Mitotic and Meiotic Spindles from Two Insect Orders, Lepidoptera and Diptera, Differ in Terms of Microtubule and Membrane Content

Mitotic and meiotic spindles from two insect orders, Lepidoptera and Diptera, differ in terms of microtubule and membrane content

KLAUS WERNER WOLF

Institut ftir Biologie der Medizinischen Universit&t zu Ltibeck, Ratzburger Allee 160, D-2400 Lilbeck 1, Federal Republic of Germany

Summary Spindles from the gonads of five insect species were tors of 1.8 to 3.0. The correlation between the amount examined after conventional preparation for elec- of spindle membranes and the microtubule content tron microscopy. The aim of the study was to deter- of the spindle indicates a functional relationship. mine (1) the range of variation of the spindle mem- Spindle membranes are believed to influence micro- branes between mitotic and meiotic cells and (2) the tubule stability via the regulation of the Ca2+ concen- correlation of possible differences with the micro- tration within the spindle area. The high microtubule tubule content of the spindles. The study involved mass in spindles from Lepidoptera spermatocytes four moth species, Ephestia kuehniella, Phragmato- may result from the membrane-dependent lowering bia fuliginosa, Orgyia thyellina, Orgyia antiqua, and of the Ca2+ level within the spindles. Finally, an one fly, Megaselia scalaris. Somatic and gonial unconventional idea on the role of intraspindle mem- mitoses in all species examined showed a sparse branes is offered. This concept is not intended to spindle membrane inventory. In contrast, spermato- challenge the function of spindle-associated mem- cytes consistently had a multilayered spindle envel- branes as Ca2+-sequestrating compartments. Intras- ope. In spermatocytes of all Lepidoptera species pindle membranes are considered as stuffing examined, but not in those of M. scalaris, diverse material in sheathed spindles. Membranous com- forms of intraspindle membranes existed in addition partments reduce the free volume within the spindle. to the spindle envelope. Microtubule counts in Thereby, monomeric tubulin is concentrated and the serially cross-sectioned spindles of E. kuehniella re- formation of microtubules is favoured. vealed an about 6-fold increase in the mass of polym- erized tubulin during the transition from spermatogonia to primary spermatocytes. The increase was 3.3-fold in O. thyellina and less than 3-fold in M. sca- Key words: mitosis, meiosis, Ephestia kuehniella, Orgyia laris. The density of intraspindle membranes in antiqua, Orgyia thyellina, Phragmatobia fuliginosa, Megaselia E. kuehniella was higher than in O. thyelhna by fac- scalaris.

