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BOR - Papers in Press, published online ahead of print October 29, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.021683
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BIOLOGY OF REPRODUCTION 70, 867–876 (2004)
DOI: 10.1095/biolreprod.103.021683
© 2004 by the Society for the Study of Reproduction, Inc.

Embryo

Expression of Nucleolar-Related Proteins in Porcine Preimplantation Embryos Produced In Vivo and In Vitro1

Bolette Bjerregaard3, Christine Wrenzycki4, Frantisek Strejcek5,6, Jozef Laurincik5,6, Peter Holm7, Robert L. Ochs8, Christine Rosenkranz9, Henrik Callesen7, Detlef Rath4, Heiner Niemann4, and Poul Maddox-Hyttel2,3

Department of Anatomy and Physiology,3 Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Denmark Department of Biotechnology,4 Institute for Animal Science (FAL), Mariensee, 31535 Neustadt, Germany Constantin the Philosopher University,5 SK-949 74 Nitra, Slovak Republic Research Institute of Animal Production,6 SK-949 74 Nitra, Slovak Republic Section of Reproductive Biology,7 Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, 8830 Tjele, Denmark Precision Therapeutics,8 Pittsburgh, Pennsylvania 15213 Veterinary University,9 Vienna, A-1210 Wien, Austria


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-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 RT-PCR up to the blastocyst stage. Toward the end of third cell cycle, the nuclei in non-{alpha}-amanitin-treated, in vivo-produced embryos displayed different stages of transformation of the nuclear precursor bodies (NPBs) into fibrillogranular nucleoli associated with autoradiographic labeling. However, on culture with {alpha}-amanitin, NPBs were not transformed into a fibrillogranular nucleolus during this cell cycle, demonstrating that embryonic nucleogenesis requires de novo mRNA transcription. Moreover, immunolocalization of RNA Pol I, but not of UBF, and the mRNA expression of PAF53 and UBF were significantly reduced or absent after culture with {alpha}-amanitin, indicating that RNA Pol I, PAF53, and presumably, UBF are derived from de novo embryonic transcription. Embryonic genomic activation was delayed in porcine embryos produced in vitro compared to the in vivo-derived counterparts with respect to mRNAs encoding PAF53 and UBF. Moreover, differences existed in the mRNA expression patterns of pRb between in vivo- and in vitro-developed embryos. These findings show, to our knowledge for the first time, a nucleolus-related gene expression in the preimplantation porcine embryo, and they highlight the differences in quality between in vivo and in vitro-produced embryos.

developmental biology, early development, embryo, gene regulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Initial development of the mammalian preimplantation embryo is regulated by gene transcripts and polypeptides produced by, and stored in, the oocyte during its development [1]. However, following one to three cleavage divisions, the embryonic genome gains control of development, and the maternally derived transcripts and proteins are gradually degraded [2–4]. This transition from maternal to embryonic control of development is a gradual event [5], and in pigs, the major transcriptional activation occurs during the third cell cycle (i.e., the 4-cell stage) [6–9].

The major transcriptional activation of the embryonic genome includes formation of a functional fibrillogranular nucleolus signaling activation of ribosomal RNA (rRNA) genes and de novo ribosomal synthesis [7, 10, 11]. The functionally active nucleolus consists of fibrillar centers (FCs) that are surrounded by the dense fibrillar component (DFC), which is embedded in the granular component (GC) [12]. The FCs house the enzymatic apparatus for the transcription, and the DFC carries the primary nascent transcript. The GC represents processed transcripts associated with proteins in the form of preribosomal particles.

Recently, the key proteins involved in rRNA gene transcription, such as topoisomerase I, RNA polymerase I (RNA Pol I), and upstream binding factor (UBF), as well as in early (fibrillarin) and late (nucleolin and nucleophosmin) rRNA processing have been localized to the developing nucleolus in porcine embryos during in vivo development [9] and in vitro production (IVP) [13]. The IVP embryos displayed a substantial delay in, or even lack of, development of functional nucleoli, indicating an aberrant activation of rRNA genes [13]. This is in contrast to cattle, in which IVP embryos do not display disturbances in rRNA gene activation as measured by nucleolus development [14, 15].

Information regarding gene transcription patterns in porcine preimplantation embryos is scarce [16]. With the exception of genes encoding the estrogen receptor [17], the epidermal growth factor (EGF), and the EGF receptor [18], to our knowledge no mRNAs derived from the newly activated porcine embryonic genome have been identified. The quantity of cdc25c and cyclin B1 gene transcripts has been analyzed both before and at the time of genomic activation in 4-cell in vivo-derived and IVP embryos. No difference was observed between the mRNA levels of cyclin B1 or cdc25C in the two types of embryos, and inhibition with {alpha}-amanitin did not change the levels significantly [19–21].

The available data concerning gene expression in preimplantation embryos are predominately of bovine and murine origin and clearly indicate that mRNA phenotypes obtained from IVP embryos differ from those obtained from in vivo-developed embryos [22, 23]. Moreover, several studies have shown changes in mRNA expression of developmentally important genes involved in metabolism (glucose transporter-1), compaction and cavitation (connexin 43), stress adaptation (heat shock protein 70), and mRNA processing (poly[A] polymerase) during murine and bovine preimplantation development depending on the composition of the culture medium [24–27].

The first aim of the present study was to unravel embryonic genome activation in detail in in vivo-derived, porcine preimplantation embryos cultured with and without {alpha}-amanitin by employing transmission-electron microscopy, immunocytochemistry, and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of key nucleolar proteins. The second aim was to compare the chronology of the major transcriptional activation of the embryonic genome via semiquantitative RT-PCR of two key nucleolar proteins (RNA Pol I-associated factor PAF53 and UBF) in preimplantation porcine embryos and to elucidate potential differences between IVP and in vivo-derived embryos.

