The Bysl gene product, bystin, is essential for survival of mouse embryos

Human bystin is a cytoplasmic protein directly binding to trophinin, a cell adhesion molecule potentially involved in human embryo implantation. The present study shows that bystin is expressed in luminal and glandular epithelia in the mouse uterus at peri-implantation stages. In fertilized embryos, bystin was not seen until blastocyst stage. Bystin expression started during hatching and increased in expanded blastocyst. However, bystin apparently disappeared from the blastocyst during implantation. After implantation bystin re-appeared in the epiblast. Targeted disruption of the mouse bystin gene, Bysl, resulted in embryonic lethality shortly after implantation, indicating that bystin is essential for survival of mouse embryos.     

Quantification of basigin mRNA in mouse oocytes and preimplantation embryos by competitive RT-PCR

Basigin is a member of the immunoglobulin superfamily and a key molecule related to mouse blastocyst implantation. Whether preimplantation mouse embryos express basigin mRNA is still unknown. The aim of this study was to use a quantitative competitive polymerase chain reaction to assess quantitatively the levels of basigin mRNA in mouse oocyte and preimplantation embryos. Basigin mRNA was detected in the oocyte and all the stages of preimplantation embryos. The levels of basigin mRNA were 0.0606 +/- 0.0282 in the oocyte, 0.0102 +/- 0.0036 in the zygote, 0.0007 +/- 0.0003 in the 2-cell embryo, 0.0031 +/- 0.0017 in the 4-cell embryo, 0.0084 +/- 0.0024 in the 8-cell embryo, 0.0537 +/- 0.0121 in the morula and 0.0392 +/- 0.0161 attomoles in the blastocyst, respectively. The levels of basigin mRNA in the oocyte, morula and blastocyst were significantly higher than those in the zygote and embryos at the 2-cell, 4-cell and 8-cell stages. The high level of basigin expression in the blastocyst may play a role during embryo implantation.     

Effect of gossypol and its metabolite on in vitro early development of mouse embryos

The purpose of this study was to examine the effect of gossypol and its metabolite on early in vitro mouse embryo development. One hundred and thirty-eight excellent quality mouse blastocysts were randomly assigned to five different treatments. Culture media were supplemented with 10% (V/N) normal steer serum. The embryos were cultured at 37 degrees C with an atmosphere of 5% O(2), 5% CO(2) and 90% N(2), and embryo development was examined and recorded at 12-h intervals for 72 h. The percentage of embryos that developed to expanded blastocyst (92%), hatching blastocyst (84%), and hatched blastocyst (76%) stages in control Ham's F-10 media was not different from that of embryos cultured in media containing 0.1 and 5 mug of gossypol; however, none of the embryos treated with 265 ng of gossypol metabolite (GM) developed beyond the blastocyst stage. A substantial decrease in the percentage of embryos reaching hatching blastocyst (29%) and hatched blastocyst (29%) stages was observed in the embryos cultured with 5.3 ng of GM. At both light and electron microscopic levels, the embryos appeared to be affected even by a lower concentration of GM in vitro. Our results suggest that GM has a much greater potency than the parent gossypol in inhibiting the early development of mouse embryos in vitro.     

Paracrine effects of bFGF and KGF on the process of mouse blastocyst implantation

