精子介导GFP基因在小鼠早期胚胎中的表达

利用 DIG 末端标记技术和免疫组化技术分析了小鼠精子体外结合内化外源DNA的效率。试验结果表明,不同小鼠个体的精子结合外源DNA的阳性率有明显差异（P<0.01）,平均为13%。利用考马斯亮蓝染色评价了小鼠精子顶体反应发生的情况,筛选出TYH培养液为较合适的体外受精液。利用小鼠体外受精技术,将体外转染GFP基因并获能的小鼠精子与成熟卵母细胞进行体外受精,受精卵进行体外培养,表达GFP胎的阳性率为4.7%。验证了精子介导制备转基因小鼠胚胎的可行性,并建立了利用精子载体法制备转基因小鼠胚胎的平台。     

以精子为载体的体外转基因猪胚胎研究

去精清精子与外源DNA共孵育,使其携带外源DNA,再与成熟卵母细胞进行体外受精,生产转基因猪胚胎,为通过胚胎移植生产转基因猪奠定基础。结果:通过PCR从不同时期胚胎中检测出携带外源DNA的阳性胚,说明精子与外源DNA共孵育能使精子携带外源DNA,并通过体外受精技术使外源DNA进入早期胚胎。     

外源基因对精子的影响及其在山羊早期胚胎中的表达

摘要: 在前期实验中发现, 山羊精子可自发结合外源DNA, 但结合能力在不同动物个体之间差异显著。挑选结合能力明显不同的3只公羊, 进一步探讨了外源DNA对精子的影响及其在早期胚胎中的表达, 结果发现: 外源基因与精子共同孵育后, 精子的活率、顶体反应发生率和受精能力均呈下降态势, 其降幅与精子的结合能力密切相关。利用与DNA共育后的精子进行体外受精, 外源基因可被导入卵母细胞并在早期胚胎中获得表达, 但胚胎阳性率因精子供体不同而差异显著(P<0.05); 其中来源于高、中结合能力供体生产的胚胎, 分别有16.2%(25/154)和5.3%(4/76)可检测到外源基因存在, 但表达仅见于高结合能力供体生产的早期胚胎, 表达率为6.5%(10/154); 低结合能力供体生产的胚胎无外源基因。研究表明, 在以精子载体方法生产转基因动物的实验过程中, 筛选对DNA结合能力较强的精子供体是提高转基因效率的前提, 但需要考虑外源DNA对精子受精能力的影响。     

利用卵胞浆精子注射（ICSI）技术生产转基因小鼠

在掌握小鼠ICSI技术的基础上,进行了ICSI技术生产转基因小鼠的研究。来自成年KM小鼠附睾尾的精子,使用未加抗冻剂的HEPES-CZB溶液,在液氮中冻融1次后,用于本实验。解冻精子与DNA混匀1min后,精子头被显微注射到B6D2F1小鼠成熟卵母细胞质中。精子头与pEGFP-N1环状DNA共注射生产的ICSI受精卵,在CZB溶液中培养至囊胚期时,39.1%(9/23)的囊胚表达GFP基因。精子注射后6h,直接移植ICSI受精卵后,7只妊娠受体一共产仔30只,效率为23.8%(30/126)。Southernblot分析其中16只小鼠发现,3只(18.8%)转基因小鼠同时整合了GFP和Neomycin基因,它们全部来自精子和线性DNA混合的实验组(阳性效率为33.3%,3/9),相反,精子与环状质粒DNA共注射生产的7只ICSI后代中,没有检测到外源基因。转基因小鼠整合的外源基因能够传递给它们后代。结果说明,利用ICSI技术可以高效地生产转基因小鼠,宿主基因组可能更容易整合线性化的外源基因。     

通过睾丸内注射转染外源DNA在小鼠精子的表达

为研究睾丸内注射外源DNA法生产转基因小鼠(Mus musculus)的可行性,并探讨注射DNA的最佳浓度。将环形的质粒DNA pEGFP-N1与脂质体混合制备DNA-脂质体复合物,按DNA浓度不同分为0.08μg/μl、0.12μg/μl和0.24μg/μl3组,分别注射入成年SPF级昆明小鼠睾丸内,同时设空白对照;每组处理公鼠2只,注射5d后每只与3只成年母鼠同笼,20d后在荧光显微镜下检测公鼠附睾精子,并制作睾丸石蜡切片,检测绿色荧光蛋白(GFP)的表达;PCR法检测各组后代阳性率。结果显示,3组小鼠附睾荧光精子比例分别为9.09%、47.06%和27.78%。3组小鼠的睾丸石蜡切片中均可看到不同强度的GFP表达。后代经PCR检测阳性率分别为17.26%、47.61%和22.11%。本实验证实了睾丸注射法能使外源DNA整合进入精子基因组,并能在自身和后代中得到表达,本研究中外源DNA注射浓度以0.12μg/μl效果为最佳。     

