Post-implantation Embryos Research Articles

Direct ovarian - uterine transfer of progesterone increases embryo survival in gilts

This study employed a unilateral ovariectomy model to investigate the relevance of the local supply of progesterone (ovary) compared with the systemic supply of progesterone, in terms of embryo survival in the ipsilateral uterine horn as opposed to the contralateral uterine horn. Thirty gilts were unilaterally ovariectomised (ULO) during the luteal stage of their first oestrous cycle. Half of the ULO gilts were fed at 1.2 maintenance requirement (M), while the other half were fed at 2.4M. Across ULO gilts 0.8 more embryos survived in the ipsilateral horn compared with the contralateral horn at Day 35 of gestation (P<0.05). In ULO gilts on the 2.4M feed level the difference (+1.3; P<0.05) between the ipsi- and contralateral horn was more pronounced than on the 1.2M feed level (+0.4; NS). The higher feed level reduced circulating levels of systemic progesterone on Day 5 of pregnancy but not embryo survival at Day 35. However, post-implantation embryo survival was lower on the low feed level. In conclusion, these data indicate that local progesterone supply from the ovaries to the uterus contributes to the probability of embryo survival.

Stem cell therapeutics—reality versus hype and hope

In the last decade medical science has started to make the first steps towards a paradigm shift from repair to regeneration [1]. The potential use of a range of stem cells in regenerative medicine is currently one of the most intensively researched areas worldwide [2]. In particular, embryonic stem cells (ESC) and more recently Induced Pluripotent Stemcells (iPS) [3] have provoked a lot of interest as a potential source of stem cells in the treatment of diseases such as Alzheimer’s and Parkinson’s diseases. Nevertheless, these hopes have not yet been fulfilled with either ESC or iPS. Murine ESC were first described in 1981 [4] and subsequent studies showed the regenerative potential of murine stem cells derived from both preand postimplantation embryos [5–7]. This early murine work was followed by the development of human ESC in 1998 [8]. Both murine and human pre-implantation ESC are derived from the inner cell mass (ICM) of the blastocyst at day 5/6 of growth. The ICM consists of primitive ectoderm cells, which subsequently develop into epiblast cells in postimplantation embryos. Despite these ontogenic similarities human and murine ESC do not resemble each other in many aspects [9]. For example, murine ESC rely on leukemia inhibitory factor (LIF) and bone morphogenic protein-4 (BMP4) for maintenance of pluripotency [10, 11], whilst human ESC are dependent on TGFβ/Activin/Nodal pathway activity and fibroblast growth factor-2 (FGF2) [12–17] and thus resemble epiblast stem cells (EpiSC). Usually EpiSC cannot be reverted to a state such that they respond to LIF and BMP4. Furthermore, primordial germ cells, which originate from pluripotent epiblast cells in mice, respond in a similar way to LIF and BMP4, such as murine ESC derived from the ICM [18]. Additionally, murine ESC can be cultured as single cells while human ESC are particularly sensitive to dissociation and should be cultured in clumps [19]. There are clearly many basic concepts still to be explored in the biology of ESC of various species. Thorough understanding of these cells is still in its’ infancy despite 20 years of basic research. Bearing this is mind there should be an air of caution when proposing future clinical applications. Can or should we move forward into thinking about clinical applications when the fundamentals Capsule Human cord blood stem cells have a realistic and proven potential for the treatment of various diseases compared to embryonic stem cells / iPS cells.

Direct reprogramming of fibroblasts into epiblast stem cells

Epiblast stem cells (EpiSCs) derived from epiblast tissue of post-implantation embryos are pluripotent and can give rise to all three germ layers in teratoma assays. Introduction of the four transcription factors Oct4, Sox2, Klf4 and c-Myc into somatic cells has been shown to generate induced pluripotent stem cells (iPSCs) that are very similar to embryonic stem cells (ESCs) in a number of characteristics. However, generation of EpiSCs by the direct reprogramming of somatic cells using these transcription factors has not been shown to date. Here, we show that these transcription factors can be used to directly generate induced EpiSCs (iEpiSCs) under EpiSC culture conditions. iEpiSCs resemble EpiSCs in morphology, gene expression pattern, epigenetic status and chimaera-forming capability. This study demonstrates that the culture environment in transcription factor-mediated reprogramming determines the cell fate of the reprogrammed cell. We therefore hypothesize that it will eventually be possible to shape the identity of a directly reprogrammed cell simply by modulating culture conditions.

