换液培养对猪卵母细胞的成熟及胚胎体外发育的影响

通过后期去除激素培养比较了卵丘卵母细胞成熟及构建克隆胚后的发育情况,同时孤雌胚胎和体外受精胚胎体外培养48 h后全量换液,比较了其卵裂率和囊胚率之间的差异。结果显示,卵丘卵母细胞培养22h后换成不含激素的成熟培养液继续培养至44 h,其成熟率与对照组无显著差异(P0.05);后期克隆胚胎卵裂率和囊胚率与对照组均无显著差异(P0.05)。孤雌胚胎换液培养卵裂率高于未换液组,囊胚率低于未换液组,但差异均不显著(P0.05)。体外受精胚胎换液培养卵裂率以及囊胚率和未换液组差异不显著(P0.05)。本实验结果说明换液培养对卵母细胞的成熟及后续的体细胞克隆胚发育无显著影响,孤雌和体外受精胚胎换液培养对后期发育也无显著影响。     

论人类胚胎基因编辑技术之应用条件

在人类胚胎基因编辑技术尚未完全成熟的前提下,只要满足了以下三项条件,仍然能够应用于人:一是当事人的重大切身利益遭受了重大损失或面临重大威胁;二是成熟技术无力挽回当事人的重大切身利益所面临的重大损失或重大威胁;三是得到了当事人或其法定代理人的同意。至于这种技术完全成熟后,基因编辑技术的应用就可以由必须型扩大至加强型,只是后者的应用出于社会公平和国家竞争力的考量,必须覆盖全民。     

哺乳动物胚胎着床前的基因表达与调控

综述近年来哺乳动物胚胎着床前这一发育阶段基因表达与调控的研究进展。早期胚胎发育受母源型 ,即卵母细胞内 m RNA和蛋白质的调控 ,随着母型 m RNA的消耗 ,合子型基因取而代之进行合子型调控 ;其中合子型基因激活是调控过渡的关键事件。     

黄牛卵母细胞和早期胚胎组蛋白乙酰化转移酶1表达模式研究

为探讨卵母细胞成熟及早期胚胎发育过程中组蛋白乙酰基转移酶(HAT1)的表达规律,研究应用实时定量PCR技术,检测了广西本地黄牛卵母细胞和附植前胚胎HAT1基因的表达情况。结果表明:HAT1基因在黄牛生发泡期(GV)卵母细胞、第2次减数分裂中期(MⅡ)卵母细胞、体外受精(IVF)胚胎2～4细胞、8～16细胞、桑葚胚和囊胚中的相对表达量分别为1.00、0.56、0.08、0.55、0.43和0.31,在孤雌激活(PA)胚胎的2～4细胞、8～16细胞、桑葚胚和囊胚中的相对表达量分别为0.55、0.55、0.48和0.46。HAT1在GV期相对表达量最高,在IVF胚胎中2～4细胞表达量最少(P<0.05)。由此可见,HAT1基因在黄牛卵母细胞成熟和早期胚胎阶段均有表达,GV期HAT1基因的表达最高,PA胚胎HAT1基因的表达较稳定。     

哺乳动物早期胚胎中的基因表达

本文总结最新的相关研究资料,对影响早期胚胎发育中的基因表达进行综述,主要包括原癌基因、Xist基因、性别决定基因、生长因子、生长发育及凋亡调节基因、白血病抑制因子等。     

Effect of Days Post-Partum, Breed and Ovum Pick-Up Scheme on Bovine Oocyte Recovery and Embryo Development

The objective of this study was to investigate (i) the effect of two different ovum pick-up (OPU) schemes (once vs twice weekly aspirations) on oocyte recovery rate, quality and subsequent in vitro embryo development, (ii) the influence of days post-partum on oocyte recovery and (iii) possible differences in OPU results from two different herds. In group A, OPU was performed twice weekly in two Holstein Friesian (HF) and three Danish Red and White (DRW) cows from a private herd. In the research herd, two groups of eight HF cows were investigated: group B (OPU once weekly) and group C (OPU twice weekly). The collected oocytes were subsequently submitted to in vitro embryo production. More oocytes were recovered from the private herd when compared with the research herd. In the research herd, the twice weekly scheme aspirated more oocytes than the once weekly scheme. The quality of the retrieved oocytes was significantly different between groups B and C but not between groups A and C, and HF cows yielded higher quality oocytes than DRW cows (p = 0.029). Oocytes from group C showed higher level of embryonic development than group B oocytes. No differences in blastocyst rates were observed between groups A and C. Session affected the number of retrieved oocytes and subsequent developmental rates, with these being lower in the first compared with the last sessions. Finally, there was no significant effect of days post-partum in the number and quality of the retrieved oocytes, likely because of the small group size and high variation between sessions.     

