Søren W. Rasmussen

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 μ at zygotene/pachytene to at least 300 μ 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.

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.

The Hansenula polymorpha PEX14 gene encodes a novel peroxisomal membrane protein essential for peroxisome biogenesis

We have cloned the Hansenula polymorpha PEX14 gene by functional complementation of the chemically induced pex14‐1 mutant, which lacked normal peroxisomes. The sequence of the PEX14 gene predicts a novel protein product (Pex14p) of 39 kDa which showed no similarity to any known protein and lacked either of the two known peroxisomal targeting signals. Biochemical and electron microscopical analysis indicated that Pex14p is a component of the peroxisomal membrane. The synthesis of Pex14p is induced by peroxisome‐inducing growth conditions. In cells of both pex14‐1 and a PEX14 disruption mutant, peroxisomal membrane remnants were evident; these contained the H.polymorpha peroxisomal membrane protein Pex3p together with a small amount of the major peroxisomal matrix proteins alcohol oxidase, catalase and dihydroxyacetone synthase, the bulk of which resided in the cytosol. Unexpectedly, overproduction of Pex14p in wild‐type H.polymorpha cells resulted in a peroxisome‐deficient phenotype typified by the presence of numerous small vesicles which lacked matrix proteins; these were localized in the cytosol. Apparently, the stoichiometry of Pex14p relative to one or more other components of the peroxisome biogenesis machinery appears to be critical for protein import.

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.

Chromosome pairing in triploid females of Bombyx mori analyzed by three dimensional reconstructions of synaptonemal complexes

Chromosome pairing in triploid females of Bombyx mori was analyzed by serial sectioning and three dimensional reconstruction of synaptonemal complexes and unpaired lateral components in 11 nuclei covering the period from early to late pachytene. Two distinct phases of chromosome pairing with synaptonemal complex formation were revealed; one phase mediates pairing between homologous chromosomes and is followed by a second phase which is responsible for non-homologous pairing of univalents. Trivalent pairing of three homologues as well as bivalent interlocking appear to be corrected by dissolution and reassembly of the central regions of the synaptonemal complex before the initiation of or during the second pairing phase.

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.

Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome‐deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Δpex1 or Δpex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co‐sedimented with the peroxisomal membrane protein HpPex3p in both wild‐type cells and in Δpex4, Δpex8 or Δpex14 cells. Both proteins are loosely membrane‐bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co‐overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p and HpPex6p remained predominantly membrane‐bound, but only partially co‐localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. Copyright

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.