity of the fluorescent spot at the anterior pole of the following stages. A prominent fluorescent spot, however, spermatid, produced with the antibody against γ-tubulin, remains to be seen at the opposite, i.e. anterior, pole of the decreases (Fig. 3m-o). MTOCs in Tenebrio molitor 3859

a b c d e f g h i j k l m n o

Fig. 3. Telophase II and spermatids. (a,d,g,j,m) Anti-β-tubulin immunofluorescence image; (b,e,h,k,n) anti-γ-tubulin immunofluorescence image of the same cell. (c,f,i,l,o) Visualization of the chromatin of the cell using DAPI. Bar, 5 µm. (a-c) Late telophase II. Each spindle pole shows a prominent fluorescent spot with the antibody against γ-tubulin (arrows in b). Daughter nucleus (D). (d-f) Spermatid. There is faint labelling with the antibody against γ-tubulin at the base of the flagellum (compare long arrows in d and e) situated at the posterior pole of the nucleus (N). The antibody detects also a prominent block of material lateral to the nucleus (short arrow in e). (g-i) Spermatid. With the antibody against γ-tubulin, label is hardly detectable at the flagellar base (compare long arrows in g-i), whereas the anterior pole of the nucleus (N) shows a prominent fluorescent spot (short arrow in h). The antibody against β-tubulin reveals that some cytoplasmic microtubules originate at the anterior pole of the nucleus (g). (j-l) Spermatid. Note the prominent fluorescent spot (arrow in k), visualized with the antibody against γ- tubulin, at the anterior pole of the nucleus (N). The mass of cytoplasmic microtubules originating there has increased (j). (m-o) Spermatid. The nucleus (N) has assumed a lancet shape. The antibody against γ-tubulin generates weak fluorescence (arrow) at the anterior pole of the nucleus.

Electron microscopy density interspersed between the nuclear envelope and the Flagellar outgrowth is advanced in late telophase of the second acrosome precursor (Fig. 4e). MTs are not detectable around meiotic division of T. molitor in comparison with the acrosome precursor. The connection between the flagellum prometaphase primary spermatocytes (compare Figs 2e,f and and the nucleus is as in the previous stage. 4a). The flagellar vesicle has fused with the plasma membrane In spermatids at a more advanced stage of nuclear and a short membrane-covered flagellar stub has developed. elongation, manchette MTs are assembled. These originate The distal end of the forming flagellum is bulbous. The close to the acrosome precursor, which appears as a shallow proximal end of the basal body is tightly attached to the outer cup-shaped element covering the anterior pole of the nucleus layer of the nuclear envelope. Only a thin layer of PCM is (Fig. 5a-l). In places, manchette MTs approach the plasma visible around the basal body (Fig. 4a). Young spermatids with membrane (Fig. 5c,d,e,g). Sheets of the endoplasmic reticulum spherical nuclei and largly decondensed chromatin possess (ER) occur in the cytoplasm lateral to the manchette MTs. elongated flagella and two mitochondrial threads, the When nuclear elongation progresses, the acrosome precursor nebenkern derivatives (for terminology of insect sperm, the transforms into a deeper cup, attached with its open end to the reader is referred to Baccetti, 1972), arranged parallel to the anterior pole of the nucleus (Fig. 6). The analysis of serial cross axoneme. The basal body is located in a shallow invagination sections through the anterior tip of spermatids at that stage of the nucleus and PCM is hardly detectable around the basal reveals that manchette MT originate in the narrow space body (Fig. 4b). between the membrane surrounding the acrosome precursor Slightly advanced spermatids, and the stage approximately and a layer of smooth ER. The MT ends are not embedded in corresponds to that shown in Fig. 3d-f, show the onset of distinct material (Fig. 6b-i). The restructuring of the nuclear chromatin condensation within the spherical nuclei. The area in telophase II spermatocytes to spermatids with elongated flagellar base is embedded in electron-dense material, which is nuclei with emphasis on the flagellar base and the behaviour traditionally referred to as the centriolar adjunct (Baccetti, of the acrosome precursor is depicted schematically in Fig. 7. 