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BOR - Papers in Press, published online ahead of print December 20, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.055798
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BIOLOGY OF REPRODUCTION 76, 628–638 (2007)
DOI: 10.1095/biolreprod.106.055798
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Proper Chromatin Condensation and Maintenance of Histone H3 Phosphorylation During Mouse Oocyte Meiosis Requires Protein Phosphatase Activity1

Jason E. Swain 3 6, Jun Ding 4, David L. Brautigan 7, Emma Villa-Moruzzi 8, and Gary D. Smith 2 3 4 5 6

Department of Experimental Pathology,8 University of Pisa, 52126 Pisa, Italy Departments of Molecular and Integrative Physiology,3 Obstetrics and Gynecology,4 Urology,5 and Reproductive Sciences Program,6 University of Michigan, Ann Arbor, Michigan 48109 Center for Cell Signaling,7 University of Virginia School of Medicine, Charlottesville, Virginia 22908

ABSTRACT

We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 µM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.

gamete biology, kinases, meiosis, oocyte development, phosphatases

INTRODUCTION

It is estimated that 10%–25% of all human conceptions are chromosomally abnormal due to an error in chromosome segregation during meiosis [1, 2]. Many of these abnormalities are lethal and result in spontaneous abortion within the first trimester, and thus they may not be readily apparent. Furthermore, studies indicate more than 95% of this human aneuploidy is attributable to defects within the machinery of the oocyte [2, 3]. The regulatory mechanisms involved in chromosome remodeling during oocyte maturation is a complex process but one that must be fully understood to circumvent chromosomal nondisjunction, infertility, and embryonic aneuploidy and resulting congenital defects.

Chromatin condensation is an essential prerequisite to ensure subsequent fidelity of chromosome segregation into daughter cells. This is accomplished via resolution of chromosomes, which allows unencumbered movement and reduces the likelihood of entrapment, entanglement, or breakage of genetic material [4]. Thus, chromatin condensation is of great importance when considering causes of oocyte-derived embryonic aneuploidy. Chromatin condensation is the result of long strands of DNA coiling around an octamer of regulatory proteins known as histones: H2A, H2B, H3, and H4 [5]. These coiled structures of DNA and histones are referred to as nucleosomes. Additionally, a family of linker histones, known as H1 histones, functions to fold DNA into a higher ordered structure [5]. Mammalian oocytes contain a specific H1 subtype [6]. Phosphorylation of histones has long been implicated in the regulation of various aspects of the cell cycle, including chromatin condensation [7, 8]. More recent reports demonstrated that specific phosphorylation of histone H3 at Ser10 and Ser28 is tightly coupled to chromatin condensation during both mitosis [9–11] and meiosis [10, 12]. However, additional findings indicate that histone H3 phosphorylation is required only for the initiation rather than the maintenance of the condensed state of chromatin [13], whereas other evidence indicates phosphorylation of histone H3 is not required for meiotic chromatin condensation [14, 15]. Considering contradictions in the literature, it is becoming increasingly evident that differences in the role for histone phosphorylation exist depending on the organism and/or type of cellular division examined [16], and demonstrates the need for further study.

Reversible phosphorylation of proteins represents a highly conserved mechanism that regulates numerous cellular events. This process is dependent upon both phosphoprotein kinase and phosphatase (PPP) activity. Classification of PPPs is based on substrate specificity and sensitivity to defined inhibitors and results in four main types of PPPs: protein phosphatase 1C (PPP1C), PPP2CA, PPP2CB, and PPP2CC [17]. In addition, four isoforms of the PPP1C catalytic subunit exist, varying at their extreme amino and carboxyl termini: PPP1CA, PPP1CB, PPP1CC1, and PPP1CC2 (formerly known as alpha, beta/delta, gamma1, and gamma2, respectively) [18–21]. Of these PPPs, PPP1CA and PPP2CA have been previously identified in oocytes [22, 23].

An important discovery in PPP research was identification of the cell-permeable inhibitor of PPPC1 and PPP2CA, okadaic acid (OA) [24]. Microinjection or extended culture of oocytes in the presence of OA induces premature germinal vesicle breakdown (GVBD) and causes microtubule and cytoplasmic abnormalities, preventing the majority of oocytes from progressing to metaphase II (MII) [25, 26]. It has been determined that PPP1CA is the OA-sensitive PPP participating in regulation of oocyte GVBD [23]. However, arrested development of OA-treated oocytes suggests additional roles for PPPs during oocyte maturation after GVBD. These OA-treated oocytes appear to be arrested at an MI-like state, although normality of homologue condensation remains in question. Interestingly, transient exposure of oocytes to PPP inhibitors allows oocytes to undergo GVBD, progress to MII, and undergo normal fertilization [27]. Collectively, these data indicate that inhibition of a PPP can stimulate meiotic resumption and GVBD, but some PPP activity is required for completion of meiosis. The specific roles for PPP1C and PPP2CA in regulation of oocyte maturation after GVBD remain unknown.

