Premeiotic S-phase Research Articles

Cdc6 synthesis regulates replication competence in Xenopus oocytes.

The early division cycles of an embryo rely on the oocyte's ability to replicate DNA. During meiosis, oocytes temporarily lose this ability. After a single round of pre-meiotic S-phase, oocytes enter meiosis and rapidly arrest at prophase of meiosis I (G2). Upon hormonal stimulation, arrested oocytes resume meiosis, re-establish DNA replication competence in meiosis I shortly after germinal vesicle breakdown (GVBD), but repress replication until fertilization. How oocytes lose and regain replication competence during meiosis are important questions underlying the production of functional gametes. Here we show that the inability of immature Xenopus oocytes to replicate is linked to the absence of the Cdc6 protein and the cytoplasmic localization of other initiation proteins. Injection of Cdc6 protein into immature oocytes does not induce DNA replication. However, injection of Cdc6 into oocytes undergoing GVBD is sufficient to induce DNA replication in the absence of protein synthesis. Our results show that GVBD and Cdc6 synthesis are the only events that limit the establishment of the oocyte's replication competence during meiosis.

Mitotic replication initiation proteins are not required for pre-meiotic S phase.

Initiation of mitotic DNA replication in eukaryotes requires conserved factors, including Cdc18/CDC6 and minichromosome maintenance (MCM) proteins. We show here that these proteins are not essential for meiotic DNA replication or subsequent meiotic divisions in fission yeast. In addition, vegetative replication checkpoint genes are not required for the arrest of meiotic divisions in response to pre-meiotic S-phase delays. Genes essential for other aspects of vegetative DNA replication, however, including polymerases and DNA ligase, are also required for pre-meiotic DNA synthesis. Our results indicate that the process of replication initiation and checkpoint control may be fundamentally different in mitotic and meiotic cells.

Colchicine effects on meiosis in the male mouse. I. Meiotic prophase: synaptic arrest, univalents, loss of damaged spermatocytes and a possible checkpoint at pachytene.

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.

Micronucleus induction in germ and somatic cells of the mouse after exposure to the butadiene metabolites diepoxybutane and epoxybutene

The genotoxicity of diepoxibutane (DEB) and epoxybutene (EB), two of the main metabolites of 1,3-butadiene, was tested in the germ and somatic cells of the mouse by applying an MN assay in early spermatids, and in peripheral blood reticulocytes of a subgroup of the same animals. DEB (0.17 and 0.35 mmol/kg) and EB (0.35, 0.70 and 1.04 mmol/kg) were administered i.p. In the germ cell assay, significant increases of MN were observed after treatment of premeiotic S-phase cells with both butadiene metabolites, but DEB was shown to be more powerful than EB in the induction of chromosomal damage. A weak effect of the same compounds was also found after treatment of late spermatocytes, approaching the meiotic divisions. From the MN assay in peripheral blood reticulocytes, a statistically significant increase of the frequency of MN was detected at each dose tested for both chemicals. However, the results have again shown that DEB is much more efficient than EB in inducing chromosome damage.

Synthesis report of the step project detection of germ cell mutagens

The project ‘Detection of Germ Cell Mutagens’ was designed with three major goals: (1) Detection and characterization of germ-cell mutagens; (2) standardization and validation of new germ-cell tests; and (3) development of a data base on germ-cell mutagenicity. All three goals were achieved. The classical germ-cell tests were applied to characterize the genetic effects of acrylamide (AA), 1,3-butadiene (BD), trophosphamide (TP) and urethane (UR). All but UR were found to cause heritable genetic damage. The experimental data obtained for AA and BD were the basis for genetic risk evaluations during the EC/US Workshop on Risk Assessment ‘Human Genetic Risk from Exposure to Chemicals, Focusing on the Feasibility of the Parallelogram Approach’. Nine chemicals were employed to validate the spermatid micronucleus assay with mice and rats: AA, BD and its metabolites 1,2-epoxybutene-3 and 1,2:3,4-diepoxybutane, chlorambucil, mitomycin C, methylnitrosourea, TP and UR. The spermatid micronucleus test was combined with micronucleus tests in somatic cells such as bone marrow or peripheral blood erythrocytes, and splenocytes which allowed a comparison of effects in somatic and germinal cells. Improvements of the spermatid micronucleus test included BrdU-labelling of premeiotic S-phase for the determination of stage sensitivity and fluorescence in situ hybridization with pancentromeric DNA-probes to distinguish between clastogenic and aneugenic events. The results indicate that the spermatid micronucleus test with its improvements is an adequate procedure to detect germ-cell clastogenicity and to compare the activity of chemicals in different tissues and between species, i.e., rats and mice. Other germ cell methods under study were the flow cytometric measurement of testicular sperm DNA and the cytogenetic analysis of preimplantation embryos for chromosomal aberrations and micronuclei. The collection of a reliable germ-cell data base was accomplished through a critical evaluation of the literature and with the data obtained in the present project. Remarkable concordance between responses of germ cell tests to chemical mutagens was the most striking conclusion to be drawn from the present data base.

