EGF and TGF-alpha supplementation enhances development of cloned mouse embryos

In this study, we sought to determine the extent to which mitogenic growth factors affect the survival and development of cloned mouse embryos in vitro. Cloned embryos derived by intracytoplasmic nuclear injection (ICNI) of cumulus cell nuclei into enucleated oocytes were incubated in culture media supplemented with EGF and/or TGF-alpha for 4 days. Compared to control, treatment with either growth factor significantly increased the blastocyst formation rate, the total number of cells per blastocyst, the cell ratio of the inner cell mass and the trophectoderm (ICM:TE ratio), and EGF-R protein expression in cloned embryos. In most instances these effects were enhanced in cloned embryos when EGF and TGF-alpha were combined. Although fewer blastocysts developed from cloned than from fertilized one-cell stage embryos, growth factor treatment appeared to have the greatest effect on cloned embryos. These results demonstrate that mitogenic growth factors significantly enhance survival and promote the preimplantation development of cloned mouse embryos.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Effect of culture media on in vitro development of cloned mouse embryos

The effect of simple and sequential embryo culture media on the preimplantation development of mouse nuclear transfer (NT) embryos reconstructed with cumulus cell nuclei using a mechanical NT technique was studied. Blastocyst formation rate was evaluated using CZB medium and the sequential media G1/G2 and KSOM/G2. Arrested two- and three-cell NT embryos were Hoechst-stained to check for nuclear abnormalities. Nonmanipulated and sham-manipulated parthenogenetic embryos served as controls for, respectively, the medium and the handling technique. Rates of blastocyst formation for medium and handling control embryos were similar in CZB (58% and 61%), in G1/G2 (94% and 85%), and in KSOM/G2 (88% and 84%). Development of NT embryos was significantly impaired from the two-cell stage onwards, reaching the blastocyst stage at a rate of 5% in CZB, 14% in G1/G2, and 28% in KSOM/G2. Arrested two- and three-cell stage NT embryos showed a high rate of binucleation. These data demonstrate not only that NT embryos are more sensitive to in vitro culture conditions than parthenogenetic control embryos but also that selection of culture media can influence the preimplantation development of NT embryos.     

Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos.

We show that a gene introduced into cells of mouse embryos by a retrovirus can serve as a heritable marker for the study of cell lineage in vivo. We constructed a defective recombinant retrovirus in which the Escherichia coli beta-galactosidase (lacZ) gene is inserted in the genome of a Muloney murine leukemia virus (M-MuLV). Expression of lacZ was detected with a histochemical stain that can be applied to cultured cells and embryonic tissue. Infection of cultured cells showed that lacZ has no detectable deleterious effects on cell viability or growth, that the enzyme is stably expressed in the progeny of infected cells for many generations in the absence of selective pressure, and that the virus can induce lacZ in a variety of cell types. Following injection of the virus into mid-gestation mouse embryos, clones of lacZ-positive cells were detected in skin, skull, meninges, brain, visceral yolk sac, and amnion. We identified the cell types comprising a series of lacZ-positive clones in the visceral yolk sac and skin to learn the lineage relationships of the labelled cells. In each tissue, we obtained evidence that several cell types have a pluripotential ancestor and that cell fate is progressively restricted as development proceeds.     

Analysis of post-implantation mouse embryos after maternal heat stress during meiotic maturation

The effects of heat stress during oocyte maturation were studied in post-implantation mouse embryos. Virgin ICR mice were exposed to 35 +/- 1 degree C and 65 +/- 3% RH for 12.5 h beginning immediately after synchronization of ovulation with PMSG and hCG. Embryos of heat-stressed dams were developmentally heterogeneous and showed significant delays in development with as much as 48 h delayed development. Nearly 6% of these embryos were triploid, and another 2% were hyper-diploid. Development of triploid embryos was delayed more than 24 h. Nine embryos with severe developmental delay had heterogeneous chromosome constitutions. Embryo mortality before and after implantation was higher in heat-stressed dams than in controls.     

Early development and X-chromosome inactivation in mouse parthenogenetic embryos.

