Assessment of a new in vitro maturation system for mouse and human cumulus-enclosed oocytes: three-dimensional prematuration culture in the presence of a phosphodiesterase 3-inhibitor.

BACKGROUND: Controlling nuclear maturation during oocyte culture might improve nuclear-cytoplasmic maturation synchrony. In the present study, the quality of mouse and human cumulus-enclosed oocytes (CEOs) was examined after a two-step culture consisting of a three-dimensional prematuration culture (3D-PMC), followed by in vitro maturation (IVM). METHODS: Mouse and human CEOs were embedded in an extracellular matrix (collagen-gel Type I). The gels containing the CEOs were cultured in medium with a phosphodiesterase 3-inhibitor (PDE3-I; cilostamide 1 microM) for 24 h. Afterwards, CEOs were removed from the gel and washed away from inhibitor then underwent IVM. The optimal concentration of collagen (diluted 1:2 versus not-diluted) was first determined in the mouse model. Cytoplasmic maturation after IVM of human and mouse oocytes was assessed in relation to fertilization and embryonic developmental capacity. RESULTS: The diluted form of collagen was better for supporting the structure of the expanding CEOs and meiotic competence of the oocytes. Electron microscopy in combination with Lucifer Yellow dye coupling assay revealed that oocyte-cumulus cell connections could be preserved during 3D-PMC. Percentages of mouse 2-cell embryos after IVF were higher in the 3D-PMC group compared with in vitro controls and 2D-PMC oocytes, but lower compared with in vivo controls. In the human model, percentages of polar body-extruded oocytes were significantly higher in the 3D-PMC group compared with conventionally matured oocytes. The 3D-PMC also had a beneficial effect on embryonic development on Day 3 post-ICSI. CONCLUSIONS: Applying a 3D-PMC in the presence of a PDE3-I preserves oocyte-cumulus cell connections and influences oocyte developmental capacity.

Characterisation of copia-like retrotransposons in oil palm ( Elaeis guineensis Jacq.).

The work aimed to isolate and characterise copia-like sequences from Elaeis guineensis. Thirty-two different RT (reverse transcriptase) sequences were isolated from a single oil palm genome using degenerate primers. Extreme sequence heterogeneity was observed. The DNA and protein sequences were assigned to three different classes (A, B, C) on the basis of bootstrapping. We estimated the copy number of the three different classes by using a dot-blot analysis. The comparative results suggest that class-B RT sequences occur at a higher copy number in Cocos nucifera than in Elaeis guineensis and E. oleifera. Class-C RT sequences, which comprise the bulk of isolated sequences, occur in much higher copy number in Elaeis guineensis than in Cocos nucifera, and E. oleifera. Class-A sequences, which have low copy number in Elaeis guineensis and Cocos nucifera, are not present in E. oleifera. Our preliminary results suggest that class-C sequences represent about 5% of the E. guineensis genome, class B, 1% and class A, 0.1%. The methylation status of genomic domains specified by the RT probes was analysed using two pairs of restriction enzymes, each pair having the same recognition sites but different methylation sensitivities ( MspI, HpaII and Sau3AI, MboI). Results from these experiments showed clearly that the genomic domains specified by the RT probes are methylated. These also suggest that a higher copy number appears to correlate to a higher degree of methylation. Our preliminary results suggest that copia-like retrotransposons, because of their ubiquity and diversity, have great potential as genetic markers for plant genome and biodiversity analysis in E. guineensis.

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast.

Yeast cells growing in the presence of glucose or a related rapidly-fermented sugar differ strongly in a variety of physiological properties compared to cells growing in the absence of glucose. Part of these differences appear to be caused by the protein kinase A (PKA) and related signal transduction pathways. Addition of glucose to cells previously deprived of glucose triggers cAMP accumulation, which is apparently mediated by the Gpr1-Gpa2 G-protein coupled receptor system. However, the resulting effect on PKA-controlled properties is only transient when there is no complete growth medium present. When an essential nutrient is lacking, the cells arrest in the stationary phase G0. At the same time they acquire all characteristics of cells with low PKA activity, even if there is ample glucose present. When the essential nutrient is added again, a similar PKA-dependent protein phosphorylation cascade is triggered as observed after addition of glucose to glucose-deprived cells, but which is not cAMP-mediated. Because the pathway involved requires a fermentable carbon source and a complete growth medium, at least for its sustained activation, it has been called "fermentable growth medium (FGM)-induced pathway."

A specific mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K176M, eliminates glucose- and acidification-induced cAMP signalling and delays glucose-induced loss of stress resistance.

