Fertilization differently affects the levels of cyclin B1 and M-phase promoting factor activity in maturing and metaphase II mouse oocytes

Fertilization affects levels of cyclin B1 and M-phase promoting factor (MPF) activity in maturing and metaphase II mouse oocytes in two distinct ways. In metaphase II oocytes, it leads to a Ca(2)(+)-dependent, continuous degradation of cyclin B1 and inactivation of cyclin dependent kinase (CDC2A)-cyclin B1 complex (MPF). In this paper, we show that neither mono- nor polyspermic fertilization of prometaphase I and metaphase I oocytes triggered degradation of cyclin B1. However, polyspermic fertilization of prometaphase I oocytes led to a transient decrease in MPF activity that lasted for 2 h. The inactivation of MPF in polyspermic prometaphase I oocytes did not depend on the fertilization-induced increase in the cytoplasmic concentration of free Ca(2)(+) ions, but was caused, at least in part, by dephosphorylation of CDC2A at threonine 161 (Thr161). We found that polyspermic fertilization did not affect glutathione levels in prometaphase I oocytes, and concluded that the decrease in MPF activity and dephosphorylation of CDC2A at Thr161 in polyspermic prometaphase I oocytes were not caused by a change in the redox status of the cell induced by an introduction of excessive amount of sperm protamines. Instead, we propose that inactivation of MPF activity in polyspermic maturing oocytes is caused by a change in nucleo-cytoplasmic ratio that leads to a 'titration' of kinases and phosphatases responsible for keeping MPF in an active state. This idea is supported by the finding that oocytes fused with thymocytes rather than spermatozoa also showed a transient decrease in MPF activity.     

An active protein kinase A (PKA) is involved in meiotic arrest of rat growing oocytes

Reinitiation of meiosis in meiotically competent, fully grown mammalian oocytes is governed by a fall in intraoocyte cAMP concentrations and the subsequent inactivation of protein kinase A (PKA). A similar reduction in intraoocyte cAMP concentrations in growing, meiotically incompetent rat oocytes not leading to resumption of meiosis, questions the involvement of PKA in the regulation of meiosis at this early stage of oocyte development. We examined the possibility of whether PKA activity maintains growing oocytes in meiotic arrest and further explored the mode of activation of PKA under conditions of relatively low cAMP concentrations. Our experiment demonstrated that inactivation of PKA stimulates growing rat oocytes to resume meiosis, and elevates the activity of their maturation-promoting factor (MPF). We also found that the expressions of type I and type II regulatory subunits (RI and RII) of PKA are higher in growing and fully grown oocytes, respectively. In addition, we revealed that the common 1:1 ratio between the regulatory (R) and catalytic (C) subunits of PKA is apparently not abrogated and, in accordance PKA activity in growing oocyte-cell extract is fully dependent on cAMP. Finally, we identified in growing oocytes, the A kinase anchoring protein (AKAP) 140, which was previously depicted in fully grown oocytes. We conclude that an active PKA prevents growing oocytes from resuming meiosis. Our findings further suggest that relatively high abundance of the PKAI isoform and/or its subcellular compartmentalization, through interaction with AKAP140, could possibly account for the high basal PKA activity at relatively low intraoocyte cAMP concentrations.     

Fate of centrosomes following somatic cell nuclear transfer (SCNT) in bovine oocytes

Cloning mammalians by somatic cell nuclear transfer (SCNT) remains inefficient. A majority of clones produced by SCNT fail to develop properly and of those which do survive, some exhibit early aging, premature death, tumors, and other pathologies associated with aneuploidy. Alterations of centrosomes are linked to aberrant cell cycle progression, aneuploidy, and tumorigenesis in many cell types. It remains to be determined how centrosomes are remodeled in cloned bovine embryos. We show that abnormalities in either distribution and/or number of centrosomes were evident in approximately 50% of reconstructed embryos following SCNT. Moreover, centrosome abnormalities and failed 'pronuclear' migration which manifested during the first cell cycle coincided with errors in spindle morphogenesis, chromosome alignment, and cytokinesis. By contrast, nuclear mitotic apparatus protein (NuMA) exhibited normal expression patterns at metaphase spindle poles and in 'pronucleus' during interphase. The defects in centrosome remodeling and 'pronuclear' migration could lead to chromosome instability and developmental failures associated with embryo production by SCNT. Addressing these fundamental problems may enhance production of normal clones.     