昆明小鼠精子冷冻的研究(简报)

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受体,生长发育并产出幼仔。胚胎体外培养获得足够的晚桑葚胚和早期囊胚是胚胎干细胞建立与制备嵌合体小鼠的成功关键,暂时不用的胚胎应仍能耐受冷冻保存,待复苏移植,建立新的繁殖群。但小鼠精子冷冻研究在我国开展的十分有限,缺乏相关资料数据。本实验对不同周龄SPF级昆明(KM)小鼠进行精子冷冻及冻融精子IVF,并将IVF获得的2-细胞胚胎分别进行胚胎移植、体外培养、胚胎冷冻及复苏后移植,应用冻融小鼠精子进行胚胎工程实践。     

精子介导法生产转基因牦牛杂种胚胎的研究

研究外源DNA转染对牦牛精子体外受精(IVF)、早期胚胎发育以及绿色荧光蛋白(GFP)基因在早期胚胎表达的影响。用构建融合人乳铁蛋白基因绿色荧光蛋白表达载体(pAcGFP1-hLF)转染的牦牛精子IVF黄牛卵母细胞,生产的胚胎在荧光显微镜下观察GFP表达情况。结果显示,用环形质粒、线性质粒pAcGFP1-hLF转染的精子及未转染精子IVF后的卵裂率分别为56.4%、54.8%和61.0%,线性转染的囊胚率显著低于环形转染(17.7%与35.7%)。环形质粒转染的精子用DNaseⅠ消化、不消化及未转染的精子IVF后卵裂率分别为44.8%、55.2%和56.3%,囊胚率分别为33.3%、33.3%和30.2%。未经DNaseⅠ消化洗涤的环形质粒组GFP胚胎阳性率显著高于线性质粒组(99.3%与76.6%);DNaseⅠ消化组GFP胚胎阳性率极显著低于不消化组(25.4%与99.3%)。结果表明,用环形质粒DNA转染牦牛精子不会影响其受精及早期胚胎发育能力,但DNaseⅠ消化洗涤显著降低GFP胚胎阳性率。     

利用精子介导法向蚕卵导入外源基因的研究

为建立家蚕转基因中切实可行、操作简便的外源基因导入方法,进行了精子介导法探索,以精子介导法的三种方式向家蚕导入所构建质粒pFbGFP,并通过PCR扩增和DNA印迹等手段,已连续两代从基因组DNA检测到导入外源基因GFP的存在,其中的一种导入方式到第二代阳性率约30% .结果表明该法可有效进行家蚕转基因的外源基因导入.     

睾丸内注射pEGFP-N1体内转染精子并在山羊早期胚胎成功表达

目的探讨睾丸内注射法pEGFP-N1在精细胞的整合和在早期胚胎中表达.方法选择4头本地山羊,双侧睾丸注射不同剂量质粒DNA pEGFP-N1,注射后PCR和Southern杂交检测pEGEP-N1在精子中的整合.结果pEGFP-N1整合到精子基因组中,转染效率最高发生在注射后第40天,转染阳性率最高为81%;绿色荧光蛋白在精子及其体外受精的部分胚胎中表达,胚胎阳性率最高的达66.7%.结论通过睾丸内注射pEGFP-N1能整合进入精子基因组,并能通过体外受精在山羊早期胚胎中表达;睾丸内注射法可能是一种可行、简单并利于推广的制备转基因山羊的方法.     

睾丸注射法制备携带山羊H-FABP基因的转基因小鼠

为探究睾丸注射法制备转基因动物的可能性,文章将携带有山羊心脏型脂肪酸结合蛋白(H-FABP)和绿色荧光蛋白标签的重组载体经脂质体包裹后随机打点注射小鼠睾丸。对实验小鼠进行睾丸切片、精子荧光检测以及精子DNA检测,证实外源基因在亲代小鼠体内成功表达。睾丸注射后小鼠与正常母鼠交配产生的F1代,以及F1代自交产生的F2代在不同水平均可检测到外源基因的成功表达,阳性率分别为4%和30.23%。研究结果说明睾丸注射是一种制备转基因动物行之有效的方法,且外源基因可以稳定遗传。该方法的完善和成熟对于动物转基因以及动物性状改良和育种具有理论和实践意义。     

Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.     

