A calcium-rich intraspindle membrane system in spermatocytes of wolf spiders

The meiotic spindle of spermatocytes of two wolf spiders contains a highly organized system of ER-like membranes. In cells observed ultrastructurally at early prometaphase, these membranes completely invest each bivalent and are present in the periphery of the spindle in association with the centrosomes. By metaphase each bivalent and its kinetochore fibers are completely encased in a tube of this membrane. We have treated living spermatocytes with the permeant, fiuorescent-chelate probe, chlorotetracycline (CTC) to determine whether or not the intraspindle membrane system is rich in associated Ca²⁺. Spider testes were dissected into PIPES-buffered saline containing 200 M CTC and were kept in this solution for 10 min. Autofluorescence controls were prepared by incubation in saline without CTC, and nonspecific effects of CTC were assessed by incubation for 10 min in 200 M oxytetracycline (OTC). Neither unstained nor OTC-treated spermatocytes emit significant fluorescence. In contrast, CTC treatment yields bright, punctate fluorescence, which coincides with the distribution of the mitochondria. The plasma membrane is only weakly fluorescent, while the nuclear envelope exhibits prominent fluorescence. The chromosomes are not fluorescent during prophase, but after nuclear envelope breakdown, they become outlined by dim, but distinct fluorescence. As spindle formation commences, the CTC signal from the intraspindle membrane system becomes strong. In some cells, thin lines of CTC fluorescence are apparent in the metaphase half spindle; this fluorescence pattern mimics the distribution of the intraspindle membrane system and suggests that it is rich in associated Ca²⁺. We suggest that the intraspindle membrane system functions in the regulation of cytosolic Ca²⁺during meiosis through sequestration of the cation.     

The ultrastructure of an intraspindle membrane system in meiosis of spider spermatocytes

An extensive system of membranes was found in the spindles of spermatocytes of two wolf spider species, Lycosa georgicola and L. rabida. Serial section reconstructions of this membrane system revealed that each meiotic bivalent is encased in a tube of membrane, which encloses both kinetochore microtubule bundles and approaches to within a few microns of the centriolar complex. The membrane tube is open at the polar ends. The membrane composing the tube is doubled and resembles smooth endoplasmic reticulum (ER). No evidence of nuclear pore complexes has been found in the intraspindle membrane system, but typical pores are present on the nuclear envelope of prophase cells. The membrane tubes are fenestrated and microtubules sometimes penetrate these fenestrae. Besides its possible function in the regulation of chromosome movement, the intraspindle membrane system may participate in the nonrandom segregation of the sex chromosomes at meiosis in these spiders.     

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

A comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.     

Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin''s spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo''s negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.     

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes

To assess the role of γ-tubulin in spindle assembly in vivo, we have followed meiosis progression by immunofluorescence and time-lapse video microscopy in γTub23C^PI mutant spermatocytes. We have found that centrosomes associate with large numbers of astral microtubules even though γ-tubulin is severely depleted; bipolar meiotic spindles are never assembled; and later in meiosis, the microtubules get organized into a conical structure that is never observed in wild-type cells. Several lines of evidence suggest that these cones may be related to wild-type central spindles. First, they are assembled midway through meiosis and elongate during anaphase. Second, they are constricted during late meiosis, giving rise to a pointed end similar to those that form in each half of the wild-type spindle midzone. Third, Klp3A and Polo, two markers of the wild-type central spindle are also found around the pointed end of the mutant cones. Finally, ectopic cytokinesis furrows are often formed at the distal end of the cone. Our results suggest that microtubule polymerization or stabilization from the centrosome may be possible in a γ-tubulin-independent manner in Drosophila spermatocytes. However, γ-tubulin seems to be essential for spindle assembly in these cells. Finally, our results show that at least part of the central spindle and constriction-ring assembly machinery can operate on microtubule bundles that are not organized as bipolar spindles.     

The relationship between nuclear envelope-derived annulate lamellae and the asymmetric division of primary spermatocytes in aphids

