Studies on the behavior of meiotic protoplasts IV. Protoplasts isolated from microsporocytes of liliaceous plants

Enzymatic isolation of protoplasts from microsporocytes of various species of liliaceous plants is described along with some of the features of the isolated meiotic protoplasts. Protoplasts are produced with a high viability from coherent filaments of cells preculture for 24 hr during premeiosis and early meiotic prophase, but with low survival rates from free cells at late prophase. The suspensions of protoplasts contain multinucleate cells produced by spontaneous fusion at various frequencies up to 40%. In the enzyme solution meiotic protoplasts adhere to one another. When isolated at meiotic prophase, protoplasts may be cultured through the meiotic cycle.     

Hybridization between tetraploid species of Alopecurus L. (Poaceae): chromosome behaviour of natural and artificial hybrids

The meiotic behaviour of plants from mixed populations of Alopecurus pratensis × A. geniculatus and of A. pratensis × A. arundinaceus ( 2n = 4×(= 28) is reported together with observations on some artificially produced hybrids. This meiotic behaviour is correlated with the degree of hybridity as shown by the hybrid index values of the plant. In A. pratensis × A. geniculatus hybrids there are marked differences between populations in the degree of meiotic disturbance and in one population there was an almost complete breakdown of meiosis. Alopecurus pratensis × A. arundinaceus hybrids show fairly regular meiotic pairing but in most plants there are a few univalent chromosomes at metaphase I. Artificially produced hybrids of A. pratensis × A. aequalis gave meiotic configurations that suggest that the genomes of the parent species are very similar. Taken together with the results from artificially produced hybrids of A. pratensis × A. geniculatus , it is suggested that bivalent-promoting mechanisms reduce multivalent formation both in the species and the hybrids. The interaction of different genotypes involved in the control of meiosis may account for the variation in meiotic behaviour in the different populations of hybrids. Pollen fertility is reduced in most populations of hybrids and likely to be an important factor permitting introgressive hybridization.     

Evolution of meiotic patterns of oogenesis and spermatogenesis in centric diatoms

The evolution of meiotic patterns of the oogenesis and spermatogenesis in centric diatoms was inferred according to the parsimony principle. The pattern provisionally named type 1, in which one of two daughter nuclei becomes pycnotic, no cytokinesis occurs at meiosis II and two eggs are produced, was inferred to be the most primitive among extant meiotic patterns of oogenesis. It was also inferred that the pattern provisionally named 4‐2EC, in which equal cytokinesis occurs after each nuclear division and four functional haploid cells are produced, is the most primitive among extant meiotic patterns of spermatogenesis. The evolution of meiotic pattern suggests that bipolar or multipolar forms are primitive among centric diatoms.     

Positional control of the fates of nuclei produced after meiosis in Paramecium caudatum: Analysis by nuclear transplantation

The survival or degeneration of nuclei produced by meiosis in Paramecium caudatum depends on their position in the cytoplasm. The surviving nucleus always lies in the special region of the cytoplasm called the paroral region, which is the region around the cytostome. The remaining three degenerate outside the region. The mechanism controlling the survival or degeneration of meiotic nuclei was analyzed microsurgically. When the nucleus in the early conjugating cells (stage II) was transplanted into the cell at the stage when three meiotic nuclei were degenerating, it did not degenerate, but divided. When one of the meiotic nuclei which was outside the paroral region and destined to degenerate was transplanted into the cell in the meiotic prophase (stage IV), it did not survive but degenerated. When the surviving nucleus in the paroral region was removed microsurgically, one of the three meiotic nuclei lying outside the paroral region and destined to degenerate moved into the paroral region and survived. These results suggest that the nuclei after meiosis are destined to degenerate but can be rescued from degeneration by the special environment of the paroral region.     

Nitric oxide and meiotic competence of porcine oocytes

Reproductive biotechnology such as in vitro fertilization, the creation of transgenic animals or cloning by nuclear transfer depends on the use of fully grown, meiotically competent oocytes capable of completing meiotic maturation by reaching the stage of metaphase II. However, there exists only a limited quantity of these oocytes in the ovaries of females. In view of their limited number, growing oocytes without meiotic competence represent a possible source. The mechanisms controlling the acquisition of meiotic competence, however, are still not completely clear. A gas with a short half-life, nitric oxide (NO), produced by NO-synthase (NOS) enzyme can fulfill a regulatory role in this period. The objective of this study was to ascertain the role of NO in the growth phase of pig oocytes and its influence on the acquisition of meiotic competence with the help of NOS inhibitors, NO donors and their combinations. We demonstrated that the selective competitive iNOS inhibitor aminoguanidine and also the non-selective NOS inhibitor l-NAME block meiotic maturation of oocytes with partial or even full meiotic competence at the very beginning. NOS inhibitors influence even competent oocytes in the first stage of meiotic metaphase. However, blockage is less effective than at the beginning of meiotic maturation. The number of parthenogenetically activated competent oocytes greatly increased in a pure medium after inhibitor reversion. A large quantity of NO externally added to the in vitro cultivation environment disrupts the viability of oocytes. The effectiveness of the inhibitor can be reversed in oocytes by an NO donor in a very low concentration. However, the donor is not capable of pushing the oocytes farther than beyond the first stage of meiotic metaphase. The experiments confirmed the connection of NO with the growth period and the acquisition of meiotic competence. However, it is evident from the experiments that NO is not the only stimulus controlling the growth period.     

Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot.     

SEXUAL REPRODUCTION AND MEIOSIS IN PERIDINIUM INCONSPICUUM LEMMERMANN (DINOPHYCEAE)

In Peridinium inconspicuum Lemmermann, sexual reproduction occurs in both nitrogen-enriched and nitrogen-deficient media. In this homothallic strain, protoplasmic fusion begins between two thecate gametes; but zygote formation is completed in a space outside the fusing pair. This diploid cell can form a plated theca which is shed as the cell enlarges. This spherical zygote then forms a new non-plated theca. The process of ecdysis and the formation of a new non-plated theca is repeated several times. During this process the zygote gradually elongates and by cytoplasmic infurrowing becomes peanut-shaped. Eventually two cells are formed. The first and second meiotic divisions are greatly separated in time. The first meiotic division occurs in the spherical non-thecate zygote. The second meiotic division can occur in the peanut-shaped zygote before it completes cytokinesis. This meiotic division may not be synchronous, occasionally resulting in a trinucleate stage. Eventually four flagellated, haploid products are produced.     

Impaired meiotic competence in putative primordial germ cells produced from mouse embryonic stem cells

There are still several unanswered questions and problems about the recently claimed possibility of producing functional germ cells in vitro from pluripotent embryonic stem cells (ESCs). In the present paper, we compared by single-cell analysis the capability of putative primordial germ cells (PGCs), produced in vitro from ESCs, and that of endogenous PGCs isolated from embryos, to enter and progress through meiotic prophase I. Using a protocol previously reported to be suitable to produce female germ cells from mouse ESC monolayers, we first identified putative PGCs by analysing the expression pattern of several markers such as SSEA1, APase, OCT4, NANOG, MVH and SCP3 of pre- and post migratory PGCs. Next, after isolation of such cells from culture, we tested their meiotic capability. The evaluation at 2-5 days of culture of the number of cells showing meiotic nuclear SCP3 staining in cytospreads showed that it remained nearly constant in the putative PGCs, whereas it increased markedly in endogenous PGCs. Moreover, we observed that in putative PGCs, the nuclear distribution or expression of SCP3 and other meiotic markers such as DMC1, gH2AX and SCP1 were always highly abnormal in comparison to that observed in endogenous cultured PGCs. We conclude that although the formation of cells showing characteristics of PGCs can occur efficiently from ESCs in vitro, these cells possess impaired capability to enter and progress through meiotic prophase I.     

Localization and development of kinetochores and a chromatid core during meiosis in grasshoppers

Positive staining of kinetochores and a chromatid core has been achieved using a simplified silver staining method in squash preparations from meiotic chromosomes of grasshoppers. This technique permits the exact localization of kinetochores on the chromosomes whether metacentric, acrocentric or ‘telocentric’. The sister kinetochores can be observed from mid-diplotene stages but they are not differentiated during first meiotic metaphase. However they can be observed again at the onset of anaphase 1. The existence of a positively stained chromatid core in meiotic divisions is also reported. This core appears well defined inside each chromatid from diplotene to the end of the second meiotic division. The visualization of these cores in first meiotic metaphase clearly shows the points at which the chiasmata took place.     

Initiation of meiotic recombination by double-strand DNA breaks in S. pombe

Mitotic gene conversion and reciprocal recombination have recently been shown to be efficiently initiated by double-strand DNA breaks (DSBs) in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. We tested whether DSBs could also initiate meiotic recombination at the mat1 locus in S. pombe. The mat1 switching-mechanism-generated DSB found in mitotically growing cells can be repaired without mat1 switching, since strains deleted for both donor loci (mat2-P and mat3-M) have the break but do not produce inviable cells. A (mat1-P X mat1-M) cross produced a high frequency (20%) of 3:1 gene conversions of mat1 in meiotic tetrads. Gene conversion events were associated with the recombination of flanking markers. Strains lacking the DSB failed to convert. Thus, the DSB at mat1 promotes efficient meiotic recombination in fission yeast.     

Production and Characterization of Radiation-Sensitive Meiotic Mutants of Coprinus Cinereus

We have isolated four gamma-ray-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1; rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the few viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants.     

