Intravital selection of early mouse embryos by sex and karyotypic characteristics

The possibility of live karyotyping by a single blastomere isolated at the 2-, 4-, and 8-cell stage has been investigated. It is expedient to use to this end a single blastomere isolated from a 4-cell embryo. Three rest blastomeres formed normal morulae or blastocysts upon cultivation during 44 or 68 hrs. When the isolated blastomeres were cultivated for 14-16 hrs in a medium 0.5 micrograms/ml colcemide, 97% of blastomeres were at the metaphase stage and 72% of chromosome plates were suitable for karyotyping. The prospects of the method proposed in experimental embryology and for selection of the early embryos of farm animals by sex are discussed.     

ROCK inhibition prevents early mouse embryo development

ROCK is a Rho-GTPase effector that is important for actin assembly and is involved in various cellular functions, including cell contraction, migration, motility, and tumor cell invasion. In this study, we investigated ROCK expression and function during early mouse embryo development. Inhibiting ROCK by Y-27632 treatment at the zygote stage resulted in first cleavage failure, and most embryos failed to develop to the 8-cell stage. When adding Y-27632 at the 8-cell stage, embryos failed to undergo compaction and could not develop into blastocysts. In addition, fluorescence staining intensity analysis indicated that actin expression at blastomere membranes was significantly reduced. After ROCK inhibition, two or more nuclei were observed in a cell, which indicated possible cytokinesis failure. Moreover, after ROCK inhibition with Y-27632, the phosphorylation levels of LIMK1/2, a downstream molecule of ROCK, were decreased at blastomere membranes. Thus, our results showed conserved roles for ROCK in this mammalian embryo model and indicated that a ROCK-LIMK1/2-actin pathway might regulate cleavage and blastocyst formation during early mouse embryo development.     

Development of single mouse blastomeres enlarged to zygote size in conditions of nucleo-cytoplasmic synchrony

The following blastomeres were enlarged to the size of the zygote by one, two or three rounds of blastomere enucleation and electrofusion: (1) from the 2-cell stage (referred to as 2/1 embryos), (2) from the 4-cell stage (referred to as 4/1 embryos), (3) from the 8-cell stage (referred to as 8/1 embryos). Such single enlarged blastomeres developed into blastocysts in vivo in 55.5% (2/1), 28% (4/1) and 6.6% (8/1) of cases. Their mean cell numbers were 45.3, 24.5 and 13.0 in 2/1, 4/1 and 8/1 embryos, respectively. When a blastomere nucleus from another mouse strain (heterologous nucleus) was substituted for a blastomere's own (homologous) one, then fewer blastocysts were formed from 2/1 embryos (34.6%), but not from 4/1 and 8/1 embryos. Five young (10.4%) were born from 2/1 embryos with a homologous nucleus, and nine (8.3%) from 2/1 embryos with heterologous nuclei. Four young (7.1%) were born from 4/1 embryos with heterologous nuclei. No young were obtained from 8/1 embryos. Incorrect cavitation resulting in trophoblastic vesicles and false blastocyst formation was common in 4/1 embryos (18.7% of those with homologous nuclei and 41.3% with heterologous nuclei) and in 8/1 embryos (53.3% and 43.7%, respectively). The results show that neither enlargement to zygote size nor nucleo-cytoplasmic synchrony improve postimplantation development of 4- and 8-cell stage blastomeres when compared with less enlarged non-synchronous ones; therefore, it appears that an insufficient number of inner cell mass cells in blastocysts and not too small a size of isolated blastomeres precludes their postimplantation development.     

Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo

Porcine embryos produced in vitro have a small number of cells and low viability. The present study was conducted to examine the morphological characteristics and the relationship between actin filament organization and morphology of porcine embryos produced in vitro and in vivo. In vitro-derived embryos were produced by in vitro maturation, in vitro fertilization (IVF), and in vitro development. In vivo-derived embryos were collected from inseminated gilts on Days 2-6 after estrus. In experiment 1, in vitro-derived embryos (相似文献   

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Potential of hypertonic medium treatment for embryo micromanipulation: II. Assessment of nuclear transplantation methodology, isolation, subzona insertion, and electrofusion of blastomeres to intact or functionally enucleated oocytes in rabbits

