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 wereexamined after conventional preparation for elec-tron microscopy. The aim of the study was to deter-mine (1) the range of variation of the spindle mem-branes between mitotic and meiotic cells and (2) thecorrelation of possible differences with the micro-tubule content of the spindles. The study involvedfour moth species, Ephestia kuehniella, Phragmato-bia fuliginosa, Orgyia thyellina, Orgyia antiqua, andone fly, Megaselia scalaris. Somatic and gonialmitoses in all species examined showed a sparsespindle membrane inventory. In contrast, spermato-cytes consistently had a multi- layered spindle envel-ope. In spermatocytes of all Lepidoptera speciesexamined, but not in those of M. scalaris, diverseforms of intraspindle membranes existed in additionto the spindle envelope. Microtubule counts inserially cross-sectioned spindles of E. kuehniella re-vealed an about 6-fold increase in the mass of polym-erized tubulin during the transition from spermato-gonia to primary spermatocytes. The increase was3.3-fold in O. thyellina and less than 3-fold in M. sca-laris. The density of intraspindle membranes inE. kuehniella was higher than in O. thyelhna by fac-
tors of 1.8 to 3.0. The correlation between the amountof spindle membranes and the microtubule contentof the spindle indicates a functional relationship.Spindle membranes are believed to influence micro-tubule stability via the regulation of the Ca2+ concen-tration within the spindle area. The high microtubulemass in spindles from Lepidoptera spermatocytesmay result from the membrane-dependent loweringof the Ca2+ level within the spindles. Finally, anunconventional idea on the role of intraspindle mem-branes is offered. This concept is not intended tochallenge the function of spindle-associated mem-branes as Ca2+-sequestrating compartments. Intras-pindle membranes are considered as stuffingmaterial in sheathed spindles. Membranous com-partments reduce the free volume within the spindle.Thereby, monomeric tubulin is concentrated and theformation of microtubules is favoured.
The dominant component of spindles is the microtubularcytoskeleton. Microtubules (MTs) most probably play arole in chromosome migration (for reviews, see Nicklas,1988; Mclntosh, 1989; Mitchison, 1988). During the lastdecade, membranous components moved from the fringesto the focus of studies aimed at spindle structure andfunction (for reviews, see Paweletz, 1981; Hepler andWolniak, 1984; Hepler, 1989a). Alhough, as yet, a satisfy-ing conceptual framework has not been derived from theavailable data, spindle membranes seem to be involved inCa2+ transients and spindle dynamics (for detailed dis-cussions, see Wolniak, 1988; Hepler, 19896). Three types ofmembrane systems linked with spindles can be dis-tinguished. (1) Intraspindle membranes occur togetherwith microtubules (MTs) and chromosomes within thespindle domain. They are often arranged parallel to thekinetochore MTs. (2) Perispindle membranes surround thespindle apparatus. (3) Astral membranes or lamellae arearranged parallel to MTs radiating out from the spindlepoles.
The function of spindles is to supply the daughter cellswith a euploid set of chromosomes. In mitotic anaphase,chromatids migrate towards the spindle poles, whereas inanaphase I half-bivalents segregate. The total mass ofmigrating material does not differ between mitosis andmeiosis I. On this premise, prominent structural differ-ences between mitotic and meiotic spindles are not readilyanticipated. However, the comparison of spindles in sper-matogonia and spermatocytes of the Hemiptera speciesDysdercus intermedius (Motzko and Ruthmann, 1984)revealed striking differences in the membrane inventory.Therefore it seemed worthwhile to concentrate on theissue of spindle dimorphism in higher eukaryotes.
In the present study, dividing mitotic cells and sper-matocytes from five insect species are compared, with afocus on two key components: membranes associated withthe spindle and the microtubular cytoskeleton. Four Lepi-doptera species, Orgyia thyellina (n=l l ; Cretschmar,1928), Orgyia antiqua (n=14; Cretschmar, 1928), Ephestiakuehniella (n=30, Traut and Mosbacher, 1968) and Phrag-matobia fuliginosa, have been examined. The karyotype ofthe latter species contains a giant sex chromosome pair.
91
Chromosome races with haploid numbers of 28 and 29chromosomes are known in this species (Seiler, 1925). Theindividuals used in the present study have a haploidchromosome number of 29. Observations on the phorid flyMegaselia scalaris are also presented. In addition to theregular three chromosome pairs (see Johnson et al. 1988,and references therein), the karyotype of this speciescontains varying numbers of centromere-like elementswithout chromosome arms (Wolfed al. 1988).
The spindle membrane inventory and the MT mass werefound to differ between mitotic cells and spermatocytes inall species examined.
Materials and methods
Laboratory strains of the Mediterranean mealmoth, E. kuehniella(Pyralidae), were raised on rolled oats. One strain, L, has ahaploid chromosome number of 30. A second strain used, W10,contains a small heterochromatic fragment in addition to the 30chromosomes. This fragment is derived from the W-chromosome(Traut et al. 1986). Spindle morphology does not vary between thetwo strains and for the present purpose no distinction is madebetween them. Testes and imaginal disks of the wings from larvaein the last instar were prepared for electron microscopy accordingto Wolf (1987).
The same protocol was followed for gonads and imaginal disksof the wings from last instar larvae of O. thyellina and 0. antiqua(Lymantriidae). Larvae of these two species were kindly providedby Sir C. Clarke (Liverpool, UK). In addition, eggs of O. antiquawere collected in Sandhausen (FRG). The larvae from this isolatehatched in the laboratory and were fed with bramble leaves.
Larvae of P. fuliginosa (Arctiidae) were collected in Bergedorfnear Hamburg (FRG) and raised on leaves of Plantago. Testesfrom last instar larvae of the third and fourth laboratory gener-ation were processed for electron microscopy as described pre-viously (Wolf, 1987).
