Whole mount electron microscopy of meiotic chromosomes and the synaptonemal complex

The mechanism by which homologous chromosomes pair and crossover has been a major unsolved problem in genetics. Thin section electron microscopy of the synaptonemal complex has not provided enough details to allow any significant insight into this problem. Whole mount preparations of the testis of mice, quail, crayfish, and frogs provided a striking improvement in visualization of the morphological features of meiotic chromosomes. These studies, when combined with the use of deoxyribonuclease and trypsin allowed the following conclusions. 1. The synaptonemal complex (lateral and central elements with connecting L-C fibers) is composed of protein. 2. Contrary to common speculation the central element is not the pairing surface of homologous chromosomes. 3. The L-C fibers, averaging 75–100 Å in width, extend from the lateral elements and meet to form the central element which is usually composed of four fibers. 4. During leptotene, homologous axial elements, although unpaired for most of their length, attach next to each other at the nuclear membrane. 5. Short segments of the chromatin fibers attach to the lateral elements. These points of attachment are clustered, producing the chromomeres seen by light microscopy. 6. The chromatin fibers extend out from the lateral element as loops. Lampbrush chromosomes are thus not restricted to oogenesis but are common to all meiotic chromosomes.Since the morphological features of the central element of the synaptonemal complex persist despite extensive deoxyribonuclease digestion, pairing is perhaps best visualized as a two-step process consisting of a) chromosomal pairing during which the proteinaceous synaptonemal complex pulls homologous chromosomes into approximate association with each other, and b) molecular pairing, which probably takes place in the area around the synaptonemal complex.Supported by NIH Grants GM-15886 and C-2568, and The Charles and Henrietta Detoy Research Fellowship.     

The meiotic prophase in Bombyx mori females analyzed by three-dimensional reconstructions of synaptonemal complexes

Serial sectioning followed by three dimensional reconstruction of lateral components of the synaptonemal complex have been used to follow chromosome pairing during the prophase of the achiasmatic meiotic division in the silkworm, Bombyx mori. During leptotene and early zygotene, the lateral components become attached to the nuclear envelope at a specific region, thus forming a chromosome bouquet. The attachment of lateral components to the nuclear envelope precedes the completion of the components between their attachment points. Synapsis and synaptonemal complex formation start during the period of lateral component organization in the individual nucleus. Telomeric movements on the nuclear envelope occur at two stages of the prophase: the chromosome pairing appears to be initiated by an association of unpaired ends of homologous chromosomes, the nature of this primary attraction and recognition being unknown. Secondly, the paired chromosomes become dispersed in the nucleus by shifting of attachment sites of completed synaptonemal complexes at the end of zygotene. This movement is possibly related to a membrane flow occurring during this stage. Membrane material is synthesized at the region of synaptonemal complex attachment. Later, the excess membrane material is shifted to the opposite pole where it protrudes into the lumen of the nuclei thus forming vacuoles. — Two previously undescribed features of chromosome pairing were revealed. In late zygotene, chromosome pairing and synaptonemal complex formation were frequently observed to be delayed or even prevented over a short distance by interlocking of two bivalents, both being attached to the nuclear envelope. Such interlocking of bivalents was not found in pachytene. Secondly, one nucleus was found in which two homologous chromosomes were totally unpaired while the remaining 27 bivalents were completed or in a progressed state of pairing. The lateral components of the two unpaired chromosomes had the same length and were located several microns apart, thus eliminating the possibility of a permanent association of homologous chromosomes before the onset of meiosis in Bombyx mori females. — During pachytene, one of the 8 cells belonging to the syncytial cell cluster characteristic of oogenesis continues the meiotic prophase whereas the remaining 7 cells, the nurse cells, enter a different developmental sequence, finally resulting in their degeneration. The synaptonemal complex of the oocyte develops into a sausage-like structure after pachytene by a deposition of dense material onto the lateral components, thus filling out most of the central region. The diameter of this modified synaptonemal complex reaches at least 300 nm, as compaired to a pachytene width of approximately 130 nm. Also, the length of synaptonemal complexes increases from 212 at zygotene/pachytene to at least 300 at the modified pachytene stage. In nurse cells, synaptonemal complexes are shed from the bivalents shortly after pachytene simultaneously with a condensation of the chromatin. These free synaptonemal complex fragments associate and form various aggregates, either more or less normal looking polycomplexes or various complex figures formed by reorganized synaptonemal complex subunits. Later stages have not been included in the present investigation.     

