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Regular Article |
a Department of Animal Science & Production and The Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Lyons Research Farm, Newcastle, County Dublin, Ireland
b Department of Anatomy & Physiology, Royal Veterinary & Agricultural University, DK-1870 Frederiksberg, Denmark
ABSTRACT
During the growth phase of the bovine oocyte transcripts, polypeptides and ribosomes are accumulated in the oocyte to drive and sustain future meiotic maturation, fertilization, and early embryonic development. The oocyte also furnishes the early embryo with the components required to establish a functional transcriptionally active nucleolus at the time of maternal embryonic transition. The aim of the present study was to describe the behavior of key components of the nucleolus. The temporal localization of nucleolar proteins fibrillarin, nucleophosmin, nucleolin, RNA polymerase I (RNA pol I), topoisomerase I, upstream binding factor (UBF), and coilin 5P10 was investigated in growing and fully grown immature bovine oocytes during in vitro maturation and during the first postfertilization cell cycle using whole-mount immunocytochemistry and confocal microscopy. During the oocyte growth phase, fibrillarin, nucleophosmin, nucleolin, RNA pol I, and UBF were localized to the oocyte nucleolus. On completion of the growth phase, nucleolin and nucleophosmin appeared to migrate to the periphery of the nucleolus and into the nucleoplasm, and the proportion of oocytes displaying RNA pol I localization had decreased. Topoisomerase I was not detected at any stage. Fibrillarin appeared to be localized to large foci within the nucleolus and/or nucleoplasm. Nucleophosmin and nucleolin labeling was characterized by a homogenous signal over the nucleolus. RNA pol I and UBF were characterized by the localization of the antibodies to individual or clustered foci in the nucleolus and/or nucleoplasm. Following oocyte nucleus breakdown (ONBD), the proteins appeared to disperse into the cytoplasm. All proteins were undetectable during meiotic maturation and were not relocalized until 5–10 h postinsemination (hpi). UBF was localized to the fertilizing sperm head of most zygotes at 5 hpi. By 10 hpi, all proteins were detected in most oocytes displaying two pronuclei. Nucleolar protein localization was exclusive to or more abundant in one pronucleus up to 20 hpi; thereafter, the pattern was more evenly distributed. Fibrillarin, nucleophosmin, nucleolin, UBF, and Pol I are present in the nuclei of growing and fully grown bovine oocytes until ONBD. They reappear at the late telophase stage of meiosis II and continue to be present up to the first mitotic division of embryo development.
early development, gametogenesis, gene regulation, oocyte development, ovum
INTRODUCTION
Meiosis is peculiar to eukaryotes that reproduce by the union of male and female gametes. In meiosis, a single replication is followed by two sequential divisions of the nucleus, which halves the chromosome number. During the first of these divisions, which is highly specialized and differs significantly from a mitotic division, the chromosome number is reduced to haploid, recombination takes place, and RNAs and proteins are synthesized in great quantites. The second meiotic division further reduces the DNA content to half by separating the sister chromatids, so that at fertilization the chromosome number returns to diploid. The second meiotic division proceeds much like mitosis in the same species except that it is not preceded by an S-phase.
Messenger RNAs, ribosomes, and polypeptides are produced by and accumulated in the oocyte during its growth phase, which corresponds to prophase I of meiosis. However, following fertilization and one to three of the ensuing mitotic divisions, the control of development is gradually taken over by the embryonic genome and the maternally derived transcripts and proteins are gradually degraded [1, 2]. For successful activation of the embryonic genome to occur and be maintained, the embryo must be able to establish a functional, transcriptionally active nucleolus. Therefore, the oocyte must furnish the early embryo with the components required to initiate nucleogenesis, rRNA transcription, and ribosome formation.
To produce adequate quantities of rRNA, the cell contains multiple copies of tandemly arranged rRNA genes. Because of their specific association with the nucleolus in the interphase cell, the specific chromosomal sites of the rRNA genes are referred to as the nucleolus organizer regions (NORs). The nucleolus is the site of rRNA transcription and ribosome subunit formation. The functionally active mammalian nucleolus consists of three components: the fibrillar centers (FCs), the dense fibrillar component (DFC), and the granular component (GC). An FC ranges from 100 nm to 1 µm in diameter and is composed of a loose meshwork of fibrils 4 to 8 nm thick. The FC houses the enzymatic apparatus for the transcriptional process. The DFC, which is composed of finely packed fibrils 3–5 nm thick and usually surrounds the FC, is believed to carry the newly formed transcripts. The GC is composed of granules 10–14 nm in diameter and represents processed transcripts associated with proteins in the form of preribosomal particles [3].
