Three dimensional reconstruction of the X-Y pair during pachytene in the rat (Rattus norvegicus)

The ultrastructure of the X-Y pair from rat spermatocytes has been reconstructed sterically by the study of serial sections. The X-Y pair of the rat at early pachytene contains two dense cores, a long and a short one, which form a synaptonemal complex 1.7 long at their common end. The long core (10.6 ) and the short core (4.5 ) correspond to X and Y, respectively. There is no RNA histochemically detectable in the X-Y pair. Nucleoli are independent of the X-Y pair. A low number of cells show nucleoli very near the X-Y pair but no continuity exists between these structures.     

Localized chiasmata and meiotic nodules in the tetraploid onion Allium porrum.

Allium porrum L. (cultivated leek) (2n = 4x = 32) is a fertile tetraploid that forms bivalents with pericentric chiasmata at metaphase I. To investigate the basis of this unusual behavior for a tetraploid, we describe the karyotype, axial cores, synaptonemal complexes (SCs), and meiotic nodules of A. porrum. The karyotype appears to be autotetraploid. This conclusion is also supported by presynaptic alignment of axial cores in groups of four and partner trades between pairs of SCs. Numerous early nodules are distributed all along axial cores and SCs during zygonema, but they are lost by late zygonema - early pachynema. Late (recombination) nodules (RNs) are present on SCs near kinetochores throughout the remainder of pachynema. This pattern of RNs corresponds to the pattern of pericentric chiasmata. Pachytene quadrivalents usually are resolved into bivalents because partner trades between SC lateral elements rarely occur between RNs on the same segment of SC. Thus, the patterns of crossing-over and partner trades promote balanced disjunction and high fertility in autotetraploid A. porrum. Rare quadrivalents observed at metaphase I must be due to infrequent partner trades between RNs. Polycomplexes, unusual in their number and size, were observed during zygonema. Key words : synaptonemal complex, recombination nodules, localized chiasmata, polycomplex, Allium porrum.     

Synaptonemal complex analysis of mole rats (Spalax ehrenbergi): unusual polymorphisms of chromosome 1

Two unusual structural polymorphisms in the largest chromosomal pair of the Israeli mole rat, Spalax ehrenbergi, were analyzed from surface-spread and silver-stained preparations of synaptonemal complexes. A C-band negative polymorphism for the length of the 1p arm was visible as axial length differences during late zygonema and early pachynema. This region underwent synaptic adjustment resulting in a fully paired, mid-pachytene synaptonemal complex with equalized axial lengths. The somatically variable and nonargentophilic secondary constriction in the 1q arm was evident as a distinct silver-stained thickening along the synaptonemal complex. Presence of this structure on the synaptonemal complex varied both among individuals and among cells within individuals. The intraindividual variation of this region is hypothesized to represent differential biochemical activity with its cellular visualization being regulated in a manner similar to that of nucleolus organizer regions.     

Silver staining two types of meiotic nodules.

We have developed a reliable method for silver staining nodules on synaptonemal complexes (SCs) of tomato (Lycopersicon esculentum). This technique involves hypotonically bursting primary microsporocytes, fixing SC spreads with paraformaldehyde, and incubating the spreads at 40 degrees C in a 33% aqueous silver nitrate solution covered with nylon mesh. When tomato SCs were stained by this method, nodules were observed with the same distribution and frequency as nodules stained with uranyl acetate and lead citrate. Incubation in silver nitrate at higher temperatures caused the loss of some or all nodules. The pattern of loss suggests that two types of nodules coexist during late zygonema and early pachynema and that one type becomes the late nodules of mid-pachynema through early diplonema.     

An electron microscopic study of synaptonemal complex formation at zygotene in rye

A spreading technique was used to allow ultrastructural analysis of seventeen zygotene nuclei of rye (Secale cereale). Twenty pachytene nuclei were also examined. Lateral element lengths of the haploid complements decreased from 742 m at the beginning of zygotene to 451 m at the end of zygotene. Variation in pachytene synaptonemal complex lengths was also noted. Zygotene synaptonemal complex formation in rye is characterised by: (1) existence of a bouquet, with telomeric pairing initiation earliest; (2) multiple sites of initiation in each bivalent (maximum of 76 synaptonemal complex segments seen in one nucleus); (3) the potential number of pairing initiation sites may be higher (the average spacing of 4.42 m would allow approximately 160 sites per nucleus); (4) new pairing initiations occur almost until the end of zygotene; (5) initiation of new synaptonemal complexes and extension of existing synaptonemal complexes occur simultaneously. A simple zipping up of a few initiation sites is not the case in rye. Pairing in different bivalents of a nucleus is not completely synchronised, and the NOR in particular is often late to pair. Interlocking of lateral elements and synaptonemal complexes may lead to delayed completion of pairing in portions of bivalents, but interlocks are ultimately resolved. This resolution may involve breakage and rejoining of lateral elements.     