Early development and X-chromosome inactivation were studied in ethanol-induced mouse parthenogenones. About 24% of oocytes transferred to 0.5-day pseudopregnant recipients successfully implanted. However, only 49%, 20%, and 16% of implanted parthenogenones survived 5, 6, and 7 days later, respectively. Abnormal development was evident in every parthenogenone as early as 5 days after activation with the degenerating polar trophectoderm. These embryos were destined to become either small disorganized embryos or embryonic ectoderm vesicles bounded by the visceral endoderm. Only 2 of 51 representative 6- to 8-day parthenogenones sectioned had morphology of the normal egg cylinder, although growth retardation was evident. Spontaneous LT/Sv parthenogenones shared similar morphological features. In late blastocysts, the frequency of cells with an apparently inactivated X chromosome was lower in parthenogenones than in fertilized embryos. The failure of X-inactivation in the trophectoderm seems to contribute to the defective development of parthenogenones.     

Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos

The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.     

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more na?ve states in these inter-specific chimera assays will be an important future endeavor.     

Tissue-specific RNA interference in post-implantation mouse embryos using directional electroporation and whole embryo culture

In mammals, embryonic development is more difficult to analyze than in non-mammalian species because this development occurs in utero. Interestingly, whole embryo culture allows the normal development of mouse post-implantation embryos for up to 2 days in vitro. One limitation of this technology has been the difficulty of performing loss-of-gene function studies in this system. RNA interference (RNAi), whereby double-stranded RNA molecules suppress the expression of complementary genes, has rapidly become a widely used tool for gene function analyses. We have combined the technologies of mouse whole embryo culture and RNAi to allow the molecular dissection of developmental processes. Here, we review the manipulation by topical injection followed by directional electroporation of endoribonuclease-prepared siRNA to demonstrate that this technology may be useful to knock down genes in a tissue- and region-specific manner in several organs of the developing mouse embryo.     

Development of mouse embryos after immunoneutralization of mitogenic growth factors mimics that of cloned embryos

The extent to which mitogenic growth factors influence embryo development is not well characterized. We sought to determine the effect of epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) on naturally fertilized (in vivo-derived) and in vitro-fertilized mouse embryos, compared with that on cloned (intracytoplasmic nuclear injection-derived) mouse embryos, in which EGF and TGFalpha expression is markedly reduced. Immunoneutralization of EGF, TGFalpha, and EGF receptor by using specific antibodies significantly reduced the blastocyst development rate (in vivo-derived: 66%, 63%, and 63%, respectively; in vitro-fertilized: 57%, 55%, and 56%, respectively), increased the number of apoptotic nuclei (in vivo-derived: 9%, 10%, and 9%, respectively; in vitro-fertilized: 13%, 13%, and 13%, respectively), decreased the total number of cells (in vivo-derived: 87%, 85%, and 86%, respectively; in vitro-fertilized: 86%, 85%, and 86%, respectively), and increased the inner cell mass:trophectoderm ratios (in vivo-derived: 1:2.70 +/- 0.05, 1:2.73 +/- 0.04, 1:2.71 +/- 0.06, respectively; in vitro-fertilized: 1:2.94 +/- 0.02, 1:2.96 +/- 0.02, 1:2.95 +/- 0.02, respectively). In most cases, combined treatment with neutralizing antibodies to both EGF and TGFalpha accentuated changes in these parameters. Further, the effect of combined immunoneutralization on these parameters in fertilized embryos was no different from those in cloned embryos. Therefore, normal expression of mitogenic growth factors is crucial for successful development of mouse embryos before implantation. Inhibiting the action of mitogenic growth factors causes fertilized embryos to exhibit developmental characteristics similar to those of cloned embryos, which may partially explain the poor developmental potential of cloned mammalian embryos.     

Factors influencing chromosome condensation and development of cloned rat embryos

Factors influencing premature chromosome condensation (PCC) in transferred rat nuclei have been examined. Chromosome condensation of rat cumulus cell nuclei did not occur when the cell nuclei were injected into enucleated rat oocytes. By contrast, chromosome condensation did occur after transfer to enucleated mouse oocytes or intact rat oocytes. In the first serial NT experiment, rat somatic cell nuclei were injected into enucleated mouse oocytes, and the reconstructed oocytes were activated by strontium chloride. From these reconstructed embryos, karyoplasts containing pronucleus-like vesicles were transferred into pronuclear zygote-derived cytoplasts by a DC pulse. Transfer of a total of 340 serial NT zygotes into recipient females, including 206 two-cell embryos, resulted in only seven implantation sites. In the second serial NT experiment, rat somatic cell nuclei were injected into intact rat oocytes; the recipient metaphase-plate was then aspirated under UV light from the NT oocytes in which PCC of injected nuclei was observed. After activation of the NT oocytes, karyoplasts were introduced into zygote-derived cytoplasts. Transfer of a total of 115 serial NT zygotes, including 37 two-cell embryos, resulted in four implantation sites but no live offspring. These results establish a mean of inducing chromosome condensation in rat oocytes and demonstrate that reconstructed rat zygotes can be prepared by serial NT procedures. Developmental competence of these embryos remains to be clarified.     