The objective of this research was to study efficiency of embryo development following transfer of blastomeres into the perivitelline space of oocytes. Single blastomeres from 8-, 16-, and 32-cell embryos were obtained following mucin coat and zona pellucida removal by combined treatments with pronase and acidic phosphate-buffered saline (PBS, pH = 2.5). Blastomeres were separated by pipetting with a fire-polished micropipette following incubation in Ca+(+)-free PBS for 15 min at 39 degrees C. This procedure resulted in over 97% blastomere separation. For ease of blastomere insertion, oocytes were placed in droplets of 0.5 M sucrose in PBS (SPBS) during micromanipulation. To functionally enucleate oocytes some were stained with Hoechst 33342 DNA stain and irradiated. A single 8- or 16-cell blastomere was aspirated into an injection pipette (35 microns or 25 microns at the tip, respectively) and inserted into the perivitelline space of an irradiated or non-irradiated oocyte, but not fused with the oocyte. This micromanipulation procedure did not affect development of individual blastomeres into blastocysts or trophectoderm vesicles when compared with cultured control single blastomeres (P greater than .05). When the inserted blastomere was induced to fuse with an intact non-irradiated oocyte under an electric field, 56-57% were fused and 39-45% of the fused and activated oocytes developed to morulae or blastocysts. When an inserted blastomere (from 8-32-cell embryos) was induced to fuse with a functionally enucleated oocyte treated by Hoechst 33342 staining, followed by washing and UV-light irradiation, 63-66% of them were fused, but only 15-22% developed to the morula or blastocyst stage. This research demonstrated that the use of hypertonic medium treated oocytes greatly improved the ease and success rate of blastomere subzona insertion, but the value of functionally enucleated oocytes as recipient cells for nuclear transfer requires further investigation.     

Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xeno-pus embryos activates maternal program of apoptosis as a "fail-safe" mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animal side blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4- and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16- and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8- to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continueddevelopment of the injected embryos. These results indicate that cells overexpress     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90° before first cleavage

To study the mechanisms of dorsal axis specification, the alteration in dorsal cell fate of cleavage stage blastomeres in axis-respecified Xenopus laevis embryos was investigated. Fertilized eggs were rotated 90° with the sperm entry point up or down with respect to the gravitational field. At the 8-cell stage, blastomeres were injected with the lineage tracers, Texas Red- or FITC-Dextran Amines. The distribution of the labeled progeny was mapped at the tail-bud stages (stages 35–38) and compared with the fate map of an 8-cell embryo raised in a normal orientation. As in the normal embryos, each blastomere in the rotated embryos has a characteristic and predictable cell fate. After 90° rotation the blastomeres in the 8-cell stage embryo roughly switched their position by 90°, but the fate of the blastomeres did not simply show a 90° switch appropriate for their new location. Four types of fate change were observed: (i) the normal fate of the blastomere is conserved with little change; (ii) the normal fate is completely changed and a new fate is adopted according to the blastomere's new position; (iii) the normal fate is completely changed, but the new fate is not appropriate for its new position; and (4) the blastomere partially changed its fate and the new fate is a combination of its original fate and a fate appropriate to its new location. According to the changed fates, the blastomeres that adopt dorsal fates were identified in rotated embryos. This identification of dorsal blastomeres provides basic important information for further study of dorsal signaling in Xenopus embryos.     

A quantitative analysis of cell allocation to trophectoderm and inner cell mass in the mouse blastocyst

The allocation of cells to the trophectoderm and inner cell mass (ICM) in the mouse blastocyst has been examined by labelling early morulae (16-cell stage) with the short-term cell lineage marker yellow-green fluorescent latex (FL) microparticles. FL is endocytosed exclusively into the outside polar cell population and remains autonomous to the progeny of these blastomeres. Rhodamine-concanavalin A was used as a contemporary marker for outside cells in FL-labelled control (16-cell stage) and cultured (approximately 32- to 64-cell stage) embryos, immediately prior to the disaggregation and analysis of cell labelling patterns. By this technique, the ratio of outside to inside cell numbers in 16-cell embryos was shown to vary considerably between embryos (mean 10.8:5.2; range 9:7 to 14:2). In cultured embryos, the trophectoderm was derived almost exclusively (over 99% cells) from outside polar 16-cell blastomeres. The origin of the ICM varied between embryos; on average, most cells (75%) were descended from inside nonpolar blastomeres with the remainder derived from the outside polar lineage, presumably by differentiative cleavage. In blastocysts examined by serial sectioning, polar-derived ICM cells were localised mainly in association with trophectoderm and were absent from the ICM core. In nascent blastocysts with exactly 32 cells an inverse relationship was found between the proportion of the ICM descended from the polar lineage and the deduced size of the inside 16-cell population. From these results, it is concluded that interembryonic variation in the outside to inside cell number ratio in 16-cell morulae is compensated by the extent of polar 16-cell allocation to the ICM at the next division, thereby regulating the trophectoderm to ICM cell number ratio in early blastocysts.     

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.     

