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.     

Chiasma distribution in asynaptic Locusta migratoria

The formation of chiasmata in six full sib male partially asynaptic individuals of Locusta migratoria has been studied. The mean chiasma frequency per cell was 2.3 both at diplotene and metaphase I. Chiasmata tended to be distributed evenly among the bivalents. The frequency and distribution of the chiasmata in each type of bivalent (L, M, or S) depended on the level of asynapsis and on interference between the bivalents. Short bivalents were the most affected by interference, while M bivalents associated independently of L and S bivalent behaviour.     

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

The strand relationships of adjacent pairs of chiasmata have been examined in differentially stained meiotic bivalents following in vivo BrdU substitution and FPG staining. A test for chromatid interference is described, based on comparisons of the observed numbers of various combinations of hidden and visible crossover exchanges with the numbers expected assuming no chromatid interference. A comparison based on 67 pairs of chiasmata from Locusta migratoria indicates no evidence for chromatid interference in this material.     

Induced Bivalent Interlocking and the Course of Meiotic Chromosome Synapsis

WHEN chromosomes pair at meiosis the bivalents so formed do not normally interlock. Heat-treatments can, however, induce bivalent interlocking in the locust Locusta migratoria. Only the longest bivalents interlock and usually only two are found per cell; two “rod” bivalents, with single chiasmata, two “ring” bivalents, each with two or three chiasmata, or one “rod” and one “ring” bivalent (Fig. 1a, b and c). The nature of this interlocking and the metaphase orientational and congressional properties of interlocked bivalents are analysed in detail elsewhere¹.     

Further correlations between chiasmata and U-type exchanges in rye meiosis

Earlier studies have demonstrated convincing correlations between the distribution patterns of chiasmata and of U-type exchanges within bivalents. On the basis of this evidence and other considerations it has been proposed that these contrasting meiotic exchange events are related in origin, and that U-type exchanges, giving rise to bridge and fragment configurations, arise as errors in crossing-over and chiasma formation. This hypothesis is given further consideration in the present report and further correlated distributions of chiasmata and U-type exchanges are presented. These correlations involve the distribution patterns of exchanges between bivalents and between the two arns of one particular bivalent which is consistently marked by the presence of localised neocentric activity. The relationships of these exchange distributions to chromosome length are also investigated and as a result it becomes clear that a mutual dependence of the two types of exchange on chromosome length cannot account for the observed correlations. The total evidence relating to the hypothesis of a causal connection between chiasmata and U-type exchanges is reviewed and critically assessed.     

Recombination nodules and chiasma localization in two orthoptera

The distribution of recombination nodules (RNs) is reported from observations on two-dimensional spreads of Locusta migratoria and Chloealtis conspersa spermatocytes; C. conspersa is a known example of a species with terminally localized chiasmata, while L. migratoria has nonspecific positioning of chiasmata. Measurements of the distances from 102 RNs to the ends of the synaptonemal complexes (SCs) on which they were found show the RNs to be near-terminally localized in C. conspersa and to occur along the lengths of the SCs in L. migratoria. Thus, the localization of RNs appears to reflect the localization of chiasmata. These observations are interpreted as support for the proposed recombinant function of RNs.     

Influence of age on chiasma number in male Locusta migratoria

The influence of age on chiasma formation in Locusta migratoria has been studied both in wild-type and irradiated males. Samples were obtained on the one hand from different individuals and on the other hand from the same individuals at different ages through repeated biopsies. It was observed that mean chiasmata per cell decreased in ageing animals. Chi-squared tests and correlation coefficients showed that the decrease of chiasmata with age is progressive but not continuous. Intermediate values coincide with the first mating. It seems that older individuals tend to transmit their genetic combinations unaltered, increasing the probability of transmitting gene combinations of adaptive value.     

