Post-implantation Embryos Research Articles

Developmental Toxicity of Bisphenol-A on Post-Implantation Rat Embryos Cultured in Vitro

We studied the developmental toxicity and possible mechanisms of bisphenol A (BPA) damage of rat embryos in vitro. Whole-embryo culture was used to study the damage of BPA on visceral yolk sacs (VYSs) and embryos. Micromass culture was used to investigate the effects of BPA on differentiation and proliferation of embryonic midbrain cells. Neutral red staining was used to detect cell death. Immunohistochemistry was used to evaluate the expression of inducible nitric oxide synthase (iNOS) in VYSs and embryos. Our results as following, BPA could cause damage to embryonic development and morphological differentiation in a dose-dependent manner. At the highest dose, 80 and 100 mg/l BPA delayed cardiac tube growth and differentiation of VYSs, and induced various embryonic defects. BPA also induced excessive cell death and inhibited both proliferation and differentiation of rat midbrain cells. Immunohistochemical staining showed that BPA induces abnormal expression of iNOS protein in treated VYSs and embryos. Our results indicated that BPA is a potential teratogen and has toxicity on cultured rat embryo development. BPA-related developmental toxicity is caused by damage to the VYS, excessive cell death, inhibition of cell proliferation and differentiation, and abnormal expression of iNOS.

Tubulointerstitial Nephritis Antigen-Like 1 Is Expressed in Extraembryonic Tissues and Interacts with Laminin 1 in the Reichert Membrane at Postimplantation in the Mouse1

Tubulointerstitial nephritis antigen-like 1 (Tinagl1, also known as adrenocortical zonation factor 1 [AZ-1] or lipocalin 7) has been cloned from mouse adrenocortical cells and is known to be closely associated with zonal differentiation of adrenocortical cells. In cell culture systems, TINAGL1 is a matricellular protein that interacts with both structural matrix proteins and cell surface receptors. However, the physiological roles of TINAGL1 and regulation of its expression are still not clearly understood. In the present study, the expression and localization of TINAGL1 in peri-implantation mouse embryos was examined. During preimplantation, the expression of both Tinagl1 mRNA and TINAGL1 protein was increased just prior to implantation. In blastocysts, TINAGL1 expression was localized to the trophectoderm. Using a progesterone-treated, delayed-implantation model, TINAGL1 was found to be upregulated in implantation-competent blastocysts after estrogen treatment. During postimplantation, TINAGL1 expression was restricted to extraembryonic regions. Marked expression was detected in the Reichert membrane on Embryonic Days 6.5 (E6.5) and E7.5. Colocalization of laminin 1 and TINAGL1 was also examined. Using an anti-LAMA1 antibody, colocalization of LAMA1 and TINAGL1 was observed in postimplantation embryos. Colocalization was also detected in the Reichert membrane. Immunoprecipitation analysis determined that LAMA1 and TINAGL1 interact in embryos on E7.5. These results demonstrate that after implantation, TINAGL1 is an extraembryonic tissue-specific protein. In particular, TINAGL1 is a novel component of the Reichert membrane that interacts with laminin 1. These results suggest that TINAGL1 most likely plays a physical and physiological role in embryo development at postimplantation.

A Confirmation Study of Russian and Ukrainian Data on Effects of 2450 MHz Microwave Exposure on Immunological Processes and Teratology in Rats

In a series of Russian and Ukrainian papers published from 1974-1986, it was reported that 30-day whole-body exposures to continuous-wave (CW) radiofrequency (RF) radiation at 2375 MHz and 5 W/m(2) disrupted the antigenic structure of rat brain tissue. The authors suggested that this action caused an autoimmune response in exposed animals. Moreover, these studies reported that blood serum from exposed rats injected into intact nonexposed female rats on the 10th day of pregnancy led to increased postimplantation embryo mortality and decreased fetus size and body weight. Because the results of these studies served in part as the basis for setting exposure limits in the former USSR, it was deemed necessary to perform confirmation studies, using modern dosimetric and biological methods. In our study, a new system was constructed to expose free-moving rats under far-field conditions. Whole-body and brain-averaged specific absorption rates (SARs) were calculated. All results, using ELISA and classic teratology end points, were negative in our laboratory. On the basis of this investigation, we conclude that, under these exposure conditions (2450 MHz, CW, 7 h/day, 30 days, 0.16 W/kg whole-body SAR), RF-radiation exposure had no influence on several immune and degenerative parameters or on prenatal development.