Introduction The function of spindles is to supply the daughter cells with a euploid set of chromosomes. In mitotic anaphase, The dominant component of spindles is the microtubular chromatids migrate towards the spindle poles, whereas in cytoskeleton. Microtubules (MTs) most probably play a anaphase I half-bivalents segregate. The total mass of role in chromosome migration (for reviews, see Nicklas, migrating material does not differ between mitosis and 1988; Mclntosh, 1989; Mitchison, 1988). During the last meiosis I. On this premise, prominent structural differ- decade, membranous components moved from the fringes ences between mitotic and meiotic spindles are not readily to the focus of studies aimed at spindle structure and anticipated. However, the comparison of spindles in sper- function (for reviews, see Paweletz, 1981; Hepler and matogonia and spermatocytes of the Hemiptera species Wolniak, 1984; Hepler, 1989a). Alhough, as yet, a satisfy- Dysdercus intermedius (Motzko and Ruthmann, 1984) ing conceptual framework has not been derived from the revealed striking differences in the membrane inventory. available data, spindle membranes seem to be involved in Therefore it seemed worthwhile to concentrate on the Ca2+ transients and spindle dynamics (for detailed dis- issue of spindle dimorphism in higher eukaryotes. cussions, see Wolniak, 1988; Hepler, 19896). Three types of In the present study, dividing mitotic cells and sper- membrane systems linked with spindles can be dis- matocytes from five insect species are compared, with a tinguished. (1) Intraspindle membranes occur together focus on two key components: membranes associated with with microtubules (MTs) and chromosomes within the the spindle and the microtubular cytoskeleton. Four Lepi- spindle domain. They are often arranged parallel to the doptera species, Orgyia thyellina (n=ll; Cretschmar, kinetochore MTs. (2) Perispindle membranes surround the 1928), Orgyia antiqua (n=14; Cretschmar, 1928), Ephestia spindle apparatus. (3) Astral membranes or lamellae are kuehniella (n=30, Traut and Mosbacher, 1968) and Phrag- arranged parallel to MTs radiating out from the spindle matobia fuliginosa, have been examined. The karyotype of poles. the latter species contains a giant sex chromosome pair. Journal of Cell Science 97, 91-100 (1990) Printed in Great Britain © The Company of Biologists Limited 1990 91 Chromosome races with haploid numbers of 28 and 29 Results chromosomes are known in this species (Seiler, 1925). The individuals used in the present study have a haploid Lepidoptera chromosome number of 29. Observations on the phorid fly Somatic mitosis in cells of the testicular sheath (Fig. 1) Megaselia scalaris are also presented. In addition to the and of the imaginal disks in E. kuehniella is characterized regular three chromosome pairs (see Johnson et al. 1988, by a sparsely developed spindle envelope. One, and in and references therein), the karyotype of this species places two, discontinuous layers of membranous sheets contains varying numbers of centromere-like elements surround the spindle domain. The two membrane layers without chromosome arms (Wolfed al. 1988). are not closely associated and spindle MTs are found The spindle membrane inventory and the MT mass were between them. Intraspindle membranes are very scarce found to differ between mitotic cells and spermatocytes in and form flat or spherical vesicles (Fig. 1, inset). A pair of all species examined. orthogonally arranged centrioles is connected with each spindle pole. This also applies to spindle poles in all other mitotic cells examined in this study. The centrioles are embedded in pericentriolar material of moderate density. Metaphase spindles in imaginal disks of O. antiqua are Materials and methods identical to those in E. kuehniella regarding spindle membranes. Somatic mitosis in 0. thyellina and P. fuliginosa Laboratory strains of the Mediterranean mealmoth, E. kuehniella was not studied. (Pyralidae), were raised on rolled oats. One strain, L, has a haploid chromosome number of 30. A second strain used, W10, Spermatogonia of E. kuehniella have a one- to three- contains a small heterochromatic fragment in addition to the 30 layered fenestrated spindle envelope. Large gaps occur in chromosomes. This fragment is derived from the W-chromosome the vicinity of the basal bodies (Fig. 2). The individual (Traut et al. 1986). Spindle morphology does not vary between the membranous cisternae forming the spindle envelope have two strains and for the present purpose no distinction is made little space between one another. Intraspindle membranes between them. Testes and imaginal disks of the wings from larvae in the last instar were prepared for electron microscopy according are missing. Metaphase and early anaphase spindles in to Wolf (1987). gonial cells of 0. thyellina (Fig. 3), 0. antiqua (Fig. 4), and P. fuliginosa (not shown), closely resemble those in sper- The same protocol was followed for gonads and imaginal disks of the wings from last instar larvae of O. thyellina and 0. antiqua matogonia of E. kuehniella. (Lymantriidae). Larvae of these two species were kindly provided Lepidoptera produce two types of sperm (for reviews, see by Sir C. Clarke (Liverpool, UK). In addition, eggs of O. antiqua Fain-Maurel, 1966; Silberglied et al. 1984). Eupyrene were collected in Sandhausen (FRG). The larvae from this isolate spermatocytes (for terminology, see Meves, 1903) give rise hatched in the laboratory and were fed with bramble leaves. to fertile spermatozoa. Apyrene spermatocytes develop Larvae of P. fuliginosa (Arctiidae) were collected in Bergedorf into sterile sperms. The two types of spermatocytes can be near Hamburg (FRG) and raised on leaves of Plantago. Testes readily distinguished from one another. Eupyrene sper- from last instar larvae of the third and fourth laboratory gener- ation were processed for electron microscopy as described pre- matocytes are generally larger, possess a normal spindle, viously (Wolf, 1987). and form a metaphase plate. In contrast, in apyrene Two laboratory strains of M. scalaris (Phoridae), referred to as spermatocytes diverse deviations from regular develop- Wien' and Tennessee' (Johnson et al. 1988), were examined. ment occur (see Wolf et al. 1987). This study deals exclus- Differences in spindle structure are not apparent and the two ively with eupyrene meiosis. strains are not treated separately in the present study. The larvae As a rule, spindles in spermatocytes have a larger pole- were grown on a modified Drosophila medium (Johnson et al. to-pole distance and a larger volume than mitotic spindles. 1988). Pupae were dissected in an isotonic saline (Hayes, 1953). Differences in cell size and the presence of one or two basal Subsequently the gonads were transferred into lml saline solution containing 2.5% glutaraldehyde. After 5min, 3 ml of 8% bodies per spindle pole distinguish secondary and primary tannic acid in phosphate buffer (67 nw, pH 6.8) were added. One spermatocytes in E. kuehniella (Wolf and Kyburg, 1989). hour later, the specimens were rinsed in phosphate buffer and Astral lamellae and peri- and intraspindle membranes embedded in agar (2 % in phosphate buffer) in order to facilitate exist in both spermatocyte generations. In metaphase, an the further processing of the delicate specimens. Gonads were envelope composed of irregularly shaped membranous postfixed in phosphate-buffered OsO4 (2%) for one hour and elements completely surrounds the spindle area. This rinsed again. Dehydration was performed in ethanol and propyl- sheath is 1.6—2.0/on thick at the level of the metaphase ene oxide and eventually the specimens were embedded in Epon plate. Intraspindle membranes form cisternae aligned 812. parallel to the spindle MTs (Fig. 6). The membranes are In order to obtain an estimate of the spindle volume occupied by membranous compartments, the areas surrounded by intraspin- densely packed in places. Conical areas over the poleward dle membranes were measured in a metaphase I spermatocyte of surfaces of the metaphase chromosomes are devoid of 0. thyellina and in metaphase II spermatocytes of P. fuliginosa membranes. The basal bodies are embedded in an array of and E. kuehniella. The values obtained were related to the total astral membranes. Numerous stacks of Golgi cisternae are spindle area. For that purpose, the innermost layer of the spindle located at the cytoplasmic face of the astral membranes. envelope was taken as the boundary of the spindle area. The area Some traits distinguish the membrane inventory of measurements are directly proportional to the volume (see how- spermatocytes in P. fuliginosa and E. kuehniella. The ever, the note on systematic errors in the Discussion). Measure- intraspindle membranes in spermatocytes of P. fuliginosa ments were carried out using a graphic tablet (Summagraphics Supergrid: Summagraphics Corporation, Fairfield, Connecticut) have a rather regular shape. They form tubules arranged interfaced to an IBM PS2/60 computer. The software was written parallel to the pole-to-pole axis (Figs 5,7). Astral mem- in Turbo Pascal 4.0. The final magnification of the micrographs branes, flanked by stacks of Golgi cisternae, radiate from used for measurements was x 40 000 in P. fuliginosa, x 60 000 in the spindle pole. The layer of perispindle membranes is O. thyellina, and x 65 800 in E. kuehniella. Measurements were relatively thin (about 1 /mi) at the level of the metaphase made both at the level of the metaphase plate and in the half- plate. The spindle envelope of P. fuliginosa is composed of spindle adjacent to the chromosomes. In each case, measurements stacked cisternae. from three consecutive serial cross-sections were averaged. The spindles in primary spermatocytes of O. thyellina 92 K. W. Wolf c