体外成熟对牛卵母细胞孤雌激活后发育潜力的影响

本试验在卵母细胞体外成熟的研究基础上,研究了培养用水、卵泡液和成熟时间对卵母细胞体外成熟及孤雌激活后发育潜力的影响.结果表明(1)将卵母细胞分别于蒸馏水和Milli-Q超纯水配制的成熟培养液中培养22～24 h, 孤雌激活后, 卵裂率无显著差异(P>0.05); 但孤雌卵在超纯水配制的胚胎培养液中培养,囊胚发育率为22.3%, 明显高于在蒸馏水配制的培养液中的囊胚发育率14.1%(P<0.05).(2)在成熟培养液中添加10%、20%卵泡液,成熟卵母细胞孤雌激活后的囊胚发育率(32.3%, 30.9%)明显高于添加5%和0%卵泡液组的囊胚发育率(21.8%, 22.7%,P<0.05).卵母细胞在添加20%卵泡液的培养液中成熟培养,孤雌激活后囊胚孵化率明显低于添加0、5%、10%卵泡液组的囊胚孵化率(P<0.05).(3)成熟28 h或30 h的卵母细胞孤雌激活后,其囊胚发育率(30.5%或29.4%)明显高于成熟24 h或26 h组的囊胚发育率(20.5%或22.1%,P<0.05).     

营养水平对PRKAG3基因表达量及对肉质影响的研究

挑选70kg左右的DLY猪12头,随机分为2组,分别饲喂高、低2种水平的日粮,高营养水平为DE13．81MJ／kg、CP14％,低营养水平为DE12．55MJ／kg、CP11％。体重达到100kg左右时屠宰,测定胴体性状、肉质性状和PRKAG3基因表达量。以探讨营养水平对PRKAG3基因表达量及肉质的影响。结果表明：低营养水平有促进PRKAG3基因表达的趋势（P〉0．05）;高营养水平有提高屠宰率、瘦肉量、瘦肉率、眼肌面积、L值、a值和b值的趋势（P〉0．05）,但滴水损失显著低于低营养水平（P〈0．05）;营养水平对PRKAG3基因表达量有一定的影响。猪PRKAG3基因表达量与瘦肉率、眼肌面积、a值、b值和滴水损失呈正相关,但相关性均不显著（P〉0．05）;与pH2呈显著负相关（P〈0．05）。这表明营养水平对PRKAG3基因表达量有一定的影响,进而可影响肉质。     

不同培养液及不同培养时间对犬卵母细胞体外成熟的影响

试验采集犬卵巢,用切割法回收卵泡中的卵母细胞,选取胞质均匀、2层或2层以上颗粒细胞、直径≥110μm的卵母细胞移入不同的培养液中:(A)M199+10%FBS+0.5μg/mL FSH+10 IU/mL hCG,(B)M199+0.1%PVA进行体外成熟培养。在培养不同时间(0,48,72 h)用1%醋酸地衣红染色,在显微镜下观察卵母细胞核成熟情况,探索适宜犬卵母细胞体外成熟的培养体系。结果表明:卵母细胞在A液中培养48 h MII期比例(7.84%)高于B液中的比例(P<0.01);在培养72 h MII期比例也是A液中(8.00%)高于B液,虽然二者差异不显著(P>0.05),但72 h卵母细胞退化比例高于48 h(P<0.01)。因此培养液中添加FBS,FSH和hCG有利于犬卵母细胞体外成熟,且在此培养液中培养48 h为最佳成熟时间。     