Developmental Potential of Rat Extraembryonic Stem Cells

We have previously found that certain stem cells that are derived from rat blastocysts and named extraembryonic endoderm precursor (XEN-P) cells show a unique molecular signature sharing some of the characteristics of embryonic stem cells (ES), trophoblast stem cells (TS), and extraembryonic endoderm stem cells (XEN). These XEN-P cells are positive for AP, SSEA1, Oct4, and Rex1 markers similar to ES cells and also express signature markers of TS-eomesodermin (Eomes) and XEN-Gata6. Here we show that these cells integrate into the visceral and parietal extraembryonic endoderm lineages as well as into the inner cell mass (ICM), the primitive endoderm, and the polar and mural trophectoderm (TE) of cultured embryos. In addition, we find that the XEN-P cells colonize yolk sac and contribute to trophoblast lineages of postimplantation embryos following transfer to surrogate mothers. We also find that the XEN-P cell culture propagates by shedding cell clusters into the media in addition to typical expansion of colonies. Interestingly, the cell cultures exist as mixed populations of two interconvertible phenotypes of flat and round cells with preferential expression of stem cell markers Oct4 and SSEA1 in round cells. We believe these cells represent a metastable stage during ICM cellular segregation. These results are important for developing hypotheses of cell fate plasticity in the ICM and provide a model for the study of development and differentiation along the extraembryonic lineages.

Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro

Pluripotent epiblast stem cells (EpiSCs) derived from postimplantation embryos exhibit properties that are characteristically different when compared with pluripotent embryonic stem cells (ESCs) derived from mouse blastocysts. However, EpiSCs are relatively less well characterised compared with ESCs. In particular, the relationship between EpiSCs and primordial germ cells (PGCs) is unknown, and is worthy of investigation because PGCs originate from postimplantation epiblast cells in vivo. We show that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal. These PGCs generated in vitro show appropriate transcriptional and epigenetic reprogramming events and are able to develop further into late germ cells. Notably, the PGCs can, in turn, be induced to undergo dedifferentiation into pluripotent embryonic germ cells (EGCs), which resemble ESCs and not the EpiSC from which they are derived. Our observations demonstrate intrinsic reprogramming during specification of PGCs that results in the erasure of epigenetic memory of EpiSCs following reactivation of the X-chromosome, DNA demethylation and re-expression of key pluripotency genes. This study provides novel insights into the nature and properties of EpiSCs, and introduces an in vitro model system that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells.

Centromeric heterochromatin assembly during human embryonic stem cell (hESC) differentiation: implications for aneuploidy

OBJECTIVE: Aneuploidy is elevated in both hESCs and blastomeres compared to cells in the post implantation embryo. In somatic cells, experimentally induced epigenetic defects at centromeres can increase aneuploidy. Pericentric hypermethylation is regulated by histone deacetylases (HDAC1 and 2) and DNA methyltransferases. Our objective was to characterize the pericentromeric heterochromatin and HDAC distributions in pluripotent and differentiated hESCs. DESIGN: In vitro model of hESC differentiation to neurectoderm (NE), confocal immunofluorescence microscopy, western blot analysis. MATERIALS AND METHODS: HESCs were grown on mouse embryo fibroblasts (MEF) feeders. NE was prepared from hESCs on low-density MEFs for 1–3 weeks. Pluripotent (Oct4) and differentiated (Nestin) hESC were determined by confocal microscopy. Heterochromatin content was determined by blot and by colocalization of HP1a, HP1b, histone H3 trimethylated on lysine 9 (H3K9me3) and DNA methylated on cytosine (5meC). RESULTS: Pluripotent hESCs are hypomethylated at most centromeres until after differentiation when centromeres become uniformly methylated at histones and DNA. HP1a and b locations change from diffuse to discrete foci upon hESC differentiation. H3K9me3 expression increases upon hESC differentiation and colocalizes with HP1b and 5meC. In pluripotent mitotic chromosomes H3K9me3 is nearly absent and HP1b is cytoplasmic while in NE both H3K9me3 and HP1b are present on chromosomes. HP1b is completely extractable from pluripotent hESCs but not NE. The ratio of HDAC1, HDAC2 and DNA are uncorrelated in pluripotent nuclei but highly correlated in NE. CONCLUSIONS: Pericentric heterochromatin is assembled and HDACs become highly correlated upon hESC differentiation, which might decrease aneuploidy rates during development. In pluripotent cells, insufficient pericentric heterochromatin assembly might influence centromere segregation and aneuploidy resulting from mitotic nondisjunction.

