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SummaryThe present study examines the role of RNA polymerase I (RPI)-mediated transcription, maternally inherited rRNA and nucleolar proteins in the resumption of fibrillogranular nucleoli during embryonic genome activation (EGA) in porcine embryos. Late 4-cell embryos were incubated in the absence (control) or presence of actinomycin D (AD) (0.2 μg/ml for inhibition of RPI; 2.0 μg/ml for inhibition of total transcription) and late 2-cell embryos were cultured to the late 4-cell stage with 0.2 μg/ml AD to block EGA. Embryos were then processed for reverse-transcriptase polymerase chain reaction (RT-PCR), and for autoradiography (ARG), transmission electron microscopy (TEM), fluorescence in situ hybridization (FISH), silver staining and immunofluorescence (for RPI). Embryos in the control group displayed extranucleolar and intranucleolar ARG labelling, and exhibited de novo synthesis of rRNA and reticulated functional nucleoli. Nucleolar proteins were located in large foci. After RPI inhibition, nucleolar precursors transformed into segregated fibrillogranular structures, however no fibrillar centres were observed. The localization of rDNA and clusters of rRNA were detected in 57.1% immunoprecipitated (IP) analyzed nucleoli and dispersed RPI; 30.5% of nuclei showed large deposits of nucleolar proteins. Embryos from the AD-2.0 group did not display any transcriptional activity. Nucleolar formation was completely blocked, however 39.4% of nuclei showed rRNA clusters; 85.7% of nuclei were co-localized with nucleolar proteins. Long-term transcriptional inhibition resulted in the lack of ARG and RPI labelling; 40% of analyzed nuclei displayed the accumulation of rRNA molecules into large foci. In conclusion, maternally inherited rRNA co-localized with rDNA and nucleolar proteins can initiate a partial nucleolar assembly, resulting in the formation of fibrilogranular structures independently on activation of RPI-mediated transcription.

One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

Global transcription silencing occurs in the oocyte during its final phase of growth. The particular mechanism of this silencing is not well understood. Here, we investigated the silencing of RNA polymerase II transcription in porcine oocytes. First, we investigated the transcriptional activity of germinal vesicle oocytes derived from stimulated and non-stimulated gilts, but no transcriptional activity was observed. Second, we focused on the fate of RNA polymerase II in growing and fully grown oocytes. Active and inactive forms of RNA polymerase II were detected in growing oocytes by immunofluorescence and Western blots. In contrast, only the inactive form of RNA polymerase II was detected in fully grown oocytes. To evaluate if the inactive form of RNA polymerase II is released from DNA, the oocytes were subsequently permeabilized and fixed in one step. After this modified fixation protocol, the immunofluorescent labeling was negative in fully grown oocytes, but remained unchanged (positive) in growing oocytes. These results indicate that the inactive form of RNA polymerase II is not bound to DNA during the oocyte growth. Finally, based on Western blot analysis of different stages of oocyte maturation, the inactive form of RNA polymerase II was detected in metaphase I but not in metaphase II. Our study confirmed the global transcription silencing of fully grown oocytes. Compared with other mammalian species (e.g., mouse), the mechanism of RNA polymerase II silencing in porcine oocytes seems to be similar, despite some differences in dynamics.

Common features of embryonic genome activation in mammalian and non-mammalian embryos are the colocalization of pre-assembled complexes of maternally inherited nucleolar proteins, the so-called nucleolus precursor bodies and de novo synthesized transcripts with ribosomal DNA. The de novo transcription of messenger RNA and ribosomal RNA proteins is required for the development of functional nuclei during the major activation of the embryonic genome. The aim of our work was to investigate to what extent. Autoradiography and transmission electron microscopy has been applied in in vitro produced bovine embryos. The embryos were cultured to the late 8-cell stage with: α-amanitin; a specific inhibitor of RNA-polymerases II and III transcription; actinomycin D; a specific inhibitor of RNA polymerase I transcription; and without inhibitors (control group). Nucleoplasm and nucleolar structures displayed strong autoradiographic labeling and showed the initial development of fibrillo-granular nucleoli in the control group. In α-amanitin groups, however, in both inhibited groups of embryos, lack of autoradiographic labeling and disintegrated nucleolus precursor bodies stage were observed. Our study of α-amanitin as well as in actinomycin D groups proves inhibition of transformation nucleolus precursor bodies to active nucleoli. From our results follows, actinomycin D is able to penetrate through zona pellucida, what was shown for the first time.

