Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.     

家猪减数分裂粗线期二价体与有丝分裂中期染色体的比较研究

以性成熟公猪睾丸和外周血为材料,采用长低渗、高氯仿卡诺固定液固定和外周血细胞培养制备减数分裂粗线期二价体和有丝分裂中期染色体,通过对二价体和有丝分裂中期染色体分裂指数和长度的比较研究,发现二价体的分裂指数和长度分别是有丝分裂中期染色体的5倍和3．42倍（1．87～5．98）;同时以12号染色体为例,比较了二价体上的染色粒结构带与有丝分裂中期染色体G－带,表明染色粒结构带比中期染色体G－带纹丰富,而与早中期G－带带织吻合。     

Study of the human male meiosis

Summary Results obtained from a meiotic study of 250 pachytene cells from four normal human males are presented.G-banding patterns for pachytene bivalents, obtained using trypsin treatment, are also presented; a comparative study between G-banding patterns of pachytene bivalents and G-banding patterns of mitotic chromosomes, and a study of chromomere counting and distribution, have also been made.     

Location and variation of the constitutive heterochromatin in Petunia hybrida

The location of heterochromatin in the chromosomes of Petunia hybrida (2n=14) is presented. C-banded mitotic metaphase chromosomes and carmine-stained pachytene bivalents have been studied. It is shown that the heterochromatin is predominantly located near the centromeres and at the secondary constrictions of the satellite chromosomes. The distribution of chromomeres in pachytene bivalents also reveals that heterochromatin is not restricted to distinct blocks, as is the case in tomato, but occurs in smaller chromomeres which gradually decrease in size towards the ends. Conspicuous telomeres have not been observed. Both C-banding technique and pachytene analysis demonstrate large variation of heterochromatin between different lines of Petunia. The study of pachytene morphology has been hampered by a high degree of non-specific stickiness of the bivalents. Both techniques prove to be unsuitable tools for large-scale chromosome identification of Petunia lines.     

FISH-mapping of rDNAs and Arabidopsis BACs on pachytene complements of selected Brassicas.

To improve resolution of physical mapping on Brassica chromosomes, we have chosen the pachytene stage of meiosis where incompletely condensed bivalents are much longer than their counterparts at mitotic metaphase. Mapping with 5S and 45S rDNA sequences demonstrated the advantage of pachytene chromosomes in efficient physical mapping and confirmed the presence of a novel 5S rDNA locus in Brassica oleracea, initially identified by genetic mapping using restriction fragment length polymorphism (RFLP). Fluorescence in situ hybridization (FISH) analysis visualized the presence of the third 5S rDNA locus on the long arm of chromosome C2 and confirmed the earlier reports of two 45S rDNA loci in the B. oleracea genome. FISH mapping of low-copy sequences from the Arabidopsis thaliana bacterial artificial chromosome (BAC) clones on the B. oleracea chromosomes confirmed the expectation of efficient and precise physical mapping of meiotic bivalents based on data available from A. thaliana and indicated conserved organization of these two BAC sequences on two B. oleracea chromosomes. Based on the heterologous in situ hybridization with BACs and their mapping applied to long pachytene bivalents, a new approach in comparative analysis of Brassica and A. thaliana genomes is discussed.     

Pachytene variations in Ricinus

The pachytene complement in microsporocytes was compared among ten races of Ricinus communis L., the castor plant (2n=20). Four kinds of pachytene variations were observed: (1) variation in degree of spreading of the chromosomes; (2) variation in degree of attenuation of heterochromatin; (3) variation in morphology, which appeared to be restricted to the two nucleolar organizing bivalents; (4) variation in frequency of association of each of these two bivalents with the nucleolus. It is suggested that variations in the pachytene chromosomes occur ubiquitously in this species.     

Damaging effect of antibiotics on the structure of synaptonemal complexes of meiotic chromosome of mice

The structure of synaptonemal complexes (SCs) of chromosomes of mouse primary spermatocytes were studied using electron microscopy on days 1, 10, and 36 after the completion of per os administration of drugs belonging to three groups of antibiotics: tetracyclins, macrolides, and fluoroquinolones. The antibiotics were administered to mice during ten days. At the substages of early and middle pachytene, heteromorphic SC bivalents and fragments of chromosome-core elements were detected in spermatocytes at all times studied after the administration of the antibiotics of three groups. As cells passed through the period from early to middle pachytene, the number of cells containing heteromorphic SC bivalents and the fragments of chromosome cores gradually decreased, which could be an indication of selection of cells with chromosomal aberrations. A high level of associations between the X chromosome and autosome bivalents (including heteromorphic ones) also favors this suggestion. A gradual decrease in the number of chromosomal aberrations was detected, as time elapsed from the completion of antibiotics administration. The study of sperm obtained from epididymises of males did not reveal significant differences in both morphology and motility of sperm between males of the control and experimental groups.     

