Synaptonemal complex and recombination nodules in rye (Secale cereale)

Meiotic prophase in rye was investigated by serial-section reconstruction of pollen mother cell nuclei. In the mid-late zygotene nucleus, all lateral elements were continuous from telomere to telomere, and 9–20 pairing initiation sites per bivalent were observed. Chromosome and bivalent interlockings detected during zygotene were resolved at early pachytene when pairing was completed. In the three pachytene nuclei, the relative synaptonemal complex (SC) lengths and arm ratios were found to be in good correlation with light microscopic data of pachytene bivalents. Spatial tracing of the bivalents showed that they occupy separate areas in the nucleus. Three types of recombination nodules were observed: large, ellipsoïdal and small nodules at early pachytene and irregularly shaped nodules mainly associated with chromatin at late pachytene. Their number and position along the bivalents correlated well with the number and distribution of chiasmata. The classification of the seven bivalents was based on arm ratio and heterochromatic knob distribution.     

Two kinds of "recombination nodules" in Neurospora crassa

Two morphological types of recombination nodules, termed early and late, are recognized in Neurospora crassa. Eighty nuclei at different substages were used to determine numbers of nodules per nucleus, distribution of nodules along the nucleolus-organizing chromosome, and distribution of nodules among the two largest chromosomes. Early nodules appear at the synaptonemal complex at early zygotene and increase in number during zygotene until a dramatic reduction occurs at zygotene-pachytene transition. Thereafter early nodules are steadily eliminated until they disappear by diplotene. Late nodules are also present during zygotene. Their number doubles at the zygotene-pachytene transition and stays at this level until diplotene. The total number of nodules is rather constant through zygotene and pachytene. Distribution of bivalents with 0, 1, 2, etc. nodules follows a Poisson distribution at zygotene, but not at pachytene, where variance is less than the mean, indicating positive interference. Nodules are distributed nonrandomly along the nucleolus-organizer bivalent. The pattern differs slightly in nuclei of different origin. Nuclei with unusual synaptonemal complexes sustain normal levels of recombination by having the same amount of nodules as normal nuclei. In abnormal nuclei nodules are preferentially associated with normal segments. It is proposed that early nodules do not participate in any form of recombination but have a role in finding an appropriate site for a crossing-over event. Morphological change to the late type indicates that the site has been reached and the exchange event can be mediated by the late nodule.     

Synaptonemal complexes with modified lateral elements in Sordaria humana: development of and relationship to the “recombination nodules”

Meiotic prophase in Sordaria humana has been analyzed by three-dimensional reconstructions of 3 leptotene, 2 zygotene, 10 pachytene and 3 diplotene nuclei. Several notable features emerged. The lateral components of the synaptonemal complexes (SC) are hollow tubes which show dilations of variable sizes from late leptotene to early diplotene. These bulges occur before pairing. Their number decreases as soon as the SC are completely formed, but their mean size increases. Bulges can be present in all parts of the lateral components including telomeres and nucleolar organizer region, but their distribution along bivalents is not random. The remarkably uniform width of the SC central region, normally observed in other species is not observed in S. humana. Although as a general rule the bulges rarely affect the homologous components at identical sites, they often either fill or partially cover the central region. The recombination nodules are not clearly connected with the bulges. This work provides also additional insight into the development of both SC and the nodules. At late leptotene, the homologues are aligned before SC formation. One case of interlocking has been observed at early pachytene. Nodules are present from zygotene to diplotene. They are not evenly distributed along the bivalents during pachytene. The mean number of nodules, constant from late pachytene to diplotene, is equal to the mean number of chiasmata.     

