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.     

Sequential analysis of synaptonemal complexes in the repopulating spermatocytes of Rattus norvegicus after restricting the germ cell population to spermatogonia by gossypol treatment

Synaptonemal complexes of the repopulating spermatocytes of male rats were analyzed day by day using silver-stained surface spread nuclei between 8 and 25 days after restricting the germ cell population to spermatogonia by treatment of gossypol acetic acid at 30 mg/kg body weight/day for 70 days. The method allowed sequential analysis of male meiotic prophase on successive days after the last day of treatment. The leptotene cells appeared on day 11 and were characterized by a network of lateral elements and large nucleolar bodies in a diffuse mass. On day 13 the unpaired lateral elements and short stretches of synaptonemal complexes characteristic for zygotene could be seen. Pachytene nuclei showing 20 autosomal synaptonemal complexes and XY axes appeared on day 15. The diplotene cells were defined on day 22 by the loss of a complete synaptonemal complex set and by the appearance of disjoined lateral elements and persistent segments of synaptonemal complexes.     

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.     

Differentiation of the synaptonemal complex and the kinetochore in Locusta spermatocytes studied by whole mount electron microscopy

When Locusta migratoria spermatocytes are surface-spread on various salines, the axial element of leptotene and zygotene chromosomes, and the synaptonemal complex of pachytene chromosomes are well-preserved, although, in most instances, virtually denuded of chromatin. A complex association of chromosome ends with the nuclear membrane is apparent as early as leptotene, and, as pairing is initiated, the nuclear attachment points of the partner half-bivalents fuse, apparently incorporating additional membrane material between them. The meiotic kinetochore originates in association with the axial element during early prophase, and prior to synaptonemal complex formation and chromosome condensation.     

Ultrastructural study on the meiotic prophase nucleus of rat oocytes.

Rat oocytes in the meiotic prophase are studied by means of classical techniques of electron microscopy, preferential staining methods for DNA and RNA and specific enzymatic hydrolysis. The axial cores in leptotene and the lateral arms in the pachytene synaptonemal complex are composed by fibrils that keep a positive contrast after the application of the ethylenediaminetetraacetic acid staining method. They disappear with RNAse treatment, which reveals the presence of chromatin fibrils in the zone occupied by the cores. Preferential staining for DNA corroborates this evidence. Medial arm and lateral-medial fibrils are formed by ribonucleoproteic filaments that form bridges between pairing homologues in the zygotene. In the advanced pachytene stage, the RNA becomes scarce in these structures. No DNA can be detected either in the lateral-medial fibrils or in the medial arm. During diplotene the synaptonemal complex loses its individually and the synaptic space becomes wider and irregular. At the same time, loss of chromatin and a large increase of RNA-containing particles occur. These processes lead to the typical interphasic arrangement of nuclear components seen in the dictyate stage.     

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.     

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.     

Intranuclear fibrillar material in cereal pollen mother cells

Ultrastructural studies of cereal anthers found intranuclear bundles of microfilaments in pollen mother cells (PMCs) but not elsewhere. The ultrastructure, distribution, and behaviour of this fibrillar material (FM) are described. FM was seen in all 19 genotypes studied comprising Aegilops, Triticum, Secale, Hordeum and Avena species, which together included haploid, diploid and allo-and autopolyploid, and natural and synthetic polyploid examples. Detailed studies in diploid S. cereale, and hexaploid T. aestivum and Triticale showed that FM was present in PMC nuclei during premeiotic interphase, leptotene and zygotene but not at pachytene and later meiotic stages. Moreover, it was most abundant at late premeiotic interphase in T. aestivum, and at leptotene in S. cereale and Triticale, when it occurred in up to 100% of sampled PMC nuclei in an anther. Although FM and synaptonemal complex (SC) occurred together in some PMC nuclei at later stages, FM was present long before SC, and reached its peak of abundance before SC did. Bundles of FM often formed links at their ends between either two masses of chromatin, or more rarely, between chromatin and the nuclear membrane. Individual bundles of FM varied in length but showed roughly similar ranges of lengths and widths in these three species. They were up to about 0.2 m in diameter and about 3 m in length, equivalent to about 20% of the maximum diameter of the nuclei containing them. Reconstructions of PMC nuclei indicated that FM was never associated with centromeres but was sometimes, and perhaps usually, associated with telomeric or sub-telomeric chromosome segments.The function of FM is unknown but its possible role is discussed in relation to (1) previously described intranuclear inclusions in meiocytes and (2) the cytogenetics and developmental behaviour of meiotic nuclei in the wheat comparium. As FM was a constant and characteristic structural component of PMC nuclei, its presence is probably of functional significance to the meiotic process. If so, it may function before, and over greater distances, than SC in establishing or maintaining the coorientation of chromosomes prerequisite for normal chromosome pairing. As FM was most abundant at stages when major chromosome movements occur, yet its distribution was non-centromeric, it is suggested that it may function in the attachment and movement of telomeres at the nuclear membrane formed after premeiotic mitosis. The possibility that a bundle of FM normally links corresponding sites on two homologues is considered.     

