Development of the synaptonemal complex and the “recombination nodules” during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one fixed and one randomly attached telomeres. The fixed end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of fixed telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.     

The pattern of zygotene and pachytene pairing in allotetraploid Aegilops species sharing the D genome

Chromosome pairing behaviour of the allotetraploid Aegilops species sharing the D genome, Ae. crassa (DDMM), Ae. cylindrica (DDCC) and Ae. ventricosa (DDNN), was analyzed by electron microscopy in surfacespread prophase-I nuclei. Synaptonemal-complex analysis at zygotene and pachytene revealed that synapsis in the allotetraploids was mostly between homologous chromosomes, although a few multivalents were also formed. Only homologous bivalents were observed at metaphase-I. It is concluded that the mechanism controlling bivalent formation in these species acts mainly at zygotene by restricting pairing to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in homoeologous associations. These observations are discussed in relation to mechanisms of diploidization of polyploid meiosis.     

Ultrastructure of meiotic pairing in B chromosomes of Crepis capillaris

The meiotic pairing behaviour of four B isochromosomes of Crepis capillaris was studied by synaptonemal complex (SC) surface spreading of pollen mother cells. The four B chromosomes form a tightly associated group, separate from the standard chromosomes, throughout zygotene and pachytene. All four B chromosomes are also folded around their axis of symmetry, the centromere, and the eight homologous arms are closely aligned from the earliest prophase I stages. A high frequency of multivalent pairing of the four B chromosomes is observed at pachytene, in excess of 90%, mirroring the situation observed at metaphase I but exceeding the frequency expected (76.2%) on the assumption of random pairing among the eight B isochromosome arms with a single distal pairing initiation site per arm. The higher than expected frequency of multivalents is due to the occurrence of multiple pairing initiations along the B isochromosome arms, resulting in high frequencies of pairing partner switches. Pairing of the standard chromosome set is frequently incomplete in the presence of four B chromosomes, and abnormalities of SC structure such as thickening and splitting of axes and lateral elements are also frequently seen. Similarly, B chromosomes show partial pairing failure, the extent of which is correlated with pairing failure in the standard chromosome set. The B chromosomes themselves also show abnormalities of SC structure. Both standard and B chromosomes show non-homologous foldback pairing of regions that have failed to pair homologously.by D. Schweizer     

Synaptonemal complexes with modified lateral elements in Sordaria humana: development of and relationship to the “recombination nodules”

Meiotic prophase in Sordaria humana has been analyzed by three-dimensional reconstructions of 3 leptotene, 2 zygotene, 10 pachytene and 3 diplotene nuclei. Several notable features emerged. The lateral components of the synaptonemal complexes (SC) are hollow tubes which show dilations of variable sizes from late leptotene to early diplotene. These bulges occur before pairing. Their number decreases as soon as the SC are completely formed, but their mean size increases. Bulges can be present in all parts of the lateral components including telomeres and nucleolar organizer region, but their distribution along bivalents is not random. The remarkably uniform width of the SC central region, normally observed in other species is not observed in S. humana. Although as a general rule the bulges rarely affect the homologous components at identical sites, they often either fill or partially cover the central region. The recombination nodules are not clearly connected with the bulges. This work provides also additional insight into the development of both SC and the nodules. At late leptotene, the homologues are aligned before SC formation. One case of interlocking has been observed at early pachytene. Nodules are present from zygotene to diplotene. They are not evenly distributed along the bivalents during pachytene. The mean number of nodules, constant from late pachytene to diplotene, is equal to the mean number of chiasmata.     

Pachytene pairing and metaphase I configurations in a tetraploid somatic Lycopersicon esculentum x L. peruvianum hybrid.

