CELL CYCLE 2016, VOL. 15, NO. 2, 213–224 http://dx.doi.org/10.1080/15384101.2015.1121330

REPORT

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis Qian Wangy, Haojie Weiy, Juan Du, Yan Cao, Nana Zhang, Xiaoyun Liu, Xiaoyu Liu, Dandan Chen, and Wei Ma Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University, Beijing, China

ABSTRACT

ARTICLE HISTORY

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indirectly chromosome behavior during somatic mitosis. It is not fully known about the expression and function of H3 with phosphorylated Thr3 (H3T3-P) during meiosis in oocytes. In this study, we investigated the expression and sub-cellular distribution of H3T3-P, as well as its function in mouse oocytes during meiotic division. Western blot analysis revealed that H3T3-P expression was only detected after germinal vesicle breakdown (GVBD), and gradually increased to peak level at metaphase I (MI), but sharply decreased at metaphase II (MII). Immunofluorescence showed H3T3-P was only brightly labeled on chromosomes after GVBD, with relatively high concentration across the whole chromosome axis from pro-metaphase I (pro-MI) to MI. Specially, H3T3-P distribution was exclusively limited to the local space between sister centromeres at MII stage. Haspin inhibitor, 5-iodotubercidin (5-ITu), dose- and time-dependently blocked H3T3-P expression in mouse oocytes. H3T3-P inhibition delayed the resumption of meiosis (GVBD) and chromatin condensation. Moreover, the loss of H3T3-P speeded up the meiotic transition to MII of pro-MI oocytes in spite of the presence of non-aligned chromosomes, even reversed MI-arrest induced with Nocodazole. The inhibition of H3T3-P expression distinguishably damaged MAD1 recruitment on centromeres, which indicates the spindle assembly checkpoint was impaired in function, logically explaining the premature onset of anaphase I. Therefore, Haspin-catalyzed histone H3 phosphorylation is essential for chromatin condensation and the following timely transition from meiosis I to meiosis II in mouse oocytes during meiotic division.

Received 11 September 2015 Accepted 12 November 2015

Introduction Histones H2A, H2B, H3 and H4 form nucleosomal octamers, around which DNA is wound to form the basic organizing structure of the chromosome. Post translational modifications, such as phosphorylation, acetylation, methylation and ubiquitinylation, dynamicly occur in the tail structure of these core histones. These modifications produce a high degree of combinatorial complexity, the so-called “histone code,” that forms the basis of a critical regulatory system in the control of chromatin structure and the fine-tuning of transcription.1–4 H3 tail is extensively phosphorylated and associated with chromosome condensation and subsequent separation in mitosis. Typically, H3 phosphorylation at serine 10 (Ser10) and serine 28 (Ser28) begins at the pericentromeric chromosome regions and spreads throughout the chromosomes during the G2-M phase transition, thus facilitating chromosome shaping.5–7 Aurora B kinase-mediated H3 Ser10 phosphorylation (H3S10-P) is assumed to promote the process of chromatin condensation, as well as the release of cohesin and ISWI (imitation switch) chromatin-remodeling ATPases.8 The site mutation of Ser10 can perturb chromatin condensation and chromosome segregation during both meiosis and mitosis.9,10 H3 Ser28 phosphorylation

CONTACT Wei Ma [email protected] 10 Xitoutiao, Youanmen, Beijing 100069, China. y Authors equally contributed to this work. © 2016 Capital Medical University

KEYWORDS

GVBD; H3T3-P; meiotic progression; MAD1; oocytes

is also involved in mitotic chromosome shaping.11 Whether the modification of Ser10 or Ser28 is essential for chromatin condensation during oocyte meiotic maturation is still on debate. A early report demonstrated a strong relationship between H3 Ser10 phosphorylation and chromosome condensation in porcine oocyte, but this was not supported by other research data in porcine and mouse oocytes.10 Haspin, also known as germ cell-specific gene 2 protein (GSG2), was first identified as a testis-specific gene in mice, but its expression is not truly haploid germ cell-specific, and detected in cells from many other organs.12 Up to now, Haspin’s best characterized and conserved function is to catalyze H3 threonine 3 (Thr3) phosphorylation, this process can be specially inhibited by a a highly selective inhibitor, 5-iodotubercidin (5-ITu), in many cell types.13–15 H3 with phosphorylated Th3 (H3T3-P) is temporally associated with mitotic events in turkey and mammalian cells.16,17 H3 Thr3 phosphorylation and dephosphorylation is similar to that of H3 Ser10 in timing, but H3T3-P exhibits distinct subcellular distribution in contrast to H3S10-P, H3T3-P is mainly focused at the inner centromeric regions of the chromosomes, and functionally crucial for the localization of the chromosomal passenger complex

Department of Histology and Embryology, School of Basic Medical Sciences, Capital Medical University,

