An analysis of meiotic impairment and of sex chromosome associations throughout meiosis in XYY mice

The existing XYY meiotic data for mice present a very heterogeneous picture with respect to the relative frequencies of different sex chromosome associations, both at pachytene and diakinesis/metaphase I. Furthermore, where both pachytene and diakinesis/MI data are available for the same males, the frequencies of the different configurations at the two stages are very different. In the present paper we utilise "XYY" and "XY/XYY" mosaic mice with cytologically distinguishable Y chromosomes to investigate the factors responsible for this heterogeneity between different males and between the two meiotic stages. It is concluded (1) that the initial pattern of synapsis is driven by the relatedness of the three pseudoautosomal regions (PARs); (2) that the order and extent of PAR synapsis within radial trivalents are also affected by PAR relatedness and that this leads to chiasmata being preferentially formed between closely related PARs; (3) that trivalents with a single chiasma resolve into a bivalent + univalent by the diakinesis stage; (4) that although many spermatocytes with asynapsed sex chromosomes are eliminated between pachytene and diakinesis, those that survive this phase of elimination progress to the first meiotic metaphase (MI) and accumulate in large numbers, leading to an over-representation of those with univalents as compared to radial trivalents; and (5) that the arrested MI cells are eventually eliminated, so that very few "XYY" cells contribute products to MII.     

Synaptonemal complexes and chromosome chains in the rodent Ellobius talpinus heterozygous for ten Robertsonian translocations

Synaptonemal complexes (SC) in four Ellobius talpinus males heterozygous for ten Robertsonian translocations were examined with an electron microscope using a surface-spreading technique. A total of 136 late zygotene and pachytene spermatocytes were examined. From one to three completely paired SC trivalents were found in each early pachytene spermatocyte. The lateral elements of the short arms of the acrocentric chromosomes in these trivalents were joined with an SC thus forming the third arm of the SC trivalent. At the same stage a few SC trivalents did not contain lateral elements in the pericentromeric region of the metacentric chromosomes and remained unpaired in this region up to mid pachytene. At zygotene and pachytene from two to eight SC trivalents were joined into chains due to formation of SCs between the short arms of acrocentrics of other SC trivalents. These chains are frequent at late zygotene, but are resolved during pachytene into individual trivalents. It is proposed that pairing and SC formation between the short arms of the acrocentric chromosomes results from the monosomy of the short arms and partial DNA homology between these heterochromatic regions. Since crossing over probably does not take place in these segments, the chromosomal chains may subsequently be corrected into trivalents by a dissolution of the SCs combining adjacent trivalents. The correction and disjoining of chains may not be effective in all cells. The cells in which the chains are retained are assumed to be arrested at the pachytene stage.     

Cytogenetics of collared lemmings (Dicrostonyx groenlandicus). II. Meiotic behavior of B chromosomes suggests a Y-chromosome origin of supernumerary chromosomes

The patterns of synapsis and chiasma formation of the B chromosomes of male collared lemmings (Dicrostonyx groenlandicus) were analyzed by light and electron microscopy and compared to expectations for various hypotheses for the intragenomic origin of supernumerary chromosomes. Pachytene analysis revealed a variety of synaptic configurations including B-chromosome univalents, bivalents and trivalents. In approximately one-half of the pachytene nuclei examined, B chromosomes were in synaptic associations with the normally unpaired portion of the Y chromosome. The B-chromosome configurations at pachynema, including those involving the Y chromosome, were maintained into diakinesis and metaphase I. The meiotic behavior of the B chromosomes was inconsistent with their derivation from centric-fusion products, isochromosome formation, small-autosome polysomy, or the X chromosome. However, the frequent synapsis and apparent recombination between B chromosomes and the Y chromosome implicate this sex chromosome as a possible source of the B chromosomes in collared lemmings.     

Synaptic behaviour and morphological modifications of the X and Y chromosomes during pachytene in three species of Ctenomys (Rodentia, Caviomorpha, Ctenomyidae).

