REPORT Cell Cycle 14:22, 3566--3579; November 15, 2015; © 2015 Capital Medical University
Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage Juan Du1, Yan Cao1, Qian Wang1, Nana Zhang1, Xiaoyu Liu1, Dandan Chen1, Xiaoyun Liu1, Qunyuan Xu2,*, and Wei Ma1,* 1
Department of Histology and Embryology; School of Basic Medical Sciences; Capital Medical University; Beijing, China; 2Department of Neurobiology; School of Basic Medical Sciences; Capital Medical University; Beijing, China
Keywords: mouse, MTOC, oocytes, Plk1 phosphorylation, REC8 cleavage, spindle formation Abbreviations: GV, germinal vesicle; GVBD, germinal vesicle breakdown; pro-MI, pro-metaphase I; MI, metaphase I; MII, metaphase II; MTOC, microtubule organizing center; SAC, spindle assembly checkpoint; Plk1, polo-like kinase 1; pPlk1Ser137, phosphorylated Plk1 on Serine 137; pPlk1Thr210, phosphorylated Plk1 on Threonine 210; PP2A, protein phosphatase 2A; OA, okadaic acid.
Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1Ser137 was colocalized with microtubule organizing center (MTOC) proteins, g-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed g-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1Ser137 and pPlk1Thr210, as well as the subcellular distribution of pPlk1Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1Ser137 is the action executor, in mouse oocytes during meiotic division.
Introduction Chromosome segregation errors in female meiosis can result in embryo genetic instability, which is the leading cause for infertility, spontaneous abortion and congenital birth defects. An understanding of mechanisms of chromosome segregation in female meiosis is of fundamental importance and may contribute to an understanding of human aneuploidy. During cell division, chromosome congression, alignment and segregation are driven by the microtubule-based machine, spindle apparatus. Defects in spindle structure or function will usually lead to chromosome missegregation. In somatic cells, spindle
assembly is regulated by centrosome, which is composed of a pair of orthogonal centrioles and the surrounding amorphous material, named pericentriolar material (PCM).1 Centrioles are required for centrosome structural maintenance and spindle rational orientation.2 In turn, PCM essentially directs microtubule nucleation and anchoring, depending on 2 integral components, g-tubulin and pericentrin. g-tubulin combines with several other associated proteins to form g-tubulin ring complex (gTuRC), which functions as template to initiate and regulate microtubule assembly.3-4 Pericentrin scaffolds the binding of gTuRC and other key factors to centrosome, supporting the orderly formation of microtubule array.5-7 For most mammalian
*Correspondence to: Qunyuan Xu; Email: [email protected], Wei Ma; Email: [email protected] Submitted: 06/08/2015; Revised: 09/17/2015; Accepted: 09/22/2015 http://dx.doi.org/10.1080/15384101.2015.1100770
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oocytes, the centrioles are eliminated at the early stages of oogenesis,8 and the meiotic spindle formation is regulated by a unique microtubule organizing centers (MTOCs) in a centrosome-independent manner.9 It is still not fully known about the molecular composition and functional regulation mechanism of MTOC in mammalian oocytes. It has been confirmed that g-tubulin and pericentrin are retained in MTOC during centriole elimination, and definitely required for microtubule assembly in oocytes.10-15 Collecting evidences show that some protein molecules, such as Aurora A kinase (Aur-A), 16-17 protein kinase C (PKC) 18-19 and LIM kinase 1 (LIMK1), 20 etc., are recruited to MTOC during germinal vesicle breakdown (GVBD), regulating MTOC organization and polar localization. Further investigations are needed to identify the regulatory factors facilitating MTOC maturation after GVBD, and to piece together the interactive network regulating spindle formation in mammalian oocytes. The stepwise loss of cohesion between chromosomes is another guarantee mechanism for faithful chromosome segregation in oocyte meiosis.21 Cohesion is released between chromatid arms, but sustained in the local space surrounding centromeres during anaphase I, giving rise to the separation of homologous chromosomes but not sister chromatids, and the release of centromeric cohesion permits the segregation of sister chromatids during anaphase II. Premature loss of centromeric cohesion can cause chromosome missegregation.22 The cohesion is achieved by a highly conserved ring-like complex surrounding paired chromosomes, this complex is composed of 4 cohesin subunits: REC8 (meiotic recombination protein), Stag3 (stromal antigen 3) and 2 SMC (structural maintenance of chromosomes) family proteins, SMC1b and SMC3.21,23 The cohesin complex collapses, relieving the binding force that holds chromosomes together, when REC8 is degraded by separase. In addition, REC8 must be phosphorylated by specific upstream kinase before it can be degradable.24-28 During meiosis I, REC8 phosphorylation is resisted at centromere area by protein phosphatase 2A (PP2A), so as to maintain effective REC8 accumulation between sister centromeres to ensure sister chromatids are bounded together and behave as one unit until the onset of anaphase II.29-30 It has long been controversial about the kinases responsible for REC8 phosphorylation. Up to now, polo, 26,31,32 casein kinase 1d/e (CK1)33-34 and Dbf4-dependent Cdc7 kinase (DDK) 34 are implicated in REC8 phosphorylation during meiosis in yeast and fly