O-Linked N-Acetylglucosaminyltransferase Inhibition Prevents G2/M Transition in Xenopus laevis Oocytes*

Full-grown Xenopus oocytes are arrested at the prophase of the first meiotic division in a G2-like state. Progesterone triggers meiotic resumption also called the G2/M transition. This event is characterized by germinal vesicle breakdown (GVBD) and by a burst in phosphorylation level that reflects activation of M-phase-promoting factor (MPF) and MAPK pathways. Besides phosphorylation and ubiquitin pathways, increasing evidence has suggested that the cytosolic and nucleus-specific O-GlcNAc glycosylation also contributes to cell cycle regulation. To investigate the relationship between O-GlcNAc and cell cycle, Xenopus oocyte, in which most of the M-phase regulators have been discovered, was used. Alloxan, an O-GlcNAc transferase inhibitor, blocked G2/M transition in a concentration-dependent manner. Alloxan prevented GVBD and both MPF and MAPK activations, either triggered by progesterone or by egg cytoplasm injection. The addition of detoxifying enzymes (SOD and catalase) did not rescue GVBD, indicating that the alloxan effect did not occur through reactive oxygen species production. These results were strengthened by the use of a benzoxazolinone derivative (XI), a new O-GlcNAc transferase inhibitor. Conversely, injection of O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor, accelerated the maturation process. Glutamine:fructose-6-phosphate amidotransferase inhibitors, azaserine and 6-diazo-5-oxonorleucine, failed to prevent GVBD. Such a strategy appeared to be inefficient; indeed, UDP-GlcNAc assays in mature and immature oocytes revealed a constant pool of the nucleotide sugar. Finally, we observed that cyclin B2, the MPF regulatory subunit, was associated with an unknown O-GlcNAc partner. The present work underlines a crucial role for O-GlcNAc in G2/M transition and strongly suggests that its function is required for cell cycle regulation.

Immature vertebrates oocytes, arrested at prophase I of meiosis, can resume their cell cycle, also called maturation, in response to hormonal stimulation (1,2). Studies in amphibian Xenopus laevis oocyte have greatly contributed to the knowledge of the biochemical activities of the key regulatory molecules involved in these processes (3).
Meiosis entry, analogous to G 2 /M transition, is promoted by a cytoplasmic factor called MPF. 3 MPF, made up of a catalytic subunit, Cdk1 (cyclin-dependent kinase 1) (also called Cdc2 (cell division cycle 2)), and a regulatory subunit, cyclin B, has been demonstrated to be the universal regulator of mitosis and meiosis entry. Activation of this cyclin-Cdk complex is controlled by phosphorylation and proteolysis (for reviews, see Refs. 4 and 5). Association between the regulatory subunit and Cdk1 requires phosphorylation of Cdk1 on residue Thr 161 by Cdk-activating kinase (6). Then, to be catalytically active, Cdk1 is dephosphorylated on Thr 14 and Tyr 15 residues by Cdc25, a dual specific phosphatase (6,7). Simultaneously to Cdk1 dephosphorylation, cyclin B is phosphorylated (8). Xenopus immature oocytes, which are synchronized at the diplotene stage of first meiosis, contain large amounts of inactive MPF, or pre-MPF (4,5). Inhibition of pre-MPF is provided by Myt1 that phosphorylates Cdk1 on Thr 14 and Tyr 15 and then inactivates the complex. Pre-MPF can be directly activated by Cdc25 injection (9) or through an autoamplification loop. The latter loop involves the ability of MPF to phosphorylate and activate Cdc25 (10,11) and does not depend upon protein synthesis (12).
Simultaneously to MPF activation, extracellular signal-regulated kinase-like Xp42 mpk1 is phosphorylated and activated (13). Xp42 mpk1 belongs to the mitogen-activated protein kinase (MAPK) pathway that is turned on by Mos oncoprotein synthesis in response to progesterone stimulation (14). Once activated, Xp42 mpk1 phosphorylates and activates ribosomal S6 kinase (p90 rsk ), which negatively regulates Myt1 (15). Activation of the Mos-Xp42 mpk1 pathway has been shown not to be required for MPF activation but for timely M-phase entry when oocytes are stimulated either by progesterone or insulin (16 -18). Nevertheless, this pathway has been shown to be responsible for S-Phase suppression between meiosis I and meiosis II, for metaphase II arrest, and for spindle morphogenesis (17, 19 -21).
