RESEARCH ARTICLE Molecular Reproduction & Development 81:712–724 (2014)

SUMO-1 Plays Crucial Roles for Spindle Organization, Chromosome Congression, and Chromosome Segregation During Mouse Oocyte Meiotic Maturation YI-FENG YUAN, RUI ZHAI, XIAO-MING LIU, HAMID ALI KHAN, YAN-HONG ZHEN, AND LI-JUN HUO* Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, People’s Republic of China

SUMMARY Small ubiquitin-related modifier-1 (SUMO-1)-dependent modifications of many target proteins are involved in a range of intracellular processes. Previous studies reported the localization of SUMO-1 during oocyte meiosis, and that overexpression of Sentrin/ SUMO-specific protease 2 (SENP2), a de-SUMOylation protease, altered SUMOmodified proteins, and caused defects in metaphase-II spindle organization. In this study, we detailed the consequences of SUMO-1-mediated SUMOylation by either inhibition of SUMO-1 or UBC9 with a specific antibody or their depletion by specific siRNA microinjection. Inhibition or depletion of SUMO-1 or UBC9 in germinal vesicle (GV)-stage oocytes decreased the rates of germinal vesicle breakdown and first polar body (PB1) extrusion; caused defective spindle organization and misaligned chromosomes; and led to aneuploidy in matured oocytes. Stage-specific antibody injections suggested that SUMO-1 functions before anaphase I during PB1 extrusion. Further experiments indicated that the localization of g-tubulin was disordered after SUMO-1 inhibition, and that SUMO-1 depletion disrupted kinetochore-microtubule attachment at metaphase I. Moreover, SUMO-1 inhibition resulted in less-condensed chromosomes, altered localization of REC8 and securin, and reduced BUBR1 accumulation at the centromere. On the other hand, overexpression of SUMO-1 in GV-stage oocytes had no significant effect on oocyte maturation. In conclusion, our results implied that SUMO-1 plays crucial roles during oocyte meiotic maturation, specifically involving spindle assembly and chromosome behavior, by regulating kinetochore-microtubule attachment and the localization of g-tubulin, BUBR1, REC8, and securin. Mol. Reprod. Dev. 81: 712
724, 2014. ß 2014 Wiley Periodicals, Inc. Received 9 March 2014; Accepted 29 April 2014

INTRODUCTION Post-translational SUMOylation of proteins is an important modification system that can influence the function of a given protein. To date, four mammalian SUMO proteins have been identified, including SUMO-1, 2, 3, and 4. SUMO-2 and SUMO-3 are commonly referred to as SUMO-2/3 due to their 96% similarity; however, each

ß 2014 WILEY PERIODICALS, INC.



Corresponding author: Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction Education Ministry of China College of Animal Science and Technology Huazhong Agricultural University Wuhan 430070 People’s Republic of China. E-mail: [email protected]

Grant sponsor: National Natural Science Foundation of China; Grant numbers: 31071273, 31171378; Grant sponsor: Fundamental Research Funds for the Central Universities; Grant number: 2014PY045

Published online 30 July 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22339

shares only 50% amino-acid identity to SUMO-1 (GeissFriedlander and Melchior, 2007). SUMO is activated by

Abbreviations: E1/2/3, enzyme 1/2/3; GV[BD], germinal vesicle [breakdown]; MII, meiotic metaphase II; PB1, first polar body; PIAS, protein inhibitor of activated signal transducer and activator of transcription; SUMO-1, small ubiquitin-related modifier-1; UBC9, ubiquitin-conjugating enzyme 9.

SUMO-1

enzyme 1 (E1) in an ATP-dependent manner, conjugates to its target via enzyme 2 (E2), and thereafter binds to the substrate with the help of enzyme 3 (E3) ligase (Müller et al., 2001). SUMOylation can regulate transcription, nucleo-cytoplasmic transport, protein
protein interaction, protein localization, and protein stability by the specificity of its targeting. The broader effects of its activity play important roles during the cell cycle, proliferation, differentiation, and apoptosis (Zhao, 2007). Growing evidence implicates a conserved role for the SUMO pathway in the mitotic cell cycle control. In budding yeast, AOS1/UBA2 (E1) was found to be essential for the G2/M transition (Dohmen et al., 1995; Johnson et al., 1997). Ubiquitin-conjugating enzyme 9 (Ubc9) (E2)-deficient cells derived from knockout embryos displayed defects in nuclear organization, chromosome condensation, and segregation (Nacerddine et al., 2005). Defects in Protein inhibitor of activated signal transducer and activator of transcription (PIAS) (E3) resulted in embryonic lethality in Drosophila melanogaster and Caenorhabditis elegans: fruit flies deficient in PIAS showed defects in polytene chromosome segregation (in the salivary gland) and telomere assembly (Hari et al., 2001) while PIAS knockdown in C. elegans embryos led to high sensitivity to DNA damaging reagent (Holway et al., 2005). Many SUMO substrates are also involved in mitosis: Ran GTPase-activating protein 1 (RANGAP1), for example, was the first identified target of SUMO1. SUMOylation is essential for RANGAP1 localization to the mitotic spindle and for its attachment to kinetochores (Joseph et al., 2002, 2004). Centromere protein C (CENPC) is another substrate of SUMO-1 that plays a key role at centromeres during mitosis (Everett et al., 1999; Chung et al., 2004). The SUMOylation of sister chromatid cohesion protein PDS5 peaks during anaphase, and is necessary for dissolution of cohesion during mitosis in budding yeast (Stead et al., 2003). Centromere protein E (CENP-E), a kinesin-like motor protein, has been identified as a substrate of SUMO-2/3, and its recruitment to kinetochores is dependent on its SUMOylation (Zhang et al., 2008). Finally in Saccharomyces cerevisiae, SUMOylation of Top2p (the yeast homologue of Topoisomerase II) may down-regulate centromeric cohesion (Bachant et al., 2002). Meiosis is required to produce haploid gametes for fertilization. In the mouse ovary, an oocyte is arrested at the first meiotic prophase (diplotene stage), which contains a large nucleus called germinal vesicle (GV). Following a preovulatory surge of luteinizing hormone, responsive oocytes resume meiosis and undergo germinal-vesicle breakdown (GVBD). After GVBD, chromatin condenses and microtubules are assembled into a spindle. When all chromosomes are aligned at the metaphase plate, homologous chromosomes can begin to separate during the transition from metaphase to anaphase. The oocyte then extrudes its first polar body (PB1), which contains half of the chromosomes, and thereby completes meiosis I. The oocyte continues through meiosis II, arresting at the meiotic metaphase II (MII) stage until fertilization. Soon after fertilization, sister chromatids segregate and the second polar body is extruded, completing meiosis II. Throughout these sequential events,

