Chromosomal Strategies for Adaptation to Univalency

The orientation and segregation behaviour of different types of univalents, namely sex chromosomes, B chromosomes and autosomal univalents, were analysed in living spermatocytes of eight evolutionarily distant grasshopper species. The meiotic behaviour of each univalent was characterized in terms of velocity of prometaphase movements, frequency of reorientations, types of final orientation at metaphase I and modes of segregation at anaphase I. All these features were found to vary between different univalents. Certain combinations of these traits, defining a chromosomal strategy, appear commonly together in certain chromosome types, indicating that they are the result of selection acting on the chromosomes to increase transmission effectiveness. The sex univalents show in general a strategy in which all the features favouring an eventual equational segregation at anaphase I tend to be minimized. There is much more variation in behaviour among B chromosomes than among X chromosomes, which is a reflection of their heterogeneous nature. Induced autosomal univalents are studied in Locusta migratoria. They show a very irregular behaviour, indicating their lack of adaptation to univalency.     

Meiosis in chromosomally heteromorphic goitered gazelle,Gazella subgutturosa (Artiodactyla,Bovidae)

Chromosomal-pairing behaviour was studied in the spermatocytes of individual goitered gazelles which were heteromorphic for a 14/15 Robertsonian translocation and which possessed an autosome-to-X translocation. Both translocations exhibited trivalent pairing configurations in pachytene and diakinesis/metaphase I nuclei. Synapsis of the sex chromosomes during pachynema was followed by end-to-end association of the X and Y during diakinesis/metaphase I. The only univalents identified were of the Y chromosome; Y univalency ranged from 15.9% at pachynema to 5.7% at diakinesis/metaphase I. Robertsonian trivalents exhited evidence of synaptic adjustment in the paracentromeric region. Chiasmata were formed in most bivalents and trivalents; chiasmata were restricted to the autosomal portion of the autosometo-XY trivalent. Analysis of metaphse II configurations (secondary spermatocytes) revealed no nondisjunction in individuals homozygous or heterozygous for the Robertsonian translocation. These data are consistent with the hypothesis that neither the autosomal nor the gonosomal heteromorphism reduces the meiotic fitness of male goitered gazelles.     

Meiotic behaviour of holocentric chromosomes: orientation and segregation of autosomes in Triatoma infestans (Heteroptera)

The meiotic behaviour of the holocentric chromosomes of the heteropteran species Triatoma infestans has been analysed by means of orcein staining and C-banding on squashed spermatocytes. We have focused our analysis on chromosome 3, which shows a large distal heterochromatic band at one of the ends of both homologues. At metaphase I,and independently of the chiasma position, two alternative orientations have been observed: either the hetero-chromatic or the euchromatic ends of both homologues are directed to opposite poles. At anaphase I, the kinetic activity is restricted to the same chromosome end (euchromatic or heterochromatic) of each homologue. The frequencies of these two alternatives are not random and differ significantly among the five individuals analysed. However, the euchromatic ends present kinetic activity at a higher frequency than the heterochromatic ends. At metaphase II, half-bivalents also show the kinetic activity restricted to either of the chromosome ends (euchromatic orheterochromatic). The frequencies of each alternative are inverted in anaphase II compared with those scored in anaphase I. Accordingly, those ends that present kinetic activity at anaphase I segregate reductionally during the first meiotic division and equationally during the second meiotic division. These results provide sound evidence on the meiotic behaviour of holocentric chromosomes, as regards the absence of chiasma terminalization and the modes of orientation and segregation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Meiosis in Holocentric Chromosomes: Orientation and Segregation of an Autosome and Sex Chromosomes in Triatoma infestans (Heteroptera)

