Pachytene Substages Research Articles

Induced Premature G2/M-Phase Transition in Pachytene Spermatocytes Includes Events Unique to Meiosis

Little is known about the control of events ending the lengthy prophase of meiosis I and leading to the G2/M-phase transition in mammalian spermatocytes, primarily because the relevant late pachytene, diplotene, and MI cells are present in low numbers in the testis and it is not possible to isolate them in significant numbers. We have utilized short-term cultures of pachytene spermatocytes from the mouse to study events of the G2/M cell-cycle transition induced by the protein phosphatase inhibitor okadaic acid (OA). Treatment of cultured pachytene spermatocytes with OA induced a rapid and premature onset of events leading to the M phase, visualized cytologically by nuclear envelope breakdown and chromosome condensation. After OA treatment, condensed chromosomes were seen as bivalents, not as univalents. Treatment with OA induced disassembly of synaptonemal complexes and resolution of crossovers as cytologically visible chiasmata. Chiasmata counts were similar in treated cells and control cells. Thus, surprisingly, even though the treated cells were in the pachytene substage of meiotic prophase, events of recombination were apparently completed to the point of chiasma formation in the majority of these cells. The sex chromosomes, forming the sex body of the pachytene spermatocyte, lagged behind the autosomal chromosomes in their condensation and progression toward the M phase. Treatment with OA induced an increase in histone HI kinase activity, generally used as an indicator of metaphase-promoting factor (MPF) activity; furthermore, the OA-induced cell-cycle transition does not require new protein synthesis. These results suggest that OA treatment overrides a cell-cycle checkpoint control that normally keeps pachytene spermatocytes in a lengthy prophase and that this control may be exerted by regulation of protein phosphorylation status.

Two new X-autosome Robertsonian translocations in the mouse

The influence of X-autosome Robertsonian (Rb) translocation hemizygosity on meiotic chromosome behaviour was investigated in male mice. Two male fertile translocations [Rb(X.2)2Ad and Rb(X.9)6H] and a male sterile translocation [Rb(X.12)7H] were used. In males of all three Rb translocation types, the acrocentric homologue of the autosome involved in the rearrangement regularly failed at pachytene to pair completely with its partner in the Rb metacentric. The centric end of the acrocentric autosome was found regularly to associate either with the proximal end of the Y chromosome or with the ends of nonhomologous autosomal bivalents; the proportions of cells with such configurations varied between pachytene substages and genotypes. Various other categories of synaptic anomaly, such as nonhomologous synapsis, foldback pairing and interlocks, affected the sex chromosome multivalent in a substantial proportion of cells. In one of the Rb(X.12)7H males screened, an unusual, highly aneuploid spermatocyte that contained trivalent and bivalent configurations was found. Rb translocation hemizygosity did not appear to increase to a significant extent the incidence of X-Y pairing failure at pachytene, although the incidence was elevated at metaphase I in Rb(X.12)7H animals. Overall, a comparison of the frequencies and types of chromosome pairing anomalies did not suggest that these were important factors in the aetiology of infertility in males carrying the Rb(X.12)7H translocation.

Spreading synaptonemal complexes from Zea mays

Four different inversion heterozygotes of maize were examined for the occurrence of synaptic adjustment. Three substages of pachytene were identified in synaptonemal complex (SC) spreads using side-by-side comparisons of chromosome squashes with two-dimensional spreads of SCs. In SC spreads, inversion loop frequency did not change substantially from early through late pachytene for any of the four inversion heterozygotes examined. In addition, the position and size of the inversion loops remained essentially constant throughout pachytene. These results indicate that synaptic adjustment of inversion loops does not occur during pachytene in Zea mays.

Human meiosis IX. Crossing over and chiasma formation in oocytes

Human oocytes from foetal ovaries have been analyzed by serial sectioning and three dimensional reconstruction from electron micrographs. Analysis of five late zygotene, twenty pachytene, four early and three mid diplotene nuclei allowed the following observations and conclusions: 1) Nuclei at pachytene can by morphological criteria be allocated into three substages. 2) The synaptonemal complex complement is twice as long in the oocytes as in the spermatocytes. 3) The total number of nodules decreases from a mean of 68 at late zygotene to a mean of 30 at early diplotene. During pachytene, an increasing number of nodules transforms into bars. A mean of 6 bars was observed in late zygotene nuclei compared to a mean of 17 at late pachytene. 4) Nodules and bars are distributed nonrandomly among the bivalents and bivalent arms. The observed frequency of bivalents devoid of recombination structures is only between one- and two-thirds of the frequency expected if nodules were distributed at random. 5) The distribution of recombination structures along the bivalent arms is not uniform. More nodules and bars than expected from a random distribution are located in the vicinity of telomeres. Comparison of neighbour nodule/bars distances at different substages of pachytene indicated that the elimination of recombination structures primarily affects nodules and bars close to each other. 6) The distribution of recombination structures along the individual bivalents was uneven, regions with high frequency of recombination nodules and bars alternating with regions of low frequency. This pattern is very similar to that reported for human spermatocytes, which indicates the same distribution of crossovers in the two sexes. 7) Elimination of the synaptonemal complex starts at early diplotene at a limited number of sites until about 68 intact segments are created. 8) Chiasmata are not morphologically recognizable after the elimination of the synaptonemal complex is completed at mid diplotene.