蛋白激酶B活性及其级联效应对肉品质的影响

蛋白激酶B(PKB/Akt)是真核细胞中非常重要的丝/苏氨酸蛋白激酶,也是磷脂酰肌醇-3激酶(PI3K)的关键下游信号分子。它既可以通过调控哺乳动物雷帕霉素靶蛋白(mTOR)、糖原合成酶激酶-3(GSK-3)和叉头转录因子(FoxO)的表达影响蛋白质代谢;也可以通过磷酸化FoxO和GSK-3调节糖原合成/分解;还可以通过固醇调节元件结合转录因子(SREBPs)及mTOR信号通路促进机体的脂质合成。此外,Akt可磷酸化FoxO、前体凋亡蛋白(如Bad、Bcl-2)和半胱天冬蛋白酶抑制细胞凋亡,最终影响畜禽肉的品质。综上所述,Akt及其级联效应可能成为提高肉品质的重要手段。本文阐述了Akt的活化机制、级联效应及其对肉品质的影响,以期为改善肉品质提供参考。     

The role of calcium/calmodulin-dependent protein kinase II on the inactivation of MAP kinase and p34cdc2 kinase during fertilization and activation in pig oocytes

The objective of this study was to investigate the role of calmodulin-dependent protein kinase II (CaMKII) during fertilization in the pig. Since it has been reported that CaMKII is involved in the capacitation and acrosome reaction of spermatozoa, we tested whether supplementation with the CaMKII inhibitor, KN-93, in the fertilization medium affected sperm penetration. The results showed that the addition of KN-93 in the fertilization medium significantly reduced the rate of sperm penetration into oocytes. However, pre-treatment with KN-93 before in vitro fertilization (i.v.f.) did not significantly affect sperm penetration, but it did affect pronuclear formation in a dose-dependent manner. In the oocytes pre-treated with KN-93 before i.v.f. and then co-cultured with spermatozoa without the drug, the decrease in p34cdc2 kinase and the cyclin B1 level were significantly suppressed as compared with those in penetrated oocytes without treatment with KN-93. However, the decrease in MAP kinase activity was not affected by KN-93. Additional treatment with KN-93 after Ca2+ ionophore treatment also inhibited the reduction in p34cdc2 kinase activity and the cyclin B1 level, but not MAP kinase activity. Treatment with KN-92, an inactive KN-93 analogue, did not significantly affect sperm penetration and pronuclear formation. In conclusion, the activation of CaMKII by artificial stimuli or sperm stimulated the disruption of cyclin B1 and the inactivation of p34cdc2 kinase, but did not affect MAP kinase inactivation during oocyte activation in pigs.     

Role of transition metals,Fe(II), Cr(II), Pb(II), and Cd(II) in lipid peroxidation

Cod liver oil was oxidized with Fenton-like systems containing transition metals Fe(II), Cr(II), Pb(II), and Cd(II). Malonaldehyde (MA) formed from 10 μl cod liver oil oxidized by a Fenton-like system containing each metal at levels of 0.25, 0.5, 1 and 4 μmol was analyzed by a gas chromatograph equipped with a nitrogen phosphorus detector. The MA production exhibited dose response and the greatest amount (837.0 ± 19.1 nmol) was obtained by the Fe(II) system at the level of l μmol. Generally, higher MA formation is observed in the lower the third ionization potential of the metal. The decreasing order of MA formed in the metal systems at the level of 1 μmol is Fe(II) > Cr(II)(274.1 ± 20.1 nmol) > Pb(II)(150.7 ± 13.0 nmol) > Cd(II)(95.4 ± 6.7 nmol). The amounts of MA formed in Cr(II), Pb(II), and Cd(II) systems were considerably lower than those in the Fe(II) system. The relative formations of MA in the Cr(II) and Pb(II) systems were similar to those in the Fe(II) system. The results suggest that trace amounts of metals contribute oxidative effects to lipid peroxidation followed by various diseases.     