In the tetraploid somatic hybrid between the diploid Lycopersicon species L. esculentum (tomato) and L. peruvianum, synaptonemal complexes formed quadrivalents in 73 of the 120 sets of four chromosomes (60.8%) in 10 cells studied in detail at pachytene. Of these, 43 had one pairing partner exchange, 22 had two, and 8 had three, very close to a Poisson distribution. The points of pairing partner exchange were concentrated at the middle of the two arms. The frequency per arm corresponded with physical arm length. There was a sharp drop around the centromere, and pericentric heterochromatin had a slightly lower probability of being involved in pairing partner exchange than euchromatin. The chromosomes align before pairing and there are several points of pairing initiation, with concentrations at or near the ends and the centromere. From zygotene to late pachytene the quadrivalent frequency decreased considerably. At late pachytene it was lower than expected with the observed high frequency of pairing partner exchange. Pairing affinity between species was only slightly lower than affinity within species, in spite of considerable genetic differentiation. The frequency of recombination nodules increased from early to late zygotene and then decreased strongly to full pachytene. There is a highly significant negative correlation between percent pairing and SC length. At metaphase I the frequency of quadrivalents was 0.444, and branched quadrivalents were rare, probably caused by interference and restriction of chiasma formation to distal euchromatin. Metaphase I quadrivalent frequency is a relatively good indication of pairing affinity in this material.     

SUPERNUMERARY CHROMOSOMES IN TRADESCANTIA II. EFFECT OF COLCHICINE ON MEIOTIC ISOCHROMOSOME CONFIGURATIONS

The effect of colchicine on meiotic pairing and configuration frequencies of three homologous isosupernumerary chromosomes was investigated. In the absence of colchicine, the three isochromosomes displayed a high degree of interchromosomal pairing and chiasma formation. As a consequence, a high frequency of bivalents and trivalents were observed at diakinesis-metaphase I. The unique structure of isochromosomes enables them to pair intrachromosomally (i.e., foldback pairing) yet the preferential occurrence of interchromosomal pairing suggests that all six arms of the three isosupernumerary chromosomes were in close association prior to or upon initiation of synapsis. Supernumerary chromosomes in microsporocytes treated during presynapsis or early synapsis with colchicine exhibited a significant reduction (P < 0.001) in the number of bivalents and trivalents at diakinesis. However, there was no reduction in overall chiasma frequency among supernumeraries due to the induction of increased intrachromosomal pairing and chiasma formation. A colchicine-sensitive association or alignment of homologues preceding effective pairing has been demonstrated in standard chromosomes of a number of plant species. This study provides the first evidence to indicate that at least certain supernumerary chromosomes may display presynaptic association as well. The results also support the strongly held contention that colchicine is not directly preventing or inhibiting the actual formation of chiasmata, since no reduction in chiasma frequency was observed in the isochromosomes.     

Synapsis in Robertsonian heterozygotes and homozygotes of Dichroplus pratensis (Melanoplinae, Acrididae) and its relationship with chiasma patterns

Dichroplus pratensis has a complex system of Robertsonian rearrangements with central-marginal distribution; marginal populations are standard telocentric. Standard bivalents show a proximal-distal chiasma pattern in both sexes. In Robertsonian individuals a redistribution of chiasmata occurs: proximal chiasmata are suppressed in fusion trivalents and bivalents which usually display a single distal chiasma per chromosome arm. In this paper we studied the synaptic patterns of homologous chromosomes at prophase I of different Robertsonian status in order to find a mechanistic explanation for the observed phenomenon of redistribution of chiasmata. Synaptonemal complexes of males with different karyotypes were analysed by transmission electron microscopy in surface-spread preparations. The study of zygotene and early pachytene nuclei revealed that in the former, pericentromeric regions are the last to synapse in Robertsonian trivalents and bivalents and normally remain asynaptic at pachytene in the case of trivalents, but complete pairing in bivalents. Telocentric (standard) bivalents usually show complete synapsis at pachytene, but different degrees of interstitial asynapsis during zygotene, suggesting that synapsis starts in opposite (centromeric and distal) ends. The sequential nature of synapsis in the three types of configuration is directly related to their patterns of chiasma localisation at diplotene-metaphase I, and strongly supports our previous idea that Rb fusions instantly produce a redistribution of chiasmata towards chromosome ends by reducing the early pairing regions (which pair first, remain paired longer and thus would have a higher probability of forming chiasmata) from four to two (independently of the heterozygous or homozygous status of the fusion). Pericentromeric regions would pair the last, thus chiasma formation is strongly reduced in these areas contrary to what occurs in telocentric bivalents.     

