Oscillatory change of SR-protein kinase activities during oocyte maturation meiosis in fish

The SR-protein kinase activity was analyzed and the cytological changes were observed during oocyte maturation in bisexual transparent color crucian carp ( Carassius auratus color variety). The results revealed that the SR-protein kinase activity was sensitive to the artificially induced spawning hormones, and the change of oscillatory activity was similar to that of the maturation-promoting factor (MPF) kinase that regulates meiotic cell cycle in fish.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone I-Il as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longer-lasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

INVOLVEMENT OF CA(2+) SIGNALING PATHWAY DURING OOCYTE MATURATION OF THE NORTHERN QUAHOG MERCENARIA MERCENARIA

In many molluses, it has been found that Ca2+ signaling pathway is involved in the resumption of meiotic maturation in oocytes. To better understand the possible role of Ca2+ signaling pathway in regulating meiotic maturation in oocytes of the northern quahog Mercenaria mercenaria, free extracellular Ca2+, A23187 (calcium ionophore), verapamil (calcium channel blocker), and trifluoperazin (calmodulin antagonist) were used to incubate oocytes or serotonin-induced oocytes by pharmacological methods. Results show that extracellular Ca2+ (50 similar to 200 mM) and A23187 (1 similar to 10 mu M) can stimulate the meiotic maturation. In addition, verapamil (1 similar to 100 mu M) and trifluoperazin (10 similar to 1,000 mu M) could inhibit serotonin-induced oocyte maturation. Therefore, Ca2+ is essential for the reinitiation of meiotic maturation in oocytes of the northern quahog. Moreover, an increase i [Ca2+]i can promote meiotic maturation.

葡萄果皮花色苷构成特点、遗传规律及其在果实成熟过程中的变化

通过利用高效液相色谱-质谱联用技术，研究110 个不同基因型（包括3 个种和5 个种间杂种）葡萄品种的花色苷含量和成分特点。在所有品种中，最多鉴定出29 种花色苷。对葡萄的花色苷总量来说，一般情况下，欧亚种和欧美杂交种的花色苷含量较低，而野生种和砧木品种显著高于其它的种间杂种；在同一个种内，酿酒品种高于鲜食品种；在大多数高花色苷含量的种质中，二甲基花翠素类花色苷是主要的花色苷，而在低总花色苷量的品种，花青素类和花翠素类花色苷是主要的成分。此外，在欧亚种葡萄中，仅检测到单糖苷类花色苷，而在其它葡萄种质中，既有单糖苷花色苷又有双糖苷花色苷。在欧亚鲜食葡萄中，Pn-3-glucoside 是主要的花色苷，而在欧亚酿酒葡萄中，Mv-3-glucoside 是主要的花色苷。通过主成分分析，最终根据花色苷总量的不同和单、双糖苷含量的不同，110 个品种在散点图中被明显的分成3 部分。 通过连续两年调查3 个欧亚鲜食葡萄杂交组合的亲本和后代的花色苷含量来分析花色苷的遗传特点。共鉴定出16 种花色苷，且均为单糖苷类。母本中各花色苷的比例决定了后代中花色苷含量的比例，但是后代中花色苷的绝对含量不受亲本影响。不论亲本还是后代中，Peonidin 3-O-glucoside 和Malvidin3-O-glucoside 都是含量最高的花色苷。花色苷的有或无是寡基因控制的质量性状，而含量的多少是多基因控制的数量性状。通过主成分分析可以得知：在杂交后代中， peonidin 3-O-glucoside, malvidin 3-O-glucoside, delphinidin3-O-glucoside, cyanidin 3-O-glucoside, petunidin 3-O-glucoside, peonidin3-O-(6-O-coumaryl)-glucoside 和malvidin 3-O-(6-O-coumaryl)-glucoside 是影响果皮中花色苷总量的主要种类。花色苷的含量是一种高广义遗传力的性状，而且这种性状在两年间是稳定的（0.65-0.98）。 5 个不同基因型葡萄品种在成熟过程中果实品质的变化也被研究。始熟期开始后，果粒重量继续增加，果粒较大的鲜食品种增长很慢，而果粒较小的制汁和酿酒品种增长幅度很大；果实内两种主要的糖（葡萄糖和果糖）开始快速上升，且在整个成熟过程中保持1：1；有机酸的含量开始快速下降，苹果酸下降的幅度大于酒石酸。多酚物质在果实始熟期也发生巨大变化，花色苷快速积累。 ‘北紫’和‘梅鹿辄’中的花色苷在成熟前1-2 周达到最大值，‘黑奥林’、‘康可’和‘北醇’在整个成熟过程中花色苷一直增加；对非花色苷类多酚来说，‘黑奥林’和‘梅鹿辄’在果实成熟过程中一直增加，而在另3 个品种中是下降的；花色苷之间以及与黄酮醇之间成正相关，花色苷和酚酸成负相关关系，酚酸和黄酮醇也成负相关关系，黄烷醇物质之间以及与其它类黄酮物质之间成负相关关系。

