The distribution of alternating AT sequences in eukaryotic genomes suggests a role in homologous chromosome recognition in meiosis

There are general features of chromosome dynamics, such as homologue recognition in early meiosis, which are expected to involve related sequence motifs in non-coding DNA, with a similar distribution in different species. A search for such motifs is presented here. It has been carried out with the CONREPP programme. It has been found that short alternating AT sequences (10-20 bases) have a similar distribution in most eukaryotic organisms, with some exceptions related to unique meiotic features. All other microsatellite and repeat sequences vary significantly in different organisms. It is concluded that the unique structural features and uniform distribution of alternating AT sequences indicate that they may facilitate homologous chromosome pairing in the early preleptotene stage of meiosis. They may also play a role in the compaction of DNA in mitotic chromosomes.     

Clastogenic effects of hydroquinone: induction of chromosomal aberrations in mouse germ cells

The clastogenic activity of hydroquinone (HQ) in germ cells of male mice was evaluated by analysis of chromosomal aberrations in primary spermatocytes and differentiating spermatogonia. In the first experiment with treated spermatocytes the most sensitive stage of meiotic prophase to aberration induction by HQ was determined. Testicular material was sampled for microscopic analysis of cells in diakinesis-metaphase I at 1, 5, 9, 11, and 12 days after treatment with 80 mg/kg of HQ, corresponding to treated diplotene, pachytene, zygotene, leptotene and preleptotene. The frequencies of cells with structural chromosome aberrations peaked at 12 days after treatment (p less than 0.01). This indicates that the preleptotene when DNA synthesis occurred was the most sensitive stage of meiotic prophase. In the second experiment the dose response was determined 12 days post treatment by applying 2 additional doses of 40 mg/kg and 120 mg/kg. The clastogenic effects induced by 40 and 80 mg/kg were significantly different from the controls (p less than or equal to 0.01) and higher than the results obtained with 120 mg/kg of HQ. A humped dose-effect relationship was observed. In a third experiment the same doses were used to analyse chromosomal aberrations in dividing spermatogonia of mice 24 h after treatment with HQ. All the administered doses gave results statistically different from the control values (p less than or equal to 0.01) and the data were fitted to a linear equation. HQ was found to be clastogenic in male mouse germ cells. It is concluded that the clastogenic effect in male germ cells is of the same order of magnitude as in mouse bone marrow cells.     

Effect of the topoisomerase-II inhibitor etoposide on meiotic recombination in male mice

Unlike other chemicals that have been tested in mammalian germ cells, the type-II topoisomerase inhibitor etoposide exhibits significant mutagenicity in primary spermatocytes. Because this is the cell stage during which meiotic recombination normally occurs, and because topoisomerases play a role in recombination, we studied the effect of etoposide on crossing-over in male mice. Exposure to those meiotic prophase stages (probably early to mid-pachytene) during which specific-locus deletion mutations can be induced resulted in decreased crossing-over in the p-Tyr(c) interval of mouse chromosome 7. Accompanying cytological studies with fluorescent antibodies indicated that while there was no detectable effect on the number of recombination nodules (MLH1 foci), there were marked changes in the stage of appearance and localization of RAD51 and RPA proteins. These temporal and spatial protein patterns suggest the formation of multiple lesions in the DNA after MLH1 has already disappeared from spermatocytes. Since etoposide blocks religation of the cut made by type II topoisomerases, repair of DNA damage may result in rejoining of the original DNA strands, undoing the reciprocal exchange that had already occurred and resulting in reduced crossing-over despite a normal frequency of MLH1 foci. Crossing-over could conceivably be affected differentially in different chromosomal regions. If, however, the predominant action of etoposide is to decrease homologous meiotic recombination, the chemical could be expected to increase nondisjunction, an event associated with human genetic risk. Three periods in spermatogenesis respond to etoposide in different ways. Exposure of (a) late differentiating spermatogonia (and, possibly, preleptotene spermatocytes) results in cell death; (b) early- to mid-pachytene induces specific-locus deletions and crossover reduction; and, (c) late pachytene-through-diakinesis leads to genetically unbalanced conceptuses as a result of clastogenic damage.     