Fig. 1. E. kuehniella. Longitudinal section through a metaphase spindle in the testicular sheath. In this somatic cell, the two layers of the spindle envelope (se) are distant from one another. Microtubules (arrows) occur within the spindle area and between the layers of the spindle envelope. The inset depicts an example of the scarce intraspindle membranes (arrowheads) in somatic mitosis of E. kuehniella. c, centriole; k, kinetochore. Bar, linn. and 0. antiqua have been previously described (Wolf et al. MT distribution along the spindle axis of a primary 1987; Wolf, 1990a). In brief, the perispindle membrane spermatocyte in O. thyellina has been previously pub- system consists of stacks of fiat cisternae similar to those lished (Wolf et al. 1987). The MT distribution displays a of P. fuliginosa. The intraspindle membranes form sheets maximum in each half-spindle and a minimum at the level parallel to the spindle MTs. of the chromatin. The corresponding values in E. kueh- Both Orgyia species show a lower density of the intras- niella are higher (Table 2). pindle membrane system than E. kuehniella and P. fuligi- Summing the number of MT profiles in consecutive nosa. Measurements revealed that the membrane density serial sections and multiplication by the average section is highest in E. kuehniella and lowest in O. thyellina. The thickness gives the total length of MTs contained in the factors are 1.8 at the level of the chromosomes and 3.0 in spindles. Progressing towards the spindle pole beyond the the half-spindle. A metaphase II spindle from P. fuliginosa MT maximum generally showed increasing numbers of has a membrane content intermediate between those of astral MTs in spermatocytes. Astral MTs were rare in E. kuehniella and 0. thyellina (Table 1). In E. kuehniella spermatogonia. In order to avoid an overestimate of the and in P. fuliginosa, the membrane density decreases at MT mass in spermatocytes, counting was not performed the level of the metaphase plate relative to the half- beyond 60 sections in both directions from the MT mini- spindle. Only in 0. thyellina, which has the lowest mem- mum. The bulk of MTs was contained within 45 sections in brane content in the half-spindles of all three species both directions from the MT minimum (Table 2). Beyond examined, is there a slight increase in the amount of the maxima, the number of MT profiles counted in cross- membranes at the level of the chromosomes despite the sections decreased sharply. Gonial spindles have a signifi- large volume occupied by the bivalents (Table 1). cantly lower pole-to-pole distance than spermatocytes. The In mitotic and meiotic spindles of two Lepidoptera vast majority of MT was contained within an interval of species, 0. thyellina and E. kuehniella, the mass of polym- 45 sections on both sides from the MT minimum. Thus, erized tubulin was estimated in serial cross-sections. The the values resulting from the counts represent

