Meiosis and meiotic errors (Genetics)

1. The general scheme of meiosis

The meiotic process involves two cell divisions, and is accordingly divided into two parts, meiosis I and meiosis II (Figure 1). As in mitosis, these divisions are subdivided into the stages Prophase, Metaphase, Anaphase, and Telophase, and thus a full meiotic process includes two of each of these stages. The meiotic process has been recently reviewed by Page and Hawley (2003) and Tease and Hulten (2003). The salient points of each stage are highlighted in Table 1.

Following DNA replication, cells enter meiosis, with each chromosome being composed of two chromatids held together at the centromere. Meiosis I includes a prolonged Prophase in preparation for the halving of chromosome number, this “reductional” division taking place at Anaphase I (AI). Prophase I (PI) is by tradition subdivided into Leptotene, Zygotene, Pachytene, and Diplotene, with an intermediate Diakinesis stage before Metaphase I (MI).

The preparation for the MI/AI spindle to carry its cargo of whole chromosomes (rather than chromatids as in mitosis) takes place at Prophase I and involves intimate pairing of all four chromatids of the maternal and paternal homologs. This intimate pairing (synapsis) is mediated by a proteinaceous structure, called the Synaptonemal Complex (SC). Breakage and reunion (crossing-over) between nonsister chromatids gives rise to configurations known as chiasmata. The chiasmata hold homologous chromosomes together locally during the next phase, when they separate at Diplotene. Following chromatin condensation during the intermediate Diakinesis stage, bivalents align on the MI plate.

It is generally accepted that at least one so-called obligate chiasma is necessary for a normal segregation to take place. The number of additional chiasmata required to be formed to ensure mechanical stability is associated with the length of the chromosomes (Hulten, 1974; Lynn et al., 2002; Tease et al., 2002; Tease and Hulten, 2004). Most importantly, mechanical stability in this context also involves appropriate orientation of maternal and paternal kinetochores (proteinaceous spindle attachments at centromeres) normally facing opposite spindle poles at MI. This is facilitated by the topology of bivalents, where, for example, single-chiasma bivalents appear as cross configurations, doubles as rings, and triples as figures-of-eight. It is important to note that at AI (in contrast to the situation at mitosis) centromeres of homologs are kept together. The chance selection of the paternal and maternal kinetochores of any one bivalent to be presented to the opposite spindle poles leads to their random assortment in daughter cells. Each crossover event and chiasma formation give rise to a recombinant together with a nonrecombinant chromatid, which together are passed on to the daughter cells at AI, providing the basis for additional genetic variation in offspring.

 Schematic illustration of meiosis with (a) homologous chromosome synapsis, and crossing-over at the pachytene stage of prophase I and the derivative bivalents at MI, and (b) progression through metaphase I to anaphase I, metaphase II to anaphase II, and telophase II showing the four potential haploid gametes

Figure 1 Schematic illustration of meiosis with (a) homologous chromosome synapsis, and crossing-over at the pachytene stage of prophase I and the derivative bivalents at MI, and (b) progression through metaphase I to anaphase I, metaphase II to anaphase II, and telophase II showing the four potential haploid gametes

Table 1 Salient points of meiotic stages

Stage Subdivision Features
Prophase 1 DNA replication of each chromosome to give sister chromatids held together at the centromere
Leptotene Start of chromosome condensation, formation of the lateral elements of the synaptonemal complex
Zygotene Chromosome pairing; completion of the formation of the synaptonemal complex
Pachytene Chromosomes fully paired, crossovers/chiasmata fully established
Diplotene Initiation of separation of the synapsed chromosomes
Diakinesis Chromosomes condense and become fully separated, except at points of crossing-over/chiasma formation.
Metaphase 1 Homologous chromosomes align on the equatorial plate with the kinetochores being the point of attachment to the spindles
Anaphase 1 Reductional division; homologous pairs separate, but sister chromatids remain together
Telophase 1 Formation of two daughter cells
Prophase 2 Nuclear envelope dissolves; creation of a new spindle
Metaphase 2 Chromosomes align on the spindle
Anaphase 2 Separation of centromeres; migration of sister chromatids to opposite poles
Telophase 2 Further cell division resulting in four potential haploid gametes from each parent cell

After a brief PII without DNA replication, the two daughter cells complete meiosis II to produce four end products. This “equational” division is similar to mitosis. It should be recognized though that normally at least one of the sister chromatids (potential gametes) of each chromosome now is recombinant, containing a combination of variant genes (alleles) from the maternal and paternal homologs (Figure 2).

