整合蛋白β1在人类卵母细胞和早期胚胎上分布的研究

目的：研究人类卵母细胞体外成熟和体外受精前后,以及体外受精后胚胎早期发育的不同阶段,整合蛋白β1的分布规律。方法：利用激光共聚焦显微镜和荧光标记的整合蛋白β1抗体。结果：在成熟人类卵母细胞,体外受精的合子以及2细胞阶段胚胎中,整合蛋白β1的分布不同于在小鼠胚胎中的分布,不是分布在细胞质膜的表面,而是集中分布在细胞核的附近,细胞质膜的表面分布很少。在体外培养的4细胞和8细胞胚胎中,整合蛋白β均匀分近,细胞质膜的表面分布很少,在体外培养的4细胞和8细胞胚胎中,整合蛋白β均匀分布在卵裂球中,发育至桑甚胚阶段,整合蛋白β1的分布开始出现极性,到囊胚阶段,整合蛋白β1的分布集中于滋养外胚层处。结论：整合蛋白分子被普遍认为是卵母细胞中的精子受体,但本研究提示,人类精子和卵母细胞的结合,整合蛋白β1的影响可能不大,而整合蛋白β1可能与雌雄原核融合,胚胎卵裂以及胚胎着床有关。     

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

单个卵裂球分离和培养是生产同卵双胎或一卵多胎动物的一种有效方法。在羊和牛已经获得了来源于2-细胞和4-细胞期胚胎的单个卵裂球的活体后代;在兔也获得来源于4-细胞胚胎的单个卵裂球后代;在猪已获得8-细胞单个卵裂球的后代。但多数的研究者发现,小鼠单个卵裂球培养后的体外发育率、移植妊娠率和产仔率都很低。上述均为新鲜胚移植结果,至于分离后卵裂球发育成的囊胚再进行玻璃化冷冻保存尚未见报道。本实     

小鼠-牛体细胞种间核移植

本文探讨了小鼠-牛异质胚构建的简便方法及小鼠体细胞核在牛卵母细胞中重新编程的可能性。以牛的卵母细胞为细胞质供体,用去除透明带及徒手切割的方法去核,设定电压1.5 KV/cm,脉冲时间40μsec,与小鼠皮肤成纤维细胞进行电融合的融合率为67.44%,卵裂率为30.23%。融合细胞经离子酶素-6-DMAP激活,用微滴内压制做窝的方法培养小鼠皮肤成纤维细胞异质胚,异质胚的最终发育阶段为8细胞期。结果表明,去透明带牛卵母细胞经切割法去核,可用于小鼠异质胚构建;微滴内做窝的体外培养方法可避免无透明带胚胎的聚合。     

小鼠四倍体早期胚胎基因组甲基化模式

利用小鼠抗5-甲基胞嘧啶(5MeC)单克隆抗体检测了体外培养小鼠四倍体早期胚胎的基因组甲基化模式。结果表明: 利用电融合方法制备的小鼠四倍体胚胎在体外培养体系中经历细胞质融合、细胞核融合及细胞继续分裂发育直到囊胚期的过程, 在细胞质融合的时候胚胎卵裂球同体内体外培养二倍体胚胎一样, 呈现高度甲基化状态; 在细胞核开始融合的时候, 甲基化水平急速下降, 在细胞核完全融合的时候甲基化水平达到最低点; 随着胚胎继续分裂, 胚胎甲基化水平逐渐增加, 在桑葚胚期甲基化水平最高; 但是囊胚期四倍体胚胎内细胞团同滋养层细胞甲基化荧光信号没有差别, 这与体内体外培养二倍体囊胚内细胞团细胞甲基化荧光强度高于滋养层细胞甲基化荧光强度不同。因此, 小鼠体外培养四倍体胚胎的甲基化模式是不正常的, 这可能是四倍体小鼠难以发育到妊娠足月的原因之一。这是对小鼠四倍体早期胚胎基因组甲基化模式的首次报道。     