Insect mitotic and meiotic spindles 93 er

Fig. 2. E. kueheniella. Longitudinal section through a spermatogonial metaphase spindle. Two to three closely stacked membranes form the spindle envelope (se). One spindle pole, marked by the presence of two centrioles (c), is visible within the section. Note the numerous small dense clumps within the spindle area (arrowheads). They represent remnants of the nucleolus. Bar, I/an. Pig. 3. 0. thyeUina. Longitudinal section through a spermatogonial anaphase spindle. The spindle envelope (se) is one- to three- layered. The lateral cytoplasm shows endoplasmic reticulum (er). Dense chromatin threads (ds) bridge the interzone in early anaphase. c, centriole. Arrowheads: nucleolar remnants. Bar, 1/nn.

Fig. 4. O. antiqua. Longitudinal section through an oogonial metaphase spindle. A relatively regular two-layered spindle envelope (se) exists, c, centriole. Arrowheads: nucleolar remnants. Bar, 2^m. Fig. 5. P. fuliginosa. Portion of a cross-sectioned metaphase spindle of a secondary spermatocyte. The position of this section is marked by a line in a longitudinally sectioned metphase II spindle (see Fig. 7). Cisternae of the spindle envelope (se) are visible besides tubules (t) of the intraspindle membrane system. Arrows: spindle MTs. Bar, 0.5/mi. reliable estimates of the total MT content in the pertinent I. Nevertheless, a secondary spermatocyte contains 2.8 spindles. times more MTs than a metaphase spermatogonium in the A metaphase I spermatocyte in 0. thyeUina contains 3.3 same species. times more spindle MTs than the metaphase spindle in a In order to estimate the variability of the MT mass spermatogonium. The increase from mitosis to meiosis I in between spindles of the same stage, two primary sper- E. kuehniella is even higher (5.6 to 6.6-fold). The MT matocytes were analyzed in E. kuehniella. The differences content of a metaphase II spindle in E. kuehniella is found (Table 2), were well below the differences between reduced by approximately one half relative to metaphase primary spermatocytes and spermatogonia in this moth.

94 K. W. Wolf G' , . se

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bb bb-A 7.

Fig. 6. £. kuehniella. Longitudinal section through a spindle in the transition between metaphase II and anaphase II. A thick spindle envelope (se) exists. Irregular membranous elements surround the basal body (bb). Strings of cisternae are found within the spindle area. G, stacks of Golgi cisternae. Bar, 2/tm. Fig. 7. P. fuliginosa. Longitudinal section through a metaphase spindle in a secondary spermatocyte. Closely stacked cisternae form both the spindle envelope (se) and an astral array of membranes (asterisks). Intraspindle membranes appear in the form of tubules. The line gives the level of a cross-section shown in Fig. 5. bb, basal body; f, flagellum; G, stacks of Golgi cisternae. Bar, 2/on.

Diptera gaps in the spindle envelope are to be seen only at the In ovaries of M. scalaris, cells are attached to the outer spindle poles. Primary and secondary spermatocytes are face of a basal lamina around the ovarioles. The spindle identical as regards spindle membranes. The spindle area envelope in these somatic cells is formed of one layer in spermatocytes is delimited from the cytoplasm by three (Fig. 8). Larger gaps occur. In metaphase, somatic pairing to four membrane layers. Vesicles with a transparent of the homologous chromosomes is brought about by lumen are interspersed between the membrane layers segments proximal to the centromeres. Distal portions of (Fig. 11 and Fig. 5, of Wolf, 19906). the chromosome arms are not paired and may extend MT counts were carried out in aerial cross-sections poleward into the half-spindles. through metaphase spindles in a variety of mitotic cells The metaphase spindle in oogonia is surrounded by a and two spermatocytes (Table 2). The technique was ident- double layer of membranes (Fig. 9). Small discontinuities ical to that used in Lepidoptera. However, the pole-to-pole are visible all over the spindle. Membranous components distance was smaller in the fly. Therefore, spindle MTs of unclear nature are occasionally found in the periphery were recorded within a range of ±30 (mitosis and meta- of the spindle area (Fig. 9). The MT density is highest in phase II) and ±45 sections (metaphase I) of the minimum adaxial portions of the spindle, where the centromeres are in the MT distribution. A somatic cell examined in an grouped. The centrioles are situated in polar gaps of the ovary had the smallest spindle. Two spermatogonia ana- spindle envelope. lyzed differed in MT mass. The differences were insignifi- Metaphase spermatogonia in M. scalaris have one mem- cant relative to the increase in MT content from spermato- brane layer around the spindle domain (Fig. 10). Larger gonia to primary spermatocytes (2.6 to 2.9-fold). The