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual-labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N-terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin-C promoter. mCherry is expressed in almost all tissues analyzed including pre- and post-implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals.

Hazardous Apoptotic Effects of 2-Bromopropane on Maturation of Mouse Oocytes, Fertilization, and Fetal Development

2-Bromopropane (2-BP) is used as an alternative to ozone-depleting cleaning solvents. Previously, we reported that 2-BP has cytotoxic effects on mouse blastocysts and is associated with defects in subsequent development. Here, we further investigate the effects of 2-BP on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, 2-BP induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 2-BP during in vitro maturation (IVM) resulted in increased resorption of postimplantation embryos and decreased fetal weights. Experiments with a mouse model disclosed that consumption of drinking water containing 20 μM 2-BP led to decreased oocyte maturation in vivo and fertilization in vitro, as well as impairment of early embryonic development. Interestingly, pretreatment with a caspase-3-specific inhibitor effectively prevented 2-BP-triggered hazardous effects, suggesting that embryonic impairment by 2-BP occurs via a caspase-dependent apoptotic process. A study using embryonic stem cells as the assay model conclusively demonstrated that 2-BP induces cell death processes through apoptosis and not necrosis, and inhibits early embryo development in mouse embryonic stem cells. These results collectively confirm the hazardous effects of 2-BP on embryos derived from pretreated oocytes.

Establishment of Cell Lineage-Specific DNA Methylation Profile Follows the Morphological Differentiation of Trophoblast and Embryonic Cell Lineages.

There are numerous tissue-dependent and differentially methylated regions (T-DMRs) in the mammalian genome. Cell type-specific DNA methylation status of T-DMRs constitutes DNA methylation profile of the cell, which ought to be reconstructed as cells differentiate. The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages resulting in the formation of trophectoderm (TE) and inner cell mass (ICM) in blastocyst. Although lower level of global DNA methylation in TE genome compared to ICM has been suggested, dynamics of DNA methylation profile during the TE/ICM differentiation has not been studied. In this study, we aimed to identify T-DMRs between trophoblast and embryonic cell lineages and analyzed their DNA methylation status in pre- and post-implantation embryos. Oct4 and Nanog are differentially methylated between trophoblast stem (TS) and embryonic stem (ES) cell lines (TS-ES T-DMRs). These two loci are hyper-methylated in TS cells and hypo-methylated in ES cells. Elf5 is another known TS-ES T-DMR but exhibits the opposite methylation pattern. A number of other TS-ES T-DMRs have been found so far in our laboratory. First, we addressed whether these TS-ES T-DMRs are indeed differentially methylated between trophoblast and embryonic cell lineages in vivo. Bisulfite sequencing analysis of 11 TS-ES T-DMRs revealed that the DNA methylation status of them in E6.5 extraembryonic ectoderm and epiblast coincide with those in TS and ES cells, respectively, showing that the TS-ES T-DMRs are surely cell lineage-based T-DMRs in vivo. We then collected TE and ICM cells separately from E3.5 blastocysts and subjected to bisulfite sequencing. Interestingly, genomes of TE and ICM exhibited no difference in the methylation status of all T-DMRs examined. They were in extremely hypomethylated status. Here we identified valuable T-DMRs for delineation of trophoblast/embryonic cell lineages. DNA methylation analysis of these T-DMRs suggested that the DNA methylation profiles specific to each cell lineage are established after the morphological delineation of trophoblast and embryonic cell lineages. DNA methylation may thus contribute to stabilize or fix the cell fate and the differentiation capacity. Supported by Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20062003). (platform)

Cloning efficiency following ES cell nuclear transfer is influenced by the methylation state of the donor nucleus altered by mutation of DNA methyltransferase 3a and 3b