Due to the importance of PPPs in regulation of oocyte meiotic progression, it is imperative to elucidate endogenous PPP regulators. Phosphorylation of PPPC1 at Thr320 by cyclin dependent kinase 1 (CDK1) has been identified as a means of inhibiting oocyte nuclear PPPC1 during GVBD [23, 28]. However, identification of cytoplasmic PPP regulators is of interest when considering control of events such as chromatin condensation and segregation of chromosomes. Two such cytoplasmic inhibitors are inhibitor subunit 1 (PPP1R1A; formerly known as Inhibitor-1) and subunit 2 (PPP1R2; formerly known as Inhibitor-2) [29]. Inactivation of PPP1C by PPP1R1A requires PPP1R1A phosphorylation by protein kinase A (PKA) [30], whereas PPP1R2 inhibition of PPP1C is independent of phosphorylation. Rather, phosphorylation of PPP1R2 at Thr72 in the PPP1C/ PPP1R2 heterodimer results in activation of PPP activity [31–33]. In vitro and in vivo, glycogen synthase kinase 3 (GSK3) is known to regulate PPP1R2 phosphorylation at Thr72 in somatic cells [34, 35]. Transcript and inhibitor activity for PPP1R2 is present in mouse oocytes [28]; however, cytoplasmic inhibitors of PPP1C, including PPP1R2, have not been thoroughly characterized in mammalian oocytes.

Objectives of this study were to determine whether PPPs regulate oocyte chromatin condensation and histone H3 phosphorylation, and to elucidate potential means of regulating cytoplasmic PPPs involved in meiotic chromatin remodeling.

MATERIALS AND METHODS

All procedures described here were reviewed and approved by The University Committee on Use and Care of Animals at the University of Michigan and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.

Mouse Stimulation and Oocyte Collection

Female CF1 mice (Harlan, Indianapolis, IN) 21–23 days old were injected with 10 IU eCG (Sigma, St. Louis, MO). Germinal vesicle-intact (GV-intact) oocytes were isolated by manual rupturing of antral ovarian follicles in Hepes-buffered human tubal fluid medium (HTFH; Irvine Scientific, Santa Ana, CA) supplemented with 0.3% w/v polyvinylpyrrolidone (PVP; Sigma) 42–44 h after eCG injection. Oocytes were matured for 2, 7, and 16 h to obtain GVBD, MI, and MII oocytes, respectively.

To assess the effects of PPPs and GSK3 on regulation of chromosomal condensation, GV-intact oocytes were cultured in the presence or absence of PPP inhibitors OA (1 µM; Calbiochem, La Jolla, CA) and calyculin-A (CLA; 50 nM; Calbiochem) or GSK3 inhibitor alsterpaullone (Alster; 20 µM; Calbiochem) in HTF medium supplemented with 0.3% (w/v) BSA for 7 h at 37°C in 5% CO2 in air. Control treatments consisted of culture in the presence of vehicle (dimethyl sulfoxide [DMSO]). Following treatment, oocytes were collected for chromosomal spreading. To determine the effects of PPP inhibition on Ser10 histone H3 phosphorylation, GV-intact oocytes were cultured in the presence or absence of OA (1 µM) for 3 h as described above and were collected for Western blot analysis (n = 50). This time point was chosen based on initial Western blot experiments, as longer incubation made detection of different band densities difficult. To assess the effects on histone H3 Ser28 phosphorylation, GV-intact oocytes were cultured in the presence or absence of OA for 15 or 30 min, then prepared for immunocytochemistry (ICC). Five oocytes per replicate were analyzed. Use of ICC was required due to the high number of oocytes needed to obtain a signal via Western blot. Time points were selected because longer incubation times stimulated GVBD and made comparison of signal intensity within the GV difficult. To determine the effect of purified PPP1C and PPP2CA on oocyte histone H3 phosphorylation, MI oocyte extract (n = 45) was exposed to 0.1 U purified PPP1C or PPP2CA for 10 min at 30°C as defined in the PP1/PP2A Toolbox (Upstate, Charlottesville, VA). Extracts then were subjected to Western blot. All experiments were performed in triplicate. To assess the effects of GSK3 inhibition on PPP1R2 phosphorylation, GV-intact oocytes were matured to various meiotic stages as described above (GVBD: 2 h; MI: 7 h; MII: 16 h) in the presence or absence of Alster. (20 µM). Extracts (n = 300) then were subjected to Western blot.

RNA Isolation and RT-PCR

Twenty MI oocytes were placed directly into the provided extraction buffer, and RNA was extracted using the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA). Complementary DNA was synthesized using the Superscript Preamplification System for First Strand cDNA Synthesis reagents and methodology (Gibco BRL, Gaithersburg, MD). Oligo-dT was used to prime reverse transcription reactions. Primers for mouse Ppp1cb (gi|29145011|gb|BC046832.1|[29145011]) were designed with no sequence overlap between other PPP1C isoforms (sense strand: 5'-CCCTGTTAACGCTTTAGGGA-3'; antisense strand: 3'-CCTCGGACTTCTGCTTCAGT-5'). Each PCR was performed with two oocyte equivalents of cDNA and 200 nmol of each primer added to SYBR Green PCR Master Mix (Applied BioSystems, Foster City, CA). In addition, control reactions were conducted consisting of 1) no template with primers and master mix and 2) no primers with cDNA template and cocktail. PCR entailed 1 cycle for 2 min at 95°C; 40 cycles at 95°C for 10 min, 60°C for 1 min, and 72°C for 1 min; and 1 cycle at 72°C for 2 min. Following PCR, products were isolated and run on a 2% agarose gel for 60 min at 80 V to verify the size of the amplified product. Additionally, DNA was isolated from gels using QIAquick Gel Extraction Kit (Qiagen, Chatsworth, CA) and subjected to DNA sequencing to verify identity of the product.