The nucleolus contains a variety of proteins [4]. Their functional roles are to control the transcription of the rRNA genes, to process and assemble the transcripts with other ribosomal proteins, and to transport the newly synthesized ribosomes to the cytoplasm. Previously, we described the changes in nucleolar morphology and transcriptional activity during the growth phase of the bovine oocyte [5, 6]. Our findings clearly demonstrated that the transcriptionally active fibrillogranular nucleolus found in growing oocytes is inactivated at an oocyte diameter of 110 µm, corresponding to an antral follicular diameter of approximately 3 mm, and furthermore remains dismantled until the eight-cell embryo stage [7]. Therefore, a functional ribosome-synthesizing nucleolus is lacking from the fully grown oocyte stage of development (prophase I of meiosis), throughout the two reduction divisions of meiosis, and during the first three mitotic cell cycles up to the fourth embryonic cell cycle. Thus, meiosis has a profound effect on nucleolar function. The behavior of nucleolar proteins during mitosis has been investigated extensively [8–11]. The chronology and morphology of the different stages of meiosis as they occur in the bovine oocyte during in vitro maturation have been described [12]. However, little is known about the molecular composition of oocyte nucleoli in cattle, and information on the behavior of nucleolar proteins during meiosis, particularly in mammalian oocytes, is limited. In vitro oocyte maturation and fertilization systems are ideal models for studying the behavior of these proteins during meiosis in the bovine oocyte.
In the present study, we investigated the distribution of key nucleolar proteins during bovine oocyte growth, maturation, and fertilization. Fibrillarin, nucleolin, and nucleophosmin are believed to play processing and structural roles within the nucleolus, topoisomerase I, RNA polymerase I (RNA pol I), and upstream binding factor (UBF) are thought to play roles in rRNA transcription and processing, and coilin 5P10 is considered to be involved in mRNA splicing and is thought to be a component of nucleolar precursor bodies (NPBs).
MATERIALS AND METHODS
Experimental Design
The present study included three experiments conducted to characterize the presence or absence of key nucleolar proteins during bovine oocyte growth, meiotic maturation, and fertilization.
Experiment 1 The localization of the nucleolar proteins fibrillarin, nucleolin, nucleophosmin, RNA pol I, topoisomerase I, and UBF was investigated in growing and fully grown bovine oocytes. Immature oocytes were assigned to one of the following size categories [13]: <100 µm, 100–110 µm, 110–120 µm, and >120 µm (n = 58, 50, 47, and 73, respectively).
Experiment 2 The fate of nucleolar proteins fibrillarin, nucleophosmin, and nucleolin and the presence of silver-staining nucleolar proteins (Ag NOR proteins) on the chromosomes was investigated in bovine oocytes at oocyte nucleus (ON), oocyte nucleus breakdown (ONBD), metaphase I (MI), and metaphase II (MII) stages of meiosis. Cumulus oocyte complexes (COCs) were removed from the in vitro maturation (IVM) system at 0, 6, 12, 18, and 24 h [14] (n = 39, 47, 35, 53, and 35, respectively).
Experiment 3 The initial reassembly of fibrillarin, nucleolin, nucleophosmin, RNA pol I, topoisomerase I, UBF, and coilin 5P10 was investigated during the first embryonic cell cycle. Presumptive zygotes were removed from the in vitro fertilization culture system at 0–3, 5–6, 10, 15, 20, and 24–25 h postinsemination (hpi) (n = 74, 127, 136, 192, 154, and 154, respectively) for immunocytochemistry and at 3 and 6 hpi (n = 48 and 44, respectively) for whole-mount Ag NOR silver staining.
Recovery of Growing and Fully Grown Oocytes
Growing and fully grown bovine oocytes were collected and categorized as described previously [13]. COCs were liberated from follicles by slicing ovaries with a series of razor blades secured parallel to each other. The COCs were washed in PBS, and their cumulus cells were removed by pipetting. The internal zona pellucida diameter of the denuded oocytes was measured and the oocytes were assigned to one of the following size categories: <100 µm, 100–110 µm, 110–120 µm, and >120 µm.