Impact effects of puerarin on mouse embryonic development

In this report, we examine the cytotoxic effects of puerarin, an isoflavone compound, on the blastocyst stage of mouse embryos and subsequent embryonic attachment and outgrowth in vitro and in vivo implantation by embryo transfer. Mouse blastocysts were incubated in medium with or without puerarin (2.5, 5 or 10 μM) for 24 h. Cell proliferation and growth was investigated by dual differential staining, apoptosis was analyzed by 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. We found that blastocysts treated with 5 or 10 μM puerarin showed significant increases in apoptosis and significant decreases in total cell number. Interestingly, there was no significant difference of implantation success rate between puerarin-pretreated blastocysts and controls, but in vitro treatment with 5 or 10 μM puerarin was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that in vitro exposure to puerarin induced apoptosis and after transfer to host mice retards early post-implantation development. The extent to which puerarin may have teratogenic potential in early human development is not yet known.

Steroid metabolism by rabbit embryos: time of onset of steroidogenic activity.

Twenty four hour incubations of the days 5 and 6 pre-implantation and the days 7 and 8 post-implantation rabbit embryos were carried out in the presence of 3H-labelled steroid substrates viz. dehydroepiandrosterone (DHEA), androstenedione (Ad) and testosterone (Te). While both the pre- as well as the post-implantation embryos were able to actively metabolize the various neutral steroid substrates into other neutral, mostly 5 alpha-reduced, steroid metabolites, the conversion of DHEA to Ad or that of DHEA, Ad and Te to estrogens was exhibited only by the days 7 and 8 post-implantation embryos. The findings suggest that pre-implantation blastocysts of rabbit lack the potentiality to synthesize steroids and acquire this ability only after implantation.

Cytotoxic Effects of CdSe Quantum Dots on Maturation of Mouse Oocytes, Fertilization, and Fetal Development

Quantum dots (QDs) are useful novel luminescent markers, but their embryonic toxicity is yet to be fully established, particularly in oocyte maturation and sperm fertilization. Earlier experiments by our group show that CdSe-core QDs have cytotoxic effects on mouse blastocysts and are associated with defects in subsequent development. Here, we further investigate the influence of CdSe-core QDs on oocyte maturation, fertilization, and subsequent pre- and postimplantation development. CdSe-core QDs induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryo development, but not ZnS-coated CdSe QDs. Treatment of oocytes with 500 nM CdSe-core QDs during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. To our knowledge, this is the first study to report the negative impact of CdSe-core QDs on mouse oocyte development. Moreover, surface modification of CdSe-core QDs with ZnS effectively prevented this cytotoxicity.

Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility

In humans, normal healthy children are regularly produced through fertilization of fresh oocytes with fresh spermatozoa. However, asynchrony between oocytes and spermatozoa, especially when aged oocytes are fertilized by fresh or senescent spermatozoa, will not only affect the rate of fertilization and pre- and post-implantation embryo development but also the life of the offspring. As many failures in assisted reproduction technologies (ART) are related to oocyte aging, new methods are needed to control oocyte aging to benefit modern ART. We review changes associated with decreased fertilization rates and developmental potential of aged oocytes, and we present methods and approaches that prevent or delay oocyte aging. Cellular and molecular abnormalities occur during oocyte aging, but prevention, delay or reversal is possible to various extents. Modifying existing culture conditions, or treatment of oocytes with agents such as caffeine, DL-dithiothreitol, nitric oxide or trichostatin A may correct molecular pathways that are affected by aging, and thus benefit and improve success rates in modern ART. Aging of oocytes is characterized by a sequence of molecular processes that deteriorate during aging and negatively impact fertilization and development. However, oocyte aging can be delayed or reversed by various treatments to increase success rates and produce increased numbers of healthy embryos, preventing failures or abnormalities that are frequently associated with ART using aged oocytes.