Development of the synaptonemal complex and the “recombination nodules” during meiotic prophase in the seven bivalents of the fungus Sordaria macrospora Auersw

Complete reconstruction of seven leptotene, six zygotene, three pachytene and three diplotene nuclei has permitted to follow the pairing process in the Ascomycete Sordaria macrospora. The seven bivalents in Sordaria can be identified by their length. The lateral components of the synaptonemal complexes (SC) are formed just after karyogamy but are discontinuous at early leptotene. Their ends are evenly distributed on the nuclear envelope. The homologous chromosomes alignment occurs at late leptotene before SC formation. The precise pairing starts when a distance of 200–300 nm is reached. Each bivalent has several independent central component initiation sites with preferentially pairing starting near the nuclear envelope. These sites are located in a constant position along the different bivalents in the 6 observed nuclei. The seven bivalents are not synchronous either in the process of alignment or in SC formation: the small chromosomes are paired first. At pachytene the SC is completed in each of the 7 bivalents. Six bivalents have one fixed and one randomly attached telomeres. The fixed end of the nucleolar organizer is the nucleolus anchored end. At diffuse stage and diplotene, only small stretches of the SC are preserved. The lateral components increase in length is approximately 34% between leptotene and pachytene. Their lengths remain constant during pachytene. From zygotene to diplotene the central components contain local thickenings (nodules). At late zygotene and pachytene each bivalent has 1 to 4 nodules and the location of at least one is constant. The total number of nodules remains constant from pachytene to diplotene and is equal to the mean total number of chiasmata. The observations provide additional insight into meiotic processes such as chromosome movements, initiation and development of the pairing sites during zygotene, the existence of fixed telomeres, the variations in SC length. The correspondence between nodules and chiasmata are discussed.     

The occurrence and role of DNA synthesis during meiosis in wheat and rye

The incorporation of ³H-thymidine into the DNA of rye meiocytes at zygotene, pachytene-diplotene and metaphase I to telophase II stages has been studied. Low levels of ³H were found in highly purified DNA from meiocytes at all these stages, though there was more in the DNA from pachytene-diplotene meiocytes, and it is highly likely that the zygotene groups of anthers contained a proportion at pachytene. The buoyant density distributions of the labelled DNA from zygotene and pachytene-diplotene cells were indistinguishable, in contrast to the situation in Lilium, the only other example studied so far.The DNA synthesis inhibitor 2′-deoxyadenosine halted meiotic development of anthers in culture only at late zygotene and pachytene. It did not inhibit development at early zygotene, prevent chromosome pairing as judged by light microscopy or cause extensive chromosome fragmentation during zygotene as in Lilium. These results indicate that extensive synthesis of DNA does not occur at zygotene in cereals and does not suggest that zygotene DNA synthesis is a prerequisite for chromosome pairing as in Lilium.     

Chromosomes and DNA of Mus

Using C-banded preparations of Mus dunni it is possible to study the behavior of constitutive heterochromatin in early stages of meiotic prophase. The X and the Y chromosomes, both of which contain a large amount of heterochromatin, lie apart in leptotene but move toward each other during zygotene. They then form the sex vesicle at late zygotene. In autosomes zygotene pairing appears to start from the telomeric ends. The centromere of the Y chromosome associates end-to-end with the terminal end of the long arm of the X chromosome. The autosomal heterochromatic short arms show forked morphology in certain bivalents at pachytene, suggesting probable incomplete synapsis.     

Synaptonemal complex spreading in Allium cepa and A. fistulosum

The general features and fine structure of homologous chromosome alignment and pairing have been investigated in two species of Allium (A. fistulosum and A. cepa), which have similar karyotypes but very different patterns of chiasma distribution. Although there is no support for the occurrence of a general pre-meiotic alignment of homologous chromosomes, both species show some alignment of homologues as an immediate prelude to synaptonemal complex (SC) formation. In both species pairing usually commences at sub-terminal sites and is succeeded by numerous separate intercalary initiations of pairing in interstitial and distal regions and then in proximal regions. The last parts to pair, in both species, are pericentromeric and telomeric regions. There is, therefore, no evident relationship between the sequence of pairing and chiasma distribution in these species. Regularly alternating convergences and divergences of aligned axial cores (ACs), termed multiple association sites, are frequently observed. It is proposed that these represent potential pairing initiation sites and from observations on their spatial distribution it is argued that they may be evenly distributed through most of the genome. Small spherical or ellipsoid nodules are found at association sites and between closely aligned ACs which persist in the SC segments present during zygotene, but most of them disappear abruptly at the end of zygotene. These are termed zygotene nodules (ZN) and it is proposed that they are involved in matching corresponding sites on homologous chromosomes as well as possibly having a recombinational role. Their composition, structure, mode of action and relationship to pachytene recombination nodules are at present unknown.     

