Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines.

Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using 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 nuclear transfer of mouse embryonic fibroblast (MEF) and mouse HM1 embryonic stem cells (HM1), were processed for autoradiography following (3)H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF and nucleophosmin, B23). All early 2-cell embryos showed transcriptional activity only in nucleoplasm, not over nucleolar precursor bodies (NPBs). UBF was diffusely localized to cytoplasm and B23 to cytoplasm and nucleoplasm. Late 2-cell IVF and PG embryos displayed transcription over nucleoplasm and NPBs. Ultrastructurally, the latter were developing into functional nucleoli. NT-MEF and NT-HM1 embryos displayed transcription over nucleoplasm, but not over NPBs. Development of NPBs into nucleoli was lacking. UBF was in both groups localized to nucleoplasm or distinctly to presumptive NPBs. B23 was distinctly localized to NPBs. All 4-cell embryos presented nucleoplasmic transcription and developing fibrillo-granular nucleoli. UBF and B23 were distinctly localized to nucleoli. However, whereas fully transformed reticulated fibrillo-granular nucleoli were found in IVF and PG embryos, NT-MEF and -HM1 embryos displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both of these processes were delayed.

High in vitro development after somatic cell nuclear transfer and trichostatin A treatment of reconstructed porcine embryos.

Abnormal epigenetic modification is supposed to be one of factors accounting for inefficient reprogramming of the donor cell nuclei in ooplasm after somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone deacetylase, potentially enhancing cloning efficiency. The aim of our present study was to establish the optimal TSA treatment in order to improve the development of handmade cloned (HMC) porcine embryos and examine the effect of TSA on their development. The blastocyst percentage of HMC embryos treated with 37.5 nM TSA for 22-24 h after activation increased up to 80% (control group-54%; P<0.05). TSA mediated increase in histone acetylation was proved by immunofluorescence analysis of acH3K9 and acH4K16. 2-cell stage embryos derived from TSA treatment displayed significant increase in histone acetylation compared to control embryos, whereas no significant differences were observed at blastocyst stage. During time-lapse monitoring, no difference was observed in the kinetics of 2-cell stage embryos. Compact morula (CM) stage was reached 15 h later in TSA treated embryos compared to the control. Blastocysts (Day 5 and 6) from HMC embryos treated with TSA were transferred to 2 recipients resulting in one pregnancy and birth of one live and five dead piglets. Our data demonstrate that TSA treatment after HMC in pigs may affect reprogramming of the somatic genome resulting in higher in vitro embryo development, and enable full-term in vivo development.

The role of RNA polymerase I transcription and embryonic genome activation in nucleolar development in bovine preimplantation embryos.

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.

Ribosomal RNA and nucleolar proteins from the oocyte are to some degree used for embryonic nucleolar formation in cattle and pig.

The nucleolus is the site of ribosomal RNA (rRNA) and ribosome production. In the bovine primordial follicle oocyte, this organelle is inactive, but in the secondary follicle an active fibrillo-granular nucleolus develops and proteins involved in rDNA transcription (topoisomerase I, RNA polymerase I and upstream binding factor) and early (fibrillarin) or late rRNA processing (nucleolin and nucleophosmin) localize to it. At the end of the oocyte growth phase, the nucleolus is inactivated again and transforms into a solid remnant. The nucleolar remnant is dissolved when meiosis is resumed. Upon fertilization, structures resembling the nucleolar remnant, now referred to as nucleolus precursor bodies (NPBs), are established in the pronuclei. These entities are engaged in the re-establishment of fibrillo-granular nucleoli at the major activation of the embryonic genome. This nucleolar formation can be classified into two different modes: one where nucleolus development occurs inside NPBs (internal; e.g. cattle) and the other where it occurs on the surface of NPBs (external; e.g. pig). Oocyte derived proteins engaged in late rRNA processing (nucleolin and nucleophosmin) may to some degree be re-used for nucleolar formation in the embryo, while the other nucleolar proteins require de novo embryonic transcription in order to be allocated to the developing nucleoli. Moreover, unprocessed rRNA inherited from the oocyte targets to the developing embryonic nucleoli. In conclusion, the nucleolus is important for the development of oocytes and embryos and may serve as a marker for the completion of oocyte growth and the normality of activation of the embryonic genome.

Morphological characterization of pre- and peri-implantation in vitro cultured, somatic cell nuclear transfer and in vivo derived ovine embryos.

