Effects of glutathione treatments during sperm washing and in vitro fertilization on the in vitro early development of embryos of Japanese Black cattle

This study was conducted to examine the effects of adding glutathione (1 mM) to media used for sperm washing and in vitro fertilization (IVF) on the improvement of early development of embryos produced using cryopreserved spermatozoa of the less IVF-competent bull (the one considered unqualified as spermatozoa supplier for the production of bovine blastocysts using IVF). The cryopreserved spermatozoa of this bull were characterized by normal motility and lower ATP content and blastocyst productivity than those of IVF-competent bulls. The addition of glutathione to the sperm washing medium was more effective in improving the productivity of blastocysts and ATP content than the addition of glutathione to the IVF medium or no glutathione addition at all (control). These results suggest that this simple method may be used to improve the potential of cryopreserved spermatozoa of less IVF-competent bulls to fertilize oocytes in vitro and to induce normal embryonic development after fertilization.     

苜蓿皂苷对大鼠肝脏及肝脏细胞低密度脂蛋白受体、三磷酸腺苷结合盒转运体mRNA表达的影响

本试验旨在研究苜蓿皂苷对大鼠肝脏及大鼠肝脏细胞(BRL细胞)胆固醇清除和转运途径中关键基因低密度脂蛋白受体(LDLR)、三磷酸腺苷结合盒转运体G5(ABCG5)、三磷酸腺苷结合盒转运体G8(ABCG8)mRNA表达量的影响,从个体和细胞水平初步探讨苜蓿皂苷调控胆固醇清除和转运的分子机制。采用高脂饲粮建立大鼠高脂模型,测定苜蓿皂苷对正常、高脂大鼠血清指标[总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)含量]和肝脏LDLR、ABCG5、ABCG8 mRNA表达量的影响;采用高糖DMEM培养液建立BRL细胞脂变模型,测定苜蓿皂苷浓度对BRL细胞活性的影响,测定苜蓿皂苷对正常、脂变细胞LDLR、ABCG5、ABCG8 mRNA表达量的影响。结果表明:1)苜蓿皂苷显著降低高脂大鼠血清中TG、TC和LDL-C的含量(P0.05);2)苜蓿皂苷显著上调正常大鼠肝脏LDLR、ABCG5、ABCG8及高脂大鼠ABCG5、ABCG8 mRNA表达量(P0.05);3)添加200、250μg/m L苜蓿皂苷显著提高了BRL细胞的活性(P0.05);4)苜蓿皂苷显著上调正常BRL细胞LDLR、ABCG5、ABCG8 mRNA表达量(P0.05),而对脂变BRL细胞各基因mRNA表达量无显著影响(P0.05)。结果提示,苜蓿皂苷可通过调控LDLR、ABCG5、ABCG8 mRNA的表达来增加肝细胞内胆固醇的清除和转运,从而降低机体胆固醇的含量。     

High concentration of extracellular ATP causes autophagy and apoptosis of SH-SY5Y cells

AIM：To examine the effects of high concentration of extracellular ATP on human neuroblastoma SH-SY5Y cell injury. METHODS：Cultured SH-SY5Y cells were grouped according to the concentrations of ATP and treatment time. The cell viability was detected by CCK-8 assay. The variation of autophagic vacuoles was observed with monodansylcadaverine staining. The cell apoptosis was analyzed by Hoechst 33258 staining. Meanwhile, apoptotic rate was detected by flow cytometry. The levels of caspase-3 and microtubule-associated protein 1 light chain 3-Ⅱ (LC3-Ⅱ) were determined by Western blotting. RESULTS：Compared with control group, the survival rate of SH-SY5Y cells was significantly reduced by ATP at different concentrations (3, 6, 9, 12 and 15 mmol/L for 3 h) and different treatment time (1, 2, 3 and 6 h with 6 mmol/L ATP, peaking at 3 h). The autophagic vacuoles of SH-SY5Y cells were significantly increased at 1 h with ATP treatment, trended to decrease over time and returned to control level at 6 h. The protein expression of LC3-Ⅱ was significantly increased at 1 h with ATP treatment, which was consistent with the time points of increasing autophagic vacuoles. LC3-Ⅱ expression level gradually decreased at 2~3 h with ATP treatment, and returned to control level at 6 h. Compared with control group, the apoptotic rate and the expression level of caspase-3 were enhanced synchronously. The peak of apoptotic rate occurred at 3 h, and kept until 6 h.The level of cleaved caspase-3 expression peaked at 6 h. CONCLUSION：High concentration of extracellular ATP induces the autophagy and apoptosis of SH-SY5Y cells. The increased autophagy shows up, followed by the climax of apoptosis until 6 h. With the prolonged duration of ATP, apoptosis is the main process in the cells.     