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(CPC) at centromeres, which is required for multi aspects of mitotic progression, such as chromosome condensation and biorientation, spindle assembly checkpoint (SAC) setup and cytokinesis completion.18–20 Histone H3 Thr3 is highly conserved, but the significance of its phosphorylation is not fully known during meiosis in oocytes. Recently two studies about the H3T3-P in mouse oocytes were conducted in succession.14,15 Interestingly, they presented different subcellular localization of H3T3-P in mouse oocytes from that in somatic cells and demonstrated that H3T3-P is required to recruit CPC into the chromosome. However the protein expression pattern of H3T3-P was not detected in oocyte meiotic division, and the function of H3 Thr3 phosphorylation was not fully clarified because H3T3-P inhibition was conducted by application of 5-ITu from the early stage of oocyte meiosis, particularly at the stage of germinal vesicle (GV) or early germinal vesicle breakdown (GVBD), mainly influencing chromosome structure and meiotic progression. We are worried that the inhibition of H3 Thr3 phosphorylation from the early meiotic stage may mask its other functions that are supposed to be performed at late period of meiosis, such as its possible involvement in spindle checkpoint regulation. In this study we revealed the unique protein expression pattern of H3T3-P and its subcellular localization in fine detail in mouse oocytes during meiotic maturation. We inhibited H3T3-P expression at multi stages of meiotic progression and found that H3 Thr3 modification is required to regulate chromatin condensation, spindle checkpoint signaling and the timely transition of cell cycle from metaphase I to metaphase II in mouse oocytes.

Results Unique expression pattern of H3T3-P protein during oocyte meiotic division To determine the protein expression patterns of H3T3-P, H3S10-P and Haspin in mouse oocytes during meiotic maturation, oocytes were harvested after cultured for 0, 2, 7 and 17 h, corresponding to germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI) and metaphase II (MII) stages, respectively. Western blot analysis revealed a dynamic expression pattern of H3T3-P in mouse oocytes (Fig. 1). H3T3P expression was not detected at GV, it was greatly increased upon GVBD, and sustained stably up to MI, but sharply turned to a decreased level at MII. H3S10-P was only faintly assayed at GV, but dramatically increased after GVBD, reaching to maximal level at MI and maintained stable until MII, this expression pattern is different from that of H3T3-P (Fig. 1). In addition, Haspin expression was highly probed at GV stage and remained consistently stable up to MII (Fig. 1). Subcellular distribution of H3T3-P and Haspin in oocytes during meiotic maturation Furthermore, immunofluorescence staining was conducted to reveal the subcellular localization of H3T3-P and related Haspin in mouse oocytes during meiosis. In oocytes at prophase with intact germinal vesicle, no fluorescent signal of H3T3-P (Fig. 2A, C) or H3S10-P (Fig. 3A, C) was labeled in nuclear or

Figure 1. Protein expression and subcellular localization of H3T3-(P)during meiotic maturation. Western blot analysis of the protein expression of H3T3-P, H3S10-P and Haspin in mouse oocytes during meiotic division. Each sample was composed with 50 oocytes collected at 0, 2, 7 and 17 h of maturation culture, corresponding to GV, GVBD, MI and MII stages, respectively. GAPDH was included as a protein loading control. Each protein expression was determined at least 3 times.

cytoplasm, which is logically consistent with the fact that histone H3 phosphorylation was only detected after GVBD with western blot analysis. In addition, there was no special subcellular accumulation of Haspin in GV oocytes (Fig. 2A-B), this protein must be evenly distributed throughout the cytoplasm, given the high protein expression revealed with protein gel blot analysis at GV stage. As soon as GVBD occurred, intense staining of H3T3-P was clearly observed at the periphery of condensing chromosomes (Fig. 2A, G). As oocytes progressed to pro-MI and MI, strong signal of H3T3-P was observed across the chromosomes (Fig. 2A, K-O). During anaphase I (AI) / telophase I (TI) transition, H3T3-P remained on the separating chromosomes (Fig. 2A, S). When oocytes developed to MII stage, H3T3-P was found across the aligned chromosomes as well as on the first polar body (1st PB) (Fig. 2A, W). Haspin began to be accumulated as lots of foci after GVBD, and specially colocalized with H3T3-P on the condensing chromosomes (Fig. 2A, F-H), such colocalization sustained on chromosomes from pro-MI to MI (Fig. 2A, J-P). In addition to accumulation on chromosomes, Haspin was also weakly labeled as filamentous aggregates across spindle area (Fig. 2A, N). During AI / TI transition, Haspin was translocated from chromosomes and distributed across the midbody (Fig. 2A, R). Compared to high accumulation on chromosomes at MI, Haspin was only faintly labeled on chromosomes in MII oocytes, with weak filaments of Haspin organized into spindle-like structure (Fig. 2A, V). Pronounced signal of H3S10-P was aggregated on chromosomes upon it emerged after the resumption of meiosis (Fig. 3A, G), H3S10-P was sustained across chromosomes during the following meiotic stages, even during the meiotic transition from AI to TI (Fig. 3A, K-W). To determine H3T3-P localization on chromosomes in fine detail, chromosome spreads were prepared and processed for immunofluorescent staining with antibodies to phosphorylated H3 and CREST, a special auto serum recognizing centromere.