Cloning and differential expression during the sexual cycle of a meiotic endonuclease-encoding gene from the basidiomycete Coprinus cinereus

The naturally synchronous meiosis of the fungus, Coprinus cinereus provides an ideal system for the investigation of differential gene expression in relation to meiosis and fruiting body development. We have cloned a cDNA from the fruiting body of C. cinereus encoding the 12-kDa subunit of a meiotic endonuclease (mENase). The identification of the 12-kDa subunit cDNA clone was achieved by the mENase antiserum against a τ17 cDNA expression library. It was confirmed by a direct match of the amino acid (aa) sequence obtained from purified 12-kDa polypeptide with the nucleotide sequence. Northern blot analysis using the cDNA clone as a probe showed that the mENase-encoding gene ( MenA) for the 12-kDa subunit was expressed mainly in fruiting bodies and at a very low level in the asexual vegetative mycelium. In addition, it was differentially expressed in the early meiotic stages. The MenA transcript was most abundant in fruiting body primordia prior to the premeiotic S-phase; it remained high from karyogamy to early pachytene, declined drastically by late pachytene and diplotene, and was undetectable by sterigma stage. Western blot analysis showed that the mENase protein was produced at a very low level in mycelium; it was produced in great quantity during the early meiotic stages and decreased to a low level at the end of meiosis.

Demonstration of replication patterns in the last premeiotic S-phase of male Chinese hamsters after BrdU pulse labeling

Demonstration of replication patterns in the last premeiotic S-phase of male Chinese hamsters after BrdU pulse labeling

Detection of aneuploidy in male germ cells of mice by means of a meiotic micronucleus assay

The possibility of using a micronucleus (MN) assay in mouse germ cells for the identification of aneuploidogenic agents was evaluated by comparing the pattern of effects induced by 4 chemicals with different mechanisms of action (adriamycin, ADM; mitomycin C, MMC; chloral hydrate, CH; ethylenedinirrilotetraacetic acid, EDTA). The results obtained after treatment of spermatocytes at the premeiotic S-phase (preleptotene) indicated that only clastogenic agents (ADM and MMC) were able to induce MN at this cell stage. Previous data obtained with the saine compounds demonstrated by contrast that the micronucleus spermatid assay may detect both clastogenic and aneuploidogenic effects after treatment of diakinesis/MI/MII cells. Analysis of MN size distributions, in the present and previous spermatid samples, revealed that the clastogens ADM and MMC produced relatively more small MN than CH and EDTA. These data are in agreement with the proposed mechanism of action of the chemicals tested.

Demonstration of replication patterns in the last premeiotic S-phase of male Chinese hamsters after BrdU pulse labeling.

Chromosome replication in the last premeiotic S-phase of male mammals has been previously studied by [3H]thymidine autoradiography and by a 5-bromodeoxyuridine (BrdU)/Giemsa technique. We used a recently developed BrdU-antibody technique (BAT) in this study. The following conclusions were drawn: (1) The replication patterns observed are similar to that of somatic cells. (2) The heterochromatin starts replication in early S-phase. (3) The euchromatic part of the X chromosome of the male Chinese hamster replicates together with the autosomes and therefore behaves isocyclicly and not allocyclicly as hitherto assumed. Hence, genetic inactivity of the X chromosome may be brought about by a mechanism different from that in somatic cells.

A DNA exonuclease induced during meiosis of Schizosaccharomyces pombe.

In meiotic cells of the fission yeast Schizosaccharomyces pombe, a DNA exonuclease activity increased approximately 5-fold after premeiotic S-phase and decreased to the initial level before the meiotic divisions. We have purified this activity, designated exonuclease I, to near homogeneity. The activity co-purified with a polypeptide with an apparent molecular weight of 36,000. With a linear double-stranded DNA substrate, exonuclease I degraded only the 5'-ended strand from each end to produce 3'-single-stranded tails. The enzyme also acted on nicked circular DNA with comparable affinity. The meiotic induction of exonuclease I and its mode of action, similar to that of recombination-promoting exonucleases from bacteria, suggest that exonuclease I is involved in meiotic homologous recombination in S. pombe.