Two laboratory strains of M. scalaris (Phoridae), referred to asWien' and Tennessee' (Johnson et al. 1988), were examined.Differences in spindle structure are not apparent and the twostrains are not treated separately in the present study. The larvaewere grown on a modified Drosophila medium (Johnson et al.1988). Pupae were dissected in an isotonic saline (Hayes, 1953).Subsequently the gonads were transferred into lml saline sol-ution containing 2.5% glutaraldehyde. After 5min, 3 ml of 8%tannic acid in phosphate buffer (67 nw, pH 6.8) were added. Onehour later, the specimens were rinsed in phosphate buffer andembedded in agar (2 % in phosphate buffer) in order to facilitatethe further processing of the delicate specimens. Gonads werepostfixed in phosphate-buffered OsO4 (2%) for one hour andrinsed again. Dehydration was performed in ethanol and propyl-ene oxide and eventually the specimens were embedded in Epon812.
In order to obtain an estimate of the spindle volume occupied bymembranous compartments, the areas surrounded by intraspin-dle membranes were measured in a metaphase I spermatocyte of0. thyellina and in metaphase II spermatocytes of P. fuliginosaand E. kuehniella. The values obtained were related to the totalspindle area. For that purpose, the innermost layer of the spindleenvelope was taken as the boundary of the spindle area. The areameasurements are directly proportional to the volume (see how-ever, the note on systematic errors in the Discussion). Measure-ments were carried out using a graphic tablet (SummagraphicsSupergrid: Summagraphics Corporation, Fairfield, Connecticut)interfaced to an IBM PS2/60 computer. The software was writtenin Turbo Pascal 4.0. The final magnification of the micrographsused for measurements was x 40 000 in P. fuliginosa, x 60 000 inO. thyellina, and x 65 800 in E. kuehniella. Measurements weremade both at the level of the metaphase plate and in the half-spindle adjacent to the chromosomes. In each case, measurementsfrom three consecutive serial cross-sections were averaged.
Results
LepidopteraSomatic mitosis in cells of the testicular sheath (Fig. 1)and of the imaginal disks in E. kuehniella is characterizedby a sparsely developed spindle envelope. One, and inplaces two, discontinuous layers of membranous sheetssurround the spindle domain. The two membrane layersare not closely associated and spindle MTs are foundbetween them. Intraspindle membranes are very scarceand form flat or spherical vesicles (Fig. 1, inset). A pair oforthogonally arranged centrioles is connected with eachspindle pole. This also applies to spindle poles in all othermitotic cells examined in this study. The centrioles areembedded in pericentriolar material of moderate density.Metaphase spindles in imaginal disks of O. antiqua areidentical to those in E. kuehniella regarding spindle mem-branes. Somatic mitosis in 0. thyellina and P. fuliginosawas not studied.
Spermatogonia of E. kuehniella have a one- to three-layered fenestrated spindle envelope. Large gaps occur inthe vicinity of the basal bodies (Fig. 2). The individualmembranous cisternae forming the spindle envelope havelittle space between one another. Intraspindle membranesare missing. Metaphase and early anaphase spindles ingonial cells of 0. thyellina (Fig. 3), 0. antiqua (Fig. 4), andP. fuliginosa (not shown), closely resemble those in sper-matogonia of E. kuehniella.
Lepidoptera produce two types of sperm (for reviews, seeFain-Maurel, 1966; Silberglied et al. 1984). Eupyrenespermatocytes (for terminology, see Meves, 1903) give riseto fertile spermatozoa. Apyrene spermatocytes developinto sterile sperms. The two types of spermatocytes can bereadily distinguished from one another. Eupyrene sper-matocytes are generally larger, possess a normal spindle,and form a metaphase plate. In contrast, in apyrenespermatocytes diverse deviations from regular develop-ment occur (see Wolf et al. 1987). This study deals exclus-ively with eupyrene meiosis.
As a rule, spindles in spermatocytes have a larger pole-to-pole distance and a larger volume than mitotic spindles.Differences in cell size and the presence of one or two basalbodies per spindle pole distinguish secondary and primaryspermatocytes in E. kuehniella (Wolf and Kyburg, 1989).Astral lamellae and peri- and intraspindle membranesexist in both spermatocyte generations. In metaphase, anenvelope composed of irregularly shaped membranouselements completely surrounds the spindle area. Thissheath is 1.6—2.0/on thick at the level of the metaphaseplate. Intraspindle membranes form cisternae alignedparallel to the spindle MTs (Fig. 6). The membranes aredensely packed in places. Conical areas over the polewardsurfaces of the metaphase chromosomes are devoid ofmembranes. The basal bodies are embedded in an array ofastral membranes. Numerous stacks of Golgi cisternae arelocated at the cytoplasmic face of the astral membranes.
Some traits distinguish the membrane inventory ofspermatocytes in P. fuliginosa and E. kuehniella. Theintraspindle membranes in spermatocytes of P. fuliginosahave a rather regular shape. They form tubules arrangedparallel to the pole-to-pole axis (Figs 5,7). Astral mem-branes, flanked by stacks of Golgi cisternae, radiate fromthe spindle pole. The layer of perispindle membranes isrelatively thin (about 1 /mi) at the level of the metaphaseplate. The spindle envelope of P. fuliginosa is composed ofstacked cisternae.