In vitro development and mitochondrial fate of macaca-rabbit cloned embryos

Interspecies cloning may be used as an effective method to conserve highly endangered species and to support the development of non-human primate animal models for studying therapeutic cloning and nuclear-cytoplasm interaction. The use of the monkey model for biomedical research can avoid legal, ethical, and experimental limitations encountered in a clinical situation. We describe in this study the in vitro development of macaca-rabbit embryos produced by fusing macaca fibroblasts with enucleated rabbit oocytes and examine the fate of mitochondrial DNA in these embryos. We show that macaca-rabbit cloned embryos can develop to the blastocyst stage when cultured in vitro in HECM(10) +10% FBS and that mitochondrial DNA derived from donor somatic cells was detectable in cloned embryos throughout preimplantation development. These results suggest that (1) macaca fibroblast nuclei can dedifferentiate in enucleated metaphase II rabbit oocytes; (2) HECM(10) +10% FBS can break through the development block and support the development of macaca-rabbit cloned embryos to blastocysts; and (3) donor-cell-derived mitochondrial DNA is not eliminated until blastocyst stage.     

Chromosome stability differs in cloned mouse embryos and derivative ES cells

The mechanisms that have evolved to maintain genome stability during cell cycle progression are challenged when a somatic cell nucleus is placed in a meiotic environment such as the ooplasm. Chromosomal spindle aberrations ensue in the majority of reconstructed oocytes within 2 h of transplantation, but it is not known if they recover or persist with the onset of embryonic divisions. We analyzed the chromosomal spindles and the karyotype of cumulus cell-derived mouse clones through the initial and hence most critical mitoses. Cloned embryos start out with less aneuploidy than fertilized embryos but surpass them after ES cell derivation, as measured by frequencies of chromosome trisomies and structural rearrangements. Despite the limited proportion of cloned mouse embryos that reach late gestation, a phenotypic mutation lacking a karyotypic mark was found in a newborn mouse cloned in 2002 and has been inherited since by its offspring. These data concur with a prevalent epigenetic, rather than genetic, basis for cloned embryo failure, but they also warn against the temptation to think that all conditions of clones are epigenetic and recover during gametogenesis. The cloning procedure is defenseless (no matter how technically refined) towards pre-existing or induced subchromosomal mutations that are below the experimental detection limit of the cytogenetic assay.     

Role of glucose in cloned mouse embryo development

Cloned mouse embryos display a marked preference for glucose-containing culture medium, with enhanced development to the blastocyst stage in glucose-containing medium attributable mainly to an early beneficial effect during the first cell cycle. This early beneficial effect of glucose is not displayed by parthenogenetic, fertilized, or tetraploid nuclear transfer control embryos, indicating that it is specific to diploid clones. Precocious localization of the glucose transporter SLC2A1 to the cell surface, as well as increased expression of glucose transporters and increased uptake of glucose at the one- and two-cell stages, is also seen in cloned embryos. To examine the role of glucose in early cloned embryo development, we examined glucose metabolism and associated metabolites, as well as mitochondrial ultrastructure, distribution, and number. Clones prepared with cumulus cell nuclei displayed significantly enhanced glucose metabolism at the two-cell stage relative to parthenogenetic controls. Despite the increase in metabolism, ATP content was reduced in clones relative to parthenotes and fertilized controls. Clones at both stages displayed elevated concentrations of glycogen compared with parthenogenetic controls. There was no difference in the number of mitochondria, but clone mitochondria displayed ultrastructural alterations. Interestingly, glucose availability positively affected mitochondrial structure and localization. We conclude that cloned embryos may be severely compromised in terms of ATP-dependent processes during the first two cell cycles and that glucose may exert its early beneficial effects via positive effects on the mitochondria.     

Aberrant expression of imprinted genes in post-implantation rat embryos

AimImprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males.Main methodsGene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry.Key findingsAberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos.SignificanceThe study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F₁ progeny thereby causing embryo loss.