The effect of colchicine on meiosis in Lilium speciosum cv. “Rosemede”

Chromosome pairing and chiasma frequency were studied in meiocytes at diakinesis of Lilium speciosum cv. Rosemede fixed up to 21 days after the start of either continuous or 3 day pulse colchicine treatment. The two treatments gave similar results. In pulse treated pollen mother cells (PMCs) the mean chiasma frequency per cell fell from 26.4 in controls to 8.5 after fourteen days while the mean number of univalents per cell increased from 0.05 to 17.58. There was a negative correlation between mean chiasma frequency per bivalent and per PMC in colchicine treated buds; univalents were preferentially induced in bivalents with one chiasma, and preferentially excluded in bivalents with 4 chiasmata. Some chiasmata were redistributed to surviving bivalents despite the concurrent reduction in chiasma frequency per meiocyte. — Colchicine sensitivity began in premeiotic interphase and extended to mid or late zygotene in PMCs; ongoing synapsis was unaffected. However, susceptibility to univalency was asynchronous between bivalents occurring at zygotene in short chromosomes but at late premeiotic interphase in the longest chromosomes. The number of chiasmata per bivalent could be altered by colchicine without inducing univalents, but the ultimate effect was to reduce the number of chiasmata per bivalent (or per chromosome arm) directly to zero. The major factors determining the order and extent of reduced pairing and chiasma number were total chromosome length and arm length. Pairing and chiasma formation in embryo sac mother cells were less sensitive to colchicine than in PMCs, but their behavior was otherwise similar.     

Absence of chiasmata from the heteromorphic region of chromosome I during spermatogenesis in Triturus cristatus carnifex

Analysis of squash preparations of spermatocytes from crested newts, Triturus cristatus carnifex, has shown that in most cells at least one large bivalent regularly fails to form chiasmata in one arm-pair. Feulgen microphotometry of diplotene and metaphase bivalents has shown that it is the largest bivalent in each cell which shows chiasma failure in one arm-pair. A C-banding technique which identifies chromosome I by virtue of a long, darkly stained region in its long arm, was used to confirm the absence of chiasmata from one arm-pair of the longest bivalent, and specifically from the darkly stained region. The achiasmate region which chromosome I exhibits during spermatogenesis, corresponds to the heteromorphic region of oocyte lampbrush bivalent I in which chiasmata never form. A possible correlation between the complete absence of crossing-over from the heteromorphic region and unusual cytological and molecular features which it exhibits, are discussed.     

Meiotic nondisjunction in oocytes from aged Djungarian hamsters correlates with an alteration in meiosis rate but not in univalent formation

Summary The effect of maternal ageing on the meiotic rate, on chiasma and univalent frequency as well as on heteroploidy in secondary oocytes from Djungarian hamsters was exammed. The frequency of hyperhaploid oocytes increased from 0.6% in young (8–14 weeks) to 2.8% in middle-aged (26–46 weeks) and reached 3.6% in the oldest females (49–75 weeks). On the basis of malsegregated bivalents per oocyte, nondisjunction occurred most often in the middle-aged group (5.42x10^-2 bivalents per oocyte). Hereby, the large meta- and submetacentric A-D chromosomes were preferentially involved. Furthermore, the pattern of nondisjunction was not different from that expected on the basis of chromosome length or induced by colchicine. The large A-D chromosomes did not show any alteration in chiasma or univalent frequency. Terminalized chiasmata were only detected in the E group and univalents increased slightly, but not significantly in the small chromosomes (G group). At higher ages, both chromosome group were not preferentially involved in nondisjunction. Presegregation slightly increased with age and affected more or less all bivalents, whereas the incidence of diploidy significantly decreased. With respect to the rate of meiosis in oocytes from aged females, the resumption was delayed at metaphase I. Our data suggest that failures in the control of oocyte proliferation are involved in nondisjunction rather than the production-line. Furthermore, a model is proposed to explain nondisjunction of specific bivalents at certain maternal ages.     

Analysis of exchanges in differentially stained meiotic chromosomes of Locusta migratoria after BrdU-substitution and FPG staining

In male mice the X and Y chromosomes are conjoined by a single near-terminal chiasma, but XY bivalents following incorporation of 5-bromodeoxyuridine (BrdU) and fluorescence plus Giemsa (FPG) staining show only one of the two expected configurations, which suggests a preferential involvement of certain non-sister chromatids in crossover formation. To test the possibility that nonrandom chromatid involvement is a general feature of near-terminal crossovers, we reexamined the apparently terminal associations in differentially stained autosomal bivalents of Locusta migratoria. The frequencies of the two configuration types were nearly equal, as would be expected if these terminal associations resulted from conventional near-terminal chiasmata showing the random involvement of non-sister chromatids that characterises interstitial chiasmata.     