2. Variation between the two sexes

Interestingly, there are many differences in behavior between male and female germ cells during gametogenesis and meiosis. This includes the timing and continuity of events, the pairing and crossover processes, chromosome segregation, and the actual gamete produced – two cells as diverse as the oocyte and the sperm could hardly be imagined to derive from the same process! These differences are summarized in Table 2, and some of the more interesting aspects are discussed in more detail below.

Schematic illustration of the relationship of a crossover, as viewed directly (as a chiasma or MLH1 focus) and the meiotic products (gametes). There are four chromatids, each a potential gamete, only two of which are recombinant

Figure 2 Schematic illustration of the relationship of a crossover, as viewed directly (as a chiasma or MLH1 focus) and the meiotic products (gametes). There are four chromatids, each a potential gamete, only two of which are recombinant

2.1. Timing of meiosis

Meiosis in males starts after puberty and continues throughout life. On the other hand, meiosis in females begins around the 12th week of fetal life with homolog pairing, crossing-over, and chiasma formation up until around week 20. Oocytes arrest at the Diplotene stage, and are generally thought to undergo extensive cell death with numbers dropping from around 7 million to around 2 million at birth (Baker, 1963; Tilly, 2001). Meiosis resumes after puberty, when after the midcycle Luteinizing Hormone surge, chromosomes line up on the MI plate and undergo the reductional division at AI just before ovulation. This division is asymmetrical, where most of the cytoplasm is retained by one of the daughter cells, which will form the future mature oocyte. The other daughter cell forms a small polar body, which soon degenerates. Following progression through the second prophase, oocytes then arrest at MII until fertilization occurs. At fertilization, once the sperm has entered the oocyte and caused activation, oocyte meiosis continues through AII and TII, resulting in the ejection of the second polar body.

2.2. The synaptonemal complex

Meiotic chromosome pairing at PI involves three successive developmental stages, homolog recognition, presynaptic alignment, and intimate synapsis. This precisely timed process is mediated by the proteinaceous SC. The SC forms a protein scaffold for meiotic chromosome pairing taking place during PI, that is, during the substages Leptotene, Zygotene, and Pachytene. The SC then breaks down at Diplotene.

Table 2 Summary of the main differences between male and female meiosis

Male Female
Meiosis is a continuous process from puberty throughout life, and can result in the production of around 200-300 million spermatozoa daily. A full cycle of spermatogensis takes approximately 120 days, of which 72-74 days are spent during meiosis.

Each parent cell produces 4 gametes (spermatozoa), all divisions being equal.

The testis contains a population of stem cells that give rise to the continual supply of gametes.

Chromosome synapsis is initiated very near the ends of chromosomes in karyotypically normal, fertile men.

Obligate chiasma formation is an efficient process.

Lower overall chiasmata frequency.

Synaptonemal complexes are condensed.

Tendency of chiasmata to occupy

preferential positions with hotspots near the ends of chromosomes.

Meiosis can take over 40 years from start to finish and only a few oocytes actually progress to the final stages, most being lost before birth.

The two cell divisions are unequal with most cytoplasm retained in the oocyte and only a minor part forming the first and second polar bodies, thus only one actual gamete is produced from each parent cell.

Oocyte numbers appear to be limited to those present at birth with around 350 ovulating between puberty and the menopause. However in mice, recent experimentation has suggested the existence of stem cells that may constitute a reserve (Johnson et al., 2004).

Interstitial initiation of synapsis is common in females.

Poor efficiency of obligate chiasma formation.

Higher overall chiasma frequency.

Synaptonemal complexes are relatively decondensed, having almost twice the total length per cell compared to that in males.

Chiasmata occupy more interstitial positions.

The SC is a protein lattice that resembles railroad tracks and connects paired homologous chromosomes in most meiotic systems. The two side rails of the SC, known as lateral elements, are connected by proteins known as transverse filaments (reviews in Moens et al., 2002; Page and Hawley, 2004).