The epigenetic state of donor cells plays a vital role in the nuclear reprogramming and chromatin remodeling of cloned embryos. In this study we investigated the effect of DNA methylation state of donor cells on the development of mouse embryos reconstructed with embryonic stem (ES) cell nuclei. Our results confirmed that deletion of the DNA methyltransferase 3a (Dnmt3a) and DNA methyltransferase 3b (Dnmt3b) distinctly decreases the level of DNA methylation in ES cells. In contrast to wild type ES cells (J1), Dnmt3a − / − 3b − / − (DKO) and Dnmt3b − / − (3bKO) donor cells significantly elevated the percentage of embryonic stem cell nuclear transfer (ECNT) morula, blastocysts and postimplantation embryos (P < 0.05). However, the efficiency of establishment of NT-ES cell lines derived from DKO reconstructed blastocysts was not improved, and the expression pattern of OCT4 and CDX2 in cloned blastocysts and postimplantation embryos was not altered either. Our results suggest that the DNA methylation state of the donor nucleus is an important factor in regulation of the donor nuclear reprogramming.

The embryotoxicity of sera from patients with autoimmune diseases on post-implantation rat embryos in culture persists during remission and is not related to oxidative stress

We evaluated the embryolethality and embryotoxicity of sera from patients suffering from autoimmune diseases during remission on post-implantation rat embryos cultured on these sera and determined the association between the patients' clinical history, high blood levels of specific antibodies, medications, and oxidative stress parameters. One hundred and eighty, 10.5-day-old rat embryos were cultured in their yolk sacs in 33 sera of systemic lupus erythematosus (SLE)/antiphospholipid syndrome (APS) patients, and compared with 84 embryos cultured in rat sera and 88 embryos cultured in control human sera. The sera proved to be lethal and embryotoxic but not teratogenic resulting in smaller yolk sacs and embryos, lower protein level and lower developmental score. Significantly less embryos cultured in 'toxic' SLE/APS sera had peak 2 of low molecular weight antioxidants (LMWA) wave, implying a delayed maturation of the antioxidant defense. Lower peak 1 of LMWA correlated with a history of recurrent abortions. Embryonic levels of superoxide dismutase (SOD) and catalase (CAT) did not correlate with sera toxicity, patients' clinical history or specific antibodies. We conclude that SLE/APS patients' clinical remission did not prevent death or developmental delay accompanied by later appearance of peak 2 of LMWA in post-implantation rat embryo cultures. The normal levels of the antioxidant enzymes evaluated may indicate that sera toxicity is not related to oxidative stress.

Stat3 Activation Is Limiting for Reprogramming to Ground State Pluripotency

SummaryThe cytokine leukemia inhibitory factor (Lif) sustains self-renewal of mouse embryonic and induced pluripotent stem cells by activating Jak kinase and the transcription factor Stat3. Here we investigate whether Jak/Stat3 may also contribute to induction of pluripotency. EpiSCs derived from postimplantation embryos express low levels of Lif receptor and Stat3. We introduced into EpiSCs a Jak/Stat3 activating receptor (GY118F) responsive to granulocyte colony stimulating factor (Gcsf). On transfer to ground state culture, in which MAPK signaling and glycogen synthase kinase are inhibited, Gcsf induced transcriptional resetting and functional reprogramming. Activation of a tamoxifen-regulatable fusion, Stat3ERT2, also converted EpiSCs into chimera-competent iPSCs. We exploited GY118F to increase Jak/Stat3 activity during somatic cell reprogramming. Incompletely reprogrammed cells derived from neural stem cells or fibroblasts responded to Gcsf with elevated frequencies of progression to ground state pluripotency. These findings indicate that Jak/Stat3 participate directly in molecular reprogramming and that activation of this pathway is a limiting component.

Chromosomal aberration in the post-implantation embryos sired by tamoxifen treated male rats

Tamoxifen is a synthetic non-steroidal Selective Estrogen Receptor Modulator used in the treatment of breast cancer and in treatment of male fertility. Earlier studies from our laboratory had demonstrated an increase in post-implantation embryo loss following tamoxifen treatment to adult male rats at a dose of 0.4 mg/kg/day for 60 days. The post-implantation loss occurred at around 9–10 days of gestation suggesting that paternal factors involved in embryo development were affected by tamoxifen treatment. The present study was done to determine if any chromosomal aberrations occurred in the embryos sired by tamoxifen treated male rats. Chromosomal aberrations induced by tamoxifen treatment to adult male rats in the bone marrow (F 0 males) and in the embryos sired by these males (F 1 progeny) were determined. In addition, the reproductive performance of the F 1 progeny was assessed. A significant dose dependent reduction in mitotic activity in the bone marrow and embryonic cells was observed after tamoxifen treatment. In addition, tamoxifen also induced a significant dose dependent increase in the frequency of chromosomal aberrations, mainly gaps and breaks in bone marrow and embryonic cells. However, the embryos sired by the tamoxifen treated males had no effect on developmental milestones achieved and on their reproductive performance. The present study suggests that chromosomal aberrations observed in the embryos did not the affect their development until adulthood but could make the progeny of the tamoxifen treated males vulnerable to the development of adult onset diseases later in life.

