Bisphenol A (BPA) is reported to impair male fertility. The alleviating effect of Astragalus polysaccharide (APS) on sperm oxidative damage caused by BPA exposure was analysed for the first time. In this study, the effect of APS (0.25, 0.5, 0.75, 1 mg/mL) on motility of BPA-exposed sperm, energy metabolism indexes, and antioxidant parameters was evaluated. In addition, the effects of APS supplementation on protein tyrosine phosphorylation of BPA-exposed sperm were assessed. The results showed that the addition of APS (0.5 and 0.75 mg/mL) significantly increased motility of BPA-exposed sperm by decreasing the content of malondialdehyde and improving activities of superoxide dismutase and catalase (p < .05). Administration of different doses of APS to BPA-exposed sperm improved mitochondrial membrane potential and energy production (p < .05). Moreover, APS protected and alleviated tyrosine phosphorylation protein on the principal-pieces of BPA-exposed sperm flagella. In conclusion, supplementation with APS enhanced the antioxidant capacity of BPA-exposed sperm and improved in vitro capacitation, thereby improving the reproductive capacity of sperm exposed to environmental hormones.

AbstractObjectivesThe aim of this study was to evaluate the effects of zinc chloride (ZnCl2) at 20, 50, 100, and 200 μg/mL on the quality of seminal plasma‐free boar sperm stored at 17°C for 7 days and to explore the underlying mechanisms.MethodsBoar sperm were collected and incubated in non‐capacitation/capacitation medium to analyze sperm quality.ResultsIn the non‐capacitated state, the addition of ZnCl2 at 20 and 50 μg/mL improved the survival rate and plasma membrane integrity of boar sperm (p < 0.05). Compared to the control group, the addition of ZnCl2 significantly increased total antioxidative capacity and CuZn superoxide dismutase activity, while reducing the malondialdehyde content (p < 0.05). ZnCl2 at 100 and 200 μg/mL significantly decreased sperm motility, protein kinase A (PKA) substrate phosphorylation, and tyrosine phosphorylation. These proteins were mainly located on the mid‐pieces of the flagellum. The addition of ZnCl2 at 20 and 50 μg/mL conveyed a protective effect to boar sperm stored at 17°C. Furthermore, ZnCl2 at 100 and 200 μg/mL inhibited sperm motility via tyrosine phosphorylation, thus preventing the ‘capacitation‐like’ state. In the capacitated state, there was no change in PKA substrate phosphorylation and tyrosine phosphorylation of the mid‐pieces of the flagellum compared to the control groups, indicating that the addition of Zn2+ did not negatively affect capacitation of preserved sperm.ConclusionsZnCl2 showed protective capacity to the preservation extender used for boar sperm during the process of 17°C storage, and the optimal concentration of ZnCl2 for the preservation extender was 100 μg/mL.

为了评价纳米氧化锌颗粒(ZnO NPs)对17℃猪精子保存质量的影响,本试验在17℃保存猪精子稀释液中添加20,50,100,200 mg/L ZnO NPs,对保存精子质量和功能进行分析.结果显示,与对照组比较,添加50 mg/L ZnO NPs可以显著提高保存精子的总抗氧化能力(P＜0.05),降低保存精子丙二醛含量(P＜0.05).50,100,200 mg/L ZnO NPs处理组精子膜完整性显著高于对照组(P＜0.05),骨架蛋白和膜蛋白磷酸化水平显著上调(P＜0.05),100,200 mg/L处理组精子全蛋白磷酸化水平显著上升,且定位于精子鞭毛中段和主段.ZnO NPs添加不影响cAMP/PKA信号通路.推测ZnO NPs可能通过提高抗氧化能力来有效提高猪精子活力,消除过量丙二醛,提高猪精子蛋白磷酸化水平,从而提升膜完整性.

Bisphenol A (BPA) is a synthetic estrogen compound, which widely exists in the environment, interferes with mammalian endocrine and affects the function of reproductive system of males. Taking fresh sperm of boar, 17 ℃ preservation boar sperm, and mouse sperm as test materials, we examined the effects of BPA (0, 0.1,1,10,100 μmol∙L-1) on proteins tyrosine phosphorylation in sperm and the molecular mechanism by using wes-tern blot (WB) and immunofluorescence techniques coupled to in vitro culture method. The results showed that low BPA concentration (0.1, 1 μmol∙L-1) markedly accelerated the protein tyrosine phosphorylation of fresh boar capacitated sperm. However, the tyrosine phosphorylation of boar sperm decreased in high BPA concentration (10, 100 μmol∙L-1). The tyrosine phosphorylation of the mouse sperm raised with the increases of BPA concentration. Moreover, BPA affected different kinds of proteins related to tyrosine phosphorylation modification of porcine and mouse sperm capacitation, suggesting that the effect of BPA exposure on mammalian sperm was species-specific. Furthermore, the results of immunofluorescence showed that the effects of BPA on protein tyrosine phosphorylation in sperm mainly occurred in the middle and principal piece of flagellum.