Insect mitotic and meiotic spindles 95 Fig. 8. M. scalaris. Cross-section through a metaphase spindle a membrane layer, cm, centromeres. Arrowheads: membrane in a somatic cell of the ovary. Cells of this type are found elements of unclear nature in the spindle periphery. Bar, I/an. between ovarioles that are surrounded by a basal lamina (bl). Fig. 10. M. scalaris. Longitudinal section though a The spindle envelope (se) shows gaps (arrowheads). In a spermatogonial metaphase spindle. The almost spherical metacentric chromosome, only portions proximal to the spindle area is surrounded by a one-layered spindle envelope centromeres (cm) are somatically paired. Bar, I/an. (se). c, centriole; cm, centromeres. Bar, I/an. Fig. 9. M. scalaris. Longitudinal section through an oogonial Fig. 11. M. scalaris. Longitudinal section through the metaphase spindle. The two-layered spindle envelope (se) is metaphase spindle apparatus in a secondary spermatocyte. The interrupted at the spindle poles, where a centriole (c) is located. multilayered spindle envelope (se) includes transparent Note that the centriole is separated from the cytoplasm through vesicles, bb, basal body; cm, centromeres. Bar, I/an.

metaphase spindle in a secondary spermatocyte contained spermatocytes was determined using planimetry. It about the same amount of MTs as spermatogonia in the should not be overlooked that the use of ultrathin sections same species. of finite thickness introduces systematic errors (Weibel and Paumgartner, 1978). These authors calculated that the volume occupied by the rough and the smooth endo- Discussion plasmic reticulum in rat liver cells is about 29 % and 37 % lower than that measured in random section. The endo- In the present study, mitotic and meiotic spindles from five plasmic reticulum structurally resembles the intraspindle insect species in two orders, Diptera and Lepidoptera, were membranes of Lepidoptera spermatocytes. Therefore, the compared. The mass of spindle-associated membranes was present measurements probably overestimate the areas consistently higher in spermatocytes. This seems to be the occupied by membranes and are used for comparative rule in these two orders (Table 3). purposes only. The precise determination of the volume of The content of intraspindle membranes in Lepidoptera the intraspindle membrane system requires an extensive

Table 1. Estimates of the metaphase spindle area occupied by membranes in spermatocytes of three Lepidoptera species Area of the spindle adjacent to Total area of the the metaphase plate Mean area spindle adjacent to occupied by occupied by the metaphase plate S.D. membranes S.D. membranes Species Cell type (/an2) (pan2) (/an2) (/an2) (%) E. kuehniella Spermatocyte II 17.4 1.0 5.3 0.3 30.5 P. fuligmosa Speramtocyte II 17.5 0.6 3.2 0.1 18.3 0. thyellina Spermatocyte I 18.4 1.3 1.8 0.2 10.2 Area of the spindle Total area of the at the level of the spindle at the metaphase plate Mean area Area of the Mean area level of the occupied by occupied by apindle occupied occupied by metaphase plate S.D. membranes S.D. membranes by chromosomes S.D. chromosomes (/on*) (/an2) (/an2) (/an2) (%) (/an2) (/an2) (%) E. kuehniella Spermatocyte n 19.7 1.0 4.5 0.5 22.8 5.1 0.5 25.9 P. fuiiginosa Spermatocyte II 36.2 1.3 3.2 0.1 15.7 10.1 0.1 27.8 0. thyellina Spermatocyte I 29.9 0.7 3.6 0.05 12.4 12.7 0.3 43.8 Quantitative parameters of the membrane inventory of metaphase spindles in spennatocytes from E. kuehniella, P. fuiiginosa and O. thyellina. The total area per section of the spindle was measured using the innermost layer of the spindle envelope as a boundary. Each value represents the average of measurements in three consecutive serial sections. The standard deviation (s.D.) is given. Measurements were made in the metaphase plate and in the half-spindle adjacent to the chromosomes. The area occupied by the chromosomes was also determined.

Table 2. Estimates of the microtubule mass in mitotic and meiotic spindles of three insect species Maximum number of Minimum number of Microtubule length (/on) microtubule profiles microtubule profiles in ±30, ±45 or ±60 Species Cell type in each half-spindle in the equatorial plane sections of the minimum O. thyellina Spermatogonium 280,300 210 — 1121 — O. thyellina Spermatocyte I 730', 760* 440 — — 3674 E. kuehniella Spermatogonium 290,310 180 - — 1494 E. kuehniella Spermatocyte I 1600,1620 870 - 7787 8355 E. kuehniella Spermatocyte I 1700, 1770 1080 - 8956 9805 E. kuehniella Spermatocyte II 820,860 460 - 4220 — M. scalaris Somatic cell 110, 120 90 440 — — M. scalaris Oogonium 240,250 190 707 — M. scalaris Spermatogonium (W) 160, 160 140 466 — — M. scalaris Spermatogonium (T) 170,220 170 529 — — M. scalaris Spermatocyte I 240,260 220 - 1268 — M. scalaris Spermatocyte II 140, 170 100 646 — — The microtubule inventory of metaphase spindles in O. thyellina, E. kuehniella (Lepidoptera) and M. scalaris (Diptera). Maximum and minimum numbers of microtubule profiles along the spindle axis are given. The microtubule length was determined by summing the number of microtubule profiles in the range of ±30, ±45 or ±60 sections of the minimum distribution, and multiplication of the value by the average section thickness. •From Wolf etal. (1987).