The assembly of the synaptinemal complex.

The assembly of the synaptinemal complex in the ascomycete Neottiella was studied by three-dimensional reconstruction of a late zygotene nucleus. A single banded lateral component is formed between the two sister chromatids of each homologous chromosome prior to their pairing. The central regions are pre-assembled in organized form in folds of the granular part of the nucleolus and then converted into an amorphous transport form. The latter appears to move through the nucleoplasm to sites between the lateral components of synapsing homologous chromosomes. The central region material is reorganized into blocks with a recognizable central component and attached to one lateral component. The last step in the completion of the synaptinemal complex is the association of the free surface of the organized central region with the corresponding segment of the homologous lateral component. The findings are discussed in relation to mechanisms of chromosome pairing and chiasma formation.     

Chromosome pairing in the allotetraploid Aegilops biuncialis and a triploid intergeneric hybrid.

Chromosome pairing behaviour of the natural allotetraploid Aegilops biuncialis (genome UUMM) and a triploid hybrid Ae. biuncialis x Secale cereale (genome UMR) was analyzed by electron microscopy in surface-spread prophase I nuclei. Synaptonemal-complex analysis at zygotene and pachytene revealed that synapsis in the allotetraploid was mostly between homologous chromosomes, although a few quadrivalents were also formed. Only homologous bivalents were observed at metaphase I. In contrast, homoeologous and heterologous chromosome associations were common at prophase I and metaphase I of the triploid hybrid. It is concluded that the mechanism controlling bivalent formation in Ae. biuncialis acts mainly at zygotene by restricting pairing to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in the homoeologous associations. In the hybrid the mechanism fails at both stages. Key words : Aegilops biuncialis, allotetraploid, intergeneric hybrid, pairing control, synaptonemal complex.     

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.     

Identification of a structural protein component of rat synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes.     

The synaptic behaviour of the wild forms of Triticum turgidum and T. timopheevii.

Different wild allopolyploid species of Triticeae show extensive bivalent formation at zygotene while a considerable number of multivalents is present in cultivated polyploid wheats. To study the chromosome behaviour at early meiotic stages in wild forms of tetraploid wheats Triticum turgidum and T timopheevii (2n = 4x = 28) we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late zygotene and at pachytene of wild accessions of these species. The mean number of synaptonemal complex (SC) bivalents at mid-zygotene ranged from 12.22 to 13.14 among the accessions studied indicating a strong restriction of synapsis initiation to homologous chromosomes. The mean of bivalents increased at pachytene because of the transformation of multivalents into bivalents. Ring bivalents observed at metaphase I support that SC bivalents were formed by homologous chromosomes. The average values of SC bivalents at mid-zygotene in the wild forms are much higher than the average values observed in the cultivated tetraploid wheats but similar to that of a mutant line of T turgidum with a duplication that includes Ph1, the major homoeologous pairing suppressor locus. These results suggest that the efficiency of the mechanism operating in the homologous recognition for synapsis is higher in wild wheat populations than in cultivated varieties. Apparently, a relatively detrimental modification of the pairing regulating genetic system accompanied the domestication of the wild wheat forms.     

Synaptonemal complex spreading in Allium

A surface-spreading technique for synaptonemal complexes was applied to triploid Allium sphaerocephalon L. (Liliaceae). In early pachytene two of the three axial elements of each set of three homologues are synapsed, the third is intimately aligned with and accompanies them throughout their whole length. The unsynapsed axis is attached to the synaptonemal complex of the other 2 at up to 50 association sites per trivalent. The distribution of these sites within the trivalents is not even; they are under-represented in the proximal regions. From nought to eight switches (pairing partner exchanges), where the accompanying axis joins in synapsis in exchange for one of the two other strands, occur per trivalent. Very often the telomeres of the aligned axes are attached to their synapsed counterparts by dense spherules, which makes this type of association different from the interstitial ones. Frequently the unsynapsed axes show a double structure along short distances. In late pachytene the intercalary associations are abolished, allowing the unsynapsed axes to engage in various types of non-homologous pairing. Since the association sites involve homologous chromosomes and are less abundant in the pericentric regions (which are usually the last to synapse), it is conceivable that similar structures are responsible for the pre-synaptic alignment of homologues and provide the initiation sites for synaptonemal complex formation in diploids.     