In vitro production of porcine embryos by means of in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) is limited by great inefficienciy. The present study investigated chromatin and nucleolar dynamics in porcine embryos developed in vivo (IV) and compared this physiological standard to that of embryos produced by IVF, parthenogenetic activation (PA), or SCNT. In contrast to IV embryos, chromatin spatial and temporal dynamics in PA, IVF, and SCNT embryos were altered; starting with aberrant chromatin–nuclear envelope interactions at the two-cell stage, delayed chromatin decondensation and nucleolar development at the four-cell stage, and ultimately culminating in failure of proper first lineage segregation at the blastocyst stage, demonstrated by poorly defined inner cell mass. Interestingly, in vitro produced (IVP) embryos also lacked a heterochromatin halo around nucleolar precursors, indicating imperfections in global chromatin remodeling after fertilization/activation. Porcine IV-produced zygotes and embryos display a well-synchronized pattern of chromatin dynamics compatible with genome activation and regular nucleolar formation at the four-cell stage. Production of porcine embryos under in vitro conditions by IVF, PA, or SCNT is associated with altered chromatin remodeling, delayed nucleolar formation, and poorly defined lineage segregation at the blastocyst stage, which in turn may impair their developmental capacity.

Initially, development of the zygote is under control of the oocyte ooplasm. However, it is presently unknown if and to what extent is the ooplasm able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). Here, one-cell stage embryos were processed at different points in time post activation (2 hpa, 4 hpa, 8 hpa, and 12 hpa) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualization of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). Bovine and porcine intergeneric SCNT embryos were compared to their parthenogenetic counterparts to assess the effects of the introduced somatic cell. Despite the absence of morphological remodeling (premature chromatin condensation, nuclear envelope breakdown), reconstructed embryos showed nuclear and nucleolar precursor body (NPB) morphology similar to the host ooplasm, which, together with detected posttranslational activity of somatic cell introduced into the bovine ooplasm, suggests a universal function of ooplasmic factors. However, the lack of distinct UBF localization in intergeneric embryos indicates failures in sequence-specific interactions between the ooplasm and chromatin of another genus. In conclusion, the results demonstrate a possible reason why the intergeneric SCNT embryos never reached the full term.

The early stages of embryonic development are maternally driven. As development proceeds, maternally inherited informational molecules decay, and embryogenesis becomes dependent on de novo synthesized RNAs of embryonic genome. The aim of the present study is to investigate the role of de novo transcription in the development of embryos during embryonic genome activation. Autoradiography for detection of transcriptional activity and transmission electron microscopy were applied in in vitro produced bovine embryos cultured to the late 8-cell stage with or without (control group) α-amanitin, specific inhibitor of RNA-polymerases II and III transcription. The α-amanitin (AA) groups presented three sets of embryos cultivated with AA in different time intervals (6, 9 and 12 h). In control group, nucleoplasm and nucleolar structures displayed strong autoradiographic labeling and showed initial development of fibrillo-granular nucleoli. In α-amanitin groups, lack of autoradiographic labeling and disintegrated nucleolus precursor bodies (NPBs) stage were observed. Inhibition of RNA polymerase II (RNA pol II) already in the early phases of embryonic genome activation has detrimental effect on nucleolar formation and embryo survival, what was shown for the first time.

The aim of the present study was to investigate the role of RNA polymerase I (RPI) transcription in nucleolar development during major transcriptional activation (MTA) in cattle. Late eight-cell embryos were cultured in the absence (control group) or presence of actinomycin D (AD) (RPI inhibition, AD 0.2 microg/ml; total transcriptional inhibition, AD 2.0 microg/ml). Late four-cell embryos were cultured to late eight-cell stage in 0.2 microg/ml AD (MTA prevention, ADLT (long-term total transcriptional inhibition group). Embryos were processed for autoradiography, transmission electron microscopy, fluorescent in situ hybridization (ribosomal RNA, rRNA), silver staining (nucleolar proteins), and immunofluorescence (RPI). Control embryos displayed extranucleolar and nucleolar transcription, functional nucleoli, and distinct RPI localization. Nuclei (97%) showed large rRNA clusters, in 94.1% co-localized with nucleolar proteins deposits. In AD 0.2 group, only extranucleolar transcription was detected. Segregated dense-fibrillar and granular components, but no fibrillar centers, were observed. RPI was dispersed. Nuclei (55%) presented rRNA clusters, in 38.8% co-localized with silver-stained deposits. AD 2.0 and ADLT groups displayed no transcription and disintegrating nucleolar precursors. AD 2.0 (34%) and 14% (ADLT) of nuclei presented clusters of maternally inherited rRNA. In AD 2.0 group, RPI was dispersed, but 17.2% of nuclei showed colocalization of rRNA with nucleolar proteins. In ADLT group, RPI was lacking and clustering of nucleolar proteins was hampered. In conclusion, rDNA transcription is not required for targeting of rRNA processing proteins, rRNA is maternally inherited and target to rDNA independent of transcription, and de novo transcription is required for proper nucleologenesis in cattle.