Quantitative Changes of Sialoadhesin and CD163 Positive Macrophages in the Implantation Sites and Organs of Porcine Embryos/Fetuses During Gestation

Porcine reproductive and respiratory syndrome virus (PRRSV) crosses the placenta most easily in the last third of gestation. Further, PRRSV does not replicate in preimplantation embryos but does replicate in postimplantation embryos and fetuses. In the present study, it was aimed to find an explanation for these observations by localization and quantification of the macrophages carrying two entry mediators that play a crucial role in PRRSV replication, sialoadhesin (Sn) and CD163, in the implantation sites and organs of embryos/fetuses during gestation. Uterus and embryos or organs (liver, spleen, lungs) from fetuses were obtained from pregnant PRRSV negative sows at different days of gestation (20–35, 50–60, 70–80, 114) and the Sn+ and CD163+ macrophages were quantified. In endometrium and placentas, two macrophage subsets were observed: Sn−CD163+ and Sn+CD163+. The highest number of Sn+ and CD163+ macrophages was counted at 114days of gestation. In the mid-gestation fetal placentas (50–60days of gestation), most CD163+ macrophages were Sn negative. The number of Sn+ and CD163+ macrophages in organs increased during gestation. In the liver, the Sn+ and CD163+ macrophages were most abundant (Sn+: 8.1–48.7%; CD163+: 22.0–55.0%); the lowest number of Sn+ and CD163+ macrophages was observed in the lungs (Sn+: 0–15.2%; CD163+: 4.0–19.3%). Double immunofluorescence staining revealed three macrophage subsets in the spleen: Sn+CD163−, Sn−CD163+ and Sn+CD163+; and two macrophage subsets in the lungs: Sn−CD163+ and Sn+CD163+. In the liver, due to physiological presence of biotin, the double immune-fluorescence staining could not be performed. The present results show clear changes in the quantity of Sn+ and CD163+ macrophages in the placentas and organs of embryos/fetuses during gestation which most probably have a physiological basis. The absence of Sn on macrophages in the fetal placenta at mid-gestation might explain the difficulty for PRRSV to spread transplacentally at this stage of gestation.

Impact of genistein on maturation of mouse oocytes, fertilization, and fetal development

Genistein (GNT), a natural isoflavone compound found in soy products, affects diverse cell functions, including proliferation, differentiation and cell death. An earlier study by our group showed that GNT has cytotoxic effects on mouse blastocysts and is associated with defects in their subsequent development in vitro. Here, we further investigate the effects of GNT on oocyte maturation, and subsequent pre- and postimplantation development, both in vitro and in vivo. GNT induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryo development. Treatment of oocytes with GNT during in vitro maturation (IVM) led to increased resorption of postimplantation embryos, and decreased placental and fetal weights. With the aid of an in vivo mouse model, we showed that consumption of drinking water containing GNT led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Moreover, our findings support a degree of selective inhibition of retinoic acid receptors in blastocysts treated with GNT during oocyte maturation. To our knowledge, this is the first study investigating the impact of GNT on maturation of mouse oocytes, fertilization, and sequential embryonic development.

Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1-6 microM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3-6 microM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.

Reproductive and epigenetic outcomes associated with aging mouse oocytes

Female aging entails a decline in fertility in mammals, manifested by reduced oocyte reserves and poor oocyte quality accompanied by chromosomal anomalies and reduced litter size. In addition to compromised genetic integrity, recent studies suggest that epigenetic mechanisms may be altered in aging oocytes, with age affecting the expression of DNA methyltransferases, which catalyze the important epigenetic modification, DNA methylation. Loss of DNA methylation patterns, most notably for imprinted genes, is lethal to mouse embryos. To investigate how maternal age affects embryonic development and underlying DNA methylation patterns, young and aged C57BL/6 females were mated with C57BL/6 or C57BL/6(CAST7) males to allow for the identification of parental alleles; resulting blastocysts and mid-gestation embryos and placentas were evaluated. Although pregnancy, ovulation and implantation rates were similar between age groups, an age-related increase in resorption sites, morphological abnormalities and delayed development was found. Interestingly, placental morphology was also perturbed by aging, with elevated numbers of trophoblast giant cells in aged pregnancies. Normal monoallelic expression of the imprinted genes H19 and Snrpn was unaltered in blastocysts from aged females. We failed to observe any age-related changes in methylation of the differentially methylated regions of imprinted genes Snrpn, Kcnq1ot1, U2af1-rs1, Peg1, Igf2r and H19. Restriction Landmark Genome Scanning showed no significant differences in genome-wide DNA methylation in embryos and placentas, regardless of maternal age. Our findings demonstrate that maternal age affects post-implantation embryo and placental development; however embryos capable of developing to mid-gestation appear to undergo normal acquisition and maintenance of DNA methylation patterning.