Nodules associated with axial cores and synaptonemal complexes during zygotene in Psilotum nudum

Two-dimensional spreads of synaptonemal complexes (SCs) from the lower vascular plant Psilotum nudum were examined after staining with uranyl acetate-lead citrate (UP). Staining with UP allows visualization of lateral elements/axial cores (ACs), central elements, kinetochores, and nodules. Numerous darkly stained nodules were associated with forming SCs. In addition to nodules found on the central element of SC segments, other nodules were found at points of convergence between two adjacent ACs. Of these latter nodules, some were obviously associated with a fiber that connected adjacent ACs. No central element material was visible between the ACs, and the nodule complex appeared to be the only structure holding the ACs together. Although the function(s) of nodules during zygotene is unknown, the presence of a nodule-fiber complex that connects adjacent ACs before central element formation suggests that at least some of the nodules may be involved in synaptic initiation.     

OsSPO11-1 is essential for both homologous chromosome pairing and crossover formation in rice

Spo11 is a homolog of a subunit of archaebacterial topoisomerase, which catalyzes DNA double-strand breaks and initiates homologous chromosome recombination. In the present study, we silenced the SPO11-1 gene in rice (Oryza sativa) using RNAi. Rice plants with loss-of-function of OsSPO11-1 have no apparent growth defects during vegetative development, but homologous chromosome pairing and recombination are significantly obstructed. Telomeres can be assembled as bouquet during the zygotene stage of the OsSPO11-1-deficient plants, just as that in wild type. Although the two axial-associated proteins, REC8 and PAIR2, are loaded onto the chromosomes, the depletion of PAIR2 from the chromosomes is much later than in wild type. The central element of the synaptonemal complex (SC), ZEP1, does not load onto the chromosomes normally, implying that SC formation is disturbed severely. The crossover protein, MER3, isn't efficiently assembled onto chromosomes and the lack of bivalent suggests that crossovers are also affected in the absence of OsSPO11-1. Thus, OsSPO11-1 is essential for both homologous chromosomes pairing and crossover formation during meiosis in rice.     

Random homologous pairing and incomplete sister chromatid alignment are common in angiosperm interphase nuclei

The chromosome arrangement in interphase nuclei is of growing interest, e.g., the spatial vicinity of homologous sequences is decisive for efficient repair of DNA damage by homologous recombination, and close alignment of sister chromatids is considered as a prerequisite for their bipolar orientation and subsequent segregation during nuclear division. To study the degree of homologous pairing and of sister chromatid alignment in plants, we applied fluorescent in situ hybridisation with specific bacterial artificial chromosome inserts to interphase nuclei. Previously we found in Arabidopsis thaliana and in A. lyrata positional homologous pairing at random, and, except for centromere regions, sister chromatids were frequently not aligned. To test whether these features are typical for higher plants or depend on genome size, chromosome organisation and/or phylogenetic affiliation, we investigated distinct individual loci in other species. The positional pairing of these loci was mainly random. The highest frequency of sister alignment (in >93% of homologues) was found for centromeres, some rDNA and a few other high copy loci. Apparently, somatic homologous pairing is not a typical feature of angiosperms, and sister chromatid aligment is not obligatory along chromosome arms. Thus, the high frequency of chromatid exchanges at homologous positions after mutagen treatment needs another explanation than regular somatic pairing of homologues (possibly an active search of damaged sites for homology). For sister chromatid exchanges a continuous sister chromatid alignment is not required. For correct segregation, permanent alignment of sister centromeres is sufficient.     