The meiotic behaviour of the X chromosome and one autosomal pair of the heteropteran Triatoma infestans was analysed by means of C-banding plus DAPI staining. At first metaphase, the X univalent is oriented with its long axis parallel to the equatorial plate, which suggests a holocentric interaction with the spindle fibres. After this initial orientation, kinetic activity is restricted to one of both chromatid ends. The election of the active chromatid end is random and it is independent of the end selected in the sister chromatid. At second metaphase, the X and Y chromatids associate side by side forming a pseudobivalent. After that, the kinetic activity is again restricted to either of both chromosomal ends in a random fashion. At first metaphase, the fourth autosomal bivalent shows two alternative random orientations depending on the chromosome end showing kinetic activity (DAPI positive or opposite). At second metaphase, half bivalents are oriented with their long axis parallel to the equatorial plate. Three different segregation patterns are observed. The kinetic activity can be localised: (i) in the end with the DAPI signal (46.9%), (ii) in the opposite end (44.6%) or (iii) in one DAPI-positive end in one chromatid and in the opposite end in the other one (8.5%). The existence of the last pattern indicates that the same end can show kinetic activity during both meiotic divisions. Our results provide new information on the comparative meiotic behaviour of autosomes and sex chromosomes in holocentric systems.     

Segregation of holocentric chromosomes at meiosis in the nematode,Caenorhabditis elegans

The meiotic segregation of the holocentric chromosomes ofCaenorhabditis elegans in both spermatogenesis and oogenesis is described. The extended kinetochore typical of the mitotic chromosome could not be differentiated on meiotic bivalents; instead microtubules appeared to project into the chromatin. The meiotic spindles formed during spermatogenesis contain centrioles and asters, while in oogenesis the spindles are acentriolar and barrel shaped. The formation of the acentriolar spindle was studied in fixed specimens by anti-tubulin immunofluorescence. Microtubule arrays were seen first to accumulate in the vicinity of the meiotic chromosomes prior to congression. At later stages, elongated spindle structures up to 13 in length were observed parallel to the surface of the embryo. Further development of the spindle appeared to involve its shortening into a barrel shape and rotation so that one spindle pole was opposed to the membrane. By anaphase the pole-to-pole spindle length reached a minimum of 3–4 . One end of each chromatid in the meiotic bivalent was labelled byin situ hybridization of a probe DNA to show that in oogenesis the chromatids were associated end-to-end in the bivalent. Furthermore, either the right or the left ends of the homologues could be held in association. At metaphase I the bivalents were oriented axially, such that kinetic activity was restricted to one end of each pair of sister chromatids. At metaphase II the chromosomes were also aligned axially.     

Hyperthermia induced dissociation of the X-Y bivalent in mice

The mutagenic potential of hyperthermia was examined employing a spermatocyte test on males heat stressed continuously for 2, 3, or 5 days. Stress conditions were 35 ± 1° C and 65 ± 3% relative humidity. Males were sacrificed and meiotic preparations made from the testes at various intervals following removal from the stress environment. In this way, chromosome damage could be monitored in all premeiotic and early prophase I stages of spermatogenesis. Results of this study revealed a significant increase in the incidence of X and Y univalents at metaphase I. The significance of this finding is discussed.     

A Caenorhabditis elegans cohesion protein with functions in meiotic chromosome pairing and disjunction

We have studied four Caenorhabditis elegans homologs of the Rad21/Scc1/Rec8 sister-chromatid cohesion protein family. Based on the RNAi phenotype and protein localization, it is concluded that one of them, W02A2.6p, is the likely worm ortholog of yeast Rec8p. The depletion of C. elegans W02A2.6p (called REC-8) by RNAi, induced univalent formation and splitting of chromosomes into sister chromatids at diakinesis. Chromosome synapsis at pachytene was defective, but primary homology recognition seemed unaffected, as a closer-than-random association of homologous fluorescence in situ hybridization (FISH) signals at leptotene/zygotene was observed. Depletion of REC-8 also induced chromosome fragmentation at diakinesis. We interpret these fragments as products of unrepaired meiotic double-stranded DNA breaks (DSBs), because fragmentation was suppressed in a spo-11 background. Thus, REC-8 seems to be required for successful repair of DSBs. The occurrence of DSBs in REC-8-depleted meiocytes suggests that DSB formation does not depend on homologous synapsis. Anti-REC-8 immunostaining decorated synaptonemal complexes (SCs) at pachytene and chromosomal axes in bivalents and univalents at diakinesis. Between metaphase I and metaphase II, REC-8 is partially lost from the chromosomes. The partial loss of REC-8 from chromosomes between metaphase I and metaphase II suggests that worm REC-8 might function similarly to yeast Rec8p. The loss of yeast Rec8p from chromosome arms at meiosis I and centromeres at meiosis II coordinates the disjunction of homologs and sister chromatids at the two meiotic divisions.     

