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Keywords:

  • nascent RNAs;
  • 5-fluorouridine;
  • pig oocyte

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Background: Germ cells differentiate into oocytes in females and are arrested at the first meiotic prophase. However, during arrest, oocytes undergo a growth phase leading to a dramatic increase in size, which is under control of transcription events. In the current study, we examined the transcriptional activity of growing pig oocytes using an immunocytochemical approach. Our data showed that fluorouridine (FU), a halogenated nucleotide, can be successfully incorporated into synthesizing RNAs and detected using a specific monoclonal antibody. Results: Using this method, we identified dynamic changes in transcriptional activity patterns in growing pig oocytes. Oocytes obtained from small follicles exhibited the highest level of transcription, while at the final phase of growth, transcription was no longer detected. These transcriptional changes were concomitant with chromatin compaction resulting in a tightly packed ring-like chromatin conformation surrounding the nucleolar structure. Also, FU incorporation appeared sensitive to the biochemical manipulation of transcription, because transcriptional inhibitors induced a decrease in signal intensity from FU labeling and transcriptional activation caused an increase in FU signal intensity. Conclusions: Our data collectively support that a direct link exists between chromatin configuration and transcriptional activity in pig oocytes, and support the suitability of FU for studies on transcription-related events in mammalian oocytes. Developmental Dynamics 242:16–22, 2013. © 2012 Wiley Periodicals, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In females, primordial germ cells differentiate into oocytes. Oocytes in mammals are arrested at the prophase of the first meiotic division after birth. At this point, genetic material is dispersed throughout the nucleoplasm (germinal vesicles in oocytes), and this configuration is maintained until the first surge of gonadotropins at the beginning of puberty (Zoccotti et al., 2011). The extensive use of oocytes for in vitro maturation, in vitro fertilization or nuclear transfer experiments is essential to clarify the biochemical and morphological characteristics of preovulatory oocytes. Previous studies have reported that the sizes of follicles and oocytes are correlated with meiotic competence in mammals (Yoon et al.,2000; Marchal et al.,2002). Oocytes undergo a change in chromatin shape by means of increase in size. For instance, two oocyte types are recognizable within antral follicles in mice, based on chromatin configuration. In one type, the huge nucleolus is not surrounded by chromatin (NSN type), while in the other, the nucleolus is encompassed by a dense rim of chromatin (SN type) (Bouniol-Baly et al.,1999). Several studies have suggested that the SN configuration appears after NSN with the progression of oocyte growth (Debey et al.,1993; Zoccotti et al., 1995; Bouniol-Baly et al.,1999). These oocyte types have additionally been observed in other mammalian species, including pigs (Crozet et al.,1981).

The above chromatin configuration patterns are accompanied by dramatic changes in the global transcription level in oocytes. Earlier research has shown that in mouse oocytes from large antral follicles, transcriptional activity is completely quiescent (Moore et al.,1974), while other studies have reported the incorporation of [3H] uridine in mouse germinal vesicle oocytes (Bloom and Mukherjee,1972; Rodman and Bachvarova,1976; Wassarman and Letourneau,1976; Kopecny et al.,1995). Furthermore, investigation of the transcriptional activity of preovulatory oocytes in other mammalian species, including human (Miyara et al.,2003), cattle (Fair et al.,1995), and pigs (Motlik and Fulka,1986; Tan et al.,2009) has revealed a dramatic decrease in RNA polymerase II-dependent RNA synthesis in growing oocytes.

In the nucleus, RNA synthesis by polymerases can be detected by introducing RNA precursors into the cell by means of permeabilization (Wansink et al.,1993). Conventionally, detergents such as Triton X-100 or Streptolysin O are initially used for permeabilizing cells, followed by incubation with RNA precursors. The most commonly used RNA precursor is 5-bromouridine 5′-triphosphate (BrUTP), which can also be microinjected into living cells (Bouniol-Baly et al.,1999). After treatment or microinjection, BrUTP is incorporated into newly synthesized RNA and detected with immunocytochemistry using a monoclonal antibody against bromodiuridine. However, this technique presents several limitations, because both permeabilization and microinjection approaches are detrimental to living cells. In addition, RNA polymerase I-dependent transcription is rarely detected using standard permeabilization approaches (Jackson et al.,1993).

