Ultrastructure and behavior of the achiasmatic,telosynaptic XY pair of the sand rat (Psammomys obesus)

The behavior of the X and Y chromosomes in somatic and testicular cells of the sand rat (P. obesus) has been investigated with light and electron-microscope procedures. The Y chromosome has been identified as the fourth longest of the complement, both by C-banding and by its meiotic behavior. The X chromosome is the longest of the complement and carries two major C-heterochromatic blocks, one in the distal part of the long arm and the other forming most of the short arm. During presynaptic stages in spermatocytes, separate C-heterochromatic blocks, representing the sex chromosomes, are observed in the nuclei. An XY body is regularly formed at pachytene. During first meiotic metaphase the X and Y chromosomes show variable associations, none of them chiasmatic. Second meiotic metaphases contain, as in other mammals, a single sex chromosome, suggesting normal segregation between the X and the Y. — Electron microscopic observations of the autosomal synaptonemal complexes (SCs) and the single axes of the X and Y chromosomes during pachytene permit accurate, statistically significant identification of each of the largest chromosomes of the complement and determination of the mean arm ratios of the X and Y axes. The X and Y axes always lie close to each other but do not form a SC. The ends of the X and Y axes are attached to the nuclear envelope and associate with each other in variable ways, both autologously (X with X or Y with Y) and heterologously (X with Y), with a tendency to form a maximum number (four) of associated ends. Analysis of 36 XY pairs showed no significant preference for any single specific attachment between arm ends. The eighth longest autosomal bivalent is frequently partially asynaptic during early pachytene, and only at that time is often near or touching one end of the X axis. — It is concluded that while axis formation and migration of the axes along the plane of the nuclear envelope proceed normally in the X and Y chromosomes, true synapsis (with SC formation) does not occur because the pairing region of the X chromosome has probably been relocated far from the chromosome termini by the insertion of distal C-heterochromatic blocks.     

Association between synaptonemal complexes of sex and autosomal bivalents in male tx/ty mice as a possible cause of their sterility

Electron microscopic study of total preparations of synaptonemal complexes of spermatocytes I from sterile heterozygous male mice--t12/tw18; tw5/twPa-1; twPa-1/tw18 was performed. T/tw18 and C3H/N fertile heterozygotes were used in each variant as control. The cells are karyotyped in all experiments, as based on the measurements of the length of 19 SC autosomes and SC sex complex. All sterile compounds (spermatocytes) demonstrate high frequency of different types of associations (72%) between sex chromosomes and the autosome 17 carrying a chromosomal aberration in the region of the T-locus. The heterozygotes tx/ty used in our experiments show no disruption of chromosome synapsis, when even studied under electron microscope, though some atypical changes in the ultrastructure of chromosome axes and frequent atypical associations of the axes of XY-sex bivalents in sterile heterozygous animals exist.     

Electron microscopic investigations of synaptonemal complexes in an infertile human male carrier of a pericentric inversion inv(1)(p32q42)

Summary Electron microscopic investigations of surface spread synaptonemal complexes in spermatocytes from a 37-year-old man ascertained for infertility detected a pericentric inv(1), and subsequent lymphocyte analysis placed the break-points at p32 and q42. Most spermatocytes showed a maturation arrest at mid-pachytene explaining the azoospermia. As in two other comparatively large loop-forming pericentric inversions, initiation of synapsis took place in the middle of the inverted segment. Thus there is no indication of interstitial synaptic initiation being restricted to special pairing sites along the length of the chromosome. All spermatocytes investigated at mid-pachytene showed inversion loops, none of which was fully synapsed with a specific delay in pairing of the heterochromatic block 1qh and adjacent segments. The loops were of similar size in all the cells examined and synaptic adjustment had not taken place. There was no indication of a preferential association between the inv(1) bivalent and the XY configuration, and a functional disturbance of the X seems an unlikely reason for the meiotic maturation arrest. The most likely cause may be the failure of adequate synapsis of the inverted segment and the possibly associated pairing abnormalities of other homologues, including asynapsis and/or precocious desynapsis.     

Distribution of the Rad51 recombinase in human and mouse spermatocytes.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.     

The meiotic behaviour of the XY pair in Lutreolina crassicaudata (Marsupialia: Didelphoidea)

The meiotic behaviour of the XY pair of the didelphid Lutreolina crassicaudata is analyzed by microspreading of spermatocytes for visualization of chromosomal axes and by three-dimensional reconstruction of spermatocyte nuclei from EM thin sections. The delay in pairing of sex chromosomes compared to autosomes and the absence of a synaptonemal complex between the axes of the X and Y chromosomes, already described for South American marsupials by three-dimensional reconstruction and for Australian species with synaptonemal complex microspreadings, is confirmed for this species. Sections demonstrate that at the diffuse stage and diplotene the dense plate occupies the region of the inner face of the nuclear envelope in contact with the XY body. Spreads show an structure similar in staining to the axes that becomes apparent simultaneously with the dense plate, called a balloon. The mechanism of XY pairing during meiotic prophase appears to be common to American and Australian marsupials as the same morphological pattern is found in all the species described. This mechanism is different from the way of pairing and segregation known for eutherian sex chromosomes.     