The initial cell-cycle progression during preimplantation embryonic development is independent of exogenous mitogens; that is, embryos can undergo cleavage without addition of any growth factors or serum [28]. The duration of cell cycles in the early embryo is shorter than in somatic cells because of a shortened G1 phase, except for the cell cycle in which the activation of the embryonic genome occurs [29, 30]. At present, however, the molecular mechanisms of the shortened G1 phase in preimplantation porcine embryos have not yet been clarified.

The retinoblastoma family of "pocket proteins," which includes the RB gene product (pRb) and the related p130 protein, is involved in cell-cycle regulation, differentiation, and cell death [31–33]. Progression of the cell cycle beyond the G1/S-phase transition requires the inactivation of the pRb function [34, 35]. In the G1 phase, pRb binds to and represses the E2Fs, a family of transcriptional regulators that transactivate genes important for S-phase entry [36, 37]. When pRb is inactivated by phosphorylation by cyclin D-dependent kinases, the E2Fs are released, and the cells enter the S phase [37, 38]. It is therefore postulated that pRb is absent or inactivated continuously in preimplantation embryos so that they may enter the S phase immediately after the end of the previous M phase [39]. Consequently, our third aim was to study the importance of pRb and p130 for early porcine development via semiquantitative RT-PCR of IVP or in vivo-derived embryos.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. The experiments were conducted according to the rules of the German animal welfare law and were licensed by the local authorities. This is in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

IVP of Porcine Embryos

The IVP embryos were obtained after in vitro maturation of oocytes followed by in vitro fertilization and in vitro culture as described previously [40]. Briefly, ovaries were collected from prepubertal gilts slaughtered at a nearby abattoir and transported within 1–2 h to the laboratory in a prewarmed thermos at 20–25°C. Follicles with a diameter of 2–5 mm were punctured with a blunt needle, and the follicular content was aspirated into 50-ml Falcon tubes with the flow rate set to 20 ml/min. Aspirates were allowed to settle for 10 min at room temperature. Subsequently, they were washed twice in mPBS (Dulbecco PBS supplemented with 1% newborn calf serum) and then distributed into 100-mm Petri dishes. Cumulus-oocyte complexes (COCs) were evaluated under a stereomicroscope, and only intact COCs with at least three cumulus cell layers and an evenly granulated ooplasm were selected and transferred into 50-mm Petri dishes that had been prefilled with mPBS.

The selected COCs were washed three times in prewarmed PBS and placed in maturation medium (MM): BSA-free NCSU 37 [41] supplemented with 5 mg/ml of insulin, 10% porcine follicular fluid (v/v), 50 µM ß-mercaptoethanol, 6 mM L-cysteine, 10 ng/ml of EGF, 0.065 mg/ml of penicillin G, and 0.05 mg/ml of streptomycin. For the first 24 h of maturation, 10 IU/ml of hCG and 1 mM db-cAMP were added to the MM. After 24 h of incubation at 38.5°C and 5% CO2 in humidified air, the COCs were placed into MM without hCG and db-cAMP and incubated for another 22 h.

For in vitro fertilization, matured COCs with maximally expanded, intact cumulus cells were selected and mechanically denuded before being placed into TALP (Tyrode-albumin-lactate-pyruvate) fertilization medium [40] in 90-µl drops containing 50 oocytes each. Frozen epididymal semen (0.75 ml) was thawed, diluted in 10 ml of prewarmed (38°C) Androhep (Minitüb, Tifenbach, Germany), and washed once (400 x g, 3 min). The supernatant was discharged, and the remaining pellet was resuspended in 430 µl of TALP. The final concentration was adjusted to 1500 sperm per oocyte in 10 µl of TALP. At the end of in vitro maturation and at 18 h postinsemination, approximately 10% of the oocytes and presumptive zygotes were fixed in acetic acid-alcohol (1:3, v/v) and, 24 h later, stained with aceto-orcein (1%, v/v) [42] to analyze rates of nuclear oocyte maturation and fertilization. Moreover, rates of cleavage and blastocyst formation were recorded, and during the period when the present experiments were carried out, 91% of the oocytes matured to metaphase II and the rate of polyspermy was 24%. Of the inseminated oocytes, 56% displayed monospermic fertilization, 61% cleaved, and 27% developed to blastocysts. On average, our IVP blastocysts contained 20–30 cells, whereas the in vivo-produced blastocysts contained 60–70 cells.

Presumptive zygotes were cultured in vitro in NCSU 23 [41] at 39°C in a humidified atmosphere with 5% CO2 in air into the subsequent cell cycles. Around the time of expected cleavage, the embryos were examined every second hour to detect the time of cleavage. Embryos that did not progress to the subsequent cell cycle were excluded from further experimentation. The 2-cell embryos were harvested at 5 h postcleavage (hpc) to the 2-cell stage, and 4-cell embryos were harvested at 1–4, 15, and 30 hpc to the 4-cell stage. Because of asynchrony between blastomeres, collection of the embryos during the fourth (8-cell stage) and fifth (16-cell stage) cell cycles is not precise. Thus, the 8- and 16-cell embryos are referred to as tentative developmental stages. Tentative 8-cell embryos were harvested at 10 hpc to the 8-cell stage, tentative 16-cell embryos at 10 hpc to the 16-cell stage, and blastocysts at day 5 postinsemination according to morphological evaluation.

