固有无序蛋白与蛋白质相互作用位点残基特征分析

本文对固有无序蛋白(IDPs)与其他蛋白质相互作用位点残基特征进行了研究.首先在数据库中选出满足条件的109条IDPs蛋白质链及与其他配体蛋白形成的299个IDPs-蛋白质复合物,然后提取复合物中作为相互作用位点的IDPs-蛋白质残基.这109条IDPs链中共含有50 031个氨基酸残基,其中处于作用位点的残基有4 822个.通过分析发现,20种氨基酸在形成IDPs-蛋白质相互作用位点残基时具有不同的倾向性,根据形成作用位点残基的倾向性,20种氨基酸可分成三大类:倾向型氨基酸(ILE、LEU、ARG、PHE、TYR、MET、TRP)、中间型氨基酸(GLN、GLU、THR、LYS、VAL、ASP、HIS)、非倾向型氨基酸(PRO、SER、GLY、ALA、ASN、CYS).研究结果还进一步表明,不同氨基酸在有序区域与无序区域形成IDPs-蛋白质作用位点残基的倾向性不同.其中,氨基酸TRP、LEU、ILE、CYS在有序和无序区域形成作用位点残基的差异性尤为明显,而氨基酸GLU、PHE、HIS、ALA则基本没有多大差别.对IDPs-蛋白质相互作用位点残基理化特征进行分析发现:疏水性强、侧链净电荷量较少、极性较小、溶剂可及性表面积较大、侧链体积较大、极化率较大的氨基酸比较倾向于形成作用位点残基.主成分分析结果显示,残基的极化率、侧链体积和溶剂可及表面积对作用位点残基影响最大.     

低智儿童与正常儿童中氨基酸的比较研究

我们对 30例低智儿童血清中TAU ,SER ,GLU ,GLY ,ALA ,VAL ,CYS ,MET ,ILE ,LEU ,TYR ,PHE ,TRP ,HIS ,ORN ,LYS ,ARG ,PRO ,18种游离氨基酸进行了测定。研究结果显示 :患儿血清中 11种游离氨基酸降低分别为 :TAU ,SER ,VAL ,MET ,ILE ,LEU ,TRY ,PHE ,ORN ,LYS ,TRP .氨基酸的失衡 ,儿童的蛋白质合成就将会受到严重的影响 ,因此将导致大脑的分化 ,发育受阻 ,引起智力低下。     

Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.     

Effect of Si-status on diel variation of intracellular free amino acids in Thalassiosira weissflogii under low-light intensity

Twenty-one intracellular free amino acids were analysed during a 12-12 h light-dark cycle, on duplicate axenic cultures of Thalassiosira weissflogii (clone Actin, Provasoli-Guillard CCMP) under either Si-sufficient or Si-starved conditions. Total concentrations ranged between 40 and 165 fmol/cell. Total level as well as individual levels of amino acids decreased during the dark period, and GLN/GLU ratio was lower during the dark period. All these results were correlated with the light-dark carbon metabolism of the algae and related to the protein synthesis at night. The Si-starved cultures showed a lower total level of FAA compare to the Si-sufficient cultures, especially in the light period. Silica status of the cells affected more the metabolites of the dark respiration than the photorespiratory metabolites SER and GLY. Si deprivation induced higher range of ALA and VAL, and a decrease of the TCA metabolites GLU & ASP. Additionally, the relative percentage of ASP increased under Si starvation, at the expense of GLU, and this shift was emphasized in the dark period.     

Effect of exogenous amino acids,glucose and citric acid on the patterns of short-term accumulation and loss of amino acids in the root-zone of sand-cultured forage rape (Brassica napus L.)

Amino acid loss from the roots of 25-day-old, sterile and non-sterile sand-grown forage rape plants, was determined over periods of up to 3.5 hours. Amino acid accumulation in the root-zone of sterile plants was concentration-dependent giving a convex accumulation profile. Amino acid levels in the root zone of non-sterile plants rapidly attained steady state values. Microbial assimilation of amino acids within the root zone appeared to lower amino acid concentrations, resulting in an underestimation of rates of amino acid loss from roots. The concentrations of most amino acids were higher after selected amino acids were supplied to the root zone. The response to exogenous acids was dependent on the concentration and composition of the acids added. Addition of a mixture containing ASN, GLN and GABA, each at 0.25 mM resulted in a greater increase in individual and total acid levels compared with a mixture containing ALA, SER, GLY and THR at the same concentration. Apparently, amino acids supplied exogenously competed with acids lost from the plant, by providing an alternative nutrient source for root zone micro-organisms. Addition of glucose and citric acid had a similar effect to addition of ALA, SER, GLY and THR, but were less effective than ASN, GLN and GABA at all concentrations tested. The nitrogen-rich amino acids ASN and GLN, and the -amino acid, GABA, appeared to compete more effectively with plant-derived acids than did ALA, SER, GLY and THR, the most abundent constituents of the plant-derived acids, which had the highest calculated rates of microbial consumption. Therefore, although bacterial consumption showed a dependence on amino acid concentration, a degree of selectivity for nitrogen-rich acids and gaba was also apparent.     

