The dynamics of cyclin B1 distribution during meiosis I in mouse oocytes

Cdk1-cyclin B1 kinase activity drives oocytes through meiotic maturation. It is regulated by the phosphorylation status of cdk1 and by its spatial organisation. Here we used a cyclin B1-green fluorescent protein (GFP) fusion protein to examine the dynamics of cdk1-cyclin B1 distribution during meiosis I (MI) in living mouse oocytes. Microinjection of cyclin B1-GFP accelerated germinal vesicle breakdown (GVBD) and, as previously described, overrides cAMP-mediated meiotic arrest. GVBD was pre-empted by a translocation of cyclin B1-GFP from the cytoplasm to the germinal vesicle (GV). After nuclear accumulation, cyclin B1-GFP localised to the chromatin. The localisation of cyclin B1-GFP is governed by nuclear import and export. In GV intact oocytes, cyclin export was demonstrated by showing that cyclin B1-GFP injected into the GV is exported to the cytoplasm while a similar size dextran is retained. Import was revealed by the finding that cyclin B1-GFP accumulated in the GV when export was inhibited using leptomycin B. These studies show that GVBD in mouse oocytes is sensitive to cyclin B1 abundance and that the changes in distribution of cyclin B1 contribute to progression through MI.     

Activation of Akt (protein kinase B) stimulates metaphase I to metaphase II transition in bovine oocytes

In somatic cells, the serine/threonine kinase Akt (or protein kinase B) was shown to contribute to processes linked to cellular growth, cell survival and cell cycle regulation. In contrast to these findings, the function of Akt during the meiosis of mammalian oocytes remains to be investigated. We analysed the phosphorylation pattern and the activity of Akt during meiotic maturation (transition from prophase I to metaphase II) of bovine oocytes. The oocytes were matured in vitro (IVM) for 0, 10 and 24 h to reach the germinal vesicle (GV), metaphase I (M I) and metaphase II (M II) stages respectively. The abundance and phosphorylation pattern of Akt was revealed by Western blotting using total Akt or phosphoso-Akt-specific antibodies. The activity of this particular kinase was determined by an in vitro kinase assay. Furthermore, functional properties were analysed by cultivating oocytes in the presence of the Akt inhibitor SH6. The results showed that the overall abundance of Akt did not change significantly during IVM. On the other hand, Akt became phosphorylated at Thr 308 and Ser 473, reaching its maximum at the M I phase. In the GV and M II stages, only low basal phosphorylation levels were observed on both sides. This phosphorylation profile corresponded strictly to the activity of the kinase. The cultivation of oocytes in the presence of the phosphatidylinositol analogue SH6 for 24 h showed that, with higher concentrations, up to 65% of the oocytes were arrested in the M I stage. This result indicated that Akt is involved in the M I/M II transition during the meiotic maturation of bovine oocytes. The physiological aspects of the Akt function will be discussed.     

Mad2 is required for inhibiting securin and cyclin B degradation following spindle depolymerisation in meiosis I mouse oocytes

Mad2 is a pivotal component of the spindle assembly checkpoint (SAC) which inhibits anaphase promoting complex/cyclo-some (APC/C) activity by sequestering Cdc20 thereby regulating the destruction of securin and cyclin B. During mitosis, spindle depolymerisation induces a robust Mad2-dependent arrest due to inhibition of securin and cyclin B destruction. In contrast to mitosis, the molecular details underpinning the meiosis I arrest experienced by mouse oocytes exposed to spindle depolymerisation remain incompletely characterised. Notably, the role of Mad2 and the fate of the anaphase-marker, securin, are unexplored. As shown previously, we find that spindle depolymerisation by nocodazole inhibits first polar body extrusion (PBE) and stabilises cyclin B and cyclin-dependent kinase 1 activity in mouse oocytes. Here we show that stabilisation of cyclin B in nocodazole can be sustained for several hours and is associated with stabilisation of securin. These effects are SAC-mediated as, in oocytes depleted of the majority of Mad2 by morpholino antisense, securin and cyclin B are destabilised and 15% of oocytes undergo PBE. This reflects premature APC/C activation as a mutant form of cyclin B lacking its APC/C degradation signal is stable in Mad2-depleted oocytes. Moreover, homologues do not disjoin during the prolonged meiosis I arrest (> 18 h) induced by nocodaozole indicating that a non-cleavage mechanism is insufficient on its own for resolution of arm cohesion in mammalian oocytes. In conclusion, when all kinetochores lack attachment and tension, mouse oocytes mount a robust Mad2-dependent meiosis I arrest which inhibits the destruction of securin and cyclin B.     