实验旨在分析17℃保存对获能精子蛋白酪氨酸磷酸化的影响,通过精子蛋白亚组分分离及酪氨酸磷酸化鉴定,研究精浆以及中药水提液对保存精子获能相关蛋白酪氨酸磷酸化的影响.选择获能指标-酪氨酸磷酸化分析新鲜猪精子和17℃保存猪精子,提取精子全蛋白、膜蛋白、核蛋白以及骨架蛋白,利用WB分离及鉴定酪氨酸磷酸化差异.结果显示:17℃保存条件下,获能精子在40～45、46～50 ku分子量膜蛋白酪氨酸磷酸化程度减弱;未获能精子在30～35 ku分子量胞浆蛋白磷酸化程度增强,出现似"获能"现象;精浆在保存中起到提供营养的作用但易导致细菌滋生;中药水提液对精子保存过程中的保护效果体现在精子获能培养后,鞭毛中段、主段酪氨酸磷酸化和新鲜精子无明显差异.

哺乳动物成熟精子是一种高度分化的生殖细胞,“获能”后具有受精能力,其中酪氨酸磷酸化过程是获能过程中非常重要的环节,参与调控获能相关的顶体反应和超激活运动.精子获能分子机理研究有助于解决临床男性不育以及男性避孕问题,同时为牲畜体外人工授精技术应用提供理论支持.目前哺乳动物获能精子蛋白酪氨酸磷酸化的分子机制研究主要集中在信号通路、蛋白质种类鉴定、影响因素等方面.

主要研究菌根的形成对蓝莓幼苗生长及矿质元素吸收的影响.采用分子生物学方法对3个菌株2-R-01-1-A、7-R-10-2和2-R-32-2进行分类学鉴定,再将其分别与生根蓝莓幼苗共生培养60天,测定菌根真菌的侵染率、菌根依赖性、植株生物量以及植株中矿质元素的含量.结果 表明:3个菌株分别归为毛霉属(Mucor sp.)、角担菌属(Ceratobasidium sp.)和毛壳菌属(Chaetomium sp.);3个菌株对蓝莓根均存在侵染情况,对2-R-01-1-A和7-R-10-2为弱依赖性,对2-R-32-2为中等依赖性;3株菌根真菌对蓝莓幼苗的生长和矿物质元素的吸收均没有促进作用,菌根真菌7-R-10-2和2-R-32-2提高了蓝莓幼苗的含水率.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that play important roles in the regulation of gene expression. However, the role of miRNAs in bovine mammary gland responses to heat stress is not well understood.

The sperms of mammal need normal kinetism to pass through female reproductive tract to fertilize with the egg.The hyperactivated sperm can penetrate the zona pellucida of the egg to combine with the egg to form oosperm.Activation and maintenance of the hyperactivation of sperm are accomplished through the combination of their own physiology and the biochemical environment within the female reproductive tract.From the introduction of "hyperactivation" to now,it has been one of the most important parts of the reproductive physiology research.This review discusses the relationship between hyperactivation and fertilization,the known structures of flagellum,regulatory factors,technology to analyse the movement of hyperactivated sperm,and the main investigating fields in the future to provide theoretical reference for sterility and contraception.

以廊坊市主要园林绿化树种白蜡(Fraxinus chinensis Rosb.)、法国梧桐(Platanus orientalisL.)、桑树(Morus alba L.)、臭椿[Ailanthus altissima(Mill.) Swingle]、毛白杨(Populus tomentosa Cart.)和香椿[Toona sinensis(A.Juss.) Roem.]为目标树种,2014年9月在其3年生林地中进行美国白蛾[Hyphantriacunea(Drury)]人工接种试验,并用目标树种的离体叶片饲喂美国白蛾幼虫,统计其食叶量,同时测定目标树种非自然侵染前后可溶性蛋白质含量的变化.结果表明:美国白蛾对目标树种取食量的高低顺序为桑树＞法国梧桐＞臭椿＞白蜡＞毛白杨＞香椿;美国白蛾取食后,树种(抗性和感性)叶片内可溶性糖含量的变化没有规律,抗虫性高低与可溶性糖含量的高低没有对应关系.