Hazardous Effects of Curcumin on Mouse Embryonic Development through a Mitochondria-Dependent Apoptotic Signaling Pathway

In this study, we examined the cytotoxic effects of curcumin, the yellow pigment of Curcuma longa, on the blastocyst stage of mouse embryos, subsequent embryonic attachment, and outgrowth in vitro and in vivo implantation by embryo transfer. Mouse blastocysts were incubated in medium with or without curcumin (6, 12 or 24 μM) for 24 h. Cell proliferation and growth were investigated using dual differential staining, apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and implantation and post-implantation development of embryos were measured by in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 24 μM curcumin displayed significantly increased apoptosis and decreased total cell number. Interestingly, we observed no marked differences in the implantation success rates between curcumin-pretreated and control blastocysts during in vitro embryonic development through implantation with a fibronectin-coated culture dish. However, in vitro treatment with 24 μM curcumin was associated with decreased implantation rate and increased resorption of postimplantation embryos in mouse uterus, as well as decreased fetal weight in the embryo transfer assay. Our results collectively indicate that in vitro exposure to curcumin triggers apoptosis and retards early postimplantation development after transfer to host mice. In addition, curcumin induces apoptotic injury effects on mouse blastocysts through ROS generation, and further promotes mitochondria-dependent apoptotic signaling processes to impair sequent embryonic development.

Expression of the transcription factor, TFII-I, during post-implantation mouse embryonic development

BackgroundGeneral transcription factor (TFII-I) is a multi-functional transcription factor encoded by the Gtf2i gene, that has been demonstrated to regulate transcription of genes critical for development. Because of the broad range of genes regulated by TFII-I as well as its potential role in a significant neuro-developmental disorder, developing a comprehensive expression profile is critical to the study of this transcription factor. We sought to define the timing and pattern of expression of TFII-I in post-implantation embryos at a time during which many putative TFII-I target genes are expressed.FindingsAntibodies to the N-terminus of TFII-I were used to probe embryonic mouse sections. TFII-I protein is widely expressed in the developing embryo. TFII-I is expressed throughout the period from E8-E16. However, within this period there are striking shifts in localization from cytoplasmic predominant to nuclear. TFII-I expression varies in both a spatial and temporal fashion. There is extensive expression in neural precursors at E8. This expression persists at later stages. TFII-I is expressed in developing lung, heart and gut structures. There is no evidence of isoform specific expression. Available data regarding expression patterns at both an RNA and protein level throughout development are also comprehensively reviewed.ConclusionsOur immunohistochemical studies of the temporal and spatial expression patterns of TFII-I in mouse embryonic sections are consistent with the hypothesis that hemizygous deletion of GTF2I in individuals with Williams-Beuren Syndrome contributes to the distinct cognitive and physiological symptoms associated with the disorder.

Epicatechin Gallate Decreases the Viability and Subsequent Embryonic Development of Mouse Blastocysts

Catechins, a family of polyphenols found in tea, evoke various responses including cell death. We examined the cytotoxic effects of epicatechin gallate (ECG), a polyphenol extract from green tea, on the blastocyst stage of mouse embryos, subsequent embryonic attachment, and in vitro and in vivo outgrowth implantation after embryo transfer. Mouse blastocysts were incubated in medium with or without ECG (12.5 microM, 25 microM or 50 microM) for 24 hours. Cell proliferation and growth were investigated using dual differential staining, apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and implantation and post-implantation development of embryos were measured by in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 50 microM ECG exhibited a significant increase in apoptosis and a corresponding decrease in total cell number. Importantly, the implantation success rate of blastocysts pretreated with 50 microM ECG was lower than that of controls, and in vitro treatment with 50 microM ECG was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that in vitro exposure to ECG induces apoptosis and retards early post-implantation development after transfer to host mice. The degree of teratogenic potential exerted by ECG in early human development is unknown at present and requires further investigation.