Free amino acid analysis of five microalgae

The HPLC separation of fluorescent o-phtaldialdehyde (OPA) derivatives has been applied to the assay of free amino acids from five microalgae commonly used in aquaculture: Tetraselmis suecica, Skeletonema costatum, Chaetoceros calcitrans, Thalassiosira sp. and Isochrysis galbana, as part an assessment of their potential use in cosmetic products. Thirteen free amino acids were analyzed using High Performance Liquid Chromatography. There were considerable differences between species. However, four amino acids were responsible for more than 60% total concentration in all species: ASP, GLU, ARG and TYR; the next most important (accounting for less than 30%) were: ALA, VAL, PHE and LYS. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.     

Hominoid evolution as judged by fibrinopeptide structures

Summary The fibrinopeptides A and B from gorilla, organgutan and siamang have been characterized, thereby completing a study of all six extant hominoids. The gorilla peptides were identical with the corresponding fibrinopeptides previously reported for human and chimpanzee. The orangutan peptide A was also identical with the human-chimpanzee-gorilla type A, but its fibrinopeptide B had two amino acid differences. The siamang A peptide differed from the others in one of its sixteen residues, but its peptide B was identical with the orangutan B. A cladogram based on the fibrinopeptide sequences of all six hominoids indicates that five amino acid replacements and one deletion can account for the evolution of present day sequences. It was also possible to deduce the amino acid sequence of the fibrinopeptides of the common ancestor of Old World monkeys and hominoids.Abbreviations Used PITC phenylisothiocyanate - DNS dimethylaminonaphthalene sulfonyl- - PCA pyrrolidone carboxylic acid - ASP aspartic acid - ASN asparagine - THR threonine - SER serine - GLU glutamic acid - GLN glutamine - GLY glycine - ALA alanine - VAL valine, ILE isoleucine - LEU leucine - PHE phenylalanine Supported by grants from the National Science Foundation (GB 7332) and the National Institutes of Health (GM-17, 702 and HE-12, 759).     

How do side chains orient globally in protein structures?

An angle Omega is defined to serve as a metric for global side-chain orientations, which reflects the orientation of the side chain relative to the radial vector from the center of the protein to an amino acid. The side-chain orientations of buried residues exhibit characteristically different orientations than do exposed residues, in both monomeric and dimeric structures. Overall, buried side chains point mostly inward, whereas surface side chains tend to point outward from the surface. This difference in behavior also correlates well with the residue hydrophobicity; so a global side-chain orientation can be viewed as a direct structural manifestation of hydrophobicity. When various solvent-accessible layers are considered, the behavior is relatively continuous between centrally located and exposed residues. In the case of interfacial residues between subunits, there are statistically significant differences between exposed residues and interface residues for ALA, ARG, ASN, ASP, GLU, HIS, LYS, THR, VAL, MET, PRO, and overall the interface residues have an increased tendency to point inward. Presumably, these substantial differences in orientations of side chains may be a manifestation of hydrophobic forces.     

Cysteamine or beta-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection

The present study was carried out to investigate the effect of adding 100 microM cysteamine (Cys) or 100 microM beta-mercaptoethanol (beta-ME) to a defined maturation medium on in vitro maturation (IVM), and fertilization and developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The two control media for IVM culture were modified TCM199 containing 10% (v/v) porcine follicular fluid (pFF) or 0.05% (w/v) polyvinyl alcohol (PVA), and Cys or beta-ME was supplemented to the PVA-control medium. There was no significant difference in the proportions of in vitro matured oocytes among the four treatment groups (94.5-98.4%). The percentages of pronuclear formation (51.0-64.2%) after ICSI were also not significantly different among the four groups. The cleavage rate (72.8%) in the oocytes treated with Cys showed no significant difference compared with those of the two control media containing pFF (72.2%) or PVA (61.5%), but was higher (P<0.05) than that in the oocytes treated with beta-ME (56.3%). However, the rates of blastocyst formation of Cys (36.7%), beta-ME (27.1%) and pFF (31.4%) were higher (P<0.05) than that using the control medium containing PVA (15.6%). The mean cell number of blastocysts ranged from 42 to 52 among the four groups, without significant differences. In conclusion, the addition of Cys or beta-ME to a defined maturation medium enhanced blastocyst formation after ICSI, to a level similar to that achieved by adding pFF.     

Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media

This study was conducted to develop a serum-free, defined medium for IVM of pig oocytes. Modified North Carolina State University (mNCSU)-23 media with or without supplementation with both epidermal growth factor (EGF) and gonadotrophin were used as base media. In separate experiments, each base medium was supplemented with porcine follicular fluid (pFF), polyvinyl alcohol (PVA), PVA and essential amino acids (EAA), PVA and nonessential amino acids (NEAA) or PVA with both EAA and NEAA. Averaged across these five treatments, the percentage of blastocyst formation was higher (P < 0.05) in the base medium supplemented with EGF and gonadotrophins. In both base media, the addition of NEAA yielded similar percentages of maturation (81-82% versus 75-80%), sperm penetration (89-93% versus 80-86%) and blastocyst formation (4-18% versus 4-13%) as media supplemented with pFF. Although similar benefits were found after the addition of EAA, their addition was associated with lower (P < 0.05) maturation (66%) and sperm penetration (58%) than when pFF was added to the base medium without EGF and gonadotrophins. However, decreased maturation after EAA addition was not detected in the base medium containing EGF and gonadotrophins. Within the same base medium, monospermy, male pronucleus formation, cleavage and blastocyst formation were not affected by the treatments; and combined addition of EAA and NEAA did not further improve oocyte development. In conclusion, a maturation system using a defined mNCSU-23 medium supplemented with EGF, gonadotrophins and EAA or NEAA was developed which yielded a similar number of blastocysts compared with a pFF-containing medium.     

K+-stimulated amino acid release from cultured cerebellar neurons: Comparison of static and dynamic stimulation paradigms

The release of several endogenous amino acids and adenosine from rat cerebellar neuronal cultures following elevated K⁺ exposure in the presence and absence of added Ca²⁺ was studied. The amino acids aspartate (ASP), glutamate (GLU) and GABA were released from the cultures in a dose- and Ca²⁺-dependent manner. Taurine (TAU) and the nucleoside adenosine (ADN) efflux rates were dose-dependent but Ca²⁺-independent, and basal levels increased in the absence of Ca²⁺. The K⁺ depolarization induced release of serine (SER), alanine (ALA) and proline (PRO), was not dose-dependent and in the absence of extracellular Ca²⁺ (with added Mg²⁺) higher basal release of SER and ALA, but not PRO, was noted. These findings demonstrate that in addition to known cerebellar neurotransmitters, other neuroactive and neutral amino acids are released from cultured cerebellar neurons in response to K⁺ depolarization. Their observed efflux suggests they may have as yet unidentified roles in neuronal function with different classes of efflux corresponding to: neurotransmitter-type release (ASP, GLU, GABA), and osmoregulatory, possibly neuromodulatory-type release (TAU), a Ca²⁺-insensitive, possibly neuromodulatory-type release (ADN), and a depolarization-sensitive release (SER, ALA, PRO) of which SER and ALA are partially Ca²⁺-sensitive.     

Patterns of short-term amino acid accumulation and loss in the root-zone of liquid-cultured forage rape (Brassica napus L.)

Amino acid release from roots of sterile and non-sterile, solution-grown, 7-, 21- and 60-days-old forage rape plants (Brassica napus L.), was measured over periods of up to 6 hours. With sterile plants, release of amino acids into a fixed volume of collection medium (6, 12, 70 mL) was concentration-limited, giving rise to similar convex accumulation profiles for individual acids. In contrast, amino acid accumulation in continuously circulating collection medium was not concentration limited, giving a linear accumulation pattern. The compositions of accumulating amino acids, which were similar to those measured in root extracts, did not change significantly. However, the proportions of ALA, GABA, GLU and ILE in both root extracts and root-derived amino acids increased as plants aged. Older plants released more amino acids per plant, while younger plants released more amino acids g^-1 root DW. Using non-sterile plants, the patterns of change in amino acid concentration and composition in the collection medium were completely different from those determined with sterile plants. In general, with 7-days-old plants, and 60-days-old plants that had recently become non-sterile, an initial rise in the concentration of all acids was followed by a fall to low levels. The loss of amino acids was apparently due to microbial consumption. Individual amino acids attained maximum concentration at different times during the collection process. This is attributed mainly to concentration-dependent differential assimilation of amino acids, since those with the highest initial concentrations, the major components of the mixtures released from roots, declined the earliest. When calculated rates of amino acid release from roots (R_r) and microbial consumption of amino acids (R_c) were compared (for 7-days-old plants), the highest ratios of R_c/R_r were found for ASN, ARG, GLU, GLN, and LYS. This suggests a degree of selectivity for glutamate and nitrogen-rich acids on the part of the consuming micro-organisms. With 21-days old plants and 60-days old plants grown entirely under non-sterile conditions, fluctuations in amino acid concentration were similar for all acids.     