In Vitro Maturation
Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated. COCs were recovered from ovaries by aspirating the antral follicles visible from the surface. COCs were washed in modified PBS (PBS supplemented with 36 µg/ml pyruvate, 50 µg/ml gentamycin, and 0.5 mg/ml BSA, Sigma fraction V). Following washing, groups of approximately 50 COCs were placed in 500 µl of maturation medium and cultured for up to 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity. The maturation medium was Medium 199 supplemented with 10% v/v fetal calf serum and 10 ng/ml epidermal growth factor [15].
In Vitro Fertilization
Following IVM for 24 h, COCs were washed four times in PBS and then in fertilization medium. Groups of 50 COCs were transferred to four-well plates (Nunc, Roskilde, Denmark) in 250 µl of fertilization medium (TALP [16], containing 10 µg/ml heparin; Calbiochem, San Diego, CA) per well. The same frozen ejaculate of semen was used throughout all experiments. Motile spermatozoa were obtained by centrifugation on a Percoll (Pharmacia, Uppsala, Sweden) discontinuous density gradient (2 ml at 45% over 2 ml at 90%) for 20 min at 700 x g. Viable spermatozoa collected at the bottom of the 90% fraction were washed in TALP and pelleted by centrifugation at 100 x g for 10 min. Spermatozoa were counted in a hemocytometer and diluted in the appropriate volume of TALP to give a concentration of 2 x 106 spermatozoa/ml; a 250-µl aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1 x 106 spermatozoa/ml. Plates were incubated for up to 24 h in 5% CO2 in humidified air at 39°C.
MitoTracker Labeling of Spermatozoa
To distinguish between maternal and paternal pronuclei, zygotes that were tested for localization of RNA pol I, topoisomerase I, UBF, and coilin 5P10 were initially inseminated with MitoTracker-labeled spermatozoa. A 1 mM stock solution of MitoTracker Green FM (Molecular Probes, Leiden, The Netherlands) was prepared by adding 74 µl of dimethyl sulfoxide to an ampule containing 50 µg of the MitoTracker probe. The stock solution was diluted to 400 nM with TALP. Motile sperm were prepared as described above and pelleted by centrifugation. The supernatant was removed, and the pellet was resuspended in 400 nM of MitoTracker and incubated at 37°C for 30 min. Following incubation, the MitoTracker was removed by washing twice in TALP and pelleting by centrifugation. The pellet was resuspended in TALP at the normal concentration for in vitro fertilization and inseminated as described above.
Preparation of Oocytes for Immunocytochemistry
The cumulus investments of all immature, maturing, and fertilized COCs were removed by pipetting the COCs manually. Following denudation, the zonae pellucida of all oocytes were removed using prewarmed (37°C) pronase solution (0.5% Protease). The oocytes were subsequently washed in PBS, fixed in 4% paraformaldehyde for 15 min, washed in Soerensen phosphate buffer, and stored in the same buffer containing 1% sodium azide.
Ag NOR Silver Staining
Silver staining of whole-mount oocytes was performed according to the method of Lindner [17]. Several of the factors required for rRNA transcription and preribosome processing, including nucleophosmin and nucleolin, are acidic proteins that have the ability to reduce silver nitrate. Most silver-positive nucleolar proteins adhere to the NORs during mitosis; hence, they are termed Ag NOR proteins [18]. Because this technique reveals the presence of several nucleolar proteins, it was applied in the present study to eliminate false-negative results from whole-mount immunocytochemistry due to insufficient individual antigen concentration. Slides were incubated in 1% dithiothreitol for 12 min at room temperature and subsequently rinsed with distilled water. Slides were then covered with a 3:1 dilution of 50% silver nitrate (Merck, Darmstadt, Germany):2% gelatine and 1% formic acid (aqueous solutions) in the dark for 30 min, rinsed, and mounted in mounting medium (Dako, Cambridge, UK) containing propidium iodide (PI, 125 ng/ml). Samples were subsequently analyzed using dark field and fluorescence microscopy.