A Meiotic Chromosomal Core Consisting of Cohesin Complex Proteins Recruits DNA Recombination Proteins and Promotes Synapsis in the Absence of an Axial Element in Mammalian Meiotic Cells

The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.     

Meiosis in Bombyx mori females.

Crossing over is absent in oocytes of the silkworm, Bombyx mori. Synaptonemal complexes are present during pachytene between the paired chromosomes. At leptotene, lateral components of the synaptonemal complex are attached in a bouquet to a limited region of the nuclear envelope. Before completion of lateral components, synaptonemal complex formation begins at the nuclear envelope. With synaptonemal complex formation proceeding from both ends bivalents occasionally become interlocked. After pairing is completed, the bouquet arrangement is dissolved possibly as a result of a flow of the inner membrane of the nuclear envelope thereby separating the telomeres. After the telomeres are released from the nuclear envelope, material is deposited onto the lateral components of the synaptonemal complex. The modified synaptonemal complexes are retained by the bivalents until metaphase I. It is suggested that these modified synaptonemal complexes substitute for chiasmata in order to ensure regular disjunction of homologous chromosomes in the absence of crossing over.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

Formation of cytoplasmic synaptonemal-like polycomplexes at leptotene and normal synaptonemal complexes at zygotene in Ascaris suum male meiosis

A thread-like (more than 70 cm long) testis of Ascaris suum, when examined under the light and electron microscope, reveals the linear succession of meiotic stages. Beginning from, at least, late leptotene, the spermatocytes are synchronous in their development. Thus within each transverse section of the testis all the spermatocytes are in the same stage. The spermatocytes at each stage of prophase I occupies several (4 to 10) cm of the whole testis length. — At leptotene, synaptonemal-like polycomplexes of lateral and central stacked elements are formed in the cytoplasm of spermatocytes. At late leptotene, the polycomplexes are attached to the external nuclear membrane. The polycomplexes disappear at zygotene. Slightly discernable axial cores are observed in the late leptotene chromosomes. The synaptonemal complexes (SCs) are formed at the zygotene stage, their structure being characteristically tripartite. The SCs disappear from the nuclei at the diffuse stage of prophase I. In other organisms completely developed polycomplexes of stacked lateral and central elements were never found during the presynaptic period of meiosis, although single or two parallel layers of aggregated central regions of SC were found in Neottiella meiocytes at the stage prior to chromosome pairing (Westergaard and von Wettstein, 1970, 1972). — First appearance of the polycomplexes in the cytoplasm insetead of the nucleus is also a novel fact. It is concluded that the polycomplexes at leptotene are formed by a self-assembly of the SC molecular material precociously synthesized in the cytoplasm. Two hypotheses regarding possible function and the further fate for leptotene polycomplexes are discussed.     

Silver-Stained accessory structures on human sex chromosomes

Summary Using a combination of silver-staining and light microscopic techniques on human male meiotic preparations, it is feasible to study the morphology and behavior of both autosomal synaptonemal complexes and sex chromosome axes. During leptotene and early zygotene, the X and Y chromosomes are separate; their axes appearing as thin, filamentous structures. During late zygotene/early pachytene, the sex chromosomes come close to each other and a distinct sex vesicle is formed. We confirm the existence of a short synaptonemal complex between the terminal ends of the X and Y chromosomes. In our preparations, a number of accessory structures can be seen along the axes of the sex chromosomes. These structures appear to be similar in morphology to those previously observed in several other mammalian species.     

Some morphological aspects of the synaptonemal complex in higher plants.

The synaptonemal complex is illustrated in electron micrographs from pollen mother cells (p.m.cs) of the following plants: Fritillaria lanceolata, Allium fistulosum, Tulbaghia violacea, Luzula purpurea, Phaedranassa viridiflora and the tulip cultivar Keiserkroon. The possibility that the lateral elements in synaptonemal complexes of plants are tubiform structures is discussed in relation to their fine structure and in the light of a deformity seen in them. An assessment of the evidence suggesting that both lateral and central elements in the complex are ribonucleoprotein structures is made. The effect of brief water treatment on the chromatin and synaptonemal complex at zygotene in p.m.cs of the Phaedranassa is discussed, particularly with reference to two precisely oriented axial strands then seen running between the lateral elements. Examination of stages of premeiotic interphase and early leptotene in p.m.cs of the Fritillaria, revealed that the axial cores laid down at leptotene are formed first in heterochromatic regions, which in this species are locked in chromocentres that persist until pachytene. Further, at leptotene the chromatin in these parts was singularly more decondensed (diffuse) than at any other period, including the premeiotic interphase, subsequent stages of meiosis and mitotic cycle in meristems. It is suggested that the diffuse state of the chromatin in chromocentres at the onset of leptotene, allows the necessary freedom of movement required to promote homologous pairing of the heterochromatic segments. Evidence of such a movement was indicated by a change in position of the nucleoli, which moved from a more central position at early premeiotic interphase to a peripheral one at the onset of leptotene, when they are seen adpressed to the nuclear envelope.     

Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis

Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.     

Centromere Pairing in Early Meiotic Prophase Requires Active Centromeres and Precedes Installation of the Synaptonemal Complex in Maize

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize STRUCTURAL MAINTENANCE OF CHROMOSOMES6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species.     

The fine structure of meiotic chromosome polarization and pairing in Locusta migratoria spermatocytes

At the leptotene stage of meiotic prophase in Locusta spermatocytes (2n=22 telocentric autosomes + X-chromosome), each chromosome forms an axial core. The 44 ends of the autosomal cores are all attached to the nuclear membrane in a small region opposite the two pairs of centrioles of the juxtanuclear mitochondrial mass. At later stages of meiotic prophase, the cores of homologous chromosomes synapse into synaptinemal complexes. Synapsis is initiated near the nuclear membrane, in the centromeric and the non-centromeric ends of the chromosomes. Homologous cores have their attachment points close together and some cores are co-aligned prior to synapsis. At subsequent stages of zygotene, the number of synaptinemal complexes at the membrane increases, while the number of unpaired axial cores diminishes. At pachytene, all 11 bivalents are attached to the membrane at both ends, so that there are 22 synaptinemal complexes at the membrane near the centrioles. Because each bivalent makes a complete loop, the configuration of the classic Bouquet stage is produced. The X-chromosome has a poorly defined single core at pachytene which also attaches to the nuclear membrane. These observations are based on consecutive serial sections (50 to 100) through the centriolar zone of the spermatocytes. Labeling experiments demonstrated that tritiated thymidine was incorporated in the chromatin of young spermatocytes prior to the formation of the axial cores at leptotene. It is concluded that premeiotic DNA synthesis is completed well in advance of pairing of homologous chromosomes, as marked by the formation of synaptinemal complexes.     

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.     

The structure and function of the synaptinemal complex inLilium longiflorum sporocytes

The development of meiotic prophase in pollen mother cells ofLilium longiflorum is presented through photomicrographs of squashes and sections and through electron micrographs of thick and thin sections. Emphasis is placed on the first appearance of axial cores, the participation of axial cores in the formation of synaptinemal complexes, the fine structure of the complex and the fate of the complex at the end of pachytene. It is shown that axial cores are formed in early meiotic prophase chromosomes and that the two axial cores of a set of homologous chromosomes participate in the formation of a synaptinemal complex. It is proposed that the transverse filaments of each axial core meet and interdigitate and so produce the transverse filaments of the complex. It is shown that the complex is axial to the pachytene bivalent and that the association of the complex with chromosomal material is terminated at the end of pachytene. The pairing affinity of the cores in homologous and non-homologous chromosome associations is discussed. The zygotene stage is defined in terms of the occurrence of synaptinemal complexes and the attachment of the nucleolus to the nuclear membrane during this stage is noted.     

The synaptic behaviour of the X and Y chromosomes in the marsupial Monodelphis dimidiata

The pairing behaviour of the X and Y chromosomes of Monodelphis dimidiata was studied with light and electron microscopy. Pairing of the sex chromosomes is delayed with respect to autosome synapsis. Both the X and the minute Y chromosome show an axis attached by its two ends to the nuclear envelope. Synapsis of the sex chromosomes occurs by the joining of the chromatin sheaths that surround the axes and by a small, three-layered structure close to the nuclear envelope. The X and Y chromosomes remain joined to each other during the diffuse stage and diplotene-diakinesis but they do not show a synaptonemal complex. During the diffuse stage a dense plate is formed at the boundary between the X-Y body and the nuclear envelope. During early metaphase a folded sheet is attached to the periphery of the X-Y body. This sheet is formed by a piece of the nuclear envelope carrying the dense plate and it shows transverse fibrils and a central element similar to synaptonemal-complex remains. No evidence of a non-chiasmate segregation mechanism was observed. Polarization of the axial ends of the sex chromosomes is observed after X-Y synapsis. These important departures from the X-Y pairing pattern of eutherian mammals are discussed and assumed to present a special mechanism for holding the minute Y joined to the X chromosome in this marsupial.     