生殖激素对奶水牛卵母细胞成熟及发育潜能的影响

本试验以屠宰场获取的奶水牛卵巢为试验材料,收集卵母细胞进行体外成熟培养(IVM)、体外受精(IVf)及早期胚胎培养(IVC).研究激素(FSH、LH、E2、P4)的不同浓度对奶水牛卵母细胞成熟和早期胚胎发育的影响,以期探讨奶水牛卵母细胞成熟和早期胚胎体外培养发育机制,优选不同激素的最佳浓度.结果表明:添加FSH试验组奶水牛颗粒细胞扩散率和卵裂率高于未添加试验组(P<0.05);添加LH试验组奶水牛颗粒细胞扩散率、卵裂率及8-细胞率与未添加试验组比较,差异不明显(P>0.05);17β-E2试验组(1.0μg/mL)的奶水牛颗粒细胞扩散率、卵裂率及8-细胞率高于未添加试验组(P<0.05);添加P4试验各组(0.9μg/mL、1.2μg/mL)的颗粒细胞扩散率明显低于未添加试验组(P<0.01).

In vitro construction of a recombinant human embryonic brain-derived neurotrophin-4 gene and pEGFP-N1 vector

BACKGROUND: Neurotrophin-4 (NT-4) can promote neuronal growth, development, differentiation, maturation, and survival. NT-4 can also improve recovery and regeneration of injured neurons, but cannot pass through the blood-brain barrier, which limits its ac

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and prema

牛卵泡卵母细胞体外成熟的研究

研究了卵泡大小、卵泡液和卵丘细胞对牛卵泡卵母细胞体外成熟的影响。结果表明:直径2～6 mm 卵泡中的卵母细胞成熟 率(7211 %) 最高,与直径小于2 mm(5814 %) 、6～8 mm(5614 %) 及大于8 mm(3510 %) 卵泡中的卵母细胞组差异显著;成熟培养基 中添加10 %的新鲜牛卵泡液(bFF) 对牛卵母细胞的体外成熟有促进作用,但高浓度的bFF(20 %、30 %) 则抑制牛卵母细胞的体外成 熟;卵丘- 卵母细胞复合体的质量影响卵母细胞的体外成熟,A、B、C 三级卵母细胞成熟率分别为8611 %、6613 %、3516 % ,卵裂率分 别为4218 %、3119 %、1015 % ,差异均显著( P < 0105) 。

Hepatocyte growth factor enhances the generation of high-purity oligodendrocytes from human embryonic stem cells