The onset of ribosomal RNA (rRNA) synthesis occurs during the second half of the third cell cycle, that is, at the four-cell stage, in porcine embryos developed in vivo. In the present study the onset of rRNA synthesis was investigated in porcine embryos produced in vitro (IVP) or by somatic cell nuclear transfer (SCNT) using fluorescence in situ hybridization (FISH) with an rDNA probe and subsequent visualization of the nucleolar proteins by silver staining. In the 205 IVP embryos investigated, all two-cell embryos (n = 34) were categorized as transcriptionally inactive. At the late four-cell stage (n = 45), 38% of the embryos contained 1-3 nuclei with signs of rRNA transcription, indicating an asynchronous transcription initiation. This pattern continued in the following stages, as 78% (n = 47), 47% (n = 42) and 83% (n = 37) of the embryos revealed a mixture of transcriptionally inactive and active cells at the eight-cell, 16-cell and blastocyst stage, respectively. In the 143 SCNT embryos investigated, all two-cell embryos (n = 34) and early four-cell embryos (n = 12) were also transcriptionally inactive. At the late four-cell stage (n = 33) and at the eight-cell stage (n = 24) there were equal proportions of transcriptionally active and inactive embryos and essentially all embryos that developed to the 16-cell stage (n = 21) and further to the blastocyst stage (n = 19) contained only transcriptionally active cells. In conclusion, porcine embryos produced in vitro had an asynchronous pattern of rRNA transcription initiation when compared to SCNT and in vivo developed porcine embryos.

The nucleolus formation was studied as an indirect marker of the ribosomal RNA (rRNA) genes activation in porcine embryos following oocyte maturation, fertilization, and culture in vitro. Nucleologenesis was assessed by transmission electron microscopy (TEM), light microscopical autoradiography following 20 min of 3H-uridine incubation, and immunocytochemical localization of key nucleolar proteins involved in rRNA transcription (upstream binding factor (UBF), topoisomerase I, and RNA polymerase I) and processing (fibrillarin, nucleophosmin, nucleolin) by confocal laser scanning microscopy. During the first four post-fertilization cell cycles, TEM revealed spherical nucleolus precursor bodies (NPBs), consisting of densely packed fibrils, as the most prominent intra-nuclear entities of the blastomeres. Fibrillo-granular nucleoli were observed in some blastomeres in a single embryo during the 5th cell cycle, i.e., the tentative 16-cell stage, where formation of fibrillar centres (FC), a dense fibrillar component, and a granular component on the surface of the NPBs was seen. In this embryo, autoradiographic labeling was detected over the nucleoplasm and in particular over the nucleoli. Fibrillarin was immunocytochemically localized in the presumptive NPBs of the pronuclei. This protein was again localized to the presumptive NPBs together with nucleolin from late during the 3rd cell cycle, i.e., the four-cell stage in some embryos. UBF, RNA polymerase I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the 4th cell cycle, i.e., the tentative eight-cell stage and onwards. Toposiomerase I was not localized to intra-nuclear entities even during the 5th post-fertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of other nucleolar proteins. In conclusion, porcine embryos produced in vitro display a substantial delay in or even lack of the development of functional nucleoli. Mol. Reprod. Dev. 68: 327–334, 2004. © 2004 Wiley-Liss, Inc.