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space between sister kinetochores at MII stage, in accordance with markedly decreased protein level of H3T3-P detected with western blot at this stage (Fig. 1). Weak H3T3-P was also found on centromeres which were recognized by anti-centromere auto serum CREST (Fig. 2B). By the same protocol, H3S10-P was strongly detected along the entire chromosome body, this distribution pattern was maintained stable through pro-MI to MII (Fig. 3B). These data suggest that distribution pattern of H3T3P is different from that of H3S10-P in oocytes during meiotic division, implying different functional emphasis. In addition, H3T3-P localization in meiotic oocytes is also different from that in mitotic somatic cells, in which H3T3-P is mainly concentrated at centromeric area.18,19 Suppressed H3 Thr3 phosphorylation with Haspin inhibitor 5-ITu in dose- and time-dependent manner To assess the functional significance of H3T3-P during oocyte maturation, we treated oocytes with a small molecule inhibitor

Figure 2. H3T3-(P)subcellular localization in mouse oocytes during meiotic maturation. (A) Oocytes at various stages were immunostained with antibodies against H3T3-P and Haspin. DNA was visualized in blue, H3T3-P in red and Haspin in green. Bar, 20 mM. No special accumulation of H3T3-P or Haspin was detected in oocytes at GV stage. H3T3-P was brighly detected on the condensing chromatin after GVBD, H3T3-P remained localized on chromosome as cell cycle progressed to MII, even during AI-TI transition (s). Haspin began to accumulate on chromosomes upon GVBD, and remained localized on chromosomes as oocytes developed to MI stage (n: arrow), Haspin was also distributed across spindle area (n: arrowhead; v: arrow). During AI-TI transition, Haspin was transferred to the midbody, only weak signal of Haspin was labeled on dividing chromosomes (r). (B) Oocytes chromsome spreads were prepared and immnuo-labeled with antibodies to H3T3-P and human auto serum CREST, which recognizes centromere complex. H3T3-P was visualized in red, CREST in green and DNA in blue. Bar, 10 mM. H3T3-P was localized on chromosome, particularly accumulated along the interstitial axes between chromosome arms at pro-MI and MI stages (a-j). H3T3-P aggregation was dramatically reduced and mainly confined within the local space between sister kinetochores at MII stage (l-n: arrow). In addition, H3T3-P was also weakly labeled on centromeres.

As showed in Figure. 2B, H3T3-P was accumulated on chromosome, especially focused as linear clustering along the interstitial axes between chromosome arms during pro-MI and MI stages (Fig. 2B, A-J). H3T3-P accumulation was sharply reduced on chromosomes and only distinctly localized at local

Figure 3. Subcellular localization of H3S10-P in mouse oocytes during meiotic division. (A) Immunofluorescence staining showed dynamic localization of H3S10-P in mouse oocytes during meiosis. Spindle microtubules were visualized by acetylated tubulin in green, DNA in blue and H3S10-P in red. Bar, 20 mM. No signal of H3S10P was detected in GV oocytes, H3S10-P only began to be detected on the condensing chromatin upon GVBD. H3S10-P accumulation was sustained on chromosome during meiotic progression from GVBD to MII. (B) Oocytes chromsome spreads were prepared and processed for immnuo-labeling with antibodies to H3S10-P and human auto serum CREST. H3S10-P was visualized in red, CREST in green and DNA in blue. Bar, 10 mM. H3S10-P was evenly distributed across the whole chromosome body from pro-MI to MII.

