Study on Immunochemical Assays for the Organophosphorus Insecticide Chlorpyrifos

The anti-chlorpyrifos polyclonal antibodies were obtained by using the artificial immune antigen to immune in New Zealand′s white rabbits. The enzyme-tagged antibodies were prepared by coupling horseradish peroxidase (HRP) to the purified antibody with the modified sodium periodate method. The indirect competitive enzyme linked immuno-sorbent assays (ELISA) and the HRP-taggedantibodydirect ELISA (E-Ab) were established, respectively.The limit of detection (LOD) for the indirect ELISA and E-Ab were 0.0033 and 0.0042 μg mL-1, respectively. The linear detection ranged well from 0.005 to 2.0 μg mL-1.     

小鼠冻胚分割试验

研究了分割所用溶液、冷冻前胚胎发育时期和分割后将半胚是否装入透明带对冻胚分割效果的影响。结果证明:①将解冻囊胚分割后,无论是否将半胚装入透明带对冻胚分割的效果均无显著影响(P>0.05);②在PBS中分割解冻桑椹胚和囊胚的效果无显著差异(P>0.05);③与分割解冻桑椹胚相比,在蔗糖液中分割解冻囊胚可显著提高冻胚分割效果(P<0.05)。将在蔗糖液中分割冻囊胚获得10对半胚移植于5只受体,结果有2只妊娠,共获得半胚仔鼠6只,其中2对为同卵双生。     

Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.     

基于RNA-Seq技术的牦牛体外受精胚胎发育转录组分析

【目的】探明不同发育阶段牦牛体外受精（IVF）胚胎的转录组差异,了解差异表达基因（DEG）在功能、分类和代谢通路的差异,为揭示牦牛早期胚胎发育调控机制,促进牦牛胚胎体外生产技术的发展提供理论基础。【方法】以IVF技术生产的牦牛2-细胞、4-细胞、8-细胞、桑椹胚和囊胚5个发育阶段的胚胎为样本分别提取总RNA,采用Smart-Seq2扩增技术构建5个测序文库,应用HiSeq^TM 2500高通量测序技术进行转录组测序,对获得的有效序列进行功能注释及相关生物信息学分析。【结果】牦牛IVF后的卵裂率和囊胚率分别为69.3%和26.2%。2-细胞、4-细胞、8-细胞、桑椹胚和囊胚5个发育阶段的牦牛胚胎Clean reads为47 355 570-50 855 888条,其中有85.65%-90.02%的reads能比对到牦牛参考基因组序列上;8-细胞的胚胎比对上的转录本最多（14 893）,而囊胚比对上的转录本最少（9 827）。牦牛胚胎转录本主要有5种可变剪接类型,转录起始区域可变剪接（TSS）和转录结束区域可变剪接（TTS）所占比例最大;牦牛2-细胞、4-细胞、8-细胞、桑椹胚和囊胚基因组分别有116 601、234 131、196 420、70 841和94 840个位点存在单核苷酸多态性（SNP）。在4-细胞、8-细胞、桑椹胚、囊胚期开始表达的基因分别有1 221、1 116、142和564个;随着胚胎发育的进行,BMP15、KIT、GDF9、STAT3、ZP3和ZP4等母源基因的表达量逐渐减少,而SARS、IL18、ACO2、TXN2、ATP5B、PCGF4、UBE3A、MAPK13、SNURF和JUP等胚胎基因的表达量则逐渐增加。以|log₂ratio| ≥1且Q-value ＜ 0.05为筛选标准,在2-细胞和4-细胞胚胎、4-细胞和8-细胞胚胎、8-细胞胚胎和桑椹胚以及桑椹胚和囊胚比对中分别筛选到6 922、7 601、8 071和10 555个DEGs。GO分析表明,4个发育阶段的DEGs归类注释都涉及生物过程（BP）、细胞组分（CC）和分子功能（MF）3大类62个二级条目。通过KEGG pathway数据库分析,2-细胞和4-细胞期胚胎的DEGs参与到308条通路中,显著富集剪接体、RNA转运和泛素介导的蛋白水解等11条通路;4-细胞和8-细胞胚胎的DEGs参与到310条通路,显著富集嗅觉转导、神经活性配体-受体互作和核苷酸切除修复等9条通路;8-细胞期胚胎和桑椹胚的DEGs参与到316条通路,显著富集嗅觉转导、泛素介导的蛋白水解和神经活性配体-受体互作等10条通路;桑椹胚和囊胚的DEGs参与到315条通路,显著富集剪接体和RNA转运2条通路。【结论】利用高通量测序技术对牦牛IVF胚胎不同发育阶段的转录组进行测序和分析,揭示了牦牛胚胎发育不同阶段差异表达基因的数量,获得了差异表达基因的功能、分类和代谢通路。为丰富牦牛胚胎转录组信息,揭示牦牛胚胎发育分子调控机制及完善其胚胎体外培养技术研究奠定基础。     