Production of Porcine Embryos In Vivo

Prepubertal gilts received intramuscular injections of 1500 IU of eCG (Intergonan; Intervet, Unterschleissheim, Germany), followed by 500 IU of hCG (Ekluton; Intervet) 72 h later. At 24 and 36 h after hCG, the gilts were inseminated with 3 billion spermatozoa from a fertility-tested boar. Subsequently, the gilts were slaughtered at the institute's abattoir (Mariensee) at 48–56 h after hCG injection to obtain the 1-cell stage, at 70–74 h to collect the 2- to 4-cell stages, at 94–98 h to collect the 4-cell stage, at 118–122 h to collect the 4- to 8-cell stages, and at 142–146 h to collect the 8- to 16-cell stages. The oviducts and uteri were flushed, and presumptive zygotes and embryos were recovered. The ova were then cultured in vitro in NCSU 23 [41] at 39°C in a humidified atmosphere with 5% CO2 in air into the subsequent cell cycles. Around the time of expected cleavage, the embryos were examined every second hour to detect the onset of cleavage. Embryos that did not progress to the subsequent cell cycle were excluded from further experimentation. Subsequently, 2-cell embryos were harvested at 5 hpc for RT-PCR and 4-cell embryos at 1–4, 10, 15, 20, 25, 30, 40, and 50 hpc for RT-PCR, light-microscopical autoradiography, transmission-electron microscopy, and immunocytochemistry. Tentative 8-cell embryos were harvested at 10 and 20 hpc, tentative 16-cell embryos at 10 hpc, and blastocysts at day 5 according to morphological evaluation.

Inhibition of mRNA Synthesis in Embryos Produced In Vivo

A portion of the in vivo-produced 2-cell embryos were cultured in the presence of {alpha}-amanitin (25 µg/ml) [43] into the subsequent cell cycle and processed for light-microscopical autoradiography, transmission-electron microscopy, and immunocytochemistry at 10, 20, 30, 40, and 50 hpc. For RT-PCR, the 2-cell embryos were cultured with {alpha}-amanitin into the subsequent cell cycle and harvested at 1–4, 15, and 30 hpc.

Processing of Embryos for Light-Microscopical Autoradiography and Transmission-Electron Microscopy

The 4-cell, in vivo-produced, {alpha}-amanitin-cultured embryos at 10, 20, 30, 40, and 50 hpc and untreated 4-cell embryos at 30, 40, and 50 hpc were incubated for 20 min in NCSU 23 medium supplemented with 100 µCi/ml of [3H]uridine (specific activity, 962 GBq/mmol; Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany) at a final concentration of 40 MBq/mM [7]. After incubation with precursor, the embryos were repeatedly washed in [3H]uridine-free culture medium and fixed for 1 h at 4°C in 3% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2). Subsequently, they were washed in 0.1 M sodium phosphate buffer, postfixed in 1% OsO4 in 0.1 M sodium phosphate buffer, embedded in Epon (Merck, Darmstadt, Germany), and serially sectioned into semithin sections (thickness, 2 µm). Every second section was stained with basic toluidine blue and evaluated by bright-field light microscopy. Selected semithin sections were re-embedded according to the method described by Hyttel and Madsen [44] and processed for ultrathin sectioning (thickness, 70 nm). The ultrathin sections were examined on a Philips CM100 transmission-electron microscope (Philips, Eindhoven, The Netherlands). Neighboring unstained semithin sections were processed for autoradiography for detection of total RNA synthesis and nucleolus-associated RNA synthesis. The sections were coated with Ilford K5 liquid nuclear emulsion (Ilford; Basildon, Essex, UK) and exposed for 6 wk at 4°C. Finally, the specimens were developed in Kodak D19 (Kodak, Rochester, NY) at 17°C, stained with basic toluidine blue, and evaluated by bright-field and epipolarized light microscopy.

Whole-Mount Immunolabeling of Embryos

Antibodies The following primary antibodies against key nucleolar proteins, kindly provided by Dr. R.L. Ochs, were used: mouse monoclonal antinucleolin (C23; 1:1000) [45], mouse monoclonal antinucleophosmin (B23; 1:1000) [45], human antifibrillarin (1:1000) [46], human anti-topoisomerase I (1:100) [47], human anti-RNA Pol I (1:500) [48], and human anti-UBF (1:500) [49].

Immunofluorescence For indirect immunofluorescence, in vivo-produced 4-cell embryos cultured in the presence of {alpha}-amanitin at 10, 20, 30, 40, and 50 hpc and nontreated 4-cell embryos at 30, 40, and 50 hpc were harvested. Tentative in vivo-produced 8-cell embryos were harvested at 10 and 20 hpc. Subsequently, embryos were liberated from zona pellucida by 0.5% pronase treatment and fixed in a mixture of 4% paraformaldehyde and 0.1% Triton X-100 for 3 h at 4°C. The specimens were washed in 1% Triton X-100 in PBS and preincubated for 2 h with 5% rabbit serum (DakoCytomation, Glostrup, Denmark) in PBS (blocking buffer) at room temperature. Thereafter, they were incubated with the primary antibodies diluted in blocking buffer overnight at 4°C. Excess primary antibodies were removed by extensive washing in PBS before a 4-h (at 4°C) and 1-h (at room temperature) incubation in rabbit anti-human/biotin (for anti-topoisomerase I, anti-RNA-polymerase I, anti-UBF, and antifibrillarin; DakoCytomation) or rabbit anti-mouse/biotin (for antinucleolin and antinucleophosmin; DakoCytomation) diluted in blocking buffer. The secondary antibodies were visualized by streptavidin/fluorescein isothiocyanate (DakoCytomation) in PBS. Finally, the specimens were mounted on glass slides using Dako fluorescent mounting medium (DakoCytomation) and examined on a Leica confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany). Control immunostaining of unspecific labeling by the secondary antibody was performed by omitting the primary antibodies.

RNA Isolation and RT-PCR

For analysis of mRNA expression of the RNA Pol I-associated factor PAF53, UBF, pRb, and p130 in the in vivo-developed and IVP embryos, 2-cell embryos were harvested at 5 hpc and 4-cell embryos at 1–4, 15, and 30 hpc. Tentative 8-cell embryos were harvested at 10 hpc, tentative 16-cell embryos at 10 hpc, and blastocysts at day 5 according to morphological evaluation.