In September 2014,an experiment of factitious infection by Hyphantria cunea( Drury)( Lepidoptera: Arctiidae) was done in 3-year-old woodland of target garden greening tree species,namely Fraxinus chinensis Rosb.,Platanus orientalis L.,Morus alba L.,Ailanthus altissima( Mill.) Swingle,Populus tomentosa Carr.,Toona sinensis( A. Juss.) Roem in Langfang,Hebei. The larvae of H. cunea were reared on leaves of the target species under laboratory conditions. Leaf consumption and changes of soluble protein content before and after factitious infection by H. cunea were measured. The results indicated that the order of leaf consumption by H. cunea was arranged as M. Alba Pl. orientalis A. altissima F. chinensis P. tomentosa T. sinensis. The soluble protein content ascended firstly and then descended in target species M. alba,Pl.orientalis,A. altissima and F. Chinensis with low resistance to H. cunea,but it descended firstly in P. tomentosa with high resistance to H. cunea after 2,4,6,8,10 days of feeding,respectively; but these protein contents on day 10 were lower than that before feeding by H. cunea and of the control. The results demonstrated that the effect of H. cunea on protein contents of target species varied with the extent of tree species tolerance to the moths. The resistance of trees to H. cunea was insignificantly correlated with protein content.

During the process of incubation in vivo or in vitro, the changes associated with mammalian sperm capacitaion include: cholesterol efflux, plasma membrane reorganization, ion channel regulation and changes in the phosphorylation state of many proteins. The effect of signaling pathways and corresponding signal molecules plays an important role during sperm capacitation and modification of functions, and becomes the key links of sperm capacitation to hyperactivation and fertilization completed of sperm cells. Based on some reports published in recent years, this review examines that during the process of mammalian sperm capacitation, the known signal pathways, signal molecules, regulatory factors, ion channels, the existing questions and the main investigating fields in the future to provide theoretical reference for in vitro fertilization and assisted reproduction.

In order to study the effects of the pads fermented with different combined probiotics on the digestibility of amino acid and nitrogen in pigs,the feeding experiments in the deep-litter production systems were conducted using 60 hybrid pigs(Duroc × Landrace × Yorkshire) with weight around 20 kg.The experimental animals were randomly divided into 3 experimental groups(JDM,SHC,GLD) based on the different compound probiotics used in the pads and one control group(CON).The experiment lasted for 30 days.The results showed that the content of nitrogen and apparent digestibility of amino acids among 3 experimental groups was not significant different(P0.05).However,the apparent digestibility of nitrogen and multi-amino acids in JDM group showed the tendency of increasing.Compared with CON,the apparent digestibility of Ser(84.3%),Gly(75.6%),Lys(85.4%),Phe(85.6%) increased significantly in JDM group(P0.05) and the increased percentage were 6.2,4.9,5.9 and 6.8,respectively.The apparent digestibility of Met(69.9%),Try(81.4%) in GLD group was the highest and significantly higher than that of CON group(62.3% and 73.1% for Met and Try,respectively)(P0.05).The results indicated that the deep-litter production systems were useful to increase digestibility of amino acid of diets in pigs of growing stage.The effect of probiotics type in the deep-litter production systems on the digestibility of amino acids in diets can not be neglected.In addition,the effects of compound probiotics were better than that of single probiotics in improving the digestibility of amino acids in pigs.

测定超低温冷冻保存过程能否诱导猪冻融精子产生“似凋亡”变化,探讨猪冻融精子活力降低的分子机理.利用Annexin-V/PI及JC-1荧光抗体试剂盒与流式细胞术相结合的方法,检测猪精子冷冻前后及不同培养时间冻融精子质膜中磷脂酰丝氨酸(PS)位置及线粒体膜电位(△ψm)变化;蛋白免疫印迹分析方法测定猪精子Bcl-2、Caspase-9及Caspase-3等“凋亡”因子含量变化.猪精子超低温冷冻保存后活性精子的比例(54.4％)显著低于鲜精组(87.9％)(P＜0.05),其中PS移位的精子“似凋亡”比例约为26.8％,同鲜精组(5.6％)相比增加21.2个百分点(P＜0.05);降温平衡及冷冻复苏后猪精子线粒体膜电位变化显著,其中低△ψm精子比例显著增加(P＜0.05);同新鲜精子(19.6％)相比,冷冻复苏后(79.4％)低△ψm精子比例提高59.8个百分点(P＜0.05);冻融精子中Bcl 2、Caspase-9及Caspase-3等凋亡因子活性显著高于鲜精子及降温平衡后的精子(P＜0.05),由此证明冻融过程能诱导猪精子进入凋亡状态.超低温冷冻保存能导致猪冻融精子质膜磷脂酰丝氨酸(PS)移位及线粒体膜电位降低,同时激发精子Bcl-2、Caspase-9及Caspase-3等凋亡因子活性,诱导成活冻融精子提前进入“似凋亡”状态,明显缩短冻融精子存活时间,这可能是猪冻精繁殖力降低的又一诱因.