Melosis in holocentric chromosomes: Kinetic activity is randomly restricted to the chromatid ends of sex univalents inGraphosoma italicum (Heteroptera)

We have determined the number and location of the nucleolar organizing regions in spermatocytes ofGraphosoma italicum (2n=12A+ XY/XX) by means of silver impregnation, chromomycin A₃/distamycin A staining and fluorescencein situ hybridization. The identification of only one nucleolar organizing region located at one of the X chromosome ends has provided a suitable cytological marker to analyse the segregation of this univalent and that of the XY pseudobivalent during the first and second meiotic divisions respectively. Our results clearly show that at first meiotic metaphase the chromatids of the X chromosome are orientated with their long axes perpendicular to the polar axis. Although the kinetic activity is restricted to only one end in both X chromatids during the first meiotic division, both ends of the same chromatid have the same probability of showing such kinetic activity. In this sense, we also report that the chromatid segregation maybe initiated either at the same sister chromatid ends or at opposite ends in each chromatid. Thus, this indicates a sex chromatid independence as regards to the chromatid segregation during the first meiotic division. Throughout the second meiotic division both ends of the X chromatid are involved with the same probability in the end-to-end association to conform the XY pseudobivalent. This also implies a random localization of the kinetic activity at the ends opposite to those involved in the end-to-end association.accepted for publication by J. S. (Pat) Heslop-Harrison     

Nucleolus size variation during meiosis and NOR activity of a B chromosome in the grasshopper <Emphasis Type="Italic">Eyprepocnemis plorans</Emphasis>

The number of nucleoli and nucleolar area were measured in meiotic cells from males of the grasshopper Eyprepocnemis plorans collected in three natural populations. Number of nucleoli per cell showed no significant correlation among cells in different meiotic stages, but there was strong positive correlation for nucleolar area between leptotene and interkinesis cells in individuals from distant populations (Salobreña in Spain, and Smir in Morocco). No correlation was, however, observed for both parameters between the meiotic stages analysed in individuals from the population of Torrox (Spain). The number of nucleoli at leptotene was about double the number at interkinesis, as expected from the double ploidy level at leptotene and the corresponding double number of rDNA clusters. Leptotene nucleolar area, however, was about fourfold that in interkinesis, presumably due to higher requirements for ribosome biogenesis in meiosis I than meiosis II. In Torrox, diplotene cells showed a lower number of nucleoli but larger nucleolar area than in leptotene cells, suggesting an increase in nucleolus size during prophase I. Significant differences were found among populations for nucleolar area but not for number of nucleoli, the smallest nucleolar area being observed in Torrox, which is the population harbouring the most parasitic B chromosome variant. No clear effects on nucleolar area or number of nucleoli were associated with the B-chromosome number. However, B-chromosome effects on the nucleolar area were apparent in the Torrox population when data were analysed with respect to a B-chromosome odd–even pattern in leptotene and interkinesis cells. However, in diplotene cells no odd–even pattern was observed for both nucleolar parameters, suggesting that the increase in nucleolar size from leptotene to diplotene dilutes the leptotene odd–even pattern. The rDNA distally located in the B chromosome was associated with a nucleolus in 6.5% out of the 247 diplotene cells analysed. The implications of these findings are discussed in the context of B chromosomes as stress-causing genome parasites and the nucleolus as a sensor of stress.     

From spermatocytes to sperm: meiotic behaviour of human male reciprocal translocations

BACKGROUND: Human male translocation carriers may present alterations in the meiotic process due to the presence of the translocated chromosomes. The aim of this work was to study the mechanisms that affect meiotic segregation in translocation carriers by analysing different stages of the meiotic process. METHODS: Meiotic studies using fluorescence in-situ hybridization on both spermatocytes and sperm nuclei were performed in two translocation carriers, t(11;17)(q13.1;p11.2) and t(10;14)(q24;q32). RESULTS: A ring configuration was the main type of quadrivalent found in metaphase I. Overall chiasma frequency was significantly decreased in the t(11;17) carrier. In the t(10;14) carrier, chiasma frequency within the interstitial region of chromosomes 10 and 14 was increased and the recombination pattern was modified. As expected from the frequencies of interstitial chiasmata found in metaphase I in the two subjects, the incidence of asymmetric dyads was sporadic in t(11;17) and very high in t(10;14). In both carriers, segregation frequencies observed at metaphase II were not different from the segregation data obtained in decondensed sperm nuclei. CONCLUSIONS: The concordance observed among results obtained in different spermatogenic stages indicates an absence of cellular selection based on chromosomal imbalances. Results obtained in the aneuploidy assay have not provided any evidence for an interchromosomal effect.     

