DNA topoisomerase II-dependent control of the cell cycle progression in root meristems of Allium cepa.

The catalytic ability of DNA topoisomerases (Topo) to generate short-term DNA breaks allow these enzymes to play crucial functions in managing DNA topology during S-phase replication, transcription, and chromatin-remodelling processes required to achieve commitment for the onset and transition through mitosis. Our experiments on root meristem cells of onion (Allium cepa) were designed to gain insight into the contribution of Topo II to plant-specific progression throughout interphase and mitosis. Irrespective of the position of the cell in interphase, the immunofluorescence of Topo II revealed similar nuclear labelling pattern with well defined signals dispersed in the nucleoplasm and the cortical zone of the nucleolus. Only weak labelling was detected in metaphase and anaphase chromosomes. Experiments with two potent anti-Topo II agents, doxorubicin (DOX, an anthracycline) and a bisdioxopiperazine derivative, ICRF-193, suggest that the inhibition-mediated increase in Topo II immunofluorescence may represent a compensatory mechanism, by which an up-regulated expression of the enzyme tends to counteract the drug-induced loss of indispensable catalytic and relaxation functions. γ-H2AX immunolabelling seems to indicate that both DOX- and ICRF-193-induced alterations in cell cycle progression reflect primarily the activity of the G2/M DNA damage checkpoint. Our findings provide evidence for the plant-specific cell cycle control mechanism induced by Topo II inhibitors under DNA stress conditions.

Calmodulin-dependent protein kinase gamma 3 (CamKIIgamma3) mediates the cell cycle resumption of metaphase II eggs in mouse.

Mature mammalian eggs are ovulated arrested at meiotic metaphase II. Sperm break this arrest by an oscillatory Ca(2+) signal that is necessary and sufficient for the two immediate events of egg activation: cell cycle resumption and cortical granule release. Previous work has suggested that cell cycle resumption, but not cortical granule release, is mediated by calmodulin-dependent protein kinase II (CamKII). Here we find that mouse eggs contain detectable levels of only one CamKII isoform, gamma 3. Antisense morpholino knockdown of CamKIIgamma3 during oocyte maturation produces metaphase II eggs that are insensitive to parthenogenetic activation by Ca(2+) stimulation and insemination. The effect is specific to this morpholino, as a 5-base-mismatch morpholino is without effect, and is rescued by CamKIIgamma3 or constitutively active CamKII cRNAs. Although CamKII-morpholino-treated eggs fail to exit metaphase II arrest, cortical granule exocytosis is not blocked. Therefore, CamKIIgamma3 plays a necessary and sufficient role in transducing the oscillatory Ca(2+) signal into cell cycle resumption, but not into cortical granule release.

Oxidative stress and tumor necrosis factor-alpha-induced alterations in metaphase II mouse oocyte spindle structure.

OBJECTIVE(S): To examine the effect of exogenous exposure to hydrogen peroxide (H(2)O(2)) and tumor necrosis factor (TNF)-alpha on mouse metaphase II (MII) oocyte spindle structure and to examine the potential benefits of supplementing the culture media with vitamin C. DESIGN: Prospective study. SETTING: Research laboratory in a tertiary hospital. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microtubule changes and alterations in chromosomal alignment. RESULT(S): Both concentration- and time-dependent alterations were seen in spindle structure after exposure to H(2)O(2). An H(2)O(2) concentration as low as 12.5 microM increased the odds of an oocyte with altered microtubule and chromosome alignment (score >or=3) by 93%. Significantly increased damage was seen with increasing period of incubation. Higher scores were seen after exposure to both TNF-alpha alone and in combination with H(2)O(2) compared with controls. Changes in chromosomal alignment were comparable among the three groups. Oocytes coincubated with H(2)O(2) and vitamin C at 200 microM demonstrated less damage compared with those with H(2)O(2) alone. CONCLUSION(S): Oxidative stress results in concentration and time-dependent alterations in the spindle structure and augments the effects induced by TNF-alpha. Proper oocyte handling in vitro may help reduce oxidative insult, thus improving the oocyte quality. Antioxidants may have a protective effect and need to be further evaluated.

Low concentrations of MEM vitamins during in vitro maturation of porcine oocytes improves subsequent parthenogenetic development.

