表皮生长因子对无透明带小鼠胚胎体外发育的影响

为了观察不同浓度的表皮生长因子对无透明带小鼠胚胎体外发育的影响,试验将小鼠原核期胚胎用链霉蛋白酶去除透明带后,分别置于含有不同浓度的表皮生长因子无血清胚胎体外培养液中培养,观察各期胚胎的发育情况.结果表明:除0.01 ng/mL表皮生长因子添加组胚胎的2细胞发育率和囊胚回收率与添加0.1 ng/mL表皮生长因子处理组差异不显著外(P>0.05),其体外胚胎各期的发育率、囊胚回收率和囊胚细胞数与其他处理组比较均差异显著(P<0.05).说明一定浓度的表皮生长因子可以提高无透明带小鼠胚胎体外培养各期的发育率,但是高浓度的表皮生长因子对无透明带小鼠胚胎的体外发育有一定的抑制作用.     

In vitro development of mouse pronuclear embryos to blastocysts in sequential media with and without co-culture of autologous cumulus cells

The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects.     

玉米赤霉烯酮对小鼠胸腺细胞凋亡的影响

玉米赤霉烯酮(ZEA),过去又称F-2毒素,是由禾谷镰刀菌、黄色镰刀菌、木贼镰刀菌和半裸镰刀菌等镰刀菌属真菌产生的一种非类固醇类,但具有雌激素样活性的真菌毒素[1].ZEA广泛存在于霉变的玉米、小麦、高粱等谷类作物、发霉的饲料以及被污染的肉、奶等动物性食品中.     

Effect of time of the addition of glucose to a medium on the development of 1-cell stage mouse embryos

In the present study, 1‐cell stage mouse embryos were cultured with or without glucose, and their development to the blastocyst stage was compared. Embryos cultured in a glucose‐free medium had a higher percentage of development to the 8‐cell stage, and they had higher developmental speeds compared with those cultured in a glucose‐containing medium. The percentages of embryos that developed to the early blastocyst stage, blastocyst stage and expanded blastocyst stage were much lower than those developed in a glucose‐containing medium. This suggests that the culture of 1‐cell stage embryos in a glucose‐containing medium inhibits development at the 8‐cell stage, and that glucose is necessary for blastocyst formation. Previous reports indicate that, from the 1‐cell stage to the 4‐cell stage, glucose inhibits embryo development. In the present study, exposure of early embryos to a glucose‐free medium improved subsequent embryo development, and there was no difference in the percentage of development to the stages ranging from the 8‐cell stage to the expanded blastocyst stage between embryos cultured in a glucose‐free medium from the 1‐cell stage to the 2‐cell, 4‐cell and 8‐cell stage. This indicates that embryo development is improved when a 1‐cell stage embryo is exposed to a glucose‐free medium before and during the 2‐cell stage, and glucose only has an inhibitory effect on embryo development during conversion from the 1‐cell stage to the 2‐cell stage.     

Effect of zearalenone on days 7 to 10 postmating on intrauterine environment and migration of embryos in sows

On d 7 to 10 postmating, first-litter sows were fed either a control diet or a diet containing zearalenone (ZEN; 1 mg/kg body weight). Surgery was performed on either d 9, 11 or 13 postmating to collect blastocysts and uterine flushings. The rostral and caudal portion of each uterine horn was flushed with phosphate buffered saline, and the blastocysts were separated from the recovered solution. Uterine flushings were analyzed for total Ca, Mg, Zn, estradiol-17 beta (E2 17 beta) and progesterone (P4). Administration of ZEN did not affect the number of blastocysts recovered or the position of embryos within the uterus on d 9 or 11. Blastocysts recovered on d 13 were filamentous and could not be enumerated. Total Ca in uterine flushings of control sows was higher (P less than .001) on d 11 than on d 9 or 13, but intrauterine Ca of ZEN-treated sows did not vary by sampling day (P greater than .05) and was lower (P = .01) than that of controls on d 11. Total intrauterine Mg of ZEN sows was greater (P = .002) than of control sows on d 11 and 13, and total intrauterine Zn of ZEN sows was greater than that in control sows on d 13. There were no differences in total intrauterine P4 or E2 17 beta among ZEN-treated and control sows on d 9, 11 or 13 postmating. Serum concentrations of 13, 14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) increased from d 9 to 13 in control and ZEN-treated sows, but there were no differences between treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