Implantation is a complex process that requires the interaction of the blastocyst, and subsequently, that of the developing embryos with the endometrium. Several growth factors and cytokines are involved in implantation, but the details of their actions as related to the regulation of blastocyst implantation remain unclear. In the present study, the RT-PCR method was used to determine the gene expression of basic fibroblast growth factor (bFGF), keratinocyte growth factor (KGF), FGF receptor 1 (FGFR1), FGF receptor 2 (FGFR2), and KGF receptor (KGFR) in mouse embryos and in the stromal and epithelial cells of the uterine endometrium. Basic FGF and KGF mRNA were expressed in the endometrial cells, but were not expressed in the embryos. The mRNAs of receptors for bFGF and KGF were expressed in the blastocysts and in the in vitro implanting embryos, suggesting that bFGF and KGF may exert paracrine effects on blastocyst implantation. In this mouse model of blastocyst implantation, it was found that transforming growth factor α (TGF-α) at the concentrations of 1 ng/ml and 10 ng/ml significantly enhanced the blastocyst attachment and trophoblast spreading and increased trophoblast surface area. Relatively high concentrations of bFGF (100–500 ng/ml) significantly enhanced the rates of blastocyst attachment and of trophoblast spreading and promoted the expansion of the surface area of the implanting embryos. Unlike the rates of blastocyst attachment and trophoblast spreading, the surface area of the spreading embryos was significantly increased by addition of KGF (1–100 ng/ml). These results suggest that the bFGF and KGF derived from the endometrial cells exert paracrine effects on the process of implantation by stimulating trophoblast outgrowth through their cognate receptors. Mol. Reprod. Dev. 50:54–62, 1998. © 1998 Wiley-Liss, Inc.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

小鼠早期胚胎发育期间LIF基因表达的研究(简报)

白血病抑制因子(Leukemia inhibitory fac-tor,LIF)是近年来研究较为广泛的细胞生长调节因子之一。最初发现LIF能够在体外诱导小鼠髓样白血病细胞株M1细胞向正常细胞分化,进一步分离纯化蛋白以及克隆基因后发现LIF在体外还具有多种功能,作用于不同的靶细胞时引起的生理效应也各不相同。目前已知的功能有:刺激肝脏细胞急性期反应蛋白的     

Heterologous anti-progesterone monoclonal antibody arrests early embryonic development and implantation in the ferret (Mustela putorius)

Early embryo development and implantation were arrested in ferrets passively immunized with a mouse monoclonal anti-progesterone antibody injected intraperitoneally at 72 and 96 h post coitum (p.c.) or at 72 h p.c. only. In control ferrets injected with mouse serum or 0.9% NaCl, implantation sites were found in all mated females; autopsies were carried out at Day 14 p.c. A total of 34 unimplanted embryos were recovered from the reproductive tract of antibody-treated ferrets and none of these had progressed to the blastocyst stage. When ferrets were treated with antibody at 72 h p.c. and autopsies were carried out at Day 6 p.c., only 1 of 29 embryos recovered had progressed beyond the 4-cell stage in 4 females. In 4 control animals most embryos recovered at Day 6 were at the morula (32%) or blastocyst (28%) stage. Embryos from ferrets treated with antibody were therefore developmentally arrested when recovered 72 h after antibody administration. Plasma progesterone concentrations were approximately 6-fold higher in antibody-treated ferrets with unimplanted embryos (711 +/- 132 nmol/l; 223 ng/ml) compared with control pregnant females (102 +/- 4 nmol/l; 32 ng/ml) at Day 14 p.c. The results are consistent with the hypothesis that the normal course of pregnancy is arrested as a result of antibody binding of progesterone in the circulation, presumably causing a decrease in the amount of progesterone available to target cell receptors, and that heterologous anti-progesterone antibody blocks normal cleavage and embryonic development at an early stage before cavitation.     

Effect of RU486 on different stages of mouse preimplantation embryos in vitro

17 beta-Hydroxy-11 beta(4-dimethylaminophenyl)-17 alpha-(1-propynyl)estra-4, 9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 degrees C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 micrograms/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 micrograms/mL culture medium (p less than 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 microgram/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 micrograms/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.     

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.     