The expression of nucleolar-related proteins was studied as an indirect marker of the ribosomal RNA (rRNA) gene activation in porcine embryos up to the blastocyst stage produced in vivo and in vitro. A group of the in vivo-developed embryos were cultured with α-amanitin to block the de novo embryonic mRNA transcription. Localization of proteins involved in the rRNA transcription (upstream binding factor [UBF], topoisomerase I, RNA polymerase I [RNA Pol I], and the RNA Pol I-associated factor PAF53) and processing (fibrillarin, nucleophosmin, and nucleolin) was assessed by immunocytochemistry and confocal laser-scanning microscopy, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). These findings were correlated with ultrastructural data and autoradiography following 20-min [3H]uridine incubation. Additionally, expression of the pocket proteins pRb and p130, which are involved in cell-cycle regulation, was assessed by semiquantitative R...

The immediate events of genomic reprogramming at somatic cell nuclear transfer (SCNT) are to high degree unknown. This study was designed to evaluate the nuclear and nucleolar changes during the first cell cycle. Bovine SCNT embryos were produced from starved bovine fibroblasts and fixed at 0.5, 1, 2, 3, 4, 8, 12, and 16 h postactivation (hpa). Parthenogenetic (PA) embryos were used as control. The SCNT and PA embryos were processed for lacmoid staining, autoradiography, transmission electron microscopy (TEM), and immunofluorescence localization of: upstream binding factor (UBF) and fibrillarin at 4 and 12 hpa. Likewise, starved and nonstarved fibroblasts were processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillogranular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. In conclusion, somatic cell nuclei introduced into enucleated oocytes displayed chromatin condensation, partial nuclear envelope breakdown, nucleolar desegregation and transcriptional quiescence already at 0.5 hpa. Somatic cell cytoplasm remained temporally attached to introduced nucleus and nucleolus was partially restored indicating somatic influence in the early SCNT phases. At 1-3 hpa, chromatin gradually decondensed toward the nucleus periphery and nuclear envelope reformed. From 4 hpa, the somatic cell nucleus gained a PN-like appearance and displayed NPBs suggesting ooplasmic control of 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.

The oocyte cytoplasm (ooplasm) constitutes a unique mixture of factors that are critical for successful reprogramming of haploid maternal and paternal genomes at fertilization as well as the diploid somatic cell genome after somatic cell nuclear transfer (SCNT). The 1-cell-stage embryos (produced by transfer of bovine fibroblast into porcine enucleated ooplast; interspecies SCNT, iSCNT) were processed at different time points (2, 4, 8, and 12 h post-activation, hpa) for detailed nuclear and nucleolar analysis by transmission electron microscopy (TEM; 4 to 5 embryos per group), and immunofluorescence (4 and 12 hpa) for visualization of nucleolar proteins related to transcription (upstream binding factor, UBF) and processing (fibrillarin; 8 to 9 embryos per group). The parameters of interspecies embryos were compared with porcine parthenogenetic (PA) counterparts (4 to 5 embryos per group for TEM analysis; 8 to 9 embryos per group for immunofluorescence analysis). At all evaluated time points, embryos in both groups displayed pronucleus-like nuclei with abundant euchromatin and characteristic porcine nucleolus precursor body (NPB), indicating maternal origin of nucleolar components. Fibrillarin was in both groups localized into shell-like intranuclear entities surrounding NPB. On the contrary, UBF in PA embryos was at 12 hpa colocalized with fibrillarin, whereas in iSCNT embryos, UBF staining was absent at both time points. Despite the similar morphology and localization of processing factor fibrillarin, striking differences in localization of transcription factor UBF suggest limitation of porcine ooplasm to mediate initial phases of nucleolar remodelling. This work was supported by VEGA 1/0077/11.

Ooplasm posses several, undefined factors allowing for reprogramming of somatic genome into pluripotent state. However, it is presently unknown if and to what extent the ooplasm is able to interact with a transferred somatic cell from another species in the context of interspecies somatic cell nuclear transfer (SCNT). The 1-cell stage embryos were processed at different time points (2, 4, 8, and 12 h post-activation) for detailed nuclear and nucleolar analysis by TEM, and immunofluorescence for visualisation of nucleolar proteins related to transcription (UBF) and processing (fibrillarin). The embryos produced by SCNT of porcine fibroblast into non-activated bovine ooplasm were compared to bovine parthenogenetic counterparts. In the absence of morphological remodelling features (premature chromatin condensation, nuclear envelope break-down), intergeneric embryos showed bovine nuclear and nucleolar precursor body (NPB) morphology. The somatic cell introduced into the bovine ooplasm displayed intranuclear activity at the post-translational level signalling a universal function of ooplasmic factors. The lack of distinct UBF localization in intergeneric embryos, however, suggests failures in sequence-specific interactions between the ooplasm and transferred cell from another genus. We conclude that sperm or a somatic cell of the same species is required for successful embryonic development. Clonet MRTN-CT-2006-035468; MedRat LSHG-CT-2006-518240; PluriSys FP7-HEALTH-2007-223485; DFG, Alexander von Humboldt fellowship, VEGA 1/0057/08; VEGA 1/0012/10.