Mitogenic signals orchestrate a regulatory network of proteins mainly through post-translational modifications (PTMs). Although phosphorylation is the molecular mechanism associated with the regulation of cell cycle, it is clearly not the only post-translational mechanism involved in cell cycle progression. Recent observations have suggested that the cytosolic and nucleus-specific O-linked N-acetylglucosaminylation (O-Glc-NAc) could be involved in cell cycle progression (22)(23)(24). O-GlcNAc is a highly dynamic PTM whose versatility is regulated by two enzymes (25): the O-GlcNAc transferase (OGT) that catalyzes the transfer of the GlcNAc moiety from UDP-GlcNAc and O-GlcNAcase that hydrolyzes the GlcNAc residue. Although the functions played by this single PTM remain to be determined, several studies tend to demonstrate that O-GlcNAc is tightly linked to cell cycle regulation. Microinjection of bovine galactosyltransferase inhibited Xenopus oocytes M-phase entry and blocked S-to M-phase transition (26). Slawson et al. (27) showed that perturbation of Xenopus oocyte O-GlcNAc levels, either by glucosamine or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) treatments, modified maturation kinetics. We previously reported that the G 2 -to M-phase transition was accompanied with a noticeable increase in O-GlcNAc glycosylation (22). Last, PUGNAc was used to inhibit O-GlcNAcase on somatic cultured cells; such treated cells progressed through the cell cycle more slowly than untreated cells (24). This delay was more pronounced during the S-phase progression and at the G 2 /M boundary. By contrast, inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT) with 6-diazo-5oxonorleucine (DON), resulting in low O-GlcNAc level, shortened S-phase, and G 2 /M progression. In return, GFAT inhibition delayed G 1 progression in comparison with controls or PUGNAc-treated cells. Taken together, these data demonstrate that O-GlcNAc dynamic is a key regulatory PTM in cell cycle progression. The present study pointed out a role for O-GlcNAc glycosylation as a key regulator mechanism for progression from G 2 arrest to M-phase in Xenopus oocyte, through the regulation of both MPF and MAPK pathways.
Handling of Oocytes-After anesthetizing Xenopus females by immersion in 1 g/liter MS222 solution (tricaine methane sulfonate), ovarian lobes were surgically removed and placed in ND96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2 , 5 mM HEPES-NaOH, pH 7.5). Fully grown stage VI oocytes were isolated, and follicle cells were partially removed by 1 mg/ml collagenase A treatment for 30 min followed by a manual microdissection. Oocytes were stored at 14°C in ND96 medium until experiments.
Stimulation and Analysis of G 2 /M Transition (Meiotic Resumption) in Xenopus Oocytes-Meiotic resumption was induced by incubating oocytes in ND96 medium containing 10 M progesterone or by cytoplasm microinjection. Briefly, cytoplasm of matured oocytes was collected and microinjected in immature oocytes (50 nl/oocyte) using a positive displacement digital micropipette (Nichiryo, Tokyo, Japan). GVBD was scored by the appearance of a white spot at the animal pole of the oocyte and confirmed by hemisection of oocyte after heat fixation (100°C, 5 min). Alternatively, oocytes were fixed overnight in Smith's fixative, dehydrated, and embedded in paraffin for cytological studies. 7-m sections were stained with nuclear red for the detection of nuclei and chromosomes, whereas picroindigocarmine was used to reveal cytoplasmic structures (21).
