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.     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Synaptonemal complex (SC) analysis by electron microscopy of spermatocytes in surface microspreads was carried out in mice heterozygous for two paracentric inversions: either In(1)1RK or In(2)5Rk. Characteristic SC inversion loops are formed at synapsis in bivalents carrying the rearrangements. Although all loops were observed to be eliminated by late pachytene through synaptic adjustment, every spermatocyte at early pachytene contained a fully synapsed loop. Cells in the earliest stage of pachytene contained the longest loops and thus had undergone minimal adjustment. The SC estimates of inversion lengths and breakpoint positions in such cells corresponded well with those from mitotic chromosome banding and could be correlated with genetic maps of chromosomes # 1 and # 2, thus demonstrating the basis for the mapping of pachytene chromosomes. The regularity of loop formation and reproducibility of the SC analysis are reflected in the constant relative positions of the estimated breakpoints. The method is sensitive enough to reflect small, real, interstitial length differences between meiotic and mitotic chromosomes. The results demonstrate the feasibility and precision of detection and quantitative characterization of inversions at early meiotic prophase by SC analysis.This paper is warmly dedicated to Prof. Dr. Wolfgang Beermann, on the occasion of his 60th birthday     

Synaptonemal complex analysis of mouse chromosomal rearrangements

Two paracentric inversions in the mouse, In(1)1 Rk and In(2)5 Rk, have been studied in surface microspreads of spermatocytes from heterozygotes. At zytogene, synaptic initiation occurs independently in three regions: within the inversion, and without, on either side. Synaptonemal complex (SC) formation is restricted to homologous regions, resulting in inversion loops in all early pachytene spermatocytes. An adjusting phase then occurs during pachytene in which the inversion loop is reduced by desynapsis of homologously synapsed SC, followed immediately by non-homologous synapsis with the alternate pairing partner, progressing from the ends toward the middle. Adjustment occurs during the first half of pachytene, but is not closely synchronized with sub-stage. It is complete by late pachytene, the loop having been eliminated in all cases and replaced by straight SCs in which the inverted region is heterosynapsed. Synapsis in the adjustment phase is evidently permitted only after the homosynaptic phase, and is indifferent to homology. It may lead to heterosynapsis, as in the inversion region, or to synapsis of homologous regions not synapsed at zytogene. The anaphase bridge frequency, a measure of crossing over within the inversion, is about 34% for both inversions studied, indicating that such crossovers do not block adjustment, that crossing over probably occurs before or during the adjustment period, and that there is some crossover suppression. The last could be the consequence of blocking by desynapsis/heterosynapsis. Synaptic adjustment appears to be a general phenomenon that occurs to varying extents in different forms. A hypothetical scheme for two phases of synapsis is proposed: at zytogene, a basic propensity for indifferent SC formation is limited by a restricting condition to synapsis between homologous regions. Subsequently, the restriction is lifted, whereupon synaptic instability is resolved by desynapsis, followed by resynapsis that is indifferent to homology, but that results in a topologically more stable structure.     

Heterosynapsis and axial equalization of the sex chromosomes of the northern bobwhite quail.

The pairing behavior of the Z and W chromosomes in the female northern bobwhite quail (Colinus virginianus) was analyzed by electron microscopy of silver-stained synaptonemal complexes (SCs). After autosomal pairing was completed, synapsis of the sex chromosomes initiated at the short-arm end of the W chromosome and one end of the Z chromosome. Synapsis then progressed unidirectionally, producing a sex bivalent in which the entire length of the W axis was paired with an equivalent length of the Z axis. Progressive contraction and asymmetrical twisting of the Z axis ultimately resulted in a fully paired configuration with aligned axial ends. Further contraction of the Z axis reduced the extent of asymmetrical twisting such that only the nonaligned centromeric regions distinguished the SC of the ZW bivalent from SCs of similar-sized autosomes in late-pachytene nuclei. Quantitative analyses indicated that the length of the Z axis shortened significantly during the adjustment process, whereas no significant difference occurred in the length of the W axis. The nonalignment of the centromeric regions during transitional stages of ZW synapsis indicates that direct heterosynapsis of nonhomologous segments, followed by axial equalization of the length inequality, is responsible for the length adjustment during synapsis in the sex chromosomes of the bobwhite quail.     

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.     