Centromere and telomere redistribution precedes homologue pairing and terminal synapsis initiation during prophase I of cattle spermatogenesis

Alterations in nuclear topology associated with meiotic chromosome pairing were studied in premeiotic cells and spermatocytes I of adult bovine males. To this end, we performed FISH with chromosome, pericentromeric satellite-DNA and telomere-specific probes in combination with immunostaining of synaptonemal complex proteins (SCP3, SCP1) on testis tissue sections. Nuclei of premeiotic cells (spermatogonia) exhibited a scattered telomere distribution while pericentromeres formed a few intranuclear clusters. We observed that the chromosome pairing process in cattle prophase I is preceded by repositioning of centromeres and telomeres to the nuclear periphery during preleptotene. Clustering of chromosome ends (bouquet formation) was observed during the transition from leptonema to zygonema and coincided with pairing of a sub-centromeric marker of bovine chromosomes 7. Dissolution of bouquet topology during zygonema left perinuclear telomeres scattered over the nuclear periphery at pachynema. SCP3 staining in frozen tissue sections revealed the appearance of this axial element protein in intranuclear aggregates during preleptotene, followed by extensive axial element formation during leptotene. Synapsis as revealed by SCP1 staining initiated peripherally at earliest zygotene, at this stage nuclei still contained numerous SCP3 clusters. Our observations reveal prominent non-homologous satellite-DNA associations in spermatogonia and indicate the conservation of topological features of the meiotic chromosome pairing process among mammals. The comparison of telomere dynamics in mouse and cattle prophase I suggests that a larger number of chromosomes prolongs the duration of the bouquet stage.     

Delayed termination of nuclear histone doubling after premeiotic DNA synthesis inTriturus vulgaris male meiosis

It has been shown by means of double wavelength cytophotometry of DNA (Feulgen reaction) and histone (fast green, pH 8.2) inTriturus vulgaris spermatocytes that the doubling of DNA content in nuclei terminates at the end of preleptotene to beginning of leptotene whereas the doubling of histone content begun at premeiotic interphase is delayed and proceeds till the end of leptotene to beginning of zygotene. As a result preleptotene spermatocytes contain approximately 4C DNA and only 3C histone. Histone content in leptotene amounts to 93% of 4C, and in zygotene, pachytene and metaphase I both DNA and histone contents equal 4C. Thus, the temporal pattern of nucleo-histone doubling in meiotic chromosomes ofT. vulgaris differs from the synchronous DNA and histone doubling in mitotic chromosomes of all previously studied species. The delay of histone doubling inT. vulgaris meiocytes is less pronounced than in the previously studied insectsAcheta domestica andPyrrhocoris apterus where the histone content amounts to 3C in leptotene—zygotene and the equal histone/DNA ratio is restored only in pachytene.—Responsibilities for this phenomenon and its biolgoical sinnificance are discussed in connection with recent hypotheses concerning mechanisms of homologous chromosome pairing.     

A fluorescent in situ hybridization analysis of X chromosome pairing in early human female meiosis

Fluorescent in situ hybridization (FISH) utilizing an X chromosome whole library probe was used directly to assess the rate of aneuploidy and pairing behavior of the X chromosome in human female meiosis. Over 3000 meiotic cells obtained from fetal ovaries (gestational age 13–22 weeks) were scored for meiotic stage and evaluated for pairing abnormalities. No pairing anomalies were observed in 832 pachytenes. Twenty-two percent (88/398) of cells in zygotene were partially paired, but nonhomologous pairings could not be identified. One aneuploid preleptotene oocyte, presumably from mitotic nondisjunction was detected. To our knowledge, this is the first report of the use of FISH utilizing whole chromosome probes to evaluate the pairing behavior of chromosomes in human female meiosis. The application of this technique to study the relationship between nondisjunction and chromosome pairing behavior in maternal-age-related aneuploidy is discussed.     

Premeiosis and meiosis in Lilium “Enchantment”

Various aspects of premeiosis and meiosis in Lilium Enchantment are described. There was evidence of a premeiotic slow-down but no cells in premeiotic despiralization were observed. A relationship was found between sequence of bud development, or reproductive age of the individual, and degree of preleptotene chromosome contraction. The sequence of development of microsporocytes in the anther differed from the apex-to-base order previously reported in Lilium and, in contrast to observations in L. longiflorum cultivars, the maximum degree of preleptotene contraction was found in basal, last developing microsporocytes. Delayed despiralization of telophase nuclei was observed. There were extremely rare cells in meiotic division in anthers in which all other archesporial cells had not yet reached premeiotic interphase. Extreme variation was observed among anthers in proportions of microsporocytes in mid-stages of meiosis as well as in preleptotene contraction. These observations are discussed in relation to meiosis readiness, meiotic behavior in early and late developing regions of reproductive organs and in aging individuals, synchrony of meiotic development and rates of meiotic division.     

Do male germ cells begin meiosis during fetal life?]

The existence of a preleptotene chromosome condensation and decondensation stage occuring between the last premeiotic interphase and the leptotene stage was described in numberous plants. This stage was also reported in the human fetal oocyte and in various animals (rabbit, sheep, mouse). A similar process of chromosome condensation was described in the human foetal testis, but in this latter, the decondensation phase leading to leptotene was never observed. According to this observation and to various experimental results from the literature, the preleptotene condensation stage could be related to the processes of meiotic initiation. It could represent a phase of transition between mitotic and meiotic behaviour during which the germinal cell could be sensitive to meiosis-inducing factors. It is suggested that the male germinal cell 46,XY could enter meiosis. This hypothesis is confirmed by the observation of the capacity of the XY germ cell in the mouse to become an oocyte. This capacity is normally not expressed, due to the repressive control of the adjacent Sertoli cells. Thus, stimulation of the germinal cell to enter meiosis could result from environmental factors rather than from a genetic programmation.     