Roasting process affects differently the bioactive compounds and the antioxidant activity of arabica and robusta coffees

The influence of different roasting degrees on the content of bioactive compounds and the antioxidant activity (AA) of arabica and robusta coffees was evaluated. AA was estimated by Folin–Ciocalteau, FRAP and ABTS methods. The results were analyzed by principal components analysis. While the levels of 5-CQA (5-caffeoylquinic acid), trigonelline, furfural and HMF (hydroxymethylfurfural) decreased with the degree of roasting, the level of melanoidins increased. Changes in the levels of the bioactive compounds were more intense that those observed for AA. PC 1 (principal component 1) was mainly correlated with the levels of, furfural, HMF, 5-CQA, trigonelline, and melanoidins. PC2 was mainly correlated with AA and caffeine content. Different compounds contribute to the AA of the coffee and the final result for AA is dependent on the balance of the compounds formed and degraded during the roasting process.     

Incidence of Fusarium verticillioides and levels of fumonisin B1 and B2 in corn in Turkey

A total of 100 corn samples conforming collected from local farmers and markets from districts of Samsun, Turkey, were analyzed for Fusarium verticillioides, fumonisin B₁ and B₂ contamination. Ninety-three corn samples were found to contain F. verticillioides, 52 samples fumonisin B₁, and 25 samples fumonisin B₂. Fumonisin B₁ contamination ranged from 0.05 to 25.72 mg/kg and B₂ from 0.05 to 5.7 mg/kg, respectively. This figure indicated widespread contamination of fumonisin B₁ and B₂ in maize grown in different areas of Samsun, Turkey.     

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to -7.0 ° C, induced to freeze externally, and finally cooled at 20 ° C/min to -70 ° C. IIF that occurred during the 20 ° C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were -34, -40, -35, and -25 ° C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10-15 ° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.     

Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout

Female rainbow trout (Oncorhynchus mykiss) were exposed to 4-nonylphenol (NP) at (mean measured) concentrations of 0.7, 8.3, and 85.6 micrograms/L for 18 weeks, during early ovarian development. Fish were sampled sublethally every six weeks, and terminal samples were taken at 18 weeks. NP induced an estrogenic effect (the synthesis of vitellogenin) at concentrations of 8.3 and 85.6 micrograms/L. An effect on gonadotropin synthesis and secretion was also observed. Plasma follicle stimulating hormone (FSH) levels and FSH gene expression in the pituitary were the most sensitive endpoints assessed, being reduced at the lowest dose employed (0.7 microgram NP/L). Pituitary gland luteinizing hormone (LH) content was significantly lower in fish exposed to 85.6 micrograms NP/L, and LH gene expression was suppressed in fish exposed to 8.3 and 85.6 micrograms NP/L. In contrast, plasma LH concentration increased in these fish, but by a very minor absolute amount, and returned to control levels by the final sampling time. Gonadal development ceased in the fish exposed to 85.6 micrograms NP/L, and steroidogenesis in these fish was also markedly inhibited. Although the mechanisms underlying these responses are unknown, this study demonstrates that NP has adverse effects on pituitary function that can result in inhibition of ovarian development.     

Cruciferous vegetable phytochemical sulforaphane affects phase II enzyme expression and activity in rat cardiomyocytes through modulation of Akt signaling pathway

The isothiocyanate sulforaphane (SF), abundant in Cruciferous vegetables, is known to induce antioxidant/detoxification enzymes in many cancer cell lines, but studies focused on its cytoprotective action in nontransformed cells are just at the beginning. Since we previously demonstrated that SF elicits cardioprotection through an indirect antioxidative mechanism, the aim of this study was to analyze the signaling pathways through which SF exerts its protective effects. Using cultured rat cardiomyocytes, we investigated the ability of SF to activate Akt/protein kinase B (PKB) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling pathways, which are implicated in cardiac cell survival, and to increase the phosphorylation of Nuclear factor E2-related factor 2 (Nrf2) and its binding to the antioxidant response element. By means of specific inhibitors, we demonstrated that the Phosphatidylinositol 3-kinase (PI3K)/Akt pathway represents a mechanism through which SF influences both expression and activity of glutathione reductase, glutathione-S-transferase, thioredoxin reductase, and NAD(P)H:quinone oxidoreductase-1, analyzed by western immunoblotting and spectrophotometric assay, respectively, and modulates Nrf2 binding and phosphorylation resulting in a cytoprotective action against oxidative damage. Results of this study confirm the importance of phase II enzymes modulation as cytoprotective mechanism and support the nutritional assumption of Cruciferous vegetables as source of nutraceutical cardioprotective agents.     

Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain,embryo stage,sodium and the hexose transport inhibitor,phloridzin

The uptake of myo-inositol by mouse oocytes and preimplantation embryos of a crossbred (DBA x C57BL/6) and a purebred outbred strain (MF1) was measured using [2-(3)H]myo-inositol. Uptake in crossbred embryos increased about 15-fold between the one- and two-cell stages and increased again by about sixfold at the blastocyst stage compared with the morula stage. Uptake in purebred embryos increased about 42-fold between the one- and two-cell stages and increased more than threefold at the blastocyst stage compared with the morula stage. In all stages examined, except two-cell crossbred embryos, inositol uptake was, depending on the stage, either largely or partly sodium dependent and could be inhibited by the sodium-dependent hexose transport inhibitor, phloridzin. This is consistent with the hypothesis that transport occurs via a sodium myo-inositol transporter (SMIT) protein. In addition, there was strong evidence that a sodium-independent mechanism of uptake, possibly a channel, was switched on at the two-cell stage coincident with zygotic gene activation which resulted in 141-fold and 71-fold increases in sodium-independent uptake from the one-cell to two-cell stages in crossbred and purebred embryos, respectively. This mechanism was either abolished or drastically downregulated at the blastocyst stage, whereas sodium-dependent uptake was markedly upregulated. In two-cell crossbred embryos, there was a complete abolition of sodium-dependent uptake, again possibly regulated by zygotic gene activation. The hypothesis that the changes in mechanism of inositol uptake at about the two-cell stage are due to zygotic gene activation was supported by the finding that these changes did not occur in parthenogenetic two-cell embryos.     

Caffeine promotes premature chromosome condensation formation and in vitro development in porcine reconstructed embryos via a high level of maturation promoting factor activity during nuclear transfer

When the nucleus in G0/G1 phase is transferred to an enucleated oocyte by nuclear transfer (NT), its nuclear envelope is broken, followed by condensation of chromosome structure by maturation promoting factor (MPF). This morphological remodeling of the transferred interphase nucleus seems to be essential for subsequent development of NT embryos. In this study, we treated porcine NT embryos with caffeine, which has been reported to increase MPF activity, to keep their MPF level high during NT. When 2.5 mM caffeine was added to the handling medium, the proportion of NT embryos showing condensed chromosome increased significantly (P < 0.05). In NT embryos treated with caffeine, the activity of p34(cdc2) kinase was significantly (P < 0.05) higher than in those without caffeine at 3 h post-injection. In addition, the rate of development to the blastocyst stage after activation was significantly (P < 0.05) higher in NT embryos treated with caffeine. These results indicate that caffeine treatment can increase not only the rate of chromosome condensation but also the developmental rate to the blastocyst stage of porcine NT embryos. This action is most likely due to the support/increase of MPF activity throughout the process of NT.     

Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes

With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after activation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/+) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/+ oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/+ oocytes when compared with ethanol. Compared with all other strains, NU/+ oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demonstrated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied.     

Assessment of fumonisins B1 and B2 levels in commercial maize-based food products by liquid chromatography with fluorimetric detection and postcolumn chemical derivatization

The occurrence of the fumonisins B(1) and B(2) in maize-based food products marketed in Italy was examined. A simply and reliable chromatographic method with fluorimetric detection and postcolumn o-phtalaldehyde derivatization was used for a monitoring of 100 samples (8 flours, 21 corn-meal, 16 snacks, 7 maize samples, 13 gluten-free products, and 35 corn-flakes) bought in local supermarkets during the years 2008 and 2009. The presence of both fumonisins B(1) and B(2), at a concentration higher than 15 μg/kg, was observed in all samples of corn-meal and maize-flour, in 75% of snacks, in 57% of maize samples, in 54% of gluten-free products, and in 29% of corn-flakes. A total of 7 samples including 4 corn-meals, 2 maize-flours, and 1 maize showed a value exceeding the maximum level fixed in the Regulation 1126/2007/EC; no positive sample was observed in corn-flakes, snacks, and gluten-free foods. Fumonisins contamination, on the whole range of maize-based food products analyzed, emphasizes the need of improve agricultural practices, and increase official control and monitoring studies.     

