Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes

Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.     

A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes

Factors affecting the efficiency of animal cloning remain to be elucidated. Enucleation of recipient oocytes is a critical step in cloning procedures and typically is performed by aspirating a portion of the cytoplasm underlying the first polar body. Enucleation is evaluated using epifluorescence after Hoechst staining for DNA, which may disrupt functions of the cytoplast, especially mitochondria. Mitochondrial DNA in Dolly and other cloned sheep has been shown to derive exclusively from recipient oocytes. Not only might evaluation of the aspirated karyoplast portion inadequately reflect the state of the cytoplast, it is also time consuming. Here we report a reliable, noninvasive technique for spindle imaging and enucleation of oocytes using a new microscope, the Pol-Scope. The efficiency of enucleation was 100%, and only 5.5% of the oocytes' mitochondria entered the karyoplast upon Pol-Scope-directed removal of the spindle. Moreover, Pol-Scope imaging of spindles and micromanipulation did not compromise the developmental competence of reconstituted oocytes and cytoplasts.     

Pre-loading of mouse oocytes with DNA-specific fluorochrome (Hoechst 33342) permits rapid detection of sperm-oocyte fusion

Mouse oocytes exposed to 1 microgram Hoechst 33342 (H-33342)/ml and then fertilized in vitro developed normally into blastocysts and blastocyst outgrowths. After penetration of the zona, the fertilizing spermatozoon showed intense fluorescence upon fusion with the vitelline membrane. Due to fluorochrome leakage from the perivitelline space a faint fluorescence was detected in zona-bound spermatozoa. This fluorescence of zona-bound spermatozoa intensified with increased fluorochrome concentration (10 micrograms/ml), obscuring the fluorescence of the fertilizing spermatozoa. Spermatozoa added to zona-free mouse oocytes (pre-loaded with 1 or 10 micrograms H-33342/ml) fluoresced within 10 min of insemination, provided the zonae were removed mechanically. Removal by protease digestion induced leakage of fluorochrome, so that all spermatozoa in the vicinity of an oocyte pre-loaded with 10 micrograms H-33342/ml became labelled. This leakage was not visibly apparent when protease-treated oocytes were exposed to only 1 microgram H-33342/ml. The technique could not be applied to zona-free hamster oocytes under our conditions, since the fluorochrome leaked freely from the oocytes whether the zona was removed mechanically or enzymically.     

Chemically assisted handmade enucleation of porcine oocytes

The purpose of our work was to find an efficient and reliable chemically assisted procedure for enucleation of porcine oocytes connected to the handmade cloning (HMC) technique without the potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection. After 41-42 h in vitro maturation, porcine oocytes were incubated with 0.4 microg/mL demecolcine for 45 min. Subsequently, the cumulus cells were removed and zonae pellucidae were partially digested. Oocytes with extrusion cones or oocytes only with polar body (PB) were subjected to oriented bisection. Less than half of the cytoplasm with the extrusion cone or adjacent to the PB was removed with a microblade. The remaining putative cytoplasts, containing the major part of the cytoplasm, were used as recipients for reconstruction with porcine fetal fibroblasts as nuclear donors. The overall efficiency achieved with chemically assisted enucleation was higher compared to oriented bisection without demecolcine incubation (90 +/- 3% vs. 81 +/- 4%, respectively; mean +/- absolute deviation [AD]). Reconstructed and activated embryos were cultured in vitro for 7 days. Fusion, cleavage and blastocyst rates were 87 +/- 7%, 97 +/- 6%, and 28 +/- 9%, respectively. These rates are at least as good as those achieved with normal HMC (81 +/- 4%, 87 +/- 8%, and 21 +/- 9%, respectively). For traditional, micromanipulator-based cloning, fusion and blastocyst rates were similar (81 +/- 10% and 21 +/- 6%, respectively), but the cleavage rate was lower (69 +/- 9%). In conclusion, chemically assisted handmade enucleation seems to be a simpler and potentially superior alternative to more conventional methods used for somatic cell nuclear transfer in pigs.     