The structure of dividing primary spermatocytes of Amphorophora tuberculata (Aphididae, Hemiptera) as determined by electron microscopy and serial sectioning is described. The developmental stages examined extend from late prophase I to late telophase I. We looked for any asymmetric organization that could be causally linked to the differences in chromatin behaviour between the two daughter nuclei towards the end of meiosis I of this species. In late prophase I, evaginations of the nuclear envelope in the vicinity of two neigh-bouring centrosomes develop into closed cytoplasmic compartments with a dense content. The compartments open in prometaphase I and come to lie together with fragments of the nuclear envelope within the spindle area. Since nuclear pores are preserved in the membranes, intraspindle annulate lamellae have formed. These and material of presumed nuclear origin associated with them are asymmetrically distributed within the cell. Although dispersed at stages beyond prometaphase I, the material may be largely incorporated into one of the two daughter cells and thus be decisive for further development. Some annulate lamellae form a cap at the chromosome surface opposite to the neighbouring centrosomes in prometaphase I. These membranes may prevent interaction between spindle microtubules and chromosomes until a bipolar spindle forms in metaphase I. At this stage, both the banana-shaped autosomal bivalent and the X univalent occupy the equatorial plane. This is strange, because the X univalent has microtubular connections with one spindle pole and would be expected to migrate towards that pole. Possibly, the kinetochore of the X chromosome is inactive, and remains so in anaphase I, when the X univalent remains located between the two autosomal half-bivalents.M.F. Trendelenburg     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Diversity in meiotic spindle origin and determination of cytokinetic planes in sporogenesis of complex thalloid liverworts (Marchantiopsida)

As the earliest divergent land plants, bryophytes (mosses, hornworts, and liverworts) provide insight into the evolution of the unique plant process of sporogenesis by which meiosis results in heavy walled spores. New immunohistochemical data on microtubules and γ-tubulin in four genera of complex thalloid liverworts combined with previously published data on another four genera demonstrate grades in the evolution of spindle organization in meiosis. We have discovered that all recognized forms of microtubule organizing centers (MTOCs) in plant cells (plastid MTOCs, spheroid cytoplasmic MTOCs, polar organizers, and nuclear envelope MTOCs) occur in organization of the meiotic spindle of complex thalloid liverworts. In addition, all aspects of pre-meiotic preparation for quadripartitioning of the sporocyte into a tetrad of spores occur, with the exception of pre-meiotic wall precursors found in certain simple thalloids. The preparation includes morphogenetic plastid migration, cortical bands of microtubules that mark future cytokinetic planes in pre-meiosis, quadrilobing of the cytoplasm during meiotic prophase, and quadripolar microtubule systems that are transformed into functionally bipolar metaphase I spindles. Quadripolar spindle origin is typical of bryophyte sporogenesis even though the MTOCs involved may differ. However, in certain crown taxa of complex thalloids the spindle develops with no traces of quadripolarity and placement of intersporal walls is determined after meiosis, as is typical of higher plants.     

Acetylation of α-tubulin in male meiotic spindles ofPyrrhocoris apterus,an insect with holokinetic chromosomes

Summary Kinetochore structure was examined in metaphase spermatogonia and primary spermatocytes of the red firebug,Pyrrhocoris apterus (Pyrrhocoridae, Hemiptera). Chromosome spreads were analysed using light microscopy and serial sections through spindles were studied using electron microscopy. Mitotic chromosomes were rod-shaped bodies and did not possess primary constrictions. Trilaminar kinetochores occurred throughout about 72% of the chromosomal length. Numerous microtubules (MTs) were connected with the outer plates of the kinetochores and interactions between MTs and the remainder of the chromosomal surface were rare. The bivalents formed dumbbell-shaped bodies in metaphase I spermatocytes. At that stage, MTs were found in contact with the entire poleward surface of the chromosomes. Distinct kinetochore material was, however, not detectable and some MTs penetrated deeply into the chromatin. Mitotic and meiotic chromosomes ofP. apterus are holokinetic and consequently the number of kinetochore MTs is expected to be relatively high. In the second part of the study, the question whether holokinetic chromosomes affect spindle MT dynamics is addressed. To this end, primary spermatocytes ofP. apterus were labelled with a widely used antibody, 6-11B-1, directed against acetylated -tubulin. The acetylation of -tubulin is believed to indicate the presence of long-lived MTs. MT bundles were labelled in metaphase and anaphase I spindles, while prophase and prometaphase I spermatocytes did not contain acetylated MTs. MTs in early and mid telophase spindles were not acetylated. Only late telophase I spindles possessed small amounts of acetylated -tubulin. The acetylated MT bundles of metaphase and anaphase I spindles probably represent kinetochore MTs stabilized by their association with the holokinetic chromosomes at one end and the spindle poles at the opposite end.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole · 2HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis(2-ethane sulfonic acid) - MT microtubule     

Gamma-tubulin is required for bipolar spindle assembly and for proper kinetochore microtubule attachments during prometaphase I in Drosophila oocytes

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

Summary A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 m in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.     