山羊体外受精胚胎序贯培养的研究

目的：根据早期胚胎不同发育阶段的营养需求设计体外培养体系,以建立适于山羊早期胚胎体外发育的序贯培养方法。方法：对屠宰场来源山羊卵巢卵母细胞进行体外成熟和体外受精后移入添加不同胚胎发育影响因子的培养体系中进行体外培养,显微镜观察、统计各阶段胚胎发育情况,并对培养的胚胎进行移植。结果：BSA和EGF对山羊体外受精卵具有明显促卵裂作用,培养系统中添加EGS、EGF、HTAU或p—Me可有效支持8-细胞期山羊胚胎克服发育阻滞而提高桑葚胚发育率;在不同胚胎期添加各发育影响因子进行序贯培养的卵裂率、≥8-细胞率和桑葚胚发育率分别为45．2％、60．6％和23．4％,序贯培养的胚胎移植后产羔率为11．1％。结论：宜根据早期胚胎各时期代谢特点和营养需求对山羊胚胎进行序贯培养。     

小鼠2,4,8—细胞胚胎卵裂球体外培养的研究

以ＣＺＢ为基础培养液,培养小鼠２、４、８－细胞胚胎的卵裂球,研究葡萄糖、牛磺酸和胰岛素对１／２卵裂球体外发育的影响及１／４、１／８和２／８卵裂球的体外发育规律。２－细胞胚胎卵裂球在ＣＺＢ中和在添加牛磺酸的ＣＺＢ中培养,其囊胚发育率（分别为９２％、８９％）无显差异（Ｐ＞０．０５）。胰岛素在少量葡萄糖存在的情况下,不影响卵裂球的囊胚发育率;在无葡萄糖时,卵裂球的囊胚发育率（３８％）显降低。牛磺酸在     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

激光显微照射金鱼受精卵对其胚胎发育的影响

2-细胞期胚胎经激光显微照射(功率为90毫瓦)其中一个卵裂球时能产生明显的损伤光斑,受照射的卵裂球立即停止发育。另一未受照射的卵裂球仍能正常卵裂直至孵化幼鱼,所获得的幼鱼在形态上与正常幼鱼相同。8-细胞或囊胚期胚胎经激光显微照射(功率为372毫瓦)时,则可在受照射的卵裂球上产生明显伤斑,并在照射部位溢出部分细胞内含物,绝大部分胚胎发育成不正常的胚体和各种畸形幼鱼。     

应用FRAP技术分析小鼠嵌合体和正常胚胎发育中细胞间隙连接介导通讯

应用激光扫描共聚焦显微镜的光漂白恢复(fluorescence redistribution after photobleaching,FRAP)技术分析小鼠嵌合体胚胎和正常胚胎的卵裂球之间细胞间隙连接介导通讯(gap junctional inter-cellular communica-tion,GJIC),结果发现:8-细胞期嵌合体胚胎的光漂白恢复率(24.3%)明显低于正常胚胎(64.2%),提示GJIC的降低可能是影响嵌合体胚胎发育率降低的因素之一;囊胚的光漂白恢复率也较低(22.7%),提示随着细胞分化,GJIC的水平有所降低。     

使用激光微束研究哺乳动物早期胚胎分化

近年来，一些研究者用显微操作方法证实， 哺乳动物早期胚胎至少直至8一细胞期，每个卵 裂球仍然是全能的，即还没有开始分化。1965 年Daniel用激光微束照射破坏2一细胞期兔胚中 一个卵裂球后，另一卵裂球在培养条件下发育 至桑堪胚期。此外，还有Tarkowski和Wroblewska (1967）的卵裂球分离实验，Mintz (1965) 卵裂球重聚合实验以及最近Willadsen (1979) 羊胚卵裂球分离实验等。     

Determination of protein concentration in cleaving mammalian ova by the capillary spectrophotometry method]