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.     

Activities of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are not required for the global histone deacetylation observed after germinal vesicle breakdown (GVBD) in porcine oocytes

The acetylation of nuclear core histone has been suggested to work as an epigenetic mark for transmitting gene expression patterns to daughter cells. Global histone deacetylations, presumably involved in the reprogramming of the gene expression, have been observed after germinal vesicle breakdown (GVBD) in a cell cycle-dependent manner during meiotic maturation of mouse and porcine oocytes, although the regulation mechanism of histone deacetylation has not been studied well. In the present study, we examined the involvement of a crucial cell-cycle-regulator, maturation-promoting factor (MPF), and a meiosis-related kinase, mitogen-activated protein kinase (MAPK), in the global histone deacetylation during porcine oocyte maturation. In order to know whether the activities of MPF and MAPK were required, or the breakdown of GV membrane was sufficient, for the global histone deacetylation observed after GVBD, we artificially destroyed the GV membrane of the porcine immature oocytes. The artificial GV destruction (AGVD) induced histone deacetylation without the activation of MPF and MAPK. This deacetylation after AGVD was not affected by an MPF inhibitor, roscovitine, or an inhibitor of protein synthesis, cycloheximide, but was completely prevented by an inhibitor of histone deactylases (HDACs), trichostatine A. HDAC1 was present in the GV of the immature oocytes and localized on chromosomes after GVBD and AGVD. These results suggest that the MPF and MAPK activities were dispensable and the breakdown of the GV membrane was sufficient for the global histone deacetylation, which was catalyzed by HDAC activity.     

Stage-dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes in vitro

Ribonucleic acid synthesis continues at a low rate within 1 h of germinal vesicle breakdown. To determine if this newly synthesized mRNA is required for the resumption of meiosis in cattle oocytes, cumulus-enclosed oocytes were removed from 2 to 4-mm antral follicles directly into Dulbecco's medium with or without the RNA inhibitor, alpha-amanitin, or the protein synthesis inhibitor, cycloheximide (10 micrograms/ml). They were washed twice and matured for 28 or 48 h in medium 199, with Earle's salts, 2.2 g/L NaHCO3 and L-glutamine supplemented with 20% heated fetal calf serum to which were added gonadotropins (10 micrograms/ml NIH-LH-S18; 10 micrograms/ml NIH-FSH-S8; 20 ng/ml NIH-P-S9), estradiol-17 beta (1.5 micrograms/ml), solcoseryl (30 microliter/ml), and Dibekacin sulfate (100 micrograms/ml) with or without the inhibitors. After 28 h of maturation, 95.8% of control oocytes had undergone germinal vesicle breakdown and formation of a metaphase plate compared with only 1.3% of those continuously exposed to cycloheximide and 38% of those continuously exposed to amanitin. Exposure to cycloheximide or amanitin for only the first 16 h of culture followed by 12 h of inhibitor free culture resulted in 96.6% germinal vesicle breakdown with cycloheximide but only 56.5% germinal vesicle breakdown with amanatin. We conclude newly synthesized mRNA and protein synthesis is required for both full cumulus cell expansion and germinal vesicle breakdown.     