Regarding to sensitivity of mammalian oocytes to permeabilization and microinjection, we used a cell-permeable modified RNA precursor, 5-fluorouridine (FU), to identify transcription sites and changes during pig oocyte growth. Our experiments showed that FU can be successfully incorporated in both mRNA and rRNA by simple incubation of oocytes in FU-containing culture medium. Moreover, FU incorporation appears sensitive to both RNA synthesis inhibitors and activators.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Chromatin Configurations in Growing Pig Oocytes

Initially, we characterized the changes in chromatin configuration during the course of oocyte growth in pig. COCs were collected from antral follicles (<1 mm, 1–3 mm and 3–6 mm in size) (Fig. 1A,B), and the chromatin shape was analyzed using fixed oocytes stained with DAPI, followed by confocal microscopy. Our experiments revealed four different chromatin configurations with increasing antral follicle size (Fig. 1C). In small follicles, the majority of oocytes exhibited a roughly dispersed chromatin configuration throughout the nucleoplasm (germinal vesicle), designated as nonsurrounded nucleolus (NSN). With follicle growth (1–3 mm), approximately the half the oocytes examined displayed NSN configuration and the other half were in a condensed ring-like chromatin pattern surrounding the nucleolus, denoted as surrounded nucleolus (SN). In large follicles, oocytes predominantly exhibited the SN configuration. In all experiments, intermediary chromatin shapes (partly NSN [pNSN] and partly SN [pSN]) were observed, indicative of transition from one configuration to another (Fig. 1C).

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Figure 1. Classification of pig oocytes according to follicle of origin and chromatin configuration. A: A typical pig ovary containing at least three different sizes of follicles used to obtain all sizes of antral follicles. Black, white and gray arrows indicate a 3–6, a 1–3, and a <1 mm antrum, respectively. B: A group of COCs with different sizes obtained from ovaries as shown in A. C: Four major classes of chromatin configurations, NSN, pNSN, pSN, and SN, observed in pig antral follicle oocytes. COCs were denuded immediately after collection, fixed and stained with DAPI, and examined under a confocal microscope. In total, more than 300 oocytes were examined. Scale bar = 1 cm in A; 100 μm in B; 20 μm in C.

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Detection of Transcription Sites in the Germinal Vesicle

To identify the dynamic changes in transcriptional activity of pig oocytes during the growth phase, we used a cell-permeable small molecule RNA precursor modified with a fluor residue to label the active transcription sites in germinal vesicles. Oocytes from antral follicles of different sizes were obtained and incubated with FU for 1 hr. Incorporated FU in nascent RNAs was subsequently detected using immunocytochemistry with a specific monoclonal antibody and confocal microscopy. Notably, although the signal emitted from FU labeling was present throughout the nucleoplasm of NSN and pNSN oocytes, labeling was not uniform and mainly associated with regions displaying lower DAPI staining (Fig. 2A), which is generally attributable to open chromatin area. In addition, transcription sites, especially those of RNA polymerase II, were organized in discrete foci scattered throughout the germinal vesicle. To ascertain whether signals emitted from antibodies specifically recognizing incorporated FU were not false, we replicated the experiments without FU treatment. As expected, no signal from these oocytes was evident under a confocal microscope (Fig. 2B). To ensure that FU is specifically incorporated into nascent RNA and not DNA, we treated oocytes with FU, and after fixation with paraformaldehyde, separated them into three groups. One group was treated with RNase A, a ribonuclease that cleaves single-stranded RNAs, the second group with DNase I, an endonuclease that cleaves DNA at phosphodiester bounds and acts on both single- and double-stranded DNA, and the third group was left untreated. All specimens were subjected to the same protocol as that for FU immunostaining, and examined using confocal microscopy. We observed no signal from FU labeling in the first group, whereas in the second group, signals were emitted at a level comparable with the control group (Fig. 2C). Our data clearly indicate that the signal observed in oocytes treated with FU is specifically sensitive to RNase, supporting the conclusion that FU is specifically incorporated in RNA structures.