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with high specificity for Haspin, 5-ITu, which is reported to inhibit Haspin phosphorylation of histone H3 Thr3 in somatic cells during mitosis.18,19 To test 5-ITu specificity for H3T3-P expression in oocyte meiosis, in-vitro cultured mouse pro-MI oocytes were treated with growing concentrations of 5-ITu for 1 h. The results showed that H3 phosphorylation on Thr3 was significantly blocked with 5-ITu in a dose-dependent manner. As demonstrated with protein gel blot analysis, H3T3-P protein expression was significantly reduced after 1 h treatment with 0.1 mM 5-ITu, and totally inhibited after treatment with 1 mM and 5 mM 5-ITu (Fig. 4A). Moreover, in consistence with the western blot data, immunofluorescence analysis illustrated that H3T3-P accumulation on chromosomes was significantly decreased in oocytes treated with 0.1 mM 5-ITu (Fig. 5A, D-F), and completely disappeared after treated with 1 mM and 5 mM 5-ITu (Fig. 5A, G-L). By comparison, the levels of H3S10-P protein expression remained apparently constant after 1 h incubation with 5-ITu in concentrations from 0.1 mM to 5 mM (Fig. 4A). Further immunostaining showed that H3S10-P distribution across chromosomes was unabated after 5-ITu treatment at different concentrations (Fig. 5A, D-L). In order to analyze time effects of inhibitor on H3T3-P expression, pro-MI oocytes were treated with 1 mM 5-ITu for different times. As showed by the protein gel blot results, the protein expression of H3T3-P was not significantly changed after incubation with 1 mM 5-ITu for 10 min, but obviously undetectable when checked at 30 min and 60 min of treatment (Fig. 4B). Immunofluorescence further confirmed the complete depletion of H3T3-P on chromosomes after 30 min incubation with 1 mM 5-ITu (Fig. 5B, G-L). In sharp contrast, H3S10-P protein level was consistently stable when checked at different time points during oocyte incubation with 1 mM 5-ITu (Fig. 4B), H3S10-P subcellular localization was not affected and comparable to that in control oocytes (Fig. 4B, A-L). This data indicates 5-ITu inhibits histone H3 Thr3 phosphorylation in time-dependent manner. Taken together, the results above suggest that, similar to mitotic cycling cells, Haspin activity can be specially inhibited in mouse oocytes by the application of 5-ITu, which can block H3 phosphorylation on Thr3 in dose- and time-dependent manner, with no effects on Ser10 phosphorylation. Requirement of H3T3-P for meiotic resumption and cell cycle progression to MII in oocytes Germinal vesicle breakdown (GVBD) is the first morphological event during oocyte meiotic maturation, which marks the resumption of meiosis. Since H3T3-P expression begins around the window time of GVBD, it is rational to speculate that Thr3 phosphorylation of histone H3 may be involved in the restart of meiosis. Just as we expected, the meiotic resumption was restrained by the inhibition of H3T3-P expression with 5-ITu. As showed in Figure 6B, after 1 h culture, more than half control oocytes (57.67 § 3.18%) underwent GVBD, with bright H3T3-P accumulated on condensed chromosomes and microtubules assembled around chromosomes (Fig. 6A, A-D), in contrast, the majority oocytes were still arrested at GV stage in 1 mM 5-ITu treatment group (94.67 § 0.67%) and 5 mM 5-ITu group (100%). At 2 h of culture, only very few control cells (6.67 § 1.33%) remained

Figure 4. Western blot analysis of reduced H3T3-(P)in oocytes by 5-ITu in a dose and time-dependent manner. (A) Following in vitro maturation for 5 h, oocytes were incubated for additional 1 h in the presence of 0, 0.1, 1 and 5 mM 5-ITu, respectively, prior to western blot analysis. The protein expressions of H3T3-P and H3S10-P were assayed, a control blot of GAPDH shows equal protein loading between the various samples. After 1 h incubation, H3T3-P expression was significantly reduced in 0.1 mM 5-ITu group, and totally disappeared in 1 mM 5-Itu and 5 mM 5-ITu group. The protein level of H3S10-P remained stable in oocytes after treated with different concentrations of 5-ITu. (B) Pro-MI oocytes were incubated with 1uM 5-ITu for 0, 10, 30, and 60 min, respectively, prior to protein gel blot analysis. Both H3T3-P and H3S10-P were determined after drug treatment. GAPDH level was assayed and used as a control blot. H3T3-P protein level was markedly decreased after 10 min incubation in 1 mM 5-ITu, and could not be detected after 30 min treatment. In contrast H3S10-P level sustained consistently at different time points of drug treatment.

at GV, significantly lower than that in two 5-ITu treatment groups (P < 0 .05), additionally, the number of GV oocytes was higher in 1 mM 5-ITu group than that in 5 mM 5-ITu group (1 mM 5-ITu vs 5 mM 5-ITu, 73.33 § 4.06% vs 100%; P < 0 .05). By 4 h, all the cells passed through GVBD and further developed to pro-MI stage in control group, and only a handful of oocytes were arrested at GV in 1 mM 5-ITutreated group (10.67 § 3.18%), but significantly higher than that in control (P < 0 .05), by contrast, the vast majority of cells were still blocked at GV stage by 5 mM 5-ITu treatment (95.33 § 0.67%, P < 0 .05), clearly, there was no signs of chromatin condensing, H3T3-P expression and microtubule assembly in these oocytes (Fig. 6A, E-H). These results suggest that 5-ITu incubation at early stages of the cell cycle can

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Figure 5. Immunofluorescence analysis of reduced H3T3-P in oocytes by 5-ITu in time- and dose-dependent manner. (A) The subcellular localization of H3T3-P and H3S10-P was analyzed with immunofluorescent staining in pro-MI oocytes incubated for 1 h in 0, 0.1, 1 and 5 mM 5-Itu, respectively. DNA was visualized in blue, H3T3-P or H3S10-P in red and acetylated-tubulin in green. Bar, 20 mM. At the end of 1 h incubation, the fluorescent signal of H3T3-P was significantly reduced in 0.1 mM 5-ITu group, and totally undetectable in oocytes treated with 1 mM 5-Itu and 5 mM 5-ITu. H3S10-P was brightly labeled on chromosomes in oocytes after treated with different concentrations of 5-ITu. (B) The subcellular localization of H3T3-P and H3S10-P was detected with immunofluorescent staining in pro-MI oocytes after treated with 1 uM 5-ITu for 0, 10, 30 and 60 min. DNA was visualized in blue, H3T3-P or H3S10-P in red and acetylated-tubulin in green. Bar, 20 mM. H3T3-P accumulation on chromosomes was dramatically reduced after 10 min incubation, and total undetectable in oocytes treated for 30 min. Bright H3S10-P was consistently detected in oocytes at all the time points of drug incubation.

