Preben Bach Holm

Human meiosis II. Chromosome pairing and recombination nodules in human spermatocytes

Moiosis in human spermatocytes has been analyzed by three dimensional reconstructions of 4 leptotene, 4 earlymid zygotene, 10 late zygotene, and 21 early pachytene nuclei. At leptotene, a lateral component is organized along each chromosome and the telomeres attach to the nuclear envelope. At early zygotene, the attachment sites aggregate and a chromosome bouquet is formed. Pairing and synaptonemal complex formation are initiated from the telomeres by binding of precursor material for the central region to the lateral components of the aligned homologues. In the 10 late zygotene nuclei, on the average 72% of the autosomal complement had been paired. Synaptonemal complex formation is in most cases initiated from both ends of the homologues and only in 5 cases was initiation of complex formation interstitial. Pairing of the short arms of the acrocentric bivalents and the X and Y chromosomes is delayed compared to the remainder of the genome. Irregularities such as interlockings and breaks of the lateral components of chromosomes or breaks of the synaptonemal complexes of bivalents are observed in 8 out of the 10 nuclei. Most of the breaks appeared to be the result of a resolution of interlockings. At early pachytene, all bivalents are fully paired the only exception being the secondary constrictions on bivalents present between the X and Y chromosomes. Only two interlockings and three breaks of lateral components of chromosomes were found at this stage. Recombination nodules are present in or at the central region of the synaptonemal complex from early zygotene and devidence has been obtained in favor of an attachment of nodules to precursor material for the central region at the pairing fork. Nodules can, however, also attach to a fully formed synaptonemal complex. At late zygotene, an average number of 101 nodules per nucleus is present. Assuming that all regions of the complex have equal probabilities of receiving a nodule about 144 nodules are expected to be present at themoment of complete pairing. At early pachytene, the mean number of nodules is 75. Generally, nodules appear to be distributed evenly along the bivalent arms. Nodules are present, however, in excess in the telomere regions and in the XY bivalent while the centromere regions, the secondary constrictions, and the short arms of the acrocentric bivalents are relatively depleted of nodules. Measurements of the distances between adjacent nodules and a comparison with a theoretical distribution of such distances assuming a random positioning of nodules have demonstrated that at late zygotene nodules are more frequently clustered than would be expected if they were distributed independently. At early pachytene, the reverse pattern is observed.

Chromosome pairing and chiasma formation in allohexaploid wheat, Triticum aestivum analyzed by spreading of meiotic nuclei

An ultrastructural analysis of spread, silver stained meiotic nuclei of wheat, Triticum aestivum, has been performed. Partial tracings of lateral components and synaptonemal complexes of a number of leptotene and early zygotene nuclei, and complete tracings of eleven mid zygotene, seven late zygotene, twenty pachytene, twelve early diplotene, twenty-one mid diplotene and sixteen late diplotene nuclei have permitted the following observations and conclusions: 1) The chromosomes appear to be randomly distributed within the spread leptotene and early zygotene nuclei. 2) The complement length, i.e., the lateral component length of the three genomes, decreases from a mean of 2063 μm at mid zygotene to 1806 μm at late zygotene and 1474 μm at pachytene. 3) Chromosome pairing is initiated preferentially from the telomeres. These are aggregated during zygotene whereby a chromosome bouquet is established. Synaptonemal complex formation is thereafter also initiated interstitially, the mean number of synaptonemal complex segments being 156 per nucleus in the eleven mid zygotene nuclei where on the average 61% of the complement is paired. 4) Chromosome pairing and synaptonemal complex formation remain frequently incomplete. In most cases, however, more than 95% of the complement has synapsed. 5) Interlocking of lateral components and synaptonemal complexes is frequent at zygotene. Detailed analysis of one mid zygotene nucleus revealed more than 20 interlockings, most of them being bivalent interlockings. At late zygotene there was a mean of 5 bivalent interlockings per nucleus and a few chromosome interlockings. Some interlockings persist up to pachytene and a few may remain into diplotene. 6) Multiple associations of chromosomes, probably signifying pairing between homoeologues, are seen in eight of the eleven mid zygotene nuclei, in four of the seven late zygotene nuclei, in three of the 20 pachytene nuclei and possibly in one of the early diplotene nuclei. 7) Synaptonemal complex degradation is initiated at a limited number of sites at the pachytene to diplotene transition, the number of retained synaptonemal complex segments amounting to 89, 119 and 136 at early, mid and late diplotene. This exceeds the number of chiasmata observable at metaphase I in the light microscope by a factor of two to three. 8) It is proposed that the diploid behaviour of allohexaploid wheat results from: I) A high stringency of chromosome pairing at the beginning of zygotene whereby pairing preferentially is initiated between homologues. II) Transformation of multiple association into bivalents at the end of zygotene and during pachytene before crossing over occurs and/or III) a suppression of crossing over between paired segments of homoeologous chromosomes. The possible effect of the Ph gene(s) on the long arm of chromosome 5B is discussed.

