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.     

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.     

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 0.1-0.2% 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.     

Sex-specific telomere redistribution and synapsis initiation in cattle oogenesis

The process of homolog pairing is well characterised in meiosis of male mammals, but much less information is available from female meiosis. We have therefore studied telomere dynamics by FISH and synapsis formation by immunostaining of synaptonemal complex proteins (SCP3, SCP1) on ovarian sections from 15 bovine fetuses, which covered the entire female prophase I. Telomeres displayed a dispersed intranuclear distribution in oogonia and relocated to the nuclear periphery during the preleptotene stage. Tight telomere clustering (bouquet formation) coincided with synapsis initiation at the leptotene/zygotene transition. Clustering of telomeres persisted during zygotene and even into the pachytene stage in a subset of nuclei, while it was absent in diplotene/dictyotene stage nuclei. Thus, the bouquet stage in the bovine female lasts significantly longer than in the male. Further, we observed that synapsis in the female initiated both terminally and interstitially in earliest zygotene stage oocytes, which contrasts with the predominantly terminal synapsis initiation in early zygotene spermatocytes of the bovine male. Altogether, our data disclose a sex-specific difference in telomere dynamics and synapsis initiation patterns in male and female bovine germ cells that may be related to the sex-specific differences in recombination rates observed in this and other mammalian species.     

Ribosomal gene amplification in oocytes of the lizard Podarcis sicula

In order to provide cytological evidence of amplification, Podarcis sicula oocytes were studied by cytophotometry, thymidine incorporation and in situ DNA-DNA hybridization. Our results show that DNA replication is completed during the preleptotene stage, the leptotene oocytes having the typical 4C nuclear DNA content. Between the zygotene and the mid-pachytene stages further DNA synthesis occurs with consequent increase of the ribosomal nuclear DNA content. These results and the variations in nucleolar organization observed during differentiation give clear evidence of the existence of ribosomal gene amplification in Podarcis sicula oocytes.     

Genome analysis and replication behavior of a set of repetitive sequences of Lilium longiflorum.

The four clones, pLZH47, pLZ112, pLZ113, and pLZ122, previously assumed to contain DNA sequences that replicate during zygotene (zygDNA), actually had their replication behavior tested using a replication assay. Genome analysis was also done for each clone. All of the clones seem to be members of families of dispersed repetitive DNA. The number of copies per 2C genome are as follows: pLZH47, 13 500; pLZ112, >100; pLZ113, 200; and pLZ122, 3500. The replication assays measured the amount of hybridizable sequences available at the stage of leptotene (before zygotene DNA replication occurs) and at the stage of pachytene (after zygotene replication occurs). True zygDNA clones should have twice as many sequences to hybridize to at pachytene as at leptotene. None of the clones had the expected increase. One clone, pLZ122, did show a statistically significant increase (10%). It may consist of a mixture of zygDNA and non-zygDNA sequences. Alternatively, only 10% of the pLZ122 family members may function as zygDNA.     

A timing study of DNA amplification in Xenopus laevis oocytes

The time course of meiotic amplification of nucleolar DNA in Xenopus laevis oocytes has been studied autoradiographically. We find that the process is first detectable in zygotene nuclei less than 7 days after the end of premeiotic S-phase. It is completed 3 1/2 weeks later, towards the end of pachytene. Premeiotic S-phase lasts for 1–2 weeks. We are not certain whether it is followed by a short G2 or whether leptotene commences immediately. Leptotene lasts for 5±2 days, zygotene for 7±2 days and pachytene for about 20 days before the oocyte gradually enters the extended diplotene stage. Various molecular mechanisms for amplification are discussed in the light of a 24±3 day amplification time. All are found to be potentially capable of amplifying sufficient nucleolar DNA in the time available.     

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.     

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.     

A meiotic time-course for<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

A meiotic time-course for Arabidopsis pollen mother cells has been established based on BrdU pulse-labelling of nuclear DNA in the meiotic S-phase. Labelled flower buds were sampled at intervals and the progress of labelled cells through meiosis assessed by anti-BrdU antibody detection. The overall duration of meiosis from the end of meiotic S-phase to the tetrad stage, at 18.5°C, was 33 h, which is about three times longer than the mitotic cell cycle in seedlings. The onset of leptotene was defined by reference to the loading of the axis-associated protein Asy1, and this permitted the detection of a definite G2 stage, having a maximum duration of 9 h. It is likely, from two independent sources of evidence, that the meiotic S-phase has a duration similar to that of G2. The durations of leptotene and zygotene/pachytene are 6 h and 15.3 h, respectively, but the remaining meiotic division stages are completed very rapidly, within 3 h. The establishment of a meiotic time-course provides a framework for determining the relative timing and durations of key molecular events of meiosis in Arabidopsis in relation to cytologically defined landmarks. In addition, it will be important in a broader developmental context for determining the timing of epigenetic mechanisms that are known or suspected to occur during meiosis.     

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.     

Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts

Xenopus egg extracts prepared before and after egg activation retain M- and S-phase specific activity, respectively. Staurosporine, a potent inhibitor of protein kinase, converted M-phase extracts into interphase- like extracts that were capable of forming nuclei upon the addition of sperm DNA. The nuclei formed in the staurosporine treated M-phase extract were incapable of replicating DNA, and they were unable to initiate replication upon the addition of S-phase extracts. Furthermore, replication was inhibited when the staurosporine-treated M- phase extract was added in excess to the staurosporine-treated S-phase extract before the addition of DNA. The membrane-depleted S-phase extract supported neither nuclear formation nor replication; however, preincubation of sperm DNA with these extracts allowed them to form replication-competent nuclei upon the addition of excess staurosporine- treated M-phase extract. These results demonstrate that positive factors in the S-phase extracts determined the initiation of DNA replication before nuclear formation, although these factors were unable to initiate replication after nuclear formation.     

