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.     

Analysis of imprinted gene expression in normal fertilized and uniparental preimplantation porcine embryos

In the present study quantitative real-time PCR was used to determine the expression status of eight imprinted genes (GRB10, H19, IGF2R, XIST, IGF2, NNAT, PEG1 and PEG10) during preimplantation development, in normal fertilized and uniparental porcine embryos. The results demonstrated that, in all observed embryo samples, a non imprinted gene expression pattern up to the 16-cell stage of development was common for most genes. This was true for all classes of embryo, regardless of parental-origins and the direction of imprint. However, several differentially expressed genes (H19, IGF2, XIST and PEG10) were detected amongst the classes at the blastocyst stage of development. Most interestingly and despite the fact that maternally and paternally expressed genes should not be expressed in androgenones and parthenogenones, respectively, both uniparental embryos expressed these genes when tested for in this study. In order to account for this phenomenon, we compared the expression patterns of eight imprinted genes along with the methylation status of the IGF2/H19 DMR3 in haploid and diploid parthenogenetic embryos. Our findings revealed that IGF2, NNAT and PEG10 were silenced in haploid but not diploid parthenogenetic blastocysts and differential methylation of the IGF2/H19 DMR3 was consistently observed between haploid and diploid parthenogenetic blastocysts. These results appear to suggest that there exists a process to adjust the expression status of imprinted genes in diploid parthenogenetic embryos and that this phenomenon may be associated with altered methylation at an imprinting control region. In addition we believe that imprinted expression occurs in at least four genes, namely H19, IGF2, XIST and PEG10 in porcine blastocyst stage embryos.     

TGFα Reactivates Imprinted <Emphasis Type="Italic">Igf2</Emphasis> in the Parthenogenetic Mice Embryos and Placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE), there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here, we studied effect of transforming growth factor alpha (TGFα) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5 days of gestation, 25 somites) and their placentas (PP). Using RT-PCR, we showed that TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments, it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFα treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP, both Igf2 and H19 were expressed. It seems that TGFα can play an important role as modulator of the Igf2/H19 locus.     

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.     

Karyotype Characterization of In Vivo- and In Vitro-Derived Porcine Parthenogenetic Cell Lines

Mammalian haploid cell lines provide useful tools for both genetic studies and transgenic animal production. To derive porcine haploid cells, three sets of experiments were conducted. First, genomes of blastomeres from 8-cell to 16-cell porcine parthenogenetically activated (PA) embryos were examined by chromosome spread analysis. An intact haploid genome was maintained by 48.15% of blastomeres. Based on this result, two major approaches for amplifying the haploid cell population were tested. First, embryonic stem-like (ES-like) cells were cultured from PA blastocyst stage embryos, and second, fetal fibroblasts from implanted day 30 PA fetuses were cultured. A total of six ES-like cell lines were derived from PA blastocysts. No chromosome spread with exactly 19 chromosomes (the normal haploid complement) was found. Four cell lines showed a tendency to develop to polyploidy (more than 38 chromosomes). The karyotypes of the fetal fibroblasts showed different abnormalities. Cells with 19–38 chromosomes were the predominant karyotype (59.48–60.91%). The diploid cells were the second most observed karyotype (16.17%–22.73%). Although a low percentage (3.45–8.33%) of cells with 19 chromosomes were detected in 18.52% of the fetus-derived cell lines, these cells were not authentic haploid cells since they exhibited random losses or gains of some chromosomes. The haploid fibroblasts were not efficiently enriched via flow cytometry sorting. On the contrary, the diploid cells were efficiently enriched. The enriched parthenogenetic diploid cells showed normal karyotypes and expressed paternally imprinted genes at extremely low levels. We concluded that only a limited number of authentic haploid cells could be obtained from porcine cleavage-stage parthenogenetic embryos. Unlike mouse, the karyotype of porcine PA embryo-derived haploid cells is not stable, long-term culture of parthenogenetic embryos, either in vivo or in vitro, resulted in abnormal karyotypes. The porcine PA embryo-derived diploid fibroblasts enriched from sorting might be candidate cells for paternally imprinted gene research.     

Identification of the mouse paternally expressed imprinted gene Zdbf2 on chromosome 1 and its imprinted human homolog ZDBF2 on chromosome 2

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.     

Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media

This study was conducted to develop a serum-free, defined medium for IVM of pig oocytes. Modified North Carolina State University (mNCSU)-23 media with or without supplementation with both epidermal growth factor (EGF) and gonadotrophin were used as base media. In separate experiments, each base medium was supplemented with porcine follicular fluid (pFF), polyvinyl alcohol (PVA), PVA and essential amino acids (EAA), PVA and nonessential amino acids (NEAA) or PVA with both EAA and NEAA. Averaged across these five treatments, the percentage of blastocyst formation was higher (P < 0.05) in the base medium supplemented with EGF and gonadotrophins. In both base media, the addition of NEAA yielded similar percentages of maturation (81-82% versus 75-80%), sperm penetration (89-93% versus 80-86%) and blastocyst formation (4-18% versus 4-13%) as media supplemented with pFF. Although similar benefits were found after the addition of EAA, their addition was associated with lower (P < 0.05) maturation (66%) and sperm penetration (58%) than when pFF was added to the base medium without EGF and gonadotrophins. However, decreased maturation after EAA addition was not detected in the base medium containing EGF and gonadotrophins. Within the same base medium, monospermy, male pronucleus formation, cleavage and blastocyst formation were not affected by the treatments; and combined addition of EAA and NEAA did not further improve oocyte development. In conclusion, a maturation system using a defined mNCSU-23 medium supplemented with EGF, gonadotrophins and EAA or NEAA was developed which yielded a similar number of blastocysts compared with a pFF-containing medium.     

Dietary essentiality of “nutritionally non-essential amino acids” for animals and humans

Based on growth or nitrogen balance, amino acids (AA) had traditionally been classified as nutritionally essential (indispensable) or non-essential (dispensable) for animals and humans. Nutritionally essential AA (EAA) are defined as either those AA whose carbon skeletons cannot be synthesized de novo in animal cells or those that normally are insufficiently synthesized de novo by the animal organism relative to its needs for maintenance, growth, development, and health and which must be provided in the diet to meet requirements. In contrast, nutritionally non-essential AA (NEAA) are those AA which can be synthesized de novo in adequate amounts by the animal organism to meet requirements for maintenance, growth, development, and health and, therefore, need not be provided in the diet. Although EAA and NEAA had been described for over a century, there are no compelling data to substantiate the assumption that NEAA are synthesized sufficiently in animals and humans to meet the needs for maximal growth and optimal health. NEAA play important roles in regulating gene expression, cell signaling pathways, digestion and absorption of dietary nutrients, DNA and protein synthesis, proteolysis, metabolism of glucose and lipids, endocrine status, men and women fertility, acid–base balance, antioxidative responses, detoxification of xenobiotics and endogenous metabolites, neurotransmission, and immunity. Emerging evidence indicates dietary essentiality of “nutritionally non-essential amino acids” for animals and humans to achieve their full genetic potential for growth, development, reproduction, lactation, and resistance to metabolic and infectious diseases. This concept represents a new paradigm shift in protein nutrition to guide the feeding of mammals (including livestock), poultry, and fish.     

Identification of genes showing altered expression in preimplantation and early postimplantation parthenogenetic embryos

Uniparental embryos have been instrumental in studying imprinting because contributions from the parental genomes can be determined unambiguously. In this study, we set out to identify imprinted genes showing differential expression between parthenogenetic and fertilized embryos during preimplantation and early postimplantation stages of development. We identified three genes-apolipoprotein E, pyruvate kinase-3, and protein phosphatase 1 gamma-that represent excellent candidates for imprinted genes, based on the results of the differential screen, their function in differentiation and the cell cycle, and their location within imprinted chromosomal regions. In addition, two novel genes expressed in trophoblast were identified, 1661 and RA81. These genes, together with four known imprinted genes, H19, Igf2r, Igf2, and Snrpn, showed evidence of expression from both parental alleles in early stage embryos, indicating a role for postfertilization processes in regulating imprinted gene function. © 1995 Wiley-Liss, Inc.     

Asb4, Ata3, and Dcn are novel imprinted genes identified by high-throughput screening using RIKEN cDNA microarray

Genes differentially expressed between parthenogenetic and androgenetic embryos are candidates for the identification of imprinted genes, which are expressed specifically from the maternal or paternal allele. To search for genes differentially expressed between parthenogenetic and androgenetic embryos, we used the RIKEN full-length enriched mouse cDNA microarray. The 25 candidates obtained included 8 known imprinted genes (such as IgfII, Snrpn, and Neuronatin) and 3 new ones--Asb4 (ankyrin repeat and SOCS box-containing protein 4), Ata3 (amino acid transport system A3), and Decorin--which were confirmed by using normal diploid embryos from the reciprocal F1 crosses of B6 and JF1 mice. The 25 candidates also included genes that showed no imprinting-associated expression in normal diploid embryos. We describe a feasible high-throughput method of screening for novel imprinted genes by using the RIKEN cDNA microarray.     

Expression of imprinted <Emphasis Type="Italic">Igf2</Emphasis> and <Emphasis Type="Italic">Peg1/Mest</Emphasis> genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor α in vitro

The effect of transforming growth factor α (TGFα) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA × C57BL/6)F₁ mice during the postimplantation period of embryogenesis. The PEs were treated with TGFα in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf₂and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf₂ before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFα) PEs, neither gene was expressed at the same 21-to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30-to 35-somite stages, while the Peg1/Mes t expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30-to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFα-treated PEs at the 40-to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40–45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFα can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.