Three-dimensional reconstruction of chromosome pairing during the zygotene stage of meiosis in Lilium longiflorum (thunb.)

Chromosome pairing in early zygotene in Lilium longiflorum has been studied by three-dimensional reconstructions at the electron microscopical level. One nucleus has been completely reconstructed, and the pairing behaviour and the length of the 12 partially synapsed bivalents was analysed and measured. The total length of the 24 chromosomes comprises 7,4 mm, and pairing is initiated at several sites along the chromosomes. Analysis of the pattern and temporal sequence of pairing suggests that all regions of the lateral component contain the required specificity for site-to-site matching. Measurements of the length of the unpaired homologous regions demonstrate that identical chromosome length is secured for both homologues before pairing is initiated. Pairing is accomplished by the attachment of a piece of central region to one of the lateral components after rotation of the associated chromatin, followed by subsequent binding of the central region to the homologous lateral component. The majority of the telomeres are attached via a special substance to a restricted portion of the nuclear envelope. The occurrence of chromatin associated structures at the nuclear envelope in combination with the highly polarized distribution of nuclear pores suggests that considerable membrane activity of possible significance for chromosome movements takes place during this interval. The observations are discussed in relation to current hypotheses on the structure and biochemistry of early meiotic chromosomes and the processes of alignment and pairing.

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.

Chromosome pairing, recombination nodules and chiasma formation in diploid Bombyx males

Serial sectioning and three dimensional reconstruction of 82 nuclei divided into 15 well characterized substages covering the period from early zygotene to metaphase I in diploid Bombyx spermatocytes have permitted the following observations and conclusions: 1) At late zygotene, a mean of four chromosome and bivalent interlockings was found per nucleus. 2) During the transition from zygotene to pachytene, all interlockings resolve by breakage and reunion of chromosomes or bivalents. 3) Recombination nodules first appear at early zygotene, reach a maximum of 91 per nucleus by late zygotene, decrease to 55 per nucleus at mid pachytene and increase again to 70–75 towards the end of pachytene. 4) At late zýgotene and pachytene, nodules are essentially distributed at random among bivalents as well as along bivalents. 5) Small, but important deviations from randomness were, however, revealed: Three percent of the bivalents were without nodules at late zygotene, 8% at early pachytene but at mid and late pachytene, only 2% were devoid of nodules. 6) From mid pachytene, an increasing fraction of the recombination nodules becomes larger and more irregular in shape. These are termed chromatin nodules and at the pachytene-diplotene transition, nearly all nodules are of this type. 7) During early diplotene, the bulk of the chromatin decondenses leaving only about 60 major condensed domains. These domains originate from chromatin nodules and are frequently associated with a piece of synaptonemal complex which in its central region contains a dense core resembling a recombination nodule. 8) At late diplotene, the condensed domains of the bivalents consist of two dense chromatin segments, one on each of the homologues, and bridged by a complex circular structure giving the regions a characteristic tripartite appearance. 9) At mid diakinesis, the circular structures are located in chromatin bridges — chiasmata —between the homologous chromosomes. 10) The number of chiasmata per nucleus is constant from mid diplotene to mid diakinesis and is distributed among the bivalents similar to the distribution of recombination nodules at mid and late pachytene. 11) The circular chiasma components are eliminated from late diakinesis until metaphase I. 12) Finally, each metaphase I bivalent contains four distinct localized centromeres, two by two facing opposite spindle poles.