Electron microscopic study of preleptotene-stage oocyte nuclei in the prophase of meiosis in the hen

The morphology of oocyte nuclei at preleptotene and early leptotene stages of meiotic prophase I in the chick embryo was examined by electron microscopy and light cytochemistry. The intrenuclear fibrillar body (IFB) of proteinaceous nature is described. The IFB has a spherical or irregular form and consists of the disoriented fibrils ranging from 3 to 18 nm in diameter and of globules lying along the course of fibrils. The condensed chromatin in the oocyte nuclei at the early--late preleptotene stages and the thread-like chromosomes in the oocyte nuclei at the middle preleptotene--early leptotene stages either closely adjoin to IFB, localized in the centre of the nucleus, or are situated at some distance from it, and then the fibrils or fibrillar filaments are seen between chromosome material and IFB. The chromosomes are attached to IFV by the telomer or interstitial segment. The chromosomes lose the connection with IFB after the attachment of both the telomeres to the nuclear envelope (middle--late leptotene), and IFB removes from the centre of the nucleus to its periphery. It is supposed that IFB represents a nuclear skeleton element and takes part in the spatial organization of the chromosomes in the oocyte nuclei at the early stages of meiotic prophase I.     

Mammalian meiotic telomeres: protein composition and redistribution in relation to nuclear pores

Mammalian telomeres consist of TTAGGG repeats, telomeric repeat binding factor (TRF), and other proteins, resulting in a protective structure at chromosome ends. Although structure and function of the somatic telomeric complex has been elucidated in some detail, the protein composition of mammalian meiotic telomeres is undetermined. Here we show, by indirect immunofluorescence (IF), that the meiotic telomere complex is similar to its somatic counterpart and contains significant amounts of TRF1, TRF2, and hRap1, while tankyrase, a poly-(ADP-ribose)polymerase at somatic telomeres and nuclear pores, forms small signals at ends of human meiotic chromosome cores. Analysis of rodent spermatocytes reveals Trf1 at mouse, TRF2 at rat, and mammalian Rap1 at meiotic telomeres of both rodents. Moreover, we demonstrate that telomere repositioning during meiotic prophase occurs in sectors of the nuclear envelope that are distinct from nuclear pore-dense areas. The latter form during preleptotene/leptotene and are present during entire prophase I.     

Separation of DNA replication from the assembly of break-competent meiotic chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.     

Preleptotene chromosome contraction in Lilium longiflorum “Croft”

A period of chromosome contraction between premeiotic interphase and leptotene was regularly observed in three samples of Lilium longiflorum Croft. Extensive preleptotene chromosome contraction was also observed in L. longiflorum Ace and in the Lilium hybrid Enchantment. Although the stage resembles late mitotic prophase, microsporocytes never develop to metaphase, but despiralize to leptotene, and regular alignment, pairing and chiasma formation follow. As preleptotene chromosome contraction is discovered in an increasing number of organisms it becomes less likely that it represents a true reversion to mitosis. However its absence in many organisms and its extreme variability in others do not support the concept of preleptotene chromosome contraction as a regular meiotic stage. It is suggested that the line of demarcation between mitosis and meiosis is often imprecise, and meiocytes may fluctuate to some extent between these states before a final transition to meiosis is made. The occurrence and extent of this fluctuation may possibly be related to some externally produced substances required for the orderly development of meiotic prophase.     

The effect of retinoic acid on the meiosis in the chick embryo (Gallus gallus domesticus)

In this study it was shown that the injection of retinoic acid (RA) into incubated eggs on day 9 or 14 induced entry the males germ cells into preleptotene stage of prophase I on day 17, which are absent in the control embryos. At the same time the meiosis marker SCP3 was detected in the germ cells. Which was also absent at control embryos. On day 19 in male embryos the number of male germ cells at the stage preleptoteny increased, but there were no germ cells in the following stages of the prophase of meiosis. In 20-day-old chicks meiotic germ cells were absent. Thus, white it is shown that the influence of RA on the developing chicken embryos induces the entry of germ cells into preleptotene stage of prophase I meiosis. However, further meiotic transformations don't occur. Thus RA is only one of many factors providing meiotic cell division.     