甜菜夜蛾细胞分裂期染色体的观察

以精巢组织为材料,采用空气干燥法制备染色体标本,对甜菜夜蛾Spodopteraexigua有丝分裂和减数分裂染色体形态行为进行了研究。结果表明：甜菜夜蛾的染色体数目为n=31;染色体为弥散着丝粒染色体,2条染色体上存在次缢痕;晚偶线期出现染色体互锁现象;从早粗线期到晚粗线期联会复合体逐渐伸长;终变期同源染色体形成环状、端部交叉、尾尾相对的结构。     

Spreading the synaptonemal complex of Neurospora crassa

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.     

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.     

The effect of colchicine on synapsis and chiasma formation in microsporocytes of Lilium

Microsporocytes of Lilium that are exposed to colchicine as late as early zygotene show reduced chiasma frequencies and the presence of univalents at Division I. These effects are preceded at pachytene by the appearance of pairing gaps (light microscopy) and by a relatively high ratio of uncomplexed lateral elements/synaptonemal complexes (EM). Chiasma formation thus appears to be reduced by failures in synapsis. Unlike the behavior of wheat, colchicine can disrupt chiasma formation in Lilium after cells have entered meiosis.     

Meiotic Structures in the Animal-Parasitic Nematode Ascaris megalocephala: Synaptonemal Complexes,Recombination Nodules,and Centrioles

Two synaptonemal complexes (SCs) were present in the pachytene nuclei of Ascaris megalocephala. The SC was tripartite and comprised of two lateral elements (25 nm) with a striated central element (25 nm) and a central region of 65 nm. Spherical recombination nodules were observed to be associated only with the central element, although they are non-existent in the related A. lumbricoides var. suum (Goldstein, 1977). The SCs were attached to the nuclear envelope at only one end, while the other end was free in the nucleoplasm. This lack of bouquet formation of the chromosomes is consistent with all other nematodes studied. Morphologically distinct sex chromosomes were not observed, which differs from the presence of five Y-chromosomes present in A. lumbricoides var. suum (Goldstein and Moens, 1976). Centrioles (0.2 µm wide) reproduced by budding off the parental centriole. The centrioles consisted of nine singlet microtubules connected by an electron-dense proteinaceous ring. This structure is consistent with centrioles described in other nematodes, yet distinctly different from the centriole structure observed in most organisms in which it consists of nine triplet microtubules without any connecting ring. Multiple synaptonemal complexes, or polycomplexes, are found in A. megalocephala and A. lumbriocoides var. suum. They appear as stacked SC and are present inside the nucleus during zygotene and in the cytoplasm at pachytene.     

THE TIMING OF MEIOSIS AND DNA SYNTHESIS DURING EARLY OOGENESIS IN THE TOAD, XENOPUS LAEVIS

Recently metamorphosed female Xenopus laevis toads were injected with tritiated thymidine. Animals were kept at 20°C and were sacrificed 1–23 days after isotope injection. Radio-autographs of squash preparations of the ovaries were made. The progress of labeled germ cell nuclei was followed to obtain information on the time course of early meiosis and extra-chromosomal DNA synthesis. Premeiotic S was estimated to take not more than 7 days. Leptotene takes 4 days, zygotene takes 5 days, and pachytene was estimated to be completed in about 18 days. The major period of amplification of the extrachromosomal DNA occurs in pachytene and takes about 13 days. A low level of synthesis was observed before and after this period, in zygotene and late pachytene-early diplotene, extending the total time for extrachromosomal DNA synthesis during meiosis to about 18 days. These data allowed the calculation to be made that one round of replication of the amplified DNA takes between 1.2 and 3.0 days. It was also found that in both oogonial and premeiotic interphases, the nucleolus-associated DNA shows asynchronous (probably late) labeling with respect to the chromosomes.     