On the significance of tetrahedral tetrads of Precambrian algal cells

Tetrahedral tetrads of coccoid cells are known from two Precambrian localities; one example has been reported from the late Proterozoic Bitter Springs Formation and a second example, morphologically different from the first, is here described from the middle Proterozoic Amelia Dolomite. Among modern algae, sheath- or wall-enclosed tetrahedral tetrads are regularly produced only by eukaryotes, either during meiotic or mitotic reproduction. In evaluating the significance of Precambrian tetrads, consideration should be given to the following: whether the tetrad geometry is truly tetrahedral; if so, whether the tetrahedral configuration is original or artifactual; and if original, whether the tetrad was produced during meiotic or mitotic reproduction. We suggest several kinds of evidence which may assist in making these determinations. Considering the Amelia Dolomite specimen, we conclude that it most closely resembles a mitotically produced green algal autospore tetrad, although this interpretation cannot yet be confirmed because of the difficulty in ascertaining whether the tetrahedral configuration is original. Considering the Bitter Springs specimen, we conclude that the organism probably was eukaryotic but that its mode of origin (whether through meiosis or mitosis) cannot be unequivocally ascertained from data presently available.     

Growth of cells on solid culture medium

Summary For precise experiments with yeast (or other) cells stationary populations are produced by growth on the surface of a solid nutrient medium. The energy supply to these cells is well known from a former publication. The oxygen supply during growth is analysed here in detail. Different types of cell populations can be produced in this way dependent on the thickness of nutrient medium.If such cells are transferred into a liquid buffer solution cell multiplication can be initiated without any nutrient flux into the cell. This new type of initiation of the cell cycle of G₁-cells has to be distinguished from the usual initiation by nutrient supply and from the mechanism of meiotic cell division. The dependence of this cell growth on cell volume, pH-value, oxygen concentration and osmotic pressure is analysed and possibilities to avoid this kind of cell multiplication reaction are discussed.     

Maintenance of murine oocyte meiotic arrest: uptake and metabolism of hypoxanthine and adenosine by cumulus cell-enclosed and denuded oocytes

To analyze the potential mechanisms by which hypoxanthine and adenosine maintain meiotic arrest in mouse oocytes this study focused on: the uptake and metabolism of hypoxanthine and adenosine; the effect of inhibitors of inosine monophosphate (IMP) dehydrogenase on purine-mediated meiotic arrest; and the role of adenosine metabolism on the maintenance of meiotic arrest. Although the denuded oocyte can take up radiolabeled hypoxanthine and adenosine, an intact cumulus oophorus greatly augments uptake of these molecules (and/or metabolites). Both of these compounds were completely metabolized during incubation in vitro: hypoxanthine was apparently metabolized to uric acid and adenosine was metabolized to ADP; a small amount of each compound was also converted to inosine by cumulus cells and transferred to the oocyte. The IMP dehydrogenase inhibitors, bredinin and mycophenolic acid (MA), induced, in a dose-dependent manner, the resumption of maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine but had no effect on denuded oocytes. MA did not induce maturation when meiotic arrest was maintained by guanosine. Nor did MA alter the uptake of hypoxanthine by cumulus cell-enclosed oocytes. The poorly metabolized analog of adenosine, 2-chloroadenosine, was as effective as adenosine in its synergistic action with hypoxanthine in maintaining meiotic arrest. It is concluded that hypoxanthine and adenosine are metabolized within the oocyte-cumulus cell complex; xanthyl and/or guanyl compounds are produced by oocyte-cumulus cell complexes in the presence of hypoxanthine and play an important role in the maintenance of meiotic arrest; and adenosine need not be metabolized to act synergistically with hypoxanthine in maintaining meiotic arrest.     

DIVERGENCE OF MEIOTIC DRIVE-SUPPRESSION SYSTEMS AS AN EXPLANATION FOR SEX-BIASED HYBRID STERILITY AND INVIABILITY

Two empirical generalizations about speciation remain unexplained: the tendency of the heterogametic sex to be sterile or inviable in F₁ hybrids (Haldane's rule), and the tendency of the X chromosome to harbor the genetic elements that cause this sex bias in hybrid fitness. I suggest that divergence of meiotic drive systems on the sex chromosomes can explain these observations. The theory follows from two simple facts. First, sex chromosomes are particularly susceptible to the forces of meiotic drive. Second, divergence of meiotic drive systems can cause hybrid sterility and in viability. The main objection to the theory is that meiotic drive is apparently rare, whereas the observed pattern of hybrid fitness is widespread. I answer this objection by showing that divergence of meiotic drive systems can explain the two generalizations even if large departures from Mendelian segregation are rarely observed.     