Whole-Mount Immunolabeling of Oocytes
The following primary antibodies were used: human monoclonal anti-fibrillarin (1:1000) [19, 20], mouse monoclonal anti-nucleophosmin (1:1000), mouse monoclonal anti-nucleolin (1:1000) [21], human anti-toposisomerase I (1:100), human anti-RNA pol I (1:500), human anti-UBF (1:500), and mouse anti-coilin P510 (1:10). The prepared oocytes were washed for 1 h in PBS containing 1% Triton-X (washing buffer) and blocked for 2 h in washing buffer supplemented with 5% rabbit serum (blocking buffer). Oocytes were subsequently incubated overnight at 4°C with one of the primary antibodies. The following day, excess primary antibodies were removed by extensive washing in PBS. The signal was amplified by incubation with biotinylated secondary antibodies (rabbit anti-human IgG biotin and rabbit anti-mouse IgG biotin; Dako) diluted with blocking buffer 1:500 for 4 h at 4°C and 1 h at room temperature. The signal was labeled by incubating with streptavidin fluorescent isothyocyanate (FITC; Dako). The processed oocytes were mounted on clean glass slides in fluorescent mounting medium (Dako) supplemented with 1 µl/ml 4',6-diamidino-2-phenylindole*dihydrochloride hydra (DAPI) or 125 ng/ml PI for chromatin visualization and covered with a clean coverslip that was fixed to the slide with nail polish. The slides were stored at 4°C in the dark.
As a positive control, Day 8 blastocysts [21] and cumulus oocyte complexes were included in the Ag NOR silver staining experiments and whole-mount immunocytochemical experiments. As a negative control, the primary antibody was omitted during the processing of a sample of oocytes, zygotes, and Day 8 blastocysts in each replicate.
Evaluation of Stage of Fertilization
The stages of fertilization and pronucleus formation are described according to the classifications presented by Laurincik et al. [22]. Zygotes displaying a metaphase plate and an uncondensed sperm head in the ooplasm were termed PN1. Zygotes displaying a decondensing sperm head and extruded polar body were termed PN2. At PN3, the chromatin was further decondensed, at PN4 the chromatin had completed decondensation, at PN5 the pronuclei had enlarged in size, and at PN6 the pronuclei had reached their maximum size and had become apposed.
Microscopy and Image Processing
Immunolabeled oocytes were analyzed initially using an upright fluorescent microscope (BX60 system microscope; Olympus, Tokyo, Japan). The presence of FITC-labeled nucleolar proteins was detected using an Olympus U-MNB filter (excitation band pass 470–490 nm and emission barrier 515 nm). The DAPI- or PI-labeled chromatin was visualized through Olympus U-MNU and U- MWG filters, respectively, (excitation band passes 360–370 and 510–550 nm and emission barriers 420 and 590 nm, respectively). A selection of samples was subsequently reanalyzed using a confocal laser scanning microscope system (BioRad, Hemelhempstead, Herts., UK). The fluorochrome was excited using the appropriate combination of excitation and barrier filters and argon/krypton laser lines for FITC (488 nm) and PI (515 nm). Images were obtained using a 40x water immersion objective (NA 1.15) and a 63x oil immersion objective (NA 1.4) with the zoom function.
RESULTS
With respect to the controls, Day 8 blastocysts and COCs were positively labeled by the primary antibodies under investigation and by Ag NOR silver staining. Samples that were processed without primary antibodies were unlabeled.
Nucleolar Proteins in Immature Oocytes
The localization of fibrillarin, nucleophosmin, nucleolin, topoisomerase I, RNA pol I, and UBF to the nuclei of oocytes in all size categories was investigated. In general, nucleophosmin and nucleolin were characterized by a diffuse pattern of localization. Fibrillarin was characterized by localization to large foci, and RNA pol I and UBF were characterized by localization to small foci that were scattered or clustered. All proteins except topoisomerase I were detected immunocytochemically. The location of the labeling is referred to as nucleolar (on the nucleolus) or nuclear (outside the nucleolus in the surrounding nucleoplasm). The results are summarized in Table 1 and are illustrated in Figure 1.
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Fibrillarin Almost all of the growing oocytes, i.e., <110 µm in diameter, displayed localization of fibrillarin to large foci throughout the nucleoli. In addition, half of these oocytes also displayed small labeled foci in the nucleoplasm. Oocytes >110 µm in diameter displayed localization of fibrillarin to large foci at the periphery of the nucleoli. Furthermore, the proportion of oocytes displaying large labeled foci in the nucleoplasm increased and the proportion of oocytes displaying labeled nucleoli decreased as the oocytes increased in size.
Nucleophosmin The majority of growing oocytes displayed a diffuse pattern of localization to their nucleoli, and approximately half of the oocytes also displayed diffuse labeling over the nucleoplasm. As oocyte diameter increased to >110 µm, labeling of the nucleoli became more peripheral, and the proportion of oocytes displaying diffusely labeled nucleoplasm increased.