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Figure 6. H3 Thr3 phosphorylation involvement in meiotic resumption and cell cycle progression to MII in oocytes. (A) Immunofluorescence analysis demonstrated that non-condensed chromatin after GV oocytes were incubated for 4 h in 5 mM 5-ITu. DNA was visualized in blue, H3T3-P in red and acetylated-tubulin in green. Bar, 20 mM. The nuclear envelope was still intact (e) in 5-ITu-treated oocytes with no signal of H3T3-P expression (g) and microtubule assembly (f), in contrast, GVBD occurred and chromatin was properly condensed into individual chromosomes (a) in control oocytes with bright fluorescent signal of H3T3-P (c) and assembling microtubules (b). (B) Statistical analysis indicated that the meiotic resumption was delayed in GV oocytes when H3T3-P expression was inhibited with 5-ITu. GV oocytes were cultured for 1, 2, and 4 h, respectively, in maturation medium added with 1 and 5 mM 5-Itu, then collected for immunofluorescence staining to judge and score oocytes arrested at GV stage. Data are presented as mean § SEM of 3 repeated experiments. (C) Statistical analysis showed the meiotic progression to MII was affected when oocytes were incubated in 5-ITU for 15 h after GVBD. GV oocytes were initially cultured for 2 h in normal maturation medium, then incubated for additional 15 h in the same medium supplemented with 0, 0.1, 1 and 5 mM 5-ITu. After incubation, oocytes were collected for immunofluorescence staining to determine the developmetal stages of oocytes. I indicates interphase-specific chromosome configuration, C indicates chromosomes collapsed into “ball” structure. Data are presented as mean § SEM of 3 repeated experiments.

inhibit meiotic resumption of mouse oocytes in a dose-denpendent manner, essentially implying Haspin-catalyzed histone H3 phosphorylation on Thr3 is required for the timely restart of meiosis in oocytes, consistent with a reported delay in the G2-M transition in HeLa cells depleted of Haspin.19 However, as reported previously, the majority oocytes could finally break through the inhibition and resume the meiotic

progression if sufficient incubation time applied, but did not reach MII stage, instead, being arrested at MI stage.14 In order to further determine the effect of drug application time, GV oocytes were initially cultured in normal maturation medium for 2 h, by which time oocytes putatively pass through GVBD, and then incubated for additional 15 h in the same medium with the presence of 5-ITu in concentrations of 0, 0.1,

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1 and 5 mM 5-ITu, respectively. We found that oocyte progression to MII was delayed (Fig. 6C), the number of oocytes at MII stage was significantly lower in 1 mM 5-ITu group (51.00 § 8.08%) than that in control (85.33 § 3.28%, P < 0 .01) and 0.1 mM 5-ITu group (89.00 § 2.52%, P < 0 .01). Most extremely low MII proportion (6.00 § 2.65%, P < 0 .01) was counted in 5 mM 5-ITu group, the subject population were arrested at MI stage, and in quite a few oocytes (10.67 § 6.33%), chromosomes were collapsed and presented as “ball” structure, and even configuration specific for interphase stage. These data suggest that H3T3-P inhibition around GVBD may undermine the process of chromatin shaping into chromosomes in right structure, resulting in chromosome disability and meiotic arrest. Essential involvement of H3T3-P in timely MI/MII transition in oocyte meiosis In mitosis H3T3-P is crucial for functional setup of spindle assembly checkpoint (SAC), which guarantees the timely start of anaphase until all chromosomes are properly connected to spindle mictotubules, ensuring the accuracy of chromosomes separation.18–19 We conducted 2 distinct assays to test whether H3T3-P involvement in regulating anaphase onset is conserved during meiotic division in oocytes. Firstly, GV oocytes were cultured for 5 h, allowing oocytes to develop to pro-MI stage, about this time, chromosomes are proper condensed, SAC system is putatively active and blocks the start of anaphase. These pro-MI oocytes were processed for an additional incubation with 1 mM 5-ITu for different time periods. As showed in Figure 7A, at the time point of 1 h incubation, no control oocytes underwent anaphase I (AI), while approaximately 8.21% 5-ITu-treated oocytes precociously left meiosis I, and progressed to AI, telophase I (TI) and even MII stage, this number increased to 30.33%, 36.25% and 43.27%, respectively, at 2, 3 and 4 h of 5-ITu incubation, significantly higher than that in control at each time points (2 h, 11.28 § 1.75%; 3 h, 19.59 § 1.30%; 4 h, 28.83 § 3.55%; P < 0 .05). Clearly, this data shows 5-ITu incubation causes premature start of AI in spite of the possibility that chromosomes may be not properly attached by spindle microtubules, ascribed to a lack of functional SAC, and potentially indicates H3T3-P participating in the regulation of SAC function. The mini concentration of Nocodazole (NOCO), 50 ng/ml, is safe for the maintenance of intact spindle structure in MI oocytes, but strong enough to disrupt the connection between spindle microtubules and chromosome kinetochores, inspiring SAC proteins recruiting on kinetochores to block anaphase onset.21 We continued to determine whether NOCO-induced oocyte arrest at MI can be reversed by 5-ITu. MI oocytes were collected after 7 h maturation culture and processed for additional culture in presence of 50 ng/ml NOCO, 1 mM 5-ITu and a combination of 50 ng/ml NOCO with 1 mM 5-ITu for 1, 2 and 3 h, respectively, and then the meiotic progression was evaluated by immunofluorescence analysis at each time points. MI oocytes are manifested with properly aligned chromosomes and established interaction between kinetochores and microtubules from orderly formed MI spindle, satisfying SAC requirement and ready for the transition to anaphase. With the

