6-DMAP和胚胎干细胞代数对异种重构卵体外发育的影响(简报)

核移植技术已经广泛应用于动物克隆,但是克隆动物的成活率仍然很低。许多克隆胚胎死于妊娠期,少部分能发育到期,正常出生,但多数在出生后由于心肺和消化道的问题,很快就夭折,有些克隆动物有异常表型,如出生时体重和胎盘过大等。研究发现,在同种克隆动物实验中用胚胎干细胞(Embryonic stem cell,ES细胞)作为核供体,发育到期的克隆动物比例明显高于体细胞,并且用杂交一代的小鼠ES细胞为核供体,绝大多数克隆仔     

体细胞来源及培养代数对核移植重构胚发育的影响

为探讨体细胞来源及培养代数对核移植重构胚发育的影响,实验采用电融合法将小鼠2—细胞胚胎卵裂球、胚胎干细胞(ES)、胎儿成纤维细胞、耳成纤维细胞、尾尖成纤维细胞、睾丸支持细胞和精原细胞以及不同培养代次的胎儿成纤维细胞进行了核移植。结果显示：2—细胞胚胎卵裂球供核重构胚发育最好,囊胚率为7．4％;ES细胞重构胚虽然发育率低,但仍有囊胚出现,比例为0．7％;胎儿成纤维细胞重构胚最高发育阶段为桑椹胚,比例为0．2％;精原细胞重构胚只能发育到8-细胞阶段,比例为0．3％;其他几类细胞重构胚则仅能发育至4-细胞阶段。不同培养代数的胎儿成纤维细胞重构胚除第3代外都可发育到8-细胞阶段,且发育率差异不显著,但第一代细胞重构胚2-细胞发育率(40．7％)显著低于2、3和4代细胞重构胚。结果表明：不同分化程度的细胞核移植后,重新编程的难易程度是不一样的,分化程度越高则重新编程越难;未调整细胞周期的ES细胞由于多数处于S期,所以重构胚发育率很低;体外培养传代有利于体细胞核移植后重新编程。     

自分泌生长因子对早期胚胎体外发育的影响（简报）

Two-cell-stage embryos were flushed from the oviducts on Day 2. Zygotes were collected from oviducts on Day 1 (Fertilization In Situ, ISF) or derived from fertilization in vitro (IVF). 2-cell embryos had a high rate of blastocyst development to each embryo concentration from 1 embryo/microliter to 1 embryo/1000 microliters. The zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (P < 0.001), with approximately 82.5% of ISF zygotes developing to blastocysts at highest concentration but only 22.3% at the lowest. For IVF zygotes the corresponding results were 46.3% and 5.2%. The number of cells in each blastocyst from 2-cell embryos was significantly higher than that from ISF and IVF group. The media supplementing Platelet-activating factor (PAF) caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentration of 1 embryo/10 microliters (10 ng/ml) and 1 embryo/100 microliters (100 ng/ml). Insulin-like growth factor (IGF) (10 ng/ml) also stimulated development of IVF zygotes when they were cultured at the concentration of 1 embryo/10 microliters. Epidermal growth factor (EGF) was no effect over range of 1-1000 ng/ml to embryo development. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The PAF, IGF-I partially compensate the effects of low embryo concentration during culture and play important roles as autocrine embryotrophic factors.     

TSA和VPA处理对食蟹猴-猪异种体细胞核移植胚胎早期发育的影响

食蟹猴-猪异种体细胞核移植(Interspecies somatic cell nuclear transfer,iSCNT)研究旨在由iSCNT胚胎建立具有与人类相似遗传背景的胚胎干细胞(ESCs),作为医学和基础科学研究的实验材料。文章探讨了两种组蛋白脱乙酰化酶抑制剂(HDACi)Trichostatin A(TSA)和Valproic acid(VPA)处理浓度、时间与培养液(PZM-3和HECM-10)组合对食蟹猴-猪iSCNT胚胎早期发育的影响。结果表明,在PZM-3中添加10 nmol/L TSA处理48 h组的囊胚率显著高于对照组(22.78%vs 9.86%,P<0.05)。但是,不管在PZM-3或是HECM-10中,添加2～10mmol/L VPA处理均不能提高iSCNT胚胎早期发育能力。文章证明了TSA处理可以提高食蟹猴-猪iSCNT胚胎早期发育能力。     