96 K. W. Wolf »

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11 morphometric approach, which is beyond the scope of the branes seem to have a function in the interaction between present paper. chromosomes and MTs during spindle morphogenesis Several hypotheses on the function of spindle mem- (Motzko and Ruthmann, 1984). The mitotic chromosomes branes have been proposed. The membranes are possibly of all Hemiptera species examined so far have kinetochore involved in chromosome transport and orientation (Kubai plates. Bivalents in male meiosis are devoid of kinetochore and Ris, 1969; Kubai, 1982). In Hemiptera, spindle mem- plates. Instead, the spindle MTs end in dense spots at the

Insect mitotic and meiotic spindles 97 Table 3. The intra- and perispindle membranes in meta- and early anaphase ofmitotic cells and spermatocytes in Lepidoptera and Diptera species Intraspindle Species Cell type membranes PenBpindle membranes Lepidoptera Bombyx mori Spennatocytesb|C Irregular vesicles Thin layer of cisternae Ephestia kuehnieUa Somatic cells* Low number of vesicles Fragments of the nuclear envelope Spermatogonia*id Not detected One- to two-layered sheath Spennatocytes" Irregular cisternae Multi-layered sheath Inachis io Spermatogonia* Not detected One- to three-layered sheath Spermatocytes* Irregular cistemae Multi-layered sheath Orgyia thyellina Spermatogonia" Not detected One- to three-layered sheath Oogonia* Not detected One- to three-layered sheath Spennatocytes' Irregular cisternae Multi-layered sheath 0. antiqua Somatic cells* Low number of vesicles Fragments of the nuclear envelope Oogonia* Not detected Two-layered sheath Spermatocytes' Regular cisternae Multi-layered sheath Ostnnia nubilalis Spermatocytes1' Irregular cistemae Thin layer of vesicles Phragmatobia fuliginosa Spermatogonia* Not detected One- to three-layered sheath Spermatocytea* Membraneous tubules Multi-layered sheath Pieris brassicae Spermatocytes' Irregular cisternae Thin layer of cisternae Trichoplu&ia Spermatogoniad Not detected One layered with discontinuities Diptera Drosophila melanogaster Somatic cells' Not detected Two-layered sheath Spermatocytesk Not detected Multi-layered Bheath D. virilis Spermatocytes1 Not detected Multi-layered sheath Heteropeza pygmaea Somatic cells™ Not detected Layer of flat vesicles Megaseha scalaris Somatic cells* Not detected Fragments of the nuclear envelope Oogonia* Membranous inclusions Two-layered sheath of unclear nature Spermatogonia* Not detected One-layered sheath Spermatocytes* Not detected Three to four-layered sheath Pales ferruginea Spermatocytes" Not detected Layers of endoplasmic reticulum