Figure 7. H3 Thr3 phosphorylation was essential for timely MI/MII transition in oocyte meiosis. (A) Statistical analysis demonstrated that inhibition of H3 Thr3 phosphorylation could cause premature start of AI in pro-MI oocytes. GV oocytes were initially cultured for 5 h, then processed for an additional incubation with 1 mM 5-ITu for 1, 2, 3 and 4 h. After that, oocytes were processed for immunofluorescence staining to count the number of oocytes at AI, TI or MII stage. The data represents results of 3 replicates of experiments. (B) Statistical data indicated that H3T3-P inhibition could reverse MI arrest induced by NOCO. GV oocytes were normally cultured in vitro for 7 h, putatively reach MI stage, and then indicated for additional 1, 2, 3 h in the presence of respectively, DMSO, 1 mM 5-ITu, 50 ng/ml NOCO, or 1 mM 5-ITu C 50 ng/ml NOCO, and then processed for immunofluorescence staining to determine specific meiotic stages. Oocytes at AI, TI or MII were assumed to have happened with transition from meiosis I to meiosis II. Graphs report mean § SEM. All experiments were repeated at least 3 times.

increase in incubation time, control oocytes gradually left MI and were progressed to AI, TI and MII (1 h, 24.00 § 4.16%; 2 h, 61.00 § 10.26%; 3 h, 87.67 § 3.71%), the MI/MII transition was significantly accelerated by the addition of 5-ITu (1 h, 54.33 § 3.93%; 2 h, 84.67 § 6.17%; 3 h, 92.33 § 3.48%), instead, severely suppressed by NOCO application, with only about 10.33% oocytes moved out of MI stage after 3 h culture, however, the combined administration of 5-ITu dramatically reversed NOCO-induced MI arrest, promoting oocytes enter into MII (1 h, 15.00 § 3.06%; 2 h, 45.67 § 6.69%; 3 h, 72.67 § 4.49%). These results demonstrate that 5-ITu can speed up MI/ MII transition during meiotic progression in mouse oocytes, even override the meiotic arrest caused by NOCO. Taken together, the results of 2 assays above suggest Haspin activity, substantively the proper level of H3T3-P, is indispensable for timely meiotic transition in oocytes, fundamentally implying H3T3-P involvement in SAC functional setup. Blocked recruitment of SAC protein MAD1 to centromeres in 5-ITu-treated oocytes The recruitment of SAC proteins to kinetochore, the super structure assembled on centromeres, is considered important for optimal checkpoint operation. Therefore we next asked if

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5-ITu treatment perturbed the accumulation of SAC components at centromeres, consequently undermining SAC activity, a leading cause for precocious anaphase onset. We found MAD1, the key checkpoint component, was obviously concentrated at kinetochores in pro-MI oocytes (Fig. 8A, A-D), however this accumulation was severely disrupted by the addition of 5-ITu in pro-MI oocytes (Fig. 8A, E-L). Statistical analysis demonstrated the fluorescent signal intensity of MAD1 was significantly reduced in pro-MI oocytes after 1 h incubation of 1 mM 5-ITu (Fig. 8B). This result clearly displays 5-ITu administration can inhibit MAD1 recruitment to kinetochores, thus impair SAC function, producing premature chromosome separation in pro-MI oocytes. MAD1 aggregation on kinetochores is gradually decreased as oocytes progress from pro-MI to MI.21 We observed that MAD1 accumulation level was weak or undetectable in MI oocytes (Fig. 8C, A-D), and further weakened in the presence of 5-ITu (Fig. 8C, E-H, D). MAD1 signal was dramatically enhanced when MI oocytes were treated with NOCO (Fig. 8C, I-L, D), suggesting SAC effectively responds to the detachment of microtubules from chromosomes, however, this increasing trend was completely reversed when NOCO and 5-ITu were simultaneously used, MAD1 signal was significantly lower than that induced with NOCO alone (Fig. 8C, M-P, D), indicating MAD1 recruiting back to centromeres is blocked with combined administration of 5-ITu. Collectively, these results demonstrate 5-ITu is a rather potent SAC inhibitor, in other words, Haspin-catalyzed histone H3 phosphorylation is required for SAC components recruitment to kinetochore, ensuring functional SAC formation and timely transition from meiosis I to meiosis II.