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid‐gastrulation

Transgenic mice with a defined cell- or tissues-specific expression of Cre-recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre-expression from Cdx1 regulatory elements. The expression of Cre-recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre-mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development.

Gametic products transmitted by rcp(13q--;14q+) translocation heterozygous pigs, and resulting embryonic loss.

Among embryos and neonates produced by pigs heterozygous for the reciprocal translocation rcp(13q-;14q+) were recovered: Preimplantation embryos: (1) From a heterozygous boar mated to 10 normal gilts, 109 embryos, 36 karyotyped (14 balanced normal, 11 balanced heterozygous, 11 unbalanced). (2) From a normal boar mated to 10 heterozygous gilts, 81 embryos, 33 karyotyped (10 balanced normal, 14 balanced heterozygous, 9 unbalanced). (3) From a heterozygous boar mated to 2 heterozygous gilts, 24 embryos, 11 karyotyped (2 balanced normal, 4 balanced heterozygous, 5 unbalanced). Postimplantation embryos: (1) From a heterozygous boar mated to 3 normal gilts, 19 embryos (7 balanced normal, 12 balanced heterozygous). (2) From a normal boar mated to 4 heterozygous gilts, 17 embryos (5 balanced normal, 10 balanced heterozygous, 2 degenerated). (3) From a heterozygous boar mated to a heterozygous gilt, 4 embryos (2 balanced heterozygous, 2 degenerated). Embryonic mortality estimated from numbers of corpora lutea and of living implanted embryos in heterozygous gilts mated with a normal boar, and in normal and heterozygous gilts mated with a heterozygous boar was 72.3 %, 68 %, and 85.5 %, respectively. The average litter size in the latter two types of matings was 7 and 5.1. The results are discussed in relation to meiotic behaviour, gametic products and embryonic mortality.

LAT-1 Expression in Pre- and Post-implantation Embryos and Placenta

Objectives LAT-1 (L-type amino acid transporter 1) is a system L, Na +-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post-implantation stages. Methods LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry. Results Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGCs) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGCs at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGCs, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGCs at E9.5 and E11.5. Conclusions Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.

Role of Apoptosis in Mediating Salicylic Acid-Induced Teratogenesis In Vitro

Salicylic acid (SAL) 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 SAL 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 SAL, i.e. 10, 100, and 1000 μg/ml cultures, respectively. The growth and developmental of each embryo was evaluated and compared with control ones for the presence of any malformations. The SAL decreased all growth and developmental parameters in a concentration-dependent manner, when compared with control. However, exposure to SAL at 10 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 SAL-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 SAL in vitro.

An in vitro ES cell imprinting model shows that imprinted expression of theIgf2rgene arises from an allele-specific expression bias

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles.

Early lethality of β-1,4-galactosyltransferase V-mutant mice by growth retardation

The β-1,4-galactosyltransferase (β-1,4-GalT) V whose human and mouse genes were cloned by us has been suggested to be involved in the biosynthesis of N-glycans and O-glycans, and lactosylceramide. To determine its biological function, β-1,4-GalT V (B4galt5) mutant mice obtained by a gene trap method were analyzed. Analysis of pre- and post-implantation embryos revealed that the B4galt5−/− mice die by E10.5 while B4galt5+/− mice were born and grown normally. Histological study showed that most tissues are formed in B4galt5−/− embryos but their appearance at E10.5 is close to that of B4galt5+/− embryos at E9.0-9.5. The results indicate that the growth is delayed by one to one and half day in B4galt5−/− embryos when compared to B4galt5+/− embryos, which results in early death of the embryos by E10.5, probably due to hematopoietic and/or placental defects.