Induction of cytotoxicity and apoptosis in mouse blastocysts by silver nanoparticles

Silver nanoparticles (nanoAg) are antibacterial materials widely used in various products and medical supplies. In this report, we examined the cytotoxic effects of nanoAg on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation by embryo transfer. Blastocysts treated with 50 μM nanoAg exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Importantly, the implantation success rate of blastocysts pretreated with nanoAg was lower than that of their control counterparts. Moreover, in vitro treatment with 50 μM nanoAg was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that in vitro exposure to nanoAg induces apoptosis and retards early post-implantation development after transfer to host mice. However, nanoAg-stimulated embryonic cytotoxicity appeared lower than that induced by the Ag + ion. The results collectively show that nanoAg has the potential to induce embryo cytotoxicity. Further studies are required to establish effective protection strategies against the cytotoxic effects of these nanoparticles.

Involvement of apoptosis in mediating mitomycin C-induced teratogenesis in vitro

Mitomycin C (MMC) is among the most commonly used drugs worldwide and is known to cause congenital malformations and fetal death in animals. In this study, the effect of MMC on major organogenesis period and the role of apoptosis in mediating congenital malformations have been carried out. In the present study, post-implantation rat embryos of day 11 were cultured for 24 h with various concentrations of MMC, i.e. 1, 10, and 100 µg/ml cultures. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The MMC decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to MMC at 1 µg/ml culture did not show any significant effect on embryonic growth and development. Parallel to this, flow cytometric analysis (cell cycle and annexin V binding) and DNA fragmentation assay were carried out followed by quantitation by 3′-OH labeling of cultured rat embryos to evaluate the role of apoptosis in bringing about MMC-induced teratogenesis. All results were found to be dose-dependent and an increase in apoptosis in embryonic tissues may be related to the increased risk of congenital malformations. The data suggested that apoptosis might be involved in mediating teratogenesis of MMC in vitro.

Nuclear Transfer-Derived Epiblast Stem Cells Are Transcriptionally and Epigenetically Distinguishable from Their Fertilized-Derived Counterparts

Mouse embryonic pluripotent stem cells can be obtained from the inner cell mass at the blastocyst stage (embryonic stem cells, ESCs) or from the late epiblast of postimplantation embryos (epiblast stem cells, EpiSCs). During normal development, the transition between these two stages is marked by major epigenetic and transcriptional changes including DNA de novo methylation. These modifications represent an epigenetic mark conserved in ESCs and EpiSCs. Pluripotent ESCs derived from blastocysts generated by nuclear transfer (NT) have been shown to be correctly reprogrammed. However, NT embryos frequently undergo abnormal development. In the present study, we have examined whether pluripotent cells could be derived from the epiblast of postimplantation NT embryos and whether the reprogramming process would affect the epigenetic changes occurring at this stage, which could explain abnormal development of NT embryos. We showed that EpiSCs could be derived with the same efficiency from NT embryos and from their fertilized counterparts. However, gene expression profile analyses showed divergence between fertilized- and nuclear transfer-EpiSCs with a surprising bias in the distribution of the differentially expressed genes, 30% of them being localized on chromosome 11. A majority of these genes were downregulated in NT-EpiSCs and imprinted genes represented a significant fraction of them. Notably, analysis of the epigenetic status of a downregulated imprinted gene in NT-EpiSCs revealed complete methylation of the two alleles. Therefore, EpiSCs derived from NT embryos appear to be incorrectly reprogrammed, indicating that abnormal epigenetic marks are imposed on cells in NT embryos during the transition from early to late epiblast.