Electrophoresis and Western Blot Analysis

Groups of oocytes were placed in 2x SDS-PAGE sample loading buffer (80 mM Tris-HCl [pH 6.8], 20% glycerol, 4% SDS, 4% ß-mercaptoethanol, and 0.04% bromophenol blue), vortexed, and placed on ice for 15 min. Following sonication on ice for 10 sec, samples were denatured at 90°C for 10 min and loaded for electrophoresis. Total protein from equal numbers of mouse oocytes was loaded in each lane and separated by one-dimensional SDS-PAGE. Resolving gels were cast using 12% acrylamide; stacking gels contained 5% acrylamide. HeLa histone lysate was used as a positive control for recognizing phospho-Ser10 and phosphor-Ser28 histone H3. Gels were equilibrated and transferred to Hybond-P PVDF transfer membrane (Amersham Life Sciences, Little Chalfont, England) by Semi-Dry Electrophoretic Transfer Cell (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Blots were blocked in 5% nonfat milk in Tris-buffered saline + 0.5% Tween (TBST) at room temperature for 1 h and incubated with the appropriate primary antibody diluted in TBST + 5% nonfat milk overnight at 4°C with agitation. Antibodies included anti-phospho-Ser10 histone H3 antibody (1:500; Upstate), anti-phospho-Ser28 histone H3 (1:500; Upstate), an antibody recognizing PPP1R1A (1:1000; Chemicon, Temecula, CA) or PPP1R2 antibody (1:1000; kindly provided by Dr. Brautigan). After complete washing in TBST, blots were incubated with the appropriate horseradish peroxidase-conjugated IgG secondary antibody (diluted 1:2000) at room temperature for 1 h, washed in TBST, and developed with ECL Plus reagents (Amersham Life Sciences) according to the manufacturer's instructions. To verify equal protein loading of lanes to allow densitometric analysis, blots were stripped for 30 min at 50°C water bath with agitation in a stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM ß-mercaptoethanol, and 2% SDS). Completely stripped blots were blocked in 5% nonfat milk in TBST for 1 h at room temperature, then incubated with histone H3 antibody (diluted 1:1000; Chemicon) overnight at 4°C with agitation and processed further as described above. Band densities were assessed using imaging software Image J (National Institutes of Health, Bethesda, MD).

Immunocytochemistry

To examine localization of OA-sensitive PPPs, GV-intact oocytes were collected and cultured as described above to obtain GVBD, MI, and MII oocytes. Oocytes were attached to poly-lysine-coated coverslips and fixed in 2% (w/v) paraformaldehyde with 0.05% (v/v) Triton X-100 in PBS (pH 7.3) for 30 min. Oocytes then were blocked overnight with 2% (w/v) BSA, 0.1 M glycine, and 5% (w/v) dry milk in PBS at 4°C. Oocytes were incubated with PPP1CB antibody (1:1000; courtesy of Dr. Villa-Moruzzi), PPP2CA antibody (1:500; Upstate), PPP1CA antibody (1:500; Upstate), PPP1R2 antibody (1:100), phospho-Ser10 histone H3 (1:100), or phospho-Ser28 histone H3 (1:100) for 1 h at 37°C. Negative controls included nonimmune rabbit serum in place of primary antibodies. After three 5-min washes with blocking solution, samples were reacted with the appropriate Alexa 488-conjugated secondary antibody (Molecular Probes) at a 1:750 dilution for 1 h at 37°C. Following washing, slides were incubated with Hoechst 33342 (1 µg/ml) in PBS for 20 min at 37°C. Coverslips then were mounted on glass slides with 90% glycerol in PBS for fluorescence microscopic visualization under 1000x magnification with either a Leica DMR or an Olympus FluoView 500 laser scanning confocal microscope. Densitometry analysis of ICC images was performed using Image J imaging software. Statistical analysis was performed using a Student unpaired t-test.

Chromosomal Spreading and Analysis

After culture to MI (7 h), oocytes were collected and prepared for chromosomal spreading [36]. Briefly, zona pellucida were removed by exposure to 1% pronase in HTFH. Zona-free oocytes then were washed and fixed by carefully placing them onto a microscope slide dipped in a solution of 1% paraformaldehyde in distilled H20 (pH 9.2) containing 0.15% Triton X and 3 mM dithiothreitol. Slides then were placed into a humidified chamber overnight before being subjected to triplicate 5-min washes in PBS and air dried at room temperature.

To analyze chromosomal condensation, slides were placed into a 1% solution of Hoechst 33342 in PBS for 10 min and subjected to three more washes in PBS. To analyze PPP2CA and PPP1C association with chromatin, spreads also were treated with a PPP2CA antibody (1:100), PPP1CB antibody (1:100), or histone H3 antibody (1:100) in PBS for 1 h at 37°C, followed by three washes in PBS and treatment with a 1:750 dilution of anti-rabbit Alexa 568 secondary antibodies (Molecular Probes). Glycerol mounting solution and a coverslip were added, and slides were sealed. Chromosomal spreads were analyzed blind to treatment at 1000x magnification on a Leica DMR microscope. Statistical differences between treatment groups were analyzed using Student unpaired t-test.