Effects of constitutive heterochromatin and genotype on frequency and distribution of chiasmata in the seven individual rye bivalents

Summary Number and distribution of chiasmata were studied in the single pair of homologous rye chromosomes in 29 chromosomal F₁ hybrids between the seven disomic wheat rye addition lines of Chinese Spring/ Imperial and five selected inbred genotypes of cultivated rye by using the differential Giemsa staining technique. The results indicate that the number and position of chiasmata is independent from the amount and position of C-heterochromatin. Genotype had an effect on chiasma number, whereas chiasma distribution within bivalents appeared to be determined by morphological features of chromosomes. Late replicating DNA in constitutive heterochromatin may delay the separation of half bivalents if chiasmata are formed between them and the centromere.Supported by Deutsche Forschungsgemeinschaft, Bonn     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

At metaphase I during spermatogenesis in Mesostoma ehrenbergii ehrenbergii (2 × = 10), 3 bivalents and 4 univalents form. The bivalents each have a single distally localised chiasma. Electron microscope serial section reconstructions have shown that 3 short pieces of synaptonemal complex (SC) are present in pachytene nuclei in the testis. All the SC is contained in a lobe which is separated by an invagination from the main body of the nucleus. The SC pieces vary in length from approximately 2 m to 5 m, and the greatest amount found in one nucleus was approximately 13 m. It is proposed that the pieces of SC correspond to the paired regions of the 3 bivalents present at metaphase I. This system is therefore an example of localised pairing leading to localised chiasmata. Later prophase stage nuclei have also been reconstructed, in which stacks of polycomplex are present but no SC has been found. The polycomplex is thought to be an association of discarded SC sub-units.     

Differential meiotic behaviour of diploid and tetraploid cells in a partially asynaptic mutant

A partially asynaptic individual of the grasshopperEuchorthippus pulvinatus (2n = 16 + X) was analysed at meiosis, using a Giemsa C-banding technique. Long chromosomes formed univalents less frequently than did medium and short ones. Homologues which succeeded in forming bivalents showed reduced chiasma frequency, the long chromosomes being affected by most. Changes in chiasma distribution were also observed. The presence of univalents at metaphase I seems to affect the function of the spindle, since most cells at the second division were unreduced. Cytokinesis was also subsequently suppressed in a great number of these products of restitution, resulting in the formation of diploid and tetraploid spermatids. Fifteen tetraploid metaphase I cells were also found in which pairing level and chiasma frequency were almost twice the average value in normal diploid individuals. The nature of this mutant is discussed.     

Meiosis VII: “Detorsive bending” as a basis for geometric shapes of late prophase bivalents

A new model depending on mechanical properties of chromosomes is adduced as a basis for diplotene opening-out and for curvature occurring in grasshopper bivalents, during and subsequent to diplotene. Conditions underlying the model are: (1) rigid physical binding exists between sister chromatids, (2) each chromatid remains free of torsional strain if its pairing face is straight, i.e. the chromatid is bilaterally symmetrical, (3) reciprocal exchange together with stiff binding between sisters produces twist in each chromated before diplotene begins, (4) stiffening of the bivalent during late meiotic prophase removes the twist resulting from reciprocal exchange, (5) since sister binding prevents untwisting of chromatids about their long axes, untwisting would be achieved only in conjunction withbending of each chromated. It is shown that this bending, called detorsive bending, automatically produces opening out, not only in bivalents with one chiasma but also in those with more than one, especially if the chiasmata are interstitial.In bivalonts with two chiasmata, classes of curvature resulting when both chiasmata are interstitial (II), when one is interstitial and one terminal (IT) and when both are terminal (TT) are attributed to differences in strength of opening out at interstitial and at terminal chiasmata respectively. It is postulated that mechanisms responsible for opening out at terminal chiasmata are basically different from those at interstitial chiasmata.A theoretical basis of a method for cytological detection of chromatid interference is outlined and arguments are presented against the electrostatic hypothesis.     