oocytes. Human recombinant protein of polo-like kinase 1 (Plk1), the homolog of Drosophila polo, also promotes REC8 cleavage by separase in vitro.27 Further studies are needed to clarify the exact protein kinases that are in charge for REC8 phosphorylation in vivo during meiosis in mammalian oocytes. It is now widely appreciated that Plk1 is the ‘master regulator’ of somatic cell mitosis and involved in multiple events, including entry into mitosis, centrosome maturation, spindle assembly, the activation of spindle assembly checkpoint (SAC), and the timely destruction of cohesion between sister chromatids, as well as the proper completion of cytokinesis.35 Plk1 activity is also required in regulating the gamete meiotic progression. Data from different studies confirm alterations in Plk1 activity definitely cause severe spindle defects and chromosome missegregation during mouse
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oocytes meiotic maturation.36-37 Though Plk1 is required unambiguously for cohesin degradation in sister chromatid splitting during somatic mitosis, whether it plays similar role during germ cell meiotic division is still not clearly revealed. It was recently reported that Plk1 promotes the phosphorylation and disassembly of synaptonemal complex, including cohesin subunit REC8, in mouse spermatocytes.38 Clear evidences are still absent about the involvement of Plk1 activity in cohesin degradation in mammalian oocytes. The functional diversity of Plk1 in mitotic cells is associated with its consecutive posttranslational modification. Plk1 phosphorylation at Ser137 and Thr210 in vivo occurs with different timing, and regulates separate Plk1 actions.39-41 Thr210 phosphorylation is required for Plk1 activation39-40 and entry into mitosis,40-41 while phosphorylation of Ser137 takes place only in late mitosis, not required for initial activation of Plk1.39 An alteration in Ser137 phosphoryaltion can induce spindle assembly checkpoint failure and untimely premature onset of anaphase.41 However, it is still completely unknown about the pattern of Plk1 phosphorylation, as well as the subcellular distribution and potential function of phosphorylated Plk1 during meiotic division in oocytes. In the present study we assessed the protein expression and subcellular localization of phosphorylated Plk1 in mouse oocytes during meiotic division, and examined its function by using an ATP-competitive inhibitor, BI2536. The results indicate that Plk1 phosphorylation at Ser137 (pPlk1Ser137) and Thr210 (pPlk1Thr210) occurs in oocytes, with distinct expression and localization patterns. pPlk1Ser137 localization is sensitive to BI2536, and required for meiotic spindle assembly and REC8 cleavage during oocyte meiosis.
Results Asynchronous Plk1 phosphorylation at Ser137 and Thr210 in mouse oocytes during meiotic maturation In somatic cells, Plk1 activity is regulated by its putative phosphorylation on Ser137 and Thr210. We investigated whether this phosphorylation occurs in mouse oocytes during meiosis. Prior to exploring the features of Plk1 phosphorylation, the protein expression pattern of total Plk1 was validated in this study. A commercial anti-Plk1 antibody (ab17057, Abcam), which recognizes the peptide sequence from residue 330 to 370, was used to detect total Plk1, no matter Ser137 or Thr210 residues are phosphorylated or not. Consistent with the previous results,36 stable and consistent quantity of Plk1 was detected during oocyte meiotic progression from germinal vesicle (GV) to metaphase II (MII) stage (Fig. 1A), manifested as a single band at 68 kDa. The expression characteristics of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) were directly determined using phospho-specific antibodies. As showed in Figure 1A, pPlk1Ser137 protein was detected as a single band at about 72 kDa in mouse oocytes, which was highly expressed at GV stage and sustained stable up to MII stage. pPlk1Thr210 protein was labeled as one single band more than 72 kDa at GV
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including Ser137 and Thr210, at GV stage, and partial dephosphorylation takes place after the onset of meiotic resumption, suggesting that such modifications may function at late stages in meiotic progression. Dynamic sub-cellular localization of Plk1 during oocyte meiosis Though Plk1 protein was stable and consistent in quantity, its subcellular distribution was highly dynamic along oocyte meiotic progression from GV to MII stage. At GV stage, Plk1 was labeled as several round dots of different size in nucleus (Fig. S1A, a-d). These dots were gradually dismantled and reassembled as highly concentrated foci on centromeres as chromatin condensing into individual chromosomes after GVBD (Fig. S1A, e-h). This centromeric localization was consistently observed from pro-metaphase I (pro-MI) to MI, also at MII stage (Fig. S1A, i-x), and further displayed in greater detail by immunofluorecsence analysis on chromosome spread samples (Fig. S2). In addition, Plk1 was also found localized on spindle poles at MI and MII (Fig. S1B), and closely overlapped with MTOC components, pericentrin and g-tubulin (Data not shown). Specially, Plk1 was no longer observed on polar area but distributed over the midbody structure during anaphase I (AI) / telephase I (TI) transition (Fig. S1A, q-t). These morphological data indicates Plk1 is associated with the meiotic spindle formation, spindle assembly checkpoint function and cytokenesis completion during meiotic progression in mouse oocytes.