"This study; bHolm and Rasmussen (1980); cFriedlander and Wahrman (1970); dGassner and Klemeteon (1974); 'Wolf (unpublished); fWolf et al. (1987); « Wolf (1990); h Roth et al. (1966);' Wolf (1988); > Stafstom and Staehlin (1984); k Church and Lin (1980);' Itoh (1960); m Fux (1971); n Fuge (1971). poleward surface of the chromatin (Commings and Okada, ibly represents an attachment site for spindle MTs 1972; Ruthmann and Permantier, 1973; Rufas and Gime- (Eichenlaub-Ritter and Ruthmann, 1982; Kuck and Ruth- nez-Martin, 1986). With the development of the meiotic mann, 1985; Tucker et al. 1985). However, close examin- spindle, membranes become tightly associated with the ation of the pole-proximal endings of spindle MTs in chromosome surfaces. Only small areas remain mem- metaphase spermatocytes of E. kuehnieUa did not reveal brane-free. These sites are thought to interact with specific membrane-to-MT contacts (Wolf and Bastmeyer, spindle MTs (Motzko and Ruthmann, 1984). This sugges- in preparation). tion has been extended to Lepidoptera spermatocytes. According to the best-conceived hypothesis on the role of Lepidoptera and Hemiptera species have one property in spindle-associated membranes, these control spindle MT common: they are believed to possess holokinetic chromo- stability via the regulation of the Ca2+ concentration somes (Goodward, 1985). However, a role for spindle within the spindle area (Harris, 1975). There is conclusive membranes in directing the contacts between MTs and evidence for spindle membrane-associated Ca2+ seques- chromatin is less probable in Lepidoptera spermatocytes, tration (Silver et al. 1980; Wick and Hepler, 1980; Wise since (1) kinetochore material is visible at the poleward and Wolniak, 1984; Petzelt and Hafner, 1986; Hafner and surface of the chomosomes in metaphase I and II (Wolf, Petzelt, 1987). The mass of spindle membranes varies unpublished) and (2) membranes are not tightly attached significantly either between different species (compare to the chromosomes. Large surface areas are devoid of Fuge, 1971 and Rieder and Nowogrodzki, 1983) or between membranes in early prometaphase spermatocytes when spindles in different cell types of one species (Motzko and contacts between chromosomes and MTs already exist Ruthmann, 1984). Therefore, the suggestion has been (Wolf, unpublished). made that membranes develop preferentially within large Perispindle membranes may act as a barrier against spindles, where the distance between the center of the cytoplasmic components or, inversely, may prevent the spindle and its perimeter may be greater than the effective loss of chromosomes from the spindle. In syncytia, a diffusion limit (Hepler and Wolniak, 1984; Hepler, 1989a). membrane layer around the spindle domains (e.g. see This idea is corroborated by the present observations since Aldrich, 1969; Stafstrom and Staehlin, 1984) is considered prominent intraspindle membrane systems develop only as a means of preventing the transfer of chromosomes in the voluminous meiotic spindles of Lepidoptera and are between neighboring spindles. Since gonads also represent missing in the smaller mitotic spindles. syncytia (Gondos, 1984), the function of perispindle mem- What is the biological significance of the formation of branes as a device for keeping the chromosomes together relatively large meiotic spindles? The meiotic divisions remains a possibility (for a broader discussion of this are certainly under high evolutionary pressure. Erroneous aspect, see Wolf 1990a). segregation leads to offspring with an unbalanced karyo- Intraspindle membranes have been interpreted as a type. On the assumption that in higher eukaryotes the scaffold for the attachment of spindle MTs (Hepler and segregation fidelity increases with the MT content of Wolniak, 1984). In protozoa with a nuclear envelope the spindle, MT-rich meiotic spindles offer a selective persisting throughout mitosis, the inner membrane poss- advantage. Prominent spindle membrane systems are