The SC was first discovered in mice and was later found to be evolutionarily conserved (albeit with species- and sex-specific modifications). Many antibodies against the proteins involved are available, providing tools for SC research using immunofluorescence techniques. Fluorescence in situ hybridization (FISH) methods have also been used sequentially to highlight the behavior of some of the SC proteins involved and their interaction with chromatin components during the meiotic chromosome pairing stages in human spermatocytes and oocytes (Barlow and Hulten, 1996; Barlow and Hulten, 1997; Barlow and Hulten, 1998a; Codina-Pascual et al., 2004; Hartshorne et al., 1999; Prieto et al., 2004; Sun et al., 2004; Lenzi et al., 2005).

Schematic illustration of the SC

Figure 3 Schematic illustration of the SC

Even before meiosis is initiated, some “meiosis-specific” proteins, members of the cohesin complex (such as REC8 and STAG3) appear as diffuse aggregations within the cell nuclei. Subsequently, these cohesins function to stick chromatids together by interacting with the chromatin (DNA and proteins) of chromosomes to form initial cohesion axes.

During the initial development of the SC, most of the DNA form large loops emanating from the sides of the SC in a “chromatin cloud” (Figure 3). The chromatin is attached to the initially unpaired axial, and later, paired lateral elements of the SC by the cohesin proteins and the protein SYCP3 acting as “glue” between chromatids. These cohesion axes become “sandwiched” between chromatin and the SC axial elements marked by SYCP3. Pairing is subsequently stabilized at intimate synapsis, with transverse filaments developing between the lateral elements of the SC, forming a central element composed of proteins including SYCPI.

2.3. Chiasma formation

It is now generally accepted that initiation of crossing-over and chiasma formation in mammals takes place via double strand breaks (DSBs) induced by the protein SPO11 at Leptotene; and a range of proteins are involved in subsequent synapsis and recombination events (Bannister and Schimenti, 2004; Roig et al., 2004; Svetlanov and Cohen, 2004; Lenzi et al., 2005). Numerous DSBs (visualized by the y-H2XA antibody) are initially created in DNA loops located outside the SC. Only a minority of this DNA subsequently becomes tethered to the core of the SC, exposing them to the recombinatorial machinery, associated with successive conversion and crossover events mediated by proteins such as RAD51 and MLH1 (Barlow et al., 1997).

The application of anti-MlH1 (MutL homolog1) to human SC preparations shows a labeling pattern consisting of distinct foci, always precisely associated with the SC and never in closely juxtaposed pairs (Figure 4). The frequency distribution of male MLH1 foci agrees with that of chiasmata at Diakinesis/MI (Figure 5); and there is now no doubt that MLH1 is an appropriate marker for chiasma formation (Marcon and Moens, 2003).

MLH1 foci on SCs of spermatocyte (left) and oocyte (right). Note the near-telomere preference in the spermatocyte and the higher number of foci in the oocyte

Figure 4 MLH1 foci on SCs of spermatocyte (left) and oocyte (right). Note the near-telomere preference in the spermatocyte and the higher number of foci in the oocyte

The average crossover/recombination frequency estimated from MLH1 analysis in human males is around 50 with a range of 40-60 (Barlow and Hulten, 1998b; Lynn et al., 2002; Tease et al., 2002; Hulten and Tease, 2003a; Sun et al., 2004; Tease and Hulten, 2004), while that of females is around 70 but with a much larger variation between individuals than in males (Barlow and Hulten, 1998b; Tease et al., 2002; Roig et al., 2004; Tease and Hulten, 2004; Lenzi et al., 2005).

2.4. Patterns of chiasma distribution

A large database of patterns of chiasmata has accumulated over the last three decades by the study of Diakinesis/MI spermatocytes in preparations from testicular biopsy samples in human males, demonstrating that chiasma formation is a dynamic process, varying between chromosomes, cells, and subjects (see Article 11, Human cytogenetics and human chromosome abnormalities, Volume 1 and Article 20, Cytogenetics of infertility, Volume 1). We have quite a good knowledge on the basic patterns and its variation between individual chromosomes, cells, and subjects in normal fertile men, as well as changes associated with infertility, and in carriers of structural chromosome rearrangements (review in Hulten and Tease, 2003a,b; Oliver-Bonet et al., 2004). On the other hand, our knowledge on the underlying mechanisms of regulation of this basic pattern and its variations is still very poor. For technical reasons, the same type of information on chiasmata at the Diakinesis/MI stage is not available in human females.

