Major Genome Activation Research Articles

Single-cell RNA sequencing reveals developmental heterogeneity of blastomeres during major genome activation in bovine embryos

Embryonic development is initially controlled by maternal RNAs and proteins stored in the oocyte, until gene products gradually generated by the embryo itself take over. Major embryonic genome activation (EGA) in bovine embryos occurs at the eight- to 16-cell stage. Morphological observations, such as size of blastomeres and distribution of microvilli, suggested heterogeneity among individual cells already at this developmental stage. To address cell heterogeneity on the transcriptome level, we performed single-cell RNA sequencing of 161 blastomeres from 14 in vitro produced bovine embryos at Day 2 (n = 6) and Day 3 (n = 8) post fertilization. Complementary DNA libraries were prepared using the Single-Cell RNA-Barcoding and Sequencing protocol and sequenced. Non-supervised clustering of single-cell transcriptome profiles identified six clusters with specific sets of genes. Most embryos were comprised of cells from at least two different clusters. Sorting cells according to their transcriptome profiles resulted in a non-branched pseudo-time line, arguing against major lineage inclination events at this developmental stage. In summary, our study revealed heterogeneity of transcriptome profiles among single cells in bovine Day 2 and Day 3 embryos, suggesting asynchronous blastomere development during the phase of major EGA.

69 BLASTOCYSTS DEVELOPED FROM EMBRYOS THAT SPENT UP TO 2-CELL STAGE IN VIVO EXHIBITED MASSIVE DNA METHYLATION DYSREGULATION INCLUDING IMPRINTED GENES AND DNA METHYLTRANSFERASES

Suboptimal culture condition before minor or major genome activation is believed to affect the quality and the transcriptome landscape of the resulting blastocysts. Thus, we hypothesised that exposure of bovine embryos to suboptimal culture condition before minor embryonic genome activation could affect the genome methylation patterns of the resulting blastocysts. Therefore, here we aimed to investigate the genome wide DNA methylation patterns of blastocysts derived from embryos developed up to 2-cell stages in vivo followed by in vitro culture. For this, Simmental heifers were superovulated and artificially inseminated. The 2-cell stage embryos were then flushed using a state-of-the-art nonsurgical endoscopic early-stage embryo flushing technique and in vitro cultured until the blastocyst stage. The DNA methylation patterns of these blastocysts were then determined with reference to blastocysts derived from embryos developed completely under in vivo condition. For this, the genomic DNA isolated from each blastocyst group were fragmented, and unmethylated genomic regions were cleaved using methylation sensitive restriction enzymes. The samples were then amplified using ligation mediated PCR and labelled either with Cy-3 or Cy-5 dyes in a dye-swap design using the ULS Fluorescent genomic DNA labelling kit (Kreatech Biotechnology) and hybridized on an EmbryoGENE DNA Methylation Array as described previously (Saadi 2014 BMC Genomics 15, 451; Salilew-Wondim 2015 PLoS ONE 10, e0140467). Array hybridization was performed for 40 h at 65°C, and 4 hybridizations were preformed to represent 4 biological replicates. The slides were scanned using Agilent’s High-Resolution C Scanner (Agilent Technologies, Santa Clara, CA, USA), and Agilent’s Feature Extraction software (Agilent Technologies) was used to extract data features. Differentially methylated regions with fold change ≥1.5 and P-value < 0.05 were identified using linear modelling for microarray and R software. The results have shown that including imprinted genes (PEG3, IGF1, RASGRF1, IGF2R, GRB10, SNRPN, and PLAGL1) and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), a total of 10,388 genomic regions were differentially methylated, of which 6393 genomic regions were hypermethylated in blastocysts derived from 2-cell flush compared with the complete vivo group. In addition, comparative analysis of the current DNA methylation data with our previous transcriptome profile data have shown that including DNMT3A, CTSZ, ElF3E, and PPP2R2B, the expression patterns of 603 genes was inversely correlated with the methylation patterns. Moreover, canonical pathways including gap junction, adherens junction, axon guidance, focal adhesion, and calcium signalling were affected by differentially methylated regions. Therefore, this study indicated that exposure of embryos to suboptimal culture condition before embryonic genome activation would lead to a massive dysregulation of methylation pattern of genes involved in developmentally relevant pathways in the resulting blastocysts.

Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development.

Utilizing 3D structured illumination microscopy, we investigated the quality and quantity of nuclear invaginations and the distribution of nuclear pores during rabbit early embryonic development and identified the exact time point of nucleoporin 153 (NUP153) association with chromatin during mitosis. Contrary to bovine early embryonic nuclei, featuring almost exclusively nuclear invaginations containing a small volume of cytoplasm, nuclei in rabbit early embryonic stages show additionally numerous invaginations containing a large volume of cytoplasm. Small-volume invaginations frequently emanated from large-volume nuclear invaginations but not vice versa, indicating a different underlying mechanism. Large- and small-volume nuclear envelope invaginations required the presence of chromatin, as they were restricted to chromatin-positive areas. The chromatin-free contact areas between nucleolar precursor bodies (NPBs) and large-volume invaginations were free of nuclear pores. Small-volume invaginations were not in contact with NPBs. The number of invaginations and isolated intranuclear vesicles per nucleus peaked at the 4-cell stage. At this stage, the nuclear surface showed highly concentrated clusters of nuclear pores surrounded by areas free of nuclear pores. Isolated intranuclear lamina vesicles were usually NUP153 negative. Cytoplasmic, randomly distributed NUP153-positive clusters were highly abundant at the zygote stage and decreased in number until they were almost absent at the 8-cell stage and later. These large NUP153 clusters may represent a maternally provided NUP153 deposit, but they were not visible as clusters during mitosis. Major genome activation at the 8- to 16-cell stage may mark the switch from a necessity for a deposit to on-demand production. NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina. To our knowledge, the present study demonstrates for the first time major remodeling of the nuclear envelope and its underlying lamina during rabbit preimplantation development.

82 STRUCTURAL REMODELLING OF THE NUCLEAR ENVELOPE IN BOVINE PRE-IMPLANTATION EMBRYOS

In the present study, we investigated the changes of the nuclear envelope and its underlying lamina, as well as features of higher order chromatin organisation in bovine embryos generated by in vitro fertilization during pre-implantation development. We used super-resolution, 3-dimensional structured illumination microscopy combined with 2-colour immunostaining of the nucleoporin Nup153 and lamin B serving as markers for nuclear pore complexes (NPC) and the nuclear lamina, respectively. DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). We examined 20 nuclei for the zygote (10 male pronuclei and 10 female pronuclei; n = 10) and the blastocyst (10 trophectoderm and 10 inner cell mass nuclei; n = 1) stage, and 10 nuclei for each the 2-cell (n = 5), 4-cell (n = 3), 8-cell (n = 2), 19-cell (n = 1), and morula (n = 1) stages. We report 4 major findings: (1) At the onset of major genome activation (MGA) nuclei showed a peripheral location of chromosome territories (CT), separated by wide IC channels and surrounding a major lacuna depleted of chromatin. The NPC were exclusively present at sites where DAPI-stained DNA contacted the nuclear lamina, whereas extended lamina regions without such contacts lacked NPC. In post-MGA nuclei, the CT formed a higher order chromatin network distributed throughout the entire nuclear space and the major lacuna disappeared. In line with a switch to a ubiquitous lining of DNA at the lamina, NPC were also uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions that control the integration of NPC into the nuclear envelope. (2) The switch from maternal to embryonic production of mRNA was accompanied by an increased amount of nuclear lamina invaginations covered with NPC, which may serve the increased demands of mRNA export and protein import. (3) Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Because an increase in these lamin B positive structures occurred in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size throughout bovine pre-implantation development. (4) Throughout the cytoplasm, randomly distributed extranuclear clusters of Nup153 without associated lamin B were frequently observed from the zygote stage up to MGA. These clusters may represent a deposit of maternal Nup153 and likely other nucleoporines not studied here. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced RNA before MGA, which increased strongly at the initiation of embryonic NUP153 expression at MGA. After MGA, these clusters were exclusively located at or near the nuclear border and were no longer present at the morula stage and later. In conclusion, our findings demonstrate the dynamic adaptation of the nuclear envelope to the special needs of bovine pre-implantation development and show the necessity of chromatin association for the integration of nuclear pores into the nuclear envelope.

Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos

Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.

54 EXPRESSION PATTERN OF DNMT1 AND DNMT3a GENES DURING INTERGENERIC SOMATIC CELL NUCLEAR TRANSFER EMBRYO DEVELOPMENT

One of the most-discussed reasons for developmental incompetence of embryos constructed by the cloning procedure is inadequate reprogramming of the transferred nucleus to a state equivalent to that of an early embryonic nucleus. Previous studies have shown species-dependent expression patterns of DNA methyltransferase (DNMT) genes in mammalian oocytes and preimplantation embryos, and also a correlation between incomplete DNA methylation and the lack of NT success in mammals. In the present study, the expression pattern of DNMT1 and DNTM3a genes at the 2-cell and 4-cell stages of bovine versus porcine intergeneric nuclear transfer (iSCNT) embryos was observed by reverse-transcriptase (RT) PCR. All pools were done in triplicate and contained 10 iSCNT embryos. The species-specific primers for DNMT1 and DNMT3a genes were designed for determination of de novo synthesis of epigenetic enzymes. As positive controls, porcine and bovine parthenogenetic embryos were used. Gene transcription for bovine DNMT1 (bDNMT1) and DNMT3a (bDNMT3a) was not observed in 2- and 4-cell stage embryos generated by bovine fibroblast transfer into the porcine ooplasm; however, using primers for pig DNMT1 (pDNMT1) and DNMT3a (pDNMT3a), positive results were obtained. In the 2- and 4-cell-stage embryos constructed using porcine fibroblast and bovine ooplasm, only the bovine-specific primers showed positive signals. Based on the different timing of major genome activation during embryonic development in bovine and porcine embryos, the strong influence of ooplasm on introduced fibroblast was expected. Despite the mRNA presence of DNMT1 and DNMT3a enzymes of oocyte origin, de novo transcription of somatic DNMT1 and DNMT3a genes was not detected and iSCNT embryos did not develop beyond the 4-cell stage. These results strongly suggest species-specific and maternally driven regulation of epigenetic reprogramming during early embryogenesis. This work was supported by VEGA 1/0077/11.