In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embyo yield in vitro

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P 相似文献   

Supplementation with vascular endothelial growth factor during in vitro maturation of porcine cumulus oocyte complexes and subsequent developmental competence after in vitro fertilization

The aim of the present study was to investigate whether the effects of vascular endothelial growth factor (VEGF) on porcine cumulus oocyte complexes (COCs) and subsequent blastocyst formation following in vitro fertilization are attributable to improved fertilization and cytoplasmic maturation. Porcine COCs were cultured for 42 h in TCM199 medium with 5 ng/mL human recombinant VEGF, and the resultant metaphase II oocytes were fertilized in vitro. COCs without VEGF supplementation served controls. Supplementation with VEGF during in vitro maturation (IVM) significantly (P < 0.05) improved the blastocyst formation rate and total cell number (46.7 ± 3.1% and 82.8 ± 6.7, respectively) compared with controls (32.5 ± 3.4% and 64.1 ± 5.6, respectively). On day 2, the percentage of four-cell stage embryos was significantly higher in the VEGF-matured group (49.1 ± 2.7%) than in the control (33.1 ± 5.8%), and the percentage of two-cell stage embryos was significantly higher in the control group (10.4 ± 1.4%) than in the VEGF-matured group (6.6 ± 0.9%). At 10 h after the onset of in vitro fertilization (IVF), oocytes with two pronuclei were considered as monospermically or normally fertilized, and oocytes with more than two pronuclei were considered as polyspermically fertilized. Monospermy was significantly higher in VEGF-matured oocytes (47.2 ± 4.3%) than in the control (20.0 ± 2.4%), and polyspermy and sperm penetration per oocyte were significantly higher in the control group (54.4 ± 3.8% and 2.3 ± 0.1, respectively) than in the VEGF-matured oocytes (43.9 ± 3.6% and 1.8 ± 0.1, respectively). Supplementation with VEGF during IVM significantly (P < 0.05) improved male pronuclear formation as compared with the control (91.1 ± 1.9 vs 74.4 ± 3.8%). Type III cortical granule distribution in oocytes was more common in VEGF-matured oocytes (78.0%) than in the control (52.1%). These results suggest that VEGF supplementation during IVM enhanced the developmental potential of porcine IVF embryos through higher male pronuclear formation and higher monospermic fertilization rates as a consequence of improved cytoplasmic maturation.     

High oxygen tension during in vitro oocyte maturation improves in vitro development of porcine oocytes after fertilization

This study was conducted to evaluate the effect of oxygen tension during IVM and/or IVC on developmental competence of porcine follicular oocytes. Prospective, randomized experiments were designed, and oocytes were matured, inseminated and cultured in vitro in the designated condition. In experiment 1, either high (20%) or low (7%) oxygen tension was used for IVM. The high oxygen significantly improved blastocyst formation (23% versus 13%; P<0.01) after IVF than the low oxygen. Such treatment, however, did not significantly (P>0.05) improve the rates of nuclear maturation (89% in each treatment), sperm penetration (62-72%), monospermic fertilization (56-67%), pronuclear formation (90-96%), cleavage (49-53%) and blastocyst cell number (31-32 cells). In experiment 2, the combined effect of oxygen tension during IVM and IVC of embryos was evaluated by a 2 x 2 factorial arrangement. Again, the high oxygen tension during IVM supported blastocyst formation more efficiently (P<0.01) than the low oxygen, and this was independent of oxygen tension during IVC (26-28% versus 15-16%). In oocytes matured under the high oxygen, a tendency to increase blastomere number (P=0.0630) was found, when the low oxygen was used for IVC after insemination (39-45 cells/blastocyst). In conclusion, the use of high oxygen tension (20% maintained by exposure to 5% CO2 in air) for IVM of porcine oocytes promoted blastocyst formation in vitro.     