Chromosome pairing, recombination nodules and chiasma formation in the basidiomycete coprinus cinereus

Meiosis in the basidiomycete Coprinus cinereus was analyzed by three dimensional reconstructions of nuclei covering the period from leptotene to telophase II. Crosses involving three different strains (JR52, PR2301 and E991) were used.The analysis of 94 completely reconstructed nuclei arranged in a temporal sequence according to the morphology of the synaptonemal complex, the centromeres and the centrosomes permitted the following observations and conclusions: (1) The haploid chromosome number of Coprinus cinereus is 13. (2) Reciprocal translocations have been identified in strains PR2301 and E991. In the former strain, the translocation is between chromosomes 3 and 5 and in the latter between chromosomes 1 and 9. (3) Interlockings and chromosome breaks are present during zygotene but at a lower frequency than in organisms with longer chromosomes. The translocation quadrivalents are more often than normal bivalents involved in interlockings and have more chromosome breaks. (4) Transformation of a translocation quadrivalent into two heteromorphic bivalents was only observed once in agreement with the contention that the turnover of the synaptonemal complex required for this transformation is prevented in bivalent regions where crossing over has occurred. (5) Correction of interlockings by the «breakage-reunion» mechanism is complete before mid-late pachytene. (6) The presence of two apparently normal bivalents replacing the translocation quadrivalent in at least one, possibly several, cases suggests that a «retranslocation» has taken place, possibly by a mechanism similar to that responsible for the resolution of interlockings. The implications of this possibility are discussed. (7) During early diplotene the synaptonemal complexes are eliminated from the bivalent arms while synaptonemal complex constituents often remain associated with the centromeres and the chiasmata until late diplotene. (8) Homologous centromere regions remain fused at least until early diakinesis. It is the suggested that this association may serve the same function as chiasmata in maintaining the bivalent configuration up to metaphase I and hence improve the chances of a regular disjunction in bivalents without chiasmata. (9). Recombination nodules are readily identified in the central region of the synaptonemal complex from early zygotene to late diplotene. The total number of nodules expected upon completion of synaptonemal complex formation at late zygotene amounts to 37 and is the same as that observed at early pachytene. The total number of nodules is reduced to 26 before midlate pachytene, a reduction similar to that reported in other organisms. (10) An increasing fraction of the nodules becomes larger and surrounded by chromatin during pachytene—diplotene and by late diplotene, all nodules are replaced by small chromatin condensations—chiasmata. (11) The distribution of nodules among and along the bivalents has been analyzed by comparing the observed distributions and those produced by computer simulation of a random positioning of nodules. The analysis reveals a nearly random distribution at late zygotene while during early pachytene and especially pronounced at mid-late pachytene, the distribution of nodules deviates from a random distribution. The comparison furthermore indicates that the placement of recombination nodules on the bivalents of a nucleus is controlled by nodule/bivalent (bivalent arm) interactions while the interaction between nodules appears to be of less importance.

Chromosome pairing in autotetraploid Bombyx females. Mechanism for exclusive bivalent formation

Meiosis in tetraploid Bombyx mori oocytes has been analyzed by three dimensional reconstructions of 1 mid zygotene, 7 early pachytene and 11 mid-late pachytene nuclei. The general pattern of chromosome pairing was found to be essentially similar to that of diploid oocytes. The zygoene stage is recognizable by incomplete pairing and a distinct chromosome bouquet, early pachytene by complete or almost complete pairing and a distinct bouquet and mid-late pachytene by dissolution of the bouquet followed by release of chromosome ends from their attachment sites on the nuclear envelope. At early pachytene the nuclei contained 5–12 quadrivalents, 0–7 univalents, 0–3 trivalents and 27–44 bivalents. The corresponding figures for mid-late pachytene were 0–2 quadrivalents, 0–3 univalents, 0–2 trivalents and 48–56 bivalents. The remarkable decrease in the number of quadrivalents and the corresponding increase in the number of bivalents between early and mid-late pachytene is the result of a correction of the zygotene pairing. In the absence of crossing over and chiasma formation, the pairing of the randomly located leptotene chromosomes into the maximal number of bivalents consists thus of two phases: 1) A specific zygotene pairing with synaptonemal complex formation restricted to homologous chromosome regions and 2) a correction of irregularities in this pairing by partial dissolution of the central region of the complex succeeded by or coinciding with a second round of synaptonemal complex formation, permitting also nonhomologous pairing. It is suggested that in organisms with crossing over and chiasma formation the correction process is impeded by the occurrence of crossovers.