After washing three times in PBS containing 0.1% polyvinyl alcohol, the embryos from each of the seven developmental stages defined above were stored as single embryos or as pools of five embryos at -80°C in a minimum volume (<5 µl) of medium until experimental use.

Before RNA isolation, 1 pg of rabbit globin RNA (Gibco, Invitrogen, Carlsbad, CA) was added to the samples as an internal standard. Subsequently, Poly(A)+ RNA was isolated from single embryos or pools of five embryos using Dynabeads (Dynal Biotech, Oslo, Norway) as described previously [27] and was then used immediately for RT, which was carried out in a total volume of 20 µl using 2.5 µM random hexamers (Perkin-Elmer, Vaterstetten, Germany) to get the widest array of cDNA. The RT reaction mixture consisted of 1 x RT buffer (pH 8.3, 50 mM KCl, 10 mM Tris-HCl), 5 mM MgCl2, 1 mM each dNTP, 20 IU of RNase inhibitor (Perkin-Elmer), and 50 IU of MuLV reverse transcriptase (Perkin-Elmer). The RT reaction was carried out at 25°C for 10 min and 42°C for 1 h, followed by a denaturation step at 99°C for 5 min and flash cooling on ice.

The PCR primers were designed from the coding regions of each gene sequence using the Primer 3 software (http://broad.mit.edu/cgi-bin/primer/primer3_www.cgi). For each pair of gene-specific primers, semilog plots of the fragment intensity as a function of cycle number were used to determine the range of cycle numbers over which linear amplification occurred, and the number of PCR cycles was kept within this range [27]. Because the total efficiency of amplification for each set of primers during each cycle is not known, such an assay can only be used to compare relative abundances of one mRNA among different samples [50].

The PCR-reaction was performed with a gene-specific cDNA embryo equivalent and 50 fg of globin RNA in a final volume of 50 µl of 1 x PCR buffer (20 mM Tris-HCl [pH 8.4], 50 mM KCl), 1.5 mM MgCl2, 200 µM each dNTP, 1 µM each sequence-specific primer, and 0.5 µM each globin primer using a PTC-200 thermocycler (MJ Research, Waltham, MA). To ensure specific amplification, a "hot-start" PCR was employed by adding 1 IU of Taq DNA polymerase (Gibco) at 72°C.

The PCR program employed an initial step of 97°C for 2 min and 72°C for 2 min (hot start), followed by different cycle numbers of 15 sec each at 95°C for DNA denaturation, 15 or 25 sec at different temperatures for annealing of primers, and 15 sec at 72°C for primer extension. The last cycle was followed by a 5-min extension at 72°C and cooling to 4°C. As negative controls, tubes were prepared in which RNA or reverse transcriptase was omitted during the RT reaction (data not shown). The sequences of the primers used, the annealing temperatures, the gene-specific embryo equivalents, and the fragment sizes are summarized in Table 1. The primer pair to detect these mRNAs was first designed from the homologue sequence, and the product was then sequenced. The resulting "porcine-specific" sequence was used to create the primer pair employed to detect the transcript of interest.


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  		TABLE 1. Primers used for PCR

The RT-PCR products were subjected to electrophoresis on a 2% agarose gel in 1 xTBE buffer (pH 8.3, 90 mM Tris, 90 mM borate, 2 mM EDTA) containing 0.2 µg/ml of ethidium bromide. Additional ethidium bromide at the same concentration was added to the running buffer. The image of each gel was recorded using a CCD camera (Quantix; Photometrics, München, Germany) and the IPLab Spectrum program (Scanalytics, Fairfax, VA). The intensity of each band was assessed by densitometry using an image-analysis program (IPLab Gel, Scanalytics). The relative amount of the mRNA of interest was calculated by dividing the intensity of the band for each developmental stage by the intensity of the globin band for the corresponding stage. Experiments were repeated with at least eight embryos for each mRNA.

The general RNA recovery rate was estimated as the ratio between the intensity of the globin band with and without RNA preparation procedure, starting with an equivalent of 50 fg of globin in the PCR reaction. On average, 46% of poly(A)+-tailed RNA was recovered using the Dynabeads oligo-d(T) mRNA isolation method, which corresponds well to other published yields [51]. The number of replicates was calculated to get an acceptable repeatability of the assay (0.90). The average repeatability (precision) of the assay varied from 0.60 to 0.70. Therefore, a minimum of 10 replicates was performed.

Statistical Analysis

Relative abundances were analyzed using the SigmaStat 2.0 (Jandel Scientific, San Rafael, CA) software package. After testing for normality (Kolmogorov-Smirnov test with Lilliefor correction) and for equal variance (Levene median test), an ANOVA followed by multiple pairwise comparisons using the Tukey test was employed. Differences of P <= 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Autoradiography and Ultrastructure

Nontreated in vivo-produced embryos Nontreated in vivo-produced 4-cell embryos at 30 hpc (n = 3), 40 hpc (n = 3), and 50 hpc (n = 3) displayed autoradiographic labeling dispersed over the entire nucleoplasm and concentrated over the nucleoli in all blastomeres (Fig. 1a). At the ultrastructural level, nuclei displayed different stages of nucleolus formation, ranging from nucleolus precursor bodies (NPBs), appearing as electron-dense spheres of densely packed fibrillar material, to fibrillogranular nucleoli, presenting semilunar formations of FCs, DFC, and GC on the surface of the NPBs that were more or less encapsulated (Fig. 1c). Different stages of nucleolus development were observed within the same nucleus.