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Figure 2. Detection of transcription in pig GV oocyte. A: Fluorouridine (FU) incorporated into nascent RNA structures and immunocytochemically labeled with a monoclonal antibody recognizing FU and fluorescein isothiocyanate-conjugated anti-mouse IgG in a pNSN oocyte. Notably, transcription sites are distributed as discrete foci throughout the germinal vesicle. B: GV oocytes untreated with FU (negative control) but immunostained with the same antibody as A. No FU signal was detected in these oocytes. C: Sensitivity of FU incorporation to nucleases was examined through exposure of FU-treated oocytes to RNase A or DNase I after fixation with paraformaldehyde. Oocytes were subsequently immunostained with the same antibody as A. The signal from FU labeling was specifically observed in DNase-treated oocytes. In A, B, and C, DNA was counterstained with DAPI (red color) for better visualization. Scale bar = 10 μm in A; 30 μm in B,C.

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Dynamic Changes in the Transcription Level in Growing Oocytes

We detected dynamic transcriptional activity in pig GV oocytes derived from different size follicles. As shown in Figure 3A, the levels and patterns of distribution of transcription sites were distinct in four representative GV oocytes. For instance, in NSN oocytes, the transcription level appeared higher and more evenly distributed throughout the nucleoplasm. For quantification of transcription, we simultaneously measured fluorescent signal intensities emerging from FU labeling along with the signal from DAPI staining in the four types of oocytes. Interestingly, transcriptional activity was higher in oocytes obtained from small follicles. This high level of transcription coincided with the NSN configuration in which chromatin formed a diffused net-like distribution throughout the germinal vesicle (Fig. 3B). In contrast, oocytes obtained from large follicles exhibited very weak (if any) transcriptional activity, possibly as a result of chromatin compaction observed in the SN configuration.

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Figure 3. Changes in transcriptional Activity in growing GV oocytes. A: Oocytes of different sizes were treated with 5-fluorouridine (FU) for 1 hr and immunolabeled with a monoclonal antibody recognizing FU. B: Relative intensities of fluorescence signals from both FU and DNA. Samples from all stages were simultaneously processed for immunostaining and images obtained at the same laser power, enabling direct comparison of signal intensities. The results represent mean values from at least seven oocytes. The fluorescence intensities at the NSN stage and DAPI signal from SN stage have been set as 100% for FU and DNA respectively. The DAPI signal turned to red for better visualization. Scale bar = 30 μm.

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Effects of Transcriptional Inhibition and Activation on FU Incorporation

To establish that FU labeling is applicable for investigating changes in transcriptional activity, we initially determined the effects of the RNA polymerase inhibitors, α-amanitin and actinomycin-D (AcD), on transcriptional activity in GV oocytes. The α-amanitin is one of the most potent and specific RNA polymerase II inhibitors that affects RNA polymerases in a dose-dependent manner. RNA polymerase II is irreversibly inhibited by 10–100 μg/ml of α-amanitin, while RNA polymerase I and III are not affected at this dose range. For inhibition of RNA Pol III, two- to fourfold higher concentrations of α-amanitin are required, while RNA polymerase I activity is suppressed only in the presence of 1,000-fold higher concentration of the drug. AcD has been shown to inhibit transcription by binding to DNA at the transcription initiation complex and preventing elongation of the RNA chain by RNA polymerase. A low concentration of AcD (100 nM) is sufficient to inhibit all RNA polymerases within the cell nucleus. Considering these properties, we designed an experiment to address whether or not FU is incorporated during transcription by RNA polymerase I and II. To this end, we used 20 μg/ml of α-amanitin to specifically inhibit RNA polymerase II and 100 nM AcD to inhibit both RNA polymerases I and II in pig GV oocytes. Confocal microscopy revealed a dramatic decrease in the level of FU incorporation in α-amanitin-treated oocytes (Fig. 4A). In these oocytes, a rim of FU signal surrounding the nucleolar areas, that can be attributable to RNA polymerase I-dependent transcription, remained at a comparable level to that in untreated control oocytes (highlighted with arrow in Fig. 4A). In AcD-treated oocytes, a global decline in FU incorporation was observed relative to untreated and α-amanitin-treated oocytes. In this case, both the signal observed throughout the nucleoplasm of untreated oocytes and the signal rim in α-amanitin-treated oocytes disappeared. Fluorescence intensity measurements revealed two peaks corresponding to the area surrounding the nucleolus in α-amanitin-treated oocytes (Fig. 4C). In AcD-treated oocytes, DAPI intensity increased (possibly due to chromatin condensation) while FU-related intensity remained at the background level, suggesting that transcriptional activity was abolished (Fig. 4B,C). These findings strongly indicate that FU is incorporated into both nascent mRNAs and rRNAs in growing pig oocytes.