Spermatogenesis in two patients with the fragile X syndrome

Summary Chromosomes at first meiosis from two males with the fra(X) form of mental retardation were studied using pachytene surface spreads and air-dried preparations. The pachytene sex bivalents showed no discontinuation of the synaptonemal complex in the terminal part of Xq corresponding to band Xq27–28 of the mitotic chromosomes. In both cases the frequency of a secondary association of Xq and Yq appeared to be increased compared with controls. The pairing behavior of autosomal bivalents in pachytene and the frequency and distribution of chiasmata in diakinesis were normal. The impairment of spermatogenesis found in these males may not be caused by a meiotic disorder, but could be related to peritubular or intratubular pressure effects on germ cells.     

Oocyte meiosis in a trisomy 18 fetus. Behavior of the supernumerary chromosome and identification of the 18 bivalent]

Associations of the three n degrees 18 chromosomes were studied in a trisomy 18 fetus (the chromosomal constitution of which had been identified by amniocentesis). The three classes of associations observed were those observed in other trisomic organisms:trivalent, trivalent presenting an important asynaptic region, and bivalent accompanied by a univalent. In addition, the sequence was established of chromomeres, the number of which varied from 18 to 23 depending on the degree of chromosome contraction. In elongated pachytene oocyte bivalents each G-band of mitotic metaphase chromosomes could be subdivided into several sub-bands.     

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.     

Damaging Effect of Antibiotics on the Structure of Synaptonemal Complexes of Meiotic Mouse Chromosomes

The structure of synaptonemal complexes (SCs) of chromosomes of mouse primary spermatocytes were studied using electron microscopy on days 1, 10, and 36 after the completion of per os administration of drugs belonging to three groups of antibiotics: tetracyclins, macrolides, and fluoroquinolones. The antibiotics were administered to mice during ten days. At the substages of early and middle pachytene, heteromorphic SC bivalents and fragments of chromosome-core elements were detected in spermatocytes at all times studied after the administration of the antibiotics of three groups. As cells passed through the period from early to middle pachytene, the number of cells containing heteromorphic SC bivalents and the fragments of axial cores gradually decreased, which could be an indication of selection of cells with chromosomal aberrations. A high level of associations between the X chromosome and autosome bivalents (including heteromorphic ones) also favors this suggestion. A gradual decrease in the number of chromosomal aberrations was detected, as time elapsed from the completion of antibiotics administration. The study of sperm obtained from epididymises of males did not reveal significant differences in both morphology and motility of sperm between males of the control and experimental groups.     

Complete pachytene chromomere karyotypes of human spermatocyte bivalents

Summary Well-spread human pachytene spermatocyte bivalents were obtained allowing specific identification of each bivalent within its total complement according to its chromomere sequence combined with further staining of its centromeric heterochromatin. The total number of chromomeres was found to be related to the degree of bivalent contraction: 396 in condensed bivalents and 511 in decondensed bivalents. A striking correspondance between chromomeres and mitotic G-bands was observed; on account of the variability of bivalent contraction, condensed bivalents corresponded to prometaphase somatic chromosomes and decondensed bivalents to mid/late prophase chromosomes.     

Synapsis in grasshopper bivalents heterozygous for centric shifts.

Analysis of surface-spread synaptonemal complexes of zygotene and pachytene spermatocytes was carried out on centric-shift heterozygotes of grasshoppers. These rearrangements affected the M7 chromosome in Chorthippus vagans and the M6 and S8 chromosomes in Chorthippus apricarius. The shifts in the latter two chromosomes were also associated with C-heterochromatin variations between homologous chromosomes. Rearranged chromosomes proceeded directly to heterosynapsis without an apparent intervening homosynaptic phase in M7 bivalents of Ch. vagans and M6 bivalents of Ch. apricarius. In the latter case, axial equalization of the heterochromatin polymorphism was also achieved. On the other hand, asynapsis of the intercentromeric regions throughout pachytene was the rule in the centric shift involving the S8 chromosome of Ch. apricarius. In the three cases analysed, the production of unbalanced gametes in the heterozygotes is precluded either by the lack of chiasma formation in heterosynapsed rearranged segments or by the lack of pairing between such segments. Chiasmata were limited to the homologous regions of the heteromorphic bivalents.     