RESULTS

To determine whether the block in oocyte MII progression due to inhibition of PPPs via OA and CLA treatment described in previous reports could be due to defects in chromosome condensation, GV-intact oocytes were cultured in the presence or absence of 1 µM OA or 50 nM CLA for 7 h to reach MI. Chromosomal spreads were performed, and condensation of homologues was analyzed. At the light microscope level, inhibition of oocyte PPPs had no effect on oocyte development to MI, and oocytes in all treatments had condensed metaphase chromosomes. However, chromosomal spreading indicated that inhibition of PPPs results in significant abnormal condensation of chromatin (P < 0.0001), as evidenced by smearing and failure to observe distinct resolution of bivalents. Control spreads displayed 90% (25 of 39) normal chromatin spreading, whereas OA and CLA had 3% (1 of 30) and 0% (0 of 35) normal spreading of chromosomes, respectively (Fig. 1).


Figure 01
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  		FIG. 1. Inhibition of oocyte PPP1C and/or PPP2CA via treatment with OA (1 µM) or CLA (50 nM) for 7 h resulted in MI oocytes with significantly greater defects in chromosome condensation, as evidenced by inability to resolve bivalents, compared with controls (P < 0.0001). Images are representative micrographs of chromosome spreads. Original magnification x1000.

To begin to elucidate mechanisms of how oocyte PPPs regulate bivalent formation, initial experiments determined the phosphorylated state of histone H3 at Ser10 and Ser28 residues during meiotic progression. As assessed by ICC, GV-intact oocytes (0 h) lacked phosphorylation of histone H3 at Ser10. At or around GVBD (2 h), histone H3 became phosphorylated at Ser10 and remained phosphorylated at MI (7 h) and MII (16 h) in association with condensed chromatin (Fig. 2A). ICC indicated that Ser28 of histone H3 is phosphorylated in GV-intact oocytes and remains phosphorylated and associated with condensed chromatin throughout oocyte meiosis (Fig. 2B). Western blot analysis confirmed the pattern of Ser10 and Ser28 histone H3 phosphorylation (Fig. 2C).


Figure 02
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  		FIG. 2. Representative micrographs of histone H3 phosphorylation in oocytes at various stage of meiotic progression. A) Phosphorylation of histone H3 at Ser10 (green) was absent in GV-intact oocytes and became phosphorylated at times coinciding with chromatin condensation, as evidenced by ICC. This phosphorylation colocalized (yellow) with chromatin stained with Hoechst (blue). PB, polar bodies. B) Phosphorylation of histone H3 at Ser28 was present in oocytes at all meiotic stages examined. C) Top panel: Western blot analysis confirmed Ser10 phosphorylation pattern (n = 40 oocytes per lane). Bottom panel: Western blot analysis confirmed Ser28 phosphorylation pattern (n = 360 oocytes per lane). Original magnification x1000.

To determine whether aberrant oocyte chromosome condensation due to OA/CLA treatment could be a result of changes in the phosphorylated state of histone H3, we assessed the effects of PPP inhibition on Ser10 and Ser28 phosphorylation. Western blot followed by densitometric analysis of a representative blot revealed that inhibition of oocyte PPPs in GV-intact oocytes treated with OA for 3 h results in an approximate 2.2-fold increase in phosphorylation of histone H3 at Ser10 (Fig. 3A). Due to the high number of oocytes needed to obtain a signal for Ser28 histone H3 phosphorylation via Western blot, we used ICC to assess the effect of PPP inhibition on Ser28 histone H3 phosphorylation. Culture of GV-intact oocytes for 15 or 30 min in the presence of OA (1 µM) resulted in increased Ser28 histone H3 phosphorylation within the GV compared with controls (Fig. 4). Densitometric analysis of oocytes from a representative replicate revealed that OA treatment for 15 min resulted in a significant 2.6-fold increase in Ser28 histone H3 phosphorylation compared with controls, P < 0.0001 (42.5 ± 3.1 vs. 109.4 ± 2.3, respectively). Treatment of oocytes with OA for 30 min resulted in a significant 1.9-fold increase in Ser28 histone H3 phosphorylation compared with controls, P < 0.003 (52.9 ± 11.4 vs. 103.1 ± 3.1, respectively).


Figure 03
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  		FIG. 3. A) Culture of GV-intact oocytes (n = 50) for 3 h in the presence of 1 µM OA resulted in increased phosphorylation of histone H3 at Ser10 compared with untreated controls. B) Densitometric analysis on a representative blot was used to graphically display this increase in phosphorylation. Controls were normalized to 100 arbitrary units, and phosphorylation following treatment with OA from a representative blot is displayed as an increase compared with untreated controls.


Figure 04
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  		FIG. 4. Representative micrographs of histone H3 Ser28 phosphorylation in oocytes following PPP inhibition. Culture of GV-intact oocytes for 15 or 30 min in the presence of 1 µM OA resulted in increased phosphorylation of histone H3 at Ser28 within the germinal vesicle of oocytes compared with untreated controls. N, nucleus; n, nucleoli. Original magnification x1000.

Inhibitor studies in intact cells using OA or CLA do not allow differentiation between PPP1C and PPP2CA activity. Therefore, to verify that PPP1C could in fact dephosphorylate histone H3, MI oocyte lysate (n = 50) was exposed to purified PPP1C or PPP2CA. Western blot analysis of treated lysates verified that Ser10 histone H3 is a substrate for purified PPP1C (Fig. 5).