1972). Not far away from the centriolar adjunct, the acrosome precursor docks to the nuclear envelope. The acrosome Controls precursor consists of a cone-shaped vesicle connected with its Although there can be no doubt that the poles of meiotic blunt basis to the outer layer of the nuclear envelope. The spindles in males of T. molitor are rich in γ-tubulin, staining lumen of the acrosome precursor contains material of moderate of the spindles proper is a matter of interpretation and we felt electron density in its portion distant from the nucleus. MTs the necessity to display our controls. Isolated spindles were are missing in the vicinity of the acrosome precursor (Fig. 4c). labelled, with omission of the antibody to γ-tubulin. The result In spermatids with egg-shaped nuclei, the acrosome is demonstrated in a telophase spindle of the first division. In precursor is found within the slender cytoplasmic cleft between the FITC-canal, fluorescent spots are missing at the spindle the plasma membrane and the nucleus at the anterior pole of poles, but the interzone spindle is evenly labeled throughout at the cell (Fig. 4d). The acrosome precursor is a platelet-like low intensity (Fig. 8a-c). Clearly this represents background element evenly filled with material of moderate electron staining. Inclusion of the antibody to γ-tubulin produced a sig- density. The analysis of ultrathin sections at high magnifica- nificantly more intense label in particular in the vicinity of the tion reveals a thin layer of material of moderate electron daughter nuclei (compare Fig. 1n). For the sake of complete- 3860 K. W. Wolf and H. C. Joshi

Fig. 4. Electron micrographs of ultrathin sections through a spermatocyte II and spermatids at f different developmental stages of Tenebrio molitor. (a) Telophase II. The daughter nucleus (D) is completely surrounded by the nuclear envelope (NE). A basal body (arrow) continuous with a short flagellar stub (F) is attached to the outer membrane of the nuclear envelope. Bar, 0.5 µm. (b) Spermatid. Within the roughly spherical nucleus (N), the decondensed chromatin is unevenly distributed. Electron- dense bodies of unknown nature are present. The nucleus a b possesses a shallow invagination. There, a basal body (arrow) into which a long flagellum (F) is inserted. Mitochondrial threads (M), the nebenkern derivatives, are arranged parallel to the flagellum. Bar, 1 µm. (c) Spermatid. The nuclear lumen (N) appears homogeneous. A shallow invagination of the nuclear envelope contains a basal body (arrow) surrounded by the centriolar adjunct. A acrosome precursor (asterisk) is attached to the cytoplasmic side of the nuclear envelope. Flagellum (F), mitochondrion (M). Bar, 1 µm. (d) Spermatid. The egg-shaped nucleus (N) contains homogeneously staining condensed chromatin throughout. At the posterior pole of the d nucleus, a basal body (arrow) is attached. Note the centriolar adjunct surrounding it. At the anterior pole of the nucleus, the acrosome precursor (asterisk) is c e located. F, flagellum. Bar. 0.5 µm. (e) Spermatid. This detailed micrograph of the anterior nuclear pole shows the acrosome precursor (asterisk). Note the layer of moderately electron-dense material (arrowheads) interspersed between the outer nuclear membrane and the membrane of the acrosome precursor. Bar, 100 nm. ness we show also control micrographs of spermatids. In early cytoskeleton and its relationship with γ-tubulin-containing spermatids, the antibody to β-tubulin reveals the onset of cellular elements was described during spermatogenesis of a manchette MT formation close to the apical poles of the nuclei beetle, T. molitor. We used an antibody that was raised against (Fig. 8d-f and