Nucleolin The majority of oocytes of <110 µm in diameter displayed diffusely labeled nucleoli and unlabeled nucleoplasm. However, nucleoplasmic labeling was observed in at least half of all oocytes >110 µm in diameter. Furthermore, in oocytes >120 µm in diameter the proportion of oocytes displaying nucleolin labeling had decreased.
RNA pol I RNA pol I was localized to small nucleolar foci in varying numbers in all oocytes <120 µm in diameter. Thereafter, the proportion of oocytes displaying RNA pol I localization decreased to half.
UBF UBF was localized either diffusely or to small foci in the nucleoli of oocytes from all size groups. Large labeled foci were observed in the nucleoplasm of all oocytes >110 µm in diameter.
Nucleolar Proteins During Meiosis
Removing the oocytes from the IVM system at 0, 6, 12, 18, and 24 h gave a representative sample of each stage of meiosis: ON, ONBD, MI, telophase I, and MII, respectively. At 0 h of IVM, the whole-mount immunocytochemistry results were identical to those observed in experiment 1 in oocytes >120 µm in diameter. Once oocytes underwent ONBD, which was generally observed at 6 h of IVM, fibrillarin and nucleolin could no longer be detected. Nucleophosmin appeared to be associated with the condensing chromatin at ONBD but was not detected thereafter. Similarly, Ag NOR staining was not detected on oocytes once they had undergone ONBD.
Nucleolar Proteins in Zygotes
The localization of fibrillarin, nucleophosmin, nucleolin, RNA pol I, topoisomerase I, UBF, and coilin 5P10 during the first embryonic cell cycle after in vitro fertilization was examined. Zygotes investigated for localization of RNA pol I, topoisomerase I, UBF, and coilin 5P10 were inseminated with MitoTracker-labeled sperm to identify the paternal pronuclei. The results of monospermic zygotes are presented in Table 2 and illustrated in Figure 2.
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During the first 3 hpi, the majority of specimens were at MII stage of meiotic maturation and the spermatozoa were enmeshed in the cumulus cells or attached to the zona pellucida. None of the proteins were detected by whole-mount immunocytochemistry either in the sperm heads or within the oocytes. In agreement with these results, at 3 and 5–6 hpi the oocytes and sperm heads were also negative for Ag NOR silver staining proteins.
Fibrillarin Apart from one zygote that displayed localization to the fertilizing sperm head at 5–6 hpi, the zygotes first displayed fibrillarin localization at 10 hpi. Both pronuclei displayed small labeled foci; however, the number of labeled foci was generally higher in one of the pronuclei. At 15 hpi, all zygotes displayed labeled foci in both pronuclei, but the size and abundance of the foci was greater in one of the pronuclei. By 20 hpi, the pattern of localization had become more synchronous; large and small labeled foci were distributed equally between both pronuclei. This pattern of localization continued up to 25 hpi. However, zygotes undergoing synkaryosis at this time were unlabeled.
Nucleophosmin One zygote displayed nucleophosmin localization to the fertilizing sperm head at 5–6 hpi. By 10 hpi, one pronucleus in the majority of zygotes showed small labeled foci and light diffuse labeling. At 15 hpi, the majority of zygotes displayed ring-shaped labeled foci in both pronuclei; however, the size and abundance of the foci were usually greater in one of the pronuclei. By 20 hpi, large and small labeled foci were distributed equally between both pronuclei. At 25 hpi, numerous small labeled foci were observed in both pronuclei. In zygotes with closely apposed pronuclei, the labeling was localized to the areas of close apposition.
Nucleolin Two zygotes displayed a labeled fertilizing sperm head at 5–6 hpi. By 10 hpi, the majority of zygotes displayed small labeled foci in one pronucleus. At 15 hpi, both pronuclei contained labeled foci; however, the size and abundance of the foci were usually greater in one of the pronuclei. At 20 and 25 hpi, large and small labeled foci, occasionally with tails, were observed in equal abundance in both pronuclei.
UBF Small foci on the fertilizing sperm heads of all zygotes was first detected at 5–6 hpi. At 10 and 15 hpi, the majority of zygotes displayed UBF localization to small foci. These foci appeared either exclusively or more abundantly in the paternal pronucleus. However, by 20 hpi the proportion of zygotes showing UBF localization had started to decrease, and by 25 hpi less than half of the zygotes displayed labeled pronuclei.
RNA pol I Small labeled foci were first detected at 10 hpi, either exclusively or more abundantly in the paternal pronucleus of the majority of zygotes. However, by 15 hpi only one-third of the zygotes displayed labeled pronuclei, and by 25 hpi labeling was no longer detected.