Oocyte Treatments-Before stimulation of meiotic resumption, oocytes (15-20 oocytes/condition) were incubated overnight with alloxan concentrations ranging from 1 to 5 mM, 5 mM uracil, 500 M Me 2 SO-solubilized benzoxazolinone derivative, 40 -80 units of catalase, 150 units of SOD, or 0.1-5 mM hydrogen peroxide. For GFAT inhibition experiments, 100 M DON or 20 M azaserine were directly injected into oocytes before progesterone treatment. For O-GlcNAcase inhibition, PUGNAc (concentrations ranging from 100 to 400 M) was also microinjected in immature oocytes before meiotic resumption stimulation. 5-10 oocytes were taken at the end of the experiment respecting the white spot ratio, and stored at Ϫ20°C until further biochemical analysis.
Enrichment of O-GlcNAc-bearing Proteins with WGA Immobilized on Agarose Beads-These experiments were performed in two conditions as previously described (29): in smooth conditions, which allows the recovery of all O-GlcNAc-modified proteins and their associated partners, and in more stringent conditions in which all protein-to-protein interactions are broken.
WGA Enrichment in Smooth Conditions-Batches of 20 immature or matured oocytes were taken and lysed in 200 l of homogenization buffer (60 mM ␤-glycerophosphate, 15 mM paranitrophenylphosphate, 25 mM MOPS, 15 mM EGTA, 15 mM MgCl 2 , 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitors, pH 7.2). After centrifugation at 20,000 ϫ g, supernatants were collected and diluted with phosphate-buffered saline. Samples were then incubated for 90 min at 4°C with 50 l of WGA-agarose beads. Proteins bound to WGA-beads were collected by centrifugation, washed four times with phosphate-buffered saline, resuspended in 50 l of Laemmli buffer, and boiled for 10 min.
Measurement of UDP-GlcNAc Pools by High Performance Anion Exchange Chromatography-Ten oocytes were lysed in 1 ml of hypotonic buffer (10 mM Tris/HCl, 10 mM NaCl, 15 mM 2-mercaptoethanol, 1 mM MgCl 2 , and proteases inhibitors, pH 7.2). 50 l of 1 M HCl were then added to the lysate, and the mixture was passed through a 1.5-ml Dowex 50WX2-400 column. The column was washed with 10 ml of bipermuted water. The unbound fraction and washes were collected on ice and adjusted to pH 8.0 with 500 l of 1 M Tris/HCl. 250 l of the diluted fraction was injected using a ProPAC-PA1 column (4 ϫ 250 mm) on a Dionex (Jouy en Josas, France) high performance liquid chromatography system. The elution was achieved as follows: Tris/HCl (20 mM, pH 9.2) (solution A) for 1 min; elution gradient for 29 min with 85% A and 15% NaCl at 2 M (solution B); plateau of 5 min in the same conditions; 10-min elution gradient until 100% B was reached; plateau at 100% B for 5 min. The column was then re-equilibrated in 100% A. The flow rate was 1 ml/min. Detection was performed using a Spectroflow 757 UV spectrophotometer (Kratos-Analytical, Shimadzu, Champs sur Marne, France) at a wavelength of 256 nm.

The OGT Inhibitor Alloxan Blocks O-GlcNAc Increase and
Progesterone-induced G 2 /M Transition-In a previous report, we demonstrated that Xenopus oocyte meiotic resumption was accompanied with a burst in O-GlcNAc content (22). In order to understand the biological significance of this glycosylation increase, we inhibited OGT using the uracil analogue alloxan (ALX) (31). Recently, alloxan has been successfully used for its OGT-inhibitory effect, in isolated neonatal ventricular car- diomyocytes (32), in intact rat heart (33), and in C2C12 myoblasts (34). Oocytes were incubated with progesterone to trigger G 2 /M transition, together with increasing amounts of alloxan, and a dose-dependent inhibition of GVBD was observed (Fig. 1A). Used as a negative control, uracil had no effect on meiosis resumption induced by hormonal stimulation (third lane). A significant inhibitory effect of alloxan was observed for concentrations ranging from 3 to 5 mM, whereas lower doses (1 and 2 mM) had no or little effect on G 2 /M transition. Prevention of M-phase by alloxan appeared to be correlated with an O-GlcNAc level reduction: O-GlcNAc progressively decreased from 3 to 5 mM alloxan-treated oocytes to