The processes of cellular differentiation were studied in somatic cell nuclear transfer (SCNT), in vitro cultured (IVC) and in vivo developed (in vivo) ovine embryos on days 7, 9, 11, 13, 17 and 19. SCNT embryos were constructed from in vitro matured oocytes and granulosa cells, and IVC embryos were produced by in vitro culture of in vivo fertilized zygotes. Most SCNT and IVC embryos were transferred to recipients on day 6 while some remained in culture for day 7 processing. In vivo embryos were collected as zygotes, transferred to intermediate recipients and retransferred to final recipients on day 6. All embryos were processed for examination by light and transmission electron microscopy or immunohistochemical labelling for alpha-1-fetoprotein and vimentin. Overall, morphological development of in vivo embryos was superior to IVC and SCNT embryos. Day 7 and particularly day 9 IVC and SCNT embryos had impaired hypoblast development, some lacking identifiable inner cell masses. On day 11, only in vivo and IVC embryos had developed an embryonic disc, and gastrulation was evident in half of in vivo embryos and one IVC embryo. By day 13, all in vivo embryos had completed gastrulation whereas IVC and SCNT embryos remained retarded. On days 17 and 19, in vivo embryos had significantly more somites and a more developed allantois than IVC and SCNT embryos. We conclude that IVC and particularly SCNT procedures cause a retardation of embryo development and cell differentiation at days 7-19 of gestation.

Chronological appearance of apoptosis in bovine embryos reconstructed by somatic cell nuclear transfer from quiescent granulosa cells.

Efficiency of cloning has remained low and in spite of attempts to improve this technology, many reconstructed embryos do not implant or are lost during early pregnancy. Chromosomal aberrations, deviant gene expression patterns and abnormal regulation of cell death may be involved in this increased early embryonic loss. Here, we investigate the chronological onset of both apoptotic changes in nuclear morphology and DNA degradation [detected by transferase-mediated dUTP nick-end labelling (TUNEL) reaction] in bovine two-cell- to blastocyst-stage embryos. Such embryos were generated either by reconstruction with nuclear transfer from quiescent granulosa cells or by regular in vitro embryo production. Nuclear condensation was observed from the two-cell stage and TUNEL labelling was observed from the six-cell stage in reconstructed embryos, whereas nuclear condensation was evident from the eight-cell stage and TUNEL labelling from the 13-cell stage in embryos derived in vitro. Furthermore, reconstructed embryos displayed elevated ratios of embryos containing apoptotic nuclei at pre-compaction stages and higher indices of apoptotic nuclei in morula and blastocyst stages when compared with in vitro-produced embryos.

Renal lipofuscinosis in Danish slaughter cattle.

A study was performed to characterize dark brown or black discoloured kidneys ("black kidneys") in Danish slaughter cattle and to investigate the aetiology and pathogenesis. In 133 939 cattle entering four abattoirs, 359 cases of "black kidneys" were recorded. Of these, 57 cases were submitted for macroscopical, microscopical, and ultrastructural examination. A pigment with characteristics similar to those of lipofuscin was found in secondary lysosomes in epithelial cells of the proximal tubules. Pigment accumulation was the cause of discoloration, with a positive correlation between the discoloration of the renal cortex and the degree of pigment accumulation. Cases occurred only in cattle of the Holstein breed or the Red Danish Dairy breed and mainly in animals aged 3 years or older. In these breeds, prevalences of 0.44% and 2.51% were found, respectively. Epidemiological analyses indicated that affected animals aged 4.5 to 6.5 years or 7.5 to 8.5 years were culled more frequently than unaffected cattle. Epidemiological and genealogical analyses strongly indicated a genetic aetiology with simple autosomal recessive inheritance.

Regulation of ribosomal RNA gene expression in porcine oocytes.

In vitro production (IVP) of porcine embryos including in vitro maturation (IVM) of oocytes followed by in vitro fertilization (IVF) and in vitro culture (IVC) of the resultant embryos may result in live offspring, but it is still associated with great inefficiencies probably due to incomplete cytoplasmic maturation of the oocytes in vitro. Therefore, fundamental knowledge on the regulation of transcription during the oocyte growth phase when the messengers and protein synthetic machinery necessary for oocyte developmental competence are formed, is of great importance. In mammals, synthesis of RNA, up to 60-70% of which is ribosomal (rRNA), increases during oocyte growth and reaches a peak at the beginning of follicular antrum formation. In oocytes at the end of the growth phase, acquisition of full meiotic competence coincides with a markedly decreased rRNA transcriptional activity in the gametes. Our recent studies on the porcine oocyte growth phase have revealed a deeper molecular and biological insight into the complex regulation of rRNA transcription at different stages of follicular development. The data indicate that the so-called pocket protein, p130, is involved in the down-regulation of rRNA transcription at the end of the oocyte growth phase through an inhibition of the action of upstream binding factor (UBF). The latter protein is necessary for the function of RNA polymerase I (RNA Pol I), which is the actual enzyme driving rRNA gene transcription. Moreover, rRNA transcription also appears to be down-regulated by a decrease in the expression of mRNA encoding PAF53, an RNA Pol I-associated factor also required for the polymerase to exert its action. At the ultrastructural level, these molecular changes are paralleled by marginalization of the fibrillar centres of the oocyte nucleolus followed by compaction of the nucleolus into an inactive sphere of fibrils.