Human meiosis V. Substages of pachytene in human spermatogenesis

The pachytene stage of human spermatogenesis has been investigated by serial sectioning of spermatocytes and three dimensional reconstruction of nuclei from electron micrographs. The analysis of 73 completely reconstructed normal nuclei from late zygotene to early diplotene revealed a continuous sequence of changes in the fine structure and morphology. The developmental sequence permits the recognition of 7 substages on the basis of morphological changes of autosomal chromatin, centromeric, chromatin, the XY bivalent, the secondary constriction on bivalents 1,9 and 16, the nucleolus and the centrioles. Of particular interest is the observation that the disappearance of the synaptonemal complex segment between the X and Y chromosomes at mid pachytene is only temporary, a distinct complex segment being present in nearly all analysed XY bivalents by the end of pachytene. The number of recombination nodules and recombination bars in nuclei of the different substages is recorded. The total number of nodules decreases from 75 at the beginning of pachytene to 1 or none at the end of this state. The bars appear at early pachytene, reach a number of 35 at mid pachytene and disappear following the nodules towards the end of pachytene. The morphology of pachytene nuclei at various stages of degeneration as well as the structural characteristics of two tetraploid spermatocytes are described.

Synaptonemal complex analysis of mouse chromosomal rearrangements. I. Cytogenetic observations on a tandem duplication.

Electron microscopy of surface-spread spermatocytes from mice heterozygous for a tandem duplication shows the heteromorphic synaptonemal complex (SC) to comprise two lateral elements of unequal length, the longer of which is buckled out in a characteristic loop, representing the unsynapsed portion of the duplication. The loop is a regular feature of late zygotene-early pachytene nuclei; it is longest at these early stages, but, through equalization of the two axes as a consequence of synaptic adjustment, it is replaced by a normal appearing SC at late pachytene. Because equalization, as indicated by a decrease in the percent difference between axes, may begin shortly after completion of synapsis, estimates of duplication segment length are restricted to a sample selected for least adjustment. --Although the mean position of the loop is constant at various pachytene substages, individual positions vary widely from cell to cell, consistent with the behavior expected of a duplication, but not of a deletion or an inversion. The length of the segment that is duplicated is estimated to be 22% of the normal chromosome, the midpoint of the segment is mapped at 0.61 of the chromosome distal to the kinetochore, and the ends of the segment are mapped at 0.50 to 0.72. Measurements of G-banded mitotic chromosomes give comparable values: duplication length, 24%; midpoint, 0.60, and segment ends, 0.48 and 0.71. This agreement constitutes further validation of the SC/spreading method for detecting and analyzing chromosomal rearrangements at pachytene and substantiates the fidelity with which the axes and SCs represent the behavior of chromosomes in synapsis.

Synaptosomal 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 micrometer. Six morphological types of XY pairs 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 mid-pachytene, 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 A 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 micrometer. The distribution along the SCs of 923 bars shows that near-termini locations are preferred (SC length per bar, 2.98 micrometer) and centromere regions are avoided (SC length per bar, 16.9 micrometer). --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.

Change in length of synapsed bivalents during pachytene

Total pachytene bivalent length was measured in 93 nuclei from plants of an inbred line of maize (KYS). These nuclei are thought to represent samples of substages from the portion of pachytene which begins with the opening of the synizetic knot and ends with the initiation of loss of the synaptonemal complex at diplotene. Pachytene substages were ranked on the basis of the length of the anther in which they occurred, with longer anthers generally expected to contain more advanced stages. The data strongly suggest that maize synapsed bivalents shorten significantly during this period.

Human meiosis I. The human pachytene karyotype analyzed by three dimensional reconstruction of the synaptonemal complex

The synaptonemal complexes of the 22 autosomal bivalents have been reconstructed in 22 human spermatocyte nuclei at pachytene. The mean total length of the autosomal synaptonemal complexes has been measured and amounts to 231 μm (s.d.=16μm). On the basis of absolute and relative lengths and the position of the centromeric heterochromatin it was possible to identify 14 of the 22 autosomal bivalents and to allocate each of the remaining 8 bivalents unequivocally to a major group. The relative synaptonemal complex lengths and centromere indices of the autosomal bivalents exhibit a good correlation with light microscopical data on relative lengths and centromere indices of bivalents at diakinesis and of somatic metaphase chromosomes. Significant differences in total mean length were not found, neither among nuclei from the five individuals analyzed nor amng nuclei in different substages of pachytene. The absolute lengths of the individual bivalents were however found to vary among different nuclei, the maximum difference exceeding the estimated reconstruction and measuring error. It is furthermore shown that each of the five acrocentric bivalents is capable of organizing a nucleolus but that in most cases less than five nucleoli are present in each nucleus. A short piece of synaptonemal among nuclei in different substages of pachytene. The absolute lengths of the individual bivalents were however attachment site of the telomeres on the nuclear envelope.

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus). II. Morphology of the XY pair in spread preparations.

The X and Y chromosome axes have a distinctive morphology at pachytene and are clearly distinguishable from autosomal SCs. The X and Y are totally unpaired at late zygotene when most of the autosomes are synapsed; however, their attachment points at the presumptive SC end are closely apposed. The X and Y axes pair to form a length of SC that is somewhat shorter than the unpaired portion of the Y. Unpaired axes may appear thin and sometimes double, or may thicken to form fusiform bulges that are sometimes hollow: two on the X and one on the Y. Discrete differentiations, tentatively identified as kinetochores, are often visible at the proximal end of the SC on the Y axis, and also between the fusiform differentiations on the X axis. Additional differentiations, in the form of loops and densely staining granular excrescences form on the X axis and sometimes on the Y. A further differentiation appears as a cloud-like sheath around the distal end of the X axis, often in association with a bi-lobed dense body. At late pachytene, the XY-SC remains intact, but the unpaired X and Y axes develop side branches. Just before diplotene, while the autosomal SCs are still intact, the XY-SC is lost, although the axes persist and remain together at the attachment point of the SC. The X and Y differentiations form a logical temporal sequence when grouped according to progressive structural complexity. The morphological types may serve as markers for the sub-stages of pachytene.