Frequency and distribution of chiasmata in Syrian hamster spermatocytes studied by the BrdU antibody technique

The frequency and distribution of chiasmata and the nature of terminal "associations" was re-examined in Syrian hamster spermatocytes using the 5-bromo-2'-deoxyuridine (BrdU) antibody technique (BAT) for differential chromatid labelling. Differential chromatid substitution was achieved by BrdU incorporation at the penultimate pre-meiotic S-phase followed by one of three different staining protocols: (i) fluorescence plus Giemsa (FPG), (ii) acridine orange staining or (iii) BAT. For analysis of chiasmata frequency and localization in the diplotene/diakinesis stages the resolution of FPG and acridine orange staining was comparable to that of BAT. In metaphase II chromosomes BAT was more informative than FPG and acridine orange staining and revealed small, terminal crossover exchanges. This finding proves that many terminal associations of meiotic chromosomes actually represent chiasmata at the end of the first meiotic division. Some crossover exchanges were localized in the constitutive heterochromatin of autosomes. Using BAT we also detected crossover exchanges in close vicinity to each other. This observation is reminiscent of the fact that crossing over interference means a reduction in frequency and does not imply total inhibition.

The effect of temperature on recombination activity in testes of rodents

An extractable enzyme system capable of catalyzing recombination in vitro was described in murine spermatocytes [Hotta et al. (1985) Chromosoma 93, 140–151]. The system is specific to meiosis, its activity increasing 400-fold between the premeiotic S-phase and mid-pachytene. The present study examines the effect of temperature on this system since the elevation of testicular temperature is one of the major factors causing impairment of testicular function. A strong depression of in vitro recombination activity occurred immediately after raising the testicular temperature in vivo by translocating the testes into the abdominal cavity (cryptorchid). The in vitro study also showed that the extract from spermatocytes prefered lower temperatures (30–32°C) than somatic cells (37°C) for maximal activity of recombination. These results suggest that the strong depression of recombination activity may be an important factor which causes degeneration of testes by heat.

Correlation of endonuclease activity at meiotic prophase and cofactor-induced genetic recombination in the basidiomycete Coprinus cinereus

An endonuclease was purified from the basidiocarps of the fungus Coprinus cinereus during late premeiotic S-phase, karyogamy, and pachytene stages of meiosis. This endonuclease causes single-strand nicks on supercoiled PM2 DNA. Its activity is dependent on cofactors such as Mg2+ and Ca2+, and is enhanced by hexaminecobalt chloride and dimethylsulfoxide. Treatments with increasing cofactor concentration elicit increasing enzyme activity in vitro and increasing genetic recombination in vivo. The percent recombination that can be induced by cofactor treatment during late premeiotic S-phase, karyogamy and pachytene is correlated with the amount of endonuclease that can be extracted from the respective stages. These results suggest that Mg2+ and Ca2+ induce genetic recombination by increasing the endonuclease activity that creates nicked and gapped DNA substrates for ensuing recombination events. We believe that the meiotic endonuclease of Coprinus is involved in the early phase of genetic recombination. Cold temperature treatment is known to retard repair activity at pachytene and causes a threefold increase in recombination. Double treatments involving Mg2+ and Ca2+ at karyogamy followed by cold temperature at pachytene result in an additive (more than fourfold) increase in recombination frequency. The peak concentration of the nicking endonuclease is found at late premeiotic S and early karyogamy, whereas the peak activity of the repairing DNA polymerase b is found at late pachytene. The staging of these two enzymes is in good accord with the genetic data.Key words: Coprinus, endonuclease, meiosis, genetic recombination, Z-DNA.

Regular and irregular divisions of Lepidoptera spermatocytes as related to the speed of meiotic prophase

Lepidoptera males bear two kinds of meiotic divisions. One is regular (eupyrene) and leads to nucleate, fertilizing spermatozoa. The other (apyrene) shows metaphase I chromosomes clumping together into irregular masses which later split forming daughter cells with unbalanced sets of chromosomes which are eventually extruded from the cells; hence, the spermatids develop into anucleate spermatozoa of unknown function. The apyrene divisions are induced by a haemolymph factor which becomes functional towards pupation. Using incorporation of tritiated thymidine at the premeiotic S-phase as a marker for timing, it was found that the prophase of the apyrene spermatocyte is shorter than that of the eupyrene spermatocyte. It is proposed that meiosis-specific proteins cannot be synthesized during the shortened apyrene prophase and that this is correlated with the irregular chromosome behaviour during the subsequent metaphase-telophase of these spermatocytes.

Meiosis-specific transcripts of a DNA component replicated during chromosome pairing: Homology across the phylogenetic spectrum

In meiotic cells of Lilium, a group of single or low copy number DNA sequences that constitute about 0.10.2% of the genome do not replicate during the premeiotic S-phase but do so at zygotene in coordination with chromosome pairing. An appreciable fraction of these sequences has now been found to be transcribed into poly(A) + RNA when chromosomes initiate the pairing process. This “zygRNA” has not been detected in nonmeiotic tissues. Even within the meiocytes, zygRNA is not detectable prior to leptotene or beyond midpachytene. S1 nuclease digestion of mouse spermatocyte nuclei selectively released zygDNA, which hybridizes with lily zygRNA, zygRNA has not been detected in mouse somatic tissues. The profile of zygRNA formation and disappearance in mouse spermatocytes is very similar to that of zygRNA in lily meiocytes.