g-i). In both cells, there is no signal detectable a peptide unique to γ-tubulin (Table 1). Thus, cross-reactions in the FITC-canal at that location, when the primary antibody with α- and β-tubulin are not likely. Microtubule nucleation to γ-tubulin is omitted (Fig. 8e,h). appears to occur in the absence of immunologically detectable γ-tubulin and through direct insertion of MTs into γ-tubulin- rich material. The relationship between γ-tubulin and the DISCUSSION nucleation of manchette MTs is more complex, because the involvement of apical portions of the plasma membrane in this In the present study, the restructuring of the microtubular process has to be considered as well. The findings suggest also MTOCs in Tenebrio molitor 3861

Fig. 5. Electron micrographs of ultrathin sections through a spermatid of Tenebrio molitor. (a-l). Twelve consecutive longitudinal serial sections through the anterior pole of the spermatid. A portion of the elongating nucleus (N) and the acrosome precursor (asterisks) are visible. The lateral cytoplasm shows manchette microtubules and cisternae of smooth endoplasmic reticulum. Some manchette microtubules have their anterior ends (arrows) close to the plasma membrane (PM). Bar, 200 nm. that γ-tubulin is able to incorporate into MTs. Taken together, (Wolf and Hellwage, 1995) revealed that the non-centrosomal in spermatogenesis of a beetle, MTs may and may not be MTs are arranged parallel to mitochondrial threads. Conspic- nucleated from regions with high γ-tubulin content, and these uous and distinct elements which could be correlated with their may and may not correlate with the location of canonical cen- organization were missing in ultrathin sections inspected with trosomes. the electron microscope. In that study, spermatocytes were also probed with an antibody against acetylated MTs, 6-11B-1 Microtubule organization without detectable (Piperno and Fuller, 1985). Acetylated MTs are generally con- involvement of γ-tubulin sidered relatively stable compared to non-actylated MTs (e.g. Late prophase primary spermatocytes of T. molitor show a Webster and Borisy, 1989). The non-centrosomal MTs in late prominent array of cytoplasmic microtubules at a location prophase primary spermatocytes of T. molitor were not labeled distant from the γ-tubulin containing centrosomes. As found in with the 6-11B-1 antibody. We have, therefore, to assume that other Coleoptera species (Juberthie-Jupeau et al., 1983), these they represent MTs whose disassembly may lead to the consist of pairs of centrioles situated close to the plasma complete destruction of individual MTs. These ﬁndings render membrane. The non-centrosomal cytoplasmic MTs do not the question of how the MTs are organized all the more possess γ-tubulin at their ends or incorporated into their lattice. pressing. A ﬁne structural study of late prophase primary spermatocytes What possible mechanisms explain the origin of non-cen- 3862 K. W. Wolf and H. C. Joshi

b c d e

a f g h i

Fig. 6. Electron micrographs of ultrathin sections through a spermatid (a) A longitudinal section through the anterior pole shows a portion of the nucleus (N), the cup-shaped acrosome precursor (asterisk), manchette microtubules (arrows) and the plasma membrane (PM). Sheets of smooth endoplamic reticulum are visible lateral to the manchette microtubules. Bar, 0.5 µm. (b-i) Eight consecutive serial cross sections through the anterior pole of a spermatid at approximately the same developmental stage as that shown in a. The anterior-most section is depicted in (i). Cross-sectioned manchette microtubules are visible between the boundary of the acrosome precursor and cisternae of the smooth endoplasmic reticulum. The manchette microtubules start without apparent material at their anterior ends. Arrows in (b) and (d ) indicate the lumen of the cup-shaped acrosome precursor. Arrowhead in (h) points to a tubule which is not yet present in the preceding section (i). PM, plasma membrane. Bar, 200 nm.