不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Fertilization of mouse oocytes from in vitro-matured preantral follicles using classical in vitro fertilization or intracytoplasmic sperm injection

Early preantral mouse follicles with a diameter of 110-160 microm were cultured in vitro for 10 or 12 days. Mature oocytes were retrieved following hCG, and fertilization was attempted either by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Two-cell and blastocyst formation rates and blastocyst cell numbers were compared between 10-day and 12-day in vitro-matured oocytes versus in vivo-matured oocytes. Uncleaved IVF oocytes were subjected to chromosome analysis. The 2-cell formation rate was significantly improved by ICSI compared with IVF both in 10-day (72.1% versus 56.1%; P = 0.03) and 12-day cultures (74.1% versus 54.5%; P = 0.028). Cytogenetic analysis of uncleaved MII oocytes following IVF showed that about 30% of MII oocytes showed no sign of sperm penetration. The blastocyst formation rate was significantly lower in 12-day versus 10-day cultures, whether fertilization was by IVF (40.7% versus 62.4%, P = 0.016) or by ICSI (32.5% versus 57.1%, P = 0.035). Blastocyst cell numbers from IVF and ICSI 10-day groups were similar and both significantly higher (P < 0.001) than from IVF 12-day cultures. All above expressed values were significantly higher for in vivo-matured oocytes. In conclusion, fertilization of oocytes from in vitro-matured mouse preantral follicles can be optimized with ICSI, giving significantly higher 2-cell formation rates than IVF. Blastocyst formation rate was not influenced by the technique of fertilization but rather by the extent of the in vitro culture period. Best results on preimplantation development of oocytes for in vitro-matured preantral follicles were obtained with ICSI on oocytes from 10-day in vitro cultures.     

Treatment of sperm with extracellular adenosine 5'-triphosphate improves the in vitro fertility rate of inbred and genetically modified mice with low fertility

In vitro fertilization (IVF) is one of the most important techniques used for assisted reproduction in mouse colony management. As with natural mating, where mice have varying fertility indices, fertility rates of genetically modified (GM) [transgenic (Tg), knock out (KO) and congenic (Cg)] mice are influenced by their genetic background. Lines of GM mice that have poor fertility have a concomitant poor IVF outcome. Treatment of mouse sperm with extracellular adenosine 5'-triphosphate (ATPe) enhanced in vitro fertilization rates in outbred and hybrid mice. The objective of this study was to analyze the effects of using extracellular adenosine 5'-triphosphate-treated sperm for IVF of inbred wild type, and genetically modified mouse lines, for which standard IVF did not work well. The IVF was performed using the GM mice on C57BL/10SnJ, C57BL/6J, BALB/cJ and NFS/N background strains and wild type (WT) mice such as C57BL/6N, BALB/cAnN, and B6129SF1 strains. Oocytes from superovulated females were fertilized in vitro with sperm from the same background strain, and either treated or not treated with ATPe. The ATPe treatment enhanced IVF outcome in most of the GM and some WT strains, as indicated by the percentage of embryos that progressed to the two-cell stage. There was no marked difference between ATPe treated and control groups for the development rate of two-cell embryos to blastocysts in culture, or in the number of pups born after transfer of two-cell embryos into recipient females. The observed improvement of the IVF results following ATPe treatment of transgenic and KO mouse sperm were a potential solution for improving the outcome of assisted reproduction techniques used for rederivation or for gamete banking.     

Reduced polyspermic penetration in porcine oocytes inseminated in a new in vitro fertilization (IVF) system: straw IVF

High incidence of polyspermy is still a major problem in the in vitro fertilization (IVF) of porcine oocytes matured in vitro. This study was designed to examine whether embryo cryopreservation straws can be used to conduct IVF in porcine oocytes. The efficiency of this system was further compared with traditional microdrop IVF. Immature oocytes were aspirated from antral follicles and matured in vitro. After maturation, oocytes were inseminated either in straws or in microdrops with frozen-thawed boar spermatozoa. For straw IVF, sperm concentration and the presence of air columns between insemination segment and oil column were examined. Sperm-oocyte binding and cortical granules (CGs) before and after sperm penetration were examined by confocal microscopy. When various sperm concentrations were used for IVF in the straws with air columns, it was found that 5 x 106 cells/ml of sperm concentration was the optimal concentration; a high penetration rate (94.0%) and normal fertilization (oocytes with both male and female pronuclei) rate (38.2%) were obtained. Increasing sperm concentration to 10 x 106 cells/ml increased polyspermic penetration (61.9%) without affecting sperm penetration (86.9%). Reducing sperm concentration to 1 x 106 cells/ml reduced polyspermic penetration (25.6%), but sperm penetration rate (69.9%) was also reduced. When IVF was conducted in the straws with or without air columns, and in the microdrops, it was found that sperm penetration in the straws with air columns (96.5%) was significantly (p < 0.05) higher than that in the straws without air columns (81.7%) and in the microdrop (72.9%). However, the incidence of polyspermic penetration in the straws with air columns (34.2%) and without air columns (36.6%) was significantly (p < 0.05) lower than that (52.4%) in the microdrops. The number of spermatozoa bound to the oocytes was increased gradually in the straws but not in the microdrops in which more spermatozoa bound to the oocytes soon after insemination. CG exocytosis was more complete and faster in the oocytes inseminated in the straws than in the microdrops. These findings indicate that IVF of porcine oocytes in the straws provides a better condition in which more oocytes are fertilized normally than that in the microdrop IVF.     