Chromosome pairing in allotetraploid hybrids of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> revealed by genomic <Emphasis Type="Italic">in situ</Emphasis> hybridization (GISH)

Genomic in situ hybridization (GISH) was used to make a detailed study of chromosome pairing at metaphase I (MI) of meiosis in six F(1) hybrid plants of the allotetraploid Festuca pratensis x Lolium perenne (2n = 4x = 28; genomic constitution FpFpLpLp). The mean chromosome configurations for all hybrids analysed were 1.13 univalents + 11.51 bivalents + 0.32 trivalents + 0.72 quadrivalents, and the mean chiasma frequency was 21.96 per cell. GISH showed that pairing was predominantly intragenomic, with mean numbers of L. perenne (Lp/Lp) and F. pratensis (Fp/Fp) bivalents being virtually equal at 5.41 and 5.48 per cell, respectively. Intergenomic pairing between Lolium and Festuca chromosomes was observed in 33.3% of Lp/Fp bivalents (0.62 per cell), in 79.7% of trivalents - Lp/Lp/Fp and Lp/Fp/Fp (0.25 per cell), and in 98.4% of quadrivalents - Lp/Lp/Fp/Fp and Lp/Lp/Lp/Fp (0.71 per cell). About 4.0% of the total chromosome complement analysed remained as univalents, an average 0.68 Lp and 0.45 Fp univalents per cell. It is evident that in these hybrids there is opportunity for recombination to take place between the two component genomes, albeit at a low level, and this is discussed in the context of compromising the stability of Festulolium hybrid cultivars and accounting for the drift in the balance of the genomes over generations. We speculate that genotypic differences between hybrids could permit selection for pairing control, and that preferences for homologous versus homoeologous centromeres in their spindle attachments and movement to the poles at anaphase I could form the basis of a mechanism underlying genome drift.     

卵母细胞骨架与染色体行为及质膜表面Con A结合位点关系的研究

用体外培养和荧光双重标记技术研究小鼠卵母细胞骨架，染色体及细胞质膜表面（微绒毛，Ｃｏｎ Ａ结合位点）三者间相互关系。结果表明，不同的细胞骨架成分对染色体行为及质膜表面变化影响不同，中期微管主要聚合成纺锤体，构成减数分裂器的支架，影响染色体在赤道板上有序地排列和分离，微丝在卵母细胞皮质区呈极性分布，它是牵引减数分裂器（或染色体团）从细胞中央移到细胞皮质区的关键成分，同时微丝也参与后期和末期成对染色体     

Neocentric activity of rye 5RL chromosome in wheat

The neocentric activity of a constriction located on the long arm of rye 5R chromosome (5RL) was analysed. It is not observed in normal rye but it is unusually stretched in bivalents involving 5RL telosomes in wheat–ditelosomic 5RL addition lines. In 20% of metaphase I cells, the 5RL bivalent presents the centromeres oriented to one pole and the constrictions oriented towards the opposite pole with a strong tension. In 5% of the cells, the constriction was able to orient the bivalent to the poles without tension in the centromeres. Sister chromatid cohesion, which is one of the distinct features of centromeric function, is persistent at the constriction in delayed 5RL chromosomes at anaphase I. Neither the elongation of the constriction nor the neocentric activity was observed at second meiotic division or mitosis. FISH studies showed that the 5RL constriction lacked detectable quantities of two repetitive DNA sequences, CCS1 and the 180-bp knob repeat, present at cereal centromeres and neocentromeres, respectively. We propose that, under special conditions, such as the wheat background, the normally non-centromeric DNA present at this region of 5RL acquires a specific chromatin structure, differentiated as an elongated constriction, which is able to function as a centromere. This revised version was published online in July 2006 with corrections to the Cover Date.     