IL-6促进小鼠胚胎干细胞多能性并以发育依赖的方式调控心肌分化（英文）

白细胞介素6 (interleukin 6, IL-6)是心脏微环境的重要组成部分。在不同模型中,IL-6通过促进心肌细胞再生帮助心脏修复。胚胎干细胞是心脏修复中心肌再生的良好来源。本研究旨在探讨IL-6对小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)及其心肌分化的影响。IL-6处理mESCs两天,用CCK-8法检测mESCs增殖情况,用实时荧光定量PCR(quantitative real-time PCR, qPCR)检测干性和胚层分化基因mRNA表达水平,用Western blot检测干细胞相关信号通路的磷酸化水平,用siRNA干扰STAT3磷酸化功能。通过检测搏动拟胚体(embryoid bodies, EBs)比例和qPCR检测心脏前体细胞标记物和心肌细胞离子通道mRNA表达水平来评估mESCs的分化能力。从胚胎干细胞分化第1天(EB0)开始使用IL-6中和抗体阻断内源性IL-6的作用,分别在EB7、EB10和EB15检测心肌细胞分化;在EB15用免疫组织化学染色法示踪心肌细胞,并用Western blot检测干细胞相关信号通路的磷酸化情况...     

模拟微重力环境对昆明小鼠早期胚胎体外发育的影响（简报）

In order to investigate the development of Kunming mouse preimplantation embryos cultured in vitro under simulated microgravtiy, one-cell and 4-cell embryos of Kunming mouse (Fertilization In Situ) were cultured in CZB or KSOM media under simulated microgravity or normal gravity environment respectively. The results showed that under normal gravity, the percentage of passing 2-cell development block embryos was not different in CZB with in KSOM, but the percentage of blastocysts was lower in CZB than that in KSOM significantly. The percentages of passing 2-cell development block embryos and blastocysts in CZB or KSOM were lower under simulated microgravity than those under normal gravity. It is suggested that the frequency of early embryonic lethality is possibly increased by simulated microgravity.     

不同激活方法对牛体细胞核移植胚胎体外发育的影响（简报）

Effects of different activation methods on the cleavage and in vitro development of bovine somatic cloned embryos constructed by intracytoplasmic nuclear injection were compared. The results show that the cleavage and in vitro development rate were not different significantly for constructed embryos cultured in 6-DMAP comparing with those in 6-DMAP + cytochalasin B (CCB) after activation with Ionomycin. Culture duration (3 to 4 h) in 6-DMAP or 6-DMAP + CCB had no significant effects on the cleavage and in vitro development ability of reconstructed embryos. CCB addition in the activation medium was benefit to the development of constructed embryos, although the effect wasn't significant. Within 1 to 4 h, the longer interval duration of nuclear injection and reconstructed embryo activation was, the higher cleavage and the blastocyst development rate of reconstructed embryos were.     

ABA和6—BA对干旱玉米幼苗PEP羧化酶活性的影响（简报）

玉米幼苗ＰＥＰ羧化酶活性随着土壤含量的下降而逐渐下降,复水后其活性虽然有回升,但未恢复到干旱前水平,叶面喷施１０＾－５ｍｏｌ／Ｌ ６－ＢＡ可提高玉米幼苗ＰＥＰ羧化酶活性,而同样浓度的ＡＢＡ则抑制ＰＥＰ羧化酶活性。６－ＢＡ还可增加干旱条件下玉米幼苗的光合速率,叶水势和叶绿素含量。     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.     