Figure 05
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  		FIG. 5. Representative Western blot of histone H3 Ser10 phosphorylation following treatment with protein phosphatases. Recombinant PPP1C reduced phosphorylation of Ser10 of histone H3 in lysates of mouse MI oocytes (n = 45) compared with untreated controls. HeLa cell extract was used as a positive control. Membranes were stripped and reprobed with histone H3 antibody to verify equal protein content in each lane.

Based on localization data in somatic cells [37], PPP1CB is a likely candidate for the oocyte PPP regulating chromatin condensation and histone H3 phosphorylation. However, this PPP1C isoform has not been identified in oocytes. Using primers designed against mouse Ppp1cb, RT-PCR verified the presence of Ppp1cb transcript in MI oocytes. Products were run on an agarose gel and gave the expected 148-bp product corresponding to the amplified region (Fig. 6). Sequencing of DNA confirmed that the amplified sequence showed 100% similarity to mouse Ppp1cb.


Figure 06
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  		FIG. 6. Presence of Ppp1cb transcript in mouse oocytes was verified via agarose gel electrophoresis of PCR-amplified MI oocyte cDNA. Lanes A and F: PCR ladder; lane B: mouse brain and primers (positive control); lane C: primers, no cDNA; lane D: oocyte cDNA, no primers; lane E: oocyte cDNA and primers.

Spatial juxtapositioning of kinases/PPPs with their phosphoprotein substrates is important for direct functional regulation of substrates in vivo [38]. To begin to elucidate which OA- or CLA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, ICC was conducted using specific PPP1C isoform and PPP2CA antibodies. Microscopic analysis revealed that PPP1CB (green) colocalized to condensing chromatin (red) in GVBD, MI, and MII oocytes (Fig. 7A). No localization of PPP1CA to chromatin in GVBD, MI, or MII oocytes was observed (data not shown). In agreement with a previous report [39], PPP2CA colocalized with oocyte microtubules (data not shown). However, using conventional ICC, it was not clear whether PPP2CA also colocalized with condensed chromatin. Therefore, chromosomal spreads were prepared and probed with PPP2CA antibody to determine whether the PPP associates with oocyte-condensed chromatin. Chromosomal spreads did not demonstrate any localization of PPP2CA to chromatin; however, PPP1CB did colocalize with condensed chromatin (Fig. 7B). To further validate the absence of PPP2CA localizing to oocyte-condensed chromatin and rule out the finding as an artifact of the chromosomal spreading technique, MI oocytes were cultured in nocodazole (5 µM in culture for 2 h) or cold treated (4°C in HTFH for 15 min) to depolymerize microtubules and prevent localization near chromatin. These methods verified that PPP2CA did not localize to oocyte-condensed chromatin (data not shown).


Figure 07
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  		FIG. 7. A) Representative micrographs of oocytes at various meiotic stages demonstrating localization of PPP1CB to condensed chromatin using ICC. PB, polar bodies. B) Chromosomal spreads of MI oocytes followed by treatment with specific antibodies confirms PPP1CB localization to condensed chromatin, whereas PPP2CA showed no such localization. Histone H3 spreads were performed as a positive control to verify accuracy of the technique. Original magnification x1000.

Finally, if PPPs are important in regulating oocyte chromatin condensation and histone H3 phosphorylation, then regulation of PPP activity also is important. Western blot analysis confirmed the presence of PPP1C inhibitor PPP1R2 in oocytes at various meiotic stages, whereas inhibitor PPP1R1A was absent (Fig. 8A). Interestingly, PPP1R2 blots showed a doublet, suggesting a possible change in phosphorylation and, therefore, inhibitory activity of PPP1R2. It appeared that the intensity of the slower migrating band and, therefore, phosphorylation of PPP1R2 increased as oocytes progressed to GVBD, MI, and MII. Additionally, ICC indicated that PPP1R2 showed diffuse staining in GV-intact oocytes and colocalized with condensed chromatin at GVBD, MI, and MII (Fig. 8B).


Figure 08
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  		FIG. 8. A) Representative Western blots of PPP1C inhibitors in oocytes at various meiotic stages. Protein expression of PPP1R1A is absent in oocytes (n = 320), whereas the presence of a doublet in PPP1R2 blots was confirmed in all stages examined (n = 320). Interestingly, the slower migrating band in PPP1R21 blots increased in intensity as oocytes progressed to MII. This may represent a phosphorylated form of PPP1R2. B) ICC confirmed PPP1R2 presence and demonstrated PPP1R2 localized to condensed chromatin in MI and MII stage oocytes. Original magnification x1000.

Knowing that GSK3 regulates PPP1R2 inhibitory activity through regulation of phosphorylation at Thr72 in somatic cells [34, 35], the role of GSK3 in regulation of oocyte PPP1R2 phosphorylation was examined. GV-intact oocytes were cultured to various meiotic stages in the presence or absence of GSK3 inhibitor Alster. Inhibition of oocyte GSK3 resulted in a shift of the PPP1R2 observed band to a faster migrating form, indicating a reduction in PPP1R2 phosphorylation (Fig. 9). To verify the band shift was indicative of phosphorylation, treatment of oocyte lysate with alkaline phosphatase was conducted. Resulting disappearance of the upper band following PPP treatment confirmed phosphorylation of PPP1R2 in oocytes (data not shown).