Maize meiotic mutants with improper or non-homologous synapsis due to problems in pairing or synaptonemal complex formation

During meiotic prophase homologous chromosomes find each other and pair. Then they synapse, as the linear protein core (axial element or lateral element) of each homologous chromosome is joined together by a transverse central element, forming the tripartite synaptonemal complex (SC). Ten uncloned Zea mays mutants in our collection were surveyed by transmission electron microscopy by making silver-stained spreads of SCs to identify mutants with non-homologous synapsis or improper synapsis. To analyse the mutants further, zyp1, the maize orthologue of the Arabidopsis central element component ZYP1 was cloned and an antibody was made against it. Using antibodies against ZYP1 and the lateral element components AFD1 and ASY1, it was found that most mutants form normal SCs but are defective in pairing. The large number of non-homologous synapsis mutants defective in pairing illustrates that synapsis and pairing can be uncoupled. Of the ten mutants studied, only dsy2 undergoes normal homologous chromosome recognition needed for homologous pairing. The dsy2 mutation fails to maintain the SC. ZYP1 elongation is blocked at zygotene, and only dots of ZYP1 are seen at prophase I. Another mutant, mei*N2415 showed incomplete but homologous synapsis and ASY1 and AFD1 have a normal distribution. Although installation of ZYP1 is initiated at zygotene, its progression is slowed down and not completed by pachytene in some cells and ZYP1 is not retained on pachytene chromosomes. The mutants described here are now available through the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu/).     

The initiation of homologous chromosome synapsis in mouse fetal oocytes is not directly driven by centromere and telomere clustering in the bouquet

We investigated the behaviour of centromeres and distal telomeres during the initial phases of female meiosis in mice. In particular, we wished to determine whether clustering of centromeres and telomeres (bouquet formation) played the same crucial role in homologous chromosome pairing in female meiosis as it does in the male. We found that synapsis (intimate homologous chromosome pairing) is most frequently initiated in the interstitial regions of homologous chromosomes, apparently ahead of the distal regions. The proximal ends of the chromosomes appear to be disfavoured for synaptic initiation. Moreover, initiation of synapsis occurred in oocytes that showed little or no evidence of bouquet formation. A bouquet was present in a substantial proportion of cells at mid to late zygotene, and was still present in some pachytene oocytes. This pattern of bouquet formation and pairing initiation is in stark contrast to that previously described in the male mouse. We propose that although dynamic movements of centromeres and telomeres to form clusters may facilitate alignment of homologues or homologous chromosome segments during zygotene, in the female mouse positional control of synaptic initiation is dependent on some other mechanism.     

Synaptonemal complex antigen location and conservation

The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Defective pairing and synaptonemal complex formation in a Sordaria mutant (spo44) with a translocated segment of the nucleolar organizer

The recessive meiotic mutant spo44 of Sordaria macrospora, with 90% ascospore abortion, exhibits striking effects on recombination (67% decrease), irregular segregation of the almost unpaired homologues, and a decrease in chiasma frequency in the few cases where bivalents are formed. Three-dimensional reconstructions of ten prophase nuclei indicate that pairing, as judged by the absence of fully formed synaptonemal complexes (SC), is not achieved although lateral elements (LE) assemble. The pairing failure is attributable to defects in the alignment of homologous chromosomes. The leptotene alignment seen in the wild type before SC formation was not observed in the spo44 nuclei. Dense material, considered to be precursor of SC central elements, was found scattered among the LE in two nuclei. The behaviour of spo44 substantiates the hypothesis that chromosome matching and SC formation are separable events. — The total length of the LE in the mutant is the same as in the wild type, but due to variable numbers and length of the individual LE, homologues cannot be lined up. Light microscopic observations indicate that the irregular length and number of LE is due to extensive chromosome breakage. The wild-type function corresponding to spo44 is required for both LE integrity and chromosome matching. Reconstructions of heterozygous nuclei reveal the presence of a supernumerary nucleolar organizer in one arm of chromosome 7. It is suggested that rDNA has been inserted into a gene whose function is involved in pairing or into a controlling sequence that interacts with the pairing process.