Figure 1. Plk1 phosphorylation on Ser137 and Thr210 occurs differently in mouse oocytes during meiosis. (A) Western blot analysis was used to detect pPlk1Ser137, pPlk1Thr210 and Plk1 protein level. For each sample, 100 oocytes were collected after 0, 2, 4, 8 and 17 h culture, corresponding to GV, GVBD, MI and MII stages, respectively. GAPDH was used as a protein loading control. Each protein was assayed at least 3 times. Plk1 was detected as one 68 kDa band in oocytes and expressed consistently stable among all stages during meiotic maturation. pPlk1Ser137 was detected as a single bind at about 72 kDa and expressed in high and stable level from GV to MII stage. pPlk1Thr210 was labeled as a single band, which was larger than 72 kDa, at GV stage, and assayed as 2 bands after GVBD, with the lower band at about 72 kDa and the upper one same as that at GV stage, both bands were sustained in stable level up to MII stage. (B) Lambda protein phosphatase (l-PP) was used to determine the specificity of pPlk1T210 antibody. For each sample, 100 oocytes were collected and incubated in the absence (-l-PP) or presence of l-PP (Cl-PP) for 30 min at 30 C. The resulting sample were used for western blot analysis with pPlk1Thr210 antibody. l-PP treatment greatly reduced the protein signals of these 2 bands.
stage, and as 2 smaller bands after GVBD, which maintained in high and stable levels until MII stage. After being treated with lambda protein phosphatase (l-PP), both the large band at GV and 2 small bands after GVBD were cleared away (Fig. 1B), suggesting they represent phosphorylated protein signals. The western blot results confirmed that Plk1 phosphorylation on Ser137 and Thr210 happens in oocyte meiosis in vivo. Overphosphorylation of Plk1 may occur on multi residue sites,
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Co-localization of pPlk1Ser137 with MTOC proteins on spindle poles Since it was definitely ascertained by western blot results that Plk1 Ser137 and Thr210 phosphorylation happens in mouse oocyte meiosis, we then characterized the sub-cellular distribution patterns of pPlk1Ser137 and pPlk1Thr210 throughout meiotic division. In order to determine the intracellular localization of pPlk1Ser137, oocytes at every typical stage of meiotic process were collected and processed for double immunofluroescent staining with pPlk1Ser137 and acetylated-tubulin. As showed in Figure 2A, in oocytes arrested at GV stage, pPlk1Ser137 was accumulated as multiple bright aggregates (foci) in germinal vesicle (Fig. 2A, a-d), and then gathered as discrete foci close to condensing chromosomes upon meiotic resumption (Fig. 2A, e-h). During pro-MI when microtubules were assembled and organized into meiotic spindle around condensed chromosomes, pPlk1Ser137 foci mass were assorted into 2 groups, migrated in the opposite directions toward the 2 poles of the forming spindle (Fig. 2A, i-l), and finally localized at the polar area of barrel-like spindle at MI stage, in an “O” or “C”-shaped configuration (Fig. 2B, c,g) with positive signals radiating outward (Fig. 2A, m-p). pPlk1Ser137 was translocated to the midzone of the spindle during AI / TI (Fig. 2A, q-t), and again focused on poles of newly formed spindle as oocytes developed to MII stage. In addition, multiple discrete foci of pPlk1Ser137 were found in the cytoplasm as oocytes underwent meiotic maturation (Fig. 2A, u-x). Signal of pPlk1Ser137 was also observed across chromosomes, especially obvious at MI and MII stages (Fig. 3A).
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pPlk1Ser137 localization on spindle poles at MI and MII stages implied its probable association with MTOCs. We then performed immunofluorescent colocalization analysis of pPlk1Ser137 with crucial components of MTOCs, pericentrin and g-tubulin. As we expected, pPlk1Ser137 was specifically co-localized with g-tubulin and pericentrin on the meiotic spindle poles and at cytoplasmic MTOCs at MI (Fig. 2B), as well as other typical stages of meiosis I (data not show). Specially, filamentous aggregates of pPlk1Ser137 was radiated outward from the poles and overlapped with g-tubulin filaments. The distinct association of pPlk1Ser137 with MTOCs suggests pPlk1Ser137 may be a component of meiotic acentriolar MTOCs, thus involve in meiotic spindle assembly in oocyte meiosis.