Figure 09
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  		FIG. 9. Representative Western blot of PPP1R2 following inhibition of oocyte GSK3 via treatment with 20 µM Alster (Alst.) during oocyte maturation. Inhibition of GSK3 in faster migration of PPP1R2 is indicative of a reduction in phosphorylation (n = 300).

Previous reports indicate that GSK3 inhibition causes abnormal oocyte chromosome segregation [40]. This could be due in part to abnormal chromatin condensation. To verify that the GSK3 signaling pathway could indeed be involved in regulating chromatin condensation, GV-intact oocytes were cultured to MI in the presence or absence of Alster. Inhibition of oocyte GSK3 resulted in aberrant chromatin condensation, as evidenced by the same inability to resolve distinct bivalents through chromosomal spreading observed following OA or CLA treatment. Untreated oocytes displayed 98% normal spreads (23 of 24), whereas Alster-treated oocytes only displayed 7% normal spreads (2 of 29; Fig. 10).


Figure 10
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  		FIG. 10. Representative micrograph of oocyte chromosomal spreads following inhibition of GSK3 via treatment with 20 µM Alster (Alsterpaullone). Inhibition of oocyte GSK3 resulted in aberrant chromosome condensation, as evidenced by the inability to resolve bivalents. Original magnification x1000.

DISCUSSION

Oocyte meiotic disjunction is a leading cause of embryonic aneuploidy and resulting infertility [1]. Since proper chromatin condensation is a prerequisite for ensuring subsequent fidelity of chromosomal separation and segregation by preventing tangling and breakage of genetic material, elucidating factors regulating oocyte chromatin condensation may be beneficial in developing therapies for oocyte-derived infertility. OA inhibition of oocyte PPPs has been shown to induce GVBD and chromosome condensation, arresting oocytes at an MI-like stage [25, 26]. However, methods used to assess chromosome condensation lacked refinement. Oocytes' inability to progress to MII in the presence of OA, coupled with findings that show that transfer of oocytes to an OA-free environment results in MII development with aneuploidy [41], calls into question normality of chromatin condensation under PPP inhibition. Using a chromosome spreading technique, we showed that OA/CLA inhibition of oocyte PPPs resulted in abnormal chromosome remodeling, manifesting as smeared chromatin unable to resolve distinct bivalents. This is suggestive of abnormal chromatin condensation, as control spreads clearly showed distinct, compact tetrads. OA does not interfere with the ability of oocyte microtubules to polymerize [42], and emerging research indicates that chromosomes direct the formation of the spindle apparatus [43, 44]. Thus, this lack of proper chromatin condensation is problematic, not only because of potential problems with aneuploidy due to aberrant separation/segregation of homologues, but also because without normal formation of condensed bivalents, formation of a functional meiotic spindle may be negatively affected and meiotic progression impeded.

To begin to determine how PPPs are regulating oocyte chromatin condensation, we examined the phosphorylated state of histone H3. Phosphorylation of histone H3 at Ser10 and Ser28 is coupled to mitotic [9–11] and meiotic chromosome condensation [10, 12]. It is thought that phosphorylation of the N-terminal tails of histone H3 may act as a receptor or recruitment factor for condensation factors [45]. Alternatively, phosphorylation of the amino termini may reduce the affinity of histone H3 for DNA and make the relatively compact chromatin fiber more readily accessible to condensation factors [45], such as the condensin complex. Condensins are multisubunit protein complexes that play a central role in chromosome compaction and condensation. Interestingly, components of the condensin complex colocalize and bind with phosphorylated histone H3 [46, 47]. Two condensins have been identified. Condensin II participates in early stages of chromosome condensation within the prophase nucleus, whereas condensin I regulates later condensation events once it gains access to chromosomes following nuclear envelope breakdown [48, 49]. Interestingly, in our study Ser28 of histone H3 was phosphorylated at all cell stages examined during mouse oocyte meiosis, including in GV-intact oocytes. Chromatin condensation occurs within the GV of mouse oocytes, as evidenced by nucleolar rimming [50]. Additionally, early stages of chromatin condensation within the GV of pig oocytes have been described [51]. However, Western blot and ICC demonstrated that histone H3 was unphosphorylated at Ser10 in GV-intact oocytes. Upon GVBD, histone H3 became phosphorylated at Ser10 and localized to condensing chromatin. This pattern persisted through MI and MII. This is in agreement with two recent reports on histone H3 phosphorylation at Ser10 in pig oocytes [52, 53]. These phosphorylation patterns indicate possible differential functions for histone H3 phosphorylation at Ser10 and Ser28. Phosphorylation of Ser28 may be required for initial aspects of chromatin condensation within the GV, whereas Ser10 phosphorylation may be instrumental in later aspects of chromatin condensation following GVBD, possibly through differential recruitment of condensin II and condensing I, respectively.