Sucrose pretreatment for enucleation: an efficient and non-damage method for removing the spindle of the mouse MII oocyte

Oocytes enucleated at metaphase II stage can support reprogramming of transferred nucleus and further developing to term. However, the first polar body in mice sometimes migrates away from the original place of expulsion, so the chromosomes of the oocyte will displace from the first polar body. Thus, it is not always possible to successfully enucleate according to the position of the first polar body. Here we use sucrose treatment to visualize metaphase spindle fibers and chromosomes with standard light microscopy. In the manipulation medium containing 3% sucrose, oocytes of poor quality become shrunken, deformed or fragmented, while oocytes of good quality in the same medium would show a swelling around the metaphase chromosomes and a transparent spindle area, shaped like "infinity" and "0". So it is easy to remove the well-distinguished spindle and chromosomes in oocytes of good quality. Re-examined by Hoechst 33342 stain under the UV light, the enucleation rate was 100%. There was no significant difference in IVF and cleavage rates between the sucrose treatment and the control group. In conclusion, this study demonstrated that 3% sucrose pretreatment can give a method for evaluating embryo quality and more importantly, it can, under a common microscope, allow the visualization of the spindle and chromosomes in oocytes of good quality and hence efficiently improve enucleation rate without any harm.     

Optimization strategies for production of mammalian embryos by nuclear transfer

In order to optimize each of the individual steps in the nuclear transfer procedure, we report alternative protocols useful for producing recipient cytoplasts and for improving the success rate of nuclear transfer embryos in cattle, rhesus monkey, and hamster. Vital labeling of maternal chromatin/spindle is accomplished by long wavelength fluorochromes Sybr14 and rhodamine labeled tubulin allowing constant monitoring and verification during enucleation. The use of Chinese hamster ovary (CHO) donor cells expressing the viral influenza hemagglutinin fusion protein (HA-300a+), to adhere and induce fusion between the donor cells and enucleated cow, rhesus and hamster oocytes was examined. Cell surface hemagglutinin was activated with trypsin prior to nuclear transfer and fusion was induced by a short incubation of a newly created nuclear transfer couplet at pH 5.2 at room temperature. Donor cell cytoplasm was dynamically labeled with CMFDA, or further transfected with the green fluorescence protein (GFP) gene, so that fusion could be directly monitored using live imaging. High rates of fusion were observed between CHO donor cells and hamster (100%), rhesus (100%), and cow recipient cytoplasts (81.6%). Live imaging during fusion revealed rapid intermixing of cytoplasmic components between a recipient and a donor cell. Prelabeled donor cytoplasmic components were uniformly distributed throughout the recipient cytoplast, within minutes of fusion, while the newly introduced nucleus remained at the periphery. The fusion process did not induce activation as evidenced by unchanged distribution and density of cortical granules in the recipient cytoplasts. After artificial activation, the nuclear transfer embryos created in this manner were capable of completing several embryonic cell divisions. These procedures hold promise for enhancing the efficiency of nuclear transfer in mammals of importance for biomedical research, agriculture, biotechnology, and preserving unique, rare, and endangered species.     

Exposure of in vitro-matured porcine oocytes to SYBR-14 and fluorescence impairs their developmental capacity

Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7 μg/mL) of SYBR-14 at different incubation times (5, 10 and 30 min) in a 4 × 3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5 μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18 h after exposure to SYBR-14 (5 μg/mL for 10 min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.     

Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells

Nuclear transfer (NT) techniques have advanced in the last few years, and cloned animals have been produced from somatic cells in several species including pig. In this study we examined the feasibility of using granulosa-derived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% serum, 7 days), injected directly into cytoplasm of enucleated oocytes and the oocytes were electrically activated. The reconstructed embryos were cultured for 7 days and stained with Hoechst 33342 to determine the number of nuclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7+/-3.7% in NT and 97.4+/-4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5+/-7.2% in NT and 79.3+/-5.6% in controls. The blastocysts formation rates were 4.9+/-2.4% in NT and 26.8+/-3.8% in controls. To examine the effect of activation time on development of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post-injection. At 18 h post-activation the pronuclear formation rates were higher (62.5+/-7.3%) in the 3-4 h group as compared to the 0-0.5 h (22.0+/-12.5%) or 1-2h (44.5+/-6.3%) groups (P<0.05). However, the cleavage rates (9.6+/-4.6 to 10.7+/-4.2%) and the blastocysts formation rates (1.2+/-2.4 to 4.9+/-3.7%) were not different among treatments (P>0.05). The mean cell number of blastocysts was 15.7+/-5.7 in NT and 25.3+/-24.7 in controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that granulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocytes, and that the reconstructed embryos can develop to the blastocyst stage. In addition, EGFP can be used as a marker for gene expression of donor nuclei.     

Comparison of Hoechst 33342 and propidium iodide as fluorescent markers for sperm fusion with hamster oocytes.