The spindles in primary spermatocytes of O. thyellina
92 K. W. Wolf
c
se
Fig. 1. E. kuehniella. Longitudinal section through a metaphase spindle in the testicular sheath. In this somatic cell, the two layersof the spindle envelope (se) are distant from one another. Microtubules (arrows) occur within the spindle area and between thelayers of the spindle envelope. The inset depicts an example of the scarce intraspindle membranes (arrowheads) in somatic mitosis ofE. kuehniella. c, centriole; k, kinetochore. Bar, linn.
and 0. antiqua have been previously described (Wolf et al.1987; Wolf, 1990a). In brief, the perispindle membranesystem consists of stacks of fiat cisternae similar to thoseof P. fuliginosa. The intraspindle membranes form sheetsparallel to the spindle MTs.
Both Orgyia species show a lower density of the intras-pindle membrane system than E. kuehniella and P. fuligi-nosa. Measurements revealed that the membrane densityis highest in E. kuehniella and lowest in O. thyellina. Thefactors are 1.8 at the level of the chromosomes and 3.0 inthe half-spindle. A metaphase II spindle from P. fuliginosahas a membrane content intermediate between those ofE. kuehniella and 0. thyellina (Table 1). In E. kuehniellaand in P. fuliginosa, the membrane density decreases atthe level of the metaphase plate relative to the half-spindle. Only in 0. thyellina, which has the lowest mem-brane content in the half-spindles of all three speciesexamined, is there a slight increase in the amount ofmembranes at the level of the chromosomes despite thelarge volume occupied by the bivalents (Table 1).
In mitotic and meiotic spindles of two Lepidopteraspecies, 0. thyellina and E. kuehniella, the mass of polym-erized tubulin was estimated in serial cross-sections. The
MT distribution along the spindle axis of a primaryspermatocyte in O. thyellina has been previously pub-lished (Wolf et al. 1987). The MT distribution displays amaximum in each half-spindle and a minimum at the levelof the chromatin. The corresponding values in E. kueh-niella are higher (Table 2).
Summing the number of MT profiles in consecutiveserial sections and multiplication by the average sectionthickness gives the total length of MTs contained in thespindles. Progressing towards the spindle pole beyond theMT maximum generally showed increasing numbers ofastral MTs in spermatocytes. Astral MTs were rare inspermatogonia. In order to avoid an overestimate of theMT mass in spermatocytes, counting was not performedbeyond 60 sections in both directions from the MT mini-mum. The bulk of MTs was contained within 45 sections inboth directions from the MT minimum (Table 2). Beyondthe maxima, the number of MT profiles counted in cross-sections decreased sharply. Gonial spindles have a signifi-cantly lower pole-to-pole distance than spermatocytes. Thevast majority of MT was contained within an interval of45 sections on both sides from the MT minimum. Thus,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 membranesform the spindle envelope (se). One spindle pole, marked by the presence of two centrioles (c), is visible within the section. Note thenumerous 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 earlyanaphase. 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 ismarked by a line in a longitudinally sectioned metphase II spindle (see Fig. 7). Cisternae of the spindle envelope (se) are visiblebesides tubules (t) of the intraspindle membrane system. Arrows: spindle MTs. Bar, 0.5/mi.
reliable estimates of the total MT content in the pertinentspindles.
A metaphase I spermatocyte in 0. thyeUina contains 3.3times more spindle MTs than the metaphase spindle in aspermatogonium. The increase from mitosis to meiosis I inE. kuehniella is even higher (5.6 to 6.6-fold). The MTcontent of a metaphase II spindle in E. kuehniella isreduced by approximately one half relative to metaphase
I. Nevertheless, a secondary spermatocyte contains 2.8times more MTs than a metaphase spermatogonium in thesame species.
In order to estimate the variability of the MT massbetween spindles of the same stage, two primary sper-matocytes were analyzed in E. kuehniella. The differencesfound (Table 2), were well below the differences betweenprimary spermatocytes and spermatogonia in this moth.
94 K. W. Wolf
G ' , .
se
* ..«•', /-^^e
bb
7.bb-A
Fig. 6. £. kuehniella. Longitudinal section through a spindle in the transition between metaphase II and anaphase II. A thickspindle envelope (se) exists. Irregular membranous elements surround the basal body (bb). Strings of cisternae are found within thespindle 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 formboth 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.
DipteraIn ovaries of M. scalaris, cells are attached to the outerface of a basal lamina around the ovarioles. The spindleenvelope in these somatic cells is formed of one layer(Fig. 8). Larger gaps occur. In metaphase, somatic pairingof the homologous chromosomes is brought about bysegments proximal to the centromeres. Distal portions ofthe chromosome arms are not paired and may extendpoleward into the half-spindles.
The metaphase spindle in oogonia is surrounded by adouble layer of membranes (Fig. 9). Small discontinuitiesare visible all over the spindle. Membranous componentsof unclear nature are occasionally found in the peripheryof the spindle area (Fig. 9). The MT density is highest inadaxial portions of the spindle, where the centromeres aregrouped. The centrioles are situated in polar gaps of thespindle envelope.
Metaphase spermatogonia in M. scalaris have one mem-brane layer around the spindle domain (Fig. 10). Larger
gaps in the spindle envelope are to be seen only at thespindle poles. Primary and secondary spermatocytes areidentical as regards spindle membranes. The spindle areain spermatocytes is delimited from the cytoplasm by threeto four membrane layers. Vesicles with a transparentlumen are interspersed between the membrane layers(Fig. 11 and Fig. 5, of Wolf, 19906).