Discussion In the present study we demonstrate the unique protein expression and subcellular localization of histone H3 with phosphorylated Thr3 (H3T3-P) in mouse oocytes during meiotic division. Haspin-catalyzed Thr3 modification is required for oocyte meiotic resumption, chromatin condensation and the functional setup of spindle assembly checkpoint, therefore ensuring the accurate chromosome separation during cell cycle transition from meiosis I to meiosis II in oocytes. We for the first time quantitatively tracked H3T3-P expression pattern during meiotic progression in mouse oocytes. Thr3 phosphorylation is initially detected in high level upon GVBD, which is sustained stable to MI, but surprisingly turned to a sharply decreased level at MII. By contrast, Ser10 modification remains stable in high level up to MII stage after it emerges at GVBD. Compared to even distribution of H3S10-P across chromosomes, H3T3-P localization is dynamicly aggregated between chromosome arms from GVBD to MII stage, and different from that in somatic cells during mitosis, in which H3T3-P is mainly concentrated on centromeres.18,19 H3T3-P is accumulated along the inter-chromatid axis (ICA) from prometaphase to MI stage, but confined to local space between sister kinetochores at MII stage. The increasing levels of H3 phosphorylation on Thr3 and Ser10 around GVBD may be involved in the regulation of meiotic resumption, and the general different expression patterns suggest these 2 modification may have individual functional emphasis in oocytes. Chromosome

condensation is the first visible process occurring at the beginning of oocyte maturation, which is essential for the correct packaging of chromatin fibers into chromosomes and subsequent fidelity of chromosome segregation into daughter cells.22,23 H3 Ser10 phosphorylation by Aurora B kinase is a hallmark event in mitosis and is closely associated with chromosome condensation in various eukaryotic organisms,24–26 however, it has long been unclear about the exact role of Ser10 modification in chromatin condensation until a latest study, which proves that H3 Ser10 phosphorylation assists the deacetylation of histone H4 lysine 16 (K16), so to release the H4 tail to interact with histone H2A and therefore promote chromatin fiber condensation.24 It is believed that H3 Thr3 phosphorylation promotes Aurora B recruitment to the tail of H3 to phosphorylate Ser10.19,27,28 In yeast somatic cells, deletion of Haspin, the upsteam kinase mediating Thr3 phosphorylation, can weaken Ser10 phosphorylation, undermining the process of chromatin condensation.24 However there is still debate over whether Thr3 phosphorylation is a prerequisite for Ser10 modification. When Thr3 phosphorylation is strongly inhibited in mammalian cells using multiple Haspin inhibitors, including 5ITu, both the protein level and the subcellular localization of H3S10-P is not distinguishably changed,19 this is in line with our data that H3S10-P is not affected, both in amount and spatial distribution, in 5-ITu-treated oocytes, in spite of the significantly-blocked Thr3 phosphorylation. A growing body of research indicates that H3 Thr3 phosphorylation is indispensable for the full activity of Aurora B toward centromeric targets, such as mitotic centromere-associated kinesin (MCAK) and centromere protein A (CENP-A), which regulating kinetochore-microtubule attachments and spindle checkpoint signaling, but significantly less essential for Aurora B activity on chromosome arms, which putatively required for H3S10 phosphorylation.19,27,28 There is also other signal pathway that contributes to Aurora B kinase activity across chromosome arms.28 In the current study, we found that the meiotic resumption and chromatin condensation are significantly delayed in 5-ITutreated oocytes, despite undisturbed H3S10-P expression, this demonstrates that Ser10 phosphorylation is not a sufficient condition for chromatin condensation, and that other factors must be involved, with Thr3 phosphorylation as a prerequisite. The condensin complex is required for chromatin fiber packaging into compact rod-like chromosome, and importantly, this complex uploading to chromatin is dependent on phosphorylated Thr3 in H3 tail.10,24 It is gradually assumed that the properly uploaded condensins, together with H4 K16 deacetylation, a step promoted by H3 Ser10 phosphorylation, is responsible for shaping chromosome structure.24 In mitotic cells, H3T3-P is restricted to centromeres and serves as a ligand for Survivin, a component of the chromosomal passenger complex (CPC), mediating the kinase subunit of CPC Aurora B localization on chromosome, particulay at the centromere.20 Dysregulation of H3T3-P expression can affect Aurora B positioning, thus disturb several crucial mitotic events, especially the attachment between microtubules and kinetochores, as well as the functional setup of the spindle checkpoint, leading to severe abnormallity in cell cycle progression and chromosome separation.19,27 Mammalian oocytes contain both Aurora B and Aurora C, and in distinct difference,