Synaptic behaviour and the progression of morphological differentiation of the XY chromosome pair during pachytene was studied for the first time in three species of the South American subterranean rodents of the genus Ctenomys (tuco-tucos). In general, synapsis progression in the sex pair could be subdivided into four substages: (i) initial partial synapsis of the X and Y chromosome axes and beginning of the differentiation of the unsynapsed regions; (ii) complete or almost complete synapsis of the Y axis accompanied with morphological differentiation of the unsynapsed region of the X chromosome; (iii) a novel stage exclusive to Ctenomys perrensi consisting in a retraction of the free X axis, associated with the formation of a homogeneous and dense structure along the synaptic region, which leads to the achievement of full synapsis between sex chromosomes; or (iv) an increase in morphological complexity involving extreme splitting of the XY pair. The implications of the peculiar synaptic behaviour displayed by sex chromosomes in C. perrensi, a species complex highly polymorphic for Robertsonian translocations, are discussed in relation to both the triggering of the pachytene checkpoint and the avoidance of non-homologous associations between sex chromosomes and the asynaptic pericentromeric regions of trivalents in translocation heterozygotes.     

Sex chromosome configurations in pachytene spermatocytes of an XYY mouse

Karyotypic investigation of a phenotypically normal but sterile male mouse showed the presence of an XYY sex chromosome constitution. The synaptic behaviour of the three sex chromosomes was examined in 65 pachytene cells. The sex chromosomes formed a variety of synaptic configurations: an XYY trivalent (40%); an XY bivalent and Y univalent (38.5%); an X univalent and YY bivalent (13.8%); or X, Y, Y univalence (7.7%). There was considerable variation in the extent of synapsis and some of the associations clearly involved nonhomologous pairing. These observations have been compared with previously published information on chromosome configurations at metaphase I from other XYY males.     

Meiotic association between the XY chromosomes and unpaired autosomal elements as a cause of human male sterility

Intimate association between autosomal translocation trivalents and XY bivalents at pachytene was observed in a majority of cells of two men ascertained through primary sterility and found to be heterozygous for a 14;21 Robertsonian translocation. The association, studied by light and electron microscopy of spread first spermatocytes, was between the unpaired short arms of the normal chromosomes of the translocation trivalent and the differential axes of the XY chromosomes. In a minority of cells, this contact was not established, or not maintained, as alternative combinations between the elements available for non-homologous pairing were realized. Following a suggestion of Lifschytz and Lindsley (1972), sterility in these patients was attributed to spermatogenic arrest caused by physical contact of sex chromosomes with autosomal material and consequent interference with the normal metabolism of the sex chromosomes. Autosomal aberrations and polymorphisms, which lead to the presence of unpaired segments at meiosis, may thus play a critical role in a general mechanism of chromosomally-derived male sterility. It is proposed that such a mechanism may also be instrumental in the initiation of reproductive barriers in nature.     

Identification of putative sex chromosomes in the blue tilapia, Oreochromis aureus, through synaptonemal complex and FISH analysis

Sex determination in the blue tilapia, Oreochromis aureus, is primarily a ZW female-ZZ male system. Here, by analysis of the pachytene meiotic chromosomes of O. aureus, we demonstrate the presence of two distinct regions of restricted pairing present only in heterogametic fish. The first, a subterminal region of the largest bivalent is located near to the region of unpairing found in the closely related species O. niloticus, while the second is in a small bivalent, most of which was unpaired. These results suggest that O. aureus has two separate pairs of sex chromosomes.     

Synaptonemal complex analysis in spermatocytes of diploid and triploid Chinese shrimp Fenneropenaeus chinensis

A modified surface spreading technique for synaptonemal complex (SC) analysis was tested to assess the process of chromosome synapsis in spermatocytes of diploid and induced triploid Fenneropenaeus chinensis. Spermatocytes of diploid shrimp showed typical morphological characteristics of eukaryote SC, with complete synapsis of bivalents. No recognizable bivalent associated with sex chromosomes was observed in spermatocytes of diploid shrimp. However, differences in morphology of SC, including unsynapsed univalents, bivalents, totally paired trivalents with non-homologous synapsis, partner switches and triple synapsis were identified at early pachytene stage of triploid spermatocytes. Triple synapsis was especially common at late pachytene stage in spermatocytes of triploid shrimp. The observed abnormal synapsis behavior of chromosomes in spermatocytes indicated that triploid male shrimp may find it difficult to develop normal haploid sperm.     