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the cell division machinery must work with high precision to retain the integrity of the resulting haploid genome (Maro and Verlhac, 2002; Xiong et al., 2008); error in any of these processes can result in aberrant chromosome numbers (aneuploidy), which can cause any number of multiple reproductive defects such as infertility or birth defects (Duncan et al., 2012; Luciano et al., 2013). The full-grown oocyte is transcriptionally inactive, therefore translational and post-translational modification serve as key regulators of meiosis (Josefsberg and Dekel, 2002). Regarding regulation by SUMOylation, SUMO-1 and SUMO-2/3 are expressed in the mouse oocyte and are associated with the spindle pole or chromosomes at different stages of oogenesis. Interestingly, SUMO-1 and proteins that covalently associate with SUMO-1 localize to the spindle poles and around the chromosomes during oocyte meiosis, while SUMO-2/3 is mainly found near the centromeres. Overexpression of Sentrin/SUMO-specific protease 2 (SENP2), a SUMO-specific isopeptidase, altered the localization of SUMO-modified proteins in oocytes, and led to defects in MII spindle organization (Wang et al., 2010). Septin, a conserved GTP-binding protein that can be modified by SUMO-1, is required for chromosome congression in mouse oocytes, implying that SUMOylation also plays a role during oocyte meiosis (Zhu et al., 2010). Bub1-related kinase, or MAD3/Bub1b (BUBR1), is a spindle-assembly checkpoint protein necessary for homologous chromosome alignment that may be SUMOylated by SUMO-1 (Wei et al., 2010; Yang et al., 2012). Thus, key meiotic proteins are targets of SUMOylation, although the detailed meiotic processes involving SUMOylation are still unclear. Specific-antibody or siRNA microinjection was employed in this study to inhibit or deplete the endogenous SUMO-1 or UBC9 activity in oocytes in order to identify the roles of SUMO-1 during oocyte meiotic maturation, spindle organization, and chromosome alignment and segregation. The results indicated that inhibition or depletion of SUMO-1 or UBC9 significantly reduced the percentage of GVBD and PB1 extrusion, caused abnormal spindle organization, and led to chromosome misaligned, segregation defects, and aneuploidy in matured oocytes. SUMO-1 depletion or inhibition disrupted kinetochore-microtubule attachment and the localization of g-tubulin in meiotic spindle poles. Further study indicated that SUMO-1 functions before anaphase I, affecting PB1 extrusion. Moreover, inhibition of SUMO-1 reduced the degree of chromosome condensation and altered the localization of REC8, securin, and BUBR1. Together, these results indicated that SUMO-1-mediated SUMOylation is involved in oocyte meiotic maturation, microtubule
chromosome interaction, and spindle organization, and appears to play important roles in chromosome behavior.

RESULTS Inhibition or Depletion of SUMO-1 or UBC9 Disrupted Oocyte Meiotic Maturation In order to assess the roles of SUMO-1 or UBC9 on oocyte meiotic maturation, SUMO-1- or UBC9-specific

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antibody was microinjected into GV-stage oocytes to block the function of SUMO-1 or UBC9, respectively. Inhibition of SUMO-1 or UBC9 significantly reduced the percentage of GVBD (85.9  3.2% [n ¼ 12] and 87.3  4.7% [n ¼ 4] in non-injected and IgG-injected groups, respectively; 70.4  12.4% [n ¼ 6] and 68.7  1.9% [n ¼ 5] in SUMO-1 and UBC9 antibody-injected groups, respectively; P < 0.05) [‘‘n’’ represents the number of experiments conducted, unless stated otherwise] (Fig. 1A). Antibody injection also significantly reduced PB1 extrusion 14 hr after microinjection (22.4  8.6% [n ¼ 6] and 29.2  10.9% [n ¼ 5] in SUMO-1 and UBC9 groups, respectively), which was significantly lower than that in non-injected or IgGinjected groups (50.9  2.6% [n ¼ 12] and 50.8  4.8% [n ¼ 4], respectively; P < 0.05). Depletion of SUMO-1 or UBC9 by Sumo-1 or Ubc9 siRNA also significantly disrupted GVBD (77.5  5.3%, 50.8  13.7%, and 35.0  4.4% [n ¼ 3 per group] in control, Sumo-1, and Ubc9 siRNA microinjection groups, respectively; P < 0.05) and PB1 extrusion (48.9  1.1%, 22.6  7.4%, and 18.9  6.8% [n ¼ 3 per group] in control, Sumo-1, and Ubc9 siRNA microinjection groups, respectively; P < 0.05) (Fig. 1B). The efficiency of SUMO-1 or UBC9 knockdown by specific siRNAs was confirmed by immunofluorescence staining and Western blot: Fluorescence signal for both proteins were significantly reduced in the GV, while Western blots showed a significant reduction in the SUMO-1 or UBC9 knockdown groups compared to the control group (Fig. 1C). Neither inhibition or depletion of SUMO-1 completely blocked GVBD and PB1, however, implying that SUMO-1 might not be essential for either process; rather, it is involved in GVBD and PB1 extrusion. SUMO-2/3 or SUMO-4 might compensate for the loss of SUMO-1 activity in some cases.