Effect of serum on cell-to-cell associations during in vitro development of preimplantation mouse embryos

We have identified an activity which alters the morphology and developmental timing of post-compaction mouse embryos. A 15-min exposure of 4- and 8-cell mouse embryos to sera containing this activity induced monolayer formation, changing the normal positions of blastomeres at the 16- to 64-cell stages. Recovered embryos form normal blastocysts, based on morphology and in vitro production of trophectoderm and inner cell mass derivatives. These results suggest that under certain circumstances blastomeres remain developmentally labile as late as the sixth or seventh cleavage cycle.     

In vitro development and cell allocation following aggregation of split embryos with tetraploid or developmentally asynchronous blastomeres in rhesus monkeys

Production of genetically identical pairs of monkeys would have tremendous implications for biomedical research, particularly immunological studies and vaccine trials. Specific aims of this study were to (1) determine whether aggregation of embryos split into halves or quarters with equal numbers of either developmentally asynchronous or tetraploid blastomeres would enhance their developmental potential in vitro and increase total cell numbers in resulting blastocysts, and (2) determine the allocation of tetraploid and developmentally asynchronous blastomeres in resulting blastocysts. Results demonstrated that development into blastocysts was greater (p < 0.05) for embryos split into pairs (39.8%) than for those split into quadruplet sets (17.4%) and similar (p > 0.05) to that of nonmanipulated controls (59.6%). Creation of chimeras from aggregation of a single 4-cell and four 16-cell stage blastomeres resulted in blastocyst formation (69.2%) similar to that of nonmanipulated control embryos (66.9%). However, neither development nor total cell numbers in resulting blastocysts differed between aggregate chimeras and those split into quadruplet sets at the 16-cell stage. Blastocysts resulting from the aggregate chimeras were derived strictly from the 16-cell stage blastomeres, with complete exclusion of the 4-cell stage blastomeres. Aggregation of split embryos with equal numbers of tetraploid blastomeres doubled (p < 0.05) both the proportion developing into blastocysts and the total cell numbers in resulting blastocysts. Tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm of resulting blastocysts. In conclusion, due to exclusion of the less advanced cells, aggregation of developmentally asynchronous blastomeres did not improve the developmental competence or cell numbers of split rhesus embryos. Reconstitution of split embryos with equal numbers of tetraploid blastomeres enhanced their developmental potential and cell numbers in resulting blastocysts. However, tetraploid blastomeres were allocated to both the inner cell mass and trophectoderm.     

Spatial arrangement of individual 4-cell stage blastomeres and the order in which they are generated correlate with blastocyst pattern in the mouse embryo

In the unperturbed development of the mouse embryo one of the 2-cell blastomeres tends to contribute its progeny predominantly to the embryonic and the other to the abembryonic part of the blastocyst. However, a significant minority of embryos (20-30%) do not show this correlation. In this study, we have used non-invasive lineage tracing to determine whether development of blastocyst pattern shows any correlation with the orientation and order of the second cleavage divisions that result in specific positioning of blastomeres at the 4-cell stage. Although the orientation and order of the second cleavages are not predetermined, in the great majority (80%) of embryos the spatial arrangement of 4-cell blastomeres is consistent with one of the second cleavages occurring meridionally and the other equatorially or obliquely with respect to the polar body. In such cleaving embryos, one of the 2-cell stage blastomeres tends to contribute to embryonic while the other contributes predominantly to abembryonic part of the blastocyst. Thus, in these embryos the outcome of the first cleavage tends to correlate with the orientation of the blastocyst embryonic-abembryonic axis. However, the order of blastomere divisions predicts a specific polarity for this axis only when the earlier 2-cell blastomere to divide does so meridionally. In contrast to the above two groups, in those embryos in which both second cleavage divisions occur in a similar orientation, either meridionally or equatorially, we do not observe any tendency for the 2-cell blastomeres to contribute to specific blastocyst parts. We find that all these groups of embryos develop to term with similar success, with the exception of those in which both second cleavage divisions occur equatorially whose development can be compromised. We conclude that the orientations and order of the second cleavages are not predetermined; they correlate with the development of blastocyst patterning; and that the majority, but not all, of these cleavage patterns allow equally successful development.     