Hamster oocytes were loaded with the DNA dyes Hoechst 33342 or propidium iodide. Oocytes incubated in 10 mumol Hoechst 333421(-1) showed intracellular fluorescence within 10-20 s of exposure, as did hamster and guinea-pig spermatozoa. Impaled oocytes to which acrosome-intact hamster spermatozoa were bound before injection of Hoechst 33342 showed dye transfer to adhering spermatozoa within 2 min of injection. Oocytes loaded passively with Hoechst 33342 showed dye transfer to bound, acrosome-intact hamster spermatozoa within 10 min. On ultra-structural examination, no bound, acrosome-intact hamster spermatozoa (n = 311) were found to be fused. By contrast, oocytes incubated with 10 mumol propidium iodide l-1 showed no intracellular fluorescence after 2 h, although in approximately 50% of oocytes, fluorescence developed rapidly in the first polar body. Oocytes injected with propidium iodide showed intracellular fluorescence but no dye transfer to bound, acrosome-intact hamster spermatozoa. Oocytes impaled on pipettes containing propidium iodide showed no dye transfer to unlabelled oocytes with which they were brought into contact, whereas in similar experiments using Hoechst 33342 detectable dye transfer to an adjacent oocyte occurred within 10 min. Oocytes loaded with propidium iodide transferred propidium iodide to fusion-competent guinea-pig spermatozoa during in vitro fertilization. Normally, between 20 and 40 spermatozoa bound per oocyte, and the percentage of spermatozoa showing dye transfer varied between 0 and 41%. Dye transfer occurred within 5-45 min. Only those nuclei that showed propidium iodide transfer subsequently decondensed, suggesting that dye transfer is correlated with fusion. The presence of fused spermatozoa was confirmed by ultrastructural examination of oocytes. In separate experiments, hamster and guinea-pig spermatozoa showed detectable fluorescence from propidium iodide within 20 s of osmotic rupture or membrane stripping by detergent, suggesting the lag in dye transfer to sperm nuclei during fertilization reflects a delay in sperm-oocyte fusion following adhesion. This evidence suggests that Hoechst 33342 could be an unreliable marker for sperm-oocyte fusion in fertilization because of its capacity for passive movement from oocyte to spermatozoon. This problem can be overcome using oocytes injected with propidium iodide. With this technique, it was possible to show that fusion-competent guinea-pig spermatozoa that are held in pipettes will fuse with hamster oocytes when placed mechanically against the oocyte surface.     

Rigidity of the nucleus during nuclear rotation in 3T3 cells

Using near infrared microscopy and ultraviolet fluorescence microscopy of living 3T3 cells stained with the fluorochrome Hoechst 33342, we have demonstrated that the nucleoli and Hoechst 33342-stained chromocenters in the nucleus maintain a fixed pattern during nuclear rotation. We conclude that the term "nuclear rotation" refers to rotation of the entire nucleus in the cytoplasm of interphase cells, and that nuclear rotation is not an expression of karyoplasmic streaming. In conjunction with earlier results on nuclear rotation the data imply that the interface of nuclear rotation is located either between the two nuclear membranes or in the adjacent cytoplasm.     

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress,kinetics of cortical cytoskeletal interactions,and second polar body extrusion

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.     

Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer

The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4°C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning. Mol. Reprod. Dev. 49:29–36, 1998. © 1998 Wiley-Liss, Inc.     

Differential sensitivity of mouse pronuclei and zygote cytoplasm to Hoechst staining and ultraviolet irradiation

The exposure of mouse zygotes pre-stained with Hoechst 33342 to u.v. irradiation for 20-30 sec significantly or completely inhibited development to blastocysts in vitro. However, development to the blastocyst stage of enucleated eggs receiving pronuclei from untreated eggs was as good as that of control reconstituted eggs when the cytoplasm originated from eggs exposed to u.v. irradiation for 20-30 sec, but was significantly lower when the cytoplasm was from eggs exposed for 40 sec. The chromosomes at the second metaphase stage could be removed with 15 sec of exposure to u.v. irradiation under a fluorescence microscope. Most eggs enucleated at the second metaphase that received a single inner cell mass nucleus (75%) showed pronuclear formation 6 h after activation; 23% of them developed to morphologically normal 2-cell eggs and 5% developed to blastocysts. These results demonstrate that the cytoplasm of mouse zygotes is more resistant to u.v. irradiation after Hoechst staining. Eggs at the second metaphase, from which chromosomes have been removed under a fluorescence microscope, can therefore be used as cytoplasm recipients for nuclear transplantation of inner cell mass nuclei.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