254 REGULATORY microRNA IN THE BIDIRECTIONAL COMMUNICATION OF BOVINE OOCYTES AND THE SURROUNDING CUMULUS CELLS

MicroRNA (miRNA) are small molecules (˜22 nucleotide in length) that influence the expression of hundreds of genes for numerous biological processes including development. In this study we aimed to investigate the presence and role of miRNA in the bidirectional communication of oocyte and cumulus cells. For this, triplicate pools each containing 1600 immature and mature oocytes and their corresponding cumulus cells were used for miRNA isolation using miRNeasy® Mini Kit (Qiagen, Hilden, Germany). From each oocyte and cumulus cell group, 50 ng of small RNA was used for reverse transcription using RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD, USA). The resulting small RNA cDNA was used as a template to profile 88 human miRNA related to cell development and differentiation using SYBR green-based real-time PCR system. Data analysis was preformed using the comparative Ct method after normalization using endogenous control RNA (SNORD44, SNORD47, SNORD48, and U6). In addition, miR-205 and miR-210 were used for localization in pre-implantation stages of embryo using 3′digoxigenin labeled, LNA- modified in situ oligonucleotide probes (Exiqon, Vedbaek, Denmark). The result of the PCR array revealed a total of 34 and 49 miRNA to be greatly abundant in immature and mature oocyte, respectively, compared with the corresponding cumulus cells, whereas only 5 and 4 miRNA were enriched in cumulus cells compared with immature and matured oocytes, respectively. Based on expression intensity, 6 oocyte enriched (miR-205, miR-150, miR-96, miR-122, miR-146a, and miR-146b-5p) and 2 cumulus-cell enriched (miR-452 and miR-210) were selected for expression analysis in pre-implantation-stage embryos and in oocyte and cumulus cells matured with or without cumulus and oocyte factors, respectively. All oocyte-specific miRNA were found to be greatly abundant in early stages of embryo development and drop after 4-cells until the blastocyst stage, following a typical maternal transcript profile. Similar results were obtained by localization of miR-205 in pre-implantation-stage embryos, in which signals were greater until the 4-cell stage and reduced thereafter. However, miR-210 and miR-452 showed no defined profile. miR-205, miR-150, miR-122, miR-146a, miR-146b-5p, and miR-452 were found to be abundant at a greater level (P < 0.05) in oocytes matured without cumulus cells compared with those matured in the presence of cumulus cells. The expression of miR-205, miR-150, and miR-122 in cumulus cells was greater in the presence of oocyte cytoplasm during maturation, whereas 16-fold increases in relative abundance of miR-210 were observed in oocyte- optimized cumulus cells. These results evidenced that oocyte and cumulus cells have a distinct set of miRNA, which is dependent on the bidirectional communication of the oocyte and the surrounding cumulus cells. Moreover, maternal miRNA were found to persist until the major genome activation in bovine.

127 REGULATION OF H3K27me3 AND H3K4me3 DURING INITIAL PORCINE EMBRYONIC DEVELOPMENT

During mammalian development, gene expression is partly regulated by posttranslational modifications of histones. In particular, H3K27me3 and H3K4me3 are involved in transcriptional repression and activation, respectively. In this study, we examined the global levels of H3K27me3 and H3K4me3, as well as the expression levels of their specific methylases and demethylases during porcine pre-implantation embryonic development. Global histone methylation was analyzed by immunocytochemical quantification within in vivo porcine embryos at 1-cell (Day 1), 4-cell (Day 3), morula (Day 5), and late blastocyst (containing the epiblast; Day 9). The numbers of embryos analyzed for H3K27me3 at the 1-cell, 4-cell, morula, and late blastocyst stage were 7, 8, 6, and 5, respectively, and for H3K4me3 at these four stages were 7, 6, 6, and 5, respectively. At the same developmental stages, mRNA expression of methylases (EZH2, EED, and SUZ12, three core components of PRC2) and demethylases (JMJD3 and UTX) of H3K27me3 was performed on pooled embryos (n = 10), as well as expression of methylases (MLL1 and ASH1L) and demethylase (RBP2) of H3K4me3, by comparative RT-PCR. Expression was compared with pooled embryos from the limb bud stage (Day 21). GAPDH was used as the reference gene, and expression was normalized to Day 21 embryos. Our results show that the levels of global histone methylation of H3K27me3 and H3K4me3 decrease gradually from 1-cell to morula, but both were increased in late blastocysts. The levels of H3K27me3 methylase (EZH2, EED, and SUZ12) transcripts increased from 1-cell to late blastocyst stage. Low expression of the H3K27me3 demethylase JMJD3 was found at 1-cell stage and high expression at the 4-cell stage from when it decreased gradually to the late blastocyst. UTX expression was low but peaked at the 4-cell stage. Expression of H3K4me3 methylase MLL1, was low, whereas ASH1L expression was high at the 4-cell stage. RBP2, a demethylase of H3K4me3, was highly expressed at the late blastocyst stage. In conclusion, at the major genome activation (the 4-cell stage), H3K27me3 and H3K4me3 have decreased to moderate levels, which apparently balance each other with respect to gene repression and activation allowing for genome activation. At the 4-cell stage the activation of H3K4me3 is favored as a consequence of low levels of H3K27 methylases and high levels of H3K27 demethylases combined with high levels of H3K4 methylases and low levels of H3K4 demethylases. Interestingly, at the late blastocyst stage of development, high expression of H3K27me3 methylases and the H3K4me3 demethylase, RBP2, are observed, indicating repression of gene expression, which is counterintuitive to accelerating development. We speculate other factors, such as microRNA or other kinds of epigenetic mechanisms, might play a critical role at this developmental stage. Thus, further research is required to explain these phenomena occurred during early porcine development.