MT counts were carried out in aerial cross-sectionsthrough metaphase spindles in a variety of mitotic cellsand two spermatocytes (Table 2). The technique was ident-ical to that used in Lepidoptera. However, the pole-to-poledistance was smaller in the fly. Therefore, spindle MTswere recorded within a range of ±30 (mitosis and meta-phase II) and ±45 sections (metaphase I) of the minimumin the MT distribution. A somatic cell examined in anovary had the smallest spindle. Two spermatogonia ana-lyzed differed in MT mass. The differences were insignifi-cant relative to the increase in MT content from spermato-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 spindlein a somatic cell of the ovary. Cells of this type are foundbetween ovarioles that are surrounded by a basal lamina (bl).The spindle envelope (se) shows gaps (arrowheads). In ametacentric chromosome, only portions proximal to thecentromeres (cm) are somatically paired. Bar, I/an.Fig. 9. M. scalaris. Longitudinal section through an oogonialmetaphase spindle. The two-layered spindle envelope (se) isinterrupted at the spindle poles, where a centriole (c) is located.Note that the centriole is separated from the cytoplasm through
a membrane layer, cm, centromeres. Arrowheads: membraneelements of unclear nature in the spindle periphery. Bar, I/an.Fig. 10. M. scalaris. Longitudinal section though aspermatogonial metaphase spindle. The almost sphericalspindle area is surrounded by a one-layered spindle envelope(se). c, centriole; cm, centromeres. Bar, I/an.Fig. 11. M. scalaris. Longitudinal section through themetaphase spindle apparatus in a secondary spermatocyte. Themultilayered spindle envelope (se) includes transparentvesicles, bb, basal body; cm, centromeres. Bar, I/an.
metaphase spindle in a secondary spermatocyte containedabout the same amount of MTs as spermatogonia in thesame species.
Discussion
In the present study, mitotic and meiotic spindles from fiveinsect species in two orders, Diptera and Lepidoptera, werecompared. The mass of spindle-associated membranes wasconsistently higher in spermatocytes. This seems to be therule in these two orders (Table 3).
The content of intraspindle membranes in Lepidoptera
spermatocytes was determined using planimetry. Itshould not be overlooked that the use of ultrathin sectionsof finite thickness introduces systematic errors (Weibeland Paumgartner, 1978). These authors calculated thatthe volume occupied by the rough and the smooth endo-plasmic reticulum in rat liver cells is about 29 % and 37 %lower than that measured in random section. The endo-plasmic reticulum structurally resembles the intraspindlemembranes of Lepidoptera spermatocytes. Therefore, thepresent measurements probably overestimate the areasoccupied by membranes and are used for comparativepurposes only. The precise determination of the volume ofthe intraspindle membrane system requires an extensive
Table 1. Estimates of the metaphase spindle area occupied by membranes in spermatocytes of three Lepidoptera species
Species
E. kuehniellaP. fuligmosa0. thyellina
E. kuehniellaP. fuiiginosa0. thyellina
Cell type
Spermatocyte IISperamtocyte IISpermatocyte I
Spermatocyte nSpermatocyte IISpermatocyte I
Total area of thespindle adjacent to
the metaphase plate(/an2)
17.417.518.4
Total area of thespindle at the
level of themetaphase plate
(/on*)
19.736.229.9
S.D.(pan2)
1.00.61.3
S.D.
(/an2)
1.01.30.7
Area of thespindle adjacent to
the metaphase plateoccupied bymembranes
(/an2)
5.33.21.8
Area of the spindleat the level of themetaphase plate
occupied bymembranes
(/an2)
4.53.23.6
S.D.
(/an2)
0.30.10.2
S.D.
(/an2)
0.50.10.05
Mean areaoccupied bymembranes
(%)
30.518.310.2
Mean areaoccupied bymembranes
(%)
22.815.712.4
Area of theapindle occupiedby chromosomes
(/an2)
5.110.112.7
S.D.
(/an2)
0.50.10.3
Mean areaoccupied by
chromosomes(%)
25.927.843.8
Quantitative parameters of the membrane inventory of metaphase spindles in spennatocytes from E. kuehniella, P. fuiiginosa and O. thyellina. Thetotal area per section of the spindle was measured using the innermost layer of the spindle envelope as a boundary. Each value represents the averageof measurements in three consecutive serial sections. The standard deviation (s.D.) is given. Measurements were made in the metaphase plate and inthe 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
Species Cell type
Maximum number ofmicrotubule profilesin each half-spindle
The microtubule inventory of metaphase spindles in O. thyellina, E. kuehniella (Lepidoptera) and M. scalaris (Diptera). Maximum and minimumnumbers of microtubule profiles along the spindle axis are given. The microtubule length was determined by summing the number of microtubuleprofiles 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
»
'.a.fe;
c9
se
11
morphometric approach, which is beyond the scope of thepresent paper.
Several hypotheses on the function of spindle mem-branes have been proposed. The membranes are possiblyinvolved in chromosome transport and orientation (Kubaiand Ris, 1969; Kubai, 1982). In Hemiptera, spindle mem-
branes seem to have a function in the interaction betweenchromosomes and MTs during spindle morphogenesis(Motzko and Ruthmann, 1984). The mitotic chromosomesof all Hemiptera species examined so far have kinetochoreplates. Bivalents in male meiosis are devoid of kinetochoreplates. 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 inLepidoptera and Diptera species
Species Cell typeIntraspindlemembranes PenBpindle membranes
Not detectedNot detectedNot detectedNot detectedNot detected
Membranous inclusionsof unclear nature
Not detectedNot detectedNot detected
Thin layer of cisternaeFragments of the nuclear envelope
One- to two-layered sheathMulti-layered sheath
One- to three-layered sheathMulti-layered sheath
One- to three-layered sheathOne- to three-layered sheath
Multi-layered sheathFragments of the nuclear envelope
Two-layered sheathMulti-layered sheathThin layer of vesicles
One- to three-layered sheathMulti-layered sheath
Thin layer of cisternaeOne layered with discontinuities
Two-layered sheathMulti-layered BheathMulti-layered sheathLayer of flat vesicles
Fragments of the nuclear envelopeTwo-layered sheath
One-layered sheathThree to four-layered sheath
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,1972; Ruthmann and Permantier, 1973; Rufas and Gime-nez-Martin, 1986). With the development of the meioticspindle, membranes become tightly associated with thechromosome surfaces. Only small areas remain mem-brane-free. These sites are thought to interact withspindle MTs (Motzko and Ruthmann, 1984). This sugges-tion has been extended to Lepidoptera spermatocytes.Lepidoptera and Hemiptera species have one property incommon: they are believed to possess holokinetic chromo-somes (Goodward, 1985). However, a role for spindlemembranes in directing the contacts between MTs andchromatin is less probable in Lepidoptera spermatocytes,since (1) kinetochore material is visible at the polewardsurface of the chomosomes in metaphase I and II (Wolf,unpublished) and (2) membranes are not tightly attachedto the chromosomes. Large surface areas are devoid ofmembranes in early prometaphase spermatocytes whencontacts between chromosomes and MTs already exist(Wolf, unpublished).
