Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves

Placental deficiencies are linked with developmental abnormalities in cattle produced by somatic cell nuclear transfer (SCNT). To investigate whether the aberrant expression of imprinted genes in placenta was responsible for fetal overgrowth and placental hypertrophy, quantitative expression analysis of six imprinted genes (H19, XIST, IGF2R, SNRPN, PEG3, and IGF2) was conducted in placentas of: 1) deceased (died during perinatal period) transgenic calves (D group, n = 4); 2) live transgenic calves (L group, n = 15); and 3) conventionally produced (control) female calves (N group, n = 4). In this study, XIST, PEG3 and IGF2 were significantly over-expressed in the D group, whereas expression of H19 and IGF2R was significantly reduced in the D group compared to controls. The DNA methylation patterns in the differentially methylated region (DMR) from H19, XIST, and IGF2R were compared using Bisulfite Sequencing PCR (BSP) and Combined Bisulfite Restriction Analysis (COBRA). In the D group, H19 DMR was significantly hypermethylated, but XIST DMR and IGF2R ICR were significantly hypomethylated compared to controls. In contrast, there were no noticeable differences in the expression and DNA methylation status of imprinted genes (except DNA methylation level of XIST DMR) in the L group compared to controls. In conclusion, altered DNA methylation levels in the DMRs of imprinted genes in placentas of deceased transgenic calves, presumably due to aberrant epigenetic nuclear reprogramming during SCNT, may have been associated with abnormal expression of these genes; perhaps this caused developmental insufficiencies and ultimately death in cloned transgenic calves.     

Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves

Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3’UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied.     

Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression.The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.     

Leukocyte DNA as Surrogate for the Evaluation of Imprinted Loci Methylation in Mammary Tissue DNA

There is growing interest in identifying surrogate tissues to identify epimutations in cancer patients since primary target tissues are often difficult to obtain. Methylation patterns at imprinted loci are established during gametogenesis and post fertilization and their alterations have been associated with elevated risk of cancer. Methylation at several imprinted differentially methylated regions (GRB10 ICR, H19 ICR, KvDMR, SNRPN/SNURF ICR, IGF2 DMR0, and IGF2 DMR2) were analyzed in DNA from leukocytes and mammary tissue (normal, benign diseases, or malignant tumors) from 87 women with and without breast cancer (average age of cancer patients: 53; range: 31–77). Correlations between genomic variants and DNA methylation at the studied loci could not be assessed, making it impossible to exclude such effects. Methylation levels observed in leukocyte and mammary tissue DNA were close to the 50% expected for monoallellic methylation. While no correlation was observed between leukocyte and mammary tissue DNA methylation for most of the analyzed imprinted genes, Spearman''s correlations were statistically significant for IGF2 DMR0 and IGF2 DMR2, although absolute methylation levels differed. Leukocyte DNA methylation levels of selected imprinted genes may therefore serve as surrogate markers of DNA methylation in cancer tissue.     

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.     

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

Background  

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2) and a repetitive sequence (art2) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).     

The role and interaction of imprinted genes in human fetal growth

Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown–rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (−132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects.     

A functional screen for genes involved in Xenopus pronephros development

In Xenopus, the pronephros is the functional larval kidney and consists of two identifiable components; the glomus, the pronephric tubules, which can be divided into four separate segments, based on marker gene expression. The simplicity of this organ, coupled with the fact that it displays the same basic organization and function as more complex mesonephros and metanephros, makes this an attractive model to study vertebrate kidney formation. In this study, we have performed a functional screen specifically to identify genes involved in pronephros development in Xenopus. Gain-of-function screens are performed by injecting mRNA pools made from a non-redundant X. tropicalis full-length plasmid cDNA library into X. laevis eggs, followed by sib-selection to identify the single clone that caused abnormal phenotypes in the pronephros. Out of 768 egg and gastrula stage cDNA clones, 31 genes, approximately 4% of the screened clones, affected pronephric marker expression examined by whole mount in situ hybridization or antibody staining. Most of the positive clones had clear expression patterns in pronephros and predicted/established functions highly likely to be involved in developmental processes. In order to carry out a more detailed study, we selected Sox7, Cpeb3, P53csv, Mecr and Dnajc15, which had highly specific expression patterns in the pronephric region. The over-expression of these five selected clones indicated that they caused pronephric abnormalities with different temporal and spatial effects. These results suggest that our strategy to identify novel genes involved in pronephros development was highly successful, and that this strategy is effective for the identification of novel genes involved in late developmental events.     