Topoisomerase I Topoisomerase I was first detected at 10 hpi as a light diffuse labeling, which was exclusive to or more abundant in the male pronucleus. By 15 hpi, both pronuclei in all zygotes showed labeling; however, the paternal pronucleus was more intensely labeled and displayed one or two large foci. By 20 hpi, the pattern of localization had become more diffuse, although some pronuclei displayed small foci in chromatin-free areas. This labeling pattern was unchanged at 25 hpi.
Coilin 5P10 Coilin 5P10 was first detected at 10 hpi and localized to small foci within both pronuclei; however, the number of labeled foci was higher in the paternal pronucleus. This pattern of localization persisted until 15 hpi; thereafter, the proportion of zygotes displaying localization decreased to less than one third.
DISCUSSION
In the present series of experiments, we investigated the localization of major nucleolar proteins from prophase I of meiosis in oocytes up to the first mitotic cell division following fertilization. The main findings were that nucleolar proteins are readily detectable by immunocytochemistry in the bovine oocyte during the oocyte growth phase (prophase I). These proteins are subsequently dispersed into the ooplasm at the resumption of meiosis and reappear as aggregates at late telophase in the pronuclei following fertilization (Fig. 3).
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During the bovine oocyte growth phase, gene transcripts, polypeptides, and ribosomes are produced by and stored in the oocyte to sustain future early embryonic development [23]. The nucleus of a growing oocyte, i.e., an oocyte with a diameter of <110 µm, is characterized by the presence of one to three transcriptionally active fibrillogranular nucleoli [5, 6]. Such nucleoli are prominent structures within the nucleoplasm and are approximately 7 µm in diameter. They contain a prominent GC, which is interspersed with vacuoles and numerous FCs. At an oocyte diameter of 110 µm, rRNA transcription decreases and is coincident with the migration of the FCs to the surface of the nucleolus, the extrusion of granules into the surrounding nucleoplasm, and the formation of a central vacuole. By the time the oocyte reaches 120 µm, the nucleolus compacts to a dense fibrillar sphere attached to a lentiform FC. This nucleolar remnant is approximately 3 µm in diameter. As a final step, fragmentation of the nucleolar remnant into dense spheres of approximately 1 µm in diameter may occur [5].
In the immature oocytes, all nucleolar proteins except for topoisomerase I were localized to nucleoli or presumptive nucleolar fragments of growing and fully grown oocytes. RNA pol I, UBF, fibrillarin, nucleophosmin, and nucleolin were strongly concentrated in the nucleoli within the growing oocytes (<110 µm), and with the completion of oocyte growth the proportion of labeled oocytes decreased or the proteins were more strongly localized to the periphery of the nucleoli or nucleolar remnants.
The actual transcription of the rRNA genes is dependent on the activity of RNA pol I, which has been localized mainly to the FCs and to some degree to the DFC [24]. Therefore, the large clusters of foci of RNA pol I in nucleoli probably reflect the FCs and thus the sites of rRNA gene transcription. As the oocyte reaches a diameter of 120 µm, transcription decreases, and accordingly RNA pol I could only be detected in half of these oocytes. The binding of RNA pol I to the DNA requires several transcription factors, one of which is UBF [25]. UBF was localized to numerous small foci in the nucleoli of most oocytes in all size categories and is believed to be involved in the formation of a preinitiation complex to which RNA pol I can bind and initiate transcription [26].
Fibrillarin has been localized to the FCs and DFCs of nucleoli [19], where it is believed to be involved in primary transcript processing. In the present study, all oocytes in the smallest diameter group (<100 µm) displayed large labeled nucleolar foci throughout their nucleoli. Furthermore, as the oocyte diameter increased, the labeled foci were found at the periphery of the nucleoli. Therefore, we suggest that these large nucleolar foci correspond to the FCs and that the marginalization of the labeled foci reflects their peripheral migration in conjunction with nucleolar inactivation at the completion of oocyte growth.