鸭胚成纤维细胞培养、传代及保存方法的研究

本研究旨在建立鸭胚成纤维细胞离体培养、传代以及冷冻保存体系,为鸭胚胎或生殖干细胞的离体培养奠定基础。利用胰酶-EDTA消化鸭胚胎组织,10% FBS+DEME营养液培养,获得原代和传代成纤维细胞。分别用DMSO、甘油以及乙二醇作为冷冻保护剂对F1代鸭胚成纤维细胞进行冷冻保存,检测复苏后细胞的生长情况。利用此方法可以获得高活性的鸭胚成纤维细胞,并可成功传至第三代。其中F1代与F2代细胞活力最好,达到90%以上。3种冷冻剂保存的细胞复苏后均与F1代细胞的活力存在明显差异,均小于80%,其中用DMSO冷冻保存的细胞复苏后活力最强,生长密度保持较好。     

沙棘叶黄酮对热应激条件下肉鸡肌苷酸及基因表达的影响

试验选用健康1日龄从肉鸡公雏600只,采用单因素完全随机分组试验设计,从中随机抽取240R,分4组,每组4个重复,每个重复15只,4组分别为基础日粮组(对照组)、试验组Ⅰ(基础日粮+0.025%沙棘叶黄酮)、试验组Ⅱ(基础日粮+0.05%沙棘叶黄酮)、试验组Ⅲ(基础日粮+0.1%沙棘叶黄酮).第4周时进行热应激,温度为(36+2)℃,持续1周,然后温度恢复正常.在第3、4周末屠宰采样,利用高效液相色谱法(HPLC)检测不同组间的肌苷酸(IMP)含量,利用Real-time PCR方法检测不同组间的腺苷琥珀酸裂解酶(ADSL)基因表达.第4周试验组Ⅰ、Ⅱ、Ⅲ胸肌IMP含量分别比对照组极显著提高了33.94%、75.76%和49.09%(P<0.01);第4周试验组Ⅰ、Ⅲ肝脏ADSL mRNA表达量极显著高于对照组(P<0.01).试验表明,基础日粮中添加沙棘叶黄酮后,可提高胸肌IMP含量及其相关基因ADSL mRNA的表达量,从而缓解热应激导致的肉质风味的下降.     

鸡胚胎期和出生后Pax3基因时空表达规律的研究

为探讨鸡Pax3基因在胚胎阶段以及不同生长阶段的时空表达规律,试验选取同一批鸡胚(8、16、20 d和21 d)以及2、4、6、8、10、12周龄的如皋鸡各6只,公、母各半,采用SYBR GreenⅠ实时荧光定量PCR技术分析了Pax3基因在胸肌、腿肌中的表达规律。结果表明:胸肌和腿肌组织中公、母鸡表达水平变化趋势一致,无性别效应;在胸肌组织中,Pax3基因在8 d的鸡胚中表达水平最高,随后降到最低,出雏前缓慢上升,出雏后随着周龄的增加表达水平保持在较低状态。但在腿肌组织中,Pax3基因的表达高峰在出雏前(20 d和21 d),出雏后随周龄的增加表达量持续降低,并保持在较低的水平。表明Pax3基因表达存在组织特异性,且集中在胚胎期大量表达,出雏后表达水平较低,揭示该基因可能对鸡肌纤维生长发育存在调控作用。     

猪胎盘特异基因1(PLAC1)的克隆、组织表达及遗传方式研究

旨在研究猪胎盘特异性基因PLAC1的表达规律,并对其基因结构、生物功能和遗传方式进行初步鉴定。本研究以妊娠26、50和95天的猪胎盘组织为材料,利用RACE-PCR及RT-PCR技术克隆猪PLAC1基因及其不同转录本的全长序列,同时通过原位杂交验证其在胎盘中的表达模式。并检测猪PLAC1基因的序列变异。结果表明,猪PLAC1基因存在3种不同的转录本,3种转录本的CDS区完全相同,长525bp,编码174个氨基酸;组织表达谱和原位杂交结果显示,PLAC1基因特异性表达于猪胎盘绒毛膜上皮细胞中,并在不同妊娠时期差异表达,妊娠50和95天PLAC1mRNA的表达显著高于妊娠26天(P<0.01)。猪群中PLAC1基因的SNP基因分型检测结果表明该基因遵循半合基因的遗传方式。本试验结果为研究猪PLAC1基因在胎盘发育过程中的生物功能奠定了基础。     