A search for the synaptic adjustment phenomenon in maize

It has recently been suggested from several laboratories that complex synaptic configurations (required for homologous synapsis in the presence of heterozygosity for chromosome rearrangements, or resulting from multivalent formation in polyploids, or even resulting from interlocking of normal bivalents) may be formed initially, but are altered by dissolution of the central element of the synaptonemal complex followed by its reinstatement in such a way that only free bivalents, typical of normal sequence homozygotes in diploids, are usually found at late pachytene. It has been suggested that the synaptic adjustment inferred may be a process of widespread occurrence. Maize microsporocytes heterozygous for a paracentric inversion were studied at early and late pachytene and also at early diplotene. Clear remnants of loop configurations typical of homologous synapsis of the inverted region were found in a number of cells at early diplotene, and synaptic failure of the inverted region was common at both early and late pachytene. In maize microsporocytes a synaptic adjustment process comparable to that which has been reported in mammals seems to be absent.     

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.     

Spreading synaptonemal complexes from Zea mays

Four different inversion heterozygotes of maize were examined for the occurrence of synaptic adjustment. Three substages of pachytene were identified in synaptonemal complex (SC) spreads using side-by-side comparisons of chromosome squashes with two-dimensional spreads of SCs. In SC spreads, inversion loop frequency did not change substantially from early through late pachytene for any of the four inversion heterozygotes examined. In addition, the position and size of the inversion loops remained essentially constant throughout pachytene. These results indicate that synaptic adjustment of inversion loops does not occur during pachytene in Zea mays.     

The synaptic behaviour of the wild forms of Triticum turgidum and T. timopheevii.

Different wild allopolyploid species of Triticeae show extensive bivalent formation at zygotene while a considerable number of multivalents is present in cultivated polyploid wheats. To study the chromosome behaviour at early meiotic stages in wild forms of tetraploid wheats Triticum turgidum and T timopheevii (2n = 4x = 28) we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late zygotene and at pachytene of wild accessions of these species. The mean number of synaptonemal complex (SC) bivalents at mid-zygotene ranged from 12.22 to 13.14 among the accessions studied indicating a strong restriction of synapsis initiation to homologous chromosomes. The mean of bivalents increased at pachytene because of the transformation of multivalents into bivalents. Ring bivalents observed at metaphase I support that SC bivalents were formed by homologous chromosomes. The average values of SC bivalents at mid-zygotene in the wild forms are much higher than the average values observed in the cultivated tetraploid wheats but similar to that of a mutant line of T turgidum with a duplication that includes Ph1, the major homoeologous pairing suppressor locus. These results suggest that the efficiency of the mechanism operating in the homologous recognition for synapsis is higher in wild wheat populations than in cultivated varieties. Apparently, a relatively detrimental modification of the pairing regulating genetic system accompanied the domestication of the wild wheat forms.     

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus)

Relative length is a constant and distinctive characteristic for each autosomal SC, despite variations in absolute length from cell to cell. Arm ratio is distinctive for each SC except for two of the three sub-acrocentrics, and serves, together with relative length, for identification. The constancy of relative length and arm ratios indicates biological stability and lack of physical distortion in these spread preparations. There is a 11 relationship between relative lengths of autosomal SCs and mitotic autosomes; their arm ratios are also similar. These close parallels provide strikingly similar SC and somatic karyotypes. Variability was observed in sub-acrocentric arm ratios and in lengths of unpaired X and Y axes, correlated with the presence of constitutive heterochromatin. — Utilizing progressive differentiations of the X and Y chromosomes for staging, it is demonstrated that autosomal SCs decrease in length from late zygotene to mid-pachytene, and then increase at late pachytene. Within a nucleus, synchrony of length changes is maintained. It is concluded that the factors governing autosomal SC length are regular for any given bivalent from cell to cell, and may be related to those that control somatic autosome length relationships. — The X and Y axes differ quantitatively as well as qualitatively from autosomal SCs. The SC portion of the X and Y is constant in length through most of pachytene; the unpaired axes shorten and lengthen, but not in proportion to autosomal SCs. X and Y relative lengths and arm ratios vary throughout pachytene and do not maintain proportionality with somatic values. The evidence suggests, but does not prove, that the long arm of the X is paired with the short arm of the Y. — Twists occur in autosomal SCs at increasing frequencies throughout pachytene but cannot account for length changes. The number of twists per SC is directly proportional to SC length. Intertwining of SCs is random and proportional to SC length. End-to-end associations of autosomal SCs appear to be random; however, the ends of the X and Y are less often involved in such connections. — The length of axial material in all chromosomes at pachytene, expressed as an equivalent length of DNA double helix, represents 0.013% of the diploid DNA complement.     