Chromosome-specific desynapsis in the n = 2 race of Haplopappus gracilis (Compositae)

During cytological screening for pollen sterility in a wild population of Haplopappus gracilis (n = 2), several partially sterile plants were found that had good pachytene pairing but varying numbers of univalents. Some plants had chromosome A bivalents or A univalents, while in the same cells chromosome B had only bivalents. In other plants the reverse condition occurred; the B chromosome had B bivalents or B univalents and only A bivalents. This demonstrates a chromosome-specific effect for the desynapsis genes. Hybridization between the two homozygous mutant genotypes produced only normal bivalents; this indicates the two mutants are not alleles and each is recessive. An F2 generation showed independent assortment of the desynaptic mutations. The chromosome A bivalent is the larger of the two and normally has one or two chiasmata; the B bivalent normally has a single chiasma. Chiasmata distribution was tested in the desynaptic mutant A bivalents and showed an acceptable fit to a binomial distribution. This occurs also in heterozygous, asynaptic pairing control gene mutations. Analysis of the NOR bivalent in two hologenomic desynaptic mutations in tomato also showed a good fit to a binomial distribution of chiasmata. This indicates the same methods are applicable to diverse species.     

Experimental hybridization within the genus Triturus (Urodela: Salamandridae)

The spermatogenesis of 9 F₁ hybrids of Triturus cristatus carnifex × T. vulgaris meridionalis was studied in squash preparations of testicular fragments, treated by the C-staining method. The chromosome number of these hybrids was examined in spermatogonial metaphases and found to be diploid. The two parental sets were always recognized, which means that a regular, although heterospecific, amphimixis occurred (2n=n+n). Meiotic prophase I is greatly altered owing to a failure of typical chromosome pairing and chiasma formation. At metaphase I and/or meta-anaphase I, the effects of the hybrid combination of the 2 specific parental sets are clearly visible. Most primary spermatocytes contain only univalents. A few show chromosome associations (bivalents, trivalents and, more rarely, quadrivalent chains) besides univalents. Such associations are of 2 types: (a) intragenomal associations = associations of 2 chromosomes by a terminal (a¹) or subterminal chiasma (a²); (b) intergenomal associations = associations of 2 chromosomes by a terminal (b¹) or subterminal chiasma (b²). Univalents segregate at random while the associations often lag on the equatorial plane or migrate entire to a spindle pole. Primary spermatocytes with chromosome multivalents can encounter greater difficulties in accomplishing the first cytokinesis. Secondary spermatocytes are numerically and qualitatively unbalanced; however, some of them undergo spermiogenesis and can give rise to a small number of sperms, generally abnormal and never united in bundles. — Problems related to the occurrence of anomalous chiasmata and of intra- and inter-genomal homologies are discussed.     

Chiasma distribution in Truxaline grasshoppers

Similar patterns of chiasma distribution are found within the individual arms of the chromosome complement in four species of Truxaline grasshopper. There is a linear relationship between chiasma frequency and chromosome arm length although the telocentric elements have a consistently higher mean number of chiasmata per unit of arm length. The positions of successive chiasmata can be defined in terms of residual (r.c. and r.t.) and interference (T) distances which vary in value according to both arm length and chiasma frequency. There is a tendency for one chiasma to lie in a distal position which is accentuated when additional chiasmata form. Supernumerary B chromosomes do not appear to influence the overall control mechanism of chiasma distribution. There is no indication that bivalents within a nucleus compete for chiasmata nor does the chiasma distribution in one arm of the metacentric members influence that in the other. It is suggested that the control of chiasma formation is determined mainly by interference factors.     

Desynaptische Mutanten mit Unterschieden im Univalentenverhalten

After application of neutrons on dry seeds ofPisum sativum three recessive mutants were isolated showing irregularities in the course of meiosis. A cytogenetical analysis showed that at metaphase I, a varying number of univalents are formed, most likely as a result of reduced chiasma frequencies. At anaphase I, some univalents divide precociously in mutant 2982 but none do so in mutants 2989 and 2552. As a consequence, most cells of 2989 and 2552 built up more than two spindles at anaphase II, whereas the majority of cells in 2982 form two spindles. This situation is reflected in the frequency distribution of gones per PMC at tetrad stage. The discussion deals with the possible causes of univalent formation at meiotic prophase and the variability of univalent behaviour at anaphase I.     

The use of translocation-derived "marker-bivalents" for studying the origin of meiotic instability in female mice

Female mice of two age groups, 3--4 and 11--14 months old, homozygous for the T(1;13)70H reciprocal mouse translocation were used for cytological observations of bivalents (in primary oocytes) and metaphase II chromosomes (in secondary oocytes). Special attention was given to the behavior of the long (131) and short (113) marker chromosomes. In primary oocytes, univalents were considered "true" or "opposite". The aged females showed an eight-folded increase in "true" univalent frequency for chromosomes 113 over the young ones. A nine-fold rise for nondisjunction with regard to this chromosome was observed. For the other chromosomes, these factors were 2 and 1.7, respectively. The absolute levels of nondisjunction remained low at old age (1.42% for chromosome 113, 1.22% for all other chromosomes). The long marker bivalent 131 was used for chiasma counts. No change in chiasma number with age was observed. It is argued that poorer physiological conditions within the maturing oocytes of older females are the major cause for both the increasing frequencies of "true" and "opposite" univalents and the increased incidence for nondisjunction.     