finally reach an O-GlcNAc pattern similar to that of immature oocytes (G 2 -phase). At the biochemical level, oocytes treated with these alloxan concentrations did not exhibit the typical pattern of M-phase-entered oocytes (Fig. 1B, lanes 6 -8). Rsk, whose phosphorylation directly depends upon MAPK activity, remained unphosphorylated, in contrast to phosphorylated forms that shift in electrophoretic mobility in metaphase IIarrested control oocytes; Cyclin B2 remained unphosphorylated (proven by the presence of a doublet of isoforms), and Cdc2 phosphorylation on Tyr 15 confirmed that MPF heterodimer was under an inactive form in these oocytes (compare lanes 1, 2, and 8). Accumulation of ␤-catenin was also examined as a marker of the meiotic process. ␤-Catenin accumulates during G 2 /M transition due to inhibition of GSK3␤ (glycogen synthase kinase 3␤) that phosphorylates ␤-catenin on a PEST sequence, leading to its degradation by the proteasome system (35). In a previous work, we reported that ␤-catenin O-GlcNAc content was enhanced after progesterone stimulation (22). As expected, ␤-catenin was easily detected in oocytes incubated solely with progesterone or with low concentrations of alloxan (1 and 2 mM). Expression of ␤-catenin was dramatically reduced when alloxan was used at higher concentrations (Fig. 1B, bottom).
At the cytological level, high concentrations of alloxan prevented meiotic spindle formation, as proved by the presence of an intact germinal vesicle envelope (Fig. 2C). Meiotic spindles detected were similar in oocytes treated with uracil ( Fig. 2D) to those examined in control mature oocytes (Fig. 2B).
To check for the nontoxicity and reversibility of alloxan effect, oocytes that were incubated with 5 mM ALX were rinsed after overnight treatment and were allowed to reach the G 2 /M transition in alloxan-free ND96 medium containing progesterone (Fig. 3); in these conditions, more than half of the rinsed oocytes exhibited GVBD (57 Ϯ 8.1%) (Fig. 3A), and removal of alloxan allowed activation of both MAPK and MPF pathways,  accumulation of ␤-catenin, and increase in O-GlcNAc level (Fig. 3B, compare lanes 3 and 4).
Since alloxan could interfere with any enzyme using uridine and exert unrelated effects, a benzoxazolinone derivative (XI), another OGT inhibitor (referred to as compound number 5 in Ref. 28), has been tested for its ability to block M-phase entry. Prior to hormonal stimulation, oocytes were incubated with 5 mM alloxan (Fig. 4, lane 3) or 500 M benzoxazolinone derivative (lane 4) or without any inhibitor (lane 2). As for alloxan, the benzoxazolinone derivative blocked G 2 /M transition; MPF and MAPK pathways were not activated, and the O-GlcNAc level did not increase after stimulation with progesterone.
M-phase Entry Inhibition Induced by Alloxan Does Not Depend upon Reactive Oxygen Species Formation-Free radicals have been reported to presumably control cell cycle (e.g. through Cdc25 regulation) (36). When in solution, alloxan is in equilibrium with its reduction product, dialuric acid, which generates superoxide radicals (O 2 . ) through a redox cycle (for a review, see Ref. 37). These superoxide radicals are used by SOD to generate hydrogen peroxide (H 2 O 2 ). Within cells, hydrogen peroxide molecules sustain the Fenton reaction in the presence of Fe 2ϩ ions and are then splinted into two hydroxyl radicals (OH ⅐ ).
To counteract potentially alloxan-mediated reactive oxygen species (ROS) generation, alloxan-treated oocytes were incubated with SOD and catalase (which produces H 2 O and 1 ⁄ 2 O 2 from H 2 O 2 ) either 1 h prior to the progesterone addition (Fig. 5,  lanes 4 and 6) or overnight prior to progesterone addition (Fig.  5, lanes 8 and 10). In these conditions, the presence of the two detoxifying enzymes did not prevent the action of alloxan at concentrations ranging from 3 to 5 mM: 1) GVBD did not occur following progesterone stimulation (Fig. 5A); 2) O-GlcNAc levels did not increase; and 3) Rsk, p42 MAPK, and cyclin B2 phosphorylation patterns, as well as the ␤-catenin accumulation profile, were typical of oocytes arrested in G 2 (Fig. 5B).