玉米赤霉烯酮对体外培养猪卵巢颗粒细胞的影响

以原代培养的猪卵巢颗粒细胞为模型,设立玉米赤霉烯酮(ZEA)对照组(0.0 mg/L)及高(80.0 mg/L)、中(20.0,40.0mg/L)、低(0.2,2.0mg/L)剂量组,比较不同质量浓度ZEA对体外培养猪卵巢颗粒细胞的影响。形态学观察发现,随着ZEA质量浓度的增加,颗粒细胞呈散在生长,体积缩小变圆,数量减少;高剂量组(80.0mg/L)中,76.96%细胞均已脱壁死亡。MTT法表明,ZEA对卵巢颗粒细胞具有生长抑制作用;免疫荧光结果显示随ZEA质量浓度的增加,细胞凋亡率逐渐升高,高质量浓度组细胞增殖率仅为1.01%;Western blot检测结果显示经ZEA处理,Fas、FasL、FADD、Caspase-10的表达量均明显上调,且存在质量浓度-效应关系。结果表明,ZEA能诱导卵巢颗粒细胞凋亡,并显著上调Fas、FasL、FADD、Caspase-10的表达,可通过死亡受体通路介导卵巢颗粒细胞发生凋亡。     

Freezing mouse blastocysts. The influence of the preparations prior to freezing on the survival rate of the blastocysts

A good survival rate in culturing mouse blastocysts can be obtained in Ovum Culture Medium, enriched with 20 per cent inactivated Foetal Bovine Serum or Sheep Serum under air. The transfer of fresh blastocysts gives the best results if the recipients are on day 3 of the pseudo-pregnancy, but with 20 hours' cultured blastocysts it is better to use recipients on day 4. Exposure to 1.5 M DMSO has no harmful effect, provided that the DMSO is added at 5 degrees C in 6 steps and is removed, again in 6 steps, at 35 degrees C. The crystallization of the medium containing the embryos at -5 degrees C to -6 degrees C doet not appear te have a harmful influence on culture results of the blastocysts.     

Blastocyst production from in vitro-produced day-2 bovine embryos classified by cleavage stage, and cytogenetical evaluation of the resultant day-8 blastocysts

The present study was conducted to determine the criteria for selecting good quality embryos on Day-2 post-insemination and at the blastocyst stage. Bovine oocytes were matured, fertilized and cultured in vitro. First, Day-2 embryos were classified based on the number of blastomeres into 2-cell, 3- to 4-cell, 5- to 8-cell and >8-cell stage embryos; chromosome samples were then prepared. In the second experiment, the Day-2 embryos classified according to the number of blastomeres were cultured separately for an additional 6 days (Day 8). The resultant Day-8 blastocysts from each group of Day-2 embryos were classified into the following 3 grades based on morphology and diameter: Grade A, hatched and hatching blastocysts; Grade B, expanded blastocysts; and Grade C, unexpanded blastocysts. Chromosome samples were then prepared. The 5- to 8-cell stage Day-2 embryos had the lowest incidence of chromosomal abnormalities (13.5%, P<0.05) and the highest development rate to blastocysts (59.2%, P<0.05). Furthermore, the blastocysts derived from the 5- to 8-cell stage embryos had the largest mean number of cells (102.8+/-42.4, P<0.05), largest number of metaphases per blastocyst (9.5+/-4.8, P<0.05) and lowest incidence of chromosomal abnormalities (24.6%, P<0.05). The Grade A blastocysts had the largest mean number of cells (136.6+/-33.4, P<0.05), a large number of metaphases per blastocyst (11.9+/-5.5, P<0.05) and a low incidence of severe chromosomal abnormalities (17.3%). The results showed that, at Day 2, the 5- to 8-cell stage embryos were of better quality since they had the lowest incidence of chromosomal abnormalities and the highest blastocyst rate and the resultant blastocysts had the largest number of cells and lowest incidence of chromosomal abnormalities. In particular, selection of Grade A blastocysts can improve the development rate to term.     