The morphology of MI and MII chromosomes is very different

Figure 5 The morphology of MI and MII chromosomes is very different

One important role of chiasma formation is to link chromosome pairs in such a way that whole chromosomes are conveniently transported to daughter cells at AI. Chiasma formation allows maternal and paternal homologs to be presented together to the MI-AI spindle so that daughter cells may receive a combination of these chromosomes rather than exclusively maternal or paternal homologs. Together with the random presentation of paternal and maternal kinetochores to opposite spindle poles, this provides the basis for the “infinite” genetic variability of offspring (see Article 16, Nondisjunction, Volume 1). The 3D topology of bivalents at this stage means that chiasmata are locked in their original positions owing to the behavior of bivalents during the transition from Pachytene through Diplotene and Diakinesis to MI (Hulten, 1990). This means that chiasmata do not need to “terminalize” in order to resolve. Thus, the long-held belief, originally proposed by Darlington, that chiasmata must terminalize in order to resolve is not a valid proposition.

3. Obligate chiasma formation and implications of chiasma failure

In the normally fertile human male, one chiasma is generally formed per bivalent, irrespective of its length; this chiasma is considered to be obligate to secure “regular” segregation of the chromosomes involved, by allowing the maternal and paternal chromosomes of the bivalent to be properly orientated on the MI spindle. Failure of chiasma formation means that maternal and paternal homologs appear as disoriented univalents, leading to random rather than regular segregation. Daughter cells may thus receive a paternal, a maternal, both, or none of these (nonexchanged) chromosomes. One other complication of this situation is the potential for chromatids of univalent chromosomes to undergo precocious separation followed by disoriented segregation, leading to daughter cells receiving one, two, or no chromatids, instead of a whole (maternal, paternal, or recombinant maternal-paternal) chromosome. Univalent chromosomes are very rarely seen in male meiosis either during Pachytene (Barlow and Hulten, 1998b; Lynn et al., 2002) or at Diakinesis/MI (Hulten, 1974; Laurie and Hulten, 1985).

Efficient obligate chiasma formation in the human male stands in sharp contrast to the situation in females. Little information exists on patterns of chiasmata at the MI stage in human oocytes, however, recent SC and MLH1 observations on human fetal oocytes quite clearly demonstrate failure of synapsis and chiasma formation in a large proportion at the Pachytene stage (Tease et al., 2002).

4. Additional chiasmata, interference, and crossover hotspots

Numbers of additional chiasmata over and above the obligate are dependent on chromosome length (Hulten, 1974; Lynn et al., 2002; Tease et al., 2002; Tease and Hulten, 2004). Successive chiasmata are separated by large (many Mb) chromosome segments, a phenomenon termed chiasmata interference (Figure 6). Measurements of distances between MLH1 crossover foci on the SCs at Pachytene indicate interference distances in terms of physical SC length to be basically the same in human spermatocytes and oocytes (Tease and Hulten, 2004). This may “explain” the higher chiasma frequency in human females in comparison to males because the SCs in oocytes are relatively “decondensed” and much longer than in spermatocytes (Wallace and Hulten, 1985).

Chiasmata have a tendency for sex-specific preferential positioning (Figure 7) at crossover hotspots, occupying large (many Mb) stretches of chromosomes, which we may term megahotspots. A different type of preferential crossover positioning has more recently been revealed by DNA analysis of sperm. Comparison of certain DNA markers in somatic tissue (using e.g., blood samples) and sperm has demonstrated crossover hotspots stretching over a few kilobasepairs separated by longer noncrossover, so-called haploblock intervals, in the order of 50-100kb. Such fine-scale hotspots were originally discovered because they may occur in the vicinity of hypervariable DNA sequences (minisatellites) that have been used extensively for “DNA fingerprinting” in forensics (Jeffreys et al., 1985). It has been suggested that these types of hotspots, which we may refer to as minihotspots, represent a general situation of “punctuate” preferential crossover positioning across the whole genome (review in Jeffreys et al., 2004). The relationship of these minihotspots to the megahotspots identified by MLH1 and chiasma analysis remains unknown, and requires further study.