慢病毒载体介导的绿色荧光蛋白转基因标记法研究猪嵌合体制作

目的:通过建立慢病毒载体感染猪胚胎体系实现胚胎标记,进而研究不同发育阶段猪孤雌胚胎之间的嵌合能力,为进一步研究猪早期胚胎发育以及细胞分化奠定基础.方法:首先,通过显微注射的方法把2×109I.U./ml、2×108I.U./ml和2×107I.U./ml三个梯度的表达绿色荧光的慢病毒载体分别注射到猪1-细胞胚胎和2-细胞胚胎的透明带下,进行胚胎的GFP转基因标记,在荧光显微镜下观察比较卵裂率、阳性胚胎率、囊胚率、阳性囊胚率和囊胚细胞数.然后,采用凹窝聚合法对同步发育胚胎在不同阶段(2-细胞,4-细胞,8-细胞)进行嵌合,2-细胞胚胎与不同发育阶段(2-细胞、4-细胞、8-细胞)胚胎进行嵌合以及2-细胞胚胎卵裂球互换制作嵌合体胚胎,发育到囊胚时在荧光显微镜下检测胚胎的嵌合状态.结果:2×109I.U./ml的慢病毒感染猪2-细胞胚胎组中,体外受精和孤雌胚胎感染阳性率( 80.00％、76.36％)和阳性囊胚率(90.74％、89.56％)都显著高于其它滴度组(P＜0.05),另外,慢病毒感染的两种胚胎与对照组对卵裂率、囊胚率和囊胚细胞数三个指标没有显著影响(P＞0.05).2-细胞胚胎之间嵌合囊胚率和2-细胞卵裂球互换嵌合囊胚率( 53.85％、62.50％)显著高于2-细胞胚胎与4-细胞胚胎的嵌合率(18.60％,P＜0.05),在同步发育胚胎中8-细胞胚胎之间的嵌合率(75.00％)高于4-细胞胚胎之间和2-细胞胚胎之间的嵌合率( 65.00％、53.80％).结论:2×109I.U./ml的慢病毒感染2-细胞期胚胎效率最高,另外,慢病毒感染对猪胚胎发育没有明显影响.8-细胞间的嵌合率比较高;发育同步胚胎间的嵌合率高于发育非同步胚胎间的嵌合率.     

Fusion of Spermatozoa with Embryonic Cells and Somatic Cells in the Sea Urchin

Spermatozoa can enter the separated blastomeres of 8- and 16-cell stage embryos, the cells of blastulae and even somatic cells of the oesophagus wall of an adult sea urchin, under certain conditions. In the presence of egg jelly solution, the rate of entrance of spermatozoa is remarkably increased. In the case of the blastomere of 8-cell stage embryos, characteristic cytoplasmic protrusions are formed at the sites of sperm entry, in succession to the formation of the cytoplasmic bulge. These protrusions elongate until 4 min after insemination, and then they retract gradually. The nucleus of penetrated sperm swells and decondenses to form a pronucleus. In most cases, the pronucleus seems to fuse with the preexisting diploid nucleus of the blastomere. When the dissociated oesophagus cells were inseminated, a certain type of the cells was found to fuse with spermatozoa, although the percentage of fused cells was very low.     

Cell lineage of zebrafish blastomeres. I. Cleavage pattern and cytoplasmic bridges between cells

Patterns of cleavage and cytoplasmic connections between blastomeres in the embryo of the zebrafish, Brachydanio rerio have been described. The cell division pattern is often very regular; in many embryos a blastomere's lineage may be ascertained from its position in the cluster through the 64-cell stage. At the 5th cleavage, however, significant variability in pattern is observed, and alternative patterns of the 5th cleavage are described. The early cleavages are partial, incompletely separating blastomeres from the giant yolk cell. The tracer fluorescein-dextran (FD) was injected into blastomeres to learn the extent of the cytoplasmic bridging. It was observed that until the 10th cleavage, blastomeres located along the blastoderm margin maintain cytoplasmic bridges to the yolk cell. Beginning with the 5th cleavage, FD injected into a nonmarginal blastomere either remains confined to the injected cell, or if the injection was early in the cell cycle, the tracer spreads to the cell's sibling, through a bridge persisting from the previous cleavage. On the other hand, injected Lucifer yellow spreads, presumably via gap junctions, widely among blastomeres in a pattern unrelated to lineage.     

The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos.

During C. elegans embryogenesis an 8-cell stage blastomere, called MS, undergoes a reproducible cleavage pattern, producing pharyngeal cells, body wall muscles, and cell deaths. We show here that maternal-effect mutations in the pie-1 and mex-1 genes cause additional 8-cell stage blastomeres to adopt a fate very similar to that of the wild-type MS blastomere. In pie-1 mutants one additional posterior blastomere adopts an MS-like fate, and in mex-1 mutants four additional anterior blastomeres adopt an MS-like fate. We propose that maternally provided pie-1(+) and mex-1(+) gene products may function in the early embryo to localize or regulate factors that determine the fate of the MS blastomere.