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.     

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.     

Ultrastructural analysis of maize pachytene karyotypes by three dimensional reconstruction of the synaptonemal complexes

Aldehyde fixation followed by staining with phosphotungstic acid produces differential contrast between the synaptonemal complex and the chromatin of maize pachytene bivalents. Centromeres, heterochromatic knobs and large chromomeres are easily recognised. With this and other staining techniques the nucleolus organizer region can be differentiated into two components. — Microsporocyte nuclei at pachytene were serially sectioned and all ten bivalents reconstructed in five nuclei. An idiogram was derived from the mean chromosome (= synaptonemal complex) lengths, the arm ratios, positions of knobs and the nucleolus organizer region. The idiogram agrees well with that published from light microscopic analyses. However, bivalent lengths are only two thirds of those observed by light microscopy of squash preparations. Many telomeres of the bivalents are connected via chromatin to the nuclear envelope, but a varying number of free bivalent ends are observed in all five reconstructed nuclei. — Bivalents heterozygous for inversion 3b were reconstructed. In the presence of abnormal chromosome 10 (K10) the lateral components of the synaptonemal complex of chromosome 3 formed a typical inversion loop, while in one of the nuclei having no K10 the two lateral components of the long arms of chromosome 3 remained unpaired in the region of inversion heterozygosity. The presence of K10, which increases crossing-over frequencies and promotes intimate pairing at the light microscopic level, was thus found to permit formation of complete synaptonemal complexes in the inverted region. The extra terminal portion of the K10 chromosome folded back on itself and formed a morphologically normal synaptonemal complex in this — possibly non-homologously paired — region. The chromatin of centromeres and knobs from different bivalents were sometimes found to fuse, but the synaptonemal complexes transversing the fused centromeres or knobs retained their individuality.     

Electron microscopy of meiosis in Drosophila melanogaster females

Complete reconstruction of the synaptonemal complex in 12 pachytene (defined here as that stage in which the synaptonemal complex is continuous throughout the bivalents) nuclei from one wild-type germarium has permitted the following observations. 1) Drosophila melanogaster bivalents at pachytene exhibit a chromocentral arrangement; the pericentric heterochromatin of all bivalents lies in one region of the nucleus, the chromocenter. Telomeric ends do not appear to abutt the nuclear envelope. 2) Synaptonemal complex is present in the pericentric heterochromatin; however, it is morphologically distinct from that present in the euchromatic portion of the bivalents. 3) Length of the synaptonemal complex of the bivalent arms is greatest at early pachytene; the synaptonemal complex then becomes progressively shorter. Minimum length is approximately one-half of the maximum. 4) Decrease in length of synaptonemal complex is accompanied by an increase in thickness. Reconstruction of 20 pachytene nuclei from an additional 8 germaria suggests that these observations are typical. Correlations between these cytological observations and genetic observations (e.g., patterns of crossing-over) are discussed.     

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.     

Localised chiasmata due to partial pairing: A 3D reconstruction of synaptonemal complexes in male Stethophyma grossum

The possible role of localised pairing as a mechanism producing localised chiasmata in Stethophyma grossum spermatocytes has been examined ultrastructurally. Nuclei at four successive stages of meiosis from leptotene to pachytene were reconstructed from a series of ultrathin sections and the extent of synapsis as demonstrated by synaptonemal complex (SC) formation was calculated. On the basis of the relative lengths of SCs and condensed chromosomes it was reasoned that only the centromeric ends of the long and medium length bivalents paired, and only one end of these SCs was found attached to the nuclear envelope. Only the three shortest bivalents paired completely, and both their SC ends were attached to the nuclear envelope. Thus pairing was directly related to the distribution of chiasmata. The extent of pairing at different stages suggests that the shortest bivalent paired very quickly, the longer ones progressively slower, and that pairing proceeded zip-like from a point at or very close to the end attached to the nuclear envelope, since all stretches of SC were attached to the envelope, and there were never more than 11 pieces, one for each bivalent. Chromosome decondensation and axial core formation did not occur far in advance of SC formation, and synapsis appeared to be much slower in S. grossum than in other species with non-localised chiasmata, as a larger proportion of the meiotic cysts were in zygotene, compared to Stauroderus scalaris and Locusta migratoria, although this was not quantified.     