Effect of 6-dimethylaminopurine on electrically activated in vitro matured porcine oocytes

The effect of the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), on the maturation promoting factor (MPF) activity, pronuclear formation, and parthenogenetic development of electrically activated in vitro matured (IVM) porcine oocytes was investigated. Oocytes were activated by exposure to two DC pulses, each of 1.5 kV/cm field strength and 60 microsec duration, applied 1 sec apart. In the first experiment, subsequent incubation with 2 or 5 mM 6-DMAP for 3 hr increased the incidence of blastocyst formation compared with no treatment, whereas incubation with 2 or 5 mM 6-DMAP for 5 hr did not. In the proceeding experiments, oocytes exposed to 6-DMAP were incubated with 2 mM of the reagent for 3 hr. Assaying histone H1 kinase activity in the second experiment revealed that the levels of active MPF in electrically activated oocytes treated with 6-DMAP were depleted more rapidly and remained depleted for longer compared with electrical activation alone. The kinetics of MPF activity following 6-DMAP treatment were similar to that found in inseminated oocytes in the third experiment. The effect of 6-DMAP was correlated with an increased incidence of parthenogenetic blastocyst formation. A fourth experiment was undertaken to examine the diploidizing effect of 6-DMAP. Electrically activated oocytes treated with 6-DMAP and cytochalasin B, either alone or in combination, displayed a higher incidence of second polar body retention compared with those that were untreated or treated with cycloheximide alone. After 6 days of culture in vitro, parthenotes exposed to 6-DMAP, either alone or in combination with cytochalasin B, formed blastocysts at a greater rate compared with those exposed to cytochalasin B alone, cycloheximide alone or no treatment. The combined 6-DMAP and cytochalasin B treatment induced the highest rate of blastocyst formation (47%), but the numbers of trophectoderm and total cells in these blastocysts were lower compared with those obtained following exposure to 6-DMAP alone. These results suggest that the increased developmental potential of 6-DMAP-treated parthenotes may be attributable to the MPF-inactivating effect of 6-DMAP, rather than the diploidizing effect of 6-DMAP.     

6—DMAP对小鼠卵母细胞减数分裂启动及孤雌发育作用

小鼠卵泡卵母细胞体外培养过程中加入２ｍｍｏｌ／Ｌ６－ＤＭＡＰ可抑制卵母细胞自发的染色持浓缩和生发泡破裂（ＧＶＢＤ）。源自超排的ＭⅡ期卵母细胞则能为６－ＤＭＡＰ所激活。ｈＣＧ注射后１８－１９ｈ的卵母细胞置于２ｍｍｏｌ／Ｌ６－ＤＭＡＰ的ＣＺＢ溶液中培养０．５ｈ、１ｈ、２ｈ、３ｈ,卵母细胞的激活率分别为２６．１％、７５．２％、７５．８％、７７．３％、卵裂率分别为８８．２％、７３．２％、６７．０％、５８．     

Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro

This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen-thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development.     

In vitro development of horse oocytes reconstructed with the nuclei of fetal and adult cells

This study investigated the basic conditions required for the production of horse embryos by the transfer of the nuclei of fetal and adult fibroblast cells to enucleated oocytes. Cumulus-oocyte complexes were recovered from abattoir ovaries and matured in vitro in groups of 20-30 for 28-30 h in tissue culture medium 199 containing 20% v:v fetal bovine serum in coculture with equine oviduct epithelial cells. Fetal fibroblast cells (FFC) were derived from a 32-day-old Thoroughbred x Pony fetus, and adult skin fibroblast cells (SFC) were obtained from subdermal biopsies recovered from a 4-yr-old female Pony. The rates of fusion between the recipient cytoplasm with either FFC or SFC were significantly greater when the cells were treated with a combination of direct current (DC) pulses and Sendai virus rather than with DC pulses alone (81%-82% vs. 49%-57%, P < 0.05). There were no differences in the rates of nuclear reprogramming between FFC and SFC (88% vs. 84%), but the rate of cleavage of the resulting embryos to the 2-cell stage was higher when FFC were used (53%) than when SFC were used (35%). Blastocysts were obtained from oocytes reconstructed with both types of donor cells and after culture in vitro for 6-7 days, but the overall proportion of blastocysts produced was very low in both cases (FFC, 4%; SFC, 7%). These results demonstrate a very limited potential for in vitro development of horse embryos after nuclear reprogramming following the transfer of nuclei from either fetal or adult fibroblasts into recipient enucleated oocytes.     