98 K. W. Wolf potentially active in lowering the Ca2+ concentration presses tubulin synthesis (Ben Ze'ev et al. 1979). Spindle within the spindle domain and thereby favour MT as- structure in metaphase spermatocytes of Lepidoptera is sembly. In addition, their mere presence possibly has a characterized by the presence of perispindle membrane bearing on the formation of a large quantity of spindle layers. Their thickness, however, varies (Table 3). The MTs. This idea was derived from observations on the MT tubulin concentration within the spindle compartment and the membrane content in mitotic and meiotic meta- may control the number of MTs there. Since the spindle phase spindles from M. scalaris (Diptera), E. kuehniella has a membranous envelope, the tubulin level in the and O. thyellina (Lepidoptera). spindle area can be raised without inhibition of the The two meiotic divisions usually are only separated by cytoplasmic tubulin production by reducing the spindle a short interkinesis (Rieger et al. 1976). In E. kuehniella, volume. Preliminary experiments revealed that extensive for example, late telophase I spermatocytes develop di- MT polymerization can be induced within the spindle area rectly into prophase II spermatocytes (Wolf and Bast- in metphase spermatocytes of E. kuehniella using taxol, a meyer, unpublished data). Therefore, it is reasonable to MT-stabilizing drug, whereas in the cytoplasm MTs do not assume, that tubulin subunits required for both meiotic show up (Wolf, unpublished). Presumably, the perispindle divisions already exist in the spermatocyte I. This tubulin membrane layers act as a filter that prevents tubulin from pool, which is larger relative to that of spermatogonia, flowing out. Intraspindle membranes lower the free enables the formation of a prominent microtubular cyto- spindle space by volume exclusion. The level of monomeric skeleton in meiosis I. Since the tubulin pool is biparti- tubulin possibly increases as a function of the membrane tioned during cytokinesis, spindles in secondary spermato- density in the spindle and the formation of MT polymer is cytes should have about one half of the MT mass of favoured. In this context it is important to remember that primary spermatocytes. the mass of intraspindle membranes in spermatocytes of E. kuehniella is higher by factors of 1.8 (level of the Spindles in M. scalaris roughly follow this pattern. The metaphase plate) to 3.0 (half-spindle) than in 0. thyellina. MT mass in metaphase spindles of primary spermatocytes This observation correlates with the higher MT mass in is approximately three times larger than the MT mass in metaphase I spermatocytes of E. kuehniella relative to spermatogonia and secondary spermatocytes. Intraspindle O. thyellina. Membranes are interpreted to act as 'stuffing membranes are missing in M. scalaris. material' in metaphase spermatocytes of Lepidoptera. In contrast, Lepidoptera possess well-developed intraspindle membranes in spermatocytes (Table 3). Two Lepi- Does this interpretation of intraspindle membranes also doptera species analyzed show that the MT content in apply to species besides the Lepidoptera? The stuffing primary spermatocytes was higher, by factors of 3.3 effect brought about by membranes is considered minimal (0. antiqua) and about 6 (E. kuehniella), than in spermato- when the intraspindle membrane inventory is sparse and gonia. Moreover, the microtubular cytoskeleton in a sec- when a spindle envelope is missing. This is the case in ondary spermatocyte of E. kuehniella is significantly many plant and animal cells. (Hepler, 1980; Moll and larger than in a spermatogonium. These observations Paweletz, 1980; Wise, 1984). However, this is not the rule. deviate from the expectations on fluctuations in spindle In oocytes of the strepsipteran parasite Xenos peckii, the size during spermatogenesis (see above) and the question spindle area is densely packed with irregular vesicles and about determinants of spindle size arises. cisternae (Rieder and Nowogrodzki, 1983). In this species, Two factors, the MT nucleating activity of the spindle the metaphase spindle is surrounded by a thick layer of poles and the available pool of soluble tubulin in the cell, vesicular membrane elements. Therefore, it is conceivable will be considered as determinants of spindle MT mass. In that the large reduction in spindle volume has conse- animals, centrosomes are located at the spindle poles and quences for both tubulin concentration and the amount of seem to specifiy the number of spindle MTs (Mclntosh, tubulin polymer. 1983; Mazia, 1984). This capacity can be inferred from cyclic fluctuations in the MT-nucleating activity of the The author expresses his gratitude to Professor W. Traut for centrosomes during the cell cycle (Telzer and Rosenbaum, critically reading the manuscript, to Ms J. Kyburg for expert 1979; Kuriyama and Borisy, 1981). Anti-tubulin immuno- technical assistence, and to Mr H. G. Mertl for collecting larvae of fluorescence confirmed that centrosomes are involved in Phragmatobia fuliginosa. The funding fromth e 'Deutsche Forsch- spindle development of E. kuehniella. Numerous MTs ungsgemeinschaft' (Wo 394/1-1) for work on Megaselia scalaris is gratefully acknowledged. I thank Ms C. Keeler-Lilbeck for radiating out from the centrosomes can be seen up to early linguistic advice during the preparation of a previous version of prometaphase. During this stage, the spindle area, which this text. is delimited from the cytoplasm by membrane stacks, becomes filled with MTs. In metaphase spermatocytes, however, the connection of the MTs with the poles is lost and the vast majority of spindle MTs have their pole References proximal endings distant from the centrosomes in the half- ALDHICH, H. C. (1969). The ultrastructure of mitosis in myxamoebae and spindles (Wolf and Bastmeyer, unpublished). The polar plasmodia of Physarum flavicomum. Am. J. Bot. 56, 290-299. fenestrae of the spindle envelope are sealed by numerous BEN-ZE'BV, A., FABMBH, S. R. AND PENMAN, S. (1979). Mechanisms of irregular membranous elements. This is a typical trait of regulating tubulin synthesis in cultured mammalian cells. Cell 17, 319-325. the so-called sheathed nuclear division (Wolf, 1990a). CAHON, J. M. AND KIKSCHNER, M. W. (1986) Autoregulation of tubulin Thus, most MTs are non-centrosomal and a direct role for synthesis. BwEssays 5, 211-216. the centrosomes in the organization of the spindle is CHUHCH, K. AND LIN, H.-P. P. (1982). Meiosis in Drosopfula unlikely in metaphase of Lepidoptera spermatocytes. melanogaster. LI. The prometaphase I kinetochore microtubule bundle and kinetochore orientation in males. J. Cell Biol. 93, 365-373. It has been demonstrated in mammalian cells, and may CLEVELAND, D. W (1988). Autoregulated instability of tubulin mRNAs: a generally apply, that the level of monomeric tubulin novel eukaryotic regulatory mechanism. Trends biochem. Sci. 13, 339-343. regulates the rate of tubulin synthesis (for reviews, see COMMINGS, D. E. AND OKADA, T. A. (1972) Holocentric chromosomes in Caron and Kirschner, 1986; Cleveland, 1988). An increase Oncopeltus' Kinetochore plates are present in mitosis but absent in in the cellular tubulin concentration specifically sup- meiosis. Chromosoma 37, 177-192.

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