Transforming growth factor alpha (TGFalpha) modulates the effect of genomic imprinting and prolongs the development of parthenogenetic murine embryos]

The effect of transforming growth factor alpha (TGF alpha) on the development of diploid parthenogenetic mouse embryos (CBA x C57BL/6)F1 was studied. The embryos were in vitro treated with the TGF alpha at the stage of morula. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGF alpha was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGF alpha at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30-45 somites and had forelimb and hindlimb buds; the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGF alpha-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (42-45 somites) treated with 10 ng/ml of TGF alpha, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that endogenous TGF alpha can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Characterization of a developmentally regulated mouse embryonic antigen

Summary A mammalian embryonic cell surface glycoprotein (ESGp), whose expression and biochemical structure seem to be developmentally regulated, has been isolated and characterized. The molecule expressed in two cell through morula stage mouse embryos has a molecular weight, by electrophoretic analyses, of 90 kDa. At the blastocyst stage, however, the molecule migrates as a broad, heterogeneous band ranging from 90 to 110 kDa. Evidence obtained from studies of embryonal carcinoma (EC) cells indicates that this band is actually a composite of three distinct molecules (molecular weight 90, 95, and 105 to 110 kDa), each of which is synthesized uniquely by one of the different cell types of the blastocyst: the embryonic ectoderm and visceral and parietal endoderms, respectively. A survey of various mouse tissues and cell lines has revealed that undifferentiated cells express the low molecular weight form (90 kDa) characteristic of embryonic ectoderm, whereas differentiated cells and adult tissues express the high molecular weight form (110 kDa) characteristic of parietal endoderm. Only the EC visceral endoderm cell analogues have been shown to express the intermediate molecule (95 kDa). In embryos, the antigen is uniformly distributed over the cell surface during early cleavage stages (two to eight cell); just before compaction, however, it seems to redistribute and becomes polarized at the outside exposed edges of blastomeres. In cultured EC cells, ESGp is found only in areas of cell-to-cell contact; free-standing surfaces of cells are negative for expression. It is possible, therefore, that ESGp may be involved in the intercellular adhesion of both EC cells and compacting embryos. This work was supported by grant R01 HD23402 from the National Institutes of Health, Bethesda, MD.     

Cytoplasmic projections of trophectoderm distinguish implanting from preimplanting and implantation-delayed mouse blastocytes

A scoring scheme was devised to characterize visually the morphological differentiation of whole-mount, unfixed mouse blastocysts. Embryos were recovered from groups of intact mice (implanting embryos) and mice ovariectomized on Day 3 of pregnancy (implantation-delayed embryos) every 3 h from 18:00 h on Day 4 until 12:00 h on Day 5. Blastocyst differentiation was assessed according to the presence of a zona pellucida, the appearance of the outer margin of trophectoderm cells, the visibility of the blastocoele and the relative size of the inner cell mass. The results obtained indicate that, during this period, implanting and implantation-delayed mouse blastocysts lose the zona as well as exhibit rounded trophectoderm cells, an enlarged inner cell mass and an increasing opacity of the blastocoele. In contrast, the trophectoderm cells of implanting blastocysts only exhibit extensive cytoplasmic projections, probably due to remodelling of the intracellular cytoskeleton. Growth of the inner cell mass appeared to precede the other morphological changes in the majority of blastocysts, and thus might be a prerequisite for further differentiation. The rate of blastocyst differentiation and the survival of embryos were adversely affected by the condition of delayed implantation, induced by ovariectomy. This study suggests that the appearance of cytoplasmic projections from trophectoderm cells is central to the control of blastocyst implantation.     

Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10muM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

Acidic endomembrane organelles are required for mouse postimplantation development

Vacuolar-type H(+)-ATPase (V-ATPase) plays a major role in endomembrane and plasma membrane proton transport in eukaryotes. We found that the acidic compartments generated by V-ATPase are present from the one-cell stage of mouse preimplantation embryos. Upon differentiation of trophoblasts and the inner cell mass at the blastocyst stage, these compartments exhibited a polarized perinuclear distribution. PL16(-/-) embryos, lacking the V-ATPase 16-kDa proteolipid (c subunit), developed to the blastocyst stage and were implanted in the uterine epithelium, but died shortly thereafter. This mutant showed severe defects in development of the embryonic and extraembryonic tissues at a stage that coincided with rapid cell proliferation. When cultured in vitro, PL16(-/-) blastocysts could hatch and become attached to the surface of a culture dish, but the inner cell mass grew significantly slower and most cells failed to survive for more than 4 days. PL16(-/-) cells showed impaired endocytosis as well as organellar acidification. The Golgi complex became swollen and vacuolated, possibly due to the absence of the luminal acidic pH. These results clearly indicate that acidic compartments are essential for development after implantation.     