The distribution patterns of MLH1 foci on chromosome 21 SCs in oocytes illustrating the difference with one in comparison to two foci, which are widely spaced apart

Figure 6 The distribution patterns of MLH1 foci on chromosome 21 SCs in oocytes illustrating the difference with one in comparison to two foci, which are widely spaced apart

Genetic maps of chromosome 21 illustrating high amount of distal crossovers and the corresponding expansion of genetic length distally in the male (blue) in comparison to that in the female (red)

Figure 7 Genetic maps of chromosome 21 illustrating high amount of distal crossovers and the corresponding expansion of genetic length distally in the male (blue) in comparison to that in the female (red)

5. Construction of genetic maps

Genetic maps illustrate the frequency of crossovers that on average take place along the length of each chromosome arm. The construction of genetic maps from crossover data obtained by cytogenetic analysis in individual cells has many advantages (review in Hulten and Tease, 2003b). First of all they allow sex-specific estimates of both intra- (intercellular) and interindividual variation in patterns of meiotic recombination with respect to the whole genome, individual chromosomes, and chromosome segments. They also distinguish between reciprocal recombination (crossing-over/chiasma formation) and other types of recombination events (such as sister chromatid exchange and conversion). In addition, the direct meiotic crossover analysis allows estimates of genetic map distances as well as recombination fractions from the same observed raw data.

Haldane (1919) originally defined the Morgan unit of genetic map distance as that length of a chromatid that has experienced on average one crossover per chromatid. Each chromatid in this situation corresponds to a potential gamete

(Figure 2). As each crossover event (MLH1 focus/chiasma) may give rise to two recombinant as well as two nonrecombinant gametes, the genetic map distance (Morgan, M) is calculated as half the average number of MLH1 foci or chiasmata in the interval concerned. In other words, the genetic map distance in centimorgans (cM) is obtained by multiplying the average number by 50, thus an average of 50 autosomal chiasmata in spermatocytes corresponds to a male genetic map length of 2500 cM, while 70 MLH1 foci in oocytes translate to a female genetic map length of 3500 cM. As already mentioned, these averages, however, hide a substantial interindividual variation in crossover frequency. Effectively, we all have our own average genetic maps with further individual specificity of the particular sperm and egg transmitted to offspring.

Assuming there is no chromatid interference but successive crossover chromatids are chosen by chance, then the corresponding recombination fraction is calculated as half the proportion of cells having one or more MLH1 focus/chiasma in the interval. Most human genetic maps have been constructed by tracing DNA markers through generations where the raw data are recombination fractions between markers (e.g., Kong et al., 2002; Cullen et al., 2002). This approach has the advantage that recombination data are linked to genes or DNA sequences. On the other hand, relatively large sample sizes of families/sibships are required, and importantly the analysis recovers only 50% of recombinants from each parental crossover event and only 25% of doubles. Assumptions are also required on the risk of double crossovers within the interval studied. Double crossovers involving the same DNA strand would cancel each other out, a phenomenon that is taken into account and compensated for by the use of the so-called mapping functions, estimating crossover/chiasma interference and thus the risk of double crossovers. Earlier linkage-based genetic maps showed large discrepancies in relation to those based on cytogenetic data with a general tendency for inflation of linkage-based genetic map lengths (likely due to overcompensation for presumed double recombinants) (see Article 54, Sex-specific maps and consequences for linkage mapping, Volume 1, Article 9, Genome mapping overview, Volume 3, Article 15, Linkage mapping, Volume 3 and Article 67, History of genetic mapping, Volume 4). The most recent ones, however, are very similar, at least as regards total genetic map length for each individual chromosome (Kong et al., 2002; Cullen et al., 2002; review in Hulten and Tease, 2003b). The high density of DNA markers now available should in our view make traditional mapping processes redundant. Previous complications in estimating genetic map distance by family studies due to misinformation as regards order of loci, should also now be much less of a problem, due to the detailed information on DNA marker order available from the Human Genome Project (Collins et al., 2004; see also Article 24, The Human Genome Project, Volume 3).

6. The transition from metaphase I to anaphase I

From Pachytene until MI, all four chromatids are “glued” together along their entire length (Figure 1). At MI, each pair of sister chromatids presents as a unit with both sister kinetochores facing in the same direction. This means that both sister chromatids of a homolog are pulled to the same pole, once AI is initiated (review in Hauf and Watanabe, 2004). It appears from animal experimentation that AI chromosome segregation cannot start until all bivalents have orientated themselves on the MI plate with kinetochores of homologs facing opposite spindle poles, thus there may be a long delay before the onset of AI takes place. Once initiated however, this is seemingly a very rapid process, as indicated by, for example, the absence of any AI spermatocytes in preparations from human testicular biopsy samples.