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Figure 4. Effects of inhibition and activation of transcription on 5-fluorouridine (FU) incorporation. A: Oocytes were collected and treated with the transcriptional inhibitors, α-amanitin (20 μg/ml), actinomycin D (AcD, 100 nM), or the transcriptional activator, TSA (50 nM), for 1 hr and transferred to the same medium containing 5 mM FU for another 1 hr. Oocytes were fixed and immunostained with an antibody against FU. The arrow depicts the area resistant to α-amanitin. DAPI signal was turned to red. B: Relative intensities of fluorescence signals from both FU and DNA. Samples from all control and treatment groups were simultaneously processed for immunostaining, and images obtained at the same laser power. Results are presented as mean values from at least five oocytes. The fluorescence intensity in TSA-treated oocytes and DAPI signal from AcD-treated oocytes were set as 100% for FU and DNA, respectively. C: Examples of intensity measurements in control and drug-treated oocytes. Nuclear signals were outlined and mean fluorescence intensities measured. Encircled regions were software-dragged into the cytoplasm of the same cell, and background fluorescence subsequently measured. Each specific signal was calculated by dividing the nuclear value by the cytoplasmic value. Scale bar = 40 μm.

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Next, we ascertained whether activation of transcription influences FU incorporation. GV oocytes were treated with trichostatin A (TSA), a histone deacetylase inhibitor. It is generally accepted that TSA inhibits histone deacetylase activity at nanomolar concentrations, and the resultant histone hyperacetylation leads to the formation of relaxed chromatin and elevation of gene expression. In our experiments, a considerable increase in FU incorporation was observed in TSA-treated oocytes, compared with untreated oocytes (Fig. 4A). Fluorescence intensity measurements confirmed a higher transcription level in TSA-treated oocytes than control oocytes (Fig. 4B). Moreover, DAPI staining was lower than that of control oocytes, supporting the formation of open chromatin (Fig. 4B,C). Our observations clearly demonstrate that FU is a suitable compound to investigate the changes in global transcription as well as distinguish between RNA polymerase I and II-driven RNA synthesis in pig oocytes.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

The availability of large numbers of mature oocytes and embryos by means of in vitro maturation and fertilization is critical for agricultural and biomedical applications. Adequate growth of oocytes, followed by nuclear and cytoplasmic maturation, is an important prerequisite to obtain oocytes competent to develop to term. During oocyte growth, RNAs bearing information necessary for at least the early steps of embryo development are transcribed and stored or translated into proteins. Transcriptional activities shut down by means of germinal vesicle breakdown, and the oocyte undergoes a series of chromosomal events leading to maturation (Zoccotti et al., 2011). The main aim of this study was to analyze the organization of nuclear transcription sites in germinal vesicle pig oocytes in vitro using a run-on transcription assay based on the incorporation of 5-fluorouridine (FU) into nascent RNA. Our novel findings reveal dynamic changes in transcriptional activity in growing pig oocytes for the first time. In recent years, changes in histone modification in relation to chromatin organization in pig oocytes during growth and maturation have been a subject of focus (Prather et al.,2009). While organization and dynamic changes in chromatin and its modifications have been extensively analyzed in mammalian oocytes, direct visualization of RNA transcription is currently limited to a few studies in mice. For instance, Bouniol-Baly et al. (1999) reported both RNA polymerase I- and II-dependent transcription in NSN mouse oocytes. The group performed BrUTP microinjection into GV oocytes to identify the patterns of distribution and localization of nascent mRNAs and rRNAs. The complete lack of data in pig may be attributed to the sensitivity of porcine oocytes to microinjection and permeabilization techniques. In the current study, we applied a cell-permeable and water-soluble halogenated nucleotide modified with a Fluor residue (FU) to overcome these limitations. FU has been favored as the alternative to BrUTP in several cell biology studies over the past decade. However, to our knowledge, a transcription assay using FU incorporation has not been previously applied in mammalian oocytes. Transcriptional labeling with FU was initially developed for cultured cells (Boisverst et al., 2000). The authors showed that incorporation of FU in nascent RNAs is rapid, specific, and does not require a cell permeabilization step.