Figure 2. pPlk1Ser137 subcellular distribution in mouse oocytes during meiotic division. (A) Dynamic localization of pPlk1Ser137 is associated with spindle formation during meiosis. pPlk1Ser137 was indicated in red, acetylated-tubulin in green and DNA in blue. Scale bar D 10 mm. pPlk1Ser137 was assembled as several large dots within germinal vesicle at GV stage, and reassembled as many small foci around condensing chromatin after GVBD, these foci were gradually fused and clustered into 2 groups as cell cycle progressed to pro-MI, and concentrated on 2 opposite poles of barrel-shaped spindle at MI stage. pPlk1Ser137 was transferred to the midbody during AI-TI transition and moved back to polar areas of newly formed spindle at MII stage. Cytoplasmic pPlk1Ser137 was indicated by asterisk and pPlk1Ser137 at spindle polar area was indicated by arrow and was shown in insets with 2£ magnification. (B) pPlk1Ser137 was co-localized with g-tubulin and pericentrin at MTOCs. Oocytes at MI stage were collected after an 8 h culture for double immunostaining of pPlk1Ser137 (red) either with g-tubulin (green) (a-d) or pericentrin (green) (e-h), both of which are key components of MTOCs. DNA was visualized with DAPI in blue. Scale bar D 10 mm. At MI stage, pPlk1Ser137 concentrated in “O” or “C” shape on spindle polar area and distributed in cytoplasm and colocalized with pericentin and g-tubulin both in cytoplasm (asterisk) and spindle poles (arrow).
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pPlk1Ser137 persisted on centromeres and dynamicly located between chromosome arms during oocyte meiotic progression In order to reveal the detailed characteristics of pPlk1Ser137 localization on chromosomes, we prepared chromosome spreads from oocytes developed to pro-MI, MI and MII stage, respectively, and immune-labeled with pPlk1Ser137 antibodies and auto anti-centromere serum CREST. As showed in Figure 3A, pPlk1Ser137 was persistently concentrated on centromeres from pro-MI to MI and at MII. The relative fluorescent intensity of centromeric pPlk1Ser137 was roughly equal among these stages (Fig. 3C, pro-MI vs MI, 61.82 § 9.872 vs. 85.94 § 6.577, P > 0.05). At the same time, pPlk1Ser137 was also dynamically clustered between chromosome arms, specifically from pro-MI and MI, pPlk1Ser137 was labeled in the full space between the arms of homologous chromosomes or sister chromatids (Fig. 3A, a-h), but only confined to the local space between centromeres of sister chromatids at MII stage (Fig. 3A, i-l). Further quantitative analysis showed that the average fluorescence intensity of pPlk1Ser137 between chromosome arms was significantly higher in MI oocytes (52.99 § 2.363; P < 0.05) than that in pro-MI cells (15.16 § 4.015) (Fig. 3B). Given space between chromosomes arms is where the cohesin complex accumulated, the increasing assembly of pPlk1Ser137 between chromosome arms from pro-MI to MI highly suggested its possible involvement in the resolution of cohesion along chromosome arms. Ubiquitous distribution of pPlk1Thr210 on chromosomes during mouse oocyte meiotic division The intracellular localization of pPlk1Thr210 was examined by double immunostaining with anti-pPlk1Thr210 and CREST antiserum. There was no obvious pPlk1Thr210 assembly observed either in cytoplasm or in nucleus at GV stage (Fig. 4A, a–d). Upon GVBD, pPlk1Thr210 was constantly presented on chromosomes, in sharp contrast to the diverse subcellular localization of pPlk1Ser137 (Fig. 4A, e-h). From pro-MI to MI and at MII, high concentration of pPlk1Thr210 was labeled on whole chromosome scales, even on chromosomes of the first polar body (Fig. 4A, i-t). This distribution was more finely verified with immunostaining on chromosome spreads (Fig. 4B), which clearly illustrating the ubiquitous pPlk1Thr210 just like a “coat” covering the whole structure of chromosomes. During AI/TI transition, strong signal of pPlk1Thr210 was still observed on
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separating chromosomes, also the area of midbody (Fig. 4B, q-t). All these morphological evidences indicate that pPlk1Thr210 may be related to the establishment and maintenance of chromosome configuration during oocyte meiosis. Metaphase I arrest induced by Plk1 inhibition with BI2536 The unique distributions of phosphorylated Plk1 imply the relevance of Plk1 activity in some essential cellular events controlling chromosome division during meiotic progression. We then explored the roles of Plk1 activity by using the Plk1 inhibitor BI2536. Firstly, we took an overview of the effects of BI2536 on oocyte maturation rate. GV oocytes were matured in culture medium supplemented with either 100 nM BI2536 or same volume of DMSO, and the number of oocytes developed to MI as well as MII stage was assessed by spindle morphology and chromosome alignment at 8, 10, 12, 14 and 17 h of culture. As showed in Figure 5, when checked at 8 and 10 h of culture, the majority of oocytes were matured to MI stage, there was no significant difference in the number of MI oocytes between BI2536 treatment group and control, indicating BI2536 exacted no obvious effects Figure 3. Unique distribution pattern of pPlk1Ser137 on chromosomes. (A) pPlk1Ser137 was accumulated peron the restart of meiosis and the sistently on centeromeres and dynamically between chromosome arms. Chromosome spreads from promeiotic progression to MI. With MI, MI, and MII oocytes were processed for immunofluorescence staining with pPlk1Ser137 antibodies and the increase in incubation time, the CREST serum. Chromosome was labeled in blue, pPlk1Ser137 in red and CREST in green. Scale bar D 10 mm. portion of MI oocytes continupPlk1Ser137 was accumulated between chromosome arms from pro-MI (a-d) to MI (e-h), and only focused at local space between sister centromeres at MII stage (i-l). pPlk1Ser137 was persistently concentrated on cenously decreased while that of MII tromeres from pro-MI to MII (i-l, arrow). (B) Quantitative analysis demonstrated that higher level of oocytes, characterized by the extrupPlk1Ser137 between chromosome arms in MI oocytes than that in pro-MI cells (P
Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1Ser137 involvement in spindle formation and REC8 cleavage Juan Du1, Yan Cao1, Qian Wang1, Nana Zhang1, Xiaoyu Liu1, Dandan Chen1, Xiaoyun Liu1, Qunyuan Xu2,*, and Wei Ma1,* 1
Department of Histology and Embryology; School of Basic Medical Sciences; Capital Medical University; Beijing, China; 2Department of Neurobiology; School of Basic Medical Sciences; Capital Medical University; Beijing, China
Keywords: mouse, MTOC, oocytes, Plk1 phosphorylation, REC8 cleavage, spindle formation Abbreviations: GV, germinal vesicle; GVBD, germinal vesicle breakdown; pro-MI, pro-metaphase I; MI, metaphase I; MII, metaphase II; MTOC, microtubule organizing center; SAC, spindle assembly checkpoint; Plk1, polo-like kinase 1; pPlk1Ser137, phosphorylated Plk1 on Serine 137; pPlk1Thr210, phosphorylated Plk1 on Threonine 210; PP2A, protein phosphatase 2A; OA, okadaic acid.
Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1Ser137 with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1Thr210 was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1Ser137 was colocalized with microtubule organizing center (MTOC) proteins, g-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1Thr210 was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1Ser137 accumulation at MTOCs and between chromosome arms, consequently disturbed g-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1Ser137 and pPlk1Thr210, as well as the subcellular distribution of pPlk1Thr210, were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1Ser137 is the action executor, in mouse oocytes during meiotic division.
Introduction Chromosome segregation errors in female meiosis can result in embryo genetic instability, which is the leading cause for infertility, spontaneous abortion and congenital birth defects. An understanding of mechanisms of chromosome segregation in female meiosis is of fundamental importance and may contribute to an understanding of human aneuploidy. During cell division, chromosome congression, alignment and segregation are driven by the microtubule-based machine, spindle apparatus. Defects in spindle structure or function will usually lead to chromosome missegregation. In somatic cells, spindle
assembly is regulated by centrosome, which is composed of a pair of orthogonal centrioles and the surrounding amorphous material, named pericentriolar material (PCM).1 Centrioles are required for centrosome structural maintenance and spindle rational orientation.2 In turn, PCM essentially directs microtubule nucleation and anchoring, depending on 2 integral components, g-tubulin and pericentrin. g-tubulin combines with several other associated proteins to form g-tubulin ring complex (gTuRC), which functions as template to initiate and regulate microtubule assembly.3-4 Pericentrin scaffolds the binding of gTuRC and other key factors to centrosome, supporting the orderly formation of microtubule array.5-7 For most mammalian
*Correspondence to: Qunyuan Xu; Email: [email protected], Wei Ma; Email: [email protected] Submitted: 06/08/2015; Revised: 09/17/2015; Accepted: 09/22/2015 http://dx.doi.org/10.1080/15384101.2015.1100770
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oocytes, the centrioles are eliminated at the early stages of oogenesis,8 and the meiotic spindle formation is regulated by a unique microtubule organizing centers (MTOCs) in a centrosome-independent manner.9 It is still not fully known about the molecular composition and functional regulation mechanism of MTOC in mammalian oocytes. It has been confirmed that g-tubulin and pericentrin are retained in MTOC during centriole elimination, and definitely required for microtubule assembly in oocytes.10-15 Collecting evidences show that some protein molecules, such as Aurora A kinase (Aur-A), 16-17 protein kinase C (PKC) 18-19 and LIM kinase 1 (LIMK1), 20 etc., are recruited to MTOC during germinal vesicle breakdown (GVBD), regulating MTOC organization and polar localization. Further investigations are needed to identify the regulatory factors facilitating MTOC maturation after GVBD, and to piece together the interactive network regulating spindle formation in mammalian oocytes. The stepwise loss of cohesion between chromosomes is another guarantee mechanism for faithful chromosome segregation in oocyte meiosis.21 Cohesion is released between chromatid arms, but sustained in the local space surrounding centromeres during anaphase I, giving rise to the separation of homologous chromosomes but not sister chromatids, and the release of centromeric cohesion permits the segregation of sister chromatids during anaphase II. Premature loss of centromeric cohesion can cause chromosome missegregation.22 The cohesion is achieved by a highly conserved ring-like complex surrounding paired chromosomes, this complex is composed of 4 cohesin subunits: REC8 (meiotic recombination protein), Stag3 (stromal antigen 3) and 2 SMC (structural maintenance of chromosomes) family proteins, SMC1b and SMC3.21,23 The cohesin complex collapses, relieving the binding force that holds chromosomes together, when REC8 is degraded by separase. In addition, REC8 must be phosphorylated by specific upstream kinase before it can be degradable.24-28 During meiosis I, REC8 phosphorylation is resisted at centromere area by protein phosphatase 2A (PP2A), so as to maintain effective REC8 accumulation between sister centromeres to ensure sister chromatids are bounded together and behave as one unit until the