trosomal MTs in late prophase primary spermatocytes of T. molitor? Tubulin monomer and MTs form an equilibrium. The higher the concentration of tubulin, the more MTs are assembled (Walker et al., 1988). In preparation for the ensuing meiotic divisons, the cells under study may possess concen- trations of monomeric tubulin sufficient to initiate spontanous MT assembly. Alternatively there may be cytoplasmic factors other than accumulations of γ-tubulin responsible for MT nucleation. The presence of non-centrosomal MTs is not unique to late prophase primary spermatocytes of a beetle. In some cultured animal cells, the organization of non-centrosomal MTs promoted by proteinaceous cytoplasmic nucleating factors has been hypothesized (Bré et al., 1987). Finally, we Fig. 7. Schematic drawing of the restructuring of the nuclear area in cannot exclude the presence of γ-tubulin below the detection early spermiogenesis. The sequence starts with a secondary level of our method and that may be enough to promote MT- spermatocyte in late telophase (a). A basal body, contiguous with a nucleation. short ﬂagellum, is attached to the outer membrane of the nuclear envelope. In subsequent stages, the ﬂagellum elongates considerably, Microtubule nucleation through direct insertion into but the basal body maintains it connection with the nucleus (b-f). γ-tubulin containing material Young spermatids possess a spherical nucleus (b,c) and the acrosome precursor develops close to the nuclear envelope (c). Concomitant Spindle MTs in male meiosis of T. molitor radiate out from with the onset of nuclear elongation, the acrosome precursor compact centrosomes and numerous MTs are inserted into the migrates towards the anterior pole of the nucleus (d). Then, electron-dense PCM. In most animal cells the centrosomes manchette microtubles originate from the anterior pole of the consist of centrioles or basal bodies embedded in PCM. The spermatid close to the acrosome precursor (e). This relationship antibody against γ-tubulin detects the centrosomes in meiosis persists throughout nuclear elongation (f). of the beetle. The reactivity of the antibody is not surprising because it has been raised against a 17-amino-acid oligopep- Additionally, the presence of γ-tubulin has previously been tide, the amino-terminal 16 residues of which are conserved demonstrated in invertebrate tissue: centrosomes in a cell line among all known γ-tubulin sequences (Joshi et al., 1992). of Drosophila melanogaster reacted with an antibody against MTOCs in Tenebrio molitor 3863

Fig. 8. Control cells, where the antibody to γ-tubulin has been omitted while all other steps were identical to the regular staining procedure. Bar, 5 µm. (a-c) Primary spermatocyte in mid- telophase. The prominent interzone spindle is detected by the antibody to β- tubulin (a), but in the FITC canal only faint evenly distributed label is visible. a b c d e f g h i DAPI revels the prospective daughter nuclei (c). (d-f and g-i) Spermatids at two different stages of nuclear elongation. The ﬂagella (d,g) and the nuclei (f,i) are visualized by the antibody to β-tubulin and DAPI respectively, but distinct label is not detectable in the FITC canal.

γ-tubulin (Zheng et al., 1991). The spatial relationship between are not known. A plate consisting of material of moderate γ-tubulin in a dot-like form and the origin of MTs from these electron density, interspersed between the nuclear membrane sites is to be seen in various systems such as cultured cells in and the membrane of the acrosome precursor is probably interphase and mitosis (Stearns et al., 1991; Zheng et al., 1991; involved in the migration. Membranes possess an inherent Muresan et al., 1993), follicle cells in the ovary of Xenopus fluidity (for a review, see Lenaz, 1987) and this property has laevis (Gard, 1994) and the cytoplasm of mouse oocytes been believed to facilitate the migration of the acrosome (Gueth-Hallonet et al., 1993; Palacios et al., 