Dense bodies in silver-stained spermatocytes of the chinese hamster: behavior and cytochemical nature

From the silver staining behavior of various organelles in the nucleus we have divided meiotic prophase (leptotene to the diffuse stage) of the male Chinese hamster into five stages. Components within the nucleus, such as synaptonemal complex (SC), sex bivalent (SB), nucleolus organizer regions (NORs), chromatin and the dense bodies, showed a characteristic feature in each stage of meiotic prophase. The lampbrush chromosome stage was found to be followed by the diffuse stage. The chromatin around SC began to be organized at early pachytene and formed a brush-like structure at late pachytene. During early prophase stages a dramatic change in SB morphology occurred. Three types of morphology of SB were recognized: (1) the XY pair with long synapsis and fusiform or diffuse thickening of the unpaired portions (late zygotene and early pachytene), (2) desynapsed, thread-like axes seen at midpachytene, and (3) multistranded, branched, and anastomosed axes seen at late pachytene.Two types of the dense body were found during meiotic prophase; the double body in early stage (leptotene to early pachytene) and the single body in later stages (mid pachytene to diffuse stage). The small precursors of the double body existed at early leptotene but they increased in size and also changed the silver stainability during zygotene, becoming the characteristic double body consisted of one light body (L-body) and one dark body (D-body). These two bodies can also be recognized after Giemsa or acridine orange (AO) staining. The L-body fluoresced reddish orange after AO staining. The single body, which is probably formed by amalgamation of the D- and the L-bodies, showed a staining reaction similar to that of the D-body.Data from pancreatic lipase and protease treatments suggest that the D-body contained a lipoprotein.     

Axial shortening during pachynema unrelated to nonhomologous synapsis

The pachytene behavior of chromosomes participating in quadrivalent formation in male mice heterozygous for T(X;4)7Rl or T(X;4)8Rl was analyzed in electron micrographs of microspread spermatocytes. In each population of nuclei from the translocation heterozygotes, the longest 4X axes were approximately the proportional length expected from the respective contributions of the 4 and the X estimated from breakpoint positions in mitotic chromosomes. However, the 4X axis of these translocation quadrivalents undergoes extensive shortening. In both R7 and R8 the shortest 4X axis observed in the population of nuclei was approximately the length of the normal 4 axis. This equalization of axial lengths suggests that there may be an interchromosomal interaction between synapsed chromosomes. In R8, axial shortening of the 4X occurs as pachynema progresses. In both translocations, shortening is accompanied by twisting of the 4X around the 4. Both axial shortening and twists are characteristics exhibited by chromosomal axes of unequal length as part of the meiotic phenomenon described as "synaptic adjustment" (Moses, 1977). Synaptic adjustment involves, in addition, nonhomologous synapsis, which is delayed until the latter part of pachynema. However, axial shortening in R7 and R8 is not accompanied by nonhomologous synapsis. In R7, nonhomologous synapsis does not occur; in R8, it is confined to quadrivalents in which the 4X axis is near its maximum length (i.e., early). This behavior suggests that axial shortening and nonhomologous synapsis during the progression of pachynema (previously considered collectively under the term "synaptic adjustment") are not necessarily coupled events.     

Expression and functional analysis of <Emphasis Type="Italic">TaASY1</Emphasis> during meiosis of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Background  

Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC.     

Synapsis in single and double heterozygotes for partially overlapping inversions in chromosome 1 of the house mouse

Electron microscopic (EM) analysis of synaptonemal complexes (SC) in single and double heterozygotes for the partially overlapping inversions In(1)1Icg, In(1)1Rk and In(1)12Rk in chromosome 1 of the house mouse reveals that synapsis and synaptic adjustment are dependent on the size and location of the inversions and interaction between the latter. In(1)1Icg contains insertions of the inverted repeats Is(HSR;1C5)1Icg and Is(HSR;1D)2Icg and an inverted euchromatic region. Synaptic adjustment of the D-loops by shortening of the asynapsed segments of the lateral elements belonging to the insertions occurs at the late zytogene to early pachytene stage. Synaptic adjustment of the inversion loops takes place at early to late pachytene. A delay in adjustment was found in the double heterozygotes In(1)1Icg/In(1)1Rk and In(1)1Icg/In(1)12Rk. A correspondence between the lifespan of asynapsis in inverted regions and the probability of association of XY and heteromorphic bivalents was revealed.     

Synaptic Adjustment of Inversion Loops in Neurospora Crassa

Heterozygotes for three long inversions on chromosome 1 were analyzed by serial reconstruction from electron micrographs. Measurements of loop lengths at different meiotic prophase substages revealed that the homologous synapsis of the inverted region was gradually replaced by nonhomologous synapsis as loops were eliminated during pachytene. This synaptic adjustment was apparently not affected by crossovers which occurred within the 150- and 160-cM long loops.     