A wealth of information about the complex interaction of proteins between kinetochores and spindle microtubules at the MI to AI transition has recently accumulated (reviewed in Nasmyth, 2002; Maiato et al., 2004; Hauf and Watanabe, 2004; Firooznia et al., 2005). A range of chromosome-associated proteins (either meiosis-specific or showing meiosis-specific behavior) have been identified including meiosis-specific cohesins, and centromere/kinetochore-associated proteins. The exactly timed behavior of these proteins ensures that normally all four chromatids are “glued together” until the onset of Anaphase I and retained within the cen-tromere/kinetochore region until Anaphase II. It is also known that unattached kine-tochores cause the delay of AI initiation through the action of several “wait” signals including Mad1/Mad2, Cdc20 and Bub1, localized to kinetochores (Howell et al., 2004; Vigneron et al., 2004; Shah et al., 2004; Taylor et al., 2004); subsequent attachment of these kinetochores then involves the activation of proteins forming the Anaphase Promoting Complex (APC). On the other hand, lapse of cohesion between chromatids, located peripheral to the chiasma, is obligatory to allow regular segregation of homologs at Anaphase I; and lapse of centromere/kinetochore-specific proteins is essential for regular Anaphase II segregation.

Activation of the APC in turn leads to Separase activation (Terret et al., 2003). Once activated, Separase is able to cleave cohesin complexes, allowing chromatids to separate along chromosome arms, initiating the MI-to-AI transition. It is important to note that only chromatid arm cohesion, necessary for separation of homologs is lost at AI. Sister chromatids remain connected via the action of amongst others, the cohesin REC8, STAG3, and Shugoshin at the centromere up until MII (Lee et al., 2003; Watanabe, 2004). It is not yet clear how the absolutely crucial protection of these cohesins from cleavage at centromeres during Meiosis I is regulated. Neither is it known why Meiosis I chromosome segregation is so error-prone in human females (see further below) in relation to males (and in relation to other mammalian species such as mice).

7. MII analysis for evaluation of AI segregation

Evaluation of the efficacy of AI segregation can be made by chromosome analysis of cells at the MII stage. Information in the male may be obtained by investigation of spermatocytes at MII, prepared from testicular biopsies in a way very similar to that used for making preparations from blood lymphocytes for somatic chromosome analysis. Cells are initially exposed to a hypotonic solution and then fixed in a (3:1) mixture of acetic acid and alcohol (Hulten et al., 2001). One particular problem here is that chromatids of individual MII chromosomes in spermatocytes prepared this way often splay to the extreme and chromatids are often precociously separated. In addition they are loosely coiled and have a tendency to hook into each other (Figure 5). This makes even the counting of chromosomes problematic. Laurie et al. (1985) using stringent criteria to avoid artifacts did not detect any numerical abnormalities in 200 MII spermatocytes from six normal men with apparently normal mitotic karyotypes. Thus, there was no indication of either extra or missing chromosomes or indeed any extra or missing chromatids. To our knowledge no other studies have to date been presented on this issue. We may tentatively conclude that AI malsegregation in the human male is rare, and occurs with an incidence of less than 0.5-1% of cells.

This situation stands in sharp contrast to that in the human female. Chromosome analysis of a large population of oocytes at the MII stage has been performed. These studies demonstrate quite clearly that segregation errors involving both whole chromosomes and chromatids are common (review in Pellestor et al., 2003). It should be noted, however, that the material investigated so far consists almost exclusively of MII oocytes obtained at infertility (IVF) treatment, where spare oocytes, spontaneously arrested at MII, have been donated for research and used for karyotyping.

The morphology of oocyte MII chromosomes makes them much more amenable to karyotyping than is the case for spermatocytes; chromatids do not normally splay to the extent that they do in spermatocytes (Figure 5). Oocyte MII chromosomes are also more condensed and therefore more easily spread and separated from each other. A total of nearly 5000 human oocytes at the MII stage have been investigated with a view to obtain information on the efficacy of AI segregation as well as the occurrence of structural chromosome aberrations. Two types of methods have been used for making preparations including that of Kamiguchi et al. (1993), involving a gentle gradual fixation. Chromosome analysis has been performed by conventional (block staining as well as R-banding) methods, FISH for selected chromosomes and spectral karyotyping.

