Luca Magnani

In vitro and in vivo derived porcine embryos possess similar, but not identical, patterns of Oct4, Nanog, and Sox2 mRNA expression during cleavage development

In vitro culture conditions stress the cleavage stage mammalian embryo and can contribute to reduced developmental potential of cultured embryos. One process that may be altered during embryo culture is the establishment and maintenance of pluripotency. Pluripotency is largely controlled by three genes: Oct4, Nanog, and Sox2. The objective of this study was to determine the expression pattern of Oct4, Nanog, and Sox2 in cleavage stage porcine embryos obtained in vivo or by in vitro fertilization and parthenogenetic activation. We used quantitative, real time PCR to assess the relative amount of each transcript in cleavage stage embryos. We found that Oct4 was transiently activated at the 2‐cell stage (P‐value <0.05) while Nanog and Sox2 were activated at the 4‐cell stage (P‐value <0.05) in in vitro embryos. Embryos derived in vivo showed a similar but not identical pattern of expression of Nanog mRNA been in highest abundance both at the 4 cell and the blastocyst stage. The activation observed at the 4‐cell stage for Nanog and Sox2 was shown to be RNA polymerase II dependent (P‐value <0.05). This study showed that Oct4, Nanog, and Sox2 possess similar, but not identical, patterns of expression between in vitro and in vivo derived porcine embryos. The difference between the amount of transcripts may reflect the reduced developmental potential observed in in vitro cultured embryos. Mol. Reprod. Dev. 75: 1726–1735, 2008.

Differential Remodeling of Mono- and Trimethylated H3K27 During Porcine Embryo Development

Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage porcine embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2‐cell, 4‐cell, 8‐cell, and blastocyst stage embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage embryos produced by fertilization; the signals were faint or absent in nuclei of two‐cell through blastocyst stage embryos. In addition, EED transcripts were increased from the four‐cell stage (P < 0.05) in embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV‐stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four‐cell stage (P < 0.05) in parthenogenetic and in vivo derived embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during porcine embryogenesis. Mol. Reprod. Dev. 76: 1033–1042, 2009.

KPNA7, an oocyte- and embryo-specific karyopherin ? subtype, is required for porcine embryo development

Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin α/β heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherinαsubtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherinαsubtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherinα7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherinαsubtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherinαsubtypes and that these karyopherinαsubtypes differentially transport intracellular cargo during cleavage development.

Gene expression and development of early pig embryos produced by serial nuclear transfer.

During nuclear transfer, reprogramming makes the donor nucleus capable of directing development of the reconstructed embryo. In most cases reprogramming is incomplete, which leads to abnormal expression of early embryonic genes and subsequently, to reduced developmental potential. In the present study, we monitored the expression of Oct4, Nanog, and Sox2 in cloned porcine embryos and evaluated whether serial nuclear transfer, the transfer of nuclei of cloned embryos into enucleated oocytes, has the potential to provide a more complete reprogramming of the donor genome. The data suggested that Nanog and Sox2 expression is properly reactivated after nuclear transfer, but the relative abundance of Oct4 transcripts is abnormally low in cloned porcine blastocysts compared to control embryos produced by in vitro fertilization. When the nuclei of 8‐ to 16‐cell stage cloned embryos were introduced into enucleated oocytes to expose the chromosomes repeatedly to the ooplasmic factors, the resulting embryos showed poor developmental potential: a significantly lower percentage of embryos developed to the 4‐cell (12.0% vs. 31.8%), 8‐cell (3.1% vs. 15.0%) and blastocyst (0% vs. 8.7%) stages compared to those produced following a single round of nuclear transfer (P < 0.05). The additional time for reprogramming also did not improve gene expression. By the late 4‐cell stage, Oct4 and Sox2 expression levels were low in serial nuclear transfer embryos compared to those in embryos generated by in vitro fertilization or nuclear transfer. Overall, both developmental and gene expression data indicated that reprogramming of the donor nucleus could not be improved by serial nuclear transfer in the pig. Mol. Reprod. Dev. 76: 555–563, 2009.

Manipulation of SMARCA2 and SMARCA4 transcript levels in porcine embryos differentially alters development and expression of SMARCA1, SOX2, NANOG, and EIF1.

Epigenetic reprogramming plays a pivotal role during embryogenesis, including both covalent and non-covalent modifications to chromatin. In this study, we investigated the role of SNF2 chromatin remodeling ATPases (SMARCA2 (previously known as BRAHMA), SMARCA4 (previously known as BRG1), SMARCA5 (previously known as SNF2H), SMARCA1 (previously known as SNF2L), CHD3, and CHD5) during porcine preimplantation embryonic development. Transcript levels for these ATPases change dynamically throughout development. We also investigated the effect of altering transcript levels of SMARCA2 and SMARCA4 via mRNA injection. Overexpression of SMARCA2 and SMARCA4 severely impaired embryo development. Results from these experiments show that embryos injected with SMARCA2 mRNA arrest between the four-cell and blastocyst stages. However, embryos injected with either wild-type SMARCA4 or a dominant negative variant or SMARCA4 arrest before zygotic genome activation. No differences in transcript abundance of SOX2, POU5F1, NANOG, and EIF1 (previously known as eIF1A) were detected after injection with SMARCA2 or its dominant negative variant at 48 h post-injection. Conversely, embryos injected with wild-type SMARCA4 and its dominant negative variant possessed altered expression of these genes. Examination of SNF2-type ATPase transcript abundance across all treatment groups revealed that only SMARCA1 was altered following injection with wild-type SMARCA2 and wild-type and dominant negative SMARCA4. We conclude that the arrest in porcine embryo development observed after injection is specific to the ATPase injected. Our data strongly support the hypothesis that SMARCA2 and SMARCA4 play different but fundamental roles controlling gene expression during early mammalian embryogenesis.