显微注射针对猪ICSI胚胎的发育和EGFP表达效率的影响

本实验以猪体外成熟卵子以及冷冻解冻后失活的精子为材料,以无BSA且成分明确的TL-HEPES溶液为操作液,探讨显微注射过程中分别使用钝口针和磨口针进行注射,对猪卵子的激活、ICSI胚胎的发育及EGFP表达效率的影响。结果表明:成熟后的猪卵子在不进行精子注射和电激活的情况下,使用钝口针空注射后,卵裂率显著高于磨口针(P<0.05)。分别使用钝口针和磨口针对成熟后的猪卵子进行精子注射,不论进行或不进行电激活,使用钝口针注射,卵子的卵裂率、囊胚率和胚胎EGFP的表达效率均显著高于磨口针(P<0.05)。本实验研究发现,使用钝口针进行注射有利于猪ICSI卵子的激活,胚胎的发育以及胚胎中EGFP表达效率。     

Effects of Different Oocyte Activation Procedures on Development and Gene Expression of Porcine Pre‐Implantation Embryos

Among the factors that affect the efficiency of somatic cell nuclear transfer (SCNT) in pigs, the activation protocol is the most variable among the current SCNT procedures. The aim of this study is focused on defining an efficient activation treatment of porcine oocytes. In Experiment 1, we studied the effects of nine different oocyte activation procedures (including chemical‐ and electrical‐based treatments) on parthenogenetic embryo development. In Experiment 2, we studied the effect of the more efficient activation procedures on the gene expression profile of Oct4 and Igf2r in parthenogenetic blastocysts. In conclusion, ionomycin as a first calcium stimulus is not able to activate porcine oocytes efficiently in comparison with electric procedures. Electrical treatments with 6‐DMAP significantly increased the level of Oct4 expression, whereas the single and double pulse treatments alone maintained the same profile as the IVF group.     

Role of Fatty Acids in Energy Provision During Oocyte Maturation and Early Embryo Development

While much is known about the metabolism of exogenous nutrients such as glucose, lactate, pyruvate, amino acids by oocytes and pre-implantation mammalian embryos, the role of endogenous stores, particularly lipid, has been largely overlooked. The presence of lipid within oocytes and early embryos has been long known, and comparisons between species indicate that the amounts and types of lipid present vary considerably. Large amounts of intracellular lipid can compromise the success of cryopreservation and the removal of such lipid has been the subject of considerable effort. In this review, we present evidence that strongly suggests a metabolic role for lipid, specifically with regard to energy provision, in the late-stage oocyte and the pre-implantation embryo. We focus initially on oxygen consumption as a global indicator of metabolic activity, before reviewing different approaches that either have been designed to investigate directly, or have revealed indirectly the role of endogenous lipid in energy generation. These fall under five headings: (i) fatty acid oxidation; (ii) inhibition of triglyceride oxidation; (iii) culture in the absence of exogenous substrates; (iv) cytoplasmic organization; and (v) delipidation. On the basis of the data derived from these studies, we conclude that there is strong evidence for the utilization of endogenous lipid as an energy substrate by oocytes and early embryos.     

碱性磷酸酯酶基因在鸡胚和人乳腺癌细胞中的表达

通过PCR方法从pSEAP2-control质粒获得人胎盘碱性磷酸酯酶(PLAP)基因,并克隆到鸡输卵管特异性表达载体(pAB-Sig-SV40)卵清蛋白基因调控区下游.pSEAP2-contro1和pAB-Sig-AP-SV40质粒转染鸡胚细胞和乳腺癌细胞(MCF-7),转染48h后以对硝基苯磷酸钠(pNPP)为底物检测PLAP的活性,结果pSEAP2-contro1在鸡胚细胞和MCF-7细胞中表达PLAP,pAB-Sig-AP-SV40在鸡胚细胞中不表达,而在MCF-7细胞中表达.说明鸡胚细胞可以表达有活性的人源糖蛋白,另外也说明在雌激素受体细胞中,鸡卵清蛋白基因启动子可启动外源基因表达.     

氨基酸对基因表达的影响

氨基酸除了作为蛋白质合成底物外 ,氨基酸在体内还发挥着其它重要的营养生理功能 ,例如作为糖异生的底物、作为神经递质和信号转导的前体及蛋白质周转的调节剂等。然而 ,大量研究证实氨基酸还作为营养信号分子调控着许多调节细胞功能和氨基酸代谢的基因的表达。氨基酸主要是通过影响DNA的转录和mRNA的稳定性和翻译而调控基因的表达。但是 ,氨基酸调控基因表达的分子机制仍然不十分清楚