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  		FIG. 1. Light-microscopical autoradiographs (a and b) and transmission-electron micrographs (c and d) of nontreated (a and c) and {alpha}-amanitin-treated (b and d) porcine in vivo-produced embryos at 50 hpc to the 4-cell stage. Note the autoradiographic labeling over the nucleolus (arrowhead) and nucleoplasm in nontreated embryos (a). Correspondingly, nontreated-embryo nuclei displayed nucleolus precursor bodies (NPB) with semilunar formation of FCs, DFCs, and GC on the surface (c). {alpha}-Amanitin-treated embryos did not display autoradiographic labeling even over the NPB (arrowhead in b), and NPBs lacked any sign formation (d). NE, nuclear envelope

{alpha}-Amanitin-treated in vivo embryos None of the 4-cell embryos showed autoradiographic labeling at 10 hpc (n = 5), 20 hpc (n = 5), 30 hpc (n = 5), 40 hpc (n = 5), or 50 hpc (n = 5) after culture with {alpha}-amanitin (Fig. 1b). At the ultrastructural level, the embryos presented nuclei with one, two, or rarely, three NPBs but no formation of fibrillogranular nucleoli (Fig. 1d).

Whole-Mount Immunolabeling of Embryos

Nontreated In Vivo-Produced Embryos In 4-cell embryos (n = 180), RNA Pol I was the only protein localized to nuclear entities from 30 hpc (Table 2 and Fig. 2). The labeling of RNA Pol I was localized to small foci at the surface of the presumptive NPBs. However, nucleophosmin displayed diffuse nucleoplasmic labeling above background at 30 hpc. From 40 hpc, nucleophosmin and nucleolin were localized to shell-like structures surrounding the presumptive NPBs in addition to the nucleoplasmic labeling, which was weak at 50 hpc.


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  		TABLE 2. Numbers of porcine embryos developed in vivo displaying localization of key nucleolar proteins to nuclear entities during the third and fourth cell cycle.a



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  		FIG. 2. Confocal laser-scanning microscopy of single nuclei from in vivo-produced 4-cell embryos cultured with (+AA) or without (-AA) 25 µg/ml of {alpha}-amanitin at 30, 40, and 50 hpc labeled with antibodies against the nucleolar proteins RNA Pol I, nucleophosmin, and nucleolin. In nontreated (-AA) embryos, RNA Pol I was localized to small foci surrounding the presumptive NPBs from 30 hpc. At this time point, nucleophosmin only displayed diffuse nucleoplasm labeling. From 40 hpc, nucleophosmin and nucleolin were localized to shell-like structures surrounding the presumptive NPBs in addition to nucleoplasmic labeling. In {alpha}-amanitin-treated embryos, the labeling of RNA Pol I was not detectable throughout the 4-cell stage. Nucleophosmin displayed nucleoplasmic labeling at 40 and 50 hpc, whereas nucleolin did so at 30 and 50 hpc. At 40 hpc, nucleophosmin displayed a few densely labeled areas in addition to the nucleoplasmic labeling. Nucleolin, on the other hand, displayed the same shell-like labeling as in nontreated embryos. At 50 hpc, nucleophosmin displayed only nucleoplasmic labeling, whereas nucleolin displayed a shell-like labeling, as in nontreated embryos. The lower panel illustrates the localization of all the six nucleolar proteins examined in tentative 8-cell embryos

Topoisomerase I, fibrillarin, and UBF, however, were immunocytochemically undetectable until the early 8-cell stage (data not shown). At the 8-cell stage, labeling of fibrillarin, UBF, topoisomerase I (weak), and RNA Pol I was localized to small foci surrounding the presumptive NPBs at both 10 and 20 hpc. At the same time points, labeling of nucleolin and nucleophosmin was localized to more or less shell-like bodies, probably surrounding the NPBs. A weak nucleoplasmic labeling was observed at both sampling points with all antibodies except antifibrillarin.

{alpha}-Amanitin-treated embryos

In {alpha}-amanitin-treated 4-cell embryos (n = 161), the labeling of RNA Pol I was not detectable at any time point throughout the third cell cycle. At 30 hpc, diffuse nucleoplasmic labeling of nucleolin was seen. At 40 hpc, nucleophosmin displayed diffuse nucleoplasmic labeling and, in addition, a few densely labeled areas. Nucleolin displayed the same shell-like labeling as in nontreated embryos. At 50 hpc, nucleolin still displayed a shell-like labeling, whereas nucleophosmin showed diffuse nucleoplasmic labeling.

mRNA Expression of Nucleolar-Related Proteins

Nontreated in vivo-produced and IVP embryos To analyze whether the proteins involved in rRNA synthesis and cell-cycle regulation were regulated at the mRNA level in porcine in vivo-developed and IVP embryos, gene transcripts were examined by a semiquantitative RT-PCR assay in single embryos (n = 630) and, for pRb, in pools of five embryos (n = 160 in total). Representative gel photos and the mean value derived from 8 to 12 replicates for the calculated relative abundance of the mRNA expression of UBF, PAF53, pRb, and p130 are shown in Figures 3, 4 and 5 respectively.