Ultrastructural changes in female germ cells during the meiotic prophase in the rat: a special study of membranes after cryofracture

Ultrastructural changes in the nuclear and cytoplasmic elements in the germ cells of female rats were followed before meiotic prophase (15.50 days post-co?tum and 17.25 days post-co?tum) and during it (17.75 days post-co?tum to birth). We observed: modifications in the nuclear envelope which was thick during the oogonial stage, becoming thinner when the chromosomes entered preleptotene stage. The thinning of the envelope was due to the disappearance of the chromatin material lining it; variations in the number and distribution of germ cell nuclear pores according to stage; the pores were first scattered in small clusters of 6 to 8 over the entire nuclear membrane. From the preleptotene to zygotene stage, these clusters enriched in pores to form large areas. Finally, in the pachytene and diplotene stages, clusters of more than 100 pores were seen; nucleolar fragmentation from the preleptotene stage, followed by the formation of a new active nucleole in the diplotene; polarization of the mitochondria in the oldest oogonia just before the beginning of meiotic prophase. This polarization disappeared after the onset of the meiotic processes, then appeared again near the developing Golgi apparatus at the end of the pachytene stage; the formation of large gap junctions and numerous bands of tight junctions between the somatic cells; these formations contrasted with small gap junctions, and the tight junctions became scarce just before the meiotic process began. These observations, as well as those concerning nuclear pore distribution were made using the cryofracture technique.     

X-irradiation--induced changes in the progression of type B spermatogonia and preleptotene spermatocytes

In response to induced DNA damage, proliferating cells arrest in their cell cycle or go into apoptosis. Ionizing radiation is known to induce degeneration of mammalian male germ cells. The effects on cell-cycle progression, however, have not been thoroughly studied due to lack of methods for identifying effects on a particular cell-cycle phase of a specific germ cell type. In this study, we have utilized the technique for isolation of defined segments of seminiferous tubules to examine the cell-cycle progression of irradiated rat mitotic (type B spermatogonia) and meiotic (preleptotene spermatocytes) G1/S cells. Cells irradiated as type B spermatogonia in mitotic S phase showed a small delay in progression through meiosis. Thus, it seems that transient arrest in the progression can occur in the otherwise strictly regulated progression of germ cells in the seminiferous epithelium. Contrary to the arrest observed in type B spermatogonia and in previous studies on somatic cells, X-irradiation did not result in a G1 delay in meiotic cells. This lack of arrest occurred despite the presence of unrepaired DNA damage that was measured when the cells had progressed through the two meiotic divisions.     

DNA repair synthesis-related enzymes during spermatogenesis in the mouse

To assess whether uracil DNA glycosylase and dUTP nucleotidohydrolase (dUTPase) can be involved in repair-type DNA synthesis associated to crossing-over or induced by UV and X-ray treatments, we have studied these enzyme activities in male mouse germ cells at specific stages of differentiation.Although the highest uracil DNA glycosylase activity was observed in dividing germ cells (spermatogonia and preleptotene spermatocytes), some activity was also detected in meiotic (3.5%) and post-meiotic (1.0%) cells with a relative maximum of activity at pachytene stage (4.7%) when meiotic crossing-over takes place. These findings suggest that uracil DNA glycosylase is involved, in this biological system, in DNA replication and in repair-type DNA synthesis.dUTPase is present at all the stages of spermatogenesis studied but, unlike thymidylate synthetase which is mainly associated with replicating germ cells, dUTPase activity is maximal in spermatocytes at pachytene stages. The data reported suggest that, in this biological system, the main role of dUTPase is to degrade dUTP to prevent misincorporation of uracil into DNA during crossing-over, rather than to participate in the biosynthetic pathway of dTTP.     

Normal and BrdC-substituted chromosomes during spermatogenesis with an endomeiotic chromosome reduplication in Carausius morosus Br.

Spermatogenesis involving an additional chromosome reduplication during zygotene in sporadic males and intersexes of the thelytokous phasmid Carausius morosus Br. has been examined using differential staining of chromatids after 5-bromodeoxycytidine incorporation. After reduplication autobivalents are formed by synapsis between identical sister chromosomes. Chiasmata are only formed after reduplication; they do not occur in constitutive heterochromatin, but can be formed in facultative heterochromatin, dependent on heteropycnosis and sex. Quadrivalents and U-type exchanges occur. In spermatogonia and spermatocytes the number of differentially stained chromosomes varies considerably; sister chromatid exchanges hardly appear. Sex bivalents with differentially stained chromosomes have a lower chiasma frequency than normally stained sex bivalents. Bivalents show reduced staining of all four, two outer, or one inner chromatid. Autobivalents arise in the same way as diplochromosomes; chromatids with the oldest DNA sub-units remain together during reduplication and are thus involved in sister chromosome pairing. The additional reduplication begins 7 days after the premeiotic S-phase, first metaphase after 19 days. Spermatogenesis is abnormal from first anaphase onwards.     

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 spermatogonial stem cells of male (101/E1 x 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 (Adler, 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.