A re-examination of chiasma terminalization and chiasma frequency in male mice

The distribution and frequency of chiasmata have been analyzed in male BALB/c mice. Bivalents were classified in terms of the number of interstitial chiasmata (CH) and terminal associations (TA) present as follows; 1CH, 2CH, 1TA, 2TA, 1CH·1TA, 1CH·2TA, 2CH·1TA, 2CH-2TA and 0. We provide evidence that the TA frequently dissociates during 1st meiotic prophase. Consequently six of the observed bivalents may be derived from three basic bivalent types (namely 2CH·2TA, 1CH·2TA and 2TA) by dissociation of the TA according to the following schemas: (1) 2CH·2TA2CH·1TA2CH, (2) 1CH·2TA1CH·1TA1CH, and (3) 2TA1TA0. We also provide evidence that interstitial chiasmata do not move, which implies that a TA can not be formed by chiasma terminalization. The chiasma frequency estimated by assuming that terminal associations do not result from terminalized chiasmata is 17.2±2.4 compared to a value of 25.4±2.2 calculated on the assumption of chiasma terminalization.     

The meiotic structure and behavior of the strongly heteromorphic X/Y sex chromosomes of neotropical plethodontid salamanders of the genus Oedipina

Plethodontid salamanders in the genus Oedipina are characterized by a strongly heteromorphic sex-determining pair of X/Y chromosomes. The telocentric X chromosome and the subtelocentric Y chromosome are clearly distinguished from the autosomes and their behavior during meiosis can be sequentially followed in squash preparations of spermatocytes. In Oedipina the sex chromosomes are not obscured by an opaque sex vesicle during early meiotic stages, making it possible to observe details of sex bivalent structure and behavior not directly visible in other vertebrate groups. The sex chromosomes can first be distinguished from autosomal bivalents at the conclusion of zygotene, with X and Y synapsed only along a short segment at their non-centromeric ends, forming a bivalent that contrasts sharply with the completely synapsed autosomes. During pachytene, the XY bivalent becomes progressively shortened and more compact, disappearing as a visible structure when pachytene progresses into the diffuse stage of male meiosis. Diplotene bivalents gradually emerge from the diffuse nuclei, presumably by the return of the loops of chromatin into their respective chromomeres. During early diplotene, the X/Y bivalent is clearly visible with a single chiasma within the synapsed segment. This chiasma is terminalized by first meiotic metaphase with the X and Y appearing either in end-to-end synaptic contact or as univalents separated at opposite poles relative to the equatorially distributed autosomal bivalents. In C-banded preparations, the Y is entirely heterochromatic while the X contains a large centromeric C-band and another block of heterochromatin located at the telomeric end, in the region of synapsis with the Y. We find no cytological evidence of dosage compensation, such as differential staining of the X chromosomes or Barr bodies, in mitotic or interphase cells from female animals.     

Synapsis in Robertsonian heterozygotes and homozygotes of Dichroplus pratensis (Melanoplinae, Acrididae) and its relationship with chiasma patterns

Dichroplus pratensis has a complex system of Robertsonian rearrangements with central-marginal distribution; marginal populations are standard telocentric. Standard bivalents show a proximal-distal chiasma pattern in both sexes. In Robertsonian individuals a redistribution of chiasmata occurs: proximal chiasmata are suppressed in fusion trivalents and bivalents which usually display a single distal chiasma per chromosome arm. In this paper we studied the synaptic patterns of homologous chromosomes at prophase I of different Robertsonian status in order to find a mechanistic explanation for the observed phenomenon of redistribution of chiasmata. Synaptonemal complexes of males with different karyotypes were analysed by transmission electron microscopy in surface-spread preparations. The study of zygotene and early pachytene nuclei revealed that in the former, pericentromeric regions are the last to synapse in Robertsonian trivalents and bivalents and normally remain asynaptic at pachytene in the case of trivalents, but complete pairing in bivalents. Telocentric (standard) bivalents usually show complete synapsis at pachytene, but different degrees of interstitial asynapsis during zygotene, suggesting that synapsis starts in opposite (centromeric and distal) ends. The sequential nature of synapsis in the three types of configuration is directly related to their patterns of chiasma localisation at diplotene-metaphase I, and strongly supports our previous idea that Rb fusions instantly produce a redistribution of chiasmata towards chromosome ends by reducing the early pairing regions (which pair first, remain paired longer and thus would have a higher probability of forming chiasmata) from four to two (independently of the heterozygous or homozygous status of the fusion). Pericentromeric regions would pair the last, thus chiasma formation is strongly reduced in these areas contrary to what occurs in telocentric bivalents.     

