Somatic Cell Hybrid Mapping of Expressed Sequence Tags For Genes Showing Early Embryonic Death-Associated Changes of Expression Patterns in the Fetal Placenta of the Cow Carrying Somatic Nuclear-Derived Cloned Embryo

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.     

Somatic cell hybrid mapping of expressed sequence tags for genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo

We previously detected 368 expressed sequence tags showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo. In the present study 7 (presumed expressed sequence tags for HYPC, SPTBN1 and TNNC2, and four expressed sequence tags for unknown novel genes) out of the 368 expressed sequence tags were mapped to bovine chromosomes by analyzing deoxyribonucleic acids of bovine/murine somatic cell hybrid panel with polymerase chain reaction using primers specific for those bovine genes.     

Chromosomal assignments of eight expressed sequence tags for unknown genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.     

Chromosomal Assignments of Eight Expressed Sequence Tags for Unknown Genes Showing Early Embryonic Death-Associated Changes of Expression Patterns in the Fetal Placenta of the Cow Carrying Somatic Nuclear-Derived Cloned Embryo

Eight expressed sequence tags for unknown novel genes showing early embryonic death-associated changes of expression patterns in the fetal placenta of the cow carrying somatic nuclear-derived cloned embryo were assigned to bovine chromosomes using deoxyribonucleic acids (DNAs) of bovine/murine somatic cell hybrid panel.     

Notch2 is required for formation of the placental circulatory system, but not for cell-type specification in the developing mouse placenta

We have previously reported that a mutation in the ankyrin repeats of mouse Notch2 results in embryonic lethality by embryonic day 11.5 (E11.5), showing developmental retardation at E10.5. This indicated that Notch2 plays an essential role in postimplantation development in mice. Here, we demonstrate that whole embryo culture can circumvent developmental retardation of Notch2 mutant embryos for up to 1 day, suggesting that the lethality was primarily caused by extraembryonic defects. Histological examinations revealed delayed entry of maternal blood into the mutant placenta and poor blood sinus formation at later stages. Notch2-expressing cells appeared around maternal blood sinuses. Specification of trophoblast subtypes appeared not to be drastically disturbed and expression of presumptive downstream genes of Notch2 signaling was not altered by the Notch2 mutation. Thus, in the developing mouse placenta, Notch2 is unlikely to be involved in cell fate decisions, but rather participates in formation of maternal blood sinuses. In aggregation chimeras with wild-type tetraploid embryos, the mutant embryos developed normally until E12.5, but died before E13.5. The chimeric placentas showed a restored maternal blood sinus formation when compared with the mutant placentas, but not at the level of wild-type diploid placentas. Therefore, it was concluded that the mutant suffers from defects in maternal blood sinus formation. Thus, Notch2 is not cell autonomously required for the early cell fate determination of subtypes of trophoblast cells, but plays an indispensable role in the formation of maternal blood sinuses in the developing mouse placenta.     

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.     

Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice.     

Expression profiling of genes crucial for placental and preimplantation development in bovine in vivo, in vitro, and nuclear transfer blastocysts

Placental abnormalities and failed implantation are characterized phenotypes that occur in many species as a result of somatic cell cloning. This study examines a number of genes, critical for early placental development and reports aberrant expression patterns in a number of cloned bovine blastocysts, thus implicating a role of these genes in failed implantation. Messenger RNA (mRNA) expression of eight genes critical for early placental and preimplantation development including Acrogranin, Cdx2, Eomes, ErbB3, ERR2, Hand1, MRJ, and Rex1 were analyzed in single, in vivo, in vitro, and cloned bovine blastocysts (produced by hand-made cloning (HMC) and serial hand-made cloning (SHMC)) following complementary DNA (cDNA) amplification with a SMART cDNA synthesis kit. Aberrant expression of Acrogranin, Cdx2, and ERR2 was detected in a number of blastocysts produced by SHMC. Other genes, Eomes and Hand1, were not detectable in, in vivo bovine blastocysts, suggesting a differential expression pattern between bovine and murine embryos. A number of control marker genes including Oct4, IFN-tau, and PolyA were expressed in all single blastocysts analyzed. This is the first study to report that failure of implantation may be due to aberrant expression of genes in the preimplantation cloned embryo, which are crucial for the early regulation and differentiation of the placenta.     