SUMO-1 Mainly Functions Before Anaphase I To further define the temporal role of SUMO-1 during PB1 extrusion, we inhibited SUMO-1 by antibody injection at the GV (0 hr), pro-metaphase (6 hr), or anaphase-I stage (9.5 hr), and then cultured the oocytes for a total of 14 hr (time before plus after microinjection) to assess PB1 extrusion. PB1 extrusion was significantly inhibited when SUMO-1 antibody microinjection was performed at the GV stage (58.2  8.5% and 31.8  8.5% [n ¼ 4 per group] for rabbit IgG and SUMO-1 antibody injection groups, respectively; P < 0.05) or at the pro-metaphase stage (66.9  0.7% and 49.6  3.0% [n ¼ 3 per group] for rabbit IgG and SUMO-1 antibody injection groups, respectively; P < 0.01), but was not significantly different when SUMO-1 antibody was microinjection at anaphase I (59.8  12.6% and 59.4  11.0% [n ¼ 3 per group] for rabbit IgG and SUMO-1 antibody injection groups, respectively; P > 0.05) (Fig. 2A). Thus, SUMO-1dependent effects on PB1 extrusion occur mainly before anaphase I.

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Figure 1. Inhibition or depletion of SUMO-1/UBC9 reduced the percentage of GVBD and PB1 extrusion during mouse oocyte meiotic maturation. Fully grown GV-stage oocytes were microinjected with antibodies, siRNAs, control IgG, or control siRNAs, and then cultured for 2.5 or 14 hr for assessment of GVBD or PB1 extrusion, respectively. A: Percentage of GVBD and PB1 extrusion in oocytes after inhibition of SUMO-1/UBC9 by specific-antibody microinjection. The data represent mean  standard deviation (n > 3). Different letters denote statistical difference at a P < 0.05. B: The percentage of GVBD and PB1 extrusion in oocytes after depletion of SUMO-1/UBC9 by specific siRNA microinjection. The data represent mean  standard deviation (n ¼ 3). Different letters denote statistical difference at a P < 0.05. C: Evaluation of protein knockdown efficiency by Sumo-1 siRNA or Ubc9 siRNA, using immunofluorescence (IF) staining and Western blot (WB). Immunofluorescence images show SUMO-1 or UBC9 (green) and DNA (red). Scale bar, 50 mm. All experiments were independently repeated at least three times.

SUMO-1 is Critical for Spindle Assembly and Chromosome Organization During Meiosis I Based on the results above, SUMO-1 might be involved in spindle assembly and chromosome organization. To test this hypothesis, we assessed spindle organization and chromosome alignment in immature oocytes (non-MII)

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Figure 2. Roles of SUMO-1 in oocyte spindle organization and chromosome alignment. A: Fully grown GV-stage oocytes were cultured for 0, 6, or 9.5 hr, and then microinjection with SUMO-1 antibody or control IgG. After microinjection, oocytes were further cultured for a total period of 14 hr (before and after microinjection), and then checked for PB1 extrusion. PB1 extrusion was significantly decreased when SUMO-1 antibody was microinjection at the GV (0 hr) or pro-metaphase (6 hr) stage, but not when microinjected at anaphase I (9.5 hr) when compared with the control group. The data represent mean  standard deviation (n  3). Different letters denote statistical difference at a P < 0.05. B
F: Microinjection was performed at the GV-stage, oocytes were cultured for 14 hr, and immature oocytes were collected for analysis. (B and C) Representative spindle organization and the percentage of abnormal spindles in immature oocytes after SUMO-1- or UBC9-specific antibody microinjection. Scale bar, 50 mm. The data represent mean  standard deviation (n ¼ 3). Different letters denote statistical difference at a P < 0.05. (D and E) Phenotype of spindle and chromosome alignment, and the percentage of abnormal spindles in immature oocytes after depletion of SUMO-1 or UBC9 by siRNA microinjection. Scale bar, 50 mm. The data represent mean  standard deviation (n ¼ 3). Different letters denote statistical differences at a P < 0.05. F: The subcellular localization of g-tubulin was disordered in immature oocytes after SUMO-1 inhibition by antibody microinjection. In the IgG injection group, g-tubulin was located at the spindle poles at metaphase I, whereas SUMO-1 inhibition left a small portion of g-tubulin disassociated from the spindle poles (arrow). Scale bar, 50 mm. G: Chromosome spreads of immature oocytes after SUMO-1 inhibition by antibody microinjection at the GV stage and cultured for 8 hr. a and b: Control IgG-injected oocyte and SUMO-1 microinjected oocytes with normal 20 pairs of chromosomes, respectively; c and d: SUMO-1 antibody-microinjected oocytes exhibiting less-condensed chromosome. Scale bar, 10 mm.

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after inhibition or depletion of SUMO-1 or UBC9 in GVstage oocytes and in vitro culture. Fourteen hours of culture yielded some immature, control oocytes arrested at metaphase I with normal spindle organization (Fig. 2B, a and b). Inhibition of SUMO-1 or UBC9 caused the formation of numerous abnormal spindles, some with disordered poles or the absence of spindles in the immature oocytes (Fig. 2B, c
f). The percentage of abnormal spindles in immature oocytes after SUMO-1 or UBC9 inhibition was 67.6  6.0% and 68.6  6.1% [n ¼ 3 per group], respectively, which was significant higher than that in the control group (12.0  3.9% [n ¼ 3]; P < 0.05) (Fig. 2C). Depletion of SUMO-1 or UBC9 by specific siRNAs also resulted in defective spindles in immature oocytes (Fig. 2D, a
f), including the absence of spindles, spindles with disordered poles, or elongated spindles. The percentage of abnormal spindles after SUMO-1 or UBC9 depletion in immature oocytes were 82.4  6.4% and 67.6  6.0% [n ¼ 3 per group], respectively, which was significantly higher than that in the control group (15.0  4.1% [n ¼ 3]; P < 0.05) (Fig. 2E). We further determined the location of g-tubulin, an important regulator of spindle organization localized at the spindle poles (Lee et al., 2000), and the general morphology of chromosomes. A small portion of g-tubulin signal was disordered at the spindle poles after SUMO-1 inhibition (arrow) (Fig. 2F). Analysis of chromosome spreads revealed 20 pairs of chromosomes in control oocytes (23 oocytes tested) (Fig. 2G,a). Inhibition of SUMO-1 by SUMO-1 antibody injection, however, resulted in some oocytes with normal chromosomes morphology (Fig. 2G,b) whereas about 74.2% of the oocytes (31 oocytes tested) exhibited less-condensed chromosome (Fig. 2Gc,d).