Nucleophosmin may be involved in shuttling proteins, such as nucleolin [27] and P120 [28], into the nucleolus. It has been suggested that nucleophosmin functions with nucleolin as a preribosome assembly factor. According to Ghisolfi et al. [29], nucleolin may act in promoting the functional secondary structure of the rRNA that is necessary for the assembly of the preribosomal particles. As oocyte diameter increased, there was a slight decrease in the number of oocytes that displayed nucleophosmin-labeled nucleoli, and the number of oocytes with a stronger nucleophosmin localization signal in the nucleolar periphery and nucleoplasm increased. This finding has been reported previously in mouse oocyte nucleoli [30]. As transcriptional activity decreases with increasing oocyte diameter, proteins involved in the later stages of ribogenesis such as preribosome assembly probably migrate to the periphery of the nucleoli and the nucleoplasm where they are required. In the case of nucleolin, the nucleolar peripheral and nucleoplasmic localization of nucleolin in the fully grown oocytes coincides with the extrusion of the granular component of the nucleoli and may be reflective of preribosome subunit assembly at that time.
From the findings of the present study it is clear that the nucleoli and nucleolar remnants of fully grown oocytes contain numerous proteins, most of which are readily detectable following fertilization. However, the fate of these proteins during the resumption and progression of meiosis is unknown. In the present study, proteins that were detected up to nucleolar dispersal in late prophase I of meiosis remained undetected until late telophase II. Using immunocytochemistry, we did not detect fibrillarin, nucleophosmin, or nucleolin at any stage from ONBD until 5–10 hpi. Furthermore, these findings were confirmed by a similar lack of Ag NOR staining in oocytes investigated at the same time points. Our findings suggest that upon dismantling of the nucleolus at ONBD these nucleolar proteins are dispersed in the ooplasm, where they remain undetectable by whole-mount immunocytochemistry, possibly because of their low concentation, until fertilization and pronuclear formation, when they reaggregate. Shah et al. [31] described similar findings in Xenopus laevis oocytes; nucleolin and fibrillarin were detected in small particles immediately following ONBD but not minutes later. However, there is little other comparable information available on the behavior of nucleolar proteins during meiosis.
In contrast to meiosis, the behavior of numerous nucleolar and nuclear proteins during mitosis has been well mapped. In mitotic cells at the beginning of prophase, the nucleolus contains rRNA precursors in association with ribosomal proteins, all at various stages of preribosomal subunit formation. As prophase progresses, rRNA transcription and processing stop and the nucleolus disassembles. Much of the RNA pol I transcription machinery and DNA topoisomerase I remain associated with the NORs on NOR-bearing chromosomes [26, 32–34]. In contrast, the pre-rRNA processing components U3 small nucleolar protein, fibrillarin, nucleophosmin, nucleolin, and p52 appear to be concentrated in the perichromosomal regions [33, 35–37], or distributed throughout the cell in spherical cytoplasmic particles termed nucleolus-derived foci (NDF) [38, 39]. The rRNA precursors and processing intermediates have also been localized to the NDF [11]. At late telophase, the numbers of NDF decline coincident with the appearance of prenucleolar bodies (PNBs) containing similar material. The PNBs subsequently congregate around the NORs and contribute to the reconstitiution of the new nucleoli [21, 40]. Processing of the preexisting material resumes, and transcription of new rRNA begins.
The behavior and distribution of the nucleolar proteins during meiosis appears to be very different from that during mitosis in that the nucleolar proteins are dispersed and nondetectable for a period of up to 32 h, i.e, from prophase I until telophase II of meiosis or interphase of the first cell cycle. In mammalian embryogenesis following fertilization, two haploid compartments are formed in the zygote, a male pronucleus and a female pronucleus. During in vitro fertilization of in vitro-matured bovine oocytes, penetration of the fertilizing spermatozoon into the ooplasm occurs within 4 h after insemination, at which time pronucleus formation is initiated by chromatin decondensation and formation of the envelopes of the pronuclei. During the G1 phase of the first postfertilization cell cycle, the pronuclei enlarge and two structures appear inside them [22]. The most prominent entities are electron-dense fibrillar spheres about 1 µm in diameter. These spheres are often referred to as nucleolus precursor bodies (NPBs) because they define the sites where fibrillogranular nucleoli are formed during the fourth postfertilization cell cycle in bovine embryos [21]. The NPBs appear to be similar to PNBs; however, they are particular to mammalian embryos. On other locations in the pronuclei, clusters of electron-dense granules resembling interchromatin granules appear. During the S and G2 phases of the first cell cycle, the fibrillar spheres and the GCs become spatially associated, and at least in in vivo-derived bovine embryos the fibrillar spheres develop a central vacuole [41], which is less common in in vitro-derived zygotes [22].