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.     

Karyotype analysis ofAscaris lumbricoides var.suum

Twelve synaptonemal complexes are present in both oocyte and spermatocyte pachytene nuclei ofAscaris lumbricoides var.suum, as determined by 3-D reconstruction of the nuclear contents from electron microscopy of serial sections and therefore, n=12 in the strain ofAscaris described here. In the female the heterochromatic end of each synaptonemal complex is attached to the nuclear envelope and the other end is free in the nucleoplasm. In the male neither end ot the synaptonemal complex is attached, but there is a heterochromatic knob at one end of each complex. Five additional large heterochromatic masses are present in the spermatocyte nucleus and these may be the sex chromosomes described by earlier workers.     

Pachytene karyotype analysis of tetraploid Meloidogyne hapla females by electron microscopy

Pairing of homologous chromosomes results in the formation of 34 synaptonemal complexes (SC) at pachytene, corresponding to the 34 bivalents at metaphase I. No multivalent associations were observed and pairing occurs two-by-two. The modified SC, which lacks a central element, does not affect the pairing process. Only one end of the SC is attached to the nuclear envelope, although either end can attach. Total SC length and the number of recombination nodules in the tetraploid were about 1.5 times greater than in the diploid.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

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.     

The synaptonemal complexes of Caenorhabditis elegans

Normal synaptonemal complexes (SCs), consisting of two lateral elements and a central element, are present in wild-type, him-4 and him-8 mutant strains in both hermaphrodites and males of Caenorhabditis elegans. Thus, the increase in rate of nondisjunction in the him mutants is not related to aberrant SC morphology. The wild-type hermaphrodite has six SCs, as determined from 3-D reconstruction analysis of serial sections from electron microscopy. Thus, n = 6 and this confirms early reports based on cytological studies with the light microscope. Only one end of the SC is attached to the nuclear envelope while the other end is free in the nucleoplasm and there is no apparent bouquet formation. Either end of the SC can attach to the nuclear envelope. The pairing behavior of the XX bivalent is normal and occurs synchronously with the autosomes. Electron dense bodies, or knobs, are associated with the SC via the central element and displace the chromatin for a distance of 200 nm. Each pachytene nucleus of the wild-type hermaphrodite has six such structures that are randomly dispersed along the bivalents such that some SCs have one or two knobs while others have none. Their function is unknown.     

The Relationship between Synaptinemal Complexes, Recombination Nodules and Crossing over in NEUROSPORA CRASSA Bivalents and Translocation Quadrivalents

Reconstruction of serially sectioned zygotene and pachytene nuclei has allowed the estimation of both the number and position of central component recombination nodules in the synaptinemal complexes of two chromosomally different strains of Neurospora crassa. In both strains the number of nodules is that expected if each nodule represents one crossover event (50 map units). The distribution of nodules within the arms of bivalents shows evidence of centromeric repulsion and telomeric localization. Nodules appear quite early in the zygotene before pairing of chromosomes is complete. Evidence was found of size differences in nodules, and multiple nodules were occasionally seen. Chromosome lengths and nuclear sizes increased from early zygotene to late pachytene. The three quadrivalents present in the alcoy translocation heterozygotes were readily distinguishable in reconstructions, and their cytological dimensions were in agreement with predictions from linkage map distances.     