Molecular biology of meiosis: synapsis-associated phenomena.

In tracing the metabolism of nuclear DNA during meiosis we have identified two groups of DNA sequences, each displaying a distinctive pattern of behavior during meiotic prophase. One group, which will be referred to as zygotene DNA (“zygDNA”), is coordinated in most of its activities with chromosome pairing. The other group, referred to as pachytene DNA (“PDNA”), is coordinated in its behavior with what we believe to be the process of crossing-over. Each group has been and continues to be identified by its respective mode of replication during zygotene and pachytene, well after the apparent completion of the premeiotic S-phase. The theme of this chapter is that chromosomes of at least the larger eukaryotic genomes have evolved certain DNA sequences that are specifically addressed to particular chormosomal needs during meiosis. In this chapter we focus on the zygDNA sequences because of their apparent relationship to chromosome pairing. We have no direct evidence that this is indeed the case, but we do know that the behavior of zygDNA during meiotic prophase provides strong evidence for its playing an important role in the process. Our description of that role is necessarily conjectural and our interpretation of various findings are slanted in favor of the conjecture.

An ameiotic mutant of Coprinus cinereus halted prior to pre-meiotic S-phase

Five Coprinus cinereus monokaryons were isolated which bore a dominant mutation designated Mar. Dikaryons formed by mating Mar-bearing monokaryons with normal monokaryons produced aborted fruiting bodies in which the basidia never underwent karyogamy. The Mar mutation probably prevented pre-meiotic DNA replication; extracts of Mar-bearing dikaryons harvested at a stage equivalent to pre-meiotiv S-phase caused little net DNA synthesis in DNA polymerase assays. There was no marked incorporation of 32p into DNA (and low incorporation of 32p into RNA) of cap tissue from Mar-bearing fruiting bodies at a stage equivalent to pre-meiotic S-phase. The aborted fruiting bodies were similar to those resulting on normal cultures following treatment prior to S-phase with cycloheximide, or continuous light at 35 °C. In contrast to normal C. cinereus monokaryons, no Mar-bearing monokaryon formed fruiting bodies when subjected to nutritional stress.

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.

Meiosis in Schizophyllum commune: The effect of hydroxyurea on the frequency of recombination and mutations

The effect of exposure of nuclei at different stages of meiosis to hydroxyurea (HU) was tested by examination of its effect on the frequency of auxotrophic mutations and on intergenic recombination. The results indicate that recombination is increased most significantly if the nuclei are exposed to the drug during the premeiotic-S phase and to a lesser extent if exposed during prophase. Similarly, nuclei exposed to HU during premeiotic-S phase and prophase mutate at a frequency 10 to 50 times higher than untreated meiotic nuclei. The degree of response of the mutagenic events and recombination events is not similar and it is suggested that the mutagenic events occur at stages prior to recombination. The temporal relations of the two events in response to HU suggest that the mutagenic events precede recombination and the results obtained with HU only amplify the mutagenic events that normally occur in meiotic nuclei and are related to the meiotic effect.

Radiation-induced meiotic autosomal non-disjunction in male mice The effects of low doses of fission neutrons and X-rays in meiosis I and II of a Robertsonian translocation heterozygote

Male mice, heterozygous for the Rb(11.13)4Bnr translocation, were irradiated for 14.5 min with either a dose of 15-rad fission neutrons or 60-rad X-rays. Animals of this karyotype are known to show high levels of spontaneous autosomal non-disjunction (20–30%) after anaphase I. The effects of the irradiation on this process were determined after 2 and 3 in air-dried preparations. The length of the period from the end of meiosis I till the end of meiosis II was assessed autoradiographically, with the aid of cells showing a labelled Y chromosome only and appeared to last less than 3 h. Inter-mouse variation with regard to the duration of the period “last premeiotic S-phase till diakinesis/ methaphase I” prevented a more accurate estimate. On the basis of this 3-h datum, the induced effects were studied at intervals of 2 and 3 h after the start of the irradiation. The influence of irradiation was assessed by scoring: (1) univalents in primary spermatocytes, (2) deletions, aneuploid chromosome counts and precocious centromere separation in secondary spermatocytes, and (3) chromatid gaps and breaks in both cell types. Both radiation types induced comparable levels of chromosomal damage. A neutron-X-rays RBE value for these parameters was calculated to be 5.4 for the MI stage and 3.3 for the MII stage. The significantly higher incidence of cells showing damage at MII than at diakinesis/MI is not believed to indicate a difference in radiation sensitivity, but is believed to be merely the consequence of the different chromosomal processes taking place during the irradiation-fixation time interval.