Meiosis-like reduction during somatic embryogenesis of Arabidopsis thaliana

Summary Somatic meiosis-like reduction was observed in some cells of the embryogenic callus of Arabidopsis thaliana. Two types were identified. One type was somatic chromosome reductional grouping, in wich the chromesomes in a cell were separated direetly at either prophase or metaphase. Chromosome reductional grouping happened more frequently in polyploid cells, and the morphology of the chromosomes did not show the role of the spindle fibers. The other type was somatic meiosis which was analogous to the process of gametogenesis, characterized by the pairing and synapsis of homologous chromosomes. The roles of somatic meiosis-like reduction in somatic embryogenesis and somaclonal variations are discussed     

拟南芥（Arabidopsis thaliana）体细胞胚胎发生体系中的体细胞类减数分裂研究

在拟南芥生态型LandsbergErecta体细胞胚胎发生体系的胚性愈伤组织中观察到2种类型的体细胞减数分裂现象。一种是体细胞染色体减数分组,其中,处于前期或中期的细胞染色体分为2个或2个以上的组。其共同特点是,染色体直接分开,未观察到纺锤体,从染色体的形态也看不出纺锤体的作用。染色体减数分组较多发生于多倍体细胞中。另一种类型是体细胞减数分裂,这种类型类似于大小孢子发生过程的减数分裂,如第一次分裂前期也有染色体的联会和配对。在脱分化培养基上的胚性愈伤组织中,单倍体细胞约占3%,四倍体细胞约占4%。经体细胞类减数分裂产生的细胞都发生染色体重组。     

Nuclear and microtubule dynamics of G2/M somatic nuclei during haploidization in germinal vesicle-stage mouse oocytes

During the haploidization process, it is expected that diploid chromosomes of somatic cells will be reduced to haploid for the generation of artificial gametes. In the present study, we aimed to use enucleated mouse oocytes at the germinal vesicle-stage (G2/M) as recipients for somatic cells that are also synchronized to the G2/M stage for haploidization. The reconstructed oocytes were then induced to undergo meiosis in vitro and observed for their nuclear morphology and microtubule network formation at various expected stages of the meiotic division. Following in vitro maturation, more than half (62/119, 52.1%) of the reconstructed oocytes completed the first round of meiosis-like division, as evidenced by the extrusion of pseudopolar bodies (PBs). However, accelerated PB extrusion, approximately 3-4 h earlier than that by control oocytes occurred. Furthermore, abnormally large pseudo-PBs, as large as four times the normal PB sizes, were observed. During the process of in vitro maturation at both the expected stages of metaphase I (MI) and metaphase II (MII), condensed chromosomes were observed in 38.7% and 55.2% of oocytes, respectively. However, two other types of nuclear configurations were also observed: 1) uneven distribution of chromatin and 2) an interphase-like nucleus, indicating deficiencies in chromosome condensation. Following oocyte activation, more than half (21/33, 63.6%) of the reconstructed oocytes with pseudo-PBs formed separated pseudopronuclei (PN), suggesting formation of functional spindles. The formation of bipolar spindle-like microtubule network at both the expected MI and MII stages during in vitro maturation was confirmed by immunohistochemistry. In summary, this study demonstrated that a high proportion of G2/M somatic nuclei appear to undergo meiosis-like division, in two successive steps, forming a pseudo-PB and two separate pseudo-PN upon in vitro maturation and activation treatment. Moreover, the enucleated geminal vesicle cytoplast retained its capacity for meiotic division following the introduction of a somatic G2/M nucleus.     