Perispindle membranes may act as a barrier againstcytoplasmic components or, inversely, may prevent theloss of chromosomes from the spindle. In syncytia, amembrane layer around the spindle domains (e.g. seeAldrich, 1969; Stafstrom and Staehlin, 1984) is consideredas a means of preventing the transfer of chromosomesbetween neighboring spindles. Since gonads also representsyncytia (Gondos, 1984), the function of perispindle mem-branes as a device for keeping the chromosomes togetherremains a possibility (for a broader discussion of thisaspect, see Wolf 1990a).
Intraspindle membranes have been interpreted as ascaffold for the attachment of spindle MTs (Hepler andWolniak, 1984). In protozoa with a nuclear envelopepersisting throughout mitosis, the inner membrane poss-
ibly represents an attachment site for spindle MTs(Eichenlaub-Ritter and Ruthmann, 1982; Kuck and Ruth-mann, 1985; Tucker et al. 1985). However, close examin-ation of the pole-proximal endings of spindle MTs inmetaphase spermatocytes of E. kuehnieUa did not revealspecific membrane-to-MT contacts (Wolf and Bastmeyer,in preparation).
According to the best-conceived hypothesis on the role ofspindle-associated membranes, these control spindle MTstability via the regulation of the Ca2+ concentrationwithin the spindle area (Harris, 1975). There is conclusiveevidence for spindle membrane-associated Ca2+ seques-tration (Silver et al. 1980; Wick and Hepler, 1980; Wiseand Wolniak, 1984; Petzelt and Hafner, 1986; Hafner andPetzelt, 1987). The mass of spindle membranes variessignificantly either between different species (compareFuge, 1971 and Rieder and Nowogrodzki, 1983) or betweenspindles in different cell types of one species (Motzko andRuthmann, 1984). Therefore, the suggestion has beenmade that membranes develop preferentially within largespindles, where the distance between the center of thespindle and its perimeter may be greater than the effectivediffusion limit (Hepler and Wolniak, 1984; Hepler, 1989a).This idea is corroborated by the present observations sinceprominent intraspindle membrane systems develop onlyin the voluminous meiotic spindles of Lepidoptera and aremissing in the smaller mitotic spindles.
What is the biological significance of the formation ofrelatively large meiotic spindles? The meiotic divisionsare certainly under high evolutionary pressure. Erroneoussegregation leads to offspring with an unbalanced karyo-type. On the assumption that in higher eukaryotes thesegregation fidelity increases with the MT content ofthe spindle, MT-rich meiotic spindles offer a selectiveadvantage. Prominent spindle membrane systems are
98 K. W. Wolf
potentially active in lowering the Ca2+ concentrationwithin the spindle domain and thereby favour MT as-sembly. In addition, their mere presence possibly has abearing on the formation of a large quantity of spindleMTs. This idea was derived from observations on the MTand the membrane content in mitotic and meiotic meta-phase spindles from M. scalaris (Diptera), E. kuehniellaand O. thyellina (Lepidoptera).
The two meiotic divisions usually are only separated bya short interkinesis (Rieger et al. 1976). In E. kuehniella,for example, late telophase I spermatocytes develop di-rectly into prophase II spermatocytes (Wolf and Bast-meyer, unpublished data). Therefore, it is reasonable toassume, that tubulin subunits required for both meioticdivisions already exist in the spermatocyte I. This tubulinpool, which is larger relative to that of spermatogonia,enables the formation of a prominent microtubular cyto-skeleton in meiosis I. Since the tubulin pool is biparti-tioned during cytokinesis, spindles in secondary spermato-cytes should have about one half of the MT mass ofprimary spermatocytes.
Spindles in M. scalaris roughly follow this pattern. TheMT mass in metaphase spindles of primary spermatocytesis approximately three times larger than the MT mass inspermatogonia and secondary spermatocytes. Intraspindlemembranes are missing in M. scalaris.
In contrast, Lepidoptera possess well-developed intras-pindle membranes in spermatocytes (Table 3). Two Lepi-doptera species analyzed show that the MT content inprimary spermatocytes was higher, by factors of 3.3(0. antiqua) and about 6 (E. kuehniella), than in spermato-gonia. Moreover, the microtubular cytoskeleton in a sec-ondary spermatocyte of E. kuehniella is significantlylarger than in a spermatogonium. These observationsdeviate from the expectations on fluctuations in spindlesize during spermatogenesis (see above) and the questionabout determinants of spindle size arises.