小鼠囊胚及孵化胚胎细胞计数方法的探讨

实验以ICR小鼠体外培养的囊胚及孵化胚胎为材料,比较两种方法对小鼠胚胎细胞计数的效果。方法1为采用0.5%的柠檬酸钠低渗溶液和姬姆萨染液对囊胚及孵化胚进行压片,且不同时期胚胎低渗和染色处理时间相同;方法2染色液为苏木精染液,且不同时期胚胎低渗和染色处理时间不同。结果表明,方法2小鼠囊胚及孵化胚胎细胞球轮廓清晰,细胞间隙明显可见,其观察率分别为94.7%和96.8%;而方法1胚胎细胞球间隙不明显,一些细胞轮廓模糊,其观察率分别为70.5%和69.6%。小鼠囊胚及孵化胚胎采用压片计数法,以方法2显著好于方法1(P<0.05)。     

程序化冷冻对小鼠胚胎发育的影响

取小鼠原核(220枚)、2～4细胞胚胎(227枚)、桑椹胚(127枚),将其分为3组,每一时期胚胎分成两半,一半取出后立即冷冻复苏并体外培养至囊胚,另一半体外培养至囊胚后冷冻复苏,子宫冲取囊胚(78枚)冷冻复苏为对照组;各组囊胚均移植至于宫,比较程序化冷冻对小鼠早期胚胎存活率的影响.结果表明:原核、2～4细胞胚胎、桑椹胚体外培养至囊胚后冷冻复苏率分别为56.4%、72.2%、81.6%,移植产仔率分别为38.1%、41.6%、56.8%,原核、2～4细胞组复苏率和产仔率极显著或显著低于对照组,桑椹胚组与对照组差异不显著,显示体外培养对早期胚胎冷冻存活有影响;原核、2～4细胞胚胎、桑椹胚冷冻后体外培养至囊胚,胚胎移植后产仔率分别为25.3%、28.2%、42.1%,与对应胚胎时期体外培养至囊胚冷冻复苏后移植产仔率相比,原核、2～4细胞组均存在显著差异,而桑椹胚组差异不显著,显示原核、2～4细胞的早期胚胎体外培养至囊胚后冷冻可提高冷冻存活率.     

牛-兔种间重组胚体外发育能力的研究

 以超排的青年母兔和经产母兔卵母细胞为受体细胞,以处于休眠期和非休眠期的黑毛和牛耳成纤维细胞为核供体,共获得180枚种间重组胚,采用M199+10%FBS和RD+10%FBS2种培养液进行体外培养,有112枚发育到2-细胞期(62.2%),26枚发育到桑椹胚期(14.4%),20枚发育到囊胚期(11.1%)。结果显示,供体细胞处于休眠期与否,对重组胚的卵裂率无显著影响(P<0.05),但以休眠期细胞为核供体的重组胚8-细胞～16-细胞胚的发育率及桑椹胚发育率显著高于非休眠期细胞的重组胚(P<0.05),二者在     

牛磺酸在牛胚胎体外发育中的作用研究

研究了牛体外受精后早期胚胎体外发育时,向其基础培养液中加入牛磺酸对胚胎桑椹胚率、囊胚率和孵化囊胚率的影响,以探寻牛磺酸克服牛胚胎"体外发育阻滞"现象的作用。试验结果表明:以TCM-199 10?S为基础培养液(对照组),再加入7,14mM的牛磺酸(试验组),试验组与对照组的桑椹胚率分别为48.1%、47.4%和43.2%;囊胚率分别为26.4%、22.3%和21.0%;孵化囊胚率分别为21.8%、18.7%和0%。IVF后2细胞、4～8细胞及8～16细胞期,在基础培养液中分别添加7mM的牛磺酸时,桑椹胚率分别为48.7%、57.1%和52.0%,囊胚率分别为25.1%、30.7%和27.9%,孵化囊胚率分别为25.9%、28.6%和25.0%;而添加14mM牛磺酸组时,桑椹胚率分别为50.4%、56.4%和55.4%,囊胚率分别为26.5%、31.3%和27.7%,孵化囊胚率分别为27.5%、31.1%和29.9%。体外发育培养液中添加7mM牛磺酸可显著提高桑椹胚率和囊胚率(p<0.05),在4～8细胞期添加14mM牛磺酸最为合适。     

Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida

Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewis(x) sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewis(x) sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewis(x) sequence represents the major carbohydrate ligand for human sperm-egg binding.     

黄金鲈鱼的一种ZP基因的电子克隆与鉴定分析

[目的]克隆分析黄金鲈鱼的一种邪基因。[方法]利用生物信息学手段克隆了黄金鲈鱼的一种ZP基因的全长CDNA序列,并对其序列进行了分析。[结果]该cDNA序列包含1653bp的开放阅读框,编码550个氨基酸。氨基酸序列同源性分析和进化分析表明,黄金鲈鱼的ZP蛋白与丰滑舌鳎ZP3b和青鳝ZPC5具有较高的同源性。[结论]所获得的黄金鲈鱼的ZP基因属于ZP3类型。     

两种株型大麦籽粒灌浆特性比较

以竖叶型大麦品种驻啤3号和披叶型大麦品种豫啤1号为试验材料,从籽粒灌浆、蛋白组分和淀粉含量的积累动态变化分析了两种株型大麦籽粒干物质积累之间的差异,结果表明:豫啤1号籽粒灌浆相对较快,在花后20~35 d灌浆速率较高,而驻啤3号籽粒灌浆过程较为平稳;两者之间蛋白组分和淀粉的积累趋势基本一致,豫啤1号籽粒蛋白质含量高于驻啤3号;驻啤3号单株穗重和粒重显著高于豫啤1号,产量较高。     

Effects of Ages of Donors and Conditions of Preserving Ovaries on Porcine Oocytes Maturation in vitro and Efficiency of Parthenogenetic Activation

Effects of different ages of donors and different conditions of preserving ovaries on porcine oocytes maturation in vitro and efficiency of parthenogenetic activation were studied. The experiments included: 1) effects of different temperatures (22, 30, 37, 38.5and 40℃) of preserving ovaries on porcine oocytes maturation in vitro and developmental potential; 2) effects of periods of preserving ovaries on porcine oocytes maturation in vitro and development in vitro; 3) effects of different ages of donors on porcine oocytes maturation in vitro and developmental potential. The results of the experiment showed:1) There were no statistical differences (p＞0.05) of the parthenogenetic cleavage rate (79.64% vs 76.18%) and blastocyst rate (18.11% vs 33.82%) between oocytes from ovaries preserved at 38.5℃ and those preserved at 37℃. When the preserving temperature was increased to 40℃, the cleavage rate (21.68%) and the blastocyst rate (0) were great significantly lower than those at 37℃(p＜0.01). The cleavage rate (80.79% vs 76.18%) and blastocyst rate (29.61% vs 33.82%) were not different between 30 and 37℃(p＞ 0.05). When the preserving temperature was decreased to 22℃, the rate of cleavage was not different,but the rate of blastocyst was significantly lower, compared with that at 37℃; 2) The cleavage and blastocyst rates of the porcine oocytes collected after slaughter 2 or 6h were not different (p＞0.05); 3) The cleavage rate of oocytes from gilts and sows after maturation was not different, but the blastocyst rate of the sow group was significantly higher than that of gilt group (p＜ 0.05). The blastocyst cell number of sows and gilt showed no difference (p＞0.05).     