Meiotic Origin of Triploidy in the Frog Detected by Genetic Analysis of Enzyme Polymorphisms

A female frog heterozygous at two unlinked loci, specifying electrophoretic forms of mannosephosphate isomerase (MPI) and malate dehydrogenase (MDH) was crossed to male frogs homozygous for different alleles at each locus. In the offspring approximately ten percent proved to be triploid according to nucleolar and chromosome counts of tail tip cells. Most of these triploids had both maternal alleles at the MDH and MPI loci suggesting that the first meiotic division was repressed. Others seemed to represent a repressed second meiotic division and one animal, a pentaploid, could only have resulted from inhibition of both meiotic divisions of the egg. Densitometer tracings of starch gels stained for 6 phosphogluconate and isocitrate dehydrogenases, expected to be heterozygous in a particular cross, demonstrated that the triploids had twice as much maternal as paternal gene product for each locus, similar to patterns found in triploids produced by nuclear transplantation.     

The origin and behavior of two isodicentric bisatellited chromosomes.

Karyotyping revealed three cell lines in a boy with mental retardation and few other abnormalities. Thirty cells exhibited a normal karyotype, and 54 had an extra acrocentric chromosome of E group size with satellites on the long and short arms. The remaining 20 cells each had, in addition to the first marker (M1), a second tiny bisatellited chromosome (M2). C-banding demonstrated that both markers were dicentric. G-, C-, and Q-banding and satellite association data were consistent with the markers having originated from chromosome 15 material. We propose that M1 was formed from a meiotic breakage and a chromatid fusion in the proximal long arms of an acrocentric pair. This would have produced a symmetrical isodicentric chromosomes, plus one or two acentric fragments. M2 then could have resulted from a dicentric bridge-break-synthesis-reunion phenomenon. This model of abnormal meiotic exchange can be generalized to encompass the formation of other dicentric isochromosome cases of isochromosome X.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Meiotic arrest and aneuploidy induced by vinblastine in mouse oocytes

Young superovulated female mice were injected i.p. with single doses of vinblastine sulfate just before the onset of the first meiotic division. Secondary oocytes, fixed one by one on a slide, were cytogenetically scored. Evidence of the meiotic arresting activity of vinblastine was produced by the observation of increasing frequencies of M1-arrested oocytes and by the presence of undegenerated chromosome sets of first polar bodies. When the first meiotic division could be undertaken chromosome malsegregation occurred with high frequency, both in terms of aneuploidy and polyploidy. M1-blocked and polyploid oocytes have been interpreted as the consequence of irreversible damage to the spindle induced by vinblastine through its binding on tubulin low-affinity sites; this reaction, in fact, causes microtubule crystallization. According to this mechanism, dose-effect relationships of both phenomena show a threshold at 0.45 mg/kg. On the other hand, the incidence of aneuploid oocytes is correlated with meiotic delay, as detected by the delayed degeneration of polar bodies, and increases linearly with dose. Both phenomena are, therefore, stochastic and can be referred to the binding of the chemical on tubulin high-affinity sites, which is known to cause tubulin depolymerization in a colchicine-like way.     

Oocyte-specific expression of Gpr3 is required for the maintenance of meiotic arrest in mouse oocytes

The maintenance of meiotic prophase arrest in mouse oocytes within fully grown follicles, prior to the surge of luteinizing hormone (LH) that triggers meiotic resumption, depends on a high level of cAMP within the oocyte. cAMP is produced within the oocyte, at least in large part, by the G(s)-linked G-protein-coupled receptor, GPR3. Gpr3 is localized in the mouse oocyte but is also present throughout the follicle. To investigate whether Gpr3 in the follicle cells contributes to the maintenance of meiotic arrest, RNA interference (RNAi) was used to reduce the amount of Gpr3 RNA within follicle-enclosed oocytes. Follicle-enclosed oocytes injected with small interfering double-stranded RNA (siRNA) targeting Gpr3, but not control siRNAs, stimulated the resumption of meiosis in the majority of oocytes following a 3-day culture period. Reduction of RNA was specific for Gpr3 because an unrelated gene was not reduced by microinjection of siRNA. Meiotic resumption was stimulated in isolated oocytes injected with the same siRNA and cultured for 1 to 2 days, but at a much lower rate than in follicle-enclosed oocytes that could be cultured for longer. These results demonstrate that GPR3 specifically in the oocyte, rather than in the follicle cells, is responsible for maintenance of meiotic arrest in mouse oocytes. Furthermore, the method developed here for specifically reducing RNA in follicle-enclosed oocytes, which can be cultured for a sufficient time to reduce the level of endogenous protein, should be generally useful for targeting a wide range of other proteins that may be involved in meiotic arrest, the resumption of meiosis, fertilization, or early embryonic development.