Development of the synaptonemal complex and the “recombination nodules” during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one fixed and one randomly attached telomeres. The fixed end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of fixed telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.     

Lampbrush Chromosomes in the Japanese Quail Coturnix coturnix japonica: Cytological Map of Macrochromosomes and Meiotic Crossing-over Frequency in Females

Cytological map of lampbrush macrobivalents of the Japanese quail (Coturnix coturnix japonica) were constructed. Investigation of chiasmata allowed to estimate the frequency of reciprocal genetic recombination (crossing over) in Japanese quail female meiosis. The total chiasma number in bivalents of Japanese quail oocyte nuclei was determined to be 53–58. Macrobivalents 1–5 and Z of the Japanese quail had on average 3.3 chiasmata per bivalent, and microbivalents, 1.0–1.1 chiasmata per bivalent. The chiasmata (crossover) frequency in Japanese quail females was lower than in chicken. In macrochromosomes of Japanese quail females, one crossover occurred per 43.9 Mb, and in chicken, per 30.0 Mb. Judging from chiasma frequency, the genetic length of the Japanese quail genome is likely to be 2650–2900 cM. Crossover frequency in the species was 0.023 per Mb in macrobivalents and 0.07–0.08 Mb in microbivalents and for the total genome, 0.041 crossing over per Mb. The genetic length of one Mb (recombination rate ) in female Japanese quails was 1.14 cM in macrochromosomes, 3.60–4.12 cM in microchromosomes, and about 1.96–2.15 cM averaged over the genome.     

Experimental hybridization within the genus Triturus (Urodela: Salamandridae)

Spermatogenesis in the F₁ hybrid (2n=24=12 +12 ) between the closely related newt species T. cristatus carnifex and T. marmoratus was apparently normal up to pachytene. Many unpaired chromosomes were present at diplotene and a typical diakinesis was lacking. Primary spermatocytes at meta-and meta-anaphase contained up to 12 regular intergenomal bivalents and a corresponding number of univalents when less then 12 II. Most chiasmata were terminal or subterminal, some intercalary. Chiasmata between corresponding heterospecific chromosomes can be reported as true: real crossing over has taken place, proving the presence of primary chromosomal homologies between the 2 sets of the parental species. Evidence for recombination is based on the segregation of particular markers (i.e., subterminal C-bands and NORs) observed in certain chromosomes at metaphase II. One chromatid of single chromosomes can show the T. cristatus phenotype and the other the T. marmoratus phenotype. A few primary spermatocytes contain a certain number of irregular associations (intragenomal or intrahaploid bivalents, irregular intergenomal bivalents, chromosome multivalents) joined by chiasmata which can be defined as anomalous. Other abnormalities concern the occurrence of interlocked bivalents which occasionally show an anomalous exchange between heterologous chromatids. — Cytogenetic criteria useful to evaluate the taxonomic relationships between different species have been discussed as well as some possible trends in chromosome evolution and speciation within the genus Triturus.     

Cytological, genetic and evolutionary functions of chiasmata based on chiasma graph analysis.

The nature of the chiasma as a cytological parameter for analysing cross-over was reexamined quantitatively by an improved chiasma graph method. It was reconfirmed in Mus platythrix (n =13) that interstitial chiasmata at diakinesis are distributed randomly and almost uniformly along bivalents except for the centromere and telomere regions. The size of these chiasma blank regions was consistently 0.8% of the total length of haploid autosomes in all chromosomes. There was a minimum value of chiasma interference distance between two adjacent chiasmata, which was constantly 1.8% in all chromosomes. The chiasma frequency at diakinesis was 20.1+/-2. 0 by the conventional method including terminal chiasmata. However, the primed in situ labeling technique revealed that terminal chiasmata were mostly telomere-telomere associations. From these data and also from recent molecular data we concluded that the terminal chiasma is cytologically functional for ensuring the normal disjunction of bivalents at anaphase I, but genetically non-functional for shuffling genes. The chiasma frequency excluding terminal chiasmata was 14.6+/-1.8. Reexamination of the chiasma frequency of 106 animal species revealed that the chiasma frequency increased linearly in proportion to the haploid chromosome number in spite of remarkable difference in their genome size. The increase in chiasma frequency would be evolution-adaptive, because gene shuffling is expected to be accelerated in species with high chromosome numbers.