Our transcription assay detected tens of discrete transcription sites scattered throughout the nucleoplasm in germinal vesicles, similar to previous observations in human cell nuclei (Wansik et al., 1993). In the earlier investigation, the authors examined the distribution of transcription sites of RNA polymerase II by labeling nascent RNA with BrUTP in vitro and in vivo, and identified nascent RNA polymerase II transcripts in over 100 defined areas scattered throughout the nucleoplasm of human bladder carcinoma cells. Another study reported the presence of approximately 300 to 500 focal synthetic sites in HeLa cells (Jackson et al.,1993). These experiments revealed that BrUTP is also incorporated into RNA polymerase I rRNA transcripts, which are resistant to low concentrations of α-amanitin. Further studies disclosed that transcription factors including active polymerases coincide with these sites (Iborra et al.,1996).

We subsequently demonstrated that transcription occurs in a dynamic manner. Our experiments revealed functional differences coupled with the changing chromatin configurations in pig GV oocytes. Originally, Bouniol-Baly et al. (1999) found that SN- and pSN-type mouse oocytes are silent with regard to RNA polymerase I- and II-dependent transcription, while NSN-type oocytes transcribe actively. While chromatin configurations have been extensively studied in pig GV oocytes (Motlik and Fulka,1976; Guthrie and Garrett,2000; Sun et al.,2004), the correlations of these conformations with the levels and sites of nuclear and nucleolar transcription are unclear at present. Transcription of rRNA plays a crucial role in the maintenance of cell function, and is closely correlated with the growth and cell cycle state (Mayer and Grummt,2006). Bjerregaard et al. (2004) showed that the decrease in rRNA synthesis observed with progression of the growth phase of pig GV oocytes is regulated through and/or decrease in availability of key RNA polymerase I transcription factors. In our experiments, direct labeling of nascent RNAs indicated that the final transcriptional activity of GV oocytes could be attributable to RNA polymerase I activity in the pSN group, because only the nucleolar periphery exhibited a relatively weak FU signal. Consistent with previous studies reporting little, if any, signal intensity from RNA polymerase I transcription factors in SN oocytes, our data showed that in these oocytes, the signal emitted from FU labeling is completely abrogated.

Many anti-cancer drugs inhibit transcription, and the majority of transcriptional inhibitors have useful pharmacological properties (Bensaude,2011). Previous studies have shown that α-amanitin binds to RNA polymerase II irreversibly and promotes degradation of the largest subunit (Nguyen et al.,1996). Moreover, RNA polymerase II and other transcription factors are a maternal contribution in animal embryogenesis (Bellier et al.,1997a,b). RNA polymerase II is the most sensitive polymerase to α-amanitin (Kedinger et al.,1971; Weinmann et al.,1974) while RNA polymerase I appears almost insensitive to this drug. We selected this antibiotic to determine whether FU incorporation is sensitive to transcriptional inhibition and additionally distinguish between RNA polymerase I- and II-transcription in GV oocytes. Notably, FU incorporation was dramatically decreased in drug-treated oocytes, while a ring of FU signal surrounding the nucleolus remained almost comparable to the level in untreated cells. BrUTP incorporation into nascent rRNAs remains intact when RNA polymerase II transcription is inhibited in human cells (Jackson et al.,1993; Wansik et al., 1993). Treatment with a transcription inhibitor with a different mechanism of action confirmed that the ring is indeed a RNA polymerase I product. In general, AcD is used at concentrations of approximately 10 nM to inhibit RNA polymerase I transcription. At higher concentrations (∼100 nM), the drug affects both RNA polymerase I and II (Jao and Salic,2008). Confocal microscopy analysis disclosed that in oocytes treated with this drug, the signal from FU labeling declines to the background level. Moreover, higher intensity of DAPI staining was observed in treated oocytes, compared with untreated cells. AcD has been shown to promote chromatin condensation, and subsequently apoptosis in HeLa cells (Fraschini et al.,2005).