Ultrastructure of the synaptic autosomes and the ZW bivalent in chicken oocytes

Chromosomal axes of chicken oocytes from pre- and post-hatching chickens were analyzed with a microspreading technique for electron microscopy. At leptotene, chromosomal axes begin to be formed as discontinuous, non-polarized axial segments. During zygotene synaptonemal complex (SC) formation begins at the axial ends attached to the nuclear envelope. Polarization of axial ends is nearly simultaneous with the beginning of SC formation. The complete SC set is found at pachytene and it consists of 38 SC's and an unequal SC which has been identified as the ZW pair. This unequal SC is formed by two axes of different length. The Z and W axes represent 6.2% and 4.5% respectively of the combined length of the SC set plus the Z axis. The unpaired segment of the Z axis shortens markedly from early to mid-pachytene and becomes thicker than the lateral elements of SCs. In the paired region the Z axis forms most of the twists around a straighter W axis, suggesting some extent of non-homologous pairing between the Z and W chromosomes in this region. The existence of partial synapsis of the Z and W axes without heteropycnosis of the sex chromosomes is in marked contrast to partial synapsis in the heteropycnotic XY body of mammalian spermatocytes.     

Precocious formation of synaptonemal-like polycomplexes and their subsequent fate in female Ascaris lumbricoides var. suum

During meiotic interphase, before leptotene, synaptonemal-like polycomplexes are seen in the cytoplasm of the Ascaris lumbricoides oocytes and in the communal anucleate rachis. In some females short intranuclear synaptonemal complexes are present briefly at that early stage. The number of extranuclear complexes increases just before leptotene, some are attached to the pores of the nuclear membrane. During zygotene most polycomplexes disappear. At late pachytene they reappear in some females but not in others. The morphology, when first seen, is that of disorganized filamentous bodies, later lateral elements appear among the filaments. The dimensions of the lateral elements of the polycomplexes are variable. In the male the distribution of polycomplexes among the rachis, the cell cytoplasm, and at the nuclear envelope is similar to that observed in the female.These observations confirm the precocious occurrence of synaptonemal-like polycomplexes reported by Bogdanov (1977). Ascaris lumbricoides thus, uniquely, appears to manufacture synaptonemal complex-like material in the communal cytoplasm of the germ cells prior to the time that the full complement of synaptonemal complexes appears in the nucleus.     

The development of nuclear vacuoles during meiosis in plants

Vacuoles formed by the invagination of the inner membrane of the nuclear envelope have been observed during meiotic prophase in a wide range of plants. In the angiosperm Lycopersicon their formation was found to coincide with the completion of synaptonemal complex formation, and this timing is analogous to that observed during this stage in the silkworm Bombyx. The implications of this activity in relation to the process of chromosome movement are discussed. In the gymnosperm Pinus, the heterosporous fern Marsilea and homosporous ferns Pteridium and Dryopteris the formation of nuclear vacuoles begins much earlier, coinciding with the condensation of chromatin during leptotene. They enlarge and become more elaborate as meiosis proceeds, and may eventually become detached from the nuclear envelope. It is therefore thought unlikely that theyfulfil functions connected with chromosome movement in the manner proposed for the silkworm and the tomato. During diplotene/diakinesis they contain electron-opaque granules and fibrils, and the possible origin and significance of this material is discussed.     

The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility

During meiosis, the homologous chromosomes pair and recombine. An evolutionarily conserved protein structure, the synaptonemal complex (SC), is located along the paired meiotic chromosomes. We have studied the function of a structural component in the axial/lateral element of the SC, the synaptonemal complex protein 3 (SCP3). A null mutation in the SCP3 gene was generated, and we noted that homozygous mutant males were sterile due to massive apoptotic cell death during meiotic prophase. The SCP3-deficient male mice failed to form axial/lateral elements and SCs, and the chromosomes in the mutant spermatocytes did not synapse. While the absence of SCP3 affected the nuclear distribution of DNA repair and recombination proteins (Rad51 and RPA), as well as synaptonemal complex protein 1 (SCP1), a residual chromatin organization remained in the mutant meiotic cells.