Two factors, the MT nucleating activity of the spindlepoles and the available pool of soluble tubulin in the cell,will be considered as determinants of spindle MT mass. Inanimals, centrosomes are located at the spindle poles andseem to specifiy the number of spindle MTs (Mclntosh,1983; Mazia, 1984). This capacity can be inferred fromcyclic fluctuations in the MT-nucleating activity of thecentrosomes during the cell cycle (Telzer and Rosenbaum,1979; Kuriyama and Borisy, 1981). Anti-tubulin immuno-fluorescence confirmed that centrosomes are involved inspindle development of E. kuehniella. Numerous MTsradiating out from the centrosomes can be seen up to earlyprometaphase. During this stage, the spindle area, whichis delimited from the cytoplasm by membrane stacks,becomes filled with MTs. In metaphase spermatocytes,however, the connection of the MTs with the poles is lostand the vast majority of spindle MTs have their poleproximal endings distant from the centrosomes in the half-spindles (Wolf and Bastmeyer, unpublished). The polarfenestrae of the spindle envelope are sealed by numerousirregular membranous elements. This is a typical trait ofthe so-called sheathed nuclear division (Wolf, 1990a).Thus, most MTs are non-centrosomal and a direct role forthe centrosomes in the organization of the spindle isunlikely in metaphase of Lepidoptera spermatocytes.
It has been demonstrated in mammalian cells, and maygenerally apply, that the level of monomeric tubulinregulates the rate of tubulin synthesis (for reviews, seeCaron and Kirschner, 1986; Cleveland, 1988). An increasein the cellular tubulin concentration specifically sup-
presses tubulin synthesis (Ben Ze'ev et al. 1979). Spindlestructure in metaphase spermatocytes of Lepidoptera ischaracterized by the presence of perispindle membranelayers. Their thickness, however, varies (Table 3). Thetubulin concentration within the spindle compartmentmay control the number of MTs there. Since the spindlehas a membranous envelope, the tubulin level in thespindle area can be raised without inhibition of thecytoplasmic tubulin production by reducing the spindlevolume. Preliminary experiments revealed that extensiveMT polymerization can be induced within the spindle areain metphase spermatocytes of E. kuehniella using taxol, aMT-stabilizing drug, whereas in the cytoplasm MTs do notshow up (Wolf, unpublished). Presumably, the perispindlemembrane layers act as a filter that prevents tubulin fromflowing out. Intraspindle membranes lower the freespindle space by volume exclusion. The level of monomerictubulin possibly increases as a function of the membranedensity in the spindle and the formation of MT polymer isfavoured. In this context it is important to remember thatthe mass of intraspindle membranes in spermatocytes ofE. kuehniella is higher by factors of 1.8 (level of themetaphase plate) to 3.0 (half-spindle) than in 0. thyellina.This observation correlates with the higher MT mass inmetaphase I spermatocytes of E. kuehniella relative toO. thyellina. Membranes are interpreted to act as 'stuffingmaterial' in metaphase spermatocytes of Lepidoptera.
Does this interpretation of intraspindle membranes alsoapply to species besides the Lepidoptera? The stuffingeffect brought about by membranes is considered minimalwhen the intraspindle membrane inventory is sparse andwhen a spindle envelope is missing. This is the case inmany plant and animal cells. (Hepler, 1980; Moll andPaweletz, 1980; Wise, 1984). However, this is not the rule.In oocytes of the strepsipteran parasite Xenos peckii, thespindle area is densely packed with irregular vesicles andcisternae (Rieder and Nowogrodzki, 1983). In this species,the metaphase spindle is surrounded by a thick layer ofvesicular membrane elements. Therefore, it is conceivablethat the large reduction in spindle volume has conse-quences for both tubulin concentration and the amount oftubulin polymer.
The author expresses his gratitude to Professor W. Traut forcritically reading the manuscript, to Ms J. Kyburg for experttechnical assistence, and to Mr H. G. Mertl for collecting larvae ofPhragmatobia fuliginosa. The funding from the 'Deutsche Forsch-ungsgemeinschaft' (Wo 394/1-1) for work on Megaselia scalaris isgratefully acknowledged. I thank Ms C. Keeler-Lilbeck forlinguistic advice during the preparation of a previous version ofthis text.
ReferencesALDHICH, H. C. (1969). The ultrastructure of mitosis in myxamoebae and
plasmodia of Physarum flavicomum. Am. J. Bot. 56, 290-299.BEN-ZE'BV, A., FABMBH, S. R. AND PENMAN, S. (1979). Mechanisms of
regulating tubulin synthesis in cultured mammalian cells. Cell 17,319-325.
CAHON, J. M. AND KIKSCHNER, M. W. (1986) Autoregulation of tubulinsynthesis. BwEssays 5, 211-216.
CHUHCH, K. AND LIN, H.-P. P. (1982). Meiosis in Drosopfulamelanogaster. LI. The prometaphase I kinetochore microtubule bundleand kinetochore orientation in males. J. Cell Biol. 93, 365-373.
CLEVELAND, D. W (1988). Autoregulated instability of tubulin mRNAs: anovel eukaryotic regulatory mechanism. Trends biochem. Sci. 13,339-343.
COMMINGS, D. E. AND OKADA, T. A. (1972) Holocentric chromosomes inOncopeltus' Kinetochore plates are present in mitosis but absent inmeiosis. Chromosoma 37, 177-192.
Insect mitotic and meiotic spindles 99
CRBTSCHMAR, M. (1928). Das Verhalten der Chromosome bei derSpermatogenese von Orgyia thyellina Btl. und antiqua L. , sowie einesihrer Bastarde. Z. Zellforsch. nukrosk. Anat. 7, 291-399.