The cAMP-protein kinase A (PKA) pathway in the yeast Saccharomyces cerevisiae plays a major role in the control of metabolism, proliferation and stress resistance. Derepressed cells show a rapid increase in the cAMP level (within 1 min) after addition of glucose or after intracellular acidification. A specific mutation in adenylate cyclase, the enzyme that catalyzes the synthesis in cAMP, largely prevents both cAMP responses. The responsible mutation was originally called lcr1 (for lack of cAMP responses); lcr1 was later identified as allelic with CYR1/CDC35. The mutation was introduced into the CYR1 gene of a W303-1A wild type strain, which resulted in a large decrease in cAMP signalling. Furthermore, there was a strong reduction in GTP/Mg2+-stimulated but not in Mn2+-stimulated adenylate cyclase activity in isolated plasma membranes, which is consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the lcr1 mutant. Because of the absence of cAMP signalling during exponential growth on glucose, it was concluded that glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Activation of the PKA pathway is mediated by a G protein (either Ras1/Ras2 or Gpa2). Constitutive activation of the pathway by Ras2val19 or Gpa2val132 has a negative effect on glycogen and trehalose accumulation and heat shock survival. The lcr1 mutation partially suppresses this effect indicating that the target sites of the two G-proteins on adenylate cyclase might have at least a part in common.

Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications.

In frozen dough applications a prefermentation period during the preparation of the dough is unavoidable and might also be important to obtain bread with a good texture. A major disadvantage of the prefermentation period is that it is associated with a rapid loss of the freeze resistance of the yeast cells. A major goal for the development of new baker's yeast strains for use in frozen dough applications is the availability of strains that maintain a better freeze resistance during the prefermentation period. We have isolated mutants that retain a better stress resistance during the initiation of fermentation. Some of these showed the same growth rate and fermentation capacity as the wild type cells. These mutants are called 'fil', for deficient infermentation induced loss of stress resistance. First we used laboratory strains and heat stress treatment, given shortly after the initiation of fermentation, as the selection protocol. The first two mutants isolated in this way were affected in the glucose-activation mechanism of the Ras-cAMP pathway. The fil1 mutant had a partially inactivating point mutation in CYR1, the gene encoding adenylate cyclase, while fil2 contained a nonsense mutation in GPR1. GPR1 encodes a member of the G-protein coupled receptor family which acts as a putative glucose receptor for activation of the Ras-cAMP pathway. In a next step we isolated fil mutants directly in industrial strains using repetitive freeze treatment of doughs as selection protocol. Surviving yeast strains were tested individually for maintenance of fermentation capacity after freeze treatment in laboratory conditions and also for the best performing strains in frozen doughs prepared with yeast cultivated on a pilot scale. The most promising mutant, AT25, displayed under all conditions a better maintenance of gassing power during freeze-storage. It was not affected in other commercially important properties and will now be characterised extensively at the biochemical and molecular level.

Maturation of mouse primordial follicles by combination of grafting and in vitro culture.

Cryopreservation of ovarian cortical tissue and subsequent transplantation or in vitro culture of follicles are technologies under development with the aim to safeguard fertility in patients with gonadal failure. In the present study, we investigated whether primordial follicles could be triggered to full maturation by a combination of in vivo transplantation and in vitro culture in a mouse model. In a first step, newborn mouse ovaries containing only primordial follicles were allotransplanted under the renal capsule of ovariectomized recipient mice. The second step was to mechanically isolate growing preantral follicles from the graft and culture these in vitro to maturity. In our experiment, one newborn mouse ovary was transplanted under the renal capsule of each 8- to 12-wk-old F1 (C57Bl/6j x CBA/Ca) female ovariectomized recipient (n = 26). Two weeks after transplantation, all 26 grafts were recovered. Four grafts were processed for histology and showed that developmental stages of follicles in 14-day-old ovarian grafts were comparable to those in 14-day-old mouse ovaries. The 22 remaining grafts were used for mechanical isolation of preantral follicles. As a control group, preantral follicles isolated from ovaries of 14-day-old mice were used. The mean preantral follicle yield per ovary was 11 in the transplant group versus 33 in the control group. Follicles were cultured individually in 20-microliter droplets of alpha-MEM supplemented with 100 mIU rFSH and 5% fetal bovine serum for 12 days under an atmosphere of 5% CO(2) in air at 37 degrees C. By Day 12 of culture, 66.5% of follicles retained their oocytes in the grafting group versus 97.5% in the control group (P < 0.001). Final oocyte maturation was induced by addition of 2.5 IU/ml hCG. At 14-16 h post-HCG, the percentages of oocytes showing germinal vesicle breakdown and polar body extrusion were significantly higher in the control group (90.6% and 82.8%) compared to the grafting group (60% and 45%). The mean diameter of the mature oocytes of the grafting group (69.9 +/- 4.45 micrometer) was similar to that of oocytes from the control group (70.5 +/- 2.35 micrometer). Our results suggest that maturation of mouse primordial follicles is feasible by combination of in vivo transplantation and in vitro culture. This two-step strategy may be an attractive model for promoting the growth and maturation of primordial follicles from other species.