巨噬细胞集落刺激因子(GM-CSF)对小鼠胚胎体外发育的影响

以小鼠自然受精与体外受精胚胎为模型,探索鼠源巨噬细胞集落刺激因子(GM-CSF)在小鼠胚胎早期发育过程中的生理功能。分别使用添加不同剂量(对照组:0 ng.mL-1;试验组:0.5、2和10 ng.mL-1)GM-CSF的化学限定培养基连续培养小鼠自然受精、体外受精原核期与2细胞期胚胎,检测其胚胎着床前发育效率(卵裂率、囊胚率)及囊胚的质量(囊胚总细胞数、ICM/总细胞数的比率、凋亡指数)。结果发现,对自然受精胚胎而言,原核时期添加不同剂量的GM-CSF,其卵裂率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但10 ng.mL-1试验组的囊胚率显著低于对照组(61.6±5.1)%(P0.05);2细胞时期添加不同剂量的GM-CSF,其囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但试验组中的囊胚凋亡指数均显著低于对照组(P0.05)),并且试验组中2 ng.mL-1的囊胚凋亡指数最低,显著低于0.5 ng.mL-1(P0.05),其他试验组之间的囊胚凋亡指数没有统计学上的差异性。对体外受精胚胎来说,原核时期添加GM-CSF,其卵裂率、囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均无显著性差异;在2细胞时期,10 ng.mL-1组的囊胚率显著低于对照组及其他2个试验组(P0.05),但各组之间的囊胚总细胞数、ICM/总细胞数的比率均无显著性差异。本研究结果表明,在化学限定培养基中添加一定剂量的GM-CSF有利于改善小鼠自然受精与体外受精囊胚的质量,但剂量过高可能不利于小鼠受精胚胎的早期发育。     

砂薄地夏玉米施用包膜氮肥效果研究

在砂薄地上研究了夏玉米施用缓／控释氮肥ZP（包膜氮肥）的效果，结果表明，缓／控释氮肥ZP1次性施用、或以质量比1：1比例配合尿素施用，其增产效应和氮肥利用率与尿素分2次施用无显著差异，与尿素1次施用相比，氮肥肥效和肥料利用率均显著提高。     

印记基因XIST和H19 DNA甲基化水平与猪克隆胚胎发育效率关联分析

目的 体细胞核移植(Somatic cell nuclear transfer,SCNT)在农业、生物医学等领域应用广泛,但是克隆效率太低制约了该技术的应用和推广。本研究的目的在于探究印记基因XIST和H19 DNA甲基化水平与克隆效率的联系。方法 利用同一头猪源耳朵成纤维细胞培养获得14个细胞克隆团,分别作为供体细胞进行SCNT试验,统计比较以各克隆团为供体细胞生产克隆胚胎的囊胚率以及各囊胚的XIST和H19基因调控区的DNA甲基化水平,对XIST和H19基因差异甲基化区域DNA甲基化水平与克隆胚胎的囊胚率进行相关性分析。结果 以1号克隆团为供体细胞得到囊胚的XIST基因DNA甲基化水平最高且囊胚率最低,分别为65.04%、8.6%,以14号克隆团为供体细胞得到XIST基因囊胚的DNA甲基化水平最低但囊胚率最高,分别为16.68%、38.2%;相关性分析表明XIST基因的DNA甲基化水平与囊胚率之间存在高度负相关(|r|=0.8125>0.8)。H19基因A侧甲基化平均水平最高和最低分别为5.12%和0.61%,相关性分析表明H19基因A侧DNA甲基化水平与囊胚率间只有极弱的相关(|r|=0.1647<0.3);H19基因G侧DNA甲基化水平最高和最低分别为90.92%和72.69%,相关性分析表明H19基因G侧DNA甲基化水平与囊胚率间只有低度相关(0.3<|r|=0.3098<0.5)。结论 XIST基因DNA甲基化水平越低,则克隆团囊胚率越高。研究结果对提高猪SCNT胚胎发育效率,改善现有的猪SCNT技术体系提供了基础。     

TSA浓度及处理时间对猪体细胞核移植胚胎体外发育影响

曲古抑菌素A(Trichostatin A,TSA)是一种组蛋白去乙酰化酶抑制剂.有研究表明TSA处理可以提高核移植胚胎的发育率.为探讨TSA对猪核移植胚胎发育的作用,试验重点研究了TSA浓度及处理时间对核移植胚胎体外发育的影响,同时也探索了TSA对不同供核细胞构建的重构胚体外发育的影响.结果表明,40 nmol·L-...     