Ultrastructure of the synaptic autosomes and the ZW bivalent in chicken oocytes

Chromosomal axes of chicken oocytes from pre- and post-hatching chickens were analyzed with a microspreading technique for electron microscopy. At leptotene, chromosomal axes begin to be formed as discontinuous, non-polarized axial segments. During zygotene synaptonemal complex (SC) formation begins at the axial ends attached to the nuclear envelope. Polarization of axial ends is nearly simultaneous with the beginning of SC formation. The complete SC set is found at pachytene and it consists of 38 SC's and an unequal SC which has been identified as the ZW pair. This unequal SC is formed by two axes of different length. The Z and W axes represent 6.2% and 4.5% respectively of the combined length of the SC set plus the Z axis. The unpaired segment of the Z axis shortens markedly from early to mid-pachytene and becomes thicker than the lateral elements of SCs. In the paired region the Z axis forms most of the twists around a straighter W axis, suggesting some extent of non-homologous pairing between the Z and W chromosomes in this region. The existence of partial synapsis of the Z and W axes without heteropycnosis of the sex chromosomes is in marked contrast to partial synapsis in the heteropycnotic XY body of mammalian spermatocytes.     

Mitotic and meiotic analysis in Arctocephalus australis (Otariidae)

The karyotype with C-, G- and NOR-banding of Arctocephalus australis is reported for the first time. The chromosomal number is 2n = 36. The X chromosome, identified in G-banded metaphases from males, is metacentric and the Y chromosome is a minute chromosome, also metacentric. Pachytene spermatocytes were used for synaptonemal complexes analysis with a surface spreading technique. A total of 17 autosomal synaptonemal complexes are observed plus the XY pair. During early pachytene, the X and Y axes are thickened and remain unpaired. As pachytene advances, a short SC is formed between the gonosomes, as it is common among eutherian mammals. The particular asymmetrical appearance of the synaptonemal complex in the sex pair is described and compared to other cases among mammals.     

Synaptic behaviour and recombination nodules in the human XY pair

A sample of 90 XY pairs from men with normal karyotypes has been analyzed by measuring their morphological features in electron micrographs of microspread spermatocytes. The classification of human XY types (Solari, 1980) has been given stricter definitions. Stepwise splitting of the axes is seen in types 1 and 2. The development of axial branches and lenhthening of the X axis is seen in type 3. In the two subtypes a and b of type 4 the net-like filamentous array grows in length to a maximum (average=59.7 m) in subtype b. The location of the putative Y kinetochore defines a short arm that measures 22.34% of Y axis length, and the kinetochore of the X axis defines a short arm of 38.15% of the axial length. The average number of excrescences in the X axis is 19.9 and in the Y is 4.3. The frequency of a non-homologous, distal end-joining grows steadily from type 0 to type 3. The average length of the synaptonemal complex (SC) in 51 XY pairs of types 1 and 2 is 1.33 m (SD=0.65) and it corresponds to 25.54% of the Y axis length. Thus, the average SC covers the short arm of the Y and the pericentromeric region. Maximum lengths of this SC may reach up to 81.8% of the Y axis, 30 recombination nodules (RNs) were located in 26 XY pairs, and 90% of the nodules are located in the distal half of the short arm of the Y axis. Thus, RNs are restricted to a segment much shorter than the length of the average SC. A gradient of decreasing probability of recombination may reach up to the centromeric region of the Y chromosome. Some possible consequences of these facts are discussed.     

Persistence of two Y chromosomes through meiotic prophase and metaphase I in an XYY man

Summary Studies of spermatogenesis in an XYY male, presenting at a subfertility clinic, confirm the tendency for the germ cells to lose the second Y chromosome but for some XYY cells to reach metaphase I (MI). Light microscope studies of MI revealed the presence of YY bivalents and EM studies of microspread, silver-stained pachytene stages showed 30% of the cells to have two Y chromosomes; 13 out of 16 of these showing a YY synaptonemal complex. Strikingly, the Y axes show only partial synapsis; in no case was synapsis of the long arm heterochromatic regions apparent.     

Synaptonemal complex analysis in human male infertility

The fine structural features of human spermatocytes from carriers of some of the most frequent chromosomal abnormalities are reviewed on the basis of original data and previous reports from the literature. Special emphasis is given to the Robert-sonian translocations t (13; 14), to one specific reciprocal translocation involving chromosome 21, and to Y disomy in spermatocytes from XYY men. Synaptonemal complex analysis shows that in many carriers of chromosomal aberrations that lead to pachytene configurations having terminal asynaptic segments in autosomes, there is a gradual association of these asynaptic segments with the XY body. This associations with the XY pair is assumed to trigger a process of germ cell deterioration, presumably through the spreading of the X-chromosome inactivation towards autosomal segments. Another different process of germ cell deterioration occurs when the X chromosome becomes an univalent, as in XYY men with persistence of two Y chromosomes in the germ line. The renewed interest in the examination of spermatocytes from human testicular biopsies is commented upon.     

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.     