In vitro growth of mouse ovarian preantral follicles and the capacity of their oocytes to develop to the blastocyst stage.

Two groups of mouse preantral follicles with diameters of 125-150 and 151-175 microm were cultured individually for 6 days in a medium supplemented with FSH and fetal calf serum to determine their in vitro growth characteristics. Their oocyte capacity for maturation and development to the blastocyst stage following in vitro fertilization was also assessed. Antral formation rate at the end of culture was higher in the follicles of 151-175 microm (89%) than 125-150 microm (76%). The timing of antrum formation was different between the two follicle categories: most 151-175 microm follicles formed antra earlier than 125-150 microm follicles (days 4 and 5 vs. 5 and 6). However, follicle diameters at the time of antrum formation were the same regardless of the initial size and the culture period. Maturation rates of the oocytes derived from both categories of in vitro grown follicles (70 and 62%) were not different from those of oocytes from in vivo grown follicles (74%). The in vitro derived oocytes, however, showed less cleavage (30 and 35%) than the in vivo derived oocytes (89%). Although the oocytes from both follicle categories developed to the morula stage after in vitro fertilization, blastocysts were only obtained from oocytes derived from the 151-175 microm category. These results demonstrate that an individual follicle culture system using a medium with FSH and fetal calf serum supports in vitro growth of mouse preantral follicles with diameters of 151-175 microm to the preovulatory stage, and that their oocytes have the capability to develop to the blastocyst stage.     

超数排卵对家兔早期胚胎体外发育的影响

为探讨超数排卵对家兔早期胚胎体外发育能力的影响,收集自然发情组和超数排卵处理组交配72 h后家兔胚胎,对经筛选合格的胚胎以小鼠成纤维细胞为饲养层进行体外培养,以自然发情交配的家兔胚胎为对照,观察培养96 h贴壁情况和内细胞团传代能力.超数排卵处理的家兔交配后经筛选所获得的可用胚胎数量增多;其96 h贴壁率和成功传代内细...     

Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.     

不同培养液对小鼠受精卵体外发育的影响

小鼠卵母细胞体外受精后通过比较4种培养液(M16、mCZB、HTF、mCZB*)对小鼠受精后胚胎发育的影响,以期建立最优的体外受精培养液系统。结果表明:受精卵在mCZB*、M16、mCZB、HTF培养液中2-4细胞发育率分别为60.26%、50.00%、48.72%和52.78%。mCZB*比其他3种培养液效果显著(P<0.05),其他3个培养液之间差异不显著(P>0.05)。在mCZB*、M16、mCZB、HTF培养液中囊胚发育率分别为44.37%、27.03%、26.28%和22.22%,各培养液处理组之间差异显著(P<0.05)。本试验提示mCZB*为小鼠体外受精胚胎发育的最佳培养液。     

活性氧(ROS)对小鼠早期胚胎胚细胞分裂的影响

在体外CZB培养的不同时期添加过氧化氢(2.1μmol/L),发育至囊胚阶段,制作胚胎标本,观察其囊胚发育率、胚细胞数和分裂相数。结果显示:0~12 h组和0~72 h组的胚胎发育率显著低于其他几组(P<0.05),对照组、12~24 h组、24~36 h组、36~48 h组之间均差异不显著(P>0.05);各组之间的胚细胞数和分裂指数均无显著差异。说明胚胎在体外培养时,2细胞期胚胎对外源性H2O2最敏感,也最容易发生发育阻滞。但是经H2O2处理之后,度过2细胞期发育阻滞的胚胎能在以后的发育中发生补偿性生长,其胚细胞数和分裂指数都和体外正常发育的胚胎没有差异。     