Alloxan Blocks O-GlcNAc Glycosylation and G 2 /M Transition Induced by Egg Cytoplasm Injection-G 2 /M transition induced by progesterone depends upon protein synthesis.
However, when metaphase II-arrested oocyte (or egg) cytoplasm is injected into immature recipient oocytes, it triggers G 2 /M transition through the MPF autoamplification loop, independently of protein synthesis (12). In comparison with progesterone-treated oocytes, cytoplasm-injected oocytes exhibited no decrease in O-GlcNAc content following 3 mM alloxan incubation (Fig. 7B, top). They also exhibited a typical pattern of phosphorylation/dephosphorylation and ␤-catenin accumulation similar to those of M-phase oocytes (Fig. 7B): 1) cdc2 Tyr 15 was dephosphorylated; 2) Rsk and cyclin B2 were phosphorylated; and 3) ␤-catenin was accumulated in identical proportion to the cytoplasm-injected control oocyte alone. Complete inhibition for GVBD and activation of both MPF and MAPK pathways were obtained for 4 and 5 mM concentration (Fig. 7, A and B). Alloxan similarly blocks GVBD, MAPK, and MPF pathways activations in progesterone-treated oocytes and in cytoplasm-injected oocytes. It must be noted that compared with the first experiment in which progesterone was directly added to trigger maturation, the alloxan concentration needed to inhibit M-phase entry was higher (4 mM for egg cytoplasm injection versus 3 mM for hormonal stimulation).

O-GlcNAcase Inhibition Accelerates Xenopus Oocyte M-phase Entry-Because two OGT inhibitors were tested and prevented
Xenopus oocyte M-phase entry (Figs. 1, 2, 4, and 7), we then checked the effect of O-GlcNAcase inhibition on the maturation process.
To this end, increasing amounts of PUGNAc, an inhibitor of O-GlcNAcase (38), were microinjected in Xenopus oocytes prior to hormonal stimulation (Fig. 8). PUGNAc caused a slight acceleration in maturation kinetics (p Ͻ 0.05 for 400 M PUGNAc);  indeed, the time requested for 50% of oocytes to undergo GVBD (GVBD 50 ) was reached earlier in PUGNAc-injected oocytes than in water-injected controls (Fig. 8A), and O-GlcNAc levels were enhanced compared with injection of water (Fig. 8B, compare lanes 3, 4, and 5 with lane 2). These data reinforced those obtained with alloxan and benzoxazolinone derivative. Altogether, these results demonstrated that the O-GlcNAc level interfered with Xenopus oocyte M-phase entry.
GFAT Inhibition Fails to Prevent M-phase Entry in Xenopus Oocytes-In somatic cells, the UDP-GlcNAc pool directly depends upon glucose concentration (39). UDP-GlcNAc is generated through the hexosamine biosynthetic pathway. The hexosamine biosynthetic pathway is finely regulated by the key enzyme, GFAT. Inhibition of GFAT leads to a decrease in the UDP-GlcNAc pool and consequently to a decrease in O-Glc-NAc glycosylation. In somatic cells, such an effect may be obtained using DON and azaserine, which are well known GFAT inhibitors. Both were used in an attempt to prevent G 2 /M transition induced by progesterone in Xenopus oocytes. When injected in immature oocytes prior to progesterone treatment, DON and azaserine have no effect on hormonal stimulation-induced M-phase entry (Fig. 9A). This lack of effect of GFAT inhibitors was previously observed by Slawson et al. (27), who used DON to tentatively modify the GVBD rate. In our experiments, no delay in GVBD kinetics was observed between azaserine or DON-injected oocytes compared with the control ones (water-injected). GVBD 50 was similar to control, whatever the conditions used to inhibit GFAT (data not shown). GVBD percentages in oocytes injected either with azaserine or DON exhibited no significant differences from control oocytes (Fig. 9A). At a biochemical level, cyclin B2, Rsk, and MAPK were not phosphorylated in immature G 2 -blocked oocytes (Fig. 9B). Upon stimulation by progesterone, cyclin B2, MAPK, and Rsk were phosphorylated, and changes in electrophoretic mobility were observed for cyclin B2 and Rsk (Fig. 9B). In oocytes injected with either azaserine or DON, we observed the typical mature oocyte pattern of phosphorylation and ␤-catenin accumulation (Fig. 9B).