Expression of eukaryotic elongation initiation factor 1A differentially marks zygotic genome activation in biparental and parthenogenetic porcine embryos and correlates with in vitro developmental potential

Zygotic genome activation (ZGA) is a major event during cleavage development. In vitro manipulation of mammalian embryos (including embryo culture) can result in developmental arrest around the time of ZGA. Eukaryotic elongation initiation factor 1A (eIF1A) has been used as a marker for ZGA in some mammalian species. We hypothesised expression of eIF1A can be used to assess ZGA in the pig; we also hypothesised that the expression profile of eIF1A can be used to assess developmental potential in vitro. The aims of the present study were to determine the expression pattern of eIF1A during porcine cleavage development and to assess its expression levels in embryos of different quality. We used a real-time reverse transcription-polymerase chain reaction assay to quantify eIF1A transcripts at different time points during cleavage development in porcine embryos produced by parthenogenetic activation (PA) and in vitro fertilisation (IVF). We found that eIF1A is activated at the two-cell stage in IVF embryos and at the four-cell stage in PA embryos. We showed that the increase in transcript levels observed in parthenogenetic embryos is dependent on de novo transcription. We found altered levels of eIF1A transcripts in parthenogenetic embryos that presented as either two- or eight-cell embryos 48 h after activation compared with four-cell embryos at the same time point. Our work supports the hypothesis that eIF1A is a marker of porcine ZGA and its expression profile can be used to assess embryo quality.

Global H3K9 dimethylation status is not affected by transcription, translation, or DNA replication in porcine zygotes.

Methylation of the lysine 9 residue of histone H3 (H3K9) is linked to transcriptional repression. The observed structure of chromatin in porcine and murine embryos is different with regard to H3K9 dimethylation status, leading to our hypothesis that the intracellular mechanisms responsible for H3K9 methylation would also differ between these two species. The objectives of this study were: (1) to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes, (2) determine the extent to which the intracellular localization of individual pronuclei correlated with H3K9 dimethylation status, and (3) to determine the abundance of transcripts encoding the histone methyltransferases, with H3K9 methylation activity, in porcine oocytes and embryos. Our findings are that (1) H3K9 dimethylation status is not affected by DNA replication, transcription, or protein synthesis, (2) the location of a pronucleus does not significantly affect the H3K9 dimethylation status of the chromatin within that pronucleus, and (3) the histone methyltransferases with activity for H3K9 differ in transcript abundance in porcine oocytes and cleavage stage embyros. These results support our hypothesis that there is a difference in intracellular mechanisms affecting dimethylation status of H3K9 between porcine and murine embryos. Mol. Reprod. Dev. 77: 420–429, 2010.

Developmental capacity of porcine nuclear transfer embryos correlate with levels of chromatin‐remodeling transcripts in donor cells

Somatic cell nuclear transfer (SCNT) still retains important limitations. Impaired epigenetic reprogramming is considered responsible for altered gene expression and developmental failure in SCNT‐derived embryos. After nuclear transfer the donor cell nucleus undergoes extensive changes in gene expression that involve epigenetic modifications and chromatin remodeling. We hypothesized that SNF2‐type ATP‐dependent chromatin factors contribute to epigenetic reprogramming and the relative amount of these factors in the donor cell affects developmental potential of the reconstructed embryos. In order to test this hypothesis, we assessed the relative amount of SNF2‐type ATPases (Brahma, Brg1, SNF2H, SNF2L, CHD3, and CHD5) in three different donor cells as well as in porcine metaphase II oocytes. We performed SCNT with fetal fibroblast cells, olfactory bulb (OB) progenitor cells, and porcine skin originating sphere stem cells (PSOS). We found that OB‐NT embryos and PSOS‐NT embryos resulted in a higher morulae/blastocysts ratio as compared to fibroblast‐NT embryos (23.53%, 16.98%, and 11.63%, respectively; P < 0.05). Fibroblast cells contained a significantly higher amount of SNF2L and CHD3 transcripts while Brg1 and SNF2H were the most expressed transcripts in all the cell lines analyzed. Metaphase II oocyte expression profile appeared to be unique compared to the cell lines analyzed. This work supports our hypothesis that an array of chromatin‐remodeling proteins on donor cells may influence the chromatin structure, effect epigenetic reprogramming, and developmental potential. Mol. Reprod. Dev. 75: 766–776, 2008.