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.     

Meiotic chromosome missegregation during apyrene meiosis in the gypsy moth,Lymantria dispar,is preceded by an aberrant prophase I

The gypsy moth, Lymantria dispar, produces two structurally and genetically distinct types of spermatozoa. The eupyrene spermatozoa are genetically haploid and structurally typical. The apyrene spermatozoa are anucleate and structurally different from eupyrene spermatozoa. To understand further the events contributing to meiotic chromosome missegregation in apyrene spermatocytes, we examined the progression of meiosis in these cells with respect to their eupyrene counterparts. Chromosomal bouquet formation and fusion of nucleolar organizing regions are disrupted in apyrene nuclei. In addition, the chromatin of apyrene nuclei is prematurely and extremely condensed compared with that of eupyrene nuclei. An antibody to the conserved synaptonemal complex protein 3 (SCP3) labeled eupyrene pachytene chromosomes, but not apyrene pachytene chromosomes. In addition, apyrene meiotic spindles are missing a subset of microtubules, which likely include kinetochore microtubules. Because the condensation behavior of meiotic chromatin in apyrene spermatocytes deviates from that of eupyrene spermatocytes, we examined the appearance and distribution of the phosphorylated form of histone H3, but no significant differences in histone H3 phosphorylation were found between apyrene and eupyrene spermatocytes. We argue that because a pachytene checkpoint is not initiated in apyrene spermatocytes, this system may provide a way to understand better the underlying biochemical connections between pairing, recombination, synapsis, kinetochore assembly and segregation of chromosomes during meiosis in a higher eukaryote.     

Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild-type strain, L, normal-sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous. Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected. In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild-type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.     

组蛋白H3Ser10磷酸化在家蚕精母细胞减数分裂中的动态分布

【目的】探究组蛋白H3Ser10磷酸化(H3Ser10ph)在家蚕Bombyx mori精母细胞减数分裂中的功能。【方法】解剖并分离家蚕4龄幼虫至蛹期精巢组织,通过丙烯酰胺凝胶包埋制备处于减数分裂不同时期的精巢组织玻片,以免疫荧光标记检测H3Ser10ph抗体在精母细胞减数分裂不同时期的定位特点。【结果】在家蚕有核精子精母细胞减数分裂过程中,组蛋白H3Ser10的磷酸化发生在粗线期染色体的特定位置,双线期H3Ser10ph信号逐渐减弱,至终变期时在染色体上完全检测不到磷酸化信号。随着细胞周期的进行,磷酸化信号又开始逐渐增强,减数第一次分裂中期时达到最高水平。当细胞进入减数第二次分裂前中期时,染色体臂上的H3Ser10ph信号消失,在靠近纺锤体微管的分裂面处有弥散的H3Ser10ph抗体的信号,减数第二次分裂末期,仅剩余非常微弱的H3Ser10ph信号残留于染色体的特定位置。在无核精子精母细胞减数分裂过程中,在中期I至末期I一直在染色体上有较均一的3Ser10ph信号,后期I时纺锤丝微管与赤道面平行。【结论】组蛋白H3Ser10磷酸化与家蚕有核精子和无核精子精母细胞减数分裂中染色质的动态变化相关。     

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.     

Nonrandom chromosome segregation in Neocurtilla (Gryllotalpa) hexadactyla: an ultrastructural study

During meiosis I in males of the mole cricket Neocurtilla (Gryllotalpa) hexadactyla, the univalent X1 chromosome and the heteromorphic X2Y chromosome pair segregate nonrandomly; the X1 and X2 chromosomes move to the same pole in anaphase. By means of ultrastructural analysis of serial sections of cells in several stages of meiosis I, metaphase of meiosis II, and mitosis, we found that the kinetochore region of two of the three nonrandomly segregating chromosomes differ from autosomal kinetochores only during meiosis I. The distinction is most pronounced at metaphase I when massive aggregates of electron-dense substance mark the kinetochores of X1 and Y chromosomes. The lateral position of the kinetochores of X1 and Y chromosomes and the association of these chromosomes with microtubules running toward both poles are also characteristic of meiosis I and further distinguish X1 and Y from the autosomes. Nonrandomly segregating chromosomes are typically positioned within the spindle so that the kinetochoric sides of the X2Y pair and the X1 chromosome are both turned toward the same interpolar spindle axis. This spatial relationship may be a result of a linkage of X1 and Y chromosomes lying in opposite half spindles via a small bundle of microtubules that runs between their unusual kinetochores. Thus, nonrandom segregation in Neocurtilla hexadactyla involves a unique modification at the kinetochores of particular chromosomes, which presumably affects the manner in which these chromosomes are integrated within the spindle.     

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.     