Evidence of crossing-over inhibition in rye anthers cultured with colchicine

In order to investigate wheter colchicine affects crossing-over, rye anthers of an inbred line of rye forming bridges and fragments at anaphase I produced by erroneous chiasmata, and anthers of plants heterozygous for a conspicuous heterochromatin band, were cultured in a medium with colchicine. Anthers planted at zygotene did not show bridges at AI in the inbred line. In the heterozygotes no difference between associated chromatids in respect to the heterochromatin band, resulting from crossingover, were observed. In anthers planted at pachytene both bridges and chromosomes showing difference between associated chromatids were observed at a stage equivalent to AI with the same frequency as in anaphase I cells of untreated anthers. This demonstrates that crossing-over or a prerequisite to crossing-over is established at zygotene, and also that absence of chiasmate association at later stages is not due to precocious slipping off of chiasmata.     

Cytological studies of the two-spotted spider mite Tetranychus urticae Koch (tetranychidae,trombidiformes) II. Meiosis in growing oocytes

The morphology and behaviour of the holokinetic chromosomes of Tetranychus urticae Koch were studied in maturing oocytes from diplotene up to and including metaphase. Each of the three diplotene bivalents contains 2–3 chiasmata.During congression the dyads of the axially orientated bivalents make turning movements in opposite directions till they have reached an equatorial orientation which leads to an equational first meiotic division.It was argued that chiasmata terminalise towards both ends of the dyads and that each dyad contains two sister chromatids.It has been supposed that the inverted meiosis of organisms with holokinetic chromosomes can be brought about by the way in which chiasmata terminalise and the kinetochores are assembled subsequently on each chromatid.During congression vesicular strands form a Feulgen-negative zone round the bright plasm in which the bivalents are embedded. The question was raised whether these strands originate from the dense plasm surrounding each of the diplotene bivalents and bear some relation to the elimination chromatin present during the meiotic telophases.This investigation was supported by research grants from the Ministry of Public Health and Environment and from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

The analysis of exchanges in tritium-labelled meiotic chromosomes

The autoradiographic analysis of exchanges in tritium-labelled meiotic chromosomes is potentially a useful approach to the study of meiotic exchange events since this method differentially labels meiotic chromatids along their entire length. The main problem encountered in earlier autoradiographic studies is that of distinguishing label exchanges generated at chiasmata from label exchanges generated by sister chromatid exchange. This problem was overcome in the present study by the choice of a meiotic system (male meiosis of Stethophyma grossum) where chiasmata are limited to just one proximally localised chiasma in each bivalent. This system allows the positive identification of chiasma-generated label exchanges and demonstrates convincingly the origin of chiasmata through breakage and rejoining of homologous non-sister chromatids. Sister chromatid exchanges are also readily detected in labelled meiotic chromosomes of this species, where they occur with a mean frequency of 0.35 per chromosome. This frequency is similar to that found in mitotic spermatogonial cells and the exchanges are randomly distributed both within and between chromosomes. These features of meiotic sister chromatid exchanges suggest that they are unrelated to non-sister chiasmatic exchanges and they probably have no special meiotic significance.     

Localised chiasmata due to partial pairing: A 3D reconstruction of synaptonemal complexes in male Stethophyma grossum

The possible role of localised pairing as a mechanism producing localised chiasmata in Stethophyma grossum spermatocytes has been examined ultrastructurally. Nuclei at four successive stages of meiosis from leptotene to pachytene were reconstructed from a series of ultrathin sections and the extent of synapsis as demonstrated by synaptonemal complex (SC) formation was calculated. On the basis of the relative lengths of SCs and condensed chromosomes it was reasoned that only the centromeric ends of the long and medium length bivalents paired, and only one end of these SCs was found attached to the nuclear envelope. Only the three shortest bivalents paired completely, and both their SC ends were attached to the nuclear envelope. Thus pairing was directly related to the distribution of chiasmata. The extent of pairing at different stages suggests that the shortest bivalent paired very quickly, the longer ones progressively slower, and that pairing proceeded zip-like from a point at or very close to the end attached to the nuclear envelope, since all stretches of SC were attached to the envelope, and there were never more than 11 pieces, one for each bivalent. Chromosome decondensation and axial core formation did not occur far in advance of SC formation, and synapsis appeared to be much slower in S. grossum than in other species with non-localised chiasmata, as a larger proportion of the meiotic cysts were in zygotene, compared to Stauroderus scalaris and Locusta migratoria, although this was not quantified.     