包膜氮肥养分释放研究

通过溶出率试验和淋溶试验对研制的2种包膜肥料ZP1，ZP2的氮素释放特征进行了研究，结果表明，ZP1的初期溶出率和养分平均释放速率均高于ZP2，包膜肥料的养分释放符合缓释／控释肥料的基本要求；ZP1有一个养分释放高峰，ZP2有两个释放高峰，2种类型肥料的养分累积释放量均符合一级动力学方程。     

颗粒细胞单层对黄牛孤雌胚胎体外发育的影响

为优化颗粒细胞单层共培养体系,研究了不同类型、不同种属的颗粒细胞单层及不同时间更换培养单层对黄牛孤雌胚胎体外发育的影响.结果表明.就卵裂率、囊胚率和囊胚孵出率而言.壁颗粒细胞单层组与丘颗粒细胞单层组之间无显著差异(P>0.05);来自黄牛、猪和小鼠的颗粒细胞单层在支持黄牛孤雌胚胎体外发育方面无显著差异(P>0.05):就囊胚率而言,共培养的第3天和第6天两次更换单层分别与共培养的第4天更换单层和始终不更换单层的对照组之间无显著差异(P>0.05),但第4天更换单层与对照组之间有显著差异(P<0.05).所以.不同类型和不同种属的颗粒细胞单层都能很好地支持黄牛孤雌胚胎的体外发育.在胚胎的体外发育过程中更换培养单层可以明显提高胚胎的囊胚率和囊胚孵出率,并且在共培养的第4天更换培养单层可以得到较高的囊胚率.     

不同培养体系对绵羊体外受精胚胎发育及细胞凋亡的影响

[目的]通过SOFaaBSA和CR1 aaBSA - FBS两种培养液对体外胚胎发育率及胚胎凋亡发生的影响,筛选适合绵羊体外受精胚胎发育的培养体系,为相关研究提供一定的理论依据和研究基础.[方法]采用激光共聚焦显微镜和细胞原位凋亡检测技术(TUNEL),分析这两种胚胎体外培养系统的绵羊体外受精胚胎发育的影响及各发育阶段的细胞凋亡状况及对胚胎发育的影响.[结果]SOFaaBSA和CR1aaBSA- FBS组的卵裂率、囊胚率和囊胚孵化率均高与其它实验组,但这两组之间差异不显著(P＞0.05).同时,在两种培养液培养的2细胞、3-8细胞期的绵羊体外受精正常形态的胚胎中均未发现凋亡信号,凋亡信号在两种培养液中都是首次出现在9- 16细胞胚胎细胞中,并且随着胚胎发育至囊胚期,凋亡细胞效随着胚龄增长越来越多.在SOFaaBSA培养液培养的桑椹胚和囊胚细胞胚胎凋亡率显著低于CR1aaBSA - FBS培养液培养的桑椹胚和囊胚(P＜0.05),同时,在CR1aaBSA- FBS培养液中桑椹胚和囊胚细胞平均具有凋亡细胞的数量显著高于SOFaaBSA培养液(P＜0.05).[结论]CR1aaBSA- FBS培养系统显著的增加了绵羊晚期体外胚胎的凋亡.CR1 aaBSA - FBS能够支持绵羊体外受精胚胎的发育,但SOFaaBSA培养液更适合绵羊体外胚胎的发育.     

Deciphering the cross-talk of implantation: advances and challenges

Implantation involves a series of steps leading to an effective reciprocal signaling between the blastocyst and the uterus. Except for a restricted period when ovarian hormones induce a uterine receptive phase, the uterus is an unfavorable environment for blastocyst implantation. Because species-specific variations in implantation strategies exist, these differences preclude the formulation of a unifying theme for the molecular basis of this event. However, an increased understanding of mammalian implantation has been gained through the use of the mouse model. This review summarizes recognized signaling cascades and new research in mammalian implantation, based primarily on available genetic and molecular evidence from implantation studies in the mouse. Although the identification of new molecules associated with implantation in various species provides valuable insight, important questions remain regarding the common molecular mechanisms that govern this process. Understanding the mechanisms of implantation promises to help alleviate infertility, enhance fetal health, and improve contraceptive design. The success of any species depends on its reproductive efficiency. For sexual reproduction, an egg and sperm must overcome many obstacles to fuse and co-mingle their genetic material at fertilization. The zygote develops into a blastocyst with two cell lineages (the inner cell mass and the trophectoderm), migrates within the reproductive tract, and ultimately implants into a transiently permissive host tissue, the uterus. However, the molecular basis of the road map connecting the blastocyst with the endometrium across species is diverse (1) and not fully understood. Recent advances have identified numerous molecules involved in implantation (1-4), yet new discoveries have not yielded a unifying scheme for the mechanisms of implantation.