In vitro release of endogenous monoamines and amino acids from several CNS regions of the rat

The in vitro release of endogenous norepinephrine (NE), dopamine (DA), serotonin (5-HT), GABA, glutamate (GLU), aspartate (ASP), glycine (GLY), taurine (TAU) and alanine (ALA) from superfused slices of cerebral cortex (CTX), striatum (STR), hippocampus (HIP), hypothalamus (HYPO), midbrain (MB), thalamus (THAL), nucleus accumbens (ACC), pons-medulla (PM) and spinal cord (SC) was studied. Under resting conditions or with 60 mM K⁺ in the absence of Ca²⁺, there was little or no release of NE, DA, 5-HT, GABA, GLU or ASP from any region. In most regions, there was a measurable resting release of ALA, GLY and TAU; of these three amino acids, only GLY in the PM and SC showed an increased release in the 60 mM K⁺ plus 2.5 mM Ca²⁺ medium. In 8 of the regions studied, the release of both GABA and GLU were stimulated by 60 mM K⁺ in the presence of 2.5 mM Ca²⁺. For the amino acids, no reliable data were obtained for release from the ACC because of its small size. The highest amount of K⁺-stimulated, Ca²⁺-dependent release of GABA was found with slices from the HYPO, THAL and MB while the highest amount of GLU was released from slices of STR, HIP and CTX. In those regions where reliable levels of K⁺-stimulated, Ca²⁺-dependent release of ASP were observed (STR, CTX, THAL), the amount of ASP was at least 5-fold lower than the values for GLU. A K⁺-stimulated, Ca²⁺-dependent release of NE, DA and 5-HT was observed for all 9 CNS regions studied. The highest release of (a) DA occurred from slices of CTX, STR and ACC; (b) NE was found in the HYPO and ACC; and (c) 5-HT occurred in the HYPO. The data (a) do not support a transmitter role for ALA and TAU in the CNS; (b) support a major transmitter function for GLY only in the PM and SC; and (c) support a transmitter role for GABA, GLU, NE, DA and 5-HT in the CNS regions examined (with the exception of GABA and GLU in the ACC where no data were obtained).     

Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid

To elucidate the beneficial effects of porcine follicular fluid (pFF) added to maturation medium on the sustenance of cytoplasmic maturation responsible for the subsequent developmental competence after in vitro fertilization (IVF) of porcine oocytes, we focused on the antioxidative role of pFF in its function of protecting oocytes from reactive oxygen species (ROS)-induced cell damage. Porcine follicular fluid collected from small (2-6 mm) follicles had about 7.2-fold higher levels of superoxide dismutase (SOD) activity than that of fetal bovine serum (FBS), and this activity was markedly blocked by the CuZn-SOD inhibitor, diethyldithiocarbamate (DETC). The interruption of meiotic progression and the increasing intracellular glutathione (GSH) content throughout the maturation period, as well as an outbreak of DNA damage in oocytes and cumulus cells were difficult to detect in oocytes cultured in a medium supplemented with 10% pFF, even in the presence of ROS generated by the hypoxanthine-xanthine oxidase system, whereas cell damage encompassed by ROS was prominent in oocytes cultured with 10% FBS and 10% pFF plus 100 microM DETC. Similarly, significant enhancement to the degree of transformation of the sperm nucleus into the male pronucleus (MPN) after in vitro fertilization was shown by the addition of pFF to the maturation medium. The presence of DETC during in vitro maturation reduced the ability of oocytes to promote MPN formation to the same extent as oocytes matured with FBS. The proportion developing to the blastocyst stage was increased in oocytes that matured with pFF, but this developmental competence was significantly lowered by treatment with DETC (P < 0.05). These findings suggest that pFF plays a critical role in protecting oocytes from oxidative stress through a higher level of radical scavenging activity elicited from SOD isoenzymes, resulting in the enhancement of cytoplasmic maturation responsible for developmental competence postfertilization.     

Effects of in vitro maturation of monkey oocytes on their developmental capacity

The study of in vitro maturation (IVM) of rhesus monkey oocytes has important implications for biomedical research and human infertility treatment. In vitro-matured rhesus monkey oocytes show much less developmental potential than IVM oocytes of other species. Since about 1980 when rhesus monkey IVM, in vitro fertilization (IVF) and in vitro embryo culture (IVC) systems were established, numerous efforts have been made to improve the developmental competence of oocytes and to understand the mechanisms regulating oocyte maturation. This review describes recent progress in this area, particularly the effects of factors such as steroid hormones, energy substrates, amino acids, ovarian follicle status, maternal age and breeding season on the developmental competence, gene expression patterns and genome integrity of rhesus IVM oocytes.