Embryos aggregation improves development and imprinting gene expression in mouse parthenogenesis

Mouse parthenogenetic embryonic stem cells (PgESCs) could be applied to study imprinting genes and are used in cell therapy. Our previous study found that stem cells established by aggregation of two parthenogenetic embryos at 8‐cell stage (named as a₂PgESCs) had a higher efficiency than that of PgESCs, and the paternal expressed imprinting genes were observably upregulated. Therefore, we propose that increasing the number of parthenogenetic embryos in aggregation may improve the development of parthenogenetic mouse and imprinting gene expression of PgESCs. To verify this hypothesis, we aggregated four embryos together at the 4‐cell stage and cultured to the blastocyst stage (named as 4aPgB). qPCR detection showed that the expression of imprinting genes Igf2, Mest, Snrpn, Igf2r, H19, Gtl2 in 4aPgB were more similar to that of fertilized blastocyst (named as fB) compared to 2aPgB (derived from two 4‐cell stage parthenogenetic embryos aggregation) or PgB (single parthenogenetic blastocyst). Post‐implantation development of 4aPgB extended to 11 days of gestation. The establishment efficiency of GFP‐a₄PgESCs which derived from GFP‐4aPgB is 62.5%. Moreover, expression of imprinting genes Igf2, Mest, Snrpn, notably downregulated and approached the level of that in fertilized embryonic stem cells (fESCs). In addition, we acquired a 13.5‐day fetus totally derived from GFP‐a₄PgESCs with germline contribution by 8‐cell under zona pellucida (ZP) injection. In conclusion, four embryos aggregation improves parthenogenetic development, and compensates imprinting genes expression in PgESCs. It implied that a₄PgESCs could serve as a better scientific model applied in translational medicine and imprinting gene study.     

Effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation

Mouse embryo implantation depends on the complex interaction between the embryo trophoblast cells and the uterine environment, which deposits an extracellular matrix with abundant amounts of laminin. Intrauterine injection and blastocyst or ectoplacental cone culture models were used to study the effect of 32/67 kDa laminin-binding protein antibody on mouse embryo implantation in vivo and in vitro. Intrauterine injection of 32/67 kDa laminin-binding protein antibody (0.4 mg in 1 ml Ham's F-10 medium, 5 microl per mouse) into the left uterine horns of mice (n = 22) on day 3 of pregnancy inhibited embryo implantation significantly (P < 0.001) compared with the contralateral horns that had been injected with normal rabbit IgG. A continuous section study on day 5 after injection showed that the embryos in the control uteri implanted normally and developed healthily, but there were no embryos or the remaining embryos had disintegrated in the uteri injected with 32/67 kDa laminin-binding protein antibody. Blastocysts or ectoplacental cones were cultured in media containing 32/67 kDa laminin-binding protein antibody (0.2 mg ml(-1)) on laminin-coated dishes with normal rabbit IgG at the same concentration as in the controls. The 32/67 kDa laminin-binding protein had no effect on blastocyst or ectoplacental cone attachment, but prohibited the blastocyst or ectoplacental cone outgrowth and primary or secondary trophoblast giant cell migration. These results indicate that 32/67 kDa laminin-binding protein antibody blocked mouse embryo implantation by preventing embryo trophoblast cell invasion and migration through the uterine decidual basement membrane-like extracellular matrix which has a high laminin content.     

Evaluation of developmental competence of vitrified-warmed early cleavage stage embryos and their application for chimeric mouse production.

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.