Unequal nondisjunction frequencies of trivalent chromosomes in male mice heterozygous for two Robertsonian translocations

Robertsonian (Rb) translocation heterozygosity may cause pairing problems during prophase and segregation irregularities at anaphase of meiosis I. These stages of meiosis I were studied in male mice doubly heterozygous for the two Rb chromosomes Rb(9.19)163H and Rb(16.17)8Lub. At pachytene both Rb chromosomes similarly showed pairing irregularities like unpaired segments. However, highly different nondisjunction frequencies of chromosomes forming the respective trivalents were found. The nondisjunction frequency of the Rb8Lub trivalent chromosomes was about 40%, whereas a very low frequency of nondisjunction was found in combination with the Rb163H trivalent. Since both trivalents were together in the same cell, differences in kinetochore function are assumed to be responsible for the diverse frequency of nondisjunction.     

Distribution of Atr protein in primary spermatocytes of a mouse chromosomal mutant: a comparison of preparation techniques

In this study, we examined the suitability of a three dimensional preparation technique for studying chromosome behaviour in the first meiotic prophase in the mouse chromosomal mutant T(1;13)H/T(1;13)Wa. To preserve cellular shape, primary spermatocytes were encapsulated in a fibrin clot. Conventionally sedimented prophase nuclei served as controls. Axial elements and lateral synaptonemal complex components were subsequently stained by immunofluorescence and the presence of axial elements at the pachytene stage was highlighted with indirect immunofluorescence against the Atr protein. We compared the distribution of Atr signal in the fibrin-embedded spermatocytes with surface-spread preparations and immunohistochemically stained histological sections of seminiferous tubules. Furthermore, fluorescence in situ hybridisation of the mouse minor satellite DNA was done on fibrin-embedded spermatocytes. The Atr signal is most conspicuous in fibrin-embedded nuclei on unpaired axial elements during pachytene, both for sex chromosomal and for autosomal segments, and expanding from these elements into the surrounding chromatin. Both spread and encapsulated zygotene nuclei with extended axial element formation proved to be positive for Atr. Mid- to late zygotene nuclei were devoid of 3,3′-diaminodibenzene deposition in the histological sections. Highlighting the unpaired axial elements in the small heteromorphic 1¹³H;1¹³Wa bivalent with an Atr signal enabled meiotic analysis of this bivalent to be carried out in a three-dimensional context. Thus, proximity of this bivalent with the sex chromosomes is found more often in three-dimensional preparations than in spread preparations. Furthermore, the development of the Atr signal over the sex chromosomes as pachytene proceeds helps in substaging of this long and heterogeneous meiotic phase, in sedimented but especially in fibrin-encapsulated nuclei. Received: 22 September 1999; in revised form: 20 December 1999 / Accepted: 21 December 1999     

Meiotic chromosome pairing in fetal oocytes of trisomy 21 human females

The influence of trisomy on meiotic chromosome association and synapsis was studied in oocytes of two trisomy 21 fetuses. The patterns of association of the three chromosomes 21 were determined by analysis of late zygotene to early diplotene fetal oocytes after immunofluorescent staining of synaptonemal complexes. The identity of chromosome 21 was confirmed using FISH with either a whole chromosome 21 paint or an alpha-satellite DNA repeat probe. In both fetuses, a wide variety of configurations was present at pachytene. The most common configurations were a trivalent (35.5% and 51.6% of analyzable cells) and a bivalent plus univalent (62.9% and 45.2%). These different frequencies between the fetuses were not significant. Trivalents showed either triple synapsis or double synapsis with pairing-partner switches. The extent of triple synapsis varied from a short segment, either terminal or interstitial, to the whole chromosome length. Through use of immunofluorescent staining of the centromeres, we identified novel types of abnormal chromosome behavior in trisomy 21 fetal oocytes. Thus, we found that 6/41 trivalents had one of the chromosomes associated "out of register," i.e., in a nonhomologous fashion, with its two homologs. Likewise, we found three cells with bivalent plus univalent configurations, in which the univalent showed self-synapsis. The presence of three copies of chromosome 21 therefore results not only in the formation of complex and highly variable synaptic associations but also causes a significant increase in the occurrence of nonhomologous synapsis in human fetal oocytes.     