Attachment of kinetochores to spindle microtubules during meiosis I of Lilium microsporocytes

Kinetochores and microtubules were visualized simultaneously during spindle formation at the first meiotic division in microsporocytes of Lilium longiflorum (2n = 24) under a confocal laser-scanning microscope, after immunofluorescence staining with centromere-recognizing antiserum and tubulin-specific antibody. During early prometaphase I, each kinetochore of bivalent chromosomes appeared to be an amorphous flat structure upon its initial attachment to microtubules. It became compact and spherical with the development of the spindle. From late prometaphase I, when the bipolar spindle was nearly complete, each kinetochore resembled a double disk that was suggestive of a pair of sister kinetochores and the homologous kinetochores were oriented towards opposite poles. Thus, the bipolar spindle at metaphase I included 12 bivalent chromosomes with a total of four kinetochores each. At anaphase I, the sister kinetochores moved to the same spindle pole as a paired unit. In microsporocytes arrested at prometaphase I by colchicine treatment, the sister kinetochores also came to be distinguishable. These results suggest that the change of kinetochore structure during meiosis I may be under chromosomal control but be somewhat associated with its attachment to spindle microtubules. This revised version was published online in July 2006 with corrections to the Cover Date.     

XY chromosome behaviour in the germ-line of the human male: A FISH analysis of spatial orientation,chromatin condensation and pairing

We have used multicolour fluorescencein situ hybridization to study the behaviour of the X and Y chromosomes in relation to a representative autosome, chromosome 1, on air-dried testicular preparations from normal fertile human males. In a proportion of Sertoli cells at interphase as well as spermatogonial metaphases there is an apparent selective undercondensation of the heterochromatic block of the long arm of the Y, which may be of functional significance with respect to Y-specific gene activity, initiating and maintaining spermatogenesis; we suggest that this may involve a mechanism similar to heterochromatin position-effect variegation inDrosophila. In the supporting Sertoli as well as pre-meiotic and leptotene cells the X and Y occupy relatively restricted domains at opposite poles of the nuclear membrane, while the chromosome 1 centromere regions are located interstitially and appear prealigned. The XY pairing and sex vesicle formation comprises a complex series of spatial movement and differential condensation patterns. On the basis of these observations we propose that: the XIST/Xist gene, known to be involved in somatic X inactivation, imposes a chromatin reorganization leading to bending at the X-inactivation centre both at first meiotic prophase in males and in the soma in females; and the differential X and Y segments are protected from potentially deleterious meiotic exchanges by their separate spatial orientation. In addition, there is an indication that the timing of pairing and first meiotic segregation of the sex chromosomes is different, and precocious in comparison to the pairing and segregation of the autosomes, which may explain the high incidence of sex chromosome aneuploidy in sperm.     

Dynamic relocation of telomere complexes in mouse meiotic chromosomes

Telomeric DNA repeats as well as different specific proteins such as TRF1 and Rap1 associate in functional telomere complexes found at chromosome ends. Using spreading techniques, the presence of TRF1 and Rap1 has been reported at mammalian meiotic telomeres during prophase I. In the present study, we have analysed, by fluorescence in-situ hybridization and immunofluorescence, the appearance and location of telomere complexes during both male mouse meiotic divisions. Additionally, we have studied their relationship with different centromere/kinetochore proteins and the synaptonemal complex protein SCP3. Our results show that telomere complexes are not located at condensed meiotic chromosome tips. Therefore, a change in chromosome structure may occur from pachytene up to metaphase I involving the dynamic relocation of telomere complexes in condensed chromosomes. Moreover, we have found that proximal telomere complexes are relocated internally to kinetochores from metaphase I up to anaphase II. We discuss the functional significance of the location of telomere complexes into internal domains of condensed meiotic chromosomes.     

Comparative cytogenetic studies of the influence of phenylbutazone and cyclophosphamide on spermatogenesis in the mouse

In the germinal epithelia of mice (CFLP), sacrificed from one to twenty-one days after the administration of a single oral dose of phenylbutazone or cyclophosphamide, the relative frequency of spermatogonial and meiotic metaphases was determined, and the chromosomes at diakinesismetaphase I were examined for aberrations and univalents. After treatment with phenylbutazone (227 mg/kg), the findings were comparable with those made in the controls. In contrast cyclophosphamide (97 mg/kg) led to considerable shifts in the different types of metaphases and to a marked increase in the incidence of univalents and chromosomal aberrations; in this respect, resting spermatocytes as well as spermatogonia of type B and especially intermediate spermatogonia proved to be the most susceptible stages.     