The correlation between histone H3 phosphorylation and oocyte chromatin condensation raises the possibility that aberrations in chromatin condensation observed following PPP inhibition with OA/CLA were the result of alteration of the balance of histone H3 phosphorylation. Indeed, inhibition of the histone H3 kinase and/or PPP in yeast and nematodes results in hypophosphorylation and hyperphosphorylation of histone H3, respectively, yielding abnormal chromatin condensation [54]. Treatment of pig oocytes with PPP inhibitors induced hyperphosphorylation of histone H3 [52]. However, the effects of OA on phosphorylation of specific histone H3 amino acid residues were not examined. Our study demonstrates that treatment of oocytes with OA induced hyperphosphorylation of histone H3 on Ser10 and Ser28. Thus, altering histone H3 phosphorylation at Ser10 and Ser28 may affect the ability of oocyte chromatin to condense properly. Increased histone H3 phosphorylation may be required for initiation, but not maintenance, of chromatin condensation [13]. Our findings potentially support this theory. Inhibition of PPP with OA or CLA for short periods of time (1 h) is commonly used to condense mitotic chromosomes for karyotyping [55, 56], whereas treatment for longer periods leads to "shortened and fuzzy" chromosomes [56]. It is only with extended culture (7 h in our study) that oocyte chromatin displayed visual condensation abnormalities. Thus, although phosphorylation of histone H3 appears critical for induction of initial aspects of chromosome condensation, hyperphosphorylating histone H3 for extended periods appears to be detrimental in chromatin remodeling events crucial for formation of bivalents.

Intracellular localization of enzymes is an excellent means of regulating their activity, especially in a cell as large as the oocyte. To begin to elucidate which OA-sensitive PPP is responsible for regulating histone H3 phosphorylation and chromatin condensation, localization of PPP1C isoforms was examined. This is important, because sperm contain only a single testes-specific PPP1C isoform, PPP1CC2 [57, 58], and PPP1CC1 protein expression is not detectable in oocytes [22]. This indicates a marked difference between mitotic and meiotic cells in regards to PPP1C isoforms. PPP1CB localized to condensing chromatin in oocytes. This is the first identification of this PPP1C isoform in oocytes, and it is in agreement with intracellular localization of PPP1CB in somatic cells [37]. This localization suggests PPP1CB is the OA-sensitive PPP responsible for regulating oocyte chromatin condensation. In Drosophila it appears as if PPP1C regulates chromatin condensation [59, 60]. Furthermore, the intra-oocyte PPP2CA catalytic subunit localizes to polymerized microtubules, in agreement with a previous report [39], where it likely regulates the phosphorylated state of microtubule-associated proteins, such as Tau [61–63]. Lastly, in fibroblasts, similar to observations in oocytes, PPP1C but not PPP2CA localized to condensed chromatin [64, 65].

Inhibitor studies do not indicate whether PPP1C is acting directly on histone H3, or if inactivation of PPP1C is activating a histone H3 kinase. Studies in lower eukaryotes suggest histone H3 phosphorylation is regulated by both PPP1C and Aurora B kinase (AURKB) [54, 66]. PPP1C can directly dephosphorylate histone H3 at Ser10 [54], and inhibition of PPP1C activates AURKB activity and total histone H3 phosphorylation in Xenopus egg extracts [66]. AURKB is known to also phosphorylate Ser28 of histone H3 in mitotic cells [67], and AURKB is thought to regulate histone H3 Ser10 phosphorylation in pig oocytes [53]. Thus, a balance between both PPP1C and AURKB activity appears necessary to regulate histone H3 phosphorylation. However, although AURKB regulates Ser10 phosphorylation in pig oocytes, it does not regulate phosphorylation of Ser28 [53]. If this holds true in mouse oocytes, then our findings would suggest PPP1C may be acting directly on Ser28 or activating a kinase other than AURKB. AURKA physically interacts with the histone H3 tail and efficiently phosphorylates the protein both in vitro and in vivo [68]. Aurora A has been identified within the germinal vesicle of mouse and pig oocytes and concentrates around condensing chromatin shortly after GVBD and again shortly after completion of meiosis I [69, 70]. Recently, PPP1R2 has been reported to act as a bifunctional signaling molecule, capable of not only inhibiting PPP1C, but also activating AURKA [71]. We have identified PPP1R2 in oocytes, localized to condensed chromatin. Thus, although AURKA is thought to primarily regulate centrosome and spindle activity [69, 72], the kinase may be involved in certain aspects of early chromatin remodeling and may be acting in concert with PPP1C, via regulation by PPP1R2, to regulate phosphorylation of histone H3 on Ser28. Furthermore, it should be mentioned that neither AURKB activity nor histone H3 phosphorylation is required for chromosome condensation in pig oocytes, but they may be required for further chromosome processing necessary for alignment of chromatin to the metaphase plate [53]. However, more sensitive measures of chromatin condensation, such as the chromosomal spreading techniques used in this paper, may reveal subtle abnormalities in chromatin condensation not detected when using whole-cell ICC, demonstrating that AURKB and histone H3 phosphorylation may be required for later events in chromosome condensation necessary for bivalent resolution. Interactive studies of the AURK and PPP1C in regulation of oocyte chromatin condensation remains an area of interest.