Caffeine interactions with methyl methanesulphonate, hycanthone, benlate, and cadmium chloride in chromosomal meiotic segregation of Saccharomyces cerevisiae

Interactions of caffeine with chemicals known for their effects on chromosomal segregation during meiosis of Saccharomyces cerevisiae were studied. It appears that caffeine does interfere with the action of other compounds during the different phases of meiosis. Treatments with methyl methanesulphonate (MMS) and cadmium chloride (CdCl2) resulted in a synergistic effect consisting of an increase in the frequency of recombination. The greatest effects were found on the induction of diploid spores: MMS, hycanthone, and distamycin demonstrated strong, benlate little synergistic action. CdCl2 demonstrated antagonism to caffeine by counter-inhibiting its effect on the induction of diploids. Concerning disomic induction: caffeine reduced (or left unchanged) the effect on non-disjunction when MMS and hycanthone were used. Simple additive effects were caused in conjunction with distamycin, benlate, and (in small doses) CdCl2. 2 mg of caffeine/ml in treatments with CdCl2 resulted in a very high frequency of disomic clones.     

Effects of caffeine treatment on aged porcine oocytes: parthenogenetic activation ability, chromosome condensation and development to the blastocyst stage after somatic cell nuclear transfer

The possibility of using aged porcine oocytes treated with caffeine, which inhibits the decrease in M-phase promoting factor activity, for pig cloning was evaluated. Cumulus-oocyte complexes (COCs) were cultured initially for 36 h and subsequently with or without 5 mM caffeine for 24 h (in total for 60 h: 60CA+ or 60CA- group, respectively). As a control group, COCs were cultured for 48 h without caffeine (48CA-). The pronuclear formation rates at 10 h after electrical stimulation in the 60CA+ and 60CA- groups decreased significantly (p < 0.05) compared with the 48CA- group. However, the fragmentation rate was significantly higher (p < 0.05) in the 60CA- group than in the 60CA+ and 48CA- groups. When the stimulated oocytes were cultured for 6 days, the 60CA+ group showed significantly lower blastocyst formation and higher fragmentation or degeneration rates (p < 0.05) than the 48CA- group. However, the number of total cells in blastocysts was not affected by maturation period or caffeine treatment. When somatic cell nuclei were injected into the non-enucleated oocytes and exposed to cytoplasm for a certain duration (1-11 h) before the completion of maturation (48 or 60 h), the rate of nuclear membrane breakdown after exposure to cytoplasm for 1-2 h in the 60CA- oocytes was significantly lower (p < 0.05) than in the other experimental groups. The rate of scattered chromosome formation in the same 60CA- group tended to be lower (p = 0.08) than in the other groups. After the enucleation and transfer of nuclei, blastocyst formation rates in the 60CA+ and 60CA- groups were significantly lower (p < 0.05) than in the 48CA- group. Blastocyst quality did not differ among all the groups. These results suggest that chromosome decondensation of the transplanted somatic nucleus is affected by both the duration of exposure to cytoplasm and the age of the recipient porcine oocytes, and that caffeine treatment promotes nuclear remodelling but does not prevent the decrease in the developmental ability of cloned embryos caused by oocyte aging.     

The effect of caffeine on repair in Chlamydomonas reinhardtii: I. Enhancement of recombination repair

The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS⁺) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS⁺, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS⁺ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.     

Association of different zinc concentrations combined with a fixed caffeine dose on plasma and tissue caffeine and zinc levels in the rat

Because caffeine and tissue levels of Zn are closely related, the objectives of this study were to determine the changes in plasma caffeine levels over a period of 5 h when different concentrations of Zn combined with a fixed concentration of caffeine were injected into the femoral vein of rats and to determine the relationship between tissue levels of caffeine and Zn at 5 h postinjection. Rats were divided into three groups: group 1, 220 μg caffeine; group 2,220 μg caffeine + 8 μg Zn/g body weight (BW); group 3, 220 μg caffeine +16 μg Zn/g BW. Blood from groups 1 and 3 was collected at 3 min, 30 min, 1h, 3h, and 5h to determine the pharmacokinetics of caffeine. All groups were killed at 5 h. Caffeine and Zn concentrations of the brain, kidney, heart, and liver of all groups were determined. The plasma-caffeine curve in group 3 showed a lower concentration at 3 min and a slower caffeine-elimination rate during the first 3 h. Brain and kidney caffeine levels remained constant in all groups, whereas caffeine levels were increased in the heart in group 2 and in the liver in group 3. Zn concentrations in the brain and kidney were lower in group 2 compared with groups 1 and 3 and higher in group 3 compared to groups 1 and 2. Zn concentration in the heart was the same among the three groups but was increased in the liver in group 3 compared to groups 1 and 2. Therefore, we concluded that caffeine combined with Zn affects caffeine pharmacokinetics. With caffeine intake, levels of Zn (16 μg/g BW) that are slightly higher than the daily requirements (12 μg/g BW) may prevent a reduction of Zn in tissue. In addition, caffeine’s effects on Zn concentration among organs are different.     