The largest study so far is that of Pellestor et al. (2002) involving 1397 MII oocytes, fixed by the Kamiguchi et al. (1993) technique and analyzed by R-banding. Numerical abnormalities were detected in around one-fifth (20.1%), mainly composed of extra or missing whole chromosomes or chromatids, while structural abnormalities (breaks, deletions and, acentric fragments) were much more rare, seen in 2.1% of cells. Numerical abnormalities caused by extra or missing chro-matids were more common than aneuploidy of whole chromosomes, confirming the proposal by Angell that AI segregation errors of chromatids are common in human oocytes obtained through IVF studies (Angell, 1997). One very interesting observation in the Pellestor series is the strong positive correlation between maternal age and rate of aneuploidy, most pronounced as regards single-chromatids. The same has been seen in a small sample of fresh oocytes (not stimulated by hormone injections as part of IVF treatment) by spectral analysis (Sandalinas et al., 2002).

Another way of assessing AI segregation errors is by FISH analysis of the first polar body (review in Kuliev and Verlinsky, 2004). This has been achieved by micromanipulation of the polar bodies with subsequent FISH with 3-5 chromosome-specific probes as part and parcel of preimplantation genetic diagnosis (PGD) (see Article 21, Preimplantation genetic diagnosis for chromosome abnormalities, Volume 1 and Article 22, FISH, Volume 1). Only the “aneuploidy-free” embryos have been transferred (following FISH analysis of the second polar body as well, as described further below). The largest study here is that summarized by Kuliev et al. (2003) involving 6733 oocytes from women above the age of 35 (average 38.5 years). In this series, a large proportion of oocytes were found to be aneuploid with respect to the chromosomes tested (chromosomes 21, 22, 13, 15, and 18). Thus, 41.7% of oocytes were considered to be aneuploid because of AI malsegregation, the majority again involving chromatids rather than whole chromosomes.

8. Gametic output

Investigations of chromosomes in mature gametes allow information to be obtained on the sum total of segregation errors including both AI and AII, as well as any structural chromosome aberrations that have occurred.

Chromosome analysis of mature gametes has primarily involved sperm (see Article 25, Human sperm-FISH for identifying potential paternal risk factors for chromosomally abnormal reproductive outcomes, Volume 1). Earlier studies were performed by the so-called humster technology (human sperm and hamster ovum pseudofertilization). This type of investigation is laborious and expensive, and has been restricted to a relatively few research groups. Guttenbach et al. (1997) reviewed observations by eight groups, involving over 20 000 sperm karyotypes from healthy donor men. Aneuploidy was detected in 1-3% of sperm. Another 5-10% showed structural chromosome abnormalities, many of which are presumed to have arisen postmeiotically, during differentiation of spermatids to mature sperm.

More recently FISH has been applied for analysis of selected chromosomes on very large populations of sperm from both chromosomally normal men and carriers of structural chromosome rearrangements. Shi and Martin (2000) have reviewed the experience gained on an impressive sample of more than 5 million sperm from around 500 normal fertile men, using one-, two-, or three-probe FISH. By and large, these studies have confirmed the findings using the humster technique that around 2-3% of mature spermatozoa are aneuploid.

Investigations of individual chromosomes indicate that their average numerical abnormality rate is around 0.1-0.2%. Only chromosomes 21 and 22 and the XY pair show slightly increased rates of sperm aneuploidy. The question whether some men may be predisposed to aneuploid offspring has not yet been answered with any certainty (Martinez-Pasarell et al., 1999; Soares et al., 2001a,b; Shi et al., 2001). On the other hand, tracing of DNA markers between fathers and XXY Klinefelter sons has demonstrated a reduced XY recombination, where the implication is that this has led to XY disomy in sperm (Thomas and Hassold, 2003; see also Article 19, Uniparental disomy, Volume 1).

FISH analysis of sperm from carriers of structural chromosome rearrangements, such as translocations, has shown a high rate of imbalance for the chromosomes involved (review in Shi and Martin, 2001; Oliver-Bonet et al., 2004).