Nucleolar ultrastructure and protein allocation in in vitro produced 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.

Immunohistochemical and ultrastructural characterization of the initial post-hatching development of bovine embryos.

The problems of sustaining placenta formation in embryos produced by nuclear transfer have emphasized the need for basic knowledge about epiblast formation and gastrulation in bovine embryos. The aims of this study were to define stages of bovine post-hatching embryonic development and to analyse functional mechanisms of germ-layer formation. Embryos developed in vivo were collected after slaughter from superovulated cows on days 9, 11, 14 and 21 after insemination and processed for transmission electron microscopy (n = 26) or immunohistochemistry (n = 27) for potential germ-layer characterization (cytokeratin 8 for potential ectoderm; alpha-1-fetoprotein for potential endoderm; and vimentin for potential mesoderm). On day 9, the embryos were devoid of zona pellucida and presented a well-defined inner cell mass (ICM), which was covered by a thin layer of trophoblast cells (the Rauber's layer). Formation of the hypoblast from the inside of the ICM was ongoing. On day 11, the Rauber's layer was focally interrupted and adjacent underlying ICM cells formed tight junctions. The hypoblast, which formed a thin confluent cell layer, was separated from the ICM and the tropho-blast by intercellular matrix. The embryos were ovoid to tubular and displayed a confluent hypoblast on day 14. The epiblast was inserted into the trophoblast epithelium and tight junctions and desmosomes were present between adjacent epiblast cells as well as between peripheral epiblast and trophoblast cells. In some embryos, the epiblast was more or less covered by foldings of trophoblast in the process of forming the amniotic cavity. Cytokeratin 8 was localized to the trophoblast and the hypoblast underlying the epiblast; alpha-1-fetoprotein was localized to most hypoblast cells underlying the trophoblast; and vimentin was localized to most epiblast cells. On day 21, the smallest embryos displayed a primitive streak and formation of the neural groove, whereas the largest embryos presented a neural tube, up to 14 somites and allantois development. These embryos depicted the gradual formation of the endoderm, mesoderm and ectoderm as well as differentiation of paraxial, intermediate and lateral plate mesoderm. Cytokeratin 8 was localized to the trophoblast, the hypoblast and the surface and neural ectoderm; and alpha-1-fetoprotein was localized to the hypoblast, but not the definitive endoderm, the intensity increasing with development. Vimentin was initially localized to some, but not all, cells positioned particularly in the ventral region of the primitive streak, to presumptive definitive endoderm cells inserted into the hypoblast, and to mesoderm. In conclusion, within 2 weeks of hatching, bovine embryos complete formation of the hypoblast and the epiblast, establishment of the amniotic cavity, ingression of epiblast cells for primitive streak formation, involution of cells through the node and the streak for endoderm and mesoderm fomation, neurulation and differentiation of the mesoderm. The recruitment of cells from the epiblast to form the primitive streak as well as the endoderm and mesoderm is associated with expression of the intermediate filament vimentin.

Nucleolar proteins and ultrastructure in bovine in vivo developed, in vitro produced, and parthenogenetic cleavage-stage embryos.