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Figure 8. Destroyed recruitment of SAC protein MAD1 to centromeres in 5-ITu-treated oocytes. (A) Immunofluorescence analysis showed disrupted MAD1 accumulation at centromeres in pro-MI oocytes treated with 5-ITu. Oocytes at pro-MI stage were incubated for 1 h in 1 mM 5-ITu, and then processed for chromosome spreading and immunostaining with CREST and MAD1 antibody. DNA was visualized in blue, MAD1 in red and CREST in green. Bar, 20 mM. MAD1 was brightly labeled on centromeres in control oocytes (b-d), but faintly detected in 5-ITu-treated cells (f-h, j-l). (B) Quantization of data indicated the fluorescence intensity of MAD1 was significantly lower in 5-ITu-treated pro-MI oocytes. (C) Immunofluorescence analysis demonstrated that 5-ITu could blocked NOCO-induced MAD1 recruiment to centromeres in MI oocytes. DNA was visualized in blue, MAD1 in red and CREST in green. Bar, 20 mM. MAD1 signal was faintly detected in control MI oocytes (a-d), and undetectable in MI oocytes treated with 5-ITu (e-h). In contrast, MAD1 accumulation on centromeres was markedly increased when MI oocytes were treated with NOCO (i-l), but this increasing recruitment was completely blocked with the combined application of 5-ITu with NOCO (m-p). (D) Statistical analysis indicated that NOCO-induced MAD1 recruiting back to centromeres was blocked with combined administration of 5-ITu. The graph shows the mean § SEM of the results obtained in 3 independent experiments.

Aurora C localizes to kinetochores and the inter-chromatid axis (ICA), whereas Aurora B localizes to the spindle, in spite of their high similarity in sequence. In a great extent, Aurora C is the homolog of mitotic Aurora B in oocytes.29–31 The pattern of Aurora C subcellular localization resembles that of Survivin in mouse oocytes during meiotic division.29,32,33 Inhibition of Aurora C activity during meiosis has previously been shown to

accelerate the completion of meiosis I by inactivating SAC,29,32 phenocopying the depletion of Survivin.33 The linear distribution of H3T3-P along ICA is similar with that of both Aurora C and Survivin, and inhibition of H3T3-P specially affects the localization of Aurora C at the ICA, but not at the kinetochore, partially due to the absence of H3T3-P aggregation in the centromeric area.19 Given that H2A with phosphorylated T120 at

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centromeres has been reported to regulate the kinetochore population of Aurora B, it is also possible that Aurora C at the kinetochore might be activate by other factors in oocytes.34 We found the inhibition of H3T3-P expression evidently induces precocious anaphase initiation despite the presence of unaligned chromosomes in pro-MI oocytes or disrupted microtubule-kinetochore attachments in MI oocytes treated with nocodazole, this, combined with the destroyed recruitment of MAD1 to centromeres in 5-ITu-treated oocytes, even with the continued presence of nocodazole, suggests a defect in spindle assembly checkpoint (SAC). Given the fact that loss of Aurora kinase activity can also induce premature anaphase onset,32,35 and that H3T3-P is a prerequisite for Aurora C localization to the ICA in mouse oocytes,14 we attribute the defects of SAC to loss of Aurora C function, which is consistent with the idea that H3T3-P-dependent Aurora CPC function plays a role in SAC activation through phosphorylating and targeting SAC proteins to the kinetochore region.15,29,32,36 Consistent with recent published data,14 we observed a delay in anaphase onset in oocytes, even after 17 h incubation, when 5-ITu was administered from the early meiotic stages, either GV or GVBD, that may be due to the fact that chromatin was not fully condened into typical chromosome structure. We tend to believe that different drug application time produces different effects, suggesting H3T3-P regulates meiotic progression at multi steps. In addition, previous immunofluorescence studies show that H3T3-P is gradually reduced or even vanished on chromosomes at late anaphase and early telophase in mitotic cells,19 in contrast, we found that chromosome-associated H3T3-P persists into late telophase I. This remarkable difference implies H3T3-P may perform different roles in the establishment of segregation mechanisms between homologous chromosomes in meiosis I and sister chromatids in mitotic cells. In summary, histone H3 Thr3 phosphorylation occurs upon meiotic resumption and exhibits unique expression pattern in mouse oocytes during meiotic maturation. Thr3 phosphorylation is required for chromatin condensation, the setup and maintenance of functional spindle checkpoint system, so as to ensure timely meiotic progression and accurate chromosome separation in mammalian oocytes.

Meterials and methods Oocyte collection and culture Experimental protocols were approved by the Beijing Administration Office of Laboratory Animals, following the Administration Regulations on Laboratory Animals of Beijing Municipality. All oocytes were obtained from CB6F1 (BALB/C , £ C57BL/6

H3 Thr3 phosphorylation is crucial for meiotic resumption and anaphase onset in oocyte meiosis.

Haspin-catalyzed histone H3 threonine 3 (Thr3) phosphorylation facilitates chromosomal passenger complex (CPC) docking at centromeres, regulating indi...
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