onset of anaphase II.29-30 It has long been controversial about the kinases responsible for REC8 phosphorylation. Up to now, polo, 26,31,32 casein kinase 1d/e (CK1)33-34 and Dbf4-dependent Cdc7 kinase (DDK) 34 are implicated in REC8 phosphorylation during meiosis in yeast and fly oocytes. Human recombinant protein of polo-like kinase 1 (Plk1), the homolog of Drosophila polo, also promotes REC8 cleavage by separase in vitro.27 Further studies are needed to clarify the exact protein kinases that are in charge for REC8 phosphorylation in vivo during meiosis in mammalian oocytes. It is now widely appreciated that Plk1 is the ‘master regulator’ of somatic cell mitosis and involved in multiple events, including entry into mitosis, centrosome maturation, spindle assembly, the activation of spindle assembly checkpoint (SAC), and the timely destruction of cohesion between sister chromatids, as well as the proper completion of cytokinesis.35 Plk1 activity is also required in regulating the gamete meiotic progression. Data from different studies confirm alterations in Plk1 activity definitely cause severe spindle defects and chromosome missegregation during mouse
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oocytes meiotic maturation.36-37 Though Plk1 is required unambiguously for cohesin degradation in sister chromatid splitting during somatic mitosis, whether it plays similar role during germ cell meiotic division is still not clearly revealed. It was recently reported that Plk1 promotes the phosphorylation and disassembly of synaptonemal complex, including cohesin subunit REC8, in mouse spermatocytes.38 Clear evidences are still absent about the involvement of Plk1 activity in cohesin degradation in mammalian oocytes. The functional diversity of Plk1 in mitotic cells is associated with its consecutive posttranslational modification. Plk1 phosphorylation at Ser137 and Thr210 in vivo occurs with different timing, and regulates separate Plk1 actions.39-41 Thr210 phosphorylation is required for Plk1 activation39-40 and entry into mitosis,40-41 while phosphorylation of Ser137 takes place only in late mitosis, not required for initial activation of Plk1.39 An alteration in Ser137 phosphoryaltion can induce spindle assembly checkpoint failure and untimely premature onset of anaphase.41 However, it is still completely unknown about the pattern of Plk1 phosphorylation, as well as the subcellular distribution and potential function of phosphorylated Plk1 during meiotic division in oocytes. In the present study we assessed the protein expression and subcellular localization of phosphorylated Plk1 in mouse oocytes during meiotic division, and examined its function by using an ATP-competitive inhibitor, BI2536. The results indicate that Plk1 phosphorylation at Ser137 (pPlk1Ser137) and Thr210 (pPlk1Thr210) occurs in oocytes, with distinct expression and localization patterns. pPlk1Ser137 localization is sensitive to BI2536, and required for meiotic spindle assembly and REC8 cleavage during oocyte meiosis.
Results Asynchronous Plk1 phosphorylation at Ser137 and Thr210 in mouse oocytes during meiotic maturation In somatic cells, Plk1 activity is regulated by its putative phosphorylation on Ser137 and Thr210. We investigated whether this phosphorylation occurs in mouse oocytes during meiosis. Prior to exploring the features of Plk1 phosphorylation, the protein expression pattern of total Plk1 was validated in this study. A commercial anti-Plk1 antibody (ab17057, Abcam), which recognizes the peptide sequence from residue 330 to 370, was used to detect total Plk1, no matter Ser137 or Thr210 residues are phosphorylated or not. Consistent with the previous results,36 stable and consistent quantity of Plk1 was detected during oocyte meiotic progression from germinal vesicle (GV) to metaphase II (MII) stage (Fig. 1A), manifested as a single band at 68 kDa. The expression characteristics of phosphorylated Plk1 (pPlk1Ser137 and pPlk1Thr210) were directly determined using phospho-specific antibodies. As showed in Figure 1A, pPlk1Ser137 protein was detected as a single band at about 72 kDa in mouse oocytes, which was highly expressed at GV stage and sustained stable up to MII stage. pPlk1Thr210 protein was labeled as one single band more than 72 kDa at GV
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including Ser137 and Thr210, at GV stage, and partial dephosphorylation takes place after the onset of meiotic resumption, suggesting that such modifications may function at late stages in meiotic progression. Dynamic sub-cellular localization of Plk1 during oocyte meiosis Though Plk1 protein was stable and consistent in quantity, its subcellular distribution was highly dynamic along oocyte meiotic progression from GV to MII stage. At GV stage, Plk1 was labeled as several round dots of different size in nucleus (Fig. S1A, a-d). These dots were gradually dismantled and reassembled as highly concentrated foci on centromeres as chromatin condensing into individual chromosomes after GVBD (Fig. S1A, e-h). This centromeric localization was consistently observed from pro-metaphase I (pro-MI) to MI, also at MII stage (Fig. S1A, i-x), and further displayed in greater detail by immunofluorecsence analysis on chromosome spread samples (Fig. S2). In addition, Plk1 was also found localized on spindle poles at MI and MII (Fig. S1B), and closely overlapped with MTOC components, pericentrin and g-tubulin (Data not shown). Specially, Plk1 was no longer observed on polar area but distributed over the midbody structure during anaphase I (AI) / telephase I (TI) transition (Fig. S1A, q-t). These morphological data indicates Plk1 is associated with the meiotic spindle formation, spindle assembly checkpoint function and cytokenesis completion during meiotic progression in mouse oocytes.