SUMO-1 is Critical for Spindle Shape and Chromosome Alignment in Mature Oocytes We checked the spindle organization and chromosome alignment in 14-hr in vitro-matured oocytes (MII stage) after inhibition or depletion of SUMO-1 or UBC9 starting at the GV stage. Most oocytes extruded PB1 and formed a normal spindle in the mature oocytes of the control group (Fig. 3A, a and b). Inhibition of SUMO-1 or UBC9, however, caused numerous abnormal spindles, such as disorganized spindles lacking well-organized poles or elongated spindles, in the mature oocytes (Fig. 3A, c
f). The percentage of abnormal spindles in mature oocytes after SUMO-1 or UBC9 inhibition was 60.0  4.7% and 66.7  6.1% [n ¼ 3 per group], respectively, which was significant higher than that in the IgG injection group (11.1  4.7% [n ¼ 3]; P < 0.05) (Fig. 3B). Furthermore, depletion of SUMO-1 or UBC9 by specific siRNAs also resulted in defective spindles in mature oocytes (Fig. 3C, a
e). The percentage of abnormal spindles after SUMO-1 or UBC9 depletion in mature oocytes was also significant higher (80.0  7.8% or 66.7  0% [n ¼ 3 per group], respectively) than that in the control siRNA injection group (15.8  2.8% [n ¼ 3]; P < 0.05) (Fig. 3D). The subcellular localization of g-tubulin

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after SUMO-1 inhibition in these mature oocytes was also abnormal compared to that of control group (Fig. 3E).

Inhibition of SUMO-1 Leads to Aneuploidy in Mature Oocytes After depletion of SUMO-1 or UBC9, some matured oocytes possessed misaligned chromosomes at the spindle that appeared to dissociate from the metaphase-plate region (Fig. 3C, b0 and e0 ), and some immature oocytes showed misaligned chromosomes that could not be effectively captured and transported to the metaphase plate by microtubules (Fig. 2D, c0 and f0 ). We predicted that the misalignment of chromosome in both mature and immature oocytes could be related to the aneuploidy of matured oocytes. Therefore, we checked the chromosome numbers in the mature oocytes after SUMO-1 antibody microinjection. We found that aneuploidy was prone to occur in matured oocytes after SUMO-1 inhibition when compared to the control group, which showed 20 pairs of chromosomes and no aneuploidy (Fig. 3F). These results implied that SUMO-1 is important for accurate chromosome segregation.

SUMO-1 Depletion or Inhibition Disrupted Kinetochore-Microtubule Attachment and Reduced BUBR1 Centromere Localization As SUMO-1 inhibition or depletion resulted in chromosome misalignment and abnormal spindle organization, we hypothesized that these phenomena could be related to chromosome-microtubule attachment and the spindleassembly checkpoint. Therefore, we assessed the integrity of kinetochore-microtubule attachment by using coldtemperature treatment and then located the centromere protein CREST and a-tubulin after depletion of SUMO-1 at GV stage and cultured in vitro for 8 hr. Under these conditions, microtubules captured the kinetochores in the control-siRNA-microinjection group. Oocytes in the experimental group, however, contained some kinetochores that were disconnected and disassociated from the metaphase plate of spindles (Fig. 4A), indicating that the misalignment of chromosomes at metaphase could be a consequence of SUMO-1 depletion reducing the strength of the kinetochore-microtubule attachment. Moreover, BUBR1 levels at centromeres was significantly reduced after SUMO-1 inhibition compared with the control group (Fig. 4B and C) (5 oocytes were measured per control and SUMO-1 inhibition groups), implying that the spindle-assembly checkpoint might also be interrupted or dysfunctional.

Inhibition of SUMO-1 Affected REC8 and Securin Localization The aneuploidy observed after SUMO-1 inhibition suggested that some key proteins involved in chromosome segregation might be affected by SUMO-1 inhibition. For example, securin, an inhibitor of separase, protects REC8 from separase-dependent degradation before anaphase.

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Figure 3. SUMO-1 is critical for maintenance of spindle morphology and chromosome configuration in mature oocytes. A
E: GV-stage oocytes were microinjected, then cultured for 14 hr to mature oocytes for analysis. (A and B). Representative spindle organization and the percentage of abnormal spindles in mature oocytes after inhibition of SUMO-1 or UBC9 by specific antibody microinjection. Scale bar, 50 mm. The data represent mean  standard deviation (n ¼ 3). Different letters denote statistical difference at a P < 0.05. (C and D) Phenotype of spindle and chromosome alignment, and the percentage of abnormal spindles in mature oocytes after depletion of SUMO-1 or UBC9 by siRNA microinjection. Scale bar, 50 mm. The data represent mean  standard deviation (n ¼ 3). Different letters denote statistical difference at a P < 0.05. E: Subcellular localization of g-tubulin at the spindle poles was in mature oocytes after SUMO-1 inhibition by antibody microinjection. The immunostaining signal of g-tubulin accumulated at the poles in the IgG-injection group, but showed faint signal at the spindle poles after SUMO-1 inhibition. Scale bar, 50 mm. F: Inhibition of SUMO-1 leads to aneuploidy in mature oocytes. The control oocyte contains 20 pairs of sister chromosomes (a), but the count was more than 20 pairs (b) or less than 20 pairs after SUMO-1 inhibition (c). Scale bar, 10 mm.

Therefore, we investigated if the abundance and localization of REC8 and securin were affected by SUMO-1 inhibition. GV-stage oocytes were microinjected with SUMO-1 antibody, and then cultured in vitro for 8 hr. The abundance REC8 and securin proteins were not significantly different after SUMO-1 inhibition when compared to the control group (Fig. 5A), but the subcellular localization patterns of REC8 and securin were distinct. In the control group, REC8 mainly localized to the chromosomes while securin was found at the spindles. Following SUMO-1 inhibition, REC8 immunostaining was absent in chromosomes and the signal of securin was faint (Fig. 5B and C), suggesting

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that SUMO-1-mediated SUMOylation might directly or indirectly regulate the subcellular localization of REC8 in chromosomes and securin at spindles, thereby participating in the process of chromosome segregation.