In the present study, all of the nucleolar proteins were detected using immunocytochemistry from 10 hpi with the exception of UBF, which was detected on the fertilizing sperm at 5 hpi. Furthermore, the proteins consistently targeted the sperm head or male pronucleus first. These results differ from those of Laurincik et al. [21], who reported that with the exception of fibrillarin and UBF, which were detected after fertilization, nucleolin and nucleophosmin were not detected until the third cell cycle and topoisomerase I and RNA pol I were not detected until the fourth cell cycle.
We also localized DNA topoisomerase I and the nucleic acid binding protein coilin 5P10 to small foci or clusters of foci in one or both pronuclei from 10 to 25 hpi. The presence of DNA topoisomerase I may facilitate DNA synthesis, which, according to Laurincik et al. [22], occurs between 15 and 23 hpi. The role of coilin in the zygote may be more complex. One important function of coilin is to transport U7 small nuclear ribonucleoprotein (snRNP) into coiled bodies [42]. The snRNPs are complexes of small nuclear RNAs associated with specific proteins, and they are engaged in the splicing of pre-mRNA [43]. The coiled body is a nuclear organelle that contains essential components of the mRNA splicing machinery and fibrillarin. Previously, coilin was considered the defining protein of coiled bodies. However, coilin has been localized to the nucleoli of human antral follicular oocytes [44], interchromatin granule-associated zones [45], NPBs of the Xenopus egg [46], and precoiled bodies and coiled bodies in maternal and paternal pronuclei of one-cell mouse embryos [47]. Bell and Scheer [46] suggested that NPBs are primary assembly structures, which contribute to the formation of both nucleoli and coiled bodies. In agreement; Kopecny et al. [48] and Laurincik et al. [22] suggested that snRNPs may be involved in nucleolar formation and that NPBs may possess functions in relation to pre-mRNA splicing.
The results of the present study clearly demonstrate that the pronuclei in bovine zygotes contain a range of the molecular components necessary for nucleolar assembly and rRNA transcription. However, we can only speculate as to the functional significance of these findings. These nucleolar components may be targeted to the rRNA genes during interphase. Viuff et al. [49] demonstrated, using combined fluorescent in situ hybridization with silver staining of nucleolar proteins, that at least some of these proteins are associated with the rRNA genes during the interphase of the first postfertilization cell cycle. However, the fact that the NORs do not exhibit silver staining during metaphase until after the fourth postfertilization cell cycle [50] indicates that the association between nucleolar proteins and NORs does not reach full mitotic stability until after nucleolar activation, i.e., during the fourth cell cycle [7, 21]. These proteins probably do not exert their specific functions in relation to rRNA transcription and processing during the first cell cycle. However, although rRNA transcription is not initiated until later, the proteins could be involved in the processing of maternally derived rRNA intermediates into ribosomal subunits.
In cattle, the activation of the embryonic genome occurs gradually over several cell cycles [1, 2, 49]. The immature oocyte must carry the components required to initiate and sustain this gradual activation until the embryo becomes self-sufficient. Here, we describe for the first time in cattle how the components of rRNA gene transcription and ribosome formation are brought from the growing oocyte stage through the processes of meiotic maturation and fertilization and delivered to the one-cell embryo. Furthermore, it is highly likely that embryos that fail to progress beyond the first three cell cycles are lacking in these basic components. A failure to detect one or all of these components during the oocyte growth phase or later during interphase of the first cell cycle may serve as an indicator of developmental incompetence.
Nucleolar proteins are readily detectable in the bovine oocyte during the growth phase. They are dispersed into the oocyte cytoplasm at ONBD and cannot be detected again until late telophase following fertilization, where they initially target the paternal pronucleus.
ACKNOWLEDGMENTS
The authors thank Dr. Robert Ochs (Precision Therapeutics Inc., Pittsburgh, PA) for the generous gifts of fibrillarin, nucleophosmin, nucleolin, UBF, RNA pol I, and topoisomerase I antibodies and Dr. Judith Sleeman (Department of Biochemistry, Wellcome Trust Building, University of Dundee, Scotland) for the kind gift of coilin 5P10 antibody.
FOOTNOTES
1 Supported by the Danish Agricultural and Veterinary Research Council and Enterprise Ireland. 
2 Correspondence: Trudee Fair, Department of Animal Science & Production, Lyons Research Farm, University College Dublin, Newcastle, County Dublin, Ireland. FAX: 353 1 6288421; tfair{at}pop3.ucd.ie
Accepted: January 5, 2001.
Received: April 17, 2000.
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