The effect of chromosome 5B on synapsis and chiasma formation in wheat, triticum aestivum cv. Chinese Spring

The present paper summarizes and discusses the results obtained from a series of ultrastructural investigations of the effect of chromosome 5B on chromosome pairing, and synaptonemal complex formation in wheat, T. aestivum cv. Chinese Spring. The material comprises hexaploid wheat nullisomic for chromosome 5B, monosomic for 5B, euploid wheat, wheat where chromosomes 5B have been replaced by one, two or three copies of an isochromosome for the long arm of chromosome 5B; trihaploid wheat with and without 5B and hybrids between Chinese Spring wheat and diploid rye with chromosome 5B, without 5B or carrying an isochromosome for the long arm of 5B. It is shown that nullisomy for chromosome 5B in hexaploid wheat results in a two-to threefold increase in the number of lateral components in multiple associations already from the beginning of zygotene and in trihaploid wheat and wheat-rye hybrids in a two-to threefold increase in the number of pairing partner exchanges per lateral component involved in synapsis. Only nullisomy for chromosome 5B and triisosomy for the long arm of chromosome 5B permit crossing over between the homoeologous chromosomes of wheat. It is inferred that the product of the Ph locus on chromosome 5B affects both synapsis and crossing over. As these timewise separated processes are assumed both to be initiated by heteroduplex formation, this might provide the common link in the action of the Ph gene. The results obtained with the genotypes containing different dosage of the Ph gene disprove both the hypothesis that Ph influences a premeiotic chromosome rearrangement (13, 14, 15) and the hypothesis (18, 82) that Ph controls the timing of crossing over with respect to the process of correction of multiple pairing associations.

The premeiotic DNA replication of euchromatin and heterochromatin in Lilium longiflorum (Thunb.)

The presynaptic stages of Lilium longiflorum have been analyzed in the light microscope. Seven substages can be defined on the basis of changes in morphology of the heterochromatin and the nucleoli. The duration of the presynaptic interval and the substages has been calculated from bud length measurements and cell stage gradients in the anthers. The premeiotic and somatic DNA replication has been investigated using3H-thymidine autoradiography. The premeiotic S phase lasts 50 hours whereas the somatic S phase in root tip nuclei is six times shorter. Moreover, premeiotic S phase nuclei house 2–3 times more heterochromatin than somatic nuclei. The premeiotic DNA replication can be divided into three periods: In early S phase the DNA of euchromatin is replicated; in mid S phase synthesis is arrested for a minimum of 9 hours; and in late S phase the DNA of heterochromatin is replicated. DNA synthesis in heterochromatin is correlated with a dispersal of these chromatin regions. Determinations of volume and staining characteristics of heterochromatin after the application of C and Q banding procedures indicate that the premeiotic heterochromatin comprises C bands, Q bands and interbands. The premeiotic DNA replication is discussed in relation to the observed chromatin dynamics, and a proposal is made for the significance of the replication sequence to the formation of the leptotene chromosome and the control of crossing-over.

The pachytene karyotype of Schizophyllum commune analyzed by three dimensional reconstruction of synaptonemal complexes

Three dimensional reconstructions of 15 pachytene nuclei of Schizophyllum commune have revealed in each nucleus 11 distinct synaptonemal complexes, demonstrating a haploid chromosome number of 11. Each bivalent contained one short region of condensed chromatin marking the position of the centromere. The synaptonemal complex of, each bivalent contained from 0 to six recombination nodules. A high correlation was found between the total length of the synaptonemal complexes in a nucleus and the number of recombination nodules. Total synaptonemal complex length correlated only weakly with the nuclear volume.