Overexpression SUMO-1 Does Not Affect Oocyte Meiotic Maturation To further examine the roles of SUMO-1 during oocyte maturation, His-Sumo-1 mRNA was injected into GV-stage oocytes. Immunoblot results showed that His-Sumo-1 was expressed in the oocytes after the oocytes were arrested at

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Figure 5. Inhibition of SUMO-1 by antibody injection did not affect the abundance of REC8 and securin, but disrupted their location in oocytes. GV-stage oocytes were microinjected with SUMO-1 antibody or control IgG, and then cultured 8 hr for further analysis. A: The expression level of REC8 and securin in oocytes did not show significant differences after SUMO-1 inhibition by antibody microinjection when compared to the control group. B: Immunostaining signal of REC8 was located at the chromosomes in control oocytes, but disappeared in chromosomes after SUMO-1 inhibition by antibody microinjection. Scale bar, 10 mm. C: Securin co-localized with a-tubulin at the spindles in the control oocyte, but the signal at the spindles was faint in oocytes after SUMO-1 inhibition by antibody microinjection. Compare to the strong signal of a-tubulin in both groups. Scale bar, 50 mm.

Figure 4. Depletion or inhibition of SUMO-1 disrupted kinetochoremicrotubule attachment and reduced BUBR1 centromere localization. Microinjection was performed at the GV stage, and oocytes were cultured 8 hr for further analysis. A: Kinetochore-microtubule attachment in oocytes after depletion of SUMO-1 by Sumo-1-specific siRNA microinjection. Kinetochores (represented by CREST) were attached to microtubules in control oocytes, but could not be captured effectively by microtubules in oocytes after depletion of SUMO-1, as seen by the abnormal spindles. Scale bar, 10 mm. B: BUBR1 localization at centromeres was significantly reduced after SUMO-1 inhibition by antibody microinjection. Images were taken under the same microscope conditions. Scale bar, 10 mm. C: ImageJ measurement of BUBR1 signal intensity. The data represent mean  standard deviation (5 oocytes were measured per group). Different letters denote statistical difference at a P < 0.05.

GV stage for 7 hr (Fig. 6A). The rates of GVBD and PB1 extrusion were counted after culturing for 2.5 and 14 hr, respectively; no significant difference in GVBD and PB1 extrusion were observed between mRNA-injection groups

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(Fig. 6B). Furthermore, both the His-Sumo-1 and His mRNA injection groups showed normal spindle organization and chromosome alignment (data not shown). These results indicate that overexpressed SUMO-1 does not significantly affect meiotic maturation of oocytes.

DISCUSSION SUMO-1 is an ubiquitin-related protein involved in cellcycle control and mitosis. Previous reports on the localization and expression of SUMO-1 in oocytes indicated that SUMO-1 mediates SUMOylation during meiotic maturation (Wang et al., 2010). Its regulatory mechanism during meiosis, however, is still unclear. By inhibiting or depleting SUMO-1 or UBC9 (the E2 ligase of SUMOylation) using

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Figure 6. Overexpression of SUMO-1 had no significant effects on oocyte meiotic maturation. GV-stage oocytes were microinjected with His-Sumo-1 mRNA or His mRNA (control), arrested at the GV stage for 7 hr in DMEM/F12 medium containing 2.5 mM milrinone, and then collected for Western blot analysis or released and cultured in DMEM/F12 medium containing 10% FBS for 2.5 or 14 hr for examination of GVBD or PB1 extrusion, respectively. A: Overexpression of HisSUMO-1 in oocytes. B: Effects of His-SUMO-1 overexpression on GVBD and PB1 extrusion in oocytes. The GVBD and PB1 rate did not show significant differences between the overexpression and control groups (P > 0.05). The data represent mean  standard deviation (n ¼ 3).

specific-antibody or siRNA microinjection into GV-stage oocytes, we uncovered that SUMO-1-mediated SUMOylation is important for oocyte meiotic maturation, especially for spindle organization and chromosome alignment and segregation; is involved in the regulation of the attachment of kinetochore and microtubules; and participates in the subcellular localization of g-tubulin, BUBR1, securin, and REC8.

SUMO-1-Mediated SUMOylation is Involved in Oocyte Meiotic Maturation In order to inhibit the function of SUMO-1 or UBC9, we microinjected SUMO-1 or UBC9 antibody into GV-stage oocytes to assess their effects on oocyte meiotic maturation. These antibodies specifically bound to their endogenous antigen, and blocked the activity of each, resulting in the inhibition of GVBD and PB1 extrusion. As some oocytes still underwent GVBD (68
70%) or extruded their PB1 (22
29%), we predicted that: (1) Given the equilibration between SUMOylation and de-SUMOylation in the cells (Yeh, 2009) and the observation that only a small fraction of target proteins are SUMOylated in vivo (Geiss-Friedlander and Melchior, 2007; Andersen et al., 2009), some proteins