1993). The precursor towards the anterior pole of the nucleus in other relationship has given rise to the idea that γ-tubulin is involved systems (Troyer and Schwager, 1982). in the nucleation of MTs. The observation that microinjection It is not clear where the γ-tubulin is precisely located within of γ-tubulin antibodies into cultured mammalian cells prevents the acrosome precursor. The most plausible site would be in the regrowth of MTs after drug-induced depolymerization association with the investing membrane. This suggestion is (Joshi et al., 1992) corroborates this view. However, details of based on the fact that tubulin has been found in association the relationship between γ-tubulin and the initiation of MT with cellular membranes of a variety of systems including assembly have yet to be resolved. The morphology of the γ- mammalian brain (Feit and Barondes, 1970; Nath and Flavin, tubulin-rich MTOC may vary, but this does not influence its 1978; Zisapel et al., 1980), human platelets (Steiner, 1983), capability to apparently serve as nucleating site for MTs. In ciliary membranes of the scallop gill (Stephens, 1985) and sea contrast to the dot-like appearance of the centrosomes in a urchin embryos (Stephens, 1991, 1992). If this is true, the inte- variety of systems (see above) and dividing spermatocytes of gration of the γ-tubulin into the membrane of the acrosome a beetle (this study), spindles in female meiosis and early precursor must not rely on interaction with lipids. The prepa- cleavage divisions of the house mouse possess broad spindle rations analyzed in the present study were lysed in the presence poles containing γ-tubulin (Gueth-Hallonet et al., 1993; of a strong detergent, 1% Triton X-100, which is expected to Palacios et al., 1993). emulsify lipids. The γ-tubulin resisted this treatment. It must, therefore, be tightly bound to insoluble proteins in the The nucleation of manchette microtubules periphery of the acrosome precursor. The presence of γ-tubulin A striking finding of the present study was the observation that within the matrix of the acrosome precursor is less likely. the spermatid-specific MT system of T. molitor, the micro- When fully developed, the acrosome represents a compartment tubular manchette around the elongating nucleus, originates for a variety of hydrolases used to dissolve the egg investments from the vicinity of the acrosome precursor. This body is during fertilization (Olson and Winfrey, 1991). A role for a obviously detected by the antibody against γ-tubulin. skeletal element of the tubulin type is difficult to conceive Although only indirect evidence can be fielded, the identity there. between the acrosome precursor and the binding site of the Whereas the basal body of mouse sperm contains some γ- antibody against γ-tubulin can hardly be disputed. The corre- tubulin (Palacios et al., 1993), that of T. molitor spermatids lation between the results of our fine structure and anti-tubulin loses most of its associated γ-tubulin. The assembly of a γ- immunofluorescence analysis clearly show the formation of the tubulin-rich body in the form of the acrosome precursor occurs acrosome precursor lateral to the spermatid nucleus together subsequently lateral to the nucleus and independent of the basal with the appearance of γ-tubulin-rich material there. Distinct body in young spermatids of the beetle. This temporal elements other than the acrosome precursor which could be relationship may be taken to indicate that γ-tublin, previosuly correlated with the fluorescent spot do not exist within the bound to the basal body, is relocated to the acrosome precursor. spermatid cytoplasm. Furthermore, the simultaneous A clear-cut and direct insertion of MTs into γ-tubulin-con- movement of the fluorescent spot and the acrosome precursor taining material, as described above for canonical centrosomes, towards the anterior pole of the spermatid nucleus serves as is missing in manchette MTs