Telomerase activity alterations in sequential passages of mouse embryonic stem cells

Telomerase is associated with cell proliferation capacity, protection and stabilization of chromosomes. TA (telomerase activity) can be detected in highly replicative cells, e.g. stem and cancer cells. Most available mESC (mouse embryonic stem cell) research is done with a few cell lines. The purpose of this study has been to evaluate the TA in different passages of newly isolated mESC. TRAP (Telomeric Repeat Amplification Protocol)-ELISA method was used in a semi-quantitative evaluation of TA. Three mESC lineages were investigated (CT2, CT3 and CT4) at three different passages (P13, P15 and P19). In contrast with previous studies, these mESC lines did not show the same TA throughout their passages, having initially low TA values, followed by a subsequent rise and stabilization.     

A combined treatment with ethanol and 6-dimethylaminopurine is effective for the activation and further embryonic development of oocytes from Sprague-Dawley and Wistar rats

In nuclear-transferred or round spermatid-injected oocytes, artificial activation is required for further development in mammals. Although strontium chloride is widely used as the reagent for inducing oocyte activation in mice, the optimal method for oocyte activation remains controversial in rats because ovulated rat oocytes are spontaneously activated in vitro before artificial activation is applied. In our previous study, we found that cytostatic factor activity, which is indispensable for arrest at the MII stage, is potentially low in rats and that this activity differs greatly between two outbred rats (Slc: Sprague-Dawley (SD) and Crj: Wistar). Therefore, it is necessary to establish an optimal protocol for oocyte activation independent of strains. Given that comparative studies of the in vitro development of oocytes activated by different activation protocols are very limited, we compared four different protocols for oocyte activation (ethanol, ionomycin, strontium and electrical pulses) in two different SD and Wistar rats. Our results show that oocytes derived from SD rats have significantly higher cleavage and blastocyst formation than those from Wistar rats independent of activation regimes. In both types of rat, ethanol treatment provided significantly higher developmental ability at cleavage and blastocyst formation compared to the other activation protocols. However, the initial culture in a fertilization medium (high osmolarity mR1ECM) for 24 h showed a detrimental effect on the further in vitro development of parthenogenetic rat oocytes. Taken together, our results show that ethanol treatment is the optimal protocol for the activation of rat oocytes in SD and Wistar outbred rats. Our data also suggest that high-osmolarity media are inadequate for the in vitro development of parthenogenetically activated oocytes compared with fertilized oocytes.     

Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.     

In vitro development of bovine oocytes reconstructed with round spermatids

The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.     

In vitro differentiation of male mouse embryonic stem cells into both presumptive sperm cells and oocytes

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.     

in vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells

In the pig little information is available on cytoplasmic events during the reprogramming of oocytes reconstructed with somatic nuclei. The present study was conducted to determine the developmental potential of porcine cumulus cells (CC) and fetal fibroblasts (FF) after they were transferred into enucleated oocytes. Non-quiescent FF were fused to the enucleated oocytes using electrical pulse, whereas CC were directly injected into the oocytes. Transferred nuclei from both CC and FF underwent premature chromosome condensation (PCC), nuclear swelling and pronucleus formation. The remodeled oocytes developed to the mitotic and 2-cell stage at 18 to 24 h after nuclear transfer. The pattern of nuclear remodeling was similar regardless of the sources of karyoplasts or nuclear transfer methods. However, using FF, 24% of nuclear transferred embryos developed to the morula or blastocyst stage, whereas only 8% of those using CC developed to the morula or blastocyst stage. These results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of nuclear transferred embryos to the blastocyst stage.