As might be expected, there is much less direct cytogenetic information on gametic output including AII segregation errors in females. One intriguing way of approaching this question is FISH analysis of not only the first polar body (as described above) but also the second polar body, obtained for analysis by micromanipulation at PGD treatment in pregnancies (review in Kuliev and Verlinsky, 2004). One example of this approach concerns the FISH study, summarized by Kuliev et al. (2003) on 6733 oocytes/fertilized eggs, where the conclusion was that AII segregation errors, as regards the chromosomes tested (chromosomes 21, 22, 13, 15, 16, and 18) had occurred in a slightly lower proportion of cases than AI errors, that is, in 30.7%. Remarkably, however, more than one-quarter were found to have both AI and AII segregation errors (27.6%).

9. Mechanisms of meiotic errors

Hardly anything is so far known from direct meiotic studies on the mechanisms of origin of structural chromosome rearrangements, including, for example, extra marker chromosomes, Robertsonian and reciprocal translocations, inversion, insertions, deletions, and duplications. Some of these may arise from breakage and repair processes taking place either pre- or postmeiosis. Special attention has been paid to the origin of de novo deletions and duplications, comprising the so-called genomic disease (Shaw and Lupski, 2004). The suggestion from somatic DNA marker investigations of children and their parents is that these chromosome disorders originate by misalignment of homologs at Meiosis I Prophase, followed by “ectopic” crossing-over/chiasma formation/recombination between similar (paralogous) DNA sequences, located at a distance from each other, either within the same homolog or indeed on different homologs. To our knowledge, no investigation has yet been performed to identify such ectopic crossovers directly by investigation of SCs. However, misalignment has been clearly documented in human oocytes, in which centromere signals (by the CREST antibody) are often staggered (Hartshorne et al., 1999).

Attention has focused on aneuploidy, which is a major cause of reproductive failure and congenital disease with special reference to common chromosome disorders such as Trisomy 21 Down, Trisomy 13 Patau and Trisomy 18 Edward Syndromes as well as the sex chromosome aneuploidies XXY Klinefelter, XYY, XXX, and X Turner syndromes. Much effort has been devoted to explaining in particular the maternal age effect especially noticeable as regards Trisomy 21 Down syndrome, this being the most common genetic disorder in humans.

It is known from previous indirect studies, tracking DNA polymorphisms between parents and children, that patterns of meiotic recombination (deciding the formation of bivalents and their shape) play an important role (Hassold and Hunt, 2001). However, it is not yet clear how this may work in detail. Most segregational errors underlying these aneuploidies have been found to take place at maternal AI; and it has, for example, been suggested that certain chiasma positions, such as a single distal chiasma of bivalent 21 may constitute a specific risk factor. On the other hand, the very same position is one of the most common in spermatocytes (Hulten, 1974; Laurie et al., 1985; Hulten and Tease, 2003b), where AI segregation errors are uncommon, and there is no known paternal age effect in this respect. Clearly a so-called second hit must be of paramount importance in handling such oocyte bivalents differently from spermatocytes at a later stage. What would this be is yet unclear.

More detailed information on the intricate interaction between chromosomal and spindle proteins in oocytes at MI and AI in particular may provide some answers including an explanation for the well-known maternal age effect. Recent work indicates that knowledge obtained from the mitotic cell cycle may to some extent be extrapolated to meiosis, which opens up new avenues here. It has been suggested that age-related reduction in some proteins (such as the kinetochore-bound spindle checkpoint proteins Mad1 and Bub2 and/or the meiosis-specific cohesins) in combination with the specific spindle morphology and large size of the oocyte nucleus may play specific roles (review in Eichenlaub-Ritter, 2004). Quantitative analysis of the mRNA of some of these proteins has recently revealed an age-related degradation in human oocytes (Steuerwald et al., 2000). There is also a possibility that the morphology of the centromeres may contribute, where those with small amounts of specific (alphoid) centromeric DNA may be disadvantaged (Maratou et al., 2000). Furthermore, oocyte Anaphase I spindles have been shown to be barrel shaped rather than triangular as in spermatocytes. This may make the task of chromosome segregation more difficult. Remarkably, there are clear indications that Meiosis I spindles in oocytes obtained from older women are much more irregular than those from younger women (Battaglia et al., 1996; Volarcik et al., 1998).

Many factors may contribute to the formation of spindles including a range of spindle proteins, mitochondrial and hormonal status, follicle maturation in relation to the oocyte pool and peri-follicular microcirculation, and age-related changes in any of these factors may play a role.

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