Comparison of plasma compartment versus two methods for effect compartment--controlled target-controlled infusion for propofol.

BACKGROUND: Target-controlled infusion (TCI) systems can control the concentration in the plasma or at the site of drug effect. A TCI system that targets the effect site should be able to accurately predict the time course of drug effect. The authors tested this by comparing the performance of three control algorithms: plasmacontrol TCI versus two algorithms for effect-site control TCI. METHODS: One-hundred twenty healthy women patients received propofol via TCI for 12-min at a target concentration of 5.4 microg/ml. In all three groups, the plasma concentrations were computed using pharmacokinetics previously reported. In group I, the TCI device controlled the plasma concentration. In groups II and III, the TCI device controlled the effect-site concentration. In group II, the effect site was computed using a half-life for plasma effect-site equilibration (t1/2k(eo)) of 3.5 min. In group III, plasma effect-site equilibration rate constant (k(eo)) was computed to yield a time to peak effect of 1.6 min after bolus injection, yielding a t1/2keo of 34 s. the time course of propofol was measured using the bispectral index. Blood pressure, ventilation, and time of loss of consciousness were measured. RESULTS: The time course of propofol drug effect, as measured by the bispectral index, was best predicted in group III. Targeting the effect-site concentration shortened the time to loss of consciousness compared with the targeting plasma concentration without causing hypotension. The incidence of apnea was less in group III than in group II. CONCLUSION: Effect compartment-controlled TCI can be safely applied in clinical practice. A biophase model combining the Marsh kinetics and a time to peak effect of 1.6 min accurately predicted the time course of propofol drug effect.

Deletion of SFI1, a novel suppressor of partial Ras-cAMP pathway deficiency in the yeast Saccharomyces cerevisiae, causes G(2) arrest.

When glucose is added to Saccharomyces cerevisiae cells grown into stationary phase or on non-fermentable carbon sources a rapid loss of heat stress resistance occurs. Mutants that retain high stress resistance after addition of glucose are called 'fil', for deficient in fermentation induced loss of stress resistance. Transformation of the fil1 mutant, which harbours a point mutation in adenylate cyclase, with a yeast gene library on a single copy plasmid resulted in transformants that were again stress-sensitive. One of the genes isolated in this way was a gene of previously unknown function. We have called it SFI1, for suppressor of fil1. SFI1 is an essential gene. Combination of Sfi1 and cAMP pathway mutations indicates that Sfi1 itself is not involved in the cAMP pathway. Conditional sfi1 mutants did not show enhanced heat resistance under the restrictive condition, whereas overexpression of SFI1 rendered cells heat-sensitive. Sfi1 may be a downstream target of the protein kinase A pathway, but its precise relationship with heat resistance remains unclear. Further analysis showed that Sfi1 is required for cell cycle progression, more specifically for progression through G(2)-M transition. Cells expressing SFI1 under the control of a galactose-inducible promoter arrest after addition of glucose as doublets of undivided mother and daughter cells. These doublets contain a single nucleus and lack mitotic spindles. Sfi1 shares homology with Xenopus laevis XCAP-C, a protein required for chromosome assembly. The conserved residues between these two proteins show a strong bias for charged amino acids. Hence, Sfi1 might be required for correct mitotic spindle assembly and its precise role might be in chromosome condensation. In conclusion, we have identified an essential function in the G(2)-M transition of the cell cycle for a yeast gene of previously unknown function.

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level.

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.