Figure 1. Plk1 phosphorylation on Ser137 and Thr210 occurs differently in mouse oocytes during meiosis. (A) Western blot analysis was used to detect pPlk1Ser137, pPlk1Thr210 and Plk1 protein level. For each sample, 100 oocytes were collected after 0, 2, 4, 8 and 17 h culture, corresponding to GV, GVBD, MI and MII stages, respectively. GAPDH was used as a protein loading control. Each protein was assayed at least 3 times. Plk1 was detected as one 68 kDa band in oocytes and expressed consistently stable among all stages during meiotic maturation. pPlk1Ser137 was detected as a single bind at about 72 kDa and expressed in high and stable level from GV to MII stage. pPlk1Thr210 was labeled as a single band, which was larger than 72 kDa, at GV stage, and assayed as 2 bands after GVBD, with the lower band at about 72 kDa and the upper one same as that at GV stage, both bands were sustained in stable level up to MII stage. (B) Lambda protein phosphatase (l-PP) was used to determine the specificity of pPlk1T210 antibody. For each sample, 100 oocytes were collected and incubated in the absence (-l-PP) or presence of l-PP (Cl-PP) for 30 min at 30 C. The resulting sample were used for western blot analysis with pPlk1Thr210 antibody. l-PP treatment greatly reduced the protein signals of these 2 bands.
stage, and as 2 smaller bands after GVBD, which maintained in high and stable levels until MII stage. After being treated with lambda protein phosphatase (l-PP), both the large band at GV and 2 small bands after GVBD were cleared away (Fig. 1B), suggesting they represent phosphorylated protein signals. The western blot results confirmed that Plk1 phosphorylation on Ser137 and Thr210 happens in oocyte meiosis in vivo. Overphosphorylation of Plk1 may occur on multi residue sites,
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Co-localization of pPlk1Ser137 with MTOC proteins on spindle poles Since it was definitely ascertained by western blot results that Plk1 Ser137 and Thr210 phosphorylation happens in mouse oocyte meiosis, we then characterized the sub-cellular distribution patterns of pPlk1Ser137 and pPlk1Thr210 throughout meiotic division. In order to determine the intracellular localization of pPlk1Ser137, oocytes at every typical stage of meiotic process were collected and processed for double immunofluroescent staining with pPlk1Ser137 and acetylated-tubulin. As showed in Figure 2A, in oocytes arrested at GV stage, pPlk1Ser137 was accumulated as multiple bright aggregates (foci) in germinal vesicle (Fig. 2A, a-d), and then gathered as discrete foci close to condensing chromosomes upon meiotic resumption (Fig. 2A, e-h). During pro-MI when microtubules were assembled and organized into meiotic spindle around condensed chromosomes, pPlk1Ser137 foci mass were assorted into 2 groups, migrated in the opposite directions toward the 2 poles of the forming spindle (Fig. 2A, i-l), and finally localized at the polar area of barrel-like spindle at MI stage, in an “O” or “C”-shaped configuration (Fig. 2B, c,g) with positive signals radiating outward (Fig. 2A, m-p). pPlk1Ser137 was translocated to the midzone of the spindle during AI / TI (Fig. 2A, q-t), and again focused on poles of newly formed spindle as oocytes developed to MII stage. In addition, multiple discrete foci of pPlk1Ser137 were found in the cytoplasm as oocytes underwent meiotic maturation (Fig. 2A, u-x). Signal of pPlk1Ser137 was also observed across chromosomes, especially obvious at MI and MII stages (Fig. 3A).