GFAT Inhibition Does Not Significantly Impair O-GlcNAc and UDP-GlcNAc Contents in Xenopus
Oocytes-Strikingly, neither azaserine nor DON prevented the progesterone-induced O-GlcNAc level increase that goes with M-phase entry (Fig. 9). Increasing concentrations of azaserine (20, 60, and 100 Increasing amounts of PUGNAc were injected into immature oocytes. We also injected water as a control. Oocyte recovery was permitted overnight before progesterone treatment. A, for each condition, maturation kinetic was performed, and GVBD 50 was calculated. The histogram represents the average values Ϯ S.D. of the relative time to GVBD 50 for PUGNAc-treated oocytes compared with water-injected controls (considered as 1 for normalization). Results are from three independent experiments. B, oocytes were homogenized in lysis buffer, and Western blot analyses were performed. O-GlcNAc was assessed as described under "Experimental Procedures." Protein mass markers (kDa) are indicated to the left. WB, Western blot; Pg, progesterone.  and DON (100, 300, and 500 M) were tested, but no effect on G 2 /M transition or O-GlcNAc levels was observed (data not shown). To understand these results in apparent contrast with our previous observations, the UDP-GlcNAc pool of each condition was assayed using high performance anion exchange chromatography (Fig. 10); no significant changes in the UDP-GlcNAc pools were observed between the different conditions, even in the presence of the GFAT inhibitors. These latter observations suggest that during G 2 /M transition, oocytes run for O-GlcNAc on an existing UDP-GlcNAc pool and did not require UDP-GlcNAc synthesis for the O-glycosylation processes.

M)
Cyclin B2 Is Associated with an O-GlcNAc Partner-Since MPF is the universal key regulator of M-phase entry, we exam-ined the putative glycosylation of cyclin B2 during oocyte maturation (Fig. 11). Xenopus oocytes extracts were enriched on WGA-beads using two different conditions (29); enrichments were performed in smooth conditions (Fig. 11A) to preserve protein/protein interactions and in stringent conditions to dissociate complexes (Fig. 11B). Bound proteins were separated by SDS-PAGE and blotted using an anti-cyclin B2 antibody. Hsc/ Hsp70 were used as a positive control, since several papers related their O-GlcNAc modification (reviewed in Ref. 40). Heat-shock proteins interact with many intracellular proteins, explaining why such high quantities of Hsc/Hsp70 were obtained in smooth conditions.
In smooth conditions, we observed that the two isoforms of cyclin B2 (i.e. the active and the inactive forms) were associated with an unknown O-GlcNAc partner in immature and mature oocytes. However, in drastic conditions, staining of enriched-O-GlcNAc proteins with the anti-cyclin B2 antibody revealed that cyclin B2 was not itself O-GlcNAc-modified, since SDS does not preserve binding of cyclin B2 to WGA-beads. As a control experiment, treatment of oocyte lysates with peptide N-glycosidase F was performed in order to avoid the eventual presence of N-linked oligosaccharides (data not shown).

DISCUSSION
M-phase entry is a particularly delicate step, preparing cells for division, which requires highly organized spatio-temporal events. These events are mainly driven by post-translational modifications modulating protein expression or activities or promoting protein degradation. Among these modifications, phosphorylation and ubiquitination have been extensively studied. The highly dynamic and ubiquitous PTM O-GlcNAc has been proposed to play a role in cell cycle protein function, thus presumably regulating cell cycle progression (24) and checkpoints. Consistent with previous observations in X. laevis oocytes, which reported an overall increase in O-GlcNAc modification  during G 2 /M transition (22), we show here that O-GlcNAc is clearly requested for M-phase entry during meiosis in Xenopus oocytes.
