Whole mount electron microscopy of meiotic chromosomes and the synaptonemal complex

The mechanism by which homologous chromosomes pair and crossover has been a major unsolved problem in genetics. Thin section electron microscopy of the synaptonemal complex has not provided enough details to allow any significant insight into this problem. Whole mount preparations of the testis of mice, quail, crayfish, and frogs provided a striking improvement in visualization of the morphological features of meiotic chromosomes. These studies, when combined with the use of deoxyribonuclease and trypsin allowed the following conclusions. 1. The synaptonemal complex (lateral and central elements with connecting L-C fibers) is composed of protein. 2. Contrary to common speculation the central element is not the pairing surface of homologous chromosomes. 3. The L-C fibers, averaging 75–100 Å in width, extend from the lateral elements and meet to form the central element which is usually composed of four fibers. 4. During leptotene, homologous axial elements, although unpaired for most of their length, attach next to each other at the nuclear membrane. 5. Short segments of the chromatin fibers attach to the lateral elements. These points of attachment are clustered, producing the chromomeres seen by light microscopy. 6. The chromatin fibers extend out from the lateral element as loops. Lampbrush chromosomes are thus not restricted to oogenesis but are common to all meiotic chromosomes.Since the morphological features of the central element of the synaptonemal complex persist despite extensive deoxyribonuclease digestion, pairing is perhaps best visualized as a two-step process consisting of a) chromosomal pairing during which the proteinaceous synaptonemal complex pulls homologous chromosomes into approximate association with each other, and b) molecular pairing, which probably takes place in the area around the synaptonemal complex.Supported by NIH Grants GM-15886 and C-2568, and The Charles and Henrietta Detoy Research Fellowship.     

Mammalian protein SCP1 forms synaptonemal complex-like structures in the absence of meiotic chromosomes

Synaptonemal complexes (SCs) are evolutionary conserved, meiosis-specific structures that play a central role in synapsis of homologous chromosomes, chiasmata distribution, and chromosome segregation. However, it is still for the most part unclear how SCs do assemble during meiotic prophase. Major components of mammalian SCs are the meiosis-specific proteins SCP1, 2, and 3. To investigate the role of SCP1 in SC assembly, we expressed SCP1 in a heterologous system, i.e., in COS-7 cells that normally do not express SC proteins. Notably, under these experimental conditions SCP1 is able to form structures that closely resemble SCs (i.e., polycomplexes). Moreover, we show that mutations that modify the length of the central alpha-helical domain of SCP1 influence the width of polycomplexes. Finally, we demonstrate that deletions of the nonhelical N- or C-termini both affect polycomplex assembly, although in a different manner. We conclude that SCP1 is a primary determinant of SC assembly that plays a key role in synapsis of homologous chromosomes.     

A role for synaptonemal complex in meiotic mismatch repair

A large subset of meiotic recombination intermediates form within the physical context of synaptonemal complex (SC), but the functional relationship between SC structure and homologous recombination remains obscure. Our prior analysis of strains deficient for SC central element proteins demonstrated that tripartite SC is dispensable for interhomolog recombination in Saccharomyces cerevisiae. Here, we report that while dispensable for recombination per se, SC proteins promote efficient mismatch repair at interhomolog recombination sites. Failure to repair mismatches within heteroduplex-containing meiotic recombination intermediates leads to genotypically sectored colonies (postmeiotic segregation events). We discovered increased postmeiotic segregation at THR1 in cells lacking Ecm11 or Gmc2, or in the SC-deficient but recombination-proficient zip1[Δ21-163] mutant. High-throughput sequencing of octad meiotic products furthermore revealed a genome-wide increase in recombination events with unrepaired mismatches in ecm11 mutants relative to wildtype. Meiotic cells missing Ecm11 display longer gene conversion tracts, but tract length alone does not account for the higher frequency of unrepaired mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 when analyzing all gene conversion tracts, but is similar between wildtype and ecm11 if considering only those events with unrepaired mismatches. Thus, in both wildtype and ecm11 strains a subset of recombination events is susceptible to a similar degree of inefficient mismatch repair, but in ecm11 mutants a larger fraction of events fall into this inefficient repair category. Finally, we observe elevated postmeiotic segregation at THR1 in mutants with a dual deficiency in MutSγ crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutSγ crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates, and that absence of SC is the molecular basis for elevated postmeiotic segregation in both MutSγ crossover-proficient (ecm11, gmc2) and MutSγ crossover-deficient (msh4, zip3) strains.     