Differences in spindle association of the mitotic checkpoint protein Mad2 in mammalian spermatogenesis and oogenesis

We have investigated expression and subcellular localization of the spindle checkpoint protein Mad2 during rat and mouse spermatogenesis and in superovulated mouse oocytes. Our immunofluorescence studies demonstrate substantial differences in the localization patterns of kinetochore-associated Mad2 in these meiotic systems compared with previous studies of mitosis. In addition, the association of Mad2 with second-division-metaphase kinetochores differed significantly in male versus female meiosis. In spermatogenesis, Mad2 remained at most kinetochores throughout the entire first meiotic division and was lost only at metaphase of the second meiotic division. This result indicates that loss of kinetochore-associated Mad2 is not essential for the metaphase-to-anaphase transition during the first meiotic division. Disruption of the male meiotic spindles with the microtubule depolymerizing agent nocodazole resulted in the appearance of Mad2 at nearly all kinetochores. In contrast, the microtubule stabilizer taxol induced the loss of Mad2 from the majority of the first-division-metaphase kinetochores in which it was normally present in untreated cells. In contrast to the situation in spermatogenesis, Mad2 persisted at the kinetochores of normal, second-division oocytes at metaphase. These findings suggest that the role of the kinetochore in signaling in the spindle checkpoint may differ markedly between mammalian mitosis and meiosis, between the two meiotic divisions, and between male and female meiosis.     

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.     

The regular divisions of the spermatocytes as related to a meiotic lysine-rich protein fraction

Lepidopteran primary spermatocytes are bipotential leading first to regular (eupyrene) and later to irregular (apyrene) meiotic divisions. The kinetics of the lysine-rich proteins during this dichotomous meiosis was studied using the fluorescent dye sulfoflavine. Throughout the spermatogonial divisions, the chromatin fluoresces while the cytoplasm remains unstained. Reversely, during the meiotic prophase, the cytoplasm fluoresces strongly while the nuclei show only a few weakly fluorescing structures. From premetaphase to telophase the meiotic chromosomes fluoresce strongly again. But during this period, only in the eupyrene cells the cytoplasm remains strongly fluorescent; the fluorescence vanishs in the cytoplasm of the apyrene spermatocytes. Thus, the regular (eupyrene) meiotic divisions and the presence of a lysine-rich protein fraction in the cytoplasm of the dividing spermatocytes of Lepidoptera, are probably related.     

Centromeres put epigenetics in the driver's seat

A defining feature of chromosomes is the centromere, the site for spindle attachment at mitosis and meiosis. Intriguingly, centromeres of plants and animals are maintained by both sequence-specific and sequence-independent (epigenetic) processes. Epigenetic inheritance might enable kinetochores (the structures that attach centromeres to spindles) to maintain an optimal size. However, centromeres are susceptible to the evolution of "selfish" DNA repeats that bind to kinetochore proteins. We argue that such sequence-specific interactions are evolutionarily unstable because they enable repeat arrays to influence kinetochore size. Changes in kinetochore size could affect the interaction of kinetochores with the spindle and, in principle, skew Mendelian segregation. We propose that key kinetochore proteins have adapted to disrupt such sequence-specific interactions and restore epigenetic inheritance.     

Spermatocytes of the caddisfly Potamophylax rotundipennis (Trichoptera, Insecta): a fine structure study with emphasis on synaptonemal complex plates associated with chromatin

Electron microscopy of ultrathin sections was used to study the restructuring of primary spermatocytes in a caddisfly, Potamophylax rotundipennis (Limnephilidae). Spindle structure was also examined using light microscopy of dividing spermatocytes lysed in a microtubule-stabilizing buffer. The bulk of pachytene spermatocytes was usual in that the nuclei contained tripartite synaptonemal complexes (SCs). The SCs were attached end-on to the inner face of the nuclear envelope and loosely surrounded by electron-dense chromatin. Cells of this type gave rise to late prophase I spermatocytes, where SCs were missing and chromatin condensation was advanced. By metaphase I, a conventional bipolar spindle apparatus assembled, bivalents were aligned at the spindle equator, and membrane sheets were scattered throughout the spindle matrix. Prominent interzone spindles were typical of telophase spermatocytes. However, a subset of prophase I spermatocytes possessed unusual forms of SCs. The analysis of short series of ultrathin sections through the nuclei revealed plates composed of synaptonemal complex material. These elements will be referred to as 'SC plates'. Within the SC plates, the tripartite organization typical of regular SCs was preserved. The chromatin surrounding the SC plates was highly condensed. The SC plates ended abruptly within the nuclear lumen and did not reach the nuclear envelope. Finally, branching of SC plates was common. In light of the bizarre organization of SC material and its relation to the chromatin, and because spermatocytes with SC plates do not readily fit into the regular development of male germ cells in the caddisfly, we venture the suggestion that the SC plates are not physiological intermediates of SC disassembly. The affected cells most probably fail to complete meiosis.     

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.     