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necessary for oocyte meiotic resumption or PB1 extrusion were likely already SUMO-1-modified at the time of injection, so our methods to inhibit or deplete SUMO-1 or UBC9 (by antibody or RNAi, respectively) might not have been sufficient to completely inhibit or deplete SUMO-1’s activity. (2) The loss of SUMO-1 activity was compensated for by SUMO-2/3 or SUMO-4. And/or, (3) SUMO-1-mediated SUMOylation is not essential for GVBD and PB1 extrusion. Since inhibition or depletion of SUMO-1 or UBC9 significantly reduced the percentage of GVBD and PB1 extrusion, SUMOylation appears to be important for each process. Indeed, proteins related to GVBD and/or PB1 extrusion are putative or confirmed targets of SUMOlyation. For example, inhibition of Ran by antibody injection reduced the rate of GVBD and PB1 extrusion in the mouse oocyte (Cao et al., 2005), while its activator RanGAP1 was the first target of SUMO-1 identified. We predict that inhibition of SUMO-1 affected the ability of RanGAP1 to activate Ran, and consequently reduced the rate of GVBD and PB1 extrusion. Phosphodiesterase (PDE) 3A is a putative target, containing a conserved SUMOylation binding sequence (QAIKEEE), that is specifically expressed in oocytes and functions during GVBD (Liang et al., 2005; Li et al., 2010b). Whether or not PDE3A could be SUMO-1 modified, and if this modification alters the ability of PDE3A to degrade cAMP, thereby promoting GVBD, still need further study. Studies on D. melanogaster identified the SUMO-1 substrate CaMKII, an important regulator of mammalian oocyte meiotic progression to MII (Long and Griffith, 2000; Su and Eppig, 2002; Fan et al., 2003). SUMOylation of mitogen-activated protein-kinase kinase 1 (MEK1), another SUMOylation target (Kubota et al., 2011), could be involved in regulating the downstream effects of MAPK signaling, which is a crucial regulator of oocyte maturation and PB1 extrusion (Fan and Sun, 2004; Liang et al., 2007). In budding yeast, Smt3/ SUMO-1 is also required for anaphase-promotingcomplex/cyclosome (APC/C)-mediated proteolysis, which is necessary for the metaphase-anaphase transition (Dieckhoff et al., 2004). Based on these observations, we predict that SUMO-1mediated SUMOylation participates in GVBD and PB1 extrusion by targeting different kinds of proteins. As inhibition of SUMO-1 at specific stages of meiosis I

including GV, pro-metaphase, or anaphase-I

revealed that PB1 extrusion was not affected when SUMO-1 inhibition began at anaphase I, we hypothesize that SUMO-1-mediated SUMOylation may be needed before anaphase I.

SUMO-1-Mediated SUMOylation is Involved in Spindle Organization and Chromosome Alignment In this study, depletion or inhibition of SUMO-1 or UBC9 resulted in numerous defective spindles both in mature and immature oocytes; more importantly, the subcellular localization of g-tubulin, a spindle-pole protein, was disordered after SUMO-1 inhibition. Overexpression of SENP2 also led to defects in MII-spindle organization in mature oocytes

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(Wang et al., 2010). Some proteins of the Ran pathways, which play critical roles in spindle organization during mitosis, are also targets of SUMOylation (Joseph et al., 2002; Silverman-Gavrila and Wilde, 2006; Cesario and McKim, 2011). Nuclear mitotic-apparatus protein (NuMA) has recently been reported to be modified by SUMO-1, and SUMOylation is necessary for the NuMA-mediated formation and maintenance of mitotic-spindle poles (Seo et al., 2014). Previous work on oocyte meiosis has shown that NuMA and g-tubulin localize to the meiotic spindle poles and are important regulators of spindle organization (Lee et al., 2000). We speculate that SUMO-1 inhibition disrupted the localization of NuMA at the meiotic spindle poles and thus the ability of NuMA to regulate spindle formation. Therefore, g-tubulin localization at the meiotic spindle poles was altered, preventing the proper assembly of microtubules a normal spindle. This study also revealed that depletion or inhibition of SUMO-1 or UBC9 resulted in misaligned chromosomes both in mature and immature oocytes, with some mature oocytes containing chromosomes dissociated from the metaphase plate. Most of the abnormalities in mature oocytes originate from disruptions during meiosis I (Hassold and Hunt, 2001; Warren and Gorringe, 2006), thus defining the stage of meiosis that we could focus on. From our data, one source of chromosome misalignment associated with SUMO-1 depletion appeared to be disordered kinetochore-microtubule attachments, which may be a result of improper RanGAP1 localization to the mitotic spindle and attachment to kinetochores (Joseph et al., 2002, 2004). Another SUMO-1 target, septin2, also participates in chromosome congression during oocyte meiosis (Zhu et al., 2010). Together, these studies suggested that SUMO-1-mediated SUMOylation of specific target proteins is required for kinetochore-microtubule interaction and for the ability of spindle-pole proteins to regulate spindle organization during chromosome congression. Inhibition of SUMOylation by SUMO-1 may disrupt the subcellular localization, protein stability, and/or the function of these proteins, resulting in abnormal spindle organization and chromosome misalignment. Our results also revealed that the centromere localization signal of BUBR1 was reduced after SUMO-1 inhibition. A spindle-assembly checkpoint protein necessary for homologous-chromosome alignment, BUBR1 was reported to be SUMOylation by SUMO-1, which affected its stability as well as the integrity of chromosomal alignment and spindle organization during mitosis (Wei et al., 2010; Yang et al., 2012). We therefore predict that SUMO-1 inhibition reduced the stability and localization of BUBR1 at centromeres, leading to chromosome misalignment and spindle abnormalities during oocyte meiosis.

SUMO-1-Mediated SUMOylation is Involved in Chromosome Segregation During Oocyte Meiotic Maturation In Drosophila, mutation of Su(var)2-10, a member of the PIAS protein family and an E3 ligase utilized in

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SUMOylation, caused the improper condensation of mitotic chromosomes (less-condensed chromosomes), aberrant chromosome segregation, or chromosome fragmentation (Hari et al., 2001). SUMOylation is also involved in nondisjunction of meiosis I in Drosophila as the lesswright mutation of its UBC9 homologue could dominantly rescue the non-disjunction caused by mutant nod (no distributive disjunction). lesswright is thought to mediate the dissociation of heterochromatic regions of homologous chromosomes at the end of meiotic prophase I, perhaps keeping them together longer (Apionishev et al., 2001). Evidence for the conserved role of SUMOylation during spindle assembly comes from mice with a mutation in RanBP2, one of E3 ligase of SUMOylation. Homozygousmutant mice showed severe aneuploidy, while RanBP2 heterozygous embryonic fibroblasts exhibited centrophilic chromosomes defects (Dawlaty et al., 2008), together supporting a role for SUMOylation in the regulation of chromosome segregation and prevention of aneuploidy in mammals. Our results further support this activity as inhibition of SUMO-1 resulted in less-condensed homologous chromosomes. We therefore predict that SUMOylation modification is necessary for both the maintenance of homologous chromosomes pairs, to allow normal spindle formation and proper chromosome condensation, and their alignment, to avoid non-disjunction and aneuploidy.