EICHENLAUB-RITTBR, U. AND RUTHMANN, A. (1982). Evidence for three'classes' of microtubules in the interpolar space of the mitoticmicronucleus of a Ciliate and the particpation of the nuclear envelopein conferring stability to microtubules. Chromosoma 85, 687-706.
FAIN-MAUREL, M.-A. (1966). Acquisitions recentes sur lesspermatogeneses atypiques. Ann. Bwl. 5, 513-564.
FRIEDLANDBR, M. AND WAHRMAN, J. (1970). The spindle as a basal bodydistributor. A study in the meiosis of the male silkworm, Bombyx mon.J. Cell Sci. 7, 65-89.
FUOE, H. (1971). Spindelbau, Mikrotubuliverteilung undChromosomenstruktur wahrend der I. meiotischen Teilung derSpermatocyten von Pales ferruginea. Z. Zellforsch. nukrosk. Anat. 120,579-599.
Fux, T. (1974). Chromosome elimination in Heteropeza pygmaea. IIinfrastructure of the spindle apparatus. Chromosoma 49, 99-112.
GASSNER, G. AND KLEMETSON, D. (1974). A transmission electronmicroscope examination of hemipteran and lepidopteran gonialcentromeres. Can. J. Genet. Cytol. 16, 457-464.
GONDOS, B. (1984). Germ cell differentiation and intercellular bridges. InUltrastructure of Reproduction (ed. Van Blerkom, J. Motta, P. M.),pp. 31-45. Boston, The Hague, Drodrecht, Lancaster: Martinus NyhoffPubl.
GOODWARD, M. B. E. (1985). The kinetochore. Int Rev. Cytol. 94, 77-103.HAFNKR, M. AND PETZELT, C. (1987). Inhibition of mitosis by an antibody
to the mitotic calcium transport system. Nature 330, 264-266.HARRIS, P. (1975). The role of membranes in the organization of the
mitotic apparatus. Expl Cell Res. 94, 409-426.HAYES, R. O. (1953). Determination of a physiological saline solution for
Aedes aegyptii (L.). J. econ. Ent. 46, 624-626.HEPLBR, P. K. (1980). Membranes in the mitotic apparatus of barley
cells. J. Cell Bwl. 86, 490-499.HBPLBR, P. K. (1989a). Membranes in the mitotic apparatus. In Mitosis:
Molecules and Mechanisms (ed. Hyams, J. and Brinkley, B.R.).pp. 241-271. New York: Acad. Press.
HEPLER, P. K. (19896). Calcium transients during mitosis: Observationsin flux. J. Cell Biol. 109, 2567-2573.
HEPLER, P. K. AND WOLNIAK, S. M. (1984). Membranes in the mitoticapparatus: Their structure and function. Int. Rev. Cytol. 90, 169-238.
HOLM, P. B. AND RASMUSSEN, S. W. (1980). Chromosome pairing,recombination nodules and chiasma formation in diploid Bombyxmales. Carlsberg Res. Commun. 45, 483-548.
ITO, S. (1960). The lamellar systems of cytoplasmic membranes individing spermatogenic cells of Drosophila virilis. J. biophys. biochem.Cytol. 7, 433-442.
JOHNSON, D. S., MERTL, H. G. ANP TBAUT, W (1988). Inheritance ofcytogenetic and new genetic markers in Megaseha scalaris, a fly withan unusual sex determining mechanism. Genetica 77, 159-170.
KUBAI, D. F. (1982) Meiosis in Sciara coprophila: structure of thespindle and chromosome behavior during the first meiotic division. JCell Bwl. 93, 655-669.
KUBAI, D. F. AND RIS, H. (1969). Division in the dinoflagellateGyrodinium cohnii (Schiller). J. Cell Bwl. 40, 508-528.
KUCK, A. AND RUTHMANN, A. (1985) Micronuclear division andmicrotubular stability in the ciliate Colpoda steinu. Chromosoma 93,95-103.
KUBIYAMA, R. AND BORISY, G. G. (1981). Microtubule-nucleating activityof centrosomes in Chinese hamster ovary cells is independent of thecentnole cycle but coupled to the mitotic cycle J Cell Biol. 91,822-826.
MAZIA, D. (1984). Centrosomes and mitotic poles. Expl Cell Res. 153,1-15.
MCINTOSH, J. R. (1983). The centrosome as an organizer of thecytoskeleton. Modern Cell Bwl. 2, 115-142.
MCINTOSH, J. R. (1989). Dynamic behavior of mitotic microtubules. InCell Movement, Vol. 2, Kmesin, Dynein, and Microtubule Dynamics(ed. Warner, F. D. and Mclntoeh, J. R.), pp. 371-382. New York: AlanR Liss
MBVES, F. (1903). Ober oligopyrene und apyrene Spermien und liber ihreEntstehung, nach Beobachtungen an Paludina und Pygaera. Arch.mikrosk. Anat. EntwGesch. 61, 1-84.
MrrcHisoN, T. J. (1988). Microtubule dynamics and kinetochore functionin mitosis. A. Rev. Cell Biol. 4, 527-549.
MOLL, E. AND PAWELETZ, N. (1980). Membranes of the mitotic apparatusof mammalian cells. Eur. J. Cell Biol 21, 280-287.
MOTZKO, D. AND RUTHMANN, A. (1984). Spindle membranes in mitosisand meiosis of the heteropteran insect Dysdercus intermedius. A studyof the interrelationship of spindle architecture and the kineticorganization of chromosomes. Eur. J. Cell Bwl. 33, 205-216
NICKLAS, R. B. (1988). The forces that move chromosomes in mitosis. A.Rev. biophys. Chem. 17, 431-449.
PAWELETZ, N. (1981). Membranes in the mitotic apparatus. Cell Biol. Int.Rep. 5, 323-336.