Generation of homogeneous oligodendrocytes as donor cells is essential for human embryonic stem cell (hESC)-based cell therapy for demylinating diseases. Herein we present a novel method for efficiently obtaining mature oligodendrocytes from hESCs with high purity (79.7 +/- 6.9%), using hepatocyte growth factor (HGF) and G5 supplement(containing insulin, transferrin, selenite, biotin, hydrocortisone, basic fibroblast growth factor and epidermal growth factor) in a four-step method. We induced hESCs into neural progenitors (NP) with HGF (5 ng/ml) and G5 (1 x) supplemented medium in an adherent differentiation system. The purified NPs were amplified in suspension as neurospheres for 1 month, and terminal oligodendrocyte differentiation was then induced by G5 supplement withdrawal and HGF treatment (20 ng/ml). The cells generated displayed typical morphologies of mature oligodendrocytes and expressed oligodendrocyte markers O4 and myelin basic protein (MBP). Our result revealed that HGF significantly enhanced the proliferation of hESC-derived NPs and promoted the differentiation as well as the maturation of oligodendrocytes from NPs. Further studies suggest that HGF/c-Met signaling pathway might play an important role in oligodendrocyte differentiation in our system. Our studies provide a means for generating the clinically relevant cell type and a platform for deciphering the molecular mechanisms that control oligodendrocyte differentiation. (C) 2009 International Society of Differentiation. Published by Elsevier Ltd. All rights reserved.

Lead exposure through gestation-only caused long-term learning/memory deficits in young adult offspring

Numerous observations in clinical and preclinical studies indicate that the developing brain is particular sensitive to lead (Pb)'s pernicious effects. However, the effect of gestation-only Pb exposure on cognitive functions at maturation has not been studied. We investigated the potential effects of three levels of Pb exposure (low, middle, and high Pb: 0.03%, 0.09%, and 0.27% of lead acetate-containing diets) at the gestational period on the spatial memory of young adult offspring by Morris water maze spatial learning and fixed location/visible platform tasks. Our results revealed that three levels of Pb exposure significantly impaired memory retrieval in male offspring, but only female offspring at low levels of Pb exposure showed impairment of memory retrieval. These impairments were not due to the gross disturbances in motor performance and in vision because these animals performed the fixed location/visible platform task as well as controls, indicating that the specific aspects of spatial learning/memory were impaired. These results suggest that exposure to Pb during the gestational period is sufficient to cause long-term learning/memory deficits in young adult offspring. (C) 2003 Elsevier Inc. All rights reserved.

补充营养对柑橘凤蝶卵成熟和雄虫寿命的影响

在实验室条件下,定量地研究了蔗糖、葡萄糖、果糖和蜂蜜等4种补充营养物对柑橘凤蝶卵成熟和雄虫寿命的影响.结果表明:(1)补充蔗糖、果糖和蜂蜜水的雌虫孕成熟卵量都显著高于清水对照组,其中补充蔗糖的雌虫孕成熟卵量最高;(2)不同的补充营养物对雌虫孕亚成熟卵量和未成熟卵量的影响不显著;(3)饲喂蔗糖、果糖和蜂蜜的雄虫寿命显著高于对照组,其中饲喂果糖的雄虫寿命最长;(4)饲喂添加不同浓度NaCl溶液的蜂蜜水的雄虫寿命比仅饲喂蜂蜜水的雄虫短,当NaCl溶液浓度为0.001 mol/L,0.1 mol/L和 1 mol/L 时,雄虫寿命显著低于对照,其中饲喂0.001 mol/L NaCl溶液的雄虫寿命最短.