From Zygote to Implantation: Morphological and Molecular Dynamics during Embryo Development in the Pig

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.

Changes of higher order chromatin arrangements during major genome activation in bovine preimplantation embryos

Gene-dense chromosome territories (CTs) are typically located more interior, gene-poor CTs more peripheral in mammalian cell nuclei. Here, we show that this gene-density correlated CT positioning holds for the most gene-rich and gene-poor bovine chromosomes 19 and 20, respectively, in bovine fibroblast and lymphocyte nuclei. In order to determine the period at which this non-random CT order is established during development, we performed fluorescence in situ hybridization, on three-dimensionally preserved bovine preimplantation embryos generated by in vitro fertilization and investigated the distribution of BTA 19 and 20 CTs. Radial arrangements of CTs 19 and 20 were the same up to the 8-cell stage. At the 10- to 16-cell stage, however, a significant difference became apparent with CTs 19 localized more internally and CTs 20 more peripherally. Since major genome activation in bovine embryos occurs at the 8- to 16-cell stage, our findings demonstrate a temporal correlation between transcriptional activation and a major rearrangement of chromatin topography in blastomere nuclei.

37 REPROGRAMMING OF THE HIGHER ORDER CHROMATIN ORGANIZATION IN BOVINE NUCLEAR TRANSFER EMBRYOS

Reprogramming of nuclei after somatic cell nuclear transfer is a crucial process to reset the expression profile of the donor cell to that of a developing embryo. Reprogramming affects the epigenome of the donor nucleus, which is the complete set of epigenetic determinants that establishes and maintains a given transcriptome. These epigenetic determinants include e.g. DNA methylation, modifications of histone tails, histone variants, and other chromatin components. A less well characterized level of epigenetic gene regulation is the spatial higher order chromatin organization. In somatic cells chromosomes are organized as individual entities called chromosome territories (CTs), which localize according to their gene density with gene-rich chromosomes being in the nuclear interior, gene-poor ones at the nuclear periphery. We have shown previously that this gene density related distribution is established during major genome activation (MGA) in bovine in vitro fertilized (IVF) embryos, while embryos up to the 8-cell stage do not show a difference in their CT localization. In the present study, we investigated whether the gene density related distribution of chromosomes present in bovine fibroblasts is reprogrammed after nuclear transfer (NT), i.e. erased to restore the situation found in IVF embryos. Moreover, we analyzed if a gene density related CT arrangement is re-established at the same time point as in IVF embryos, i.e. during MGA. Therefore we analyzed the nuclear distribution of bovine chromosome 19 (19 genes Mb–1) and 20 (5 genes Mb–1) in one-, 2- and 4-cell NT embryos as pre-MGA stages and in embryos with 10–20 nuclei cells–1, representing embryos during MGA. CTs were visualized via a recently developed protocol utilizing fluorescence in situ hybridization (FISH) on three dimensionally preserved embryos. In 1- and 2-cell embryos we could not detect a gene density related CT distribution arguing for a rapid reprogramming, i.e. erasure of the positional information previously present in the donor nuclei. Surprisingly, we observed a precocious establishment of a gene density related distribution already at the 4-cell stage that became more pronounced in 10–20-cell embryos, when this specific CT distribution is established in IVF embryos. We conclude that the spatial arrangement of chromosomes is an epigenetic parameter that is rapidly reprogrammed upon nuclear transfer of a somatic nucleus, exposed to the oocyte cytoplasm. Moreover, the gene density related positioning of chromosomes in bovine NT embryos is re-established earlier than in IVF embryos, suggesting a possible involvement in early developmental failures of NT embryos. Funded by the Deutsche Forschungsgemeinschaft (ZA 425/1-2, CR 59/26-1).