不同培养基培育蛹虫草中的虫草素和腺苷含量测定

虫草素和腺苷是蛹虫草的重要活性成分,通过高效液相色谱(HPLC)法测定不同培养基培育蛹虫草中的虫草素和腺苷含量,筛选能生产高品质蛹虫草的培养基。测定结果表明,用不同培养基培育的蛹虫草子实体中,其虫草素和腺苷含量存在显著差异,总体表现为粳米+家蚕蛹浸提液培养基>糯米+家蚕蛹浸提液培养基>粳米培养基>糯米培养基,其中用粳米+家蚕蛹浸提液培养基培育蛹虫草子实体中的虫草素和腺苷质量比分别高达15.37 mg/g和39.96 mg/g。此外,相同培养基培育的蛹虫草子实体、菌丝体和培养基质中的虫草素与腺苷含量存在极显著差异,总体表现为子实体>菌丝体>基质。试验结果表明,粳米培养基中添加家蚕蛹浸提液可显著提高蛹虫草中的虫草素和腺苷含量。     

Evaluation of Root Sink Ability of Sweet Potato (Ipomoea batatas Lam.) Cultivars on the Basis of Enzymatic Activity in the Starch Synthesis Pathway

Activities of five enzymes, sucrose synthase (SUS), uridine 5'-diphosphate glucose pyrophosphorylase (UDPGPPase), fructose-1,6 bisphosphatase (FBPase), adenosine 5'diphosphate glucose pyrophosphorylase (ADPGPPase) and starch synthase (STS), in the metabolic pathway of starch synthesis, were compared between two sweet potato ( Ipomoea batatas Lam.) cultivars, Koganesengan (C.V.KOG, a recently released high yield cultivar) and Tsurunasigenji (C.V.TSU, an old, local cultivar with poor yield). The measurements were carried out using the root samples (tuberous, thick and fibrous roots) harvested at the fast tubering stage. Of the five enzymes, SUS, ADPGPPase and STS showed high activities in the tuberous root, particularly in that of C.V.KOG, and a similar trend was observed for activities of these enzymes on a protein basis. The increased activity of the three enzymes is considered to be one of the characteristics in a high yield cultivar, allowing the root to function effectively as a starch synthesis and storage organ.     

采收期对阿克苏地区灰枣cAMP含量的影响研究

[目的]研究测定灰枣cAMP含量的新方法及灰枣采收期对其cAMP含量的影响.[方法]以阿克苏地区灰枣为研究对象,建立了HPLC法测定灰枣中cAMP含量的定量测定方法,并对不同采收时期的灰枣及其制干产品的cAMP含量进行了测定.[结果]试验表明,HPLC测定cAMP含量的色谱条件为:选用Inertsil ODS-3色谱柱(4.6 ×250 ×5 μm)、以10％甲醇-90％的浓度为0.167 8％甲酸溶液为流动相、柱温30℃、流速0.8 ml/min、检测波长254nm测定灰枣中的cAMP含量,cAMP浓度在1.0～100.0 μg/ml范围内与峰面积具有良好的线性关系,R2=0.9990,且其精密性高、重现性好、加标回收率高(＞98％,RSD值为1.38％).灰枣采收时期对鲜枣及其制干产品的cAMP含量有显著影响(达到0.01显著水平).[结论]阿克苏地区灰枣在10月24日至10月31日期间采收,有利于保持较高的cAMP含量,此期采收的鲜灰枣及其干枣的cAMP含量分别为75.237和294.097μg/g左右.研究可为新疆阿克苏地区红枣产业化发展提供科学参考.     

蝙蝠蛾拟青霉高产腺苷液体培养基优化

为了提高蝙蝠蛾拟青霉菌株RCEF0936腺苷的代谢量,以腺苷含量为指标,采用高效液相色谱法(HPLC),在单因子试验的基础上通过正交试验对其高产腺苷的液体培养基进行优化研究。结果表明,在培养温度25℃、摇床转速140rpm/min、摇瓶装液量100ml/250ml、接种量5%、培养时间6d的条件下,蝙蝠蛾拟青霉菌株RCEF0936的高产腺苷的最佳液体培养基配方为:葡萄糖2%,酵母浸出粉2%,硫酸锰0.05%,硫酸锌0.05%;试验中选择的4种因子对蝙蝠蛾拟青霉菌株RCEF0936菌丝体中腺苷含量的影响由大到小依次是硫酸锌酵母浸出粉硫酸锰葡萄糖,且均达到极显著水平(P0.01)。利用优化的培养基培养的蝙蝠蛾拟青霉菌株RCEF0936菌丝体中腺苷含量可达到2.85mg/g,比优化前提高了390%,经统计学分析,两者差异极显著(P0.01)。     

Relationship between ATP content and motility in bovine spermatozoa with reference to the effects of the bull and the A.I. centre.

Deep-frozen semen from 28 bulls belonging to 6 different A.I. centres was studied after thawing and the ATP content in the spermatozoa was assayed using a bioluminescence technique. The sperm motility was subjectively estimated under a phase contrast microscope and the sperm concentration of each ejaculate was calculated in a haemocytometer. The overall mean ATP content was 16.6 nmoles ATP/spermatozoa x 10(8). There was a significant variation in ATP content between A.I. centres. Significant differences between bulls in ATP content were found as well as a significant correlation between ATP concentration and the number of motile spermatozoa. This may indicate that ATP assessment may be useful as an additional, objective laboratory test.     

应用亲水作用液相色谱法测定红花注射液腺苷含量

试验采用氨基柱(150 mm×4.6 mm,5μm粒径,10nm孔径),流动相A:CH3CN;流动相B:5 mM乙酸铵水溶液;梯度:0～5 min(5%～30%B),50～60 min(30%B);检测波长:270 nm;流速:1.0 mL/min。旨在亲水作用色谱模式下,建立测定红花注射液中腺苷含量的方法。结果表明:腺苷浓度在2.5～20μg/mL范围内,线性关系良好,平均回收率为98.56%,RSD为3.17%(n=9)。该方法简便、快速、准确,结果可靠。