Cytotoxic Effects of 2-Bromopropane on Embryonic Development in Mouse Blastocysts

2-Bromopropane (2-BP), an alternative to ozone-depleting solvents, is used as a cleaning solvent. Here, we examined the cytotoxic effects of 2-bromopropane (2-BP) on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Mouse blastocysts were incubated in medium with or without 2-BP (2.5, 5 or 10 μM) for 24 h. Cell proliferation and growth were investigated with dual differential staining, apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, and implantation and post-implantation development of embryos were assessed using in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 5 or 10 μM 2-BP displayed significantly increased apoptosis, and decreased inner cell mass (ICM) and trophectoderm (TE) cell number. Additionally, the implantation success rates of 2-BP-pretreated blastocysts were lower than those of untreated controls. In vitro treatment with 5 or 10 μM 2-BP was associated with increased resorption of postimplantation embryos, and decreased placental and fetal weights. Our results collectively indicate that in vitro exposure to 2-BP induces apoptosis, suppresses implantation rates after transfer to host mice, and retards early postimplantation development.

The role of mitochondrial DNA copy number in mammalian fertility.

Mammalian mitochondrial DNA (mtDNA) is a small, maternally inherited genome that codes for 13 essential proteins in the respiratory chain. Mature oocytes contain more than 150 000 copies of mtDNA, at least an order of magnitude greater than the number in most somatic cells, but sperm contain only approximately 100 copies. Mitochondrial oxidative phosphorylation has been suggested to be an important determinant of oocyte quality and sperm motility; however, the functional significance of the high mtDNA copy number in oocytes, and of the low copy number in sperm, remains unclear. To investigate the effects of mtDNA copy number on fertility, we genetically manipulated mtDNA copy number in the mouse by deleting one copy of Tfam, an essential component of the mitochondrial nucleoid, at different stages of germline development. We show that males can tolerate at least a threefold reduction in mtDNA copy number in their sperm without impaired fertility, and in fact, they preferentially transmit a deleted Tfam allele. Surprisingly, oocytes with as few as 4000 copies of mtDNA can be fertilized and progress normally through preimplantation development to the blastocyst stage. The mature oocyte, however, has a critical postimplantation developmental threshold of 40 000-50 000 copies of mtDNA in the mature oocyte. These observations suggest that the high mtDNA copy number in the mature oocyte is a genetic device designed to distribute mitochondria and mtDNAs to the cells of the early postimplantation embryo before mitochondrial biogenesis and mtDNA replication resumes, whereas down-regulation of mtDNA copy number is important for normal sperm function.

Methylglyoxal has injurious effects on maturation of mouse oocytes, fertilization, and fetal development, via apoptosis

Methylglyoxal (MG) is a metabolite of glucose. The serum MG level is increased in diabetic patients, and MG is implicated in diabetic complications related to embryonic development injury. We previously reported cytotoxic effects of MG on mouse embryonic stem cells and blastocysts, and a further association with defects in subsequent development. Here, we further investigate the effects of MG on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, MG induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with MG during in vitro maturation (IVM) led to increased resorption of post-implantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 10–20 μM MG led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented MG-triggered injury effects, suggesting that embryo impairment by MG occurs via a caspase-dependent apoptotic process.

CTCF binding is not the epigenetic mark that establishes post-fertilization methylation imprinting in the transgenic H19 ICR

Imprinted expression of the mouse Igf2/H19 locus is controlled by parent-of-origin-specific methylation of the imprinting control region (ICR). We previously demonstrated that when placed in a heterologous genomic context, the H19 ICR fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells. In the present study, we investigated the requirements for the CTCF-binding sites of the ICR in the acquisition of post-fertilization methylation. To this end, two mutant ICR fragments were introduced into the human beta-globin locus in a yeast artificial chromosome transgenic mouse (TgM) model: 4xMut had mutations in all four ICR CTCF-binding sites that prevented CTCF binding but retained the methylation target CpG motifs, and -9CG harbored mutations in the CpG motifs within the CTCF-binding sites but each site retained constitutive CTCF-binding activity. In TgM germ cells and pre-implantation blastocysts, the absence of CTCF-binding sites (4xMut) did not lead to hypermethylation of the transgenic H19 ICR. However, after implantation, the mutations of CTCF sites (4xMut and -9CG) affected the maintenance of methylation. These results demonstrated that although the CTCF-binding sites are indispensable for maintenance of the unmethylated state of the maternal ICR in post-implantation embryos, they are not required to establish paternal-allele-specific methylation of the transgenic H19 ICR in pre-implantation embryos.