Duration of meiotic prophase stages during oogenesis in the newt Triturus cristatus cristatus]

The duration of the early stages of meiotic prophase was determined in the oogenesis of T. cristatus cristatus by means of autoradiography. The oocytes were being investigated during 39 days from the moment of 3H-thymidine injection. It was shown that preleptotene lasts 1--2, leptotene ca. 4, zygotene 5 and pachytene 26 days. When studying the preparations obtained 1 day after the injection of 3H-thymidine, the silver grains were found to be localized over the nuclei at all stages of meiotic prophase; this suggests the amplification of rDNA which begins in leptotene-zygotene and ends in early diplotene.     

Coprinus cinereus DNA ligase I during meiotic development

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.     

Atm Inactivation Results in Aberrant Telomere Clustering during Meiotic Prophase

A-T (ataxia telangiectasia) individuals frequently display gonadal atrophy, and Atm-/- mice show spermatogenic failure due to arrest at prophase of meiosis I. Chromosomal movements take place during meiotic prophase, with telomeres congregating on the nuclear envelope to transiently form a cluster during the leptotene/zygotene transition (bouquet arrangement). Since the ATM protein has been implicated in telomere metabolism of somatic cells, we have set out to investigate the effects of Atm inactivation on meiotic telomere behavior. Fluorescent in situ hybridization and synaptonemal complex (SC) immunostaining of structurally preserved spermatocytes I revealed that telomere clustering occurs aberrantly in Atm-/- mice. Numerous spermatocytes of Atm-/- mice displayed locally accumulated telomeres with stretches of SC near the clustered chromosome ends. This contrasted with spermatogenesis of normal mice, where only a few leptotene/zygotene spermatocytes I with clustered telomeres were detected. Pachytene nuclei, which were much more abundant in normal mice, displayed telomeres scattered over the nuclear periphery. It appears that the timing and occurrence of chromosome polarization is altered in Atm-/- mice. When we examined telomere-nuclear matrix interactions in spermatocytes I, a significant difference was observed in the ratio of soluble versus matrix-associated telomeric DNA sequences between meiocytes of Atm-/- and control mice. We propose that the severe disruption of spermatogenesis during early prophase I in the absence of functional Atm may be partly due to altered interactions of telomeres with the nuclear matrix and distorted meiotic telomere clustering.     

RPA is an initiation factor for human chromosomal DNA replication

The initiation of chromosomal DNA replication in human cell nuclei is not well understood because of its complexity. To allow investigation of this process on a molecular level, we have recently established a cell-free system that initiates chromosomal DNA replication in an origin-specific manner under cell cycle control in isolated human cell nuclei. We have now used fractionation and reconstitution experiments to functionally identify cellular factors present in a human cell extract that trigger initiation of chromosomal DNA replication in this system. Initial fractionation of a cytosolic extract indicates the presence of at least two independent and non-redundant initiation factors. We have purified one of these factors to homogeneity and identified it as the single-stranded DNA binding protein RPA. The prokaryotic single-stranded DNA binding protein SSB cannot substitute for RPA in the initiation of human chromosomal DNA replication. Antibodies specific for human RPA inhibit the initiation step of human chromosomal DNA replication in vitro. RPA is recruited to DNA replication foci and becomes phosphorylated concomitant with the initiation step in vitro. These data establish a direct functional role for RPA as an essential factor for the initiation of human chromosomal DNA replication.     

Protein—nucleic acid interactions in bacteriophage φ29 DNA replication

Abstract: φ29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3' end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3'–5' exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions.     

Determination of daily sperm production in stallion testes by enumeration of germ cells in homogenates

Thirty adult stallion testes were selected with high (n = 15) and low (n = 15) Daily Sperm Production (DSP)/testis. Parenchymal samples were prepared for morphometric analysis, and the numbers of germ cells and Sertoli cells were determined. Testicular samples were homogenized, and germ cells and Sertoli cells were enumerated using phase contrast microscopy. Numbers of germ cells and Sertoli cells and potential DSP during spermatogenesis were determined. Significant correlations existed between morphometric and homogenate determinations of number per testis of preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.58; P < 0.001), pachytene plus diplotene primary spermatocytes (r = 0.67; P < 0.0001), all primary spermatocytes (r = 0.67; P < 0.0001), round spermatids (r = 0.72; P < 0.0001), and Sertoli cells (r = 0.70; P < 0.0001). Significant correlations (P < 0.0001) existed between morphometric and homogenate determination of DSP/testis based on preleptotene, leptotene plus zygotene primary spermatocytes (r = 0.78), pachytene plus diplotene primary spermatocytes (r = 0.88), and round spermatids (r = 0.85). Using morphometric determination as the standard, the sensitivity (i.e., ability to detect low DSP/testis) and specificity (i.e., ability to detect high DSP/testis) by homogenate enumeration of germ cells was 81 and 93% for round spermatids, 100 and 24% for pachytene plus diplotene primary spermatocytes, and 67 and 87% for preleptotene, leptotene plus zygotene primary spermatocytes, respectively. Enumeration of primary spermatocytes in homogenates was less accurate than enumeration of round or elongated spermatids. Enumeration of round and elongated spermatids in homogenates was a rapid and useful method for determining DSP in horses, and it may prove to be a useful technique for quantitating potential DSP from testicular biopsies.     

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.