Spermatogenesis in Tenebrio molitor (Tenebrionidae,Coleoptera): A Fine Structure and Anti-tubulin Immunofluorescence Study

Spermatogonia and both generations of spermatocytes of Tenebrio molitor possess conventional bipolar spindles with only few aster MTs. Spindles in metaphase spermatogonia are surrounded by fenestrated two-layered cisternae and do not contain intraspindle membranes. In metaphase spermatocytes, a spindle envelope is missing, but intraspindle membranes are abundant. Mitochondria form long threads lateral to the nucleus in prophase I of meiosis. The elongated mitochondria also align parallel to the spindle apparatus in prometaphase I. As a consequence, the spindles reside in a cage formed of mitochondria. This arrangement may guarantee proper bisection of the chondriome during division. Cells are tightly packed during spermatogonial divisions and in prophase I, but large intercellular spaces develop when the first meiotic spindle assembles. Then, cytoplasmic bridges which persist between the cells as a result of incomplete cytokinesis appear as slender tubes. Anti-tubulin immunofluorescence using an antibody against acetylated α-tubulin revealed intense acetylation throughout spermatogonial mitosis but a low degree of α-tubulin acetylation in meiotic spindles prior to telophase. This may indicate a high microtubule turnover in meiosis.     

What moves chromosomes, microtubules or microfilaments?

An investigation of the spindle apparatus of crane-fly (Nephrotoma suturalis) spermatocytes has been undertaken using methods that permit combined light and electron microscopy of selected cells. At the ultrastructural level, spindles contain microtubules in a granular matrix. Microtubules have been classified as kinetochore microtubules (which connect to kinetochores of chromosomes) and non-kinetochore microtubules (not attached to kinetochores). Kinetochore microtubules are distributed in densely packed bundles, which are the birefringent chromosomal fibers seen in living cells. Actin filaments were not observed in spindles of unglycerinated cells or in cells fixed in glutaraldehyde containing tannic acid, which negatively stains F-actin in situ and thus can be used to aid the localization of actin filaments in non-muscle cells. The absence of actin filaments in the spindle coupled with their presence in the "contractile ring" of spermatocytes fixed during cytokinesis is evidence against the hypothesis that chromosome movements are microfilament-based. The results are compatible with the hypothesis that microtubules are involved in the mechanism of chromosome transport. The details of that mechanism remain to be clarified.     

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.     

Asymmetric division of spindle microtubules and microfilaments during bovine meiosis from metaphase I to metaphase III

The kinetics of spindle and chromosomes during bovine oocyte meiosis from meiosis I to meiosis III is described. The results of this study showed that (1) oocytes began to extrude the first polar body (Pb1) at the early anaphase I stage and the Pb1 totally separated from the mother cell only when oocytes reach the MII stage; (2) the morphology of the spindle changed from barrel-shaped at the metaphase stage to cylinder-shaped at early anaphase, and then to a thin, long triangle-shaped cone at late anaphase and telophase stages; (3) chromosome morphology went from an individual visible stage at metaphase to a less defined chromatin state during anaphase and telophase stages, and then back to visible individual chromosomes at the next metaphase; (4) chromatin that connected with the floor of the cone became the polar bodies and expelled, and almost all of the microtubules (MTs) and microfilaments (MFs) composing the spindles moved towards and contributed to the polar bodies; and (5) the size of the metaphase I (MI) spindle was larger than the metaphase II (MII) and metaphase III (MIII) spindles. The MII spindle, however, is more barrel-shaped than the MI spindle. This study suggests that spindle MTs and MFs during bovine oocyte meiosis are asymmetrically divided into the polar bodies.     

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.     

Monastral bipolar spindles in meiosis II of male Trichosia pubescens (Sciaridae): early stages of spindle formation and chromosome orientation.

The metaphase spindle of male meiosis II in fungus gnats (Sciaridae) is one example of naturally occurring monastral bipolar spindles. To gain further insights into how the bipolar spindle is formed in the presence of only one polar center, prometaphase of male meiosis II was investigated in the sciarid Trichosia pubescens by means of anti-tubulin immunofluorescence, DAPI chromosome staining, and electron microscopy of ultrathin serial sections. The first step in spindle formation after interkinesis seems to be the organization of an astral half-spindle, probably by MTOC activity of the astral region. With the exception of the non-disjunctional X chromosome, which always lies close to the aster, the chromosomes are found to occupy various positions with respect to the astral region, revealing different orientations of their chromatid kinetochores. It was observed that some of the mal-oriented kinetochores are associated with microtubules that, due to their orientation perpendicular to the spindle axis, are unlikely to originate from the astral region. Therefore, these mal-oriented microtubules are taken as an indication of a dispersed MTOC activity near the chromosomes or at kinetochores. According to recent models of chromosome-induced spindle self-organization [e.g., Merdes et al., 1997: J. Cell Biol. 138:953-956], they could be responsible for the formation of the other (anastral) half-spindle and for amphitelic (bipolar) orientation of the chromosomes.