Constitutive glucose-induced activation of the Ras-cAMP pathway and aberrant stationary-phase entry on a glucose-containing medium in the Saccharomyces cerevisiae glucose-repression mutant hex2.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a nonfermentable carbon source triggers a rapid, transient increase in the cAMP level. The occurrence of this cAMP spike appears to be correlated inversely with the glucose-repression state of the cells. This was also observed for the hex2 mutant, which is deficient in glucose repression and which displayed the cAMP signal constitutively. When cells of the hex2 mutant were starved for nitrogen on a glucose-containing medium, they rapidly lost viability, similarly to mutants with overactivation of the Ras-adenylate cyclase pathway. Flow cytometry measurements showed that G1 arrest of the hex2 mutant under such conditions was incomplete. Trehalose accumulation, a typical feature of cells entering the stationary phase G0, was very short-lived in the hex2 mutant under the same conditions. These results are in agreement with the presence of continuous glucose-triggered activation of cAMP synthesis in hex2 cells on a glucose-containing nitrogen-starvation medium. In the course of these experiments a spontaneous suppressor mutant, shx (for suppressor of hex2), was isolated which survived nitrogen starvation on a glucose-containing medium much better than the hex2 strain. It also showed normal G1 arrest and much longer accumulation of trehalose. The suppressor mutation also caused inability to grow on nonfermentable carbon sources and absence of invertase depression, and it was epistatic to hex2 for these characteristics also. The isolation of this epistatic depression mutation supports the idea that the defect in glucose repression of the hex2 mutant is the cause of its rapid loss of viability during nitrogen starvation on a glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Control of glucose influx into glycolysis and pleiotropic effects studied in different isogenic sets of Saccharomyces cerevisiae mutants in trehalose biosynthesis.

The GGS1/TPS1 gene of the yeast Saccharomyces cerevisiae encodes the trehalose-6-phosphate synthase subunit of the trehalose synthase complex. Mutants defective in GGS1/TPS1 have been isolated repeatedly and they showed variable pleiotropic phenotypes, in particular with respect to trehalose content, ability to grow on fermentable sugars, glucose-induced signaling and sporulation capacity. We have introduced the fdp1, cif1, byp1 and glc6 alleles and the ggs1/tps1 deletion into three different wild-type strains, M5, SP1 and W303-1A. This set of strains will aid further studies on the molecular basis of the complex pleiotropic phenotypes of ggs1/tps1 mutants. The phenotypes conferred by specific alleles were clearly dependent on the genetic background and also differed for some of the alleles. Our results show that the lethality caused by single gene deletion in one genetic background can become undetectable in another background. The sporulation defect of ggs1/tps1 diploids was neither due to a deficiency in G1 arrest, nor to the inability to accumulate trehalose. Ggs1/tps1 delta mutants were very sensitive to glucose and fructose, even in the presence of a 100-fold higher galactose concentration. Fifty-percent inhibition occurred at concentrations similar to the Km values of glucose and fructose transport. The inhibitory effect of glucose in the presence of a large excess of galactose argues against an overactive glycolytic flux as the cause of the growth defect. Deletion of genes of the glucose carrier family shifted the 50% growth inhibition to higher sugar concentrations. This finding allows for a novel approach to estimate the relevance of the many putative glucose carrier genes in S. cerevisiae. We also show that the GGS1/TPS1 gene product is not only required for the transition from respirative to fermentative metabolism but continuously during logarithmic growth on glucose, in spite of the absence of trehalose under such conditions.

Polyamine metabolism and osmotic stress. I. Relation to protoplast viability.

Cereal leaves subjected to the osmotica routinely used for protoplast isolation show a rapid increase in arginine decarboxylase activity, a massive accumulation of putrescine, and slow conversion of putrescine to the higher polyamines, spermidine and spermine (HE Flores, AW Galston 1984 Plant Physiol 75: 102). Mesophyll protoplasts from these leaves, which have a high putrescine:polyamine ratio, do not undergo sustained division. By contrast, in Nicotiana, Capsicum, Datura, Trigonella, and Vigna, dicot genera that readily regenerate plants from mesophyll protoplasts, the response of leaves to osmotic stress is opposite to that in cereals. Putrescine titer as well as arginine and ornithine decarboxylase activities decline in these osmotically stressed dicot leaves, while spermidine and spermine titers increase. Thus, the putrescine:polyamine ratio in Vigna protoplasts, which divide readily, is 4-fold lower than in oat protoplasts, which divide poorly. We suggest that this differing response of polyamine metabolism to osmotic stress may account in part for the failure of cereal mesophyll protoplasts to develop readily in vitro.

Gradients of polyamines and their biosynthetic enzymes in coleoptiles and roots of corn.

The distribution of diamines, polyamines, and their biosynthetic enzymes arginine decarboxylase and ornithine decarboxylase in roots and coleoptiles of corn (Zea mays var Golden Cross Bantam) seedlings have been determined. Putrescine content, expressed on either a fresh weight or protein basis, increases from the tip to the base in both roots and coleoptiles. In roots, this gradient is paralleled by an activity gradient of arginine and ornithine decarboxylases. Spermidine is distributed equally along the length of coleoptiles; in roots, this is true only on a protein basis. Free spermine is detectable only in the root tip, but a bound form is present throughout the root and coleoptile. The results are compared with gradients in protein and DNA content and discussed in relation to the possible cellular roles of polyamines.