of T. molitor. Instead, the another strong confirmation that we are dealing with the same manchette MTs form lateral to this material, somehow in asso- element. The forces which move the acrosome precursor from ciation with the acrosome precursor. How γ-tubulin might be its lateral postion to the anterior nuclear pole of the spermatid involved in MT-nucleation is difficult to explain in light of our while attached to the outer membrane of the nuclear envelope limited knowledge of the properties of this tubulin isotype. In 3864 K. W. Wolf and H. C. Joshi the case of manchette MT organization in spermatids of T. REFERENCES molitor, however, an additional factor comes into play: some MTs end clearly adjacent to the inner face of the plasma Apostolakos, P. and Galatis, B. (1985). Studies on the development of the air membrane. This finding deserves attention because membranes pores and air chambers of Marchantia paleacea. III. Microtubule organization in preprophase-prophase initial aperture cells - formation of appear to possess the capacity to nucleate MTs. There are incomplete preprophase microtubule bands. Protoplasma 128, 120-135. several studies in flowering plants showing that membranes Baccetti, B. (1972). Insect sperm cells. Advan. Insect Physiol. 9, 315-397. serve as attachment sites for cytoplasmic MTs (Dickinson and Baccetti, B., Burrini, A. G., Dallai, R., Giusti, F., Mazzini, M., Renieri, T., Sheldon, 1984; Bakhuizen et al., 1985; van Lammeren et al., Rosati, F. and Selmi, G. (1973). Structure and function in the spermatozoon of Tenebrio molitor (The spermatozoon of Arthropoda. XX). J. 1985; Vantard et al., 1990). Plasma membrane-associated Mechanochem. Cell Motil. 2, 149-161. MTOCs were detected in ommatidia of Drosophila Bakhuizen, R., van Spronsen, P. C., Sluiman-den Hertog, F. A. J., melanogaster (Mogensen et al., 1993) and in pillar cells of the Venverloo, C. J. and Goosen-de Roo, L. (1985). Nuclear envelope mouse cochlea (Tucker et al., 1992). In mitosis, membranes radiating microtubules in plant cells during interphase mitosis transition. Protoplasma 128, 43-51. may also play a role in MT nucleation. The inner membrane Bré, M. H., Kreis, T. E and Karsenti, E. (1987). Control of microtubule of the nuclear envelope in closed micronuclear mitosis of the nucleation and stability in Madin-Darby canine kidney cells: the occurence ciliate Nyctotherus ovalis was considered responsible for the of non centrosomal, stable detyrosinated microtubules. J. Cell Biol. 105, nucleation and stabilization of spindle MTs (Eichenlaub-Ritter 1283-1296. and Ruthmann, 1982a,b). Another example for the possible Dickinson, H. G. and Sheldon, J. M. (1984). A radial system of microtubules extending between the nuclear envelope and the plasma membrane during involvement of membranes in MT initiation is found in a moss: early male haplophase in flowering plants. Planta 161, 86-90. membraneous vesicles are scattered throughout polar MTOCs Eichenlaub-Ritter, U. and Ruthmann, A. (1982a). Holokinetic composite in mitosis of Marchantia palacea (Apostolakos and Galatis, chromosomes with ‘diffuse’ kinetochores in the micronuclear mitosis of a 1985). heterotrichous ciliate. Chromosoma 84, 701-716. Eichenlaub-Ritter, U. and Ruthmann, A. (1982b). Evidence for three Thus, we are confronted with two possibilites for manchette ‘classes’ of microtubules in the interpolar space of the mitotic micronucleus MT nucleation in spermatids of T. molitor. In light of the of a Ciliate and the participation of the nuclear envelope in conferring regular association of γ-tubulin with MTOCs, a similar role of stability to microtubules. Chromosoma 85, 687-706. γ-tubulin-containing material can hardly be ignored, but in Euteneuer, U. and McIntosh, J. R. (1980). Polarity of midbody and phragmoplast microtubules. J. Cell Biol. 87, 509-515. spermatids of T. molitor apical portions of the plasma Feit, H. and Barondes, S. H. (1970). Colchicine binding activity in particulate membrane may be involved in MT initiation as well. It should fractions of mouse brain. J. Neurochem. 