Meiotic pairing and segregation of achiasmate sex chromosomes in eutherian mammals: the role of SYCP3 protein

In most eutherian mammals, sex chromosomes synapse and recombine during male meiosis in a small region called pseudoautosomal region. However in some species sex chromosomes do not synapse, and how these chromosomes manage to ensure their proper segregation is under discussion. Here we present a study of the meiotic structure and behavior of sex chromosomes in one of these species, the Mongolian gerbil (Meriones unguiculatus). We have analyzed the location of synaptonemal complex (SC) proteins SYCP1 and SYCP3, as well as three proteins involved in the process of meiotic recombination (RAD51, MLH1, and γ-H2AX). Our results show that although X and Y chromosomes are associated at pachytene and form a sex body, their axial elements (AEs) do not contact, and they never assemble a SC central element. Furthermore, MLH1 is not detected on the AEs of the sex chromosomes, indicating the absence of reciprocal recombination. At diplotene the organization of sex chromosomes changes strikingly, their AEs associate end to end, and SYCP3 forms an intricate network that occupies the Y chromosome and the distal region of the X chromosome long arm. Both the association of sex chromosomes and the SYCP3 structure are maintained until metaphase I. In anaphase I sex chromosomes migrate to opposite poles, but SYCP3 filaments connecting both chromosomes are observed. Hence, one can assume that SYCP3 modifications detected from diplotene onwards are correlated with the maintenance of sex chromosome association. These results demonstrate that some components of the SC may participate in the segregation of achiasmate sex chromosomes in eutherian mammals.     

Non-Mendelian segregation of sex chromosomes in heterospecific Drosophila males

Interspecific hybrids and backcrossed organisms generally suffer from reduced viability and/or fertility. To identify and genetically map these defects, we introgressed regions of the Drosophila sechellia genome into the D. simulans genome. A female-biased sex ratio was observed in 24 of the 221 recombinant inbred lines, and subsequent tests attributed the skew to failure of Y-bearing sperm to fertilize the eggs. Apparently these introgressed lines fail to suppress a normally silent meiotic drive system. Using molecular markers we mapped two regions of the Drosophila genome that appear to exhibit differences between D. simulans and D. sechellia in their regulation of sex chromosome segregation distortion. The data indicate that the sex ratio phenotype results from an epistatic interaction between at least two factors. We discuss whether this observation is relevant to the meiotic drive theory of hybrid male sterility.     

An analysis of univalent segregation in meiotic mutants of Arabidopsis thaliana: a possible role for synaptonemal complex

During first meiotic prophase, homologous chromosomes are normally kept together by both crossovers and synaptonemal complexes (SC). In most eukaryotes, the SC disassembles at diplotene, leaving chromosomes joined by chiasmata. The correct co-orientation of bivalents at metaphase I and the reductional segregation at anaphase I are facilitated by chiasmata and sister-chromatid cohesion. In the absence of meiotic reciprocal recombination, homologs are expected to segregate randomly at anaphase I. Here, we have analyzed the segregation of homologous chromosomes at anaphase I in four meiotic mutants of Arabidopsis thaliana, spo11-1-3, dsy1, mpa1, and asy1, which show a high frequency of univalents at diplotene. The segregation pattern of chromosomes 2, 4, and 5 was different in each mutant. Homologous univalents segregated randomly in spo11-1-3, whereas they did not in dsy1 and mpa1. An intermediate situation was observed in asy1. Also, we have found a parallelism between this behavior and the synaptic pattern displayed by each mutant. Thus, whereas spo11-1-3 and asy1 showed low amounts of SC stretches, dsy1 and mpa1 showed full synapsis. These findings suggest that in Arabidopsis there is a system, depending on the SC formation, that would facilitate regular disjunction of homologous univalents to opposite poles at anaphase I.     