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 behaviour of chromosomal axes during diplotene in mouse spermatocytes

The fate of the synaptonemal complex and its elements after pachytene has been studied by serial sectioning of diplotene nuclei in mouse spermatocytes. The lateral elements of the synaptonemal complex separate from each other during diplotene, and they form single axes, 300 Å wide, surrounded by chromatin fibrils. The single axes are continuous and end on the nuclear membrane by two different ends: the basal knob and the simple end. The single axes do not cross-over each other, but they remain approached at the convergence regions. In these regions a modified piece of synaptonemal complex is found. This piece changes into a chromatin bridge during diplotene. It has been inferred that the convergence regions represent chiasmata and that the single axes do not represent axial structures of chromatids.     

Dense bodies in silver-stained spermatocytes of the chinese hamster: behavior and cytochemical nature

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, thread-like axes seen at midpachytene, and (3) multistranded, branched, and anastomosed axes seen at late pachytene.Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body.Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.     

Chromatin organization during meiotic prophase ofBombyx mori

Chromatin organization during the early stages of male meiotic prophase inBombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200–300 Å in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5–25 of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.     

Effects of elevated temperature on meiotic chromosome synapsis in Allium ursinum

Synaptonemal complex (SC) formation in microsporocytes of Allium ursinum is severely affected by exposure of plants to 35° C for 30 h or longer. In spread preparations made from fresh and freeze-conserved material it was found that a high proportion of meiocytes is arrested at leptotene and shows no synapsis at all. In another group of nuclei synaptonemal polycomplex-like structures do occur between converging axial elements at presumed rudimentary SC initiation sites. Axial elements are virtually always thickened at these sites which seem to involve primarily heterologous chromosomes. A third situation is seen in nuclei where two or more lateral elements are engaged in the formation of longer stretches of aberrant SCs. These feature surplus material filling the central space. It may be assumed that this abnormal condition precludes crossing over and hence may be one of several ways by which elevated temperatures cause the chiasma reduction described here for A. ursinum and reported for several other organisms in the literature.     

Electronmicroscopic ammoniacal silver reaction for the synaptonemal complex of the mouse

The postformalin ammoniacal silver reaction (ASR) was applied to the synaptonemal complex in the primary spermatocyte prophase of the mouse and visualised with the electron microscope. The ASR deposits thus seen were localized on or near the lateral elements of the complex and on the chromatin of the homologous chromosomes. The central region of the synaptonemal complex was devoid of ASR deposit.     

Synaptonemal complex stability depends on repressive histone marks of the lateral element-associated repeat sequences

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.     

Abnormal meiosis in bisporic mutants of white button mushroom Agaricus bisporus (Lange) Imbach

A formerly developed method of obtaining spread preparations of mushroom basidial nuclei was applied to study of meiotic prophase I in bisporic white button mushroom (Agaricus bisporus) strains. Meiotic recombination and assemblage of axial structures (axial elements and synaptonemal complexes) of chromosomes in meiotic prophase I are interrelated. It is known that the frequency of meiotic recombination is reduced in the bisporic A. bisporus variety. We showed that formation of axial structures of meiotic chromosomes in bisporic strains of this mushroom was disrupted. The phenotypes of disruptions in spread prophase nuclei are diverse. In leptotene and early zygotene, many nuclei contain abnormal, often short, and, as a rule, few chromosomal axial elements. The abnormalities in the formation of synaptonemal complexes at the zygotene-diplotene stage are of the same kind and even more pronounced. We discovered an important feature of meiosis in A. bisporus associated with fruit-body morphogenesis. Meiosis starting in basidia (meiocytes) of young closed fruit bodies is accompanied by disruption of chromatin condensation in prophase I and, probably, is arrested. After indusium breakage, the course of meiosis normalizes. Preparations with clearly observable chromosomal axial structures can be obtained only at this stage of fruit-body development.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.