A micromodification of the Lowry's method is described which allows to measure reliably the protein content in 5-20 embryos of white rats and CBA mice. Differences in the protein content in rat embryos at the stages of 2 blastomeres and blastocyst were shown to be statistically unreliable (20.2 +/- 1.4 and 18.9 +/- 1.3 ng, resp.). The protein content in the mouse embryos at the same two stages differs reliably (19.1 +/- 1.1 and 22.0 +/- +/- 1.7 ng, resp.). The protein content in zona pellucida does not differ reliably from those in rat embryos both at the stages of 2 blastomeres (4.5 ng) and blastocyst (2.3 ng). The protein content in embryos devoid of zona pellucida decreased after 3 hours incubation in the medium 199 at 37 degrees at the stages of morula and blastocyst by 17-20% in rats and by 50% in mice. Addition of 1% serum albumin to the incubation medium did not prevent the partial "loss" of protein by the embryos. The protein content in the rat and mouse embryos at the stage of 2 blastomeres suffered no changes under long-term incubation in the medium 199.     

Polarization of blastomeres in the cleaving rabbit embryo

Cellular polarization is believed to be a crucial event in the differentiative divergence of the two cell lineages leading to the blastocyst in rodent embryos. This study was undertaken to determine if rabbit embryos exhibited cellular polarization prior to blastocyst formation and to define the embryonic stage at which polarization was first apparent. Polarity was assayed by observation of the pattern of binding of FITC-Con A to dissociated blastomeres from three stages of rabbit embryos. Scanning electron microscopy on the dissociated cells confirmed the fluorescence results. Fifty-one percent of blastomeres in 38- to 66-cell rabbit embryos exhibited an intense pole of FITC-Con A binding and a single pole of microvilli. Only 2% of blastomeres at the 17- to 34-cell stage were similarly polarized and none were polarized at the 8- to 16-cell stage. In addition, during attempts to remove the mucin coat and zona pellucida from the rabbit embryos prior to their dissociation, it was found that the protease sensitivity of these coats also changed at the 38- to 66-cell stage. Prior to this time, although the mucin coat disappeared after 30 min in 0.5% pronase, the zona required approximately 1.5-2.5 hr in pronase for even partial removal. At the 38- to 66-cell stage, pronase dissolved the mucin coat within 10 min and the zona pellucida within 20 min. The zona was resistant to 0.1% proteinase K at all stages examined.     

Effect of pronase treatment, microdissection, and zona pellucida removal on the development of porcine embryos and blastomeres in vitro

The in vitro development of porcine blastomeres and the effects of pronase treatment, microdissection, and zona pellucida removal used in the isolation procedure were investigated. Seven hundred and forty-nine two to eight-cell embryos were collected from 11 sows and 74 gilts. Zona-free porcine blastomeres (ISOL BL) were obtained by treating embryos with 2.5 or 5.0% pronase for 3.0 min and microdissecting with finely drawn siliconized glass pipettes. The effect of the pronase treatment on subsequent in vitro development was evaluated by treating two to eight-cell embryos with 5.0% pronase for 3.0 min (PTD EMB). The effect of pronase treatment and microdissection on in vitro development was evaluated by microdissecting PTD EMB, leaving one blastomere bounded by the zona pellucida (BL ZP). Untreated two to eight-cell embryos were cultured as controls (CONTROLS). Embryos and blastomeres were cultured individually in microdrops of Whitten's medium with 15 mg/ml bovine serum albumin (WM + BSA) under paraffin oil in a humidified atmosphere of 5% CO2 in air at 37 degrees C. Observations were conducted at 24-h intervals and at the cessation of division embryos were fixed, stained, nuclei enumerated, and cleavage indices assigned. Blastocysts and vesiculated embryos which developed were measured using an ocular micrometer. The incidence of blastocyst formation was greater (P less than 0.05) for ISOL BL from four-cell than from two or eight-cell embryos. The presence of the zona pellucida did not significantly affect the incidence of blastocyst formation by single blastomeres. Although ISOL BL did not develop as well as CONTROLS or PTD EMB (P less than 0.05), development of BL ZP was not significantly different from the respective PTD EMB. Blastocysts developing from blastomeres had fewer cells and were smaller than CONTROLS or PTD EMB (P less than 0.05). Although development of ISOL BL may have been impaired by the isolation procedures employed, BL ZP are capable of in vitro development comparable to their respective PTD EMB.     