Fertilization initiates the transition from anaphase I to metaphase II during female meiosis in C. elegans

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Immunocytochemical Evidence of a Tubulin Reserve at the Tip of Growing Flagella in Spermatogenesis of the Mediterranean Mealmoth,Ephestia kuehniella Z. (Pyralidae,Lepidoptera, Insecta)

Abstract The distal swellings of growing flagella in spermatocytes of Ephestia kuehniella Z. contain dense material associated with the ends of axonemal microtubules. In order to define the nature of this material, spermatocytes were lysed under microtubule-stabilizing conditions, spun onto cover-slips, probed with an antibody against β-tubulin and processed for indirect immunofluorescence. Whereas the dense material was lost from the cells when untreated spermatocytes were used, a block of stained material was visible in cold-treated spermatocytes. Most probably, cold-treatment alters the dense material and guarantees its survival during preparation of the cells for anti-tubulin immunofluorescence. The positive reaction with the antibody indicates the presence of β-tubulin. Flagellar outgrowth in spermatogenesis of the moth starts in late prophase I and continues throughout both meiotic divisions. Therefore, spindles and flagella compete for tubulin monomers. A tubulin reserve, deposited early in development at the elongating tip of axonemes, may ensure their uninterrupted growth, independent of tubulin-consuming cytoplasmic events. In order to test this hypothesis, flagellar outgrowth was studied in the spermatocytes of a long-horned beetle, Agapanthia villosoviridescens de Geer (Cerambycidae, Coleoptera) using electron microscopy. In this species, flagella begin to elongate only in telophase II, when the second meiotic spindle is just disassembling. The absence of dense material at the tip of flagellar stubs in the beetle corroborates the hypothesis formulated above.     

The reduction of chromosome number in meiosis is determined by properties built into the chromosomes

In meiosis I, two chromatids move to each spindle pole. Then, in meiosis II, the two are distributed, one to each future gamete. This requires that meiosis I chromosomes attach to the spindle differently than meiosis II chromosomes and that they regulate chromosome cohesion differently. We investigated whether the information that dictates the division type of the chromosome comes from the whole cell, the spindle, or the chromosome itself. Also, we determined when chromosomes can switch from meiosis I behavior to meiosis II behavior. We used a micromanipulation needle to fuse grasshopper spermatocytes in meiosis I to spermatocytes in meiosis II, and to move chromosomes from one spindle to the other. Chromosomes placed on spindles of a different meiotic division always behaved as they would have on their native spindle; e.g., a meiosis I chromosome attached to a meiosis II spindle in its normal fashion and sister chromatids moved together to the same spindle pole. We also showed that meiosis I chromosomes become competent meiosis II chromosomes in anaphase of meiosis I, but not before. The patterns for attachment to the spindle and regulation of cohesion are built into the chromosome itself. These results suggest that regulation of chromosome cohesion may be linked to differences in the arrangement of kinetochores in the two meiotic divisions.     

The ubiquitin ligase component Siah1a is required for completion of meiosis I in male mice

The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including beta-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

Distribution of gamma-tubulin differs in primary and secondary oocytes of Ephestia kuehniella (Pyralidae,Lepidoptera)

In a previous study, barrel-shaped spindles were found in metaphase I oocytes of Ephestia kuehniella (Pyralidae, Lepidoptera). Aster microtubules (MTs) were missing (Wolf, 1993: Cell Motil Cytoskeleton 24:200-204). This points to an acentriolar organization of the spindle apparatus. The present study was aimed at the question of whether gamma-tubulin, a newly detected member of the tubulin superfamily that has often been identified in microtubule-organizing centers, plays a role in the nucleation of MTs in meiotic spindles of the moth. To this end, the distribution of gamma tubulin was examined in oocytes of E. kuehniella using an antibody against gamma-tubulin in combination with indirect immunofluorescence. The antibody evenly decorated spindle MTs in metaphase I oocytes of the moth. Enhanced staining of the spindle poles was not detectable In subsequent stages of meiosis, gamma-tubulin was gradually lost from spindle MTs and was then found at the surface of the so-called elimination chromatin. Female meiosis in Lepidoptera is achiasmatic. The elimination chromatin, i.e., modified and persisting synaptonemal complexes, is believed to keep homologous chromosomes linked until the onset of anaphase I. In meiosis I of female Lepidoptera, the elimination chromatin persists at the spindle equa or between the segregating chromatin masses. It is plausible to assume that gamma-tubulin is involved in spindle organization in the absence of canonical centrosomes. In MTs of metaphase II spindles of E. kuehniella, gamma-tubulin was no longer detectable with our immunological approach. This points to a far-reaching change in spindle organization during transition from meiosis I to meiosis II. © 1996 Wiley-Liss, Inc.