O-GlcNAc is the most widespread glycosylation type found within the cytosolic and nuclear compartments of eukaryotic cells, and it differs from other glycosylation types by its high dynamism. Moreover, O-GlcNAc can compete with phosphorylation either at the same or at an adjacent site. Despite the intensive study and interest devoted to O-GlcNAc, many functions ruled by this glycosylation remain to be elucidated. Taking into account the most recent observations, which have pointed out the active role played by O-GlcNAc in the regulation of cell cycle (22,24,27), we explored the effects of preventing the O-GlcNAc dynamism on cell cycle progression by inhibition of OGT and O-GlcNAcase, expecting disruption of its progression. We took advantage of the Xenopus oocyte model, where G 2 /M transition may be triggered either by hormonal stimulation or by cytoplasm injection; in our hands, both were blocked when the OGT inhibitor alloxan was added in the medium.
Historically, alloxan has been used to induce experimental diabetes in animals (for a review, see Ref. 37). The mechanism by which diabetes is mimicked in animals relates to the production of ROS, such as hydroxyl radicals. Hydroxyl radicals are highly toxic for cells, especially for pancreatic beta cells, and, in conjunction with an increased flux of calcium, they lead to beta cell death and consequently to an inhibition of insulin secretion, generating diabetes. We tested the hypothesis that ROS released under alloxan treatment could affect G 2 /M transition in Xenopus oocytes. Indeed, ROS-mediated signal transduction pathways are involved in phosphorylation events by activating protein-tyrosine kinases and protein-serine/threonine kinases through a process known as "receptor transactivation." For instance, ROS inhibits Erk1/2, c-Jun N-terminal kinase, and p38 MAPK activations induced by angiotensin II and plateletderived growth factor in fibroblast (41), but ROS could alternatively sustain MAPK activity through the inhibition of MAPK phosphatases, which inactivates MAPK. ROS have been described as potent inhibitors of phosphotyrosine phosphatases, like Cdc25C (42). Thus, evidence has accumulated that ROS interfere with cell cycle progression (36,43). First, and unexpectedly, increasing concentrations of H 2 O 2 had no effects on G 2 /M transition induced by hormonal stimulation; MPF and MAPK were detected in their active forms, whereas ␤-catenin accumulated normally in H 2 O 2 -treated oocytes (Fig. 6). Second, we counteracted potential ROS production via the alloxan-dialuric acid redox cycle by the addition of SOD and catalase, two detoxifying enzymes (Fig. 5). Whatever the conditions we used (i.e. increasing the two enzyme amounts or modulating the time period post-progesterone addition), SOD and catalase did not suppress the alloxan-mediated block of G 2 /M transition. Because both SOD and catalase failed to impair alloxan effects and due to the lack of effect of a high concentration of H 2 O 2 on meiosis progression, it can be assumed that alloxan does not act on the G 2 /M transition through ROS generation. To address a potential effect of alloxan on an enzyme other than OGT, which could use uridine as a substrate, we used a derivative of benzoxazolinone, an OGT inhibitor that was recently identified (28). Since it is not a substrate for OGT, the benzox-azolinone derivative should exhibit a better selectivity. Therefore, the benzoxazolinone derivative appears to be a useful tool to probe hypo-O-GlcNAc glycosylation. Indeed, we observed that it decreased O-GlcNAc to a greater extent than alloxan, at one-tenth the concentration. In the presence of benzoxazolinone derivative (XI), reduction of O-GlcNAc levels also impaired meiotic progression as well as the activation of both MPF and MAPK pathways. This leads us to the conclusion that O-Glc-NAc modification is necessary for M-phase entry in Xenopus oocytes. O-GlcNAcase inhibition also reinforced these observations, since PUGNAc had opposite effects compared with those of alloxan and the benzoxazolinone derivative, since this chemical compound accelerated GVBD.