Synaptonemal complex analysis of heteromorphic trivalents in Lemur hybrids

Synaptonemal complexes (SCs) in surface spread pachytene spermatocytes of Lemur resemble those in other mammals and are of two types: metacentric (or submetacentric) and acrocentric, with a very short second arm. In autosomal SC and mitotic karyotypes of Lemur fulvus (2n=60) a 11 proportionality in relative length is observed as in other mammals. In an intraspecific lemur hybrid (2n=55) obtained by mating L. fulvus rufus (2n=60) x L. fulvus collaris (2n=51), G-band patterns show that 10 single acrocentric mitotic chromosomes correspond to the arms of 5 single metacentrics, implying homology. It is inferred that the metacentrics have evolved by centric (Robertsonian) fusion of the acrocentrics. In the SC karyotype of the hybrid all SCs are normal except for five which have the configurations expected of metacentric-acrocentric trivalents. Similarly, in L. f. collaris (2n= 51), with one unpaired metacentric and two unpaired acrocentrics, one such SC trivalent is present in the complement. In an SC trivalent, each of the acrocentric long axes is synapsed with an arm of the metacentric axis, confirming the homology predicted from banding similarities. At late zygotene, the acrocentric short arms, which are non-homologous, are the last to pair, demonstrating that synapsis of the homologous arms occurs first. At later pachytene the acrocentric short arms are fully synapsed, producing a short SC side arm. This subsequent non-homologous synapsis is taken to be an instance of the synaptic adjustment phenomenon which has been shown to lead to non-homologous synapsis in a duplication and several inversions in the mouse. The kinetochore of the metacentric is the same size as those of the acrocentrics, and thus is unlikely to have arisen by true centromeric fusion, but rather by a translocation. The kinetochores of the acrocentrics always lie together on the same side of the metacentric kinetochore (cis configuration), implying a single pairing face on the metacentric axis. The observed trivalent configuration may well constitute a prerequisite for proper meiotic disjunction in metacentric-acrocentric heterozygotes. Such a mechanism is consistent with fertility regularly observed in such hybrid lemurs.     

Extensive nonhomologous meiotic synapsis between normal chromosome axes of an rcp(3;6)(p14;q21) translocation in a hairless Mexican boar

Due to its low fertility, expressed as small litter size, a Mexican hairless boar was subjected to cytogenetic investigation. Analysis of G-banded mitotic chromosomes revealed a reciprocal chromosome translocation, rcp(3;6) (p14;q21). Synaptonemal complex analysis showed a regular pairing behavior of the translocation chromosome axes, always resulting in a quadrivalent configuration. However, due to extensive nonhomologous pairing between the axes of nonderivative chromosomes 3 and 6, the quadrivalent mostly had an asymmetrical cross-shaped morphology. The nonhomologous pairing occurred not only at mid and late pachytene, but also at the earliest stage of pachytene. It seems that early pachytene heterosynapsis is a common phenomenon in the pairing behavior of pig reciprocal translocations. Therefore, heterosynapsis may reduce apoptosis of germ cells due to partial absence of homologous synapsis during the pairing phase of meiosis. The frequency of spermatocytes showing quadrivalent configurations with unpaired axial segments apparently did not affect germ cell progression in the boar, since fairly normal testicular histology was noticed.     

小麂、黑麂、赤麂精母细胞联会复合体的比较研究

本工作以界面铺张——硝酸银染色技术,对小麂(Muntiacus reeuesi)、黑麂(M.crinifrons)和赤麂(M.muntjak)的精母细胞联会复合体(Syna ptonemal complex,SC)进行亚显微结构的比较研究。结果表明: 1.SC的平均相对长度和臂比指数同有丝分裂细胞相应染色体的数值有很好的一致性。根据SC的相对长度和臂比指数绘制了三种麂的SC组型图。雄性黑麂减数分裂前期形成一个复杂的易位多价体,意味着其核型的演化过程涉及两次染色体易位和一次臂间倒位。 2.在减数分裂前期,性染色体的形态和行为同常染色体的有明显差异,如性染色体嗜银性较强,配对延迟等。XY的配对起始于早粗线期,在中粗线期,Y的全长均同X配对;XY-SC开始解体于晚粗线期。 3.在粗线期,X染色体未配对区域出现自身折叠,形成“发夹”状结构。这种“发夹”结构的形成,可能是在性染色体的进化过程中,X染色体通过不对称易位得到的重复片段在减数分裂前期同源配对的一种细胞学表现。     

Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.