Earlier studies in mouse oocytes have revealed that histone deacetylases (HDACs) are essential for large-scale chromatin remodeling in the GV (De La Fuente et al.,2004). Using nucleoplasmin 2 knockout (Npm2−/−) mice in which transition into the SN configuration does not occur, the investigators showed that inhibition of HDACs with TSA induces chromatin decondensation, but does not restore transcriptional activity in mouse SN oocytes. Similarly, Bui et al. (2007) demonstrated that TSA treatment promotes chromatin decondensation and hyperacetylation in pig GV oocytes. However, the issue of whether a direct link exists between chromatin remodeling and transcription has not been addressed until now. Our experiments clearly showed that following TSA treatment, chromatin is globally decondensed, visualized as a decrease in the intensity of DAPI staining, compared with untreated counterparts. Moreover, the intensity of the FU signal increases by ∼12% on average.

In conclusion, our immunostaining and confocal microscopy analyses reveal that FU is efficiently incorporated into both mRNA and rRNA structures when made available simply in the culture medium. Evidently, the convenience of permeabilization or microinjection makes FU one of the most suitable halogenated nucleotides for use in studies on transcription events in mammalian oocytes. Additionally, for the first time, we report a correlation between chromatin condensation and transcription silencing during the final steps of pig oocyte growth with the aid of FU labeling experiments.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Collection and Culture of Pig Oocytes

Porcine ovaries were collected at a local slaughterhouse. Antral follicles were aspirated manually. Cumulus-oocyte complexes (COCs) were aspirated from follicles with different sizes and resuspended in TL-HEPES containing 0.01% polyvinyl alcohol (PVA). For classification of oocytes on the basis of chromatin configuration, follicles were separately aspirated from antra of different sizes. COCs were incubated in 500 ml of TCM-199 containing epidermal growth factor, L-cysteine, follicle stimulating hormone (FSH), luteinizing hormone (LH), and follicular fluid at 38.5°C.

Treatment and Transcription Labeling of Oocytes

To label nascent transcripts in oocytes, COCs were collected, washed with TL-HEPES, and denuded by pipeting in the same medium containing 0.5% hyaluronidase. Oocytes were subsequently washed three times with TCM-199 and placed in the same medium containing epidermal growth factor, follicular fluid, and 5 mM fluorouridine (FU, Sigma) at 38.5°C for 1 hr. For inhibition or activation of transcription, oocytes were cultured in the same medium containing 20 μg/ml α-amanitin (Sigma), 100 nM Actinomycin D (Sigma) or 50 nM Trichostatin A (Sigma) for 1 hr, and transferred to the same medium containing 5mM FU for an additional 1 hr. Oocytes were washed, fixed, and subjected to immunocytochemistry.

Immunocytochemistry and Confocal Microscopy

Oocytes were fixed in phosphate buffered saline (PBS)-PVA containing 2% paraformaldehyde, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. Next, oocytes were washed with 100 mM glycine in PBS for 10 min and blocked with 3% bovine serum albumin (BSA) in PBS, followed by 5 min in PBG (PBS containing 0.5% [w/v] BSA and 0.1% [w/v] fish skin gelatin), and stored overnight in the same solution containing anti-bromodeoxyuridine monoclonal antibody (Sigma, 1/200) at 4°C. After washing twice in PBG for 10 min each, oocytes were incubated for 1 hr at room temperature with fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody (Santa Cruz Biotechnologies). Subsequently, oocytes were washed twice with PBG for 10 min each, mounted on slides with Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA), and observed under a confocal scanning laser microscope. Images were captured using a Zeiss scanning laser confocal microscope. Serial optical sections (the Z-series) covering all the nuclear and cytoplasmic regions were collected at 1-μm intervals. The Z-series were stacked, and images depicting the staining patterns and intensities of all nuclear and cytoplasmic entities generated. All images in any particular developmental series were acquired using the same laser power output.

Fluorescence Intensity Measurement

To quantify fluorescence intensity, nuclear signals were outlined using Zeiss LSM Image Browser software and mean fluorescence intensity measured. Encircled regions were software-dragged into the cytoplasm of the same cell, and background fluorescence was subsequently measured. Each specific signal was calculated by dividing the nuclear value by cytoplasmic value.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

This work was supported by the BioGreen 21 Program of the Rural Development Administration and a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
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