Synaptonemal complexes and associated structures in microspread human spermatocytes

Human spermatocytes processed with a modified microspreading technique which involves the use of sodium dodecyl-sulphate (SDS) have been used to construct synaptonemal complex (SC) karyotypes. Twenty two pachytene spermatocytes were selected for length quantitation. The mean values of relative lengths and centromeric indexes of each SC agree closely with values obtained by three-dimensional reconstructions (Holm and Rasmussen, 1977), except for SCs #4–5, 6–7 and 19–20. Absolute lengths are consistently longer in spreads (10.7% longer than in sections, on average). The mean total length of the SC complement is 258.7 m. Six morphological types of XY paris have been described. On the basis of the relationships between the XY pair, nucleolar development and autosome behavior, these six XY types are assumed to develop in succession. Type O XY pairs occur during late zygotene, types I and II XY pairs occur during early to midpachytene, and types III, IV and V occur during later pachytene substages. Alignment of the X and Y axes is observed at late zygotene, and formation of the SC occurs in relation with type I XY pairs. Progressive desynapsis occurs in types II and III. Splitting and fusion of the X and Y axes attain a maximum in types IV and V. The breakdown of the dense bodies associated with the X and Y axes occurs during stage V. — Bar-like structures, having a mean length of 2,100 Å are associated with SCs in all the pachytene substages defined by the XY types. The average number of bars per nucleus is 46.2 (SD=8.4, N=20), and the average SC length per bar is 5.57 m. The distribution along the SCs of 923 bars shows that near-termini locations are preferred (SC length per bar, 2.98 m) and centromere regions are avoided (SC length per bar, 16.9 m). — On the basis of these data, bars are similar to recombination nodules described in other organisms. The availability of a standard SC karyotype for microspreads and a temporal sequence given by the XY pair provide a basis for rapid screening of chromosome aberrations in human testicular biopsies.     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Synaptonemal complex analysis in Talpa occidentalis spermatocytes (Insectivora, Mammalia). II. Evolution of the X-chromosome self-pairing process

Zygotene and pachytene configurations of the X chromosome were studied in whole-mount, silver-stained preparations of spermatocytes from XY males from a population of Talpa occidentalis in which sex reversal has been described. The most striking finding in this study was a self-pairing conformation of the axial (differential) element of the X chromosome. This self-pairing was markedly constant in the site of initiation, which invariably involved the distal free end of the X and the region beyond the X-Y pairing segment, so that X-Y synapsis was never disturbed. In addition, self-pairing occurred later than autosomal synapsis and was accompanied by thickening of the axes, although this seemed to occur after the formation of an ordinary synaptonemal complex. The etiology of this phenomenon may be based on residual homology, possibly due to conservation of a primitive isochromosome throughout the karyotypic evolution of this species. However, the possible relationship between self-pairing and sex reversal remains obscure.     

The chromosomes of Micromys minutus (Rodentia, Murinae). II. Pairing pattern of X and Y chromosomes in meiotic prophase

Both light and electron microscopy were used to study the pairing behavior of the sex chromosomes of the harvest mouse, Micromys minutus, in surface-spread pachytene spermatocytes. The XY pairing pattern is very exceptional in that the site of synaptic initiation is located interstitially in the short arms of the X and the Y, next to their centromeric regions. From this tiny euchromatic site, synapsis proceeds unidirectionally along the homologous heterochromatic short arms of the X and the Y toward the ends of the chromosomes. After pairing of the short arm is concluded, synapsis begins between the nonhomologous long arms of the X and the Y in the immediate vicinity of the centromeres and progresses unidirectionally toward the end of the long arm of the Y. A synaptic complex develops between the constitutive heterochromatin of the long arm of the Y and the euchromatin of the long arm of the X. Analysis of C-banded and distamycin A/DAPI-stained diakineses revealed a trefoil-like XY bivalent, which was interpreted to be the result of an interstitial chiasma occurring in the paired short arms of the X and the Y. A conspicuous, electron-dense body, about 1 micron in diameter, was found closely associated with the centromeres of the X and the Y in numerous pachytene spermatocytes. A review of the literature showed that comparable XY-associated bodies have been found in only eight other mammals to date.     

The morphological sequence of XY pairing in the Norway rat Rattus norvegicus

The sequence of XY pairing at meiotic prophase in the Norway rat, Rattus norvegicus, has been studied in spread preparations of spermatocytes obtained from pubertal males. As in most mammals, sex chromosome pairing is delayed in relation to that of the autosomes. At one stage in pachytene, the Y is fully paired in synaptonemal complex association with about one-third of the X. Observation in spread preparations at pachytene and diplotene and in air-dried metaphase I preparations indicates that the long arm of the Y pairs with the short arm of the X. Pairing of the Y with both ends of the X is seen in about 4% of pachytene spermatocytes. The possibility that XY pairing in the rat may be nonhomologous (Ashley 1983) is considered, and the view is expressed that the XY synaptonemal complex may be incomplete in fine structural detail, thus not providing for the effective pairing required in true reciprocal recombination. The same mechanism that excludes crossing over from heterochromatic regions of autosomes may also operate to minimize or prevent crossing over in the sex pair of mammals.     

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.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.