To investigate the effects of water-soluble vitamin supplementation for IVM/IVC of porcine oocytes and evaluate maturation and developmental capacity in vitro, porcine cumulus oocyte complexes (COCs) was matured in NCSU-23-based medium with water-soluble vitamins for 44 h and then cultured in PZM-3 for 7 days following activation. The COCs were allocated into five treatment groups and matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, 0.4, and 1x). Metaphase II plates of the cumulus-free oocytes were observed following Hoechest 33258 staining. The COCs were allocated into four treatment groups, matured in various concentrations of MEM vitamins (control, 0.05, 0.1, 0.2, and 0.4x) and cultured in PZM-3 following activation. Also, COCS were matured without MEM vitamins and cultured in PZM-3 with various concentrations (control, 0.1, 0.4, 1.0, and 2.0 x) of MEM vitamins. Furthermore, 2 x 2 factorial (IVM/IVC) experiments were performed in IVM medium with or without 0.05 x MEM vitamins and IVC medium with or without 0.4x MEM vitamins to examine the in vitro development of parthenogenetic embryos. Maturation rates of COCs treated with MEM vitamins did not differ significantly among groups. However, compared to the control group, oocytes matured with the addition of 0.05 x MEM vitamins developed to blastocysts at a higher percentage (P<0.05) following activation and culture in PZM-3 without MEM vitamins. Total cell number of blastocysts was significantly higher in the 0.05 x group. Addition of 0.4x MEM vitamins decreased (P<0.05) cleavage and blastocyst developmental rates compared with 0.05 x MEM vitamins-treated group. In contrast, addition of vitamins to PZM-3 medium for in vitro culture of activated porcine oocytes did not affect development. In conclusion, addition of a low concentration of MEM vitamins to IVM medium for porcine oocytes enhanced subsequent development and improved embryo quality.

A male-sterile mutation in soybean (Glycine max) affecting chromosome arrangement in metaphase plate and cytokinesis

A spontaneous male-sterile, female-fertile mutation affecting bivalent arrangement at the metaphase plate and cytokinesis was detected in line BR98-197 of the soybean breeding program developed by Embrapa - National Soybean Research Centre. Untill diakinesis, meiosis was normal with chromosome pairing as bivalents. From this phase, in several meiocytes, bivalents were not able to organize a single metaphase plate and remained scattered in the cytoplasm in a few or several groups. In these meiocytes, chromosomes segregated in both divisions giving rise to several micronuc1ei. However, the main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four nucleate coenocytic microspores were formed. In the mutant, pollen mitoses did not occur, and after engorgement by starch, pollen underwent a progressive process of degeneration

A male-sterile mutation in soybean (Glycine max) affecting chromosome arrangement in metaphase plate and cytokinesis

A spontaneous male-sterile, female-fertile mutation affecting bivalent arrangement at the metaphase plate and cytokinesis was detected in line BR98-197 of the soybean breeding program developed by Embrapa - National Soybean Research Centre. Untill diakinesis, meiosis was normal with chromosome pairing as bivalents. From this phase, in several meiocytes, bivalents were not able to organize a single metaphase plate and remained scattered in the cytoplasm in a few or several groups. In these meiocytes, chromosomes segregated in both divisions giving rise to several micronuc1ei. However, the main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four nucleate coenocytic microspores were formed. In the mutant, pollen mitoses did not occur, and after engorgement by starch, pollen underwent a progressive process of degeneration

Nuclear dispositions of subtelomeric and pericentromeric chromosomal domains during meiosis in asynaptic mutants of rye (Secale cereale L.).