Dmc1 fluorescent foci in prophase I nuclei of diploid,triploid and hybrid lilies

We examined the distribution of meiotic epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that retention of the foci until the pachytene stage requires a successful homology check and synaptonemal complex (SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to pachytene, and the triploid pachytene values are similar to the pachytene values of the diploid. The species-hybrid lacks chromosome homology, has reduced SC formation and few reciprocal genetic exchanges. In this species-hybrid the number of foci at early zygotene is similar to that in the normal diploid but is dramatically reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that SC formation is reduced. In contrast the extent of the reduction in reciprocal genetic exchange in the species-hybrid is much greater than the reduction in the number of foci. We conclude that Dmc1 protein is involved in homology checking, but the impact of failure to find homology affects SC formation and reciprocal genetic exchange differentially.     

Identification and immunochemical characterization of spermatogenic cell surface antigens that appear during early meiotic prophase

Three spermatogenic cell populations isolated from prepuberal mice--type B spermatogonia, preleptotene spermatocytes, and leptotene/zygotene spermatocytes--were used to elicit distinct polyclonal antisera. Surface binding specificities were determined for purified IgGs by indirect immunofluorescence and rosette assays on live cells. Binding activities were assayed both before and after absorptions with a variety of somatic and spermatogenic cells. Each of these antisera binds to surface antigens that are present on germ cells throughout spermatogenesis and are not shared by splenocytes, thymocytes, and erythrocytes. Only the antiserum raised against leptotene and zygotene spermatocytes (ALZ) recognizes a stage-specific subset of surface determinants. After appropriate absorptions, ALZ binds to the surface of early pachytene spermatocytes and germ cells at subsequent stages of differentiation, including vas deferens spermatozoa. Antigens which react with this absorbed IgG are not detected on the surface of spermatogonia or meiotic cells prior to pachynema, including leptotene and zygotene spermatocytes. The observed binding specificities may result from the synthesis of one or more surface molecules during the early meiotic stages, followed by delayed insertion into the plasma membrane during the pachytene stage of meiotic prophase. Stage-specific antigens recognized by ALZ, including both protein and probably lipid, have been localized immunochemically on nitrocellulose blots from one-dimensional SDS gels. A dithiothreitol-sensitive constituent (Mr approximately 39,000) recognized by ALZ has been identified as the major protein determinant present in early meiotic cells but absent in 8-day-old seminiferous cell suspensions containing spermatogonia and Sertoli cells. This determinant is present in populations of preleptotene, leptotene/zygotene, and early pachytene spermatocytes isolated from 17-day-old animals, an observation consistent with the hypothesis of delayed insertion into the plasma membrane.     

Analysis of highly expressed genes in the late zygotene to pachytene stages of meiotic prophase I in david lily

Plant meiotic prophase I is a complex process involving the late zygotene and pachytene stages, crucial for both completing synapsis and recombination. Using David lily (Lilium davidii var. Willmottiae) as research material, we performed suppressive subtractive hybridization to construct expessed sequence tag (EST) library of anthers at various stages of development by the pollen mother cells. From this library, we identified 34 genes with significantly enhanced expression during the late zygotene to pachytene stages. The cDNA fragment sequences were compared with data in GenBank by BLASTN and BLASTX, and 18 unique ESTs were shown to exhibit significant homology to the data in GenBank. They were classified into eight different groups: metabolism, protein modification, signal transduction, etc. Through the study of classification and functions of these highly expressed genes during the late zygotene to pachytene stages, we obtained much information about the complex biological progress of meiotic prophase I, especially during chromosome synapsis and recombination.     