In the present study, ribosomal RNA (rRNA) gene activation, monitored through nucleolus development, was studied by autoradiography following (3)H-uridine incubation, transmission electron microscopy, and immunofluorescence confocal laser scanning microscopy of key nucleolar proteins involved in rRNA transcription (topoisomerase I, upstream binding factor, and RNA polymerase I) and processing (fibrillarin, nucleolin, and nucleophosmin) in in vivo developed, in vitro produced, and parthenogenetic bovine embryos. In general, in vivo developed embryos displayed formation of fibrillo-granular nucleoli during the 4th post-fertilization cell cycle. During the previous stages of development, nucleolus precursor bodies (NPBs) were observed. However, on some occasions the initial steps of nucleolus formation were observed already at the 2- and 4-cell stage in cases where such embryos were collected from superovulated animals together with later embryonic stages presenting nucleolar development and autoradiographic labeling. The in vitro produced embryos displayed very synchronous formation of fibrillo-granular nucleoli and autoradiographic labeling during the 4th cell cycle. In vivo developed and in vitro produced embryos displayed allocation of nucleolar proteins to fibrillar and granular compartments of the developing nucleoli during the 4th cell cycle. The parthenogenetic embryos typically displayed formation of fibrillo- granular nucleoli during the 5th cell cycle and autoradiographic labeling was not observed until the morula stage. Moreover, the 1-, 2-, and 4-cell parthenogenetic embryos practically lacked NPBs. On the other hand, parthenogenetic embryos displayed allocation of nucleoar proteins to nuclear entities during the 4th cell cycle. In conclusion, both in vivo developed and in vitro produced bovine embryos displayed activation of transcription and nucleolar development during the 4th cell cycle. However, in vivo developed embryos flushed together with later developmental stages displayed premature activation of these processes. Parthenogenetic bovine embryos, on the other hand, displayed a delayed activation.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells.

In the present study immunofluorescence confocal laser scanning microscopy, autoradiography following (3)H-uridine incubation and transmission electron microscopy were used to evaluate the nucleolar protein localization, transcriptional activity, and nucleolar ultrastructure during genomic re-programming in bovine embryos reconstructed by nuclear transfer from granulosa cells into non-activated cytoplasts followed by activation. During the 1st cell cycle (1-cell embryos), no autoradiographic labelling was detected. Ultrastructurally, nucleoli devoid of a granular component were observed. During the 2nd cell cycle (2-cell embryos) autoradiographic labelling was also lacking and the embryos displayed varying degrees of nucleolar inactivation. During both the 3rd (4-cell embryos) and 4th (tentative 8-cell embryos), cell cycles autoradiographic labelling was lacking in some embryos, while others displayed labelling and associated formation of fibrillo-granular nucleoli. During the 5th cell cycle (tentative 16-cell embryos), all embryos displayed autoradiographic labelling and fibrillo-granular nucleoli. In some blastomeres, however, deviant nucleolar ultrastructure was observed. During the first cell cycle labelling of RNA polymerase I, fibrillarin, upstream binding factor (UBF) and nucleolin (C23) was localized to nuclear entities. During the 2nd cell cycle, only labelling of RNA polymerase I and fibrillarin persisted. During the 3rd and 4th cell cycle labelling of fibrillarin persisted, labelling of nucleophosmin (B23) appeared and that of nucleolin re-appeared. During the 5th cell cycle almost all embryos showed complete labelling of all proteins except for UBF, which lacked in more than half of the embryos. In conclusion, bovine granulosa cell nuclear transfer embryos showed re-modelling of the nucleoli to an inactive form followed by re-formation of fibrillo-granular nucleoli. The re-formation of fibrillo-granular nucleoli was initiated already during the 3rd cell cycle, which is one cell cycle earlier than in in vivo- and in vitro-derived bovine embryos. Moreover, in more than half of the embryos, UBF could not be immunocytochemically localized to the nucleolar compartment during the 5th cell cycle indicating lack of developmental potentials.

Gene expression during pre- and peri-implantation embryonic development in pigs.

Embryo technological procedures such as in vitro production and cloning by nuclear transfer are not as advanced in pigs as in cattle and cannot yet be applied under field conditions. The present paper focuses on genome activation in in vivo-derived, in vitro-produced and nuclear transfer pig embryos with special emphasis on the development of embryonic nucleoli, where the ribosomal RNA (rRNA) genes transcribed can be used as markers for genome activity. In addition, contemporary data on gene expression in in vivo-derived pig embryos are reviewed. In in vivo-derived pig embryos, pronounced transcription is initiated at the four-cell stage (the third cell cycle after fertilization), when nucleoli develop. In parallel with the development of the nucleoli as a result of rRNA gene activation, a cascade of other genes is also likely to be transcribed. However, apart from identification of transcripts for the oestrogen receptor at the blastocyst stage, reports on mRNAs resulting from initial transcription of the pig embryonic genome are lacking, in contrast to the situation in cattle and, in particular, mice. More information is available on gene expression during elongation of pig conceptuses, when the genes for steroidogenic enzymes, extracellular matrix receptors, oestrogen receptors, growth factors and their receptors, as well as retinol binding protein and retinoic acid receptors, are expressed. Nucleolus development appears to be disturbed in in vitro-produced pig embryos and in pig embryos reconstructed by nuclear transfer of granulosa cells to enucleated metaphase II oocytes produced by oocyte maturation in vivo or in vitro, which is indicative of disturbances in activation of rRNA genes.