The nuclear dispositions of subtelomeric and pericentromeric domains in pollen mother cells (PMCs) were tracked during meiosis in wildtype and two asynaptic mutants of rye (Secale cereale L.) by means of fluorescence in situ hybridization (FISH). Homozygotes for sy1 and sy9 non-allelic mutations form axial elements during leptotene of male meiosis, but fail to form synaptonemal complexes. Consequently, recombination is severely impaired, and high univalency is observed at metaphase I. Simultaneous FISH with pSc200 subtelomeric tandem repeat and CCS1 centromeric sequence revealed that at pre-meiotic interphase the two domains are in a bipolar Rabl orientation in both the PMCs and tapetal cells. At the onset of meiotic prophase, the subtelomeric regions in PMCs of wildtype and sy9 cluster into a typical bouquet conformation. The timing of this event in rye is comparable with that in wheat, and is earlier than that observed in other organisms, such as maize, yeast and mammals. This arrangement is retained until later in leptotene and zygotene when the pericentromeric domains disperse and the subtelomeric clusters fragment. The mutant phenotype of sy9 manifests itself during leptotene to zygotene, when the pericentromeric regions become distinctly more distended than in wildtype, and largely fail to pair during zygotene. This indicates that difference in the nature or timing of chromosome condensation in this region is the cause or consequence of asynapsis. By contrast, sy1 fails to form comparable aggregates of subtelomeric regions at leptotene in only half of the nuclei studied. Instead, two to five aggregates are formed that fail to disperse at later stages of meiotic prophase. In addition, the pericentromeric regions disperse prematurely at leptotene and do not associate in pairs at any subsequent stage. It is supposed that the sy1 mutation could disrupt the nuclear disposition of centromeres and telomeres at the end of pre-meiotic interphase, which could cause, or contribute to, its asynaptic phenotype.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes.

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.

Mph1, a member of the Mps1-like family of dual specificity protein kinases, is required for the spindle checkpoint in S. pombe.

The spindle assembly checkpoint pathway is not essential for normal mitosis but ensures accurate nuclear division by blocking the metaphase to anaphase transition in response to a defective spindle. Here, we report the isolation of a new spindle checkpoint gene, mph1 (Mps1p-like pombe homolog), in the fission yeast Schizosaccharomyces pombe, that is required for checkpoint activation in response to spindle defects. mph1 functions upstream of mad2, a previously characterized component of the spindle checkpoint. Overexpression of mph1, like overexpression of mad2, mimics activation of the checkpoint and imposes a metaphase arrest. mph1 protein shares sequence similarity with Mps1p, a dual specificity kinase that functions in the spindle checkpoint of the budding yeast Saccharomyces cerevisiae. Complementation analysis demonstrates that mph1 and Mps1p are functionally related. They differ in that Mps1p, but not mph1, has an additional essential role in spindle pole body duplication. We propose that mph1 is the MPS1 equivalent in the spindle checkpoint pathway but not in the SPB duplication pathway. Overexpression of mad2 does not require mph1 to impose a metaphase arrest, which indicates a mechanism of spindle checkpoint activation other than mph1/Mps1p kinase-dependent phosphorylation. In the same screen which led to the isolation of mad2 and mph1, we also isolated dph1, a cDNA that encodes a protein 46% identical to an S. cerevisiae SPB duplication protein, Dsk2p. Our initial characterization indicates that S.p. dph1 and S.c. DSK2 are functionally similar. Together these results suggest that the budding and fission yeasts share common elements for SPB duplication, despite differences in SPB structure and the timing of SPB duplication relative to mitotic entry.

Characterization of two structurally related Xenopus laevis protein tyrosine phosphatases with homology to lipid-binding proteins.

We have chosen Xenopus laevis as a model system to study how protein tyrosine phosphatases (PTPases) function in growth and development. As an initial step, we have previously isolated in a polymerase chain reaction (PCR)-based protocol cDNA fragments which correspond to sequences within the catalytic domains of PTPases (Yang, Q., and Tonks, N. K. (1993) Adv. Protein Phosphatases 7, 359-372). Two of these PCR products, designated X1 and X10, have now been used to screen a X. laevis ovary cDNA library to obtain complete coding sequences for two distinct PTPases. The X1 cDNA encodes a protein (PTPX1) of 693 amino acids (approximately 79 kDa); the X10 cDNA encodes a protein of 597 amino acids (approximately 69 kDa). Both PTPX1 and PTPX10 lack potential membrane spanning sequences and therefore can be classified as non-transmembrane/cytoplasmic PTPases. While the overall structure of these PTPases are similar, sharing segments of 95% amino acid identity, they differ in that PTPX1 contains a unique 97-amino acid insert between the N-terminal segment and C-terminal catalytic domain. The absence of complete identity between PTPX1 and PTPX10 suggests that these two sequences are the products of separate genes and not the result of alternative splicing. This conclusion is confirmed by PCR analysis of Xenopus genomic DNA. Both PTPases share sequence identities in their N-terminal segments with two lipid-binding proteins, cellular retinaldehyde-binding protein and SEC14p, a phospholipid transferase. In addition, the unique insert sequence of PTPX1 shares identity with PSSA, a protein involved in phosphatidylserine biosynthesis. Sequence comparison suggests that PTPX10 is the Xenopus homolog of the human PTPase Meg-02, while PTPX1 is a structurally related yet distinct PTPase. Intrinsic PTPase activity of PTPX1 and PTPX10 was demonstrated in lysates of Sf9 cells infected with recombinant baculoviruses encoding either enzyme. PTPX1 can be recovered in both soluble and membrane fractions from Xenopus oocytes with the membrane form exhibiting approximately 4-fold higher activity than the soluble form.