17, 1355-1364. be added that the situation described for the beetle is not the Friedländer, M. and Wahrman, J. (1971). The number of centrioles in insect rule in insects. Two further systems, Ephestia kuehniella sperm: a study of two kinds of differentiating silkworm spermatids. J. Morphol. 134, 383-398. (Pyralidae, Lepidoptera) and Pyrrhocoris apterus (Pyrrhocori- γ γ Gard, D. L. (1994). -tubulin is asymmetrically distributed in the cortex of dae, Hemiptera), were probed with the antibody against - Xenopus oocytes. Dev. Biol. 161, 131-140. tubulin and analysed in the fluorescence microscope. Sper- Gueth-Hallonet, C., Antony, C., Aghion, J., Santa-Maria, A., Lajoie- matids of both animals did not show elements rich in γ-tubulin Mazenc, I., Wright, M. and Maro, B. (1993). γ-tubulin is present in (K. W. Wolf, unpublished observations). acentriolar MTOCs during early mouse development. J. Cell Sci. 105, 157- 166. Hoffman, J. C., Vaughn, K. C. and Joshi, H. C. (1994). Structural and Microtubule stabilization through integration or immunocytochemical characterization of microtubule organizing centers in association of γ-tubulin with the microtubular lattice pteridophyte spermatogenous cells. Protoplasma 179, 46-60. Centrosomes and acrosome precursors of T. molitor contain γ- Joshi, H. C., Palacios, M. J., McNamara, L. and Cleveland, D. W. (1992). γ- tubulin in higher quantities and confined to a narrow space. tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature 356, 80-83. This does not apply to the interzone spindle in meiosis of the Joshi, H. (1994). Microtubule organizing centers and γ-tubulin. Curr. Opin. beetle, where γ-tubulin shows a diffuse distribution and its con- Cell Biol. 6, 55-62. centrations is low. Nevertheless it is detectable at the polar Juberthie-Jupeau, L., Durand, J. and Cazals, M. (1983). Spermatogenäse ends of the interzone spindle, an array of two interdigitated comparée chez les coléoptäres Bathysciinae souterains. Cytobios 37, 187- 208. MTs populations (Rattner, 1992). This reminds us of Julian, M., Lajoie-Mazenc, I., Moisand, A., Mazarguil, H., Puget, A. and mammalian telophase spindles, where γ-tubulin has been found Wright, M. (1993). γ-tubulin participates in the formation of the midbody in the same location (Julian et al., 1993). The portions of during cytokinesis in mammalian cells. J. Cell Sci. 105, 145-156. telophase spindles that are rich in γ-tubulin contain the (−) ends Kalt, A. and Schliwa, M. (1993). Molecular components of the centrosome. Trends Cell Biol. 3, 118-128. of the MTs (compare Euteneuer and McIntosh, 1980) and the Lenaz, G. (1987). Lipid fluidity and membrane protein dynamics. Biosci. Rep. presence of γ-tubulin may stabilize these MT ends. Electron- 7, 823-837. dense material comparable to the PCM of canonical centro- Liu, B., Marc, J., Joshi, H. C. and Palewitz, B. A. (1993). A γ-tubulin-related somes is missing at the (−) ends of MTs in telophase spindles protein associated with the microtubule arrays of higher plants in a cell cycle- of T. molitor. Therefore, the incorporation of γ-tubulin into dependent manner. J. Cell Sci. 104, 1217-1228. Mazia, D. (1984). Centrosomes and mitotic poles. Exp. Cell Res. 153, 1-15. MTs or its association with the MT lattice may bring about Mogensen, M. M., Tucker, J. B. and Baggaley, T. B. (1993). Multiple plasma stabilization. The staining behavior of plant cells also suggests membrane-associated MTOC systems in the acentrosomal cone cells of that γ-tubulin or a protein related to γ-tubulin is able to Drosophila ommatidia. Eur. J. Cell Biol. 60, 67-75. associate with MTs (Liu et al., 1993; Hoffman et al., 1994). Muresan, V., Joshi, H. C. and Besharse, J. C. (1993). γ-tubulin in differentiated cell types: localization in the vicinity of basal bodies in retinal photoreceptors and ciliated epithelia. J. Cell Sci. 104, 1229-1237. The authors are grateful to Professor W. Traut for critically reading Nath, J. and Flavin, M. (1978). A structural difference between cytoplasmic the manuscript and to Mrs. F. Niedereichholz for skillful technical and membrane-bound tubulin of brain. FEBS Lett. 9, 335-338. assistance. The support of the ‘Deutsche Forschungsgemeinschaft’ to Olson, G. E. and Winfrey, V. (1991). Structure-function relationships in the K.W.W. is acknowledged (Wo 394/6-1). sperm acrosome. Ann. NY Acad. Sci. 637, 240-257. MTOCs in Tenebrio molitor 3865