This study was designed in order to evaluate the global transcription and morphology of reprogramming during somatic cell nuclear transfer (SCNT). Following 20–22 h of IVM, couplets of MII cytoplasts and starved bovine fibroblasts were, after 2 h co-culture, electrically fused, chemically activated by 5 µm ionomycin for 5 min, followed by a 3–4 h incubation in 2 mm 6-DMAP, and fixed at 0.5, 1, 1.5, 2, 3, 4, 8, 12, and 16 h post-activation (hpa). The reconstructed embryos were processed for lacmoid staining, autoradiography following [3H]uridine incubation, transmission electron microscopy (TEM), and immunofluorescence (4 and 12 hpa) in order to evaluate chromatin dynamics, transcriptional activity, nuclear and nucleolar ultrastructure, and localization of nucleolar proteins (upstream binding factor (UBF) and fibrillarin), respectively, during the first cell cycle. Likewise, starved fibroblasts were fixed and processed for autoradiography and TEM. The fibroblasts displayed strong transcriptional activity and active fibrillo-granular nucleoli. None of the reconstructed embryos, however, displayed transcriptional activity. During first 3 hpa, the majority of the embryos displayed a single block of condensed chromatin surrounded by a more or less complete nuclear envelope (NE) and an abundant population of elongated somatic cell mitochondria. This somatic cell complex was located peripherally in the ooplasm. At the subsequent time points, the embryos displayed pronuclear-like structures, which from 8 hpa were located centrally in the ooplasm. From about 4 hpa, the somatic cell complex had dispersed and the elongated mitochondria could no longer be tracked. The first nucleolus-related structures were observed at 1.5 hpa and only in nuclei with a complete NE. At 1.5 to 4 hpa, the nucleolus-related structures appeared either as bodies presenting a large fibrillar center and presumptive dense fibrillar component, but no granular component, or as electron-dense nucleolus precursor bodies (NPBs). From 4 hpa and onward, only compact NPBs were observed. At 4 and 12 hpa, UBF was localized into small discrete clusters of foci enclosed in a shell-like structure labeled by fibrillarin in the nucleus. In conclusion, at SCNT, the somatic cell cytoplasm remains structurally organized in a somatic cell complex over the initial 3–4 hpa. During the same period, the somatic cell chromatin undergoes condensation and the NE is partially dissolved. Subsequently, pronucleus-like euchromatic nuclei with typical NPBs are formed, although somatic cell nucleolar components may be temporarily seen. Throughout the process, transcription is repressed. This work was supported by Marie Curie Intra-European Fellowships (MEIF-CT-2006-021629), and by grant VEGA 1/3255/06 and DFG.