Two-dimensional spreads of synaptonemal complexes from solanaceous plants

By using serial sectioning and a new hypotonic bursting technique on primary microsporocytes of tomato (Lycopersicon esculentum), relatively large numbers of recombination nodules (RNs) are observed on the synaptonemal complexes forming during zygonema. In pachynema most, but not all, of these RNs are lost. If RNs represent sites of potential crossing over during zygonema and sites of actual crossing over during late pachynema, the observed temporal and spatial distribution of RNs may provide answers for some classic cytogenetic questions such as: how is at least one crossover per bivalent assured? How are crossovers localized? What is the basis for positive chiasma interference?     

X-Y chromosome univalency in the testes of hyperthermic mice: II. Light and electron microscopic analysis of synaptonemal complexes

Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.     

Synaptonemal complex analysis of spermatocytes of Talpa occidentalis (Insectivora, Mammalia): autosomal synapsis and substaging of zygonema and pachynema

Spermatocytes from the mole, Talpa occidentalis, a species that includes both XX males and intersexes, were surface-spread and silver-stained to substage meiotic prophase from early zygonema through pachynema. In zygonema, only the Z2 and Z3 substages were found. This stage differed in comparison with such species as the Chinese hamster, laboratory mouse, and deer mouse, which belong to orders other than Insectivora. Pachynema, in which five substages were established (P1-P5), seems to be a more homogeneous stage, and remarkable differences with respect to the above-mentioned species were not found. Synaptic adjustment was demonstrated in X-Y pairing. Nonhomologous pairing was evident at the Y-centromeric region and considered likely in the proximal arm of this chromosome. In addition to sequencing the events taking place during zygonema and pachynema in males from a wild population in which some members show sex reversal, our finding represents the first attempt to substage zygonema and pachynema in an Insectivore species, thus contributing to current knowledge of the nature and degree of variability in the mammalian synaptic process.     

DNA-DEPENDENT FORMATION OF THE SYNAPTINEMAL COMPLEX AT MEIOTIC PROPHASE

Evalaution of microsporocytes cultured during discrete periods of meiotic prophase in the presence of deoxyadenosine, an inhibitor of DNA synthesis, indicate that: (1) late leptonema or early zygonema DNA synthesis is required to initiate the formation of the synaptinemal complex; (2) DNA synthesized during late zygonema is necessary for the disjunction of the paired homologs at diplonema; and (3) DNA synthesis in pachynema is a requisite for normal anaphase II separation of sister chromatids.     

THE THREE-DIMENSIONAL RECONSTRUCTION OF THE XY CHROMOSOMAL PAIR IN HUMAN SPERMATOCYTES

The spatial reconstruction of the XY pair of chromosomes from human spermatocytes has been made by the study of serial sections 1000 A in thickness. The sex pair during zygotene-pachytene forms a condensed mass of chromatin that has two filamentous, electron-opaque cores: the long and the short core. During early pachytene both cores have a common ending region, about 0.4–0.8 µ long. This common end is a synaptonemal complex, and each of the cores forms a lateral element of that complex. The cores become more convoluted during middle pachytene forming "ringlike bodies." Nucleoli from spermatocytes have three distinct regions: (a) granular; (b) dense fibrillar; and (c) clear intermediate. Occasional association of the XY pair and the heteropycnotic "basal knobs" results in apparent association of nucleoli and the sex pair in a minority of cells. The evidence presented is interpreted as a strong support of the hypothesis of homologous regions in the human XY pair.     

Synaptic adjustment in Peromyscus beatae (Rodentia: Cricetidae) heterozygous for interstitial heterochromatin

Chromosomal pairing and chiasma formation were studied two individuals of Peromyscus beatae heterozygous for the presence of a large block of interstitial heterochromatin. Although the modified chromosome was of medium size, analysis of C-banded diakinetic configurations revealed that it was the homolog of one of the smallest autosomes. Analysis of silver stained synaptonemal complexes indicated that synapsis was either unidirectional from initiation at one set of telomeres or was bidirectional from initiation at both sets of telomeres. Each pattern resulted in characteristic heteromorphic pairing configurations (interstitial asynapsis or terminally positioned unpaired segments) in early pachynema. These configurations underwent synaptic adjustment and, by mid-pachynema, the lateral elements of the polymorphic bivalent either appeared typical of homomorphic bivalents or exhibited regional heteropycnosis in one or both axes. Synaptonemal complex data for Peromyscus and many other mammalian species reflect an apparent need for fully paired, linear bivalents prior to the end of pachynema.     

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chiken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis do not appear to be species specific, but intrinsic to the meiotic process.     