Reduction of ultraviolet-induced mitotic delay by caffeine in G2-phase irradiated plasmodia of Physarum polycephalum

Synchronously mitotic surface Plasmodia ofPhysarum polycephalum were ultra-violet-irradiated at different times during G2-phase (—4 h to —20 min with respect to metaphase), and treated immediately thereafter with varying concentrations of caffeine. It was observed that ultraviolet-induced mitotic delay is reduced significantly by this methylxanthine. In plasmodia irradiated between —4 and —1 h with respect to metaphase, the effect was concentration-dependent and the need for a certain threshold dose for obtaining the reduction in delay was apparent. However, higher doses than this were fairly toxic when applied at this part of the cycle and led to more mitotic delay than that obtained with UV alone. The most striking observation made during this study was the phase-specific precipitous effect seen in those plasmodia irradiated at about 20 min before mitosis which almost eliminated the long delay due to ultraviolet-irradiation. These results are discussed in the context of some of the known effects of ultraviolet and caffeine on a mitosis-promoting factor. It is proposed that the significant reduction of ultraviolet-induced mitotic delay reported here is due to the reactivation of the ultraviolet-inactivated mitosis-promoting factor by caffeine. Alternatively, it is possible that caffeine may prevent the inactivation of this factor by ultraviolet.     

Evidence for non-disomic inheritance in a Citrus interspecific tetraploid somatic hybrid between C. reticulata and C. limon using SSR markers and cytogenetic analysis

Artificial tetraploid somatic hybrids have been developed for sterile triploid citrus breeding by sexual hybridization between diploid and tetraploid somatic hybrids. The genetic structure of diploid gametes produced by tetraploid genotypes depends on the mode of chromosome association at meiosis. In order to evaluate tetraploid inheritance in a tetraploid interspecific somatic hybrid between mandarin and lemon, we performed segregation studies using cytogenetic and single sequence repeat molecular markers. Cytogenetic analysis of meiosis in the somatic hybrid revealed 11% tetravalents and 76% bivalents. Inheritance of the tetraploid hybrid was analyzed by genotyping the triploid progeny derived from a cross between a diploid pummelo and the tetraploid somatic hybrid, in order to derive genotypes of the meiospores produced by the tetraploid. A likelihood-based approach was used to distinguish between disomic, tetrasomic, and intermediate inheritance models and to estimate the double reduction rate. In agreement with expectations based the cytogenetic data, marker segregation was largely compatible with tetrasomic and inheritance intermediate between disomic and tetrasomic, with some evidence for preferential pairing of homoeologous chromosomes. This has important implications for the design of breeding programs that involve tetraploid hybrids, and underscores the need to consider inheritance models that are intermediate between disomic and tetrasomic.     