Effect of long-term bovine somatotropin (sometribove) treatment on nitrogen (protein) distribution in Jersey milk

Twenty-six Jersey cows were assigned randomly to one of two treatments. Twelve cows received biweekly subcutaneous injection of 500 mg of sometribove, USAN (recombinant methionyl bovine somatotropin), beginning 60 +/- 3 d postpartum and continuing throughout one lactation. Fourteen control animals received injections of placebo carrier. Milk samples were taken biweekly on weeks alternate to injection when differences in milk components were expected to be greatest compared with controls. The milk samples were analyzed for total nitrogen, noncasein nitrogen, and non-protein nitrogen. The average SCC for control and treatment groups was 44,000 +/- 47,000 and 56,000 +/- 65,000. Milk from sometribove-treated cows was significantly lower in total protein (3.92, 4.12%), true protein (3.74, 3.95%), and casein (3.11, 3.34%) than that from control cows on d 8 of the 14 d injection cycle. Casein as a percentage of true protein was lower (83.38, 84.52%), and non-protein nitrogen as a percentage of total nitrogen was higher (4.61, 4.26%) in milk from treated cows. The theoretical yield of Cheddar cheese was ca. .07% less for milk from treated cows than from control cows due to ca. 1% less casein as a percentage of true protein in the former. The differences in nitrogen distribution represent the response during the middle of the injection cycle when milk output was the highest and milk protein the lowest rather than the average response for the injection cycle. The results of the study indicate minimal impact on the cheese manufacturer because in practice milk is commingled from many dairies.(ABSTRACT TRUNCATED AT 250 WORDS)     

大豆蛋白质的构造和功能特性(中)

介绍了大豆蛋白质的组成、氨基酸成分、分子结构、溶解性，着重分析了大豆蛋白的食品功能特性中的乳化性、起泡性、水化性、凝胶性的相互关系以及这些特性与组成结构的关系，对大豆蛋白产品的生产提供了一定的理论依据和参考。     

Expression and proteasomal degradation of the major vault protein (MVP) in mammalian oocytes and zygotes

Major vault protein (MVP), also called lung resistance-related protein is a ribonucleoprotein comprising a major part (>70%) of the vault particle. The function of vault particle is not known, although it appears to be involved in multi-drug resistance and cellular signaling. Here we show that MVP is expressed in mammalian, porcine, and human ova and in the porcine preimplantation embryo. MVP was identified by matrix-assisted laser-desorption ionization-time-of-flight (MALDI-TOF) peptide sequencing and Western blotting as a protein accumulating in porcine zygotes cultured in the presence of specific proteasomal inhibitor MG132. MVP also accumulated in poor-quality human oocytes donated by infertile couples and porcine embryos that failed to develop normally after in vitro fertilization or somatic cell nuclear transfer. Normal porcine oocytes and embryos at various stages of preimplantation development showed mostly cytoplasmic labeling, with increased accumulation of vault particles around large cytoplasmic lipid inclusions and membrane vesicles. Occasionally, MVP was associated with the nuclear envelope and nucleolus precursor bodies. Nucleotide sequences with a high degree of homology to human MVP gene sequence were identified in porcine oocyte and endometrial cell cDNA libraries. We interpret these data as the evidence for the expression and ubiquitin-proteasome-dependent turnover of MVP in the mammalian ovum. Similar to carcinoma cells, MVP could fulfill a cell-protecting function during early embryonic development.     

Eukaryotic elongation factor-2 (eEF-2) activity in bovine mammary tissue in relation to milk protein synthesis

The amount of protein synthesis translational elongation factor 2 (eEF-2) was estimated employing diphtheria toxin-dependent ADP-ribosylation in samples prepared from small amounts of tissue from mammary gland, skeletal muscle and liver from lactating dairy cows. A very high level of ADP-ribosylatable eEF-2 was found in mammary gland, amounting to 20-times the level found in liver and 50-times the level found in skeletal muscle. This obviously reflects the high protein synthesis activity in mammary tissue. To our knowledge, similar high activities have previously been reported only for cancer cells. A close linear relationship was found between the amount of diphtheria-toxin catalysted ADP-ribosylated eEF-2 and protein and casein output in milk from cows in late lactation. This strongly suggests that eEF-2 may be a limiting factor in milk protein synthesis.