In order to provide theoretical premise for the feasibility of the abandoned litters to be used in the future as fertilizers for farm land,in this study,the feeding experiments in pigs were conducted using the pad obtained from the farm sub products fermented with the combined probiotics.The contents of N,P,Cu,Zn and heavy metal elements of litters were analyzed based on comparing the digestibility of the mineral substances in these deep-litter fermentation systems using different probiotics.The results showed that the apparent digestibilities were not significantly different among these groups(P>0.05) during the growing and fattening stage.However,with the prolonged experimental dates,the contents of N,P,Cu,Zn and heavy metal elements including As and Pb in the litters increased gradually.After 3 weeks,the contents of N and P were about 2.28% and 1.65%,respectively.The enrichment amount of Cu,Zn,As and Pb were 266,93.3,2.86 and 0.92 mg/kg,respectively.During the feeding period,the enrichment amount of Cu was obviously higher than that of Zn and the enrichment amount of As was significantly higher than that of Pb.The contents of N,P,Cu,Zn,As and Pb among the different experimental groups were no significantly different(P>0.05).In addition,the contents of N and P in the abandoned litters from the deep-litter fermentation systems were relatively higher,which could be used in the farmland.Unfortunately,the enrichment amount of mineral substances(including Cu,Zn) and heavy metal(including As,Pb) were also higher.Thus,the fertilizer efficiency and feasibility of the abandoned litters from the deep-litter fermentation systems for future use will be further investigated.

The aim of this study was to separate subsets proteins in uncapacitation and capacitation of boar sperm cells and to identify the protein tyrosine phosphorylation,and to establish the theoretical foundation for the mammalian sperm fertilization biology research.In vitro,the boar sperms were cultivated for capacitation and separated subsets proteins.The tyrosine phosphorylated proteins in uncapacitation and capacitation sperms were identified by using SDS-PAGE and Western blot.In capacitation boar sperm,the degree of tyrosine phosphorylation on 126,108,79 ku proteins were significantly higher than them on uncapacition boar sperm.The vigor indexes and the results of SDS-PAGE of uncapacitation and capacitation sperms were different,all the vigor indexes of capacitation sperms were significantly improved and these changes were related with tyrosine phosphorylation.The molecular weight of tyrosine phosphorylated proteins on membrane were approximately 25,47 and 50 ku and on cytoplasm is about 47 ku.The degree of tyrosine phosphoproteins of 25 and 47 ku in capacitation sperm were significantly higher than them in the uncapacitation sperm(P<0.05).This phenomenon also existed in cell nucleus.In sperm nucleus,these proteins' molecular weight were 23,37 and 42-50 ku.The degree of tyrosine phosphrylated 23 ku protein in nucleus of capacitation sperms are higher.The results suggests that the main tyrosine phosphorylated proteins were on the nucleus and membrane proteins,and a few of them are plasma proteins.

Studies on protein expression,modification and interaction have become important in proteomics in the post-genomics era.Phosphorylation/dephosphorylation of proteins is the main mechanism of signal transduction and regulation of enzymes in sperm cells,what's more,it plays a key role during the sperm-egg recognition and fertilization process.Researches about function of phosphorylated proteins contribute to the understanding of the molecular mechanism of sperm capacitation,hyperactive motility and acrosome reaction process.The progress in the researches of mammalian sperm phosphoproteomics was reviewed that include the phosphoproteomics technology,phosphorylated proteins identification and quantitation,function analysis benefits in finding new important fertilization related biological markers and revealing the sperm development,reproductive potential changes and molecular mechanism of fertilization.

American white moth is a remarkable worldwide quarantine pest.By the combination of indoor incubation and field observe results of American white moth in Langfang City of Hebei Province,series prevent and control bottleneck factors for the invasive agricultural pest are analyzed,such as the main operation mode of the pest against host trees,diffusion and migration characteristics,biological characteristics,natural enemy control,pesticide prevention and so on.The research aims to search for the breakthrough point of the development of environment-friendly control techniques against American white moth,which also provides the reference for further improvement of integrated pest management system.