Localization and distribution of tissue type and urokinase type plasminogen activators and their inhibitors type 1 and 2 in human and rhesus monkey fetal membranes

Fetal membranes consist of 10 distinct layers including components of amnion, chorion and decidua, the latter being of maternal origin. They form mechanically integrated sheets capable of retaining amniotic fluid and play an essential role in protecting fetal growth and development in the pregnant uterus. The extracellular matrix, substrate for plasminogen activators (PAs), is an important supportive framework of the fetal membranes. :Fetal membranes from women with preterm premature rupture of membranes may differ in their protease activity compared with normal membranes. To identify the presence of PAs and their inhibitors (PAI) and their possible role in the process of fetal membrane rupture, this study in investigated the distribution and localization of both protein and mRNA for tissue (t) and urokinase (u) PA and their inhibitors type 1 (PAI-1) and type 2 (PAI-2) in amniochorion of human and rhesus monkey using conventional and. confocal immunofluorescence microscopy. In situ hybridization analysis showed that the distribution and localization of mRNAs for tPA, uPA, PAI-I and PAI-2 were similar in the fetal membranes of human and rhesus monkey; no obvious species difference was observed. Evidence of tPA mRNA was detected in amniotic epithelium, trophoblast cells and nearly all cells of the decidual layer. Strong expression of uPA mRNA was noted in the decidual cells which increased in intensity as the abscission point was approached. Weak staining in chorion laeve trophoblast was also detected. In situ hybridization experiments showed PAI-1 mRNA to be concentrated mainly in the decidual cells, some of which were interposed into the maternal-facing edge of the chorion laeve. Maximal labelling of the decidua occurred towards the zone of abscission. Weak expression of PAI-1 mRNA nas also noted in some cells of the chorion laeve. The distribution of PAI-2 mRNA in amniochorion was also concentrated in the cells of the decidual layer, maximum expression of the mRNA was in the level of abscission. No detectable amount of mRNAs for tPA, uPA, PAI-1 and PAI-2 was found in the fibroblast, reticular and spongy layers. Distribution of the proteins of tPA, uPA and PAI-1 in the fetal membranes of these two species was consistent with the distribution of their mRNA. Anti-PAI-2 immunofluorescence was found to be strongly concentrated in the amniotic epithelium, but PAI-2 mRNA was negative in this layer, suggesting that the epithelium-associated PAI-2 is not of epithelial origin. These findings suggest that a local fibrinolysis in fetal membranes generated by precisely balanced expression of PAs and their inhibitors via paracrine or autocrine mechanisms may play an essential role in fetal membrane development, maturation and in membrane rupture. Following an analysis of the distribution and synthesis of activators and inhibitors it was found that they may play a role in abscission during the third stage of labour. (C) 1998 W. B. Saunders Company Ltd.

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, Delta GRT1 Delta GRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in Delta GRT1 Delta GRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from Delta GRT1 Delta GRT2 cells appear less adhesive than those from the wild type.

Histone H2A Has a Novel Variant in Fish Oocytes

Histone variants and their modification have significant roles in many cellular processes. In this study, we identified and characterized the histone H2A variant h2af1o in fish and revealed its oocyte-specific expression pattern during oogenesis and embryogenesis. Moreover, posttranslational modification of H2af1o was observed that results from phosphorylation during oocyte maturation. To understand the binding dynamics of the novel core histone variant H2af1o in nucleosomes, we cloned ubiquitous gibel carp h2afx as a conventional histone control and investigated the dynamic exchange difference in chromatin by fluorescence recovery after photobleaching. H2af1o has significantly higher mobility in nucleosomes than ubiquitous H2afx. Compared with ubiquitous H2afx, H2af1o has a tightly binding C-terminal and a weakly binding N-terminal. These data indicate that fish oocytes have a novel H2A variant that destabilizes nucleosomes by protruding its N-terminal tail and stabilizes core particles by contracting its C-terminal tail. Our findings suggest that H2af1o may have intrinsic ability to modify chromatin properties during fish oogenesis, oocyte maturation, and early cleavage.

Comparative responses in rare minnow exposed to 17 beta-estradiol during different life stages

Present in the excrement of humans and animals, 17 beta-estradiol (E-2) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E-2 at environmentally relevant (5, 25, and 100 ng l(-1)) and high (1000 ng l(-1)) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l(-1) E-2 in the adult stage, but 100 ng l(-1) E-2 in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l(-1) E-2. These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E-2. The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.