24 EFFECTS OF HISTONE METHYLATION RELATED GENES ON EPIGENETIC REPROGRAMMING AND ZYGOTIC GENE ACTIVATION IN OVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

After fertilization, early embryo development is dependent upon maternally inherited proteins and protein synthesised from maternal mRNA until zygotic gene activation (ZGA) occurs. The transition of transcriptional activity from maternal to embryonic control occurs with the activation of rRNA genes and the formation of the nucleolus at the 8- to 16-cell stage that coincides with a prolonged fourth cell cycle in bovine and ovine embryos. However, previous studies have reported a shift in the longest cell cycle (fifth cell cycle) in bovine somatic cell nuclear transfer (SCNT) embryos, suggesting that the major genome activation is delayed, possibly due to incomplete changes in chromatin structure such as hypermethylation and hypoacetylation of histone (Memili and First 2000 Zygote 8, 87–96; Holm et al. 2003 Cloning Stem Cells 5, 133–142). Although global gene expression profile studies have been carried out in somatic cell nuclear transfer embryos, little is known about the expression of genes which can alter chromatin structure in early embryo development and possibly effect ZGA. To determine whether epigenetic reprogramming of donor nuclei affected ZGA and expression profiles in SCNT embryos, ZBTB33 (zinc finger and BTB domain containing 33, also known as kaiso, a methy-CpG specific repressor), BRG1(brahma-related gene 1, SWI/SNF family of the ATP-dependent chromatin remodeling complexes), JMJD1A (jumonji domain containing 1A, H3K9me2/1-specific demethylase), JMJD1C (putative H3K9-specific demethylase), and JMJD2C (H3K9me3-specific demethylase) were examined by RT-PCR at different developmental stages [germinal vesicle (GV), metaphase II (MII), 8- to 16-cell, 16- to 32-cell, and blastocyst in both parthenogenetic and SCNT embryos]. All genes were detected in parthenogenetic and SCNT blastocyts, and ZBTB33 was also expressed in all embryos at all stages tested. However, the onset of expression of JMJD1C, containing POU5F1 binding site at 5′-promoter region and BRG1 required for ZGA are delayed in SCNT embryos as compared to parthenotes (16- v. 8-cell, and blastoocyst v. 16-cell stage). Furthermore, JMJD2C containing NANOG binding sites at the 3′-flanking region was expressed in GV and MII oocytes and parthenogenetic blastocysts, whereas in SCNT embryos, JMJD2C was only observed from the 16-cell stage onwards. Interestingly, JMJD1A, which is positively regulated by POU5F1, was not detected in GV and MII oocytes but was present in blastocyst stage embryos of both groups. Taken together, these results suggest that incomplete epigenetic modifications of genomic DNA and histones lead to a delayed onset of ZGA which may affect further development and establishment of totipotency. Subsequently, aberrant expression patterns reported previously in SCNT embryos may be attributed to improper expression of histone H3K9 and H3K4 demethylase genes during early embryo development.