Production of monozygotic twin calves using the blastomere separation technique and Well of the Well culture system

The present study was conducted to establish a simple and efficient method of producing monozygotic twin calves using the blastomere separation technique. To produce monozygotic twin embryos from zona-free two- and eight-cell embryos, blastomeres were separated mechanically by pipetting to form two demi-embryos; each single blastomere from the two-cell embryo and tetra-blastomeres from the eight-cell embryo were cultured in vitro using the Well of the Well culture system (WOW). This culture system supported the successful arrangement of blastomeres, resulting in their subsequent aggregation to form a demi-embryo developing to the blastocyst stage without a zona pellucida. There was no significant difference in the development to the blastocyst stage between blastomeres separated from eight-cell (72.0%) and two-cell (62.0%) embryos. The production rates of the monozygotic pair blastocysts and transferable paired blastocysts for demi-embryos obtained from eight-cell embryos (64.0 and 45.0%, respectively) were higher than those for demi-embryos obtained from two-cell embryos (49.0 and 31.0%, P<0.05). The separated demi-embryos obtained from eight-cell embryos produced by IVM/IVF of oocytes collected by ovum pick-up (OPU) from elite cows and cultured in wells tended to have a higher pregnancy rate (78.9% vs. 57.1%) and similar monozygotic twinning rate (40.0% vs. 33.3%) compared with monozygotic twin blastocysts obtained by the conventional bisection of in vivo derived blastocysts. In conclusion, producing twins by separation of blastomeres in OPU-IVF embryos, followed by the WOW culture system, yielded viable monozygotic demi-embryos, resulting in high rates of pregnancy and twinning rates after embryo transfer.     

Use of zona drilling for safe and effective biopsy of murine oocytes and embryos

With the mouse as a model, we have used zona drilling to devise procedures for safe removal of the first polar body or one or more blastomeres from cleaving embryos. These methods require minimal disruption of the zona pellucida and little or no direct contact between microtools and the materials to be biopsied. Of 175 eggs subjected to the polar body biopsy procedure, 1 was killed, and 165/174 survivors were fertilized (94.8%). For blastomere biopsy, embryos from the 2- to 16-cell stage were incubated in a chelating medium containing 100 mM sucrose for at least 30 min. The zonae were then drilled, and one or more blastomeres were "pushed" out through the hole by pressure exerted against the zona at some distance from the drilling site. In all 85 embryos biopsied, one or more additional intact blastomeres were successfully removed. Moreover, 83/84 biposied embryos that were subsequently cultured developed into blastocysts (98.8%). Although acid Tyrode's solution was used in this study, mechanical methods of zona opening were also effective. The data indicate that oocyte and embryo biopsy assisted by zona drilling is safe and does not appear to affect fertilization or development, and as such, it is applicable to genetic diagnostic procedures.     

Developmental potential of isolated blastomeres from early murine embryos

Experiments were designed to evaluate the effect of blastomere separation on blastocoele formation and development of viable fetuses. Two-cell and four-cell murine embryos were dissociated into individual blastomeres and cultured to the blastocyst stage. For embryos of both stages, zona removal and blastomere separation reduced (P<0.05) the number of viable embryos at the onset of culture and reduced (P<0.01) the frequency of continuation of development of blastomeres to the blastocyst stage. Attempts to repeatedly split two-cell stage embryos decreased in vitro development to blastocysts. The number of cells in two-cell embryos that were cultured to blastocyst was not different for control (64.8 +/- 11.5) or for two-cell embryos cultured without the zona pellucida (60.9 +/- 10.1) but was reduced (P<0.01) for one-half embryos that were cultured to blastocysts (35.6 +/- 10.6). The cell number of blastocysts obtained from dissociated four-cell (1/4) embryos (17.4 +/- 1.4) was similarly reduced (P<0.01). In vivo development was assessed after cultured embryos were transferred to the uteri of day 3 pseudopregnant females. Zona free intact embryos (2/36, 6%) and zona free half embryos (7/36; 19%) developed less frequently (P<0.05) than intact controls (45/100). Noncultured morula briefly exposed to pronase to thin the zona had similar impaired development. Embryos with thinned zona or no zona developed less frequently (21/82, 2/72 respectively, P<0.05) than nonpronase-treated controls (50/83).     