Inhibition of SUMO-1 Affects the Normal Localization of REC8 and Securin, which Might Result in Chromosome Mis-Separation and Aneuploidy Before the initiation of anaphase, securin protects cohesion components from degradation by inhibiting separase activity (Terret et al., 2003; Lee et al., 2006; Kudo et al., 2009). During chromosome/chromatid separation, securin is cleaved by members of the ubiquitin-proteasome pathway, resulting in the activation of separase and subsequent cleavage of cohesion components. SUMOylation was previously found to be required for anaphase-promoting-complex (APC)-mediated proteolysis (Dieckhoff et al., 2004), thus linking this modification to anaphase. Protein phosphatase 2A (PP2A) is involved in chromosome segregation and is a substrate of SUMO-1 (Tang et al., 2006; Nie et al., 2009), but whether SUMOylation regulates its function during meiotic chromosome segregation needs further study. We found that SUMO-1 inhibition led to the loss of chromosomal REC8. A meiosis-specific cohesion protein with conserved SUMOylation binding sequences (RLVKREY, SREKFEE, and ERRKTEA). This phenotype may be due to abnormal localization of securin, leading to improper separase activation and the cleavage of cohesion components such as REC8 from chromosomes arms and centromeres, or to the absence of REC8 SUMOylation by SUMO-1, which destabilizes REC8 localization and/or accumulation on chromosomes. Combined with the observed disruption of kinetochore-microtubule attachments, which are important for chromosome segregation (Miller

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et al., 2012), the premature loss of chromosomal REC8 could lead to defective chromosome segregation and aneuploidy during oocyte meiosis. This outcome is consistent with the observation that aged oocytes, which have high levels of aneuploidy, also exhibit severely reduced levels of chromosome-associated REC8 and the centromeric cohesin protector Shugoshin-2 (SGO2) (Chiang et al., 2010; Lister et al., 2010). Indeed, our unpublished data indicate that SUMO-1 abundance is significantly lower in oocytes from older versus younger mice (12 months vs. 3 weeks, respectively). We thus predict that high rates of aneuploidy associated with oocytes from older females might result from reduced SUMOylation activity and subsequent abnormal activity of its target proteins. Overexpression of His-SUMO-1, on the other hand, had no significant effects on oocyte meiotic maturation, implying that excess SUMO-1 does not alter meiotic maturation in oocytes from young female mice. In aged oocytes of older mice, however, overexpression of SUMO-1 may improve meiotic maturation and/or rescue aneuploidy

but such an outcome needs further study. In conclusion, this study revealed that SUMOylation mediated by SUMO-1 is involved in multiple processes during oocyte meiotic maturation. In particular, SUMO-1 substrates participate in spindle organization and chromosome behavior; regulation of the attachment of kinetochore to microtubules; centromere localization of the spindleassembly checkpoint protein BUBR1; and the subcellular localization of spindle protein g-tubulin, cohesion component REC8 and the separase regulator securin.

MATERIALS AND METHODS Reagents and Solutions Polyclonal rabbit anti-SUMO-1 (sc-9060), polyclonal rabbit anti-UBC9 (sc-10759), and monoclonal mouse anti-b-actin (sc-69879) antibodies, Sumo-1 siRNA (sc36574), Ubc9 siRNA (sc-36774), normal (non-immunized) rabbit IgG (sc-2027), and negative-control siRNA (sc37007) were purchased from Santa Cruz Biotechnology (Dallas, TX). Human antibody against a centromere marker (CREST, HCT-0100) was obtained from Immunovision (Springdale, Arkansas). DMEM/F12 was acquired from Invitrogen (Life Technologies Corporation, Rockville, MD). Polyclonal rabbit anti-BUBR1 (11504-2-AP) and anti-REC8 (10793-1-AP) antibodies were purchased from Proteintech (Wuhan, China). Polyclonal rabbit antisecurin (BA1379) and monoclonal mouse anti-g-tubulin (BM1606) antibodies were obtained from Boster (Wuhan, China). All other chemicals were purchased from Sigma
Aldrich, unless stated otherwise.

Oocyte Collection and Culture Kunming mice (3
6 weeks old) were obtained from the Centre of Laboratory Animals of Hubei Province (Wuhan, China). This study was approved by the Ethical Committee of the Hubei Research Center of Experimental Animals

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(Approval ID: SCXK (Hubei) 2008
0005). The mice were sacrificed by cervical dislocation 48 hr after intraperitoneal injections of 10 IU pregnant-mare serum gonadotropin (PMSG). Only fully grown and immature oocytes arrested at the GV stage were selected for further experiments. All cultures were carried out at 378C in a humidified atmosphere of 5% CO2.

Antibody or siRNA Microinjection Microinjection was performed by using a micromanipulation system (Narishige, Tokyo, Japan) that was attached to an inverted microscope TE2000-U (Nikon UK Ltd., Kingston upon Thames, Surrey, UK) and completed within 45 min. About 7 pl of 100 mg/ml anti-SUMO-1 or -UBC9 antibody, or 10 mM Sumo-1 or Ubc9 siRNA was injected into the cytoplasm of GV-stage oocytes. The same amount of rabbit IgG or control siRNA was injected as a negative control. Each experiment was repeated at least three times, and at least 50 microinjected, GV-stage oocytes were used per experimental group. After microinjection of antibody, GV-stage oocytes were in vitro cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA). GV-stage oocytes microinjected with siRNA were arrested in DMEM/ F12 medium containing 2.5 mM milrinone for 24 hr, and then washed and cultured in DMEM/F12 containing 10% FBS medium for further study. GV status and PB1 extrusion were assessed under stereomicroscope at 2.5 and 14 hr after incubation. To study the temporal role of SUMO-1 during oocyte maturation, GV-stage oocytes were in vitro cultured for 0, 6, or 9.5 hr, which respectively correspond to GV, pro-metaphase, and anaphase I stages. Oocytes at these stages were collected for microinjection of SUMO-1 antibody or control rabbit IgG, cultured for a total period of 14 hr (before and after microinjection), and finally checked for PB1 extrusion.