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

Knockdown of CDKN1C (p57kip2) and PHLDA2 Results in Developmental Changes in Bovine Pre-implantation Embryos

Imprinted genes have been implicated in early embryonic, placental, and neonatal development and alterations in expression levels of these genes can lead to growth abnormalities and embryonic lethality. However, little is known about the functions of bovine imprinted genes during the pre-implantation period. Therefore, the objective of this study was to assess the influence of altered expression of imprinted genes on developmental progress of embryos using small interfering RNA (siRNA). Expression levels of 18 imprinted genes (MAGEL2, UBE3A, IGF2R, NAP1L5, TSSC4, PEG3, NDN, CDKN1C, PHLDA2, MKRN3, USP29, NNAT, PEG10, RTL1, IGF2, H19, MIM1, and XIST) were compared between embryos reaching the blastocyst stage and growth-arrested embryos (degenerates) using quantitative real-time PCR (qRT-PCR). Ten genes were found to be differentially expressed between blastocysts and degenerates. The CDKN1C gene showed the highest upregulation in blastocysts whereas PHLDA2 was highly expressed in degenerates. To assess whether the observed differential gene expression was causative or resultant of embryo degeneration, these genes were selected for functional analysis using siRNA. Injection of siRNA specific to PHLDA2 into one-cell zygotes resulted in a substantial increase in blastocyst development, whereas injection of CDKN1C-specific siRNA resulted in a 45% reduction (P = 0.0006) in blastocyst development. RNA-Seq analysis of CDKN1C-siRNA-injected vs. non-injected embryos revealed 51 differentially expressed genes with functions in apoptosis, lipid metabolism, differentiation, and cell cycle regulation. Gene ontology analysis revealed nine pathways related to cell signaling, metabolism, and nucleic acid processing. Overall, our results show that proper expression levels of the imprinted genes CDKN1C and PHLDA2 are critical for embryo development, which suggests that these genes can be used as markers for normal blastocyst formation.     

A phylogenetic approach to test for evidence of parental conflict or gene duplications associated with protein-encoding imprinted orthologous genes in placental mammals

There are multiple theories on the evolution of genomic imprinting. We investigated whether the molecular evolution of true orthologs of known imprinted genes provides support for theories based on gene duplication or parental conflicts (where mediated by amino-acid changes). Our analysis of 34 orthologous genes demonstrates that the vast majority of mammalian imprinted genes have not undergone any subsequent significant gene duplication within placental species, suggesting that selection pressures against gene duplication events could be operating for imprinted loci. As antagonistic co-evolution between imprinted genes can regulate offspring growth, proteins mediating this interaction could be subject to rapid evolution via positive selection. Supporting this, we detect evidence of site specific positive selection for the imprinted genes OSBPL5 (and GNASXL), and detect lineage-specific positive selection for 14 imprinted genes where it is known that the gene is imprinted in a specific lineage, namely for: PLAGL1, IGF2, SLC22A18, OSBPL5, DCN, DLK1, RASGRF1, IGF2R, IMPACT, GRB10, NAPIL4, UBE3A, GATM and GABRG3. However, there is an overall lack of concordance between the known imprinting status of each gene (i.e. whether the gene is imprinted or biallelically expressed in a particular mammalian lineage) and positive selection. While only a small number of orthologs of imprinted loci display evidence of positive selection, we observe that the majority of orthologs of imprinted loci display high levels of micro-synteny conservation and have undergone very few cis- or trans-duplications in placental mammalian lineages.     

Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.