Detection and expression of hepatitis B virus X gene in one and two-cell embryos from golden hamster oocytes in vitro fertilized with human spermatozoa carrying HBV DNA

The main objectives of this study were to introduce motile human sperm carrying Hepatitis B virus (HBV) DNA to golden hamster oocytes in a co-culture environment and to detect the replication and expression of the HBx gene in early embryonic cells. Zona-free hamster oocytes were inseminated with human sperm carrying pBR322-HBV DNA plasmid using the in vitro fertilization (IVF) technique. Both the one- and two-cell stages of early embryonic development were studied. PCR, RT-PCR, and Dot hybridization were performed to observe the HBx gene and its expression in these stages. "Fluorescence in situ hybridization" (FISH) was carried out to confirm the integration of HBV into the pronucleus, nucleus, and the chromosomes of embryos. The results showed that we have the ability to obtain a fertilization rate of 80%. RT-PCR showed that the HBx gene could be expressed in both one- and two-cell stages of embryonic development. The data suggested the possibility of sperm as a vector for the vertical transmission of HBV DNA to the next generation.     

Birth of twins after in vitro fertilization with flow-cytometric sorted buffalo (Bubalus bubalis) sperm

Flow-cytometric sorting of mammalian sperm for production of offspring with the desired sex is one of the most important new biotechnologies available for livestock industry. The objectives of this study were: (i) to sort the sperm into X- and Y-enriched populations and (ii) use the sorted sperm for in vitro fertilization (IVF) to produce sex-preselected embryo and offspring. The results revealed that the accuracy of sorted X- and Y-sperm was 94% and 89%, respectively. There was a decrease in blastocyst development rate in IVF with sorted sperm comparing to unsorted sperm, but the percentage of blastocysts on D6-D8 was not statistically different. Transplantation of the presumed X-embryos derived from IVF into a recipient resulted in the birth of female twins. These results indicated the feasibility of sperm sorting by flow cytometry and in vitro production of sex-preselected embryos and offspring in buffalo.     

Brief coincubation of gametes in porcine in vitro fertilization: role of sperm:oocyte ratio and post-coincubation medium

In this study, a short coincubation time of 10 min was used to determine the effect of different sperm:oocyte ratios during in vitro fertilization (IVF), and different periods of post-coincubation in a medium that is not appropriate for IVF, on fertilization parameters. In the first experiment, a total of 1624 in vitro matured oocytes, from 4 replicates, were inseminated with frozen-thawed spermatozoa at different sperm:oocyte ratios (2000, 1500, 1000 and 500 sperm:oocyte) and coincubated for 10 min or 6 h. The oocytes from 10 min of coincubation were washed in IVF medium to remove spermatozoa not bound to the zona pellucida and transferred to another droplet of the same medium (containing no spermatozoa) for 6h. The oocytes from the other group remained with the spermatozoa for 6h. Oocytes from both groups were then cultured in embryo culture medium (IVC) for 12h to assess fertilization parameters. In the second experiment, 1872 in vitro matured oocytes, in 3 replicates were inseminated with frozen-thawed spermatozoa using the same sperm:oocyte ratios as in the first experiment. The oocytes were coincubated for 10 min and transferred directly to IVC medium for 18 h (group A), to IVF medium (containing no sperm) only for 2h and then to IVC medium for 16 h (group B), or to IVF medium (containing no sperm) for 6h and then to IVC medium for 12 h (group C or control). There was an effect of sperm:oocyte ratio on all fertilization parameters in experiment 1. The efficiency of IVF (number of monospermic oocytes/total number inseminated) was higher (P<0.05) for oocytes coincubated with spermatozoa for 10 min and inseminated with 1500 and 1000 sperm:oocyte (35.8+/-3 and 37.6+/-2.7%, respectively) and for those coincubated for 6h with 500 spermatozoa per oocyte (37.2+/-3.1%). In experiment 2, the penetration and efficiency rates obtained in group A were poor (between 3 and 15%) irrespective of the sperm:oocyte ratio. However, in group B the fertilization parameters were similar to the controls and were also affected by the sperm:oocyte ratio. These results demonstrate that coincubation time may be reduced to 10 min to increase the efficiency of fertilization depending on the sperm:oocyte ratio, and that the spermatozoa bound to the zona pellucida require a maximum of 2h in an appropriate medium to penetrate the oocytes.