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  		FIG. 3. Relative abundance of the UBF gene transcript (values shown as the mean ± SEM) during porcine preimplantation development (2C, 2-cell; 4e, 4-cell 1–4 hpc; 4m, 4-cell 15 hpc; 4l, 4-cell 30 hpc; 8C, 8-cell; 16C, 16-cell; BL, blastocyst) both in vivo (black bars) and in vitro (open bars). Significant differences (P < 0.05) are indicated by an asterisk. Representative gel photograph of the UBF gene transcript in both in vivo-developed and IVP embryos is presented at the bottom. Each lane represents the RT-PCR product derived from Poly(A)+ RNA from the equivalent of 0.4 embryos



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  		FIG. 4. Relative abundance of the PAF53 gene transcript (values shown as the mean ± SEM) during porcine preimplantation development (2C, 2-cell; 4e, 4-cell 1–4 hpc; 4m, 4-cell 15 hpc; 4l, 4-cell 30 hpc; 8C, 8-cell; 16C, 16-cell; BL, blastocyst) both in vivo (black bars) and in vitro (open bars). Significant differences (P < 0.05) are indicated by an asterisk. Representative gel photograph of the PAF53 gene transcript in both in vivo- and in vitro-produced porcine preimplantation embryos is presented. Each lane represents the RT-PCR product derived from Poly(A)+ RNA from the equivalent of 1.0 embryo



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  		FIG. 5. Representative gel photographs of the RB gene transcript in both in vivo-developed (upper panel) and IVP (lower panel) embryos. Each lane represents the RT-PCR product derived from Poly(A)+ RNA from the equivalent of 5.0 embryos

The mRNA expression of PAF53 and UBF varied during preimplantation development (Figs. 3 and 4). A significant difference existed between the relative abundance of in vivo-developed compared to IVP embryos at the 8-cell and blastocyst stages. The relative abundance of PAF53 expression was 2.1 ± 0.4 and 1.0 ± 0.3 in 8-cell embryos (mean ± SEM, P < 0.05) and 5.8 ± 0.4 and 0.9 ± 0.3 in blastocysts (P < 0.001) in the in vivo-developed and IVP embryos, respectively. The relative abundance of UBF was 1.1 ± 0.1 and 0.5 ± 0.1 in 8-cell embryos (P = 0.001) and 2.1 ± 0.3 and 0.3 ± 0.1 in blastocysts (P < 0.001) in the in vivo-developed and IVP embryos, respectively.

The mRNA expression of p130 was unaffected during preimplantation development from zygote to the 16-cell stage regardless of whether the embryos were in vivo-produced or IVP (data not shown). However, at the blastocyst stage, a significant difference (P < 0.05) was observed between in vivo-developed (0.6 ± 0.2) and IVP (0.3 ± 0.2) embryos.

The mRNA expression of pRb was undetectable in single embryos and, therefore, was analyzed in pools of five embryos (Fig. 5). The relative abundance of pRb mRNA expression varied during preimplantation development in both in vivo-developed and IVP embryos, with maximum values at the 2-cell stage. In embryos produced in vivo, the relative abundance of pRb was high (3.5) at the 2-cell stage, then decreased to an undetectable level from the mid-4-cell stage before reappearing at the 16-cell and blastocyst stages (0.4 and 1.6, respectively). In the IVP embryos, the relative abundance of pRb was highest at the 2-cell and early 4-cell stages (2.7 and 2.5, respectively), then became undetectable from the mid-4-cell stage before increasing to a maximum abundance of 1.1 at the blastocyst stage.

{alpha}-Amanitin-treated in vivo-produced embryos To analyze whether the genes of interest were of maternal or embryonic origin, in vivo-developed 2-cell embryos (n = 75) were cultured with 25 µg/ml of {alpha}-amanitin and harvested at 1–4, 15, and 30 hpc into the 4-cell stage. The relative abundance of UBF, PAF53, and p130 was lower than in embryos cultured without {alpha}-amanitin (Fig. 6). The relative abundance of UBF was 1.3 in nontreated 4-cell embryos at 1–4 hpc and 0.1 in {alpha}-amanitin-treated embryos; thereafter, it became undetectable in the {alpha}-amanitin-treated specimens. However, the relative abundance of PAF53 decreased more slowly, from 0.6 (1.0 in non-{alpha}-amanitin-treated embryos) at 1–4 hpc to 0.2 and 0.1 at 15 and 30 hpc, respectively. The relative abundance of p130 was gradually reduced from 0.2 (0.3 in non-{alpha}-amanitin-treated embryos) at 1–4 hpc to 0.1 at both 15 and 30 hpc.



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  		FIG. 6. Relative abundance of the UBF, PAF53, and p130 transcripts (values shown as the mean ± SEM) in in vivo-developed porcine preimplantation embryos treated without (white bars) or with (black bars) {alpha}-amanitin


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In pigs, the major activation of the embryonic genome is observed during the third cell cycle (i.e., the 4-cell stage) [7–9, 52]. Usually, this major activation occurs in parallel with nucleolar formation, indirectly signaling transcription of the rRNA genes [7, 53]. Therefore, transcription of the rRNA genes and development of the nucleolus may serve as an indirect morphological marker of major activation of the embryonic genome, as proposed by Kopecny and Niemann [54].

During the end of the third cell cycle, the nuclei in non-{alpha}-amanitin-treated in vivo-developed embryos displayed, in both the present study and in others [9, 55], different stages of transformation of the NPBs into active fibrillogranular nucleoli showing autoradiographic labeling. However, on culture with {alpha}-amanitin, a specific inhibitor of RNA Pol II, the transformation of NPBs into fibrillogranular nucleoli was prevented. The nuclei displayed NPBs in all embryos examined, and they resembled nuclei from embryos before the embryonic genome activation. These data indicate that development of embryonic nucleoli requires de novo mRNA transcription. Moreover, porcine embryonic nucleologenesis and rRNA transcription have previously been shown to be inhibited by actinomycin D, in low concentrations a specific inhibitor of RNA Pol I [52]. This compound inhibits RNA Pol I in low concentrations and both RNA Pol I and II in higher concentrations. The concentration used in the cited investigation probably inhibited both RNA Pol I and II. Hence, in a more unspecific manner, these observations support the notion that embryonic transcription is required for nucleogenesis.