Ultrastructure,meiotic behavior,and evolution of sex chromosomes of the genus Ellobius

The whole-mount SC preparations from males of three species of the genus Ellobius (Ellobius fuscocapillus, Ellobius lutescens), and Ellobius tancrei were studied by electron microscopy. In the males of Ellobius fuscocapillus, behavioral peculiarities of the sex bivalent (viz. the normal male heterozygosity) are characterized by early complete desynapsis of sex chromosomes (X, Y), occurring at late pachytene-early diplotene. The karyotype of species Ellobius lutescens is unique for mammals. In both sexes it is characterized by an odd number of chromosomes (2n=17). At prophase I the unpaired chromosome 9 is not involved in synapsis with other chromosomes and forms a sex body at the end of pachytene.The complete Robertsonian fan has been described for superspecies Ellobius tancrei. As shown on the basis of G-band patterns the male and female sex chromosomes are cytologically indistinguishable.Analysis of whole-mount SC preparations revealed the formation of a closed sex SC bivalent and showed some morphological differences in the axes of sex chromosomes at meiotic prophase I. A number of assumptions are made about the relationship between the behavior of sex chromosomes, their evolution and the sex determination system in the studied species of genus Ellobius.

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Non-relationship between nucleolus and sex chromosome system Xyp in Chelymorpha variabilis boheman (Coleoptera: Chrysomelidae)

Nucleolar-organizer region, nucleolus and mode of association of the sex bivalent were analyzed in spermatecytes of Chelymorpha variabilis Boheman. This species (2n=10II+Xy_p) shows the typical sex chromosome system of the group Polyphaga. The results of silver staining techniques showed the nucleolar organizer region localized in a subterminal position of an autosomal bivalent. During meiotic prophase the nucleolus was distinguished with the silver staining and acridine orange fluorescence technique up to diakinesis. The independence of nucleolus and sex bivalent Xy_p during meiosis is demonstrated. The positively silver staining but negatively orange-red material found within the parachute could be involved in the regular co-orientation of both sex chromosomes. After a longer hypotonic treatment, sex bivalents were observed elongated and paired only at one end during the pachytene stage. Along these sex chromosomes, C-bands showed positive blocks located in the pericentromeric and telomeric regions. Heterochromatic association of both sex chromosomes was suggested.     

Mapping of pachytene bivalents of female codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae)

Spread pachytene nuclei of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) females of a Syrian strain (SY) were used to investigate chromomere patterns of chromosome bivalents and determine their length. The karyotype of female codling moths consists of 28 chromosome bivalents, of which seven are clearly distinguishable using chromosome length and the number and size of the chromomeres in the pachytene stage. One autosome bivalent has two nucleolar organizing regions (NORs) that are located at the opposite ends of the chromosome and appear as distinct structural landmarks. In female codling moths, the WZ sex chromosome bivalent was easily identified in pachytene oocytes according to the heterochromatic thread of the W chromosome. This study contributed to the knowledge and identification of pachytene chromosomes of female codling moths.     

Random acrocentric bivalent associations in human pachytene spermatocytes. Molecular implications in the occurrence of Robertsonian translocations

Acrocentric bivalent associations were studied in 232 human male germ cells at pachytene in order to understand better the preferential involvement of chromosomes 13, 14, and 21 in Robertsonian translocations. The tendency of each acrocentric bivalent to associate with another was not correlated with NOR activity, as measured by silver staining. Good agreement was noticed between their ability to associate and the amount of satellite DNA in human acrocentric chromosomes. The distribution of two-by-two acrocentric bivalent associations was random. In order to reconcile this result with the nonrandom distribution of Robertsonian translocations, a molecular hypothesis is proposed. The model is based on homology of recombinational sites, interspersed at regular interval in satellite DNA, which could increase the probability of accidental unequal crossing-over between two specific acrocentric chromosomes.     