昆明小鼠不同性别早期胚胎体外培养发育潜力观察

自主设计2对引物,提取已知性别小鼠肝细胞DNA,扩增雄性小鼠特有的Sry基因及雌、雄小鼠共有的ZFX基因,建立双重PCR性别鉴定方法.提取小鼠早期8细胞胚胎单卵裂球、双卵裂球DNA,分别进行双重PCR性别鉴定,选择出早期8细胞胚胎的适宜模板量;将已测性别的8细胞胚胎按照雌、雄性别分开培养,观察统计雌、雄8细胞胚胎发育至囊胚的比率.试验结果显示,与单个卵裂球DNA模板量相比,双卵裂球的模板量检出率高,两者之间差异显著(75.00%Vs 93.33%,P<0.05);经过性别鉴定的雄性8细胞胚胎比雌性8细胞胚胎发育至囊胚的比率高,两者之间差异显著(53.33%Vs 33.33%,P<0.05).结果表明,采用双重PCR性别鉴定方法,以双卵裂球的DNA量为模板能够有效的对小鼠早期8细胞胚胎进行性别鉴定;雄性胚胎在体外培养条件下比雌性胚胎具有更好的发育潜力.     

Effect of placental soluble factors on growth and differentiation of mouse ectoplacental cone in vitro.

The effect of soluble fractions derived from the placenta on the outgrowth and giant cell transformation of the ectoplacental cone (EPC) was investigated in vitro. EPCs taken from the fetal mice on day 7.5 post coitum (pc) were incubated with alpha-MEM-containing fetal calf serum (FCS) (FCS-alpha-MEM) for 24 hr. Then, the medium was exchanged with alpha-MEM alone, FCS-alpha-MEM, or alpha-MEM containing a crude extract from placental region on day 8.5, 10.5 or 12.5 pc (10 ml phosphate buffer/g placenta). Each EPC was continued to be incubated for 6 days, and the rate of EPC outgrowth and the number of trophoblastic giant cells (TGCs) were evaluated under a phase-contrast microscope. As a result, the administration of each placental extract significantly induced the cell spreading and TGC transformation of EPC as compared with the culture in alpha-MEM alone. The rate of cell-spreading rapidly increased on the 2nd day after incubation in the medium containing placental extracts. In particular, EPC outgrowth was more remarkable in the medium containing the 10.5-day placental extract than in the other media including FCS-containing medium. The number of transformed TGCs was also the largest in the 10.5-day medium among the groups examined. These results indicate that certain placental factors at mid-gestation, especially on day 10.5 pc, may facilitate the EPC differentiation.     

Time-lapse videomicrographic analyses of contractions in mouse blastocysts

Contraction has been observed in cultured blastocysts of many mammals, but little is known about the features of the contraction and its physiological role in blastocysts. The author analyzed contractions of a large number of cultured mouse blastocysts by time-lapse videomicrography. The results revealed that blastocysts repeated contractions of different degrees during the expanded stage from 10 h after blastocoel formation, and that the number of contractions was greater during the hatching period than in the periods pre- and post-hatching. The results also showed that the time needed for both contraction and re-expansion to the size before contraction tended to lengthen in blastocysts severely contracted. It was inferred that contractions of blastocysts occur physiologically in relation to myosin light chain kinase, but not due to an increase in permeability between trophectoderm cells in association with their division, or the influence of culture. Furthermore, it was inferred that re-expansion of contracted blastocysts occurs due to active transport and accumulation of Na(+) from the trophectoderm cells into blastocoelic fluid as a result of the action of Na(+)/K(+)-ATPase activated in the membrane of trophectoderm cells. Our results suggested that contractions are also present in blastocysts developed in vivo, and that weak contractions (less than 20% volume reduction) play an important role in hatching, whereas strong contractions (20% or more volume reduction) have the effect of inhibiting hatching. From our results on contractions of various blastocysts, it seems possible to evaluate the developmental ability of embryos, i.e. embryo quality, based on contractions of blastocysts.