High chromosome variability and the presence of multivalent associations in buthid scorpions

In this study, we investigated the mitotic and meiotic chromosomes of 11 Buthidae scorpion species, belonging to three genera (Ananteris, Rhopalurus and Tityus), to obtain detailed knowledge regarding the mechanisms underlying the intraspecific and/or interspecific diversity of chromosome number and the origin of the complex chromosome associations observed during meiosis. The chromosomes of all species did not exhibit a localised centromere region and presented synaptic and achiasmatic behaviour during meiosis I. Spermatogonial and/or oogonial metaphase cells of these buthids showed diploid numbers range from 2n?=?6 to 2n?=?28. In most species, multivalent chromosome associations were observed in pachytene and postpachytene nuclei. Moreover, intraspecific variability associated with the presence or absence of chromosome chains and the number of chromosomes in the complex meiotic configurations was observed in some species of these three genera. Silver-impregnated cells revealed that the number and location of nucleolar organiser regions (NORs) remained unchanged despite extensive chromosome variation; notably, two NORs located on the terminal or subterminal chromosome regions were commonly observed for all species. C-banded and fluorochrome-stained cells showed that species with conspicuous blocks of heterochromatin exhibited the lowest rate of chromosomal rearrangement. Based on the investigation of mitotic and meiotic cells, we determined that the intraspecific variability occurred as a consequence of fission/fusion-type chromosomal rearrangements in Ananteris and Tityus species and reciprocal translocation in Rhopalurus species. Furthermore, we verified that individuals presenting the same diploid number differ in structural chromosome organisation, giving rise to intraspecific differences of chromosome association in meiotic cells (bivalent-like elements or chromosome chains).     

Meiotin-1, a Meiosis-enriched Protein Present in Normal Leptotene Chromosomes and Lacking in Precociously Condensed Leptotene Chromosomes

During mitotic prophase, chromosomes progressively compact to their metaphase length. In contrast, meiotic chromosomes condense moderately until late in prophase I, then they condense more dramatically (coil) to their fully condensed state. Meiotin-1 is a meiosis-enriched, chromosomal protein. We propose that it delays coiling until after reciprocal genetic exchange. We have used immunoblotting and immunocytochemistry with normal lily cells undergoing meiosis to demonstrate that meiotin-1 is present during the early portions of prophase I, but diminishes at the time when meiotic chromosomes begin to coil. Additionally, we have examined lily meiotic nuclei undergoing the reversible phenomenon of precocious leptotene chromosome condensation (precocious coiling). The leptotene chromosomes that are precociously condensed lack meiotin-1 immunostaining. Furthermore nuclei returning to the normal state of moderate prophase I condensation acquire meiotin-1.     

Behaviour of ring bivalents in holokinetic systems: Alternative sites of spindle attachment in Pachylis argentinus and Nezara viridula (Heteroptera)

Heteropteran chromosomes are holokinetic; during mitosis, sister chromatids segregate parallel to each other but, during meiosis, kinetic activity is restricted to one pair of telomeric regions. This meiotic behaviour has been corroborated for all rod bivalents. For ring bivalents, we have previously proposed that one of the two chiasmata releases first, and a telokinetic activity is also achieved. In the present work we analyse the meiotic behaviour of ring bivalents in Pachylis argentinus (Coreidae) and Nezara viridula (Pentatomidae) and we describe for the first time the chromosome complement and male meiosis of the former (2n = 12 + 2m + X0, pre-reduction of the X). Both species possess a large chromosome pair with a secondary constriction which is a nucleolus organizer region as revealed by in-situ hybridization. Here we propose a new mode of segregation for ring bivalents: when the chromosome pair bears a secondary constriction, it is not essential that one of the chiasmata releases first since these regions or repetitive DNA sequences adjacent to them become functional as alternative sites for microtubule attachment and they undertake chromosome segregation to the poles during anaphase I.