Osborn, M. and Weber K. (1982). Immunofluorescence and microtubule-organizing material in the inner pillar cell of the mouse cochlea. immunocytochemical procedures with affinity purified antibodies: tubulin J. Cell Sci. 102, 215-226. containing structures. Meth. Cell Biol. 24, 97-132. van Lammeren, A. A. M., Keijzer, C. J., Willemse, M. T. M. and Kieft, H. Palacios, M. J., Joshi, H. C., Simerly, C. and Schatten, G. (1993). γ-tubulin (1985). Structure and function of the microtubular cytoskeleton during reorganization during mouse fertilization and early development. J. Cell Sci. pollen development in Gasteria verrucosa (Mill.) H. Duval. Planta 165, 1-11. 104, 383-389. Vantard, M., Levilliers, N., Hill, A.-M., Adoutte, A. and Lambert, A.-M. Pickett-Heaps, J. D. (1969). The evolution of the mitotic apparatus: an attempt (1990). Incorporation of Paramecium axonemal tubulin into higher plant at comparative ultrastructural cytology in dividing plant cells. Cytobios 3, cells reveals functional sites of microtubule assembly. Proc. Nat. Acad. Sci 257-280. USA 87, 8825-8829. Piperno, G. and Fuller, M. T. (1985). Monoclonal antibodies specific for an Walker, R. A., O’Brien, E. T., Pryer, N. K., Soboeiro, M. F., Voter, W. A., acetylated form of α-tubulin recognize the antigen in cilia and flagella from a Erickson, H. P. and Salmon, E. D. (1988). Dynamic instability of variety of organisms. J. Cell Biol. 101, 2085-2094. individual microtubules analyzed by video light microscopy: rate constants Rattner, J. B. (1992). Mapping the mammalian intercellular bridge. Cell Motil. and transition frequencies. J. Cell Biol. 107, 1437-1448. Cytoskel. 23, 231-235. Webster, D. R. and Borisy, G. G. (1989). Microtubules are acetylated in Stearns, T., Evans, L. and Kirschner, M. (1991). γ-tubulin is a highly domains that turn over slowly. J. Cell Sci. 92, 57-65. conserved component of the centrosome. Cell 65, 825-836. Wolf, K. W. (1994). The unique structure of Lepidopteran spindles. Int. Rev. Steiner, M. (1983). Membrane-bound tubulin in human platelets. Biochim. Cytol. 152, 1-48. Biophys. Acta 729, 17-22. Wolf, K. W. and Hellwage, J. (1995). Spermatogenesis in Tenebrio molitor Stephens, R. E. (1985). Evidence for a tubulin-containing lipid-protein (Tenebrionidae, Coleoptera): A fine structure and anti-tubulin structural complex in ciliary membranes. J. Cell Biol. 100, 1082-1090. immunofluorescence study. Acta Zool. 76, 267-279. Stephens, R. E. (1991). Tubulin in sea urchin embryonic cilia: chracterization Zheng, Y., Jung, M. K. and Oakley, B. R. (1991). γ-tubulin is present in of the membrane-periaxonemal matrix. J. Cell Sci. 100, 521-531. Drosophila melanogaster and Homo sapiens and is associated with the Stephens, R. E. (1992). Tubulin in sea urchin embryonic cilia: post- centrosome. Cell 65, 817-823. translational modifications during regeneration. J. Cell Sci. 101, 837-845. Zisapel, N., Levi, M. and Gozes, I. (1980). Tubulin: an integral protein of Troyer, D. and Schwager, P. (1982). Evidence for nuclear membrane fluidity: mammalian synaptic vesicle membranes. J. Neurochem. 34, 26-32. Proacrosome migration and nuclear pore redistribution during grasshopper spermiogenesis. Cell Motil. 4, 355-367. Tucker, J. B., Paton, C. C., Richardson, G. P., Mogensen, M. M. and (Received 21 October 1994 - Accepted, in revised form, Russell, I. J. (1992). A cell surface-associated centrosomal layer of 25 September 1995)