A knockout screen for protein kinases required for the proper meiotic segregation of chromosomes in the fission yeast Schizosaccharomyces pombe

The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation after just single round of DNA replication. To identify novel proteins required for the proper segregation of chromosomes during meiosis, we analyzed the consequences of deleting Schizosaccharomyces pombe genes predicted to encode protein kinases that are not essential for cell viability. We show that Mph1, a member of the Mps1 family of spindle assembly checkpoint kinases, is required to prevent meiosis I homolog non-disjunction. We also provide evidence for a novel function of Spo4, the fission yeast ortholog of Dbf4-dependent Cdc7 kinase, in regulating the length of anaphase II spindles. In the absence of Spo4, abnormally elongated anaphase II spindles frequently overlap and thus destroy the linear order of nuclei in the ascus. Our observation that the spo4Δ mutant phenotype can be partially suppressed by inhibiting Cdc2-as suggests that dysregulation of the activity of this cyclin-dependent kinase may cause abnormal elongation of anaphase II spindles in spo4Δ mutant cells.     

Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis

Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.     

Genetic homology and crossing over in the X and Y chromosomes of mammals

Summary The X-Y crossover model described in this paper postulates that (1) the pairing observed between the X and the Y chromosome at zygotene is a consequence of genetic homology, (2) there is a single obligatory crossover between the X and Y pairing segments, and (3) the segment of the X which pairs with the Y is protected from subsequent X inactivation. Genes distal to the proposed crossover (pseudoautosomal genes) will appear to be autosomally inherited because they will be transmitted to both male and female offspring. Some criteria for identifying pseudoautosomal genes are outlined.The existence of a single obligatory crossover between the X and Y of the mouse is strongly supported by a recent demonstration that the sex-reversing mutation Sxr, which is passed equally to XX and XY offspring by male carriers, is transmitted on the sex chromosomes. Pseudoautosomally inherited genes may also be responsible for XX sex reversal in goats and familial XX sex reversal in man.     

A method for preparation of synaptonemal complexes of meiotic chromosomes from basidial protoplasts of the common mushroom Agaricus bisporus (Lange) Imbach

For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia. It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations were obtained following this approach. The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene-diplotene stage of meiotic prophase I.     

The role of the chromocenter in nonrandom meiotic segregation of nonhomologous chromosomes in Drosophila melanogaster females

The evidence supporting universal significance of physical links between pericentromeric regions of homologous chromosomes for their bipolar orientation during the first meiotic division is discussed. The pericentromeric chiasmata between homologs or (in the absence of the latter) chromocentric links between nonhomologs, which are preserved until prometaphase, compensate for the disturbed binding between homologous pericentromeric regions in both structural or locus mutants. When the links between nonhomologs are involved, interchromosomal effects on chromosome disjunction and nonhomologous pairing were revealed by the genetic methods. An explanation suggested for genetic events observed during Drosophila meiosis conforms with the original, cytogenetically proved model of the orderly two-ring chromocenter formation and reorganization.     

The eects of a ring chromosome on the meiotic segregation of other chromosomes in Saccharomyces cerevisiae

Meiotic chromosome segregation must occur with high fidelity in order to prevent the generation of aneuploid cells. We have previously described the identification and genetic characterization of a yeast mutant with defects in meiotic sister-chromatid segregation. We attributed the phenotype in this mutant to a dominant allele, which we referred to as SID1-1. These mutants appeared to exhibit high levels of nondisjunction and precocious separation of sister-chromatids of chromosome III, as well as precocious separation of sister chromatids of chromosome VIII and a univalent artificial chromosome. We show here that the unusual meiotic behavior of chromosome III in these strains is due to the presence of a ring III chromosome, rather than a mutant gene. Additional experiments demonstrate that a ring III/rod III pair alters the meiotic segregation of a univalent artificial chromosome.