Changes in the levels of catechol oxidase and laccase activity in developing peaches

Catechol oxidase and laccase activity and the content of o-diphenols were followed in developing peaches from 2 weeks after fruit set to harvest time. Catechol oxidase activity in the soluble fraction drops sharply during the first few weeks and remains low until harvest time. Laccase activity rises from a very low level in the first 3 months to a level higher than that of catechol oxidase at harvest time. Catechol oxidase activity in a paniculate fraction and o-diphenols content per fruit, each show a peak during fruit development The peak in o-diphenols content precedes that of catechol oxidase activity.     

Aflatoxin B1 residues in eggs of laying hens fed a diet containing different levels of the mycotoxin.

The present study was carried out to evaluate the excretion of aflatoxin B1 residues in eggs of young laying hens fed aflatoxin B1-contaminated rations for 8 weeks. To this end, 96 twenty-week-old hens were randomly distributed into four experimental groups (24 birds per group) and given rations containing either 0 (controls), 100 micrograms, 300 micrograms or 500 micrograms aflatoxin B1/kg feed. Egg aflatoxin B1 residues were determined by thin layer chromatography; two samples per treatment per week were used for analysis. Egg production and average egg weights were not affected (p < 0.05) in the groups receiving aflatoxin B1-contaminated rations. Residues of aflatoxin B1 were detected only in the eggs of hens given 500 micrograms/kg feed, at levels that ranged from 0.05 to 0.16 microgram/kg (average: 0.10 microgram/kg). The results indicate that the feed to eggs aflatoxin B1 transmission ratio was approximately 5000:1, emphasizing the importance of controlling aflatoxin levels in rations for laying hens.     

益生菌Lactobacillus helveticus 130B4在牛乳中的生长特性及其促长因子研究

将具有ACE抑制活性的乳酸菌Laaobacillus helveficus 130B4用于发酵乳制品中,研究其在还原脱脂乳中的生长特性,并探讨通过添加生长促进物质对Laaobadllus helveticus 130B4生长特性以及对ACE抑制活性的影响.结果表明,Lb.helveticus 13084菌株在还原脱脂乳培养基中生长较缓慢,产酸能力较弱,37℃培养36 h后的pH值为4.31,酸度达0.8%,活菌数为8.93 g-1;72 h后的pH值为3.45,酸度可以达到1.85%,活菌数达9.1 g-1;酵母浸膏、大豆肽、核酸关联物质对该菌株的产酸能力有一定的促进作用.     

Further data on the levels of emerging Fusarium mycotoxins enniatins (A,A1, B,B1), beauvericin and fusaproliferin in breakfast and infant cereals from Morocco

Sixty-eight samples of cereals products, including breakfast cereals (n = 48) and infant cereals (n = 20), purchased from supermarkets and pharmacies in Rabat-Salé area from Morocco were analysed for the determination of six emerging mycotoxins: four enniatins ENs (ENA, ENA1, ENB and ENB1), beauvericin (BEA) and fusaproliferin (FUS). Samples were extracted with a mixture of acetonitrile:water (85:15, v/v), using an Ultra-Turrax® homogeniser. Mycotoxins were then identified and quantified by liquid chromatography (LC) with diode array detection (DAD). Positive samples were confirmed by LC–MS/MS.     

Oocytes in sheep homozygous for a mutation in bone morphogenetic protein receptor 1B express lower mRNA levels of bone morphogenetic protein 15 but not growth differentiation factor 9

The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell-oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1?mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.     

Complement factor B and the alternative pathway of complement activation in bovine milk

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2.06 microg/ml (+/- 0.18, SEM), 0.57% of the mean value found in serum (360 microg/ml). This proportion was comparable to that of serum albumin (0.63% of serum value) but less than the proportion of C3 in milk (2.71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 degrees C for 3 min or 50 degrees C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 degrees C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.