Clastogenic effects of cis-diamminedichloroplatinum II. Induction of chromosomal aberrations in primary spermatocytes and spermatogonial stem cells of mice

The clastogenic effect of the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) on meiotic prophase in primary spermatocytes and on spermatologonial stem cells of male (101/E1 × C3H/E1)F₁ mice was studied. The intraperitoneal doses of cisplatin tested were 5.0, 7.5 and 10.0 mg/kg. Chromosomal aberrations were examined at diakinesis-metaphase 1 of meiosis 1–13 days after treatment, representing cells treated at diplotene, pachytene, zygotene, leptotene an preleptotene. Reciprocal translocations were evaluated 63–70 days after treatment, representing treated stem-cell spermatogonia.Cisplatin had a toxic effect in zygotene to preleptotene of meiosis, as indicated by the significant reduction in testicular weight. At diplotene, pachytene and zygotene no enhancement of aberrations was found. An increase in aberrant cells was observed during leptotene with preleptotene being the most sensitive stage. The dose-response relationship for aberrant cells was linear on day 13 after treatment. It is concluded that, like mitomycin C (Alder, 1976), cisplatin primarily caused aberrations during the premeiotic phase of DNA synthesis. No significant increase of translocation multivalents was found after treatment of stem-cell spermatogonia.     

Colchicine effects on meiosis in the male mouse

Antimitotic agents administered at the time of synapsis (leptotene/zygotene) have been shown to induce synaptic abnormalities visible during pachytene in the male mouse. The object of this study was to test the hypothesis that cells with relatively large amounts of colchicine-induced damage to the synaptonemal complex (SC) are eliminated from prophase whereas cells with relatively small amounts of SC damage proceed through to the end of prophase. Male mice were injected with tritiated thymidine to mark a cohort of spermatocytes at premeiotic S-phase for tracking through pachytene. Forty-eight hours later, when those cells were at leptotene/zygotene, colchicine was administered intratesticularly. Whole-mount SC spreads were made from animals sacrificed at various times following colchicine administration, and prepared for autoradiography. The marked cells were examined by light and electron microscopy and the kind and number of synaptic abnormalities were scored throughout pachytene. Colchicine-induced SC damage included single axial elements (univalents), together with partially synapsed and nonhomologously synapsed SCs. The amount of SC damage (amount and type per cell and frequency of cells with damage) scored at early pachytene exceeded by three- to fivefold the amount at late pachytene. This is consistent with spermatogenic cell loss from the seminiferous tubule via colchicine-induced destruction of Sertoli cell microtubules. The presence of spermatocytes with no more than four autosomal univalents at late pachytene indicates that some cells with low amounts of synaptic damage progress to the end of pachytene. The loss of the most severely damaged cells may represent a meiotic checkpoint at early pachytene in the male mouse. Received: 24 April 1996; in revised form: 29 August 1996 / Accepted: 11 March 1997     

Small heat shock protein LimHSP16.45 protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily

Plant meiotic prophase I is a complicated process involving the late zygotene and pachytene stages, both crucial for completing synapsis and recombination. Using David Lily (Lilium davidii var. Willmottiae) as our research material, we performed suppression subtractive hybridization to construct EST library of anthers at various stages of development by the pollen mother cells. From this library, we identified small heat shock protein LimHSP16.45 was highly expressed during the late zygotene to pachytene stages. Our results also showed that LimHSP16.45 was almost specifically expressed in the anther compared with the root, stem, or leaf, and in situ expression of LimHSP16.45 mRNAs showed strong signals in the pollen mother cells and tapetal cells. LimHSP16.45 could be induced by heat and cold in lily anthers, and its ectopic expression enhanced the viability of E. coli cells under both high and low temperatures. In vitro, it acted as molecular chaperone and could help luciferase refolding after heat shock stress. All of these data suggest that LimHSP16.45, working as molecular chaperone, possibly protects pollen mother cells and tapetal cells against extreme temperatures during late zygotene to pachytene stages of meiotic prophase I in David Lily.