In attempting to understand upstream regulation of PPP1C in oocytes, we examined known cytoplasmic inhibitor PPP1R2, which was first identified as a heat-stable protein isolated from rabbit skeletal muscle that is capable of selectively inhibiting PPP1C activity [73]. Later, PPP1R2 levels were shown to fluctuate during the mitotic cell cycle, peaking at mitosis, suggesting its involvement in cell cycle regulation [74]. We have previously identified Ppp1r2 transcript and a PPP1C inhibitor with biochemical properties similar to PPP1R2 in mouse oocytes [28]. Our current findings have confirmed the presence of PPP1R2 protein expression in oocytes through ICC and Western blot analysis. PPP1R2 showed localization to condensed chromatin, coincident with PPP1CB. Nuclear localization of both PPP1R2 and PPP1CB has been reported previously in HeLa cells grown at low density [75], indicating that PPP1R2 can both localize to chromatin and interact with this PPP1C isoform. Additionally, evidence exists for a role for PPP1R2 in regulating chromatin remodeling. Yeast GLC8 (PPP1R2) interacts with Ipl1 (AURK) and GLC7 (PPP1C) to control chromosome segregation [76, 77], and knockdown of PPP1R2 in human epithelial cells, as well as in Drosophila, causes aberrant chromatin segregation (D.L. Brautigan, personal communication), possibly as the result of abnormal condensation. Three isoforms of PPP1R2 have been identified in rat, two of which appear to be testis-specific isoforms [78], although neither ovarian tissue nor oocytes were examined. It would be interesting to determine whether these alternate PPP1R2 isoforms could in fact be meiosis-specific isoforms or whether oocytes contain their own specific PPP1R2 isoforms, and whether these isoforms have differential localization and functions within the cell.

If PPP1R2 is important in regulating chromatin-associated PPP1C activity and chromosome condensation, then regulation of PPP1R2 phosphorylation and inhibitory activity is critical. The presence of a doublet in Western blots suggests a change in phosphorylation of PPP1R2, and thus a possible change in regulation of PPP1C activity. Recall, phosphorylation of PPP1R2 at Thr72 results in reactivation of PPP1C activity [31–33]. A known regulator of PPP1R2 phosphorylation at Thr72 in vivo is GSK3 [34, 35]. We report for the first time that inhibition of oocyte GSK3 activity with Alster increases eletrophoretic migration of PPP1R2, which is indicative of decreased PPP1R2 phosphorylation. However, it should be noted that we could not determine whether this phosphorylation change involved Thr72. Additionally, we report for the first time that Alster inhibition of oocyte GSK3 also induces aberrant bivalent formation similar to that observed following OA inhibition of PPPs. Inhibition of oocyte and preimplantation embryo GSK3 has been reported to induce chromatin segregation errors [40, 79]. Therefore, oocyte chromatin segregation errors due to GSK3 inhibition may be due in part to aberrant chromatin condensation. It should be noted that although Alster is highly selective for GSK3, it also can inhibit CDK1, with IC50s of 0.004 and 0.035 µM, respectively [80, 81]. CDK1 also may regulate PPP1R2 phosphorylation [34, 82, 83]. However, at the concentration used in this study (20 µM), Alster displays a much greater selectivity for GSK3 versus CDK1 in cell culture [80]. Additionally, inhibition of CDK1 in GV-intact oocytes results in meiotic arrest at the GV stage and inability to progress to MII [23, 84, 85]. Furthermore, inhibition of CDK1 with roscovitine in GVBD oocytes cultured for 5 h does not result in aberrant oocyte chromatin condensation (data not shown). This corresponds to data indicating that CDK1 is not required for chromatin condensation in pig or cow oocytes [52, 86, 87]. These dramatically different phenotypes following CDK1 inhibition suggest that defects seen in chromatin condensation in MI oocytes following Alster treatment are more likely due to inhibition of GSK3 rather than CDK1. Interestingly, GV-intact, MI, and MII oocytes contain both GSK3A and GSK3B isoforms, with higher levels of GSK3A expression [40]. Furthermore, MII oocytes display differential phosphorylation of GSKA and GSKB, which regulates GSK3 activity [79]. If oocytes do contain unique PPP1R2 isoforms, it would be interesting to determine whether GSK3 isoforms differentially regulate PPP1R2 isoforms, thus offering a means to differentially regulate oocyte PPP1C activity.

Here we report that oocytes contain PPP1CB and an endogenous PPP1C inhibitor, PPP1R2, both of which colocalize to condensed chromatin. Additionally, inhibition of oocyte PPP1C with OA/CLA induces aberrant chromatin condensation and histone H3 hyperphosphorylation. Furthermore, Alster treatment of oocytes to inhibit GSK3 results in decreased PPP1R2 phosphorylation and aberrant chromatin condensation. Thus, PPP1R2 may provide a signaling connection between oocyte GSK3 and PPP1C in regards to regulation of oocyte chromatin condensation. GSK3, PPP1R2, and PPP1C coimmunoprecipitate and form a complex in vivo [35]. However, caution must be used in making these assumptions. PPP1R2 is a multifunctioning protein, not only inhibiting PPP1C but also activating kinases, such as NEK2 and AURKA [71, 88]. Furthermore, phosphorylation of PPP1R2 at specific residues, such as Thr72, alters its binding and regulatory properties [31, 32]. We have not assessed phosphorylation of specific PPP1R2 residues. Future studies will focus on the role of PPP1R2 in regulation of oocyte chromatin dynamics. These data extend the basic knowledge of reversible phosphorylation, structural regulatory substrates, and mechanisms involved in controlling meiotic chromatin remodeling in mammalian oocytes.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Charles Bormann, Dr. Carrie-Cosola-Smith, and Jeni Chapman for critically reviewing this manuscript.

FOOTNOTES

1Supported by National Institutes of Health grants HD35125-01A1, HD046768-01A2, and GM-56362. Back

Correspondence: 2Gary D. Smith, 6428 Medical Sciences Building I, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0617. FAX: 734 936 8617; e-mail: smithgd{at}umich.edu

Received: 17 July 2006.

First decision: 9 August 2006.

Accepted: 5 December 2006.

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