To drive transcription and subsequent processing of rRNA and to accommodate a number of specific functions, the nucleolus requires a panel of proteins, including topoisomerase I, RNA Pol I, UBF, fibrillarin, nucleophosmin, and nucleolin [12]. Each of these proteins has a fairly well-defined role in rRNA gene transcription and subsequent processing of transcripts [12]. In brief, topoisomerase I uncoils the DNA, allowing transcription. Next, RNA Pol I is bound to the rRNA genes by UBF, thereby activating the actual transcription. Finally, fibrillarin is involved in early processing of the transcripts, whereas nucleophosmin and nucleolin are involved in later processing.

Previously, we examined the localization of these key nucleolar proteins up to 30 hpc in 4-cell porcine embryos developed in vivo and showed that RNA Pol I was the only protein detectable at this time point [9]. In the present study, the protein localization in 4-cell embryos at 30, 40, and 50 hpc was analyzed. RNA Pol I was again detectable from 30 hpc and was spatially allocated to foci resembling the FCs of the fibrillogranular nucleolus on the surface of the NPBs. Moreover, labeling of both nucleophosmin and nucleolin appeared at 40 hpc and showed a shell-like pattern compatible with labeling of the GCs of the nucleolus. Again, this labeling pattern is compatible with the presence of fibrillogranular nucleoli on the surface of the NPBs and with the formation of fibrillogranular nucleoli toward the end of the third cell cycle. However, the absence of labeling of topoisomerase I, UBF, and fibrillarin remains an enigma and may be caused by the limitation of the immunocytochemical sensitivity.

To investigate whether the above-mentioned key nucleolar proteins are of embryonic origin, as the ultrastructural data of the {alpha}-amanitin-treated embryos suggested, in vivo-developed porcine embryos was cultured with {alpha}-amanitin, and immunocytochemistry as well as semiquantitative RT-PCR were performed. The addition of {alpha}-amanitin to the culture medium resulted in arrest of embryonic development at the 4-cell stage, which is in accordance with previous findings [56, 57]. Moreover, collectively, our data show that RNA Pol I, the RNA Pol I-associated factor PAF53, and presumably, UBF are translated from embryonic messengers. Thus, the protein localization of RNA Pol I, the mRNA expression of PAF53, and the mRNA expression of UBF was inhibited by {alpha}-amanitin. Surprisingly, the protein localization of UBF was not significantly reduced or absent after culture with {alpha}-amanitin. However, UBF may, to a certain degree, be translated from maternal messengers. Nucleophosmin and nucleolin were still detectable in embryos treated with {alpha}-amanitin, indicating that they, at least to some degree, are of maternal origin. However, the localization of nucleophosmin changed from a distinct, shell-like structure to a diffuse nucleoplasmic labeling as a result of {alpha}-amanitin. Hence, it is possible that nucleophosmin is present but that a putative protein targeting nucleophosmin to the nucleolus is inhibited by {alpha}-amanitin. This change in the localization pattern of nucleophosmin caused by the {alpha}-amanitin treatment probably hampers nucleolar function, because the particular proteins necessary for rRNA gene transcription and subsequent processing need to be localized to discrete nucleolar compartments to exert their functions. Topoisomerase I and fibrillarin were undetectable until the 8-cell stage; thus, their origin was not resolved in the present experiment.

Previously, we have examined the nucleolar proteins and ultrastructure in preimplantation porcine in vivo-developed and IVP embryos and shown that nucleolar formation is delayed or even lacking at IVP [9, 13]. Hence, fibrillogranular nucleoli were observed in some blastomeres in a single IVP embryo during the fifth cell cycle (i.e., the tentative 16-cell stage), during which formation of FCs, a DFC, and a GC 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 third cell cycle (i.e., the 4-cell stage) in some embryos. The UBF, RNA Pol I, and nucleophosmin were localized to the presumptive NPBs in a proportion of the embryos at the fourth cell cycle (i.e., the tentative 8-cell stage) and onward. Topoisomerase I was not localized to intranuclear entities even during the fifth postfertilization cell cycle. Moreover, a considerable proportion of the blastomere nuclei apparently did not show localization of the other nucleolar proteins. We believe that the hampered nucleolar function in porcine IVP embryos strongly contributes to their decreased viability.

In the present study, key nucleolar proteins (e.g., UBF) were immunocytochemically undetectable during the period when fibrillogranular nucleoli were emerging in vivo. This enigma may result from a lack of immunocytochemical sensitivity. To examine whether, first, the genes of such proteins are expressed at least at the transcriptional level and, second, differences between in vivo-developed and IVP embryos can also be detected at this level, we analyzed the mRNA expression of UBF and PAF53 in both in vivo-developed and IVP embryos. In these analyses, we focussed on UBF and PAF53, because they are of paramount importance for transcription of the rRNA genes. The mRNA expression of UBF and PAF53 varied during preimplantation development but increased to maxima at the blastocyst stage. The initial mRNA level determined at the 2-cell stage corresponded to the level found in fully grown porcine oocytes (unpublished results). Subsequently, the relative abundance of the mRNA expression of UBF and PAF53 gradually increased toward the 8-cell stage, at which they were significantly higher in embryos developed in vivo compared to IVP embryos, indicating that the embryonic genome activation had occurred in the former embryos. However, at the 16-cell stage, the relative abundance of the mRNA expression of UBF and PAF53 were again comparable in the two types of embryos, indicating that the embryonic genome activation was established in those IVP embryos that survive to the 16-cell stage. These findings support our recent data, showing that fibrillogranular nucleoli first appear at the 16-cell stage in some porcine IVP embryos as visualized by transmission-electron microscopy and autoradiography as well as immunocytochemical labeling of RNA Pol I and UBF [13].