The nature of the 1;29 translocation in cattle as revealed by synaptonemal complex analysis using electron microscopy

Synaptonemal complex analyses were carried out by electron microscopy on surface-spread spermatocytes of one normal bull and two bulls that were heterozygous for the so-called 1;29 translocation. The autosomal bivalents of the normal karyotype, which could be arranged by size in a series, demonstrated kinetochores at the terminally located attachment plaques. One autosomal bivalent was clearly larger than the rest and apparently consisted of the long arm of the 1;29 translocation. The 1;29 translocation was the longest autosome in the set and had a kinetochore in a subtelocentric position. Some of the autosome pairs had nucleolus organizer regions in telomeric regions. The X and Y chromosomes, which were not paired at zygotene, demonstrated association in a very short segment at early pachytene; in no cells could a synaptonemal complex be seen between the X and Y. Very often the sex chromosomes were dissociated. At zygotene, a few, usually large, bivalents were unpaired proximally. This always also involved the proximal parts of the arms of the 1;29 translocation and their normal homologs. At early pachytene, the 1;29 trivalent, although to a less extensive degree, was also unpaired in the pericentric region. Configurations in which one chromosome, either 1 or 29, was completely paired with its corresponding arm in the 1;29 translocation chromosome also occurred. When unpaired proximally, the size of chromosome 1 agreed fairly well with the size of its corresponding arm, but the size of chromosome 29 was considerably larger than the corresponding arm of the 1;29 translocation chromosome. During late zygotene and early pachytene, the percent difference between chromosome 29 and its corresponding arm decreased, and at mid and late pachytene there had been a complete synaptic adjustment. The size difference and pairing behavior indicated that a deletion of the kinetochore and the most proximal segment of chromosome 29 had preceded the fusion with chromosome 1 into the 1;29 translocation. The unique structural appearance of the 1;29 translocation chromosome compared to that of other centric fusion translocations in cattle lends support to the theory of a monophyletic origin of the 1;29 translocation. The importance of the pairing behavior observed in governing recombination and chromosome disjunction is briefly discussed.     

Chromosome banding and genome compartmentalization in fishes

The diploid chromosome number of the Chinese raccoon dog varies from 54 (no B chromosomes) to 58 (4 B chromosomes). The B chromosomes are totally heterochromatic. An electron microscopic study was made of the synaptonemal complexes (SC) in spermatocytes of these animals. The SC karyotype consists of 27 regular chromosome pairs (autosomes and the sex chromosomes) plus the B chromosomes. The Bs pair effectively with one another at pachytene, but the SC axes of the B chromosomes are much denser than those of the A chromosomes. Depending on the number of Bs, both bivalents and multivalents have been observed. When three B chromosomes are present in a cell, parallel alignment of all three SCs can be seen. Formation of multivalents indicates high homology among these supernumerary heterochromatic chromosomes. Fusiform bulges are found along unpaired regions of all chromosomes which are particularly pronounced in diplotene.     

Synaptonemal complex analysis of a three-breakpoint translocation in a subfertile bull.

Somatic chromosome analysis of a subfertile Brown Swiss bull demonstrated a three-breakpoint translocation involving chromosomes 1, 8, and 9 in G- and R-banded karyotypes. Based on standard bovine chromosome nomenclature, the translocation was defined as t(1;8;9)(q43;q13;q26). Synaptonemal complex analysis of the chromosome aberration by electron microscopy revealed a hexavalent configuration in 52 of 53 pachytene cells. Twenty-seven cells (51%) had a completely paired hexavalent configuration showing distinctly nonhomologous pairings between normal and/or translocated chromosomes involved in the exchanges. Thirteen cells showed a hexavalent configuration with centrally unpaired chromosome segments but with completely paired terminal arms. In 13 cells (including one at zygotene) the translocation chromosomes formed an open hexavalent, and in one cell there were two completely paired trivalents. Thirty-two cells at diakinesis-MI demonstrated 28 configurations, including one large hexavalent. Testicular histology, testis size, and seminal characteristics were normal.     

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.