Transition from somatic to meiotic pairing and progressional changes of the synaptonemal complex in spermatocytes of Aedes aegypti

Aedes aegypti spermatocytes were reconstructed from electron micrographs. The species has tight somatic pairing of the chromosomes, and there are therefore no classical leptotene and zygotene stages, but rather a gradual transition from somatic pairing to meiotic pairing (= pachytene). The term prepachytene has been used for the transitory stage. The first visible sign of impending meiosis was a reorganization of the chromatin, which resulted in the formation of spaces (synaptic spaces) in the chromatin, about the width of the synaptonemal complexes (SCs). Diffuse material, possibly precursor material for the SC, was present in the spaces. Later short pieces of complex were formed throughout the nucleus. Late prepachytene, pachytene, and diplotene complexes were reconstructed. Each chromosome occupied a separate region of the nucleus. The complexes became progressively shorter from prepachytene (maximum complement length 289 m) to diplotene (175 m). The thickness of the SCs increased from prepachytene to pachytene and probably decreased again during diplotene. At the beginning of diplotene the lateral elements (LEs) separated, and the single LEs became two to three times thicker than the LEs of the SC. The centromeres were at all stages attached to the nuclear membrane, whereas the telomeres were free in the nucleoplasm during pachytene and diplotene. A heterochromatic marker was present on chromosome 1 near the sex determining locus, and a diffuse marker on chromosome 3 near the nucleolus organizer region. After breakdown of the complexes, polycomplexes were present in the nucleus.     

Meiotic chromosome pairing and synaptonemal complex transformation in Culex pipiens oocytes

The synaptonemal complexes of the oocytes of the mosquito Culex pipiens quinquefasciatus have been reconstructed from serial sections. A diffuse structure, probably a chromocenter composed of centromeric heterochromatin, was present during pachytene. As no synaptonemal complexes were visible inside the chromocenter the continuity of the 2 arms of a bivalent was lost. The telomeric ends were clustered in a small area of the nuclear membrane in a bouquet arrangement; they were associated in pairs, and sometimes joined through a special structure. One pair was composed of the 2 telomeres of the shortest bivalent and a ring configuration was thus formed. The other 2 chromosomes may form one or two rings. During a short transitional stage, after the disappearence of the synaptonemal complexes, several thousand annuli, 1200–1500 A in diameter, were present in the nuclei. The annuli disappeared as material originating mainly from the transverse filaments of the synaptonemal complexes formed a capsule around the chromosomes during diplotene.     

The transformation of the synaptonemal complex into the “elimination chromatin” in Bombyx mori oocytes

In Bombyx mori oocytes the synaptonemal complexes are retained in modified form from pachytene to metaphase I. At the end of pachytene the length and width of the lateral components of the complex increase, whereafter the complexes become compacted during later stages of the meiotic prophase. Ultimately, at metaphase I the modified synaptonemal complexes of individual bivalents fuse to form a more or less continuous sheet between the homologous chromosomes. This sheet corresponds to the structure historically known as the elimination chromatin. It is concluded that in the absence of crossing over and chiasma formation in Bombyx mori females the retainment and subsequent modification of the synaptonemal complex has evolved as a substitute mechanism to ensure regular disjunction of the bivalents.     

Synaptonemal complexes and associated structures in microspread human spermatocytes

Human spermatocytes processed with a modified microspreading technique which involves the use of sodium dodecyl-sulphate (SDS) have been used to construct synaptonemal complex (SC) karyotypes. Twenty two pachytene spermatocytes were selected for length quantitation. The mean values of relative lengths and centromeric indexes of each SC agree closely with values obtained by three-dimensional reconstructions (Holm and Rasmussen, 1977), except for SCs #4–5, 6–7 and 19–20. Absolute lengths are consistently longer in spreads (10.7% longer than in sections, on average). The mean total length of the SC complement is 258.7 m. Six morphological types of XY paris have been described. On the basis of the relationships between the XY pair, nucleolar development and autosome behavior, these six XY types are assumed to develop in succession. Type O XY pairs occur during late zygotene, types I and II XY pairs occur during early to midpachytene, and types III, IV and V occur during later pachytene substages. Alignment of the X and Y axes is observed at late zygotene, and formation of the SC occurs in relation with type I XY pairs. Progressive desynapsis occurs in types II and III. Splitting and fusion of the X and Y axes attain a maximum in types IV and V. The breakdown of the dense bodies associated with the X and Y axes occurs during stage V. — Bar-like structures, having a mean length of 2,100 Å are associated with SCs in all the pachytene substages defined by the XY types. The average number of bars per nucleus is 46.2 (SD=8.4, N=20), and the average SC length per bar is 5.57 m. The distribution along the SCs of 923 bars shows that near-termini locations are preferred (SC length per bar, 2.98 m) and centromere regions are avoided (SC length per bar, 16.9 m). — On the basis of these data, bars are similar to recombination nodules described in other organisms. The availability of a standard SC karyotype for microspreads and a temporal sequence given by the XY pair provide a basis for rapid screening of chromosome aberrations in human testicular biopsies.     