Inhibition of Xenopus oocyte G 2 /M transition (stimulated by either progesterone or cytoplasm injection) by alloxan was correlated to a drop in the O-GlcNAc level. Although the decrease in the O-GlcNAc content prevented MPF and MAPK activations, as well as ␤-catenin accumulation, these effects appeared to be reversible and nontoxic for oocytes, since removal of alloxan from the medium allowed oocytes still to undergo and complete G 2 /M transition in the presence of progesterone. In contrast to hormonal stimulation, cytoplasm injection drove M-phase entry independently of protein synthesis (12). It must be emphasized that the alloxan concentration required was higher to inhibit egg cytoplasm versus progesterone-induced GVBD (4 versus 3 mM, respectively). One might argue that mechanisms of protein synthesis induced by hormonal stimulation, which play a crucial role in meiotic resumption, are highly sensitive to O-GlcNAc modifications in Xenopus oocytes. In these conditions, the decrease in O-GlcNAc content also led to the absence of GBVD, MPF, and MAPK activations. This last observation demonstrates that O-GlcNAc modification is required for the activation of the MPF autoamplification loop.
An alternative strategy to decrease O-GlcNAc content is to target the enzyme responsible for UDP-GlcNAc synthesis, GFAT. Intriguingly, and has previously observed (27), azaserine and DON, which are both inhibitors of GFAT, had no effects on progesterone-induced GVBD and activation of both MPF and MAPK. Such inefficiency to affect the O-GlcNAc level might be related to an absence of GFAT activity in M-phase-entered oocytes. This hypothesis has been reinforced by UDP-GlcNAc pool assays; there was no difference in the UDP-GlcNAc content between immature and mature oocytes. Thus, the use of GFAT inhibitors appears not to be a relevant strategy to decrease O-GlcNAc content and to study its role in Xenopus oocytes. In addition to these observations, it has long been known that carbon metabolism in Xenopus oocytes is mainly directed to glycogen synthesis rather than to glycolysis (44). From these considerations, it can be assumed that in Xenopus oocytes, glucose tends to be stored rather than being directed toward the hexosamine biosynthetic pathway. Because early cleavages in Xenopus embryos rely on the use of amino acids as its main source of carbon, glycolysis starts only at the onset of gastrulation (45). Taken together, these observations lead to the conclusion that oocytes glycosylate their proteins on an existing UDP-GlcNAc pool in a process that does not need UDP-GlcNAc production. Thus, Xenopus oocytes offer a unique opportunity to uncouple inhibition of O-GlcNAc and GFAT in vivo.
Further targets and function of O-GlcNAc modification remain to be determined during the cell cycle. Although the involvement of O-GlcNAc at the G 2 /M transition (27) and at the metaphase/anaphase transition may be suspected from the observations of Slawson et al. (24,27), we report for the first time here a crucial role for OGT, the O-GlcNAc key enzyme, in M-phase entry, and association of cyclin B2 with an unidentified O-GlcNAc partner might account for unsuspected levels of regulation. If post-translational modifications, such as phosphorylation, have been described for cyclin B isoforms during G 2 /M progression, their functions remain unclear except for cyclin B1. The latter can be phosphorylated by MAPK and polokinase, regulating MPF nuclear export but not enzymatic activity of the heterodimer complex (46). In our hands, cyclin B2 appeared to be in association with an O-GlcNAc-modified partner throughout G 2 -and M-phases, because interaction was not associated within the active or inactive states of the cyclin B2-Cdk1 complex. Nevertheless, it may be hypothesized that O-GlcNAc is involved in the association between cyclin B2 and Cdc2 to form the heterodimer MPF or to play a role in the interactions between MPF and its downstream effectors. It should be also noted that O-GlcNAc level immediately increases after hormonal stimulation (2 h post-progesterone; data not shown), indicating that O-GlcNAc is necessary for the early steps of the G 2 /M transition. Because MAPK activity or cyclin B synthesis is not essential for M-phase entry in Xenopus oocytes (17,18,21,47), mechanisms sensitive to O-GlcNAc variation, which are essential for G 2 /M transition, remain to be elucidated.
As a valuable tool for studying O-GlcNAc function in the cell cycle, OGT inhibitors have opened a narrow field of investigation for deciphering the role of O-GlcNAc modification at the G 2 /M transition. Further efforts are required to seek modification of O-GlcNAc content on the key regulators of the cell cycle.