金花茶花药愈伤组织的体细胞减数分裂

金花茶的小孢子单核期花药,经培养的继代培养６个月后的愈伤组织中,发现有少量体细胞进行减数分裂。在减数第一和第二次分裂中,同源染色体的配对和分离基本正常,最后形成四分体。该愈伤组织经石革切片和压片观察,发现其主要由大量的液化细胞和贮藏细胞所组成,此外,还有少部分分生细胞。没有发现进一步的分化。其染色体数目,２ｎ＝３０者占７１．７％,其余则为非整倍体。     

Meiotic pairing constraints and the activity of sex chromosomes

The state of activity and condensation of the sex chromosomes in gametocytes is frequently different from that found in somatic cells. For example, whereas the X chromosomes of XY males are euchromatic and active in somatic cells, they are usually condensed and inactive at the onset of meiosis; in the somatic cells of female mammals, one X chromosome is heterochromatic and inactive, but both X chromosomes are euchromatic and active early in meiosis. In species in which the female is the heterogametic sex (ZZ males and ZW females), the W chromosome, which is often seen as a condensed chromatin body in somatic cells, becomes euchromatic in early oocytes. We describe an hypothesis which can explain these changes in the activity and condensation of sex chromosomes in gametocytes. It is based on the fact that normal chromosome pairing seems to be essential for the survival of sex cells; chromosomal anomalies resulting in incomplete pairing during meiosis usually result in gametogenic loss. We argue that the changes seen in the sex chromosomes reflect the need to avoid pairing failure during meiosis. Pairing normally requires structural and conformational homology of the two chromosomes, but when the regions is avoided when these regions become heterochromatinized. This hypothesis provides an explanation for the changes found in gametocytes both in species with male heterogamety and those with female heterogamety. It also suggests possible reasons for the frequent origin of large supernumerary chromosomes from sex chromosomes, and for the reported lack of dosage compensation in species with female heterogamety.     

Changes in intracellular cAMP level and activities of adenylcyclase and phosphodiesterase during meiosis of lily microsporocytes.

In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.     

Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture media from 24 hour cultures of minced adult mouse testes. In the bioassay one gonad of each fetus is cultured either in MIS medium, in control medium with forskolin, or in MIS medium with forskolin. The other gonad serves as the control and is cultured in control medium. After culture the gonads are fixed, squashed, and DNA-stained. In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis. MIS activity is defined to be present in a medium when meiosis is induced in male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media. These findings indicate that receptors for stimuli of meiotic initiation may exist in germ cells or neighbouring somatic cells. In addition to induction of meiosis, MIS media and forskolin also dose dependently increase the number of male germ cells compared to controls. This increase is correlated with induction of advanced stages of meiosis: Male germ cells seem to survive better if they are triggered to enter meiosis. Neither MIS media nor forskolin affected the growth of somatic cells. We therefore propose that MIS media has a growth factor activity with a specific effect on meiotic initiation. © 1993 Wiley-Liss, Inc.     

A screen for genes required for meiosis and spore formation based on whole-genome expression

BACKGROUND: Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS: To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS: Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.     

Luteinizing hormone causes MAP kinase-dependent phosphorylation and closure of connexin 43 gap junctions in mouse ovarian follicles: one of two paths to meiotic resumption

Luteinizing hormone (LH) acts on ovarian follicles to reinitiate meiosis in prophase-arrested mammalian oocytes, and this has been proposed to occur by interruption of a meioisis-inhibitory signal that is transmitted through gap junctions into the oocyte from the somatic cells that surround it. To investigate this idea, we microinjected fluorescent tracers into live antral follicle-enclosed mouse oocytes, and we demonstrate for the first time that LH causes a decrease in the gap junction permeability between the somatic cells, prior to nuclear envelope breakdown (NEBD). The decreased permeability results from the MAP kinase-dependent phosphorylation of connexin 43 on serines 255, 262 and 279/282. We then tested whether the inhibition of gap junction communication was sufficient and necessary for the reinitiation of meiosis. Inhibitors that reduced gap junction permeability caused NEBD, but an inhibitor of MAP kinase activation that blocked gap junction closure in response to LH did not prevent NEBD. Thus, both MAP kinase-dependent gap junction closure and another redundant pathway function in parallel to ensure that meiosis resumes in response to LH.