57 GENOME ACTIVATION IN MOUSE EMBRYOS OF DIFFERENT ORIGIN

Major genome activation is a key event in early embryonic development occurring at the late 2-cell stage in the mouse. Concomitantly occurring molecular and ultrastructural changes in the nucleolus, where the ribosomal RNA genes are transcribed and their transcripts processed, enable the use of this organelle as a sensitive marker of genome activation in embryos produced by different techniques. The aim of this study was to evaluate and compare the genome activation in mouse embryos of different origin using the nucleolus as a marker. Early and late 2-cell- and late 4-cell-stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and somatic cell nuclear transfer of mouse embryonic fibroblast (MEF), and mouse HM1 embryonic stem cells (HM1) were processed for autoradiography following 3H-uridine incubation and transmission electron microscopy (5 embryos per group) and for immunofluorescence for detection of nucleolar proteins involved in rRNA synthesis (upstream binding factor; UBF) and processing (nucleophosmin; B23) (10–21 embryos per group). Early 2-cell embryos in all groups showed transcriptional activity in the nucleoplasm, but not over nucleolar precursor bodies (NPBs). UBF was localized diffusely in the cytoplasm. B23 was, likewise, localized in the cytoplasm and, in 30% of embryos, in the nucleoplasm. Late 2-cell IVF and PG embryos displayed transcriptional labelling over nucleoplasm and NPBs, which, ultrastructurally, were in the process of transformation into fibrillo-granular nucleoli presenting fibrillar centers, a dense fibrillar component, and a granular component. MEF and HM1 embryos displayed transcriptional labelling over nucleoplasm, but not over NPBs, and the transformation into functional nucleoli was never observed at this stage of development. UBF and B23 were in all groups localized in the nucleoplasm and, in 40–50% of cases, distinctly in the developing nucleoli. At the late 4-cell stage, all embryos presented transcriptional labelling over nucleoplasm and NPBs, which were at different levels of transformation into fibrillo-granular nucleoli. UBF and B23 were distinctly localized in these developing nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli without remnants of NPBs were found in IVF and PG embryos, despite the distinct localization of nucleolar proteins, the nucleoli in MEF and HM1 embryos were not reticulated and still displayed remnants of NPBs. Conclusively, embryos reconstructed by nuclear transfer, independent of cell origin, displayed well-timed extranucleolar genomic activation, but delayed transformation of NPBs into reticulated fibrillo-granular nucleoli. Moreover, the proper nucleolar activation noted in PG embryos activated in the same manner as MEF and HM1 embryos demonstrate that somatic and embryonic stem cell factors exert an influence on nucleolar activation and may cause reduced embryo viability. This work was supported by the Specific Targeted Project (MED-RAT; contract LSHG-CT-2006-518240) and Marie Curie ResearchTraining Networks (CLONET; contract 035468-2).

Analysis of polysomal mRNA populations of mouse oocytes and zygotes: Dynamic changes in maternal mRNA utilization and function

Transcriptional activation in mammalian embryos occurs in a stepwise manner. In mice, it begins at the late one-cell stage, followed by a minor wave of activation at the early two-cell stage, and then the major genome activation event (MGA) at the late two-cell stage. Cellular homeostasis, metabolism, cell cycle, and developmental events are orchestrated before MGA by time-dependent changes in the array of maternal transcripts being translated. Many elegant studies have documented the importance of maternal mRNA (MmRNA) and its correct recruitment for development. Many other studies have illuminated some of the molecular mechanisms regulating MmRNA utilization. However, neither the complete array of recruited mRNAs nor the regulatory mechanisms responsible for temporally different patterns of recruitment have been well characterized. We present a comprehensive analysis of changes in the maternal component of the zygotic polysomal mRNA population during the transition from oocyte to late one-cell stage embryo. We observe global transitions in the functional classes of translated MmRNAs and apparent changes in the underlying cis-regulatory mechanisms.

Expression of genes encoding chromatin regulatory factors in developing rhesus monkey oocytes and preimplantation stage embryos: possible roles in genome activation.

One of the most critical events of preimplantation development is the successful activation of gene transcription. Both the timing and the array of genes activated must be controlled. The ability to regulate gene transcription appears to be reduced just prior to the time of the major genome activation event, and changes in chromatin structure appear essential for establishing this ability. Major molecules that modulate chromatin structure are the linker and core histones, enzymes that modify histones, and a wide variety of other factors that associate with DNA and mediate either repressive or activating changes. Among the latter are chromatin accessibility complexes, SWI/SNF complexes, and the YY1 protein and its associated factors. Detailed information about the expression and regulation of these factors in preimplantation stage embryos has not been published for any species. In order to ascertain which of these factors may participate in chromatin remodeling, genome activation, and DNA replication during early primate embryogenesis, we determined the temporal expression patterns of mRNA encoding these factors. Our data identify the predominant members of these different functional classes of factors expressed in oocytes and embryos, and reveal patterns of expression distinct from those patterns seen in somatic cells. Among each of four classes of mRNAs examined, some mRNAs were expressed predominantly in the oocyte, with these largely giving way to others expressed stage specifically in the embryo. This transition may be part of a global mechanism underlying the transition from maternal to embryonic control of development, wherein the oocyte program is silenced and an embryonic pattern of gene expression becomes established. Possible roles for these mRNAs in chromatin remodeling, genome activation, DNA replication, cell lineage determination, and nuclear reprogramming are discussed.