一种改进的牛体细胞核移植去核方法

为了提高传统的盲吸法牛体细胞核移植去核效率,将0.5mL离心管底部截掉,在截口处蒙上一层400目的细胞筛网,再与1 mL的离心管套在一起。实验1,将成熟的卵母细胞置于改造过的离心管内膜上,分别以1,000r/min、2,000r/min及3,000r/min离心10min,Hoechst 33342染色后,在荧光显微镜下计算第一极体与染色体的位置关系及去核效率;实验2,将卵母细胞以2,000r/min离心后去核做受体,颗粒细胞做供体进行核移植,检查重构胚胎的早期发育情况。结果表明:以2,000 r/min将卵母细胞离心10min后,有86.6%卵母细胞的极体与染色体之间夹角在20°以内,此时去核效率最高(87.4%);将卵母细胞离心后,对随后的重构胚发育无影响。因此,采用离心辅助去核的方法可以显著提高牛卵母细胞的去核效率。     

Selective Identification of Sperm Fused with the Surface of Echinoderm Eggs by DNA-Specific Bisbenzimide (Hoechst) Fluorochromes

When unfertilized echinoderm eggs are treated with the DNA-specific bisbenzimide fluorochrome Hoechst 33342 and then fertilized with unlabeled sperm, a single spermatozoan bound to the egg surface becomes fluorescent. Several lines of evidence, including correlative scanning electron microscopic studies, indicate that the fluorescent sperm is, in fact, the fertilizing sperm which acquires fluorescence as a consequence of membrane fusion between the sperm and egg. Comparative studies show that several fluorochromes structurally related to H33342 can be used to selectively identify the fertilizing sperm at the egg surface and that H33258 possesses a distinct advantage when used to visualize the male and female pronuclei in eggs fixed prior to fluorochrome exposure. Finally, none of the fluorochromes tested in these studies have any discernible effect on development from the first cell division through the pluteus larva stage. These observations suggest that the fluorochrome-transfer technique for identifying the fertilizing sperm may be useful in a wide variety of studies of gamete interaction as a simple and rapid cytological indicator for sperm-egg fusion.     

Measurement of DNA and antigen expression of live cells using three fluorochromes and two detectors.

In a multiparameter flow cytometric study, the fluorescence from the two different dyes fluorescein and dansyl-lysine were detected in the same spectral interval on the same photomultiplier tube, using two lasers to excite the fluorochromes at two separate laser foci. The two signals were split by a T-cable at the output of the photomultiplier tube and synchronized by delaying the first signal. Dansyl-lysine binds to the membrane of heat-inactivated cells and was used to distinguish between live and dead cells after a hyperthermic treatment. By gating on the dansyl-lysine signal, the DNA distribution and surface antigen expression of live and dead cells were obtained separately, using Hoechst 33342 and a monoclonal antibody conjugated to fluorescein.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

Spindle-view系统在猪卵母细胞去核中的应用

应用Spindle-view对体外成熟培养36、42、44和48h的猪体外成熟卵母细胞减数分裂纺锤体进行去核操作,并与传统去核方法(McGrath-Solter去核法,挤压去核法)相比较,结果表明:①在42~48h之间利用Spindle-view得到的猪卵纺锤体影像与极体的相对位置没有明显的变化;②Spindle-view适合用于猪体外成熟卵母细胞减数分裂纺锤体的观察及去核;去核效率与其他两种方法相比差异极显著(95.5%,42.1%,74.2%,P<0.01);③纺锤体成像是否清晰可用于猪卵母细胞的质量监控。     

Latrunculin a can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization

Although animal cloning is becoming more practicable, there are many abnormalities in cloned embryos, and the success rate of producing live animals by cloning has been low. Here, we focused on the procedure for preventing pseudo-second polar body extrusion from somatic cell nuclear transfer (SCNT)-derived oocytes. Typically, reconstructed oocytes are treated with cytochalasin B (CB), but here latrunculin A (LatA) was used instead of CB to prevent pseudo-second polar body extrusion by inhibiting actin polymerization. CB caps F-actin, LatA binds G-actin, and both drugs prevent their polymerization. When the localization of F-actin was examined using phalloidin staining, it was abnormally scattered in the cytoplasm of CB-treated 1-cell embryos, but this was not detected in LatA-treated or in vitro fertilization-derived control embryos. The spindle was larger in CB-treated oocytes than in LatA-treated or untreated control oocytes. LatA treatment also doubled the rate of full-term development after embryo transfer. These results suggest that cloning efficiency in mice can be improved by optimizing each step of the SCNT procedure. Moreover, by using LatA, we could simplify the procedure with a higher birth rate of cloned mice compared with our original method.