The development of a functional nucleolus accompanying the major embryonic genome activation (EGA) is considered a marker for embryo quality and viability. However, the use of this marker is limited by the lack of accurate knowledge of the biology of embryonic nucleologenesis. The objective of this study was to elucidate the role of RNA polymerase I (RPI) and total transcriptional activity, reflecting EGA, for nucleologenesis in in vivo-developed porcine embryos. Late 4-cell-stage embryos were cultured in the absence (control) or presence of actinomycin D (AD; 0.2 �g mL-1, 3 h for RPI inhibition; 2.0 �g mL-1, 3 h for total transcriptional inhibition). Late 2-cell-stage embryos were cultured to the late 4-cell stage with 0.2 �g mL-1 AD (long-term inhibition) to prevent EGA. Embryos were fixed at the late 4-cell stage and processed for RT-PCR (de novo synthesized rRNA), autoradiography (ARG, following culture with 3H-uridine for the last 20 min before fixation), TEM, FISH (probe-labeling rRNA and rDNA), silver staining of nucleolar proteins, and immunofluorescence for RPI. Control embryos displayed typical extranucleolar and nucleolar ARG labeling, fibrillo-granular nucleoli, and focal RPI localization signaling de novo rRNA synthesis in functional nucleoli, confirmed by RT-PCR. All nuclei showed large FISH clusters (rRNA and rDNA) that in 88% of the cases were co-localized with large foci of silver-stained nucleolar proteins. After RPI inhibition, only extranucleolar ARG labeling was detected and, instead of fibrillo-granular nucleoli, a segregated dense-fibrillar component and a granular component, but no fibrillar centers, were observed. RPI was dispersed in all nuclei, the number of nuclei presenting large FISH clusters decreased to 40%, and only 42% of nuclei showed nucleolar proteins localized to large foci. After total transcriptional inhibition and long-term inhibition, the nuclei did not display any ARG labeling and presented inactive nucleolus precursor bodies indicating lack of rRNA (RT-PCR) and total RNA synthesis. However, 40% of the nuclei in both groups presented large FISH clusters of rRNA. This rRNA is considered as partially processed residues of maternally inherited molecules, and their clustering is most likely independent of EGA. Inhibition of transcriptional activity at the time of EGA caused the dispersion of RPI (de novo synthesized) but did not influence the localization of silver-stained nucleolar proteins to large foci (41%). On the other hand, EGA inhibition caused the lack of RPI labeling and hampered the localization of nucleolar proteins to foci. Differences between these 2 groups could be due to the activation of RNA polymerase II before the 3-h AD treatment. In conclusion, RPI transcription and de novo protein synthesis are required for formation of functional nucleoli. However, the clustering of maternally inherited nucleolar transcripts is independent on transcriptional activity at the time of EGA. Failure in constituent RNA polymerase activation during EGA leads to pattern-specific changes in nucleologenesis, which may serve as a marker for early embryo quality.

In vitro production (IVP) of porcine embryos by in vitro maturation of oocytes followed by fertilization and culture in vitro is hampered by great deficiencies. Initiation of at least the major embryonic genome transcription, which includes activation of ribosomal RNA (rRNA) genes and the associated formation of a fibrillo-granular nuclealus, is normally seen during the 4-cell stage in pigs. We have investigated the activation of rRNA synthesis and the presence of silver staining nucleolar proteins in porcine IVP embryos as a marker of transcriptional activity and, thus, developmental competence. A total of 205 porcine IVP embryos from the 2-cell to the blastocyst stage were examined using sequential fluorescent in situ hybridization (FISH) to the rRNA genes and their transcripts and silver staining of nucleolar proteins as previously described (Viuff et al. 2002 Biol. Reprod. 66, 629–634). Briefly, cumulus-oocyte complexes with at least three cumulus cell layers and evenly granulated ooplasm were isolated from 2–5 mm ovarian follicles with stereomicroscopic evaluation. Subsequently, oocytes were matured in NCSU-37 and mechanically denuded followed by fertilization using frozen-thawed epididymal semen. Presumptive zygotes were then cultured in NCSU-23 at 39°C, 5% CO2. Around the time of expected cleavage, the embryos were examined every second hour to determine the time of cleavage. Embryos at the 2-cell stage were harvested at 5 h post-cleavage (hpc), 4-cell embryos late during the third cell cycle at 30 hpc, and tentative 8- and 16-cell embryos at 10 hpc. Blastocysts were harvested at Day 5 post-insemination. In general, nuclei of 2-cell embryos displayed 4 small foci of FITC labelling (presumably the rDNA), but no specific silver staining, and were consequently categorized as transcriptionally inactive. At the late 4-cell stage, 58% of the embryos resembled the 2-cell stage. However, in the remaining embryos (42%), some or all nuclei displayed large areas of FISH labelling (presumptive rDNA and rRNA) co-localized with silver staining, and were catagorized as transcriptionally active. Among the 8-cell embryos, 64% displayed a majority of transcriptionally active nuclei, whereas this was the case in 83% and 92% of the embryos in the 16-cell embryos and the blastocysts, respectively. In general, the majority of the embryos contained a mixture of transcriptionally active and inactive cells. These findings show that the porcine IVP embryos are often delayed and asynchronous with respect to activation of the rRNA genes. Table 1. Categorization of nuclei according to transcriptional activity This work was supported by grants from “Disease models, disease prevention and animal welfare improvement: The pig embryo as a model.” Danish Research Agency (Grant: 9901178), NATO (Grant: 978658), and Deutsche Forschungsgemeinschaft (DFG).