Sumo-1 mRNA In Vitro Synthesis and Microinjection For in vitro transcription, full-length Sumo-1 cDNA was subcloned into pET-28a-His. To amplify the Sumo-1 sequence containing an in-frame His tag and the T7 promoter, the pET-28a-His-SUMO-1 plasmid was used as a template for PCR using forward (50 -TAATACGACTCACTATAG GGCGA) and reverse primers (50 -AGCCAACTCAGCTT CCTTTC). The PCR product was purified using a gel extraction kit (Qiagen, Duesseldorf, Germany). A T7 Message Machine kit (Ambion, Life Technologies Corporation) was used to produce the capped His-Sumo-1 mRNA, and later this mRNA was purified using an RNeasy cleanup kit (Qiagen, German). His mRNA was obtained for controlgroup injections from pET-28a-His vector following the same procedure. For His-SUMO-1 expression, mRNA was microinjected into the cytoplasm of GV-stage oocytes. These oocytes were arrested at the GV-stage in DMEM/F12 medium

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Immunofluorescence Staining, Chromosome Spreads, and Confocal Microscopy Microinjected oocytes were collected for immunofluorescence staining after specific durations of in vitro culture. Oocytes were fixed in 4% paraformaldehyde in phosphatebuffered saline (PBS) for 30 min at room temperature. After permeabilization with 0.5% Triton X-100 in PBS at room temperature for 30 min, oocytes were incubated overnight at 48C with different primary antibodies, including: FITCconjugated monoclonal mouse anti-a-tubulin antibody (1:50, Sigma, St. Louis, MI), anti-g-tubulin (1:50), polyclonal rabbit anti-SUMO-1 (1:50), anti-UBC9 (1:50), anti-securin antibody (1:50), or human antibody against the centromere protein CREST (1:40), all diluted in PBS containing 0.1% Tween-20 and 0.01% Triton X-100. After three washes in PBS for 5 min each, the oocytes were further incubated in FITC- or Cy3-conjugated anti-rabbit IgG antibody (1:100) or Cy3-conjugated anti-human or -mouse antibody (1:100) according to the host of the primary antibody. Oocytes were then stained with propidium iodide (PI; 10 mg/ml in PBS) or 4, 6-diamidino-2-phenylindole (DAPI; 10 mg/ml in PBS) to visualize the nucleus. Primary antibody was replaced with normal IgGs as negative controls. For BUBR1 and REC8 staining, chromosome spreads were prepared as described previously (Hodges and Hunt, 2002). Briefly, after the removal of zona pellucida, the oocytes were fixed in a solution of 1% paraformaldehyde in distilled water (pH 9.0) containing 0.15% Triton X100 and 3 mM dithiothreitol. The slides were dried slowly at room temperature for several hours, and then blocked with 1% bovine serum albumin (BSA) prepared in PBS for 1 hr at room temperature. Oocytes were then incubated with rabbit anti-BUBR1 (1:50) or anti-REC8 (1:50) antibody overnight at 48C. After brief washes with washing buffer, the slide was incubated with FITC- or Cy3-conjugated anti-rabbit antibody for 2 hr at room temperature. Following DNA staining by PI or DAPI, the oocytes were mounted on glass slides with DABCO and examined with a LSM 510 META confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany). ImageJ software (National Institutes of Health, USA) was used to assess the immunostaining signal intensity of BUBR1 at centromeres. All of the green dots on chromosomes were chosen and evaluated by ImageJ software for comparison. All images were taken under the same conditions; for example, magnification, intensity of excitation light, gain, and offset. For chromosome spreads in immature oocytes, oocytes were incubated for 20 min in 1% sodium citrate at room temperature, and then fixed with fresh methanol: glacial acetic acid (3:1). Chromosomes were stained with 10 mg/ml PI or DAPI, then samples were examined with a LSM 510 META confocal laser scanning microscope.

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Western Blot Analysis Samples containing at least 150 oocytes were collected in 2 SDS loading buffer at the appropriate stages of meiosis for Western blot analysis. Samples were heated for 5 min at 1008C. After separation by SDS-PAGE, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. After transfer, the membranes were blocked in Tris-buffered saline containing 0.1% Tween-20 (TBST) containing 5% skim milk for 1 hr, followed by overnight incubation at 48C with polyclonal rabbit anti-SUMO-1, anti-UBC9, anti-REC8, or anti-securin antibody at 1:200 dilution or mouse monoclonal anti-His antibody at 1:1,000 dilution. After three washes for 10 min each in TBST, the membranes were incubated for 1 hr at 378C with 1:3,000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse IgG antibody. Finally, the membranes were developed using the enhanced chemiluminescence detection system (Amersham, Piscataway, NJ). The primary antibody was replaced with the normal rabbit or mouse IgG as a negative control.

Cold-Stable Assay for Kinetochore-Microtubule Attachments The cold-stable assay was conducted according to a previously published method (Li et al., 2010a). Briefly, after microinjection with Sumo-1 or control siRNA, GV-stage oocytes were maintained in DMEM/F12 medium containing 2.5 mM milrinone for 24 hr, and then washed and cultured in DMEM/F12 medium for 8 hr. Oocytes were then transferred to culture medium pre-cooled at 48C, then incubated for 10 min. At this point, oocytes were fixed and collected for immunofluorescence staining for kinetochores and atubulin.

Data Analysis and Statistics All experiments were performed independently at least three times, and data are presented as mean  standard deviation. Differences between groups were analyzed by one-way ANOVA followed by Tukey’s Honest Significant Difference (HSD) test using SPSS (Version 17.0; SPSS, Chicago, IL). Differences were considered significant when P < 0.05.

ACKNOWLEDGMENT This work was conducted in the Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction, Education Ministry of China, College of Animal Science and Technology, Huazhong Agricultural University. We are also grateful to Yao Hang (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University) for providing technical assistances for confocal microscopy. This study was supported by the National Natural Science Foundation of China (Grant No.31071273 and 31171378), and the Fundamental Research Funds for the Central Universities (Program No. 2014PY045).

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