Embryonic genome activation.

Genome activation is one of the first critical events in the life of the new organism. Both the timing of genome activation and the array of genes activated must be controlled correctly. Genome activation occurs in a stepwise manner, with some genes being transcribed well in advance of the major genome activation event, in which most housekeeping genes become activated. Changes in chromatin protein content, particularly histone proteins, and chromatin structure appear to regulate the availability of the genome for transcription and provide for specificity of transcription. Gene enhancers are not initially required for transcription, but become necessary as the chromatin structure is modified. Changes in transcription factor content or activity are also required, and protein synthesis is essential for genome activation during both early and later phases of transcriptional activation. Both the changes in chromatin structure and availability of transcription factors are regulated by cell cycle-dependent mechanisms, thus providing the necessary coordination between these processes and other processes such as DNA replication and cleavage.

Mouse embryos do not wait for the MBT: Chromatin and RNA polymerase remodeling in genome activation at the onset of development

In Xenopus and Drosophila embryos, activation of the zygotic genome occurs after a series of rapid nuclear divisions in which DNA replication occupies most of the cell cycle. In these organisms, it has been proposed that zygotic transcription does not begin until a threshold nucleocytoplasmic ratio has been obtained in which repressive factors are titrated out and interphase becomes long enough to allow synthesis of transcripts. In mammalian embryos, however, a model of threshold nucleocytoplasmic ratios does not seem to apply, as beginning with the 1-cell stage, there are regulated cell cycles with the expression of zygotic transcripts during the cleavage period. By taking advantage of the slower kinetics at the onset of mouse development, we have characterized changes in chromatin structure and the basal transcription machinery throughout the transition from transcriptional incompetence, to minor activation of the zygotic genome during the 1-cell stage, and through major genome activation at the 2-cell stage. Further maturation of chromatin structure continues through subsequent cleavage cycles as a foundation for the first cellular differentiations in the blastocyst. The epigenetic chromatin modifications that occur during the cleavage period may have long range and inheritable effects and are undoubtably important in the ability of the mammalian oocyte to remodel previously defined nuclear structures and cell fates. Dev. Genet. 22:31–42, 1998. © 1998 Wiley-Liss, Inc.

Mouse embryos do not wait for the MBT: chromatin and RNA polymerase remodeling in genome activation at the onset of development.

In Xenopus and Drosophila embryos, activation of the zygotic genome occurs after a series of rapid nuclear divisions in which DNA replication occupies most of the cell cycle. In these organisms, it has been proposed that zygotic transcription does not begin until a threshold nucleocytoplasmic ratio has been obtained in which repressive factors are titrated out and interphase becomes long enough to allow synthesis of transcripts. In mammalian embryos, however, a model of threshold nucleocytoplasmic ratios does not seem to apply, as beginning with the 1-cell stage, there are regulated cell cycles with the expression of zygotic transcripts during the cleavage period. By taking advantage of the slower kinetics at the onset of mouse development, we have characterized changes in chromatin structure and the basal transcription machinery throughout the transition from transcriptional incompetence, to minor activation of the zygotic genome during the 1-cell stage, and through major genome activation at the 2-cell stage. Further maturation of chromatin structure continues through subsequent cleavage cycles as a foundation for the first cellular differentiations in the blastocyst. The epigenetic chromatin modifications that occur during the cleavage period may have long range and inheritable effects and are undoubtedly important in the ability of the mammalian oocyte to remodel previously defined nuclear structures and cell fates.