番茄的CPD带型和45S rDNA位点的鉴别

采用CPD（PI和DAPI组合）染色对番茄减数分裂粗线期和有丝分裂中期染色体进行了显带分析,随后用两种不同的45S rDNA克隆在相同的分裂相进行了荧光原位杂交定位分析。CPD染色在8条粗线期染色体上显示出了10条红色的CPD带纹,在6对有丝分裂中期染色体上显示出了12条CPD带纹。有丝分裂中期染色体上的CPD带纹与粗线期染色体上显著的带纹具有对应性。用改良的CPD染色程序清晰而稳定地显示出这些特征性的CPD带纹为番茄的染色体,特别是有丝分裂中期染色体提供了新的识别标记。用番茄的一个45S rDNA克隆进行的荧光原位杂交,不仅在位于2号染色体短臂的随体上显示了强的杂交信号,而且在粗线期染色体的5个CPD带区或有丝分裂中期染色体的4对CPD带区显示了弱的杂交信号。然而,用来自小麦的45S rDNA克隆pTa71进行的原位杂交却只在随体上显示了杂交信号。鉴于所用的两个45S rDNA克隆在序列上的差异,推断在番茄基因组中只有随体含有45S rDNA单位的编码区,即番茄只有一对45S rDNA位点。     

Relationship between pachytene synapsis, metaphase I associations, and transmission of 2B and 4B chromosomes in rye.

2B rye plants selected for high (H) or low (L) B transmission rate were studied at pachytene and metaphase I of meiosis to determine the relationship between synapsis, bivalents at metaphase I, and B transmission rate. The results show that the 2 B chromosomes (Bs) form bivalents at pachytene in both the H and L lines, whereas the frequency of bivalents at metaphase I is much higher in the H than in the L line. This demonstrates that B transmission is mainly related to the proper association of Bs at metaphase I, as well as that synapsis of the 2 Bs in the L line is normal, but the bivalent is not consolidated by a chiasma in most cases. Crosses were made between 2B plants of the H and L lines in all combinations (H x H, H x L, L x H, and L x L) to obtain 4B plants. Similarly, bivalent formation at pachytene and metaphase I was studied. The results show that 4B plants of the H x H and L x L classes differ significantly at pachytene and metaphase I since the former forms more bivalents. The heterozygous 4 Bs of the H x L and L x H classes show intermediate values. The relation H x H > H x L > L x H > L x L was consistently found for the variables transmission rate, bivalents at pachytene, bivalents at metaphase I, and B mean chiasma frequency. A maternal effect was also found. Our data suggest that there are two separate mechanisms acting upon synapsis and chiasma formation in H and L B chromosomes: (i) there is variable efficiency of the control of synapsis at early stages of meiosis; and (ii) there is variable efficiency of the control of the number of chiasmata.     

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.     

Centric-fusion translocation and whole-arm heterochromatin in the karyotype of the blue fox (Alopex lagopus L.): synaptonemal complex analysis.

Conventional observations of mitotic chromosomes from two male blue foxes, revealing a centric-fusion translocation and whole-arm heterochromatin, were verified by synaptonemal complex analysis. This analysis revealed that the centric fusion had been preceded by a conspicuous loss of chromosome material in the two one-armed chromosomes involved, but the chromosomal origin of the centric-fusion kinetochore could not be established. The nontranslocated chromosomes of the trivalent, which in all cells but one were in cis configuration, had reached by early pachytene a stage in which almost complete homologous pairing and nonhomologous association or pairing of the free ends of the chromosomes could be observed. In later stages, complete pairing of the nontranslocated chromosomes with the corresponding arms of the centric-fusion translocation was seen occasionally. One to six autosomal bivalents demonstrated unpaired heterochromatic arms in early pachytene, and the heterochromatic chromosome arms were sometimes unpaired even in late pachytene. Some of them showed a distinct size heteromorphism in late zygotene and early pachytene. In most late-pachytene cells, however, the heteromorphic chromosomes were completely length-adjusted. Only a small fraction of the cells showed pairing interference between nonhomologous chromosomes.     

Differential associative behaviour of mitotic and meiotic acrocentric chromosomes

Summary A comparative study of the association of mitotic acrocentric chromosomes and acrocentric bivalents at the pachytene stage shows that at least two factors can act in the associative behaviour of these chromosomes: (1) Nor activity and (2) the presence of satellite DNA in the short arms of these chromosomes. These factors do not act with the same intensity in the two cell lines studied. In lymphocytes, Nor activity prevails, whereas satellite DNA plays the main role in the association of acrocentric chromosomes in germ cells at the pachytene stage.     

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.