Synapsis with and without recombination in the male meiosis of the leaf-footed bug <Emphasis Type="Italic">Holhymenia rubiginosa</Emphasis> (Coreidae,Heteroptera)

In organisms with chiasmatic meiosis two different relationships have been described between crossing over and synapsis: in one group of organisms synapsis depends on the initiation of meiotic recombination while in the other group it is independent of this initiation. These patterns have been observed mainly in organisms where all meiotic bivalents in the set have similar behaviors. In some heteropteran insects a pair of chromosomes named m chromosomes is known to behave differently from autosomes regarding synapsis and recombination. Here we used immunodetection of a synaptonemal complex component and acid-fixed squashes to investigate the conduct of the small m chromosome pair during the male meiosis in the coreid bug Holhymenia rubiginosa. We found that the m chromosomes form a synaptonemal complex during pachytene, but they are not attached by a chiasma in diakinesis. On the other hand, the autosomal bivalents synapse and recombine regularly. The co-existence of these variant chromosome behaviors during meiosis I add further evidence to the absence of unique patterns regarding the interdependence of synapsis and recombination.     

Synaptonemal complex and recombination nodules in rye (Secale cereale)

Meiotic prophase in rye was investigated by serial-section reconstruction of pollen mother cell nuclei. In the mid-late zygotene nucleus, all lateral elements were continuous from telomere to telomere, and 9–20 pairing initiation sites per bivalent were observed. Chromosome and bivalent interlockings detected during zygotene were resolved at early pachytene when pairing was completed. In the three pachytene nuclei, the relative synaptonemal complex (SC) lengths and arm ratios were found to be in good correlation with light microscopic data of pachytene bivalents. Spatial tracing of the bivalents showed that they occupy separate areas in the nucleus. Three types of recombination nodules were observed: large, ellipsoïdal and small nodules at early pachytene and irregularly shaped nodules mainly associated with chromatin at late pachytene. Their number and position along the bivalents correlated well with the number and distribution of chiasmata. The classification of the seven bivalents was based on arm ratio and heterochromatic knob distribution.     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

Female meiosis in Mesostoma ehrenbergii ehrenbergii is achiasmate. Electron microscope serial section reconstructions of nuclei from the germarium region of the ovary have shown that synaptonemal complex (SC) is present during the early prophase stages. The greatest amounts present were in two nuclei containing 389 and 401 respectively. The reconstructions showed that discontinuities in the SC existed along the lengths of the bivalents in these two nuclei which were taken to represent the maximally paired stage, i.e., pachytene. — Reconstructions of post pachytene nuclei showed that SC was absent from the bivalents and therefore retention of SC until metaphase I is not the mechanism used by this achiasmate species to ensure homologous chromosome segregation. An alternative mechanism for this function is proposed based on observations made on the later stages of meiosis in maturing oocytes.     

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.     

Chromosomal pairing in deer mice heterozygous for the presence of heterochromatic short arms

The pattern of chromosomal pairing was analyzed in male deer mice (Peromyscus maniculatus and Peromyscus sitkensis) heterozygous for the presence of heterochromatic short arms. G- and C-banding of somatic metaphases indicated that the presence of heterochromatic short arms increased the length of chromosome 4 by 15% in P. sitkensis and that of chromosome 8 by 9% in P. maniculatus. Analysis of silver-stained late zygotene and early pachytene nuclei revealed a low frequency of unequal axial lengths in the synaptonemal complexes corresponding to the heteromorphic bivalents. All mid- and late pachytene nuclei, however, exhibited fully paired synaptonemal complexes with equalized axial lengths. These observations suggest the existence of an adjustment mechanism which functions to equalize the lengths of the two axes of the heteromorphic synaptonemal complex.     

Etude preliminaire de stades de la méiose humaine dans les spermatocytes et les ovocytes

Summary Human meiotic chromosomes, from spermatocytes and ovocytes, are described after observations of whole mount preparations under E.M. Small testicular and ovarian fragments are put in distillated water, then macerated; the cell suspension is spread on the surface of sheet copper grids covered with formvar plus collodion films. After dehydratation interesting stages are selected under L.M. before observations under E.M.Zygotene and pachytene are the most common stages. During pachytene the chromomeres are well individualized; the synaptonemal complex may be observed; chromatin fibers connect the chromosomes to nuclear pores, interchromosomal fibers joint the bivalents. Zygotene and pachytene bivalents are very similar in the male and the feminine germ cells.     

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.