Molecular characterization of a pericentric inversion in mouse chromosome 8 implicates telomeres as promoters of meiotic recombination

A hot spot of meiotic recombination has been found in males on murine chromosome 8 using nonisotopic hybridization of a series of probes to mitotic and meiotic chromosomes. The sequences responsible for this enhanced recombination are the telomeric repeats. Mice both normal and hetero- or homozygous for a pericentric inversion, In(8) 1 Rl, were analyzed. The inversion subdivides chromosome 8 into three discreet regions: (1) a fraction of the micro short arm that contains 30–150 kb of telomeric sequences and only about one-fifth of the contiguous minor-satellite sequences (approximately 200 kb); (2) the inverted region; and (3) the noninverted distal two-thirds of the chromosome. In 70 spermatocytes from inversion heterozygotes, examined by electron microscopy, synapsis of the inverted region was complete but entirely nonhomologous. Nonhomologous synapsis persists from initiation of synaptonemal complex formation in zygonema/early pachynema until dissolution in late pachynema. This nonhomologous synapsis also suppresses crossing over within the inverted segment. The opportunity for proximal homologous recombination is thus restricted to the roughly 250 kb segment located between the short-arm break and the end of the bivalent. Nonetheless, an extreme proximal chiasma was observed in 11% of the heterozygous chromosome-8 bivalents, 34% of the normal 8 bivalents and 35% of the homozygous inversion 8 bivalents from spermatocyte preparations. Since in the normal chromosomes all minor satellite sequences are adjacent to the telomere, while in the inversion chromosomes most of these sequences are transposed to an interstitial position without a corresponding shift in chiasma position, the minor-satellite sequences can be ruled out as promoters of recombination. Instead, the data suggest that it is the telomeric sequences that promote recombination, not just within the telomeric repeat itself, but quite frequently in sequences more than 250 kb away.by T. HassoldThis paper is dedicated to the memory of Barbara McClintock, whose early contributions on meiosis were as fundamental as her later ones on transposable elements.     

Ultrastructural studies of late meiotic prophase nuclei of spermatocytes in Ascaris suum

Post pachytene stages of meiotic prophase in males of Ascaris suum have been analyzed with the electron microscope. No synaptonemal-like polycomplexes (PCs) have been observed in the nucleoplasm or cytoplasm during the period from pachytene to diakinesis. From Serially sectioned diplotene nuclei it was found that the bivalents are located near the periphery of the nuclei, the central part of the nuclei being vacant. Each nucleus contains one nucleolus. Up to 1 m long stretches of unpaired lateral elements (LEs) are found in some of the diplotene bivalents. These LEs are morphologically similar to unpaired LEs in early zygotene nuclei. Partial 3-dimensional reconstruction of two nuclei shows that the bivalents contain some small stretches of synaptonemal complex (SC) up to 1.9 m long. Some bivalents at diakinesis show remnants of SCs. At this stage chromosomes are fibrous, condensed, attached to the nuclear envelope and mostly with a rounded profile in cross section. The synchronous development of the spermatocytes and small bivalents at diplotene in A. suum make this system a good object for the study of localization of SC remnants.     

Chromosomal synapsis and the meiotic process in male mesquite lizards, Sceloporus grammicus complex.

Meiosis in males of the F5 cytotype of Sceloporus grammicus was examined through the analysis of synaptonemal complexes (SCs), diakinetic (metaphase I) nuclei, and secondary spermatocytes (metaphase II configurations). These data allowed the establishment of criteria for substaging of zygonema and pachynema, morphological characterization of the SC complement, and comparison of the orientation and segregation of the autosomes and sex chromosomes. The analysis of nuclei from all stages of meiotic prophase I (leptonema through diakinesis) provided a useful means of partitioning the temporal sequence of early meiotic events. Three substages of zygonema (Z1-Z3) were established, based on the extent of synapsis of the microchromosomal and macrochromosomal elements. Synaptic initiation of the autosomes and sex chromosomes was synchronous. Two patterns of macrochromosomal synapsis were observed. Whereas synapsis of the biarmed elements was biterminal (i.e., progressing from both ends of the homologs), synapsis of the acrocentric elements was uniterminal involving only the distal (noncentromeric) ends of the homologs. Unique sex-chromosomal characteristics were not observed in S. grammicus and, therefore, the substaging of pachynema was based on subjective criteria. Examination of diakinesis--metaphase I and metaphase II configurations indicated low levels of diakinetic irregularities with balanced segregation of the autosomal bivalents and the sex-chromosomal trivalent.