Maturation-promoting factor (MPF) is responsible for the transformation of sperm nuclei to metaphase chromosomes in maturing bovine oocytes in vitro.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett and Oliphant's medium in the presence of BSA (10 mg ml-1), caffeine (5 mmol l-1) and heparin (10 micrograms ml-1). When oocytes were transferred into tissue culture medium (TCM)-199 containing 10% fetal calf serum (FCS) with or without 6-dimethylaminopurine (6-DMAP; 2 mmol l-1) 8 h after insemination and cultured for 15-40 h at 39 degrees C in 5% CO2 in air, 74-83% of oocytes were penetrated and polyspermy (67-80%) was common. At 40 h after culture in 6-DMAP-free medium, 65% and 63% of unpenetrated and penetrated oocytes, respectively, reached metaphase II or beyond. A few (6%) oocytes were activated and contained both male and female pronuclei. Sperm metaphase chromosomes were observed in 90% of the penetrated oocytes. Penetration by more than four spermatozoa greatly retarded the meiotic maturation of the oocyte. However, sperm chromosomes were never observed in oocytes cultured in 6-DMAP supplemented medium and oocyte maturation did not proceed beyond the stage of prometaphase I. These results demonstrate the possible participation of maturation-promoting factor in metaphase chromosome formation in spermatozoa.

Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes.

The effect of hyperthermia on mammalian oocyte maturation was studied by allowing preovulatory mouse oocytes to mature spontaneously for 17 h in vitro under controlled temperature conditions. At the end of culture, oocytes were screened for their maturation stage and for chromosome morphology and number. Mild hyperthermic conditions (38.5-40.0 degrees C) during maturation specifically disturbed the process of bivalent chromosome disjunction, but not other maturation steps, by blocking oocytes at the metaphase I stage and preventing cells from entering subsequent maturation steps. Some oocytes that had reached metaphase II under hyperthermic conditions had chromosome imbalance. Oocytes matured at 40.0 degrees C displayed chromosome morphological abnormalities, including altered sister chromatid separation and nucleus/nuclei formation, at a frequency significantly higher than oocytes matured at 37.0-39.0 degrees C. When incubation temperature was raised above 40.0 degrees C, increasing fractions of oocytes were inhibited from entering initial maturation steps. We conclude that hyperthermia during mammalian oocyte maturation specifically damages the process of bivalent chromosome disjunction and induces the appearance of chromosome structural defects and imbalance in unfertilized eggs.

Effects of selenium deficiency on sperm morphology and spermatocyte chromosomes in mice.

Sperm morphology and spermatocyte chromosomes were examined in mice maintained on a Torula yeast diet for 5 weeks. In the selenium-deficient group, the proportion of abnormal sperm was high, ranging from 6.8% to 49.6%, while in the control group it ranged from only 4.0% to 15.0%. The most frequently occurring abnormalities in sperm shape were in the sperm head. There was also a tendency for abnormalities in other regions (neck, midpiece and tail) to be increased. However, in metaphase-I spermatocytes, the frequencies of various types of abnormal chromosomes (univalent chromosomes, translocations and structural anomalies) did not differ between the selenium-deficient and control groups. These findings indicate that selenium day be an essential constituent for spermatogenesis in mice.