At the blastocyst stage, the difference in the relative abundance in mRNA expression of UBF and PAF53 reappeared, and the in vivo expression was approximately sevenfold greater than in the IVP blastocysts. The difference in mRNA expression not only reflects a difference in cell number but also a difference in the expression at the single-cell level. Our data indicate that embryonic genome activation is delayed in IVP embryos and that the in vivo-produced blastocysts express higher levels of UBF and PAF53, indicating a higher capability of ribosomal biogenesis. The delayed and aberrant gene expression is believed to hamper the developmental potential of the IVP embryos significantly. Previous experiments in cattle have demonstrated that in that species, IVP embryos also display aberrant gene expression compared with their in vivo developed counterparts and that even the IVP system has an effect on the patterns of gene expression [23]. Moreover, in cattle, the process of in vitro culture of the embryos appears to have a more profound influence on the gene expression patterns than the process of in vitro oocyte maturation [58]. To our knowledge, whether a similar causal relationship exists in the pig is unknown at present.

In general, the major embryonic genome activation is associated with an increased duration of the cell cycle, as observed in the 4-cell porcine embryo. How this mechanism operates is unresolved. Therefore, we investigated the expression of key cell-cycle regulators, such as the pocket proteins pRb and p130, in porcine preimplantation in vivo-developed and IVP embryos by semiquantitative RT-PCR.

The mRNA expression of p130 remained unaffected during initial preimplantation development from the zygote to the 16-cell stage regardless of whether the embryos were developed in vivo or derived from IVP. However, at the blastocyst stage, a significant difference was found between in vivo and IVP embryos, possibly because of differences in cell numbers (the relative abundance in the in vivo-produced embryos was only twice that in IVP embryos). Moreover, the relative abundance of p130 messengers decreased in {alpha}-amanitin-treated embryos, indicating that this transcript is partly of embryonic origin.

The mRNA expression of pRb was undetectable in single porcine embryos; thus, it was analyzed in pools of five embryos. In 2-cell embryos produced in vivo, the relative abundance of pRb was similar to the one determined in fully grown oocytes (unpublished results). Subsequently, it decreased to an undetectable level from 15 hpc to the 4-cell stage and then reappeared at the 16-cell and blastocyst stages. This pattern indicates a shift in pRb messengers from maternal to embryonic origin. However, in the IVP embryos, the relative abundance of pRb was detectable at all stages except for 15 hpc to the 4-cell stage. The difference in the relative abundance of pRb between in vivo-developed and IVP embryos indicates that the cell-cycle regulation is disturbed around the time of the major genome activation in the IVP embryos.

Data from the present study and from murine preimplantation embryos [39, 59] suggest a relationship between the absence of pRb and the preimplantation embryo-specific cell-cycle progression. In mammalian preimplantation embryos, the G1 phase is substantially shortened, and DNA synthesis is initiated soon after mitosis. This indicates that all molecules required for the G1/S-phase transition are present during the preceding M phase or throughout the cell cycle. In somatic cells, expression of the molecules required for the G1/S-phase transition is regulated by the transcription factor, E2F, which is inhibited by pRb binding. The absence of pRb in the 4- to 16-cell stages in porcine embryos therefore might induce the constitutive activation of E2F and, hence, a constitutive ability for G1/S-phase transition.

Moreover, the observed difference in relative abundance of pRb between IVP and in vivo-developed embryos might be caused by a lack of degradation of maternal pRb messengers in IVP embryos. Activation of the embryonic genome is necessary for degradation of some maternal-derived mRNAs [56, 60]. Therefore, the delay in embryonic genome activation in the IVP embryos may cause a higher pRb level during the early embryonic cell cycles.

As mentioned, the rate of polyspermic fertilization during the IVP procedure was 24%. It may be argued that the deviant gene expression demonstrated in IVP versus in vivo-developed embryos may be associated with this considerable occurrence of polyspermic fertilization. It has been demonstrated that porcine zygotes with three or more pronuclei can develop to fetuses [61].

Only very few of these conceptuses, however, remain totally polyploid. Thus, in the majority, the polyploidy is eliminated. This fact supports the notion that polyspermic fertilization is not the major reason for the nucleolar abnormalities found in the porcine IVP embryos during the present investigation. Moreover, in a study of bovine IVP embryos, we clearly demonstrated that embryos displaying triploidy in all cells (indicating presumptive polyspermic fertilization) did not survive beyond the 8-cell stage (i.e., the stage of major genome activation in cattle) [62]. Hence, another possibility is that polyspermic embryos most likely are eliminated early during development (presumably before the 8-cell stage in the pig).

In conclusion, some of the key nucleolar proteins involved in development of embryonic nucleoli, such as RNA Pol I, the RNA Pol I-associated factor PAF53, and presumably, UBF, are transcribed de novo from the embryonic genome as shown by specific inhibition with {alpha}-amanitin. Moreover, porcine IVP embryos are delayed in the activation of the embryonic genome, which could be involved in the reduced developmental capacity of such embryos. Finally, the embryonic cell-cycle regulation as exerted by the pocket proteins pRb and p130 seems to be disturbed in IVP embryos compared to in vivo-produced embryos.


   ACKNOWLEDGMENTS
 
The authors are grateful to Ms. Jytte Nielsen for careful embedding and sectioning of specimens.


   FOOTNOTES
 
1 Supported by grants from "Disease models, disease prevention and animal welfare improvement: the pig embryo as a model," Danish Research Agency (9901178); NATO (978658); Deutsche Forschungsgemeinschaft (DFG); and Bundesministerium für Wissenschaft und Verkehr der Republik Österreich (GZ 45.517/I-VI/B/7a/2002). Back

2 Correspondence: Poul Maddox-Hyttel, Department of Anatomy and Physiology, Royal Veterinary and Agricultural University, Groennegaardsvej 7, DK-1870 Frederiksberg C, Denmark. FAX: 45 3528 2547; poh{at}kvl.dk Back

Received: 31 July 2003.

First decision: 19 August 2003.

Accepted: 17 October 2003.


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