Repositioning of aurora B promoted by chiasmata ensures sister chromatid mono-orientation in meiosis I

During meiosis I, kinetochores of sister chromatids are juxtaposed or fused and mono-orient, while homologous chromosomes that are paired by chiasmata (bivalents) have to biorient. In the absence of chiasmata, biorientation of sister chromatids (univalents), which carries a risk of aneuploidy, has been occasionally detected in several species, including humans. We show in fission yeast that biorientation of fused sister kinetochores predominates during early prometaphase I. Without chiasmata, this undesirable biorientation of univalents persists and eventually evades the spindle assembly checkpoint, provoking abnormal anaphase. When univalents are connected by chiasmata or by an artificial tether, this erroneous attachment is converted to monopolar attachment and stabilized. This stabilization is apparently achieved by a chromosome configuration that brings kinetochores to the outer edge of the bivalent, while bringing Aurora B, a destabilizer of kinetochore-microtubule attachment, inward. Our results elucidate how chiasmata favor biorientation of bivalents over that of univalents at meiosis I.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

Normal and BrdC-substituted chromosomes during spermatogenesis with an endomeiotic chromosome reduplication in Carausius morosus Br.

Spermatogenesis involving an additional chromosome reduplication during zygotene in sporadic males and intersexes of the thelytokous phasmid Carausius morosus Br. has been examined using differential staining of chromatids after 5-bromodeoxycytidine incorporation. After reduplication autobivalents are formed by synapsis between identical sister chromosomes. Chiasmata are only formed after reduplication; they do not occur in constitutive heterochromatin, but can be formed in facultative heterochromatin, dependent on heteropycnosis and sex. Quadrivalents and U-type exchanges occur. In spermatogonia and spermatocytes the number of differentially stained chromosomes varies considerably; sister chromatid exchanges hardly appear. Sex bivalents with differentially stained chromosomes have a lower chiasma frequency than normally stained sex bivalents. Bivalents show reduced staining of all four, two outer, or one inner chromatid. Autobivalents arise in the same way as diplochromosomes; chromatids with the oldest DNA sub-units remain together during reduplication and are thus involved in sister chromosome pairing. The additional reduplication begins 7 days after the premeiotic S-phase, first metaphase after 19 days. Spermatogenesis is abnormal from first anaphase onwards.     

The induction of orientational instability and bivalent interlocking at meiosis

The administration of 40° C heat-treatments was found to induce bivalent orientational instability and interlocking at male meiosis in the locust Locusta migratoria. Only the longest members of the complement showed orientational instability and these usually possessed single distally sited chiasmata, with near-maximal intercentromeric distances. An effect on the stability of spindle fibre microtubule association, or attachment to the chromosome, is considered to be a possible explanation of the behaviour found. Bipolar orientation was generally achieved prior to anaphase I so that chromosome segregation was usually normal. Diamphitelic bivalents provided the most common exception to this rule. They sometimes lagged at anaphase, with the separation of half-bivalents and the production of structures indistinguishable from lagging univalents. The bivalent interlocking also involved the longest members of the complement. Most combinations of rod/rod, rod/ring and ring/ring types of interlocking were found. Usually only two bivalents were interlocked in any one cell, although occasionally three were found interlocked. All types appeared to involve an effect on the regulation of chromosome pairing, although at least one of the cells found showed interlocking caused by the metaphase orientational instability. In most cells, interlocked bivalents showed stable orientation and this usually involved the unipolar orientation of each bivalent's two centromeres. Such configurations provide concrete support for the importance of physical tension in the maintenance of metaphase orientational stability. They lead to double non-disjunction at anaphase I. Interlocked bivalents showed normal congression to a mid-equatorial position with no tendency for the re-adjustment of arm ratios to equalise centromere distances from the poles. This behaviour is discussed in relation to spindle fibre dynamics and it is concluded that no hypothesis of congression currently available can satisfactorily explain all that we know of the behaviour of univalents, bivalents, multivalents and interlocked bivalents.     