Identification of genes aberrantly expressed in mouse embryonic stem cell-cloned blastocysts

During development, cloned embryos often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. The long-term effects resulting from embryo cloning procedures would manifest after birth as early death, obesity, various functional disorders, and so forth. Despite extensive studies, the parameters affecting the developmental features of cloned embryos remain unclear. The present study carried out extensive gene expression analysis to screen a cluster of genes aberrantly expressed in embryonic stem cell-cloned blastocysts. Differential screening of cDNA subtraction libraries revealed 224 differentially expressed genes in the cloned blastocysts: eighty-five were identified by the BLAST search as known genes performing a wide range of functions. To confirm their differential expression, quantitative gene expression analyses were performed by real-time PCR using single blastocysts. The genes Skp1a, Canx, Ctsd, Timd2, and Psmc6 were significantly up-regulated, whereas Aqp3, Ak3l1, Rhot1, Sf3b3, Nid1, mt-Rnr2, mt-Nd1, mt-Cytb, and mt-Co2 were significantly down-regulated in the majority of embryonic stem cell-cloned embryos. Our results suggest that an extraordinarily high frequency of multiple functional disorders caused by the aberrant expression of various genes in the blastocyst stage is involved in developmental arrest and various other disorders in cloned embryos.     

Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle

Poor success rates in somatic cell cloning are often attributed to abnormal early embryonic development as well as late abnormal fetal growth and placental development. Although promising results have been reported following chromatin transfer (CT), a novel cloning method that includes the remodeling of the donor nuclei in vitro prior to their transfer into enucleated oocytes, animals cloned by CT show placental abnormalities similar to those observed following conventional nuclear transfer. We hypothesized that the placental gene expression pattern from cloned fetuses was ontologically related to the frequently observed placental phenotype. The aim of the present study was to compare global gene expression by microarray analysis of Day 44–47 cattle placentas derived from CT cloned fetuses with those derived from in vitro fertilization (i.e. control), and confirm the altered mRNA and protein expression of selected molecules by qRT-PCR and immunohistochemistry, respectively. The differentially expressed genes identified in the present study are known to be involved in a range of activities associated with cell adhesion, cell cycle control, intracellular transport and proteolysis. Specifically, an imprinted gene, involved with cell proliferation and placentomegaly in humans (CDKN1C) and a peptidase that serves as a marker for non-invasive trophoblast cells in human placentas (DPP4), had mRNA and protein altered in CT placentas. It was concluded that the altered pattern of gene expression observed in CT samples may contribute to the abnormal placental development phenotypes commonly identified in cloned offspring, and that expression of imprinted as well as trophoblast invasiveness-related genes is altered in cattle cloned by CT.     

Molecular characterization of the neuronatin gene in the porcine placenta

Imprinted genes play important roles in placental and embryonic development. Neuronatin (NNAT), first identified as an imprinted gene in human and mouse brains, played important roles in neuronal differentiation in the brain and in glucose-mediated insulin secretion in pancreatic β cells. In the pig, NNAT was reported to be imprinted in eleven tissues. Our previous microarray hybridization study showed that NNAT was differentially expressed in Yorkshire and Meishan pig placentas, but the imprinting status and function of NNAT in the placenta have not been investigated. We demonstrated for the first time that NNAT was monoallelically expressed in the placenta. Immunochemistry analysis showed that NNAT was located in the uterine luminal and glandular epithelium in placentas. We also confirmed the differential expression of NNAT in Meishan and Yorkshire pig placentas by qPCR. Using IPA software and the published literature, we created a model network of the possible relationships between NNAT and glucose transporter genes. A dual luciferase reporter assay demonstrated that the crucial promoter region of NNAT contained a CANNTG sequence in the +210 to +215 positions, which corresponded to the E-box. Our findings demonstrated important roles of NNAT in placenta function.     

Abnormal expression of trophoblast major histocompatibility complex class I antigens in cloned bovine pregnancies is associated with a pronounced endometrial lymphocytic response

Early embryonic losses are much higher in nuclear transfer (cloned) pregnancies, and this is a major impediment to improving the efficiency of cloned animal production. In cattle, many of these losses occur around the time of placental attachment from the fourth week of gestation. We studied the potential for altered immunologic status of cloned pregnancies to be a contributing factor to these embryonic losses. Expression of major histocompatibility complex class I (MHC-I) by trophoblast cells and distribution of endometrial T-lymphocyte numbers were investigated. Six 5-wk-old cloned pregnancies were generated, and 2 others at 7 and 9 wk were also included, all derived from the same fetal cell line. All 8 cloned placentas displayed trophoblast MHC-I expression. None of the 8 controls (4-7 wk old) showed any MHC-I expression. The percentage of trophoblast cells expressing MHC-I varied in the clones from 17.9% to 56.5%. Numbers of T lymphocytes (CD3(+) lymphocytes) were significantly higher in the endometrium of the majority of cloned pregnancies compared with controls. In the cloned pregnancies, large aggregates of T cells were frequently observed in the endometrium in addition to increased numbers of diffusely spread subepithelial lymphocytes. As trophoblast MHC-I expression is normally suppressed during early gestation, the observed MHC-I expression in the cloned pregnancies is likely to have induced a maternal lymphocytic response that would be detrimental to maintaining viability of the cloned pregnancy. These findings support a role for immunologic rejection in the syndrome of early embryonic loss in cloned bovine pregnancies.