PETZELT, C. AND HAFNER, M. (1986). Visualization of the Ca2+- transportsystem of the mitotic apparatus of sea urchin eggs with a monoclonalantibody. Proc. natn. Acad. Sci. U.S-A. 83, 1719-1722.
RIEDKB, C. L. AND NOWOGHODZKI, R. (1983). Intranuclear membranes andthe formation of the first meiotic spindle in Xenos peckn Acrochismuswheelen) oocytes. J. Cell Biol. 97, 1144-1155.
RraoER, R., MICHABLIS, A. AND GREEN, M. M. (1976). Glossary of Geneticsand Cytogenetics, p 303. Berlin, Heidelberg, New York: Springer-Verlag.
ROTH, L. E., WILSON, H. J. AND CHAKBABORTY, J. (1966). Anaphasestructure in mitotic cells typified by spindle elongation. J. Ultrastruct.Res. 14, 460-483.
RUFAS, J. S. AND GIMENEZ-MARTIN, G. (1986). Ultrastructure of thekinetochore in Graphosoma Ualicum (Hemiptera: Heteroptera).Protoplasma 132, 142-148.
RUTHMANN, A AND PBRMANTIEB, Y. (1973). Spindel und Kinetochoren inder Mitose und Meiose der Baumwollwanze Dysdercus intermediusHeteroptera). Chromosoma 41, 271-288.
SBILEB, J. (1925). Zytologische Vererbungastudien an Schmetterlingen. I.Ergebnisse aus Kreuzungen von Schmetterlingsrassen mitverschiedener Chromosomenzahl. Ein Beweis fur das Mendeln vonChromosomen. Arch. Julius Klaus-Stift. VererbForsch. 1, 63-117.
SDLBERGIJED, R E., SHEPHERD, J. G. AND DICKINSON, J. L. (1984).Eunuchs: The role of apyrene sperm in Lepidoptera? Am. Nat. 123,255-265.
SILVER, R. B., COLE, R. D. AND CANDE, W. Z. (1980). Isolation of mitoticapparatus containing vesicles with calcium sequestration activity. Cell19, 505-516.
STAFSTROM, J P. AND STABHLIN, A. (1984). Dynamics of the nuclearenvelope and of nuclear pore complexes during mitosis in theDrosophila embryo. Eur. J. Cell Bwl. 34, 179-189.
TELZER, B. R. AND ROSENBAUM, J. L. (1979). Cell cycle-dependent, in vitroassembly of microtubules onto the pericentriolar material of HeLacells. J. Cell Biol. 81, 484-497.
TRAUT, W. AND MOSBACRBR, G. C. (1968). Geschlechtschromatdn beiLepidopteren. Chromosoma 25, 343-356.
TRAUT, W., WEI™, A. AND TRAUT, G. (1986). Structural mutants of theW chromosome in Ephestia kuehniella (Insecta, Lepidoptera). Genetica70, 69-79.
TUCKER, J. B , MATHEWS, S. A., HENDEY, K. A. K., MACKIE, J. B. ANDROCHE, D. L. J. (1986). Spindle microtubule differentiation anddeployment during micronuclear mitosis in Paramecium. J. Cell Bwl.101, 1966-1976.
WKIBEL, E. R. AND PAUMOAKTNEE, D. (1978). Integrated stereological andbiochemical studies on hepatocytic membranes. II Correction ofsection thickness effect on volume and surface density estimates. JCell Biol. 77, 584-597.
WICK, S. M. AND HEPLEB, P. K. (1980). Localization of Ca-containingantimonate precipitates during mitosis J. Cell Biol. 86, 500—513.
WISE, D. (1984). The ultrastructure of an intraspindle membrane systemin meiosis of spider spermatocytes. Chromosonia 90, 50—56.
WISB, D. AND WOLNLAK, S. M. (1984). A calcium-rich intraspindlemembrane system in spermatocytes of wolf spiders. Chromosoma 90,156-161.
WOLF, K. W. (1987). Cytology of Lepidoptera. I. The nuclear area insecondary oocytes of Ephestia kuehniella Z. contains remnants of thefirst division. Eur. J. Cell Biol. 43, 223-229.
WOLF, K. W. (1988). Cytology of Lepidoptera. IV. Chromatin-associated,virus-like particles in testes of Pieris brassicae L. Eur. J. Cell Bwl 47,132-137.
WOLF, K. W. (1990a). Sheathed nuclear division in primaryspermatocytes of Orgyia antiqua (Lepidoptera, Insecta) BioSystems 24(in press).
WOLF, K. W. (19906). The behavior of C-shaped microtubule endings inthe cell. Cell MoUl. Cytoskel. (in press).
WOLF, K W., BAUMGART, K. AND TBAUT, W (1987). Cytology ofLepidoptera. II Fine structure of eupyrene and apyrene primaryspermatocytes in Orgyia thyellina. Eur. J. Cell Bwl. 44, 57-67.
WOLF, K W., JOHNSON, D. S AND TRAUT, W (1988). Centromere-likeelements devoid of chromosome arms in Megaselia scalaris Diptera). InKew Chromosome Conference III (ed. Brandham, P.E.), pp 1-7London: Her Majesty's Stationery Office.
WOLF, K. W. AND KYBURG, J (1989). The restructuring of the flagellarbase and the flagellar necklace during spermatogenesis of Ephestiakuehniella Z. (Pyrahdae, Lepidoptera). Cell Tiss Res. 256, 77-86.
WOLNIAK, S. M. (1988) The regulation of mitotic spindle function.Biochem Cell Biol. 66, 490-614.
(Received 21 February 1990 - Accepted, in revised form, 21 May 1990)