Surface spreading of synaptonemal complexes in locusts

Details are given of techniques for preparing surface spreads of locust spermatocytes for light and electron microscopy. The pachytene synaptonemal complex (SC) karyotypes of Locusta migratoria and Schistocerca gregaria are analysed and compared. Up to six different SCs can be identified in Locusta migratoria based on lengths, centromere positions, and possession of nucleolar organiser regions, but only two SCs are identifiable in Schistocerca gregaria. The total SC length is significantly greater in Schistocerca gregaria than in Locusta migratoria, and this difference is almost exactly proportional to the difference in the genomic DNA contents of the two species.     

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.     

Frequency and distribution of chiasmata in Syrian hamster spermatocytes studied by the BrdU antibody technique

The frequency and distribution of chiasmata and the nature of terminal associations was re-examined in Syrian hamster spermatocytes using the 5-bromo-2-deoxyuridine (BrdU) antibody technique (BAT) for differential chromatid labelling. Differential chromatid substitution was achieved by BrdU incorporation at the penultimate pre-meiotic S-phase followed by one of three different staining protocols: (i) fluorescence plus Giemsa (FPG), (ii) acridine orange staining or (iii) BAT. For analysis of chiasmata frequency and localization in the diplotene/diakinesis stages the resolution of FPG and acridine orange staining was comparable to that of BAT. In metaphase II chromosomes BAT was more informative than FPG and acridine orange staining and revealed small, terminal crossover exchanges. This finding proves that many terminal associations of meiotic chromosomes actually represent chiasmata at the end of the first meiotic division. Some crossover exchanges were localized in the constitutive heterochromatin of autosomes. Using BAT we also detected crossover exchanges in close vicinity to each other. This observation is reminiscent of the fact that crossing over interference means a reduction in frequency and does not imply total inhibition.     

Cytological demonstration of mitotic crossing-over in man.

Quadriradial (QR) configurations from four different human lymphocyte metaphase samples have been analyzed: a patient with Fanconi's anemia; normal female cells X-irradiated with 150 or 200 R in S or G2; spontaneous QRs occurring in 13,584 metaphases; and cells from two sibs with Bloom's syndrome. That mitotic chiasmata are caused by crossing-over rather than by random breakage and reunion was concluded from the following observations: (1) In the spontaneous sample, mitotic chiasmata are about as frequent as all other QRs together. (2) The frequencies of mitotic chiasmata and of other QRs are not correlated in the different samples. (3) The break points in other QRs are situated at random relative to chromosome length, whereas the distribution of chiasmata is highly nonrandom. (4) Although the centromeres of chromatid translocations occur in alternate and adjacent positions with approximately equal frequencies, there are very few adjacent counterparts to mitotic chiasmata. These can best be interpreted as a result of an abnormal U-type rejoining of chromatids in a chiasma. (5) Chiasmata found in heteromorphic chromosome pairs show that crossing-over has, indeed, taken place.     

The B-chromosomes of Locusta migratoria I. Detection of negative correlation between mean chiasma frequency and the rate of accumulation of the B's; a reanalysis of the available data about the transmission of these B-chromosomes

B-chromosomes were studied in two Spanish populations of Locusta migratoria. Both exhibit a high frequency of B-carrying individuals (75% and 94%). In both the B-chromosomes are mitotically unstable and they accumulate in the male germ line. The mean rate of accumulation is 28.5% in Baños de la Encina and 31.9% in Carboneras. There are no significant differences in mean cell chiasma frequency and between-cell variance of chiasmata between follicles with different number of B's within individual males. Likewise, there are no significant differences in mean cell chiasma frequency and between-cell variance between males with and without supernumeraries. However, a significant negative correlation exists between mean chiasma frequency and the rate of accumulation.