Freezing of bovine embryos: Effects of a one-step addition of 1.4 M glycerol

The effects of a one-step addition of 1.4 M glycerol (method A) upon morphological appearance and developmental capacity of frozen/thawed day 7 bovine embryos were investigated and compared to a standard stepwise addition of 1.0 M glycerol (method B). With method A, the percentage of intact embryos (classified as excellent, good and poor) was 95.3% (61 out of 64) without differences between morulae (96.5%) and blastocysts (94.4%). With method B, the percentage of intact embryos was 83.0% (44 out of 53). The percentage was similar for blastocysts (89.2%) and significantly (p < 0.05) lower for morulae (68.8%) when compared to method A. The percentage of embryos with a damaged zona pellucida was considerably increased with method A (26.6%) when compared to method B (13.2%). The proportion of embryos with excluded blastomeres was similar in both methods (21.9% method A, 17.0% method B). With method A, pregnancy rates after nonsurgical transfer were 51.0% (25 out of 49) and were better than with method B (40.5%; 15 out of 37). Embryos with a damaged zona pellucida resulted in a high pregnancy rate of 66.7% (8 out of 12). A pregnancy rate of 52.9% (10 out of 17) was obtained with embryos showing some excluded blastomeres. Thus, a one-step addition of 1.4 M glycerol facilitates and accelerates the process of embryo cryopreservation and is compatible with high pregnancy rates. Damage of the zona pellucida does not impair further development of frozen/thawed bovine embryos provided blastomeres are intact.     

Production of mouse chimeras by injection of embryonic stem cells into the perivitelline space of one-cell stage embryos

Generation of mouse chimeras is useful for the elucidation of gene function. In the present report, we describe a new technique for the production of chimeras by injection of R1 embryonic stem (ES) cells into the perivitelline space of one-cell stage mouse embryos. One-cell embryos are injected with 2–6 ES cells into the perivitelline space under the zona pellucida without laser-assistance. Our embryo culture experiments reveal that ES cells injected at the one-cell stage embryo start to be incorporated into the blastomeres beginning at the 8-cell stage and form a chimeric blastocyst after 4 days. We have used this approach to successfully produce a high rate of mouse chimeras in two different mouse genetic backgrounds permitting the establishment of germ line transmitters. This method allows for the earlier introduction of ES cells into mouse embryos, and should free up the possibility of using frozen one-cell embryos for this purpose.     

Transfection of mouse eggs and embryos using DNA combined to cationic liposomes

We have used plasmid DNA in combination with cationic liposomes to transfect mouse eggs and embryos. The plasmid was rhodamine labeled, which allowed a direct visualization of the DNA uptake by the cells. Immature eggs, collected from the ovaries, were easily transfected, but once the egg was ovulated the zona pellucida (ZP) acted as a barrier and prevented transfection. Permeabilization or removal of the ZP was therefore a requirement to allow transfection. Transfected eggs were capable of being fertilized in vitro giving raise to embryos that expressed the recombinant protein. Morulae and blastocysts were also transfected when the ZP was permeabilized, but the efficiency of transfection decreased and in some cases not all the blastomeres incorporated the plasmid. Pronuclear embryos were cultured and showed expression of the transgene from the 2-cell stage. This indicates that liposome-transfection of oocytes or pronuclear embryos could be a simple and suitable method to introduce foreign genes in embryos and perhaps could be also useful to generate transgenic animals.     

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.