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pPlk1Ser137 localization on spindle poles at MI and MII stages implied its probable association with MTOCs. We then performed immunofluorescent colocalization analysis of pPlk1Ser137 with crucial components of MTOCs, pericentrin and g-tubulin. As we expected, pPlk1Ser137 was specifically co-localized with g-tubulin and pericentrin on the meiotic spindle poles and at cytoplasmic MTOCs at MI (Fig. 2B), as well as other typical stages of meiosis I (data not show). Specially, filamentous aggregates of pPlk1Ser137 was radiated outward from the poles and overlapped with g-tubulin filaments. The distinct association of pPlk1Ser137 with MTOCs suggests pPlk1Ser137 may be a component of meiotic acentriolar MTOCs, thus involve in meiotic spindle assembly in oocyte meiosis.
Figure 2. pPlk1Ser137 subcellular distribution in mouse oocytes during meiotic division. (A) Dynamic localization of pPlk1Ser137 is associated with spindle formation during meiosis. pPlk1Ser137 was indicated in red, acetylated-tubulin in green and DNA in blue. Scale bar D 10 mm. pPlk1Ser137 was assembled as several large dots within germinal vesicle at GV stage, and reassembled as many small foci around condensing chromatin after GVBD, these foci were gradually fused and clustered into 2 groups as cell cycle progressed to pro-MI, and concentrated on 2 opposite poles of barrel-shaped spindle at MI stage. pPlk1Ser137 was transferred to the midbody during AI-TI transition and moved back to polar areas of newly formed spindle at MII stage. Cytoplasmic pPlk1Ser137 was indicated by asterisk and pPlk1Ser137 at spindle polar area was indicated by arrow and was shown in insets with 2£ magnification. (B) pPlk1Ser137 was co-localized with g-tubulin and pericentrin at MTOCs. Oocytes at MI stage were collected after an 8 h culture for double immunostaining of pPlk1Ser137 (red) either with g-tubulin (green) (a-d) or pericentrin (green) (e-h), both of which are key components of MTOCs. DNA was visualized with DAPI in blue. Scale bar D 10 mm. At MI stage, pPlk1Ser137 concentrated in “O” or “C” shape on spindle polar area and distributed in cytoplasm and colocalized with pericentin and g-tubulin both in cytoplasm (asterisk) and spindle poles (arrow).
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pPlk1Ser137 persisted on centromeres and dynamicly located between chromosome arms during oocyte meiotic progression In order to reveal the detailed characteristics of pPlk1Ser137 localization on chromosomes, we prepared chromosome spreads from oocytes developed to pro-MI, MI and MII stage, respectively, and immune-labeled with pPlk1Ser137 antibodies and auto anti-centromere serum CREST. As showed in Figure 3A, pPlk1Ser137 was persistently concentrated on centromeres from pro-MI to MI and at MII. The relative fluorescent intensity of centromeric pPlk1Ser137 was roughly equal among these stages (Fig. 3C, pro-MI vs MI, 61.82 § 9.872 vs. 85.94 § 6.577, P > 0.05). At the same time, pPlk1Ser137 was also dynamically clustered between chromosome arms, specifically from pro-MI and MI, pPlk1Ser137 was labeled in the full space between the arms of homologous chromosomes or sister chromatids (Fig. 3A, a-h), but only confined to the local space between centromeres of sister chromatids at MII stage (Fig. 3A, i-l). Further quantitative analysis showed that the average fluorescence intensity of pPlk1Ser137 between chromosome arms was significantly higher in MI oocytes (52.99 § 2.363; P < 0.05) than that in pro-MI cells (15.16 § 4.015) (Fig. 3B). Given space between chromosomes arms is where the cohesin complex accumulated, the increasing assembly of pPlk1Ser137 between chromosome arms from pro-MI to MI highly suggested its possible involvement in the resolution of cohesion along chromosome arms. Ubiquitous distribution of pPlk1Thr210 on chromosomes during mouse oocyte meiotic division The intracellular localization of pPlk1Thr210 was examined by double immunostaining with anti-pPlk1Thr210 and CREST antiserum. There was no obvious pPlk1Thr210 assembly observed either in cytoplasm or in nucleus at GV stage (Fig. 4A, a–d). Upon GVBD, pPlk1Thr210 was constantly presented on chromosomes, in sharp contrast to the diverse subcellular localization of pPlk1Ser137 (Fig. 4A, e-h). From pro-MI to MI and at MII, high concentration of pPlk1Thr210 was labeled on whole chromosome scales, even on chromosomes of the first polar body (Fig. 4A, i-t). This distribution was more finely verified with immunostaining on chromosome spreads (Fig. 4B), which clearly illustrating the ubiquitous pPlk1Thr210 just like a “coat” covering the whole structure of chromosomes. During AI/TI transition, strong signal of pPlk1Thr210 was still observed on
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separating chromosomes, also the area of midbody (Fig. 4B, q-t). All these morphological evidences indicate that pPlk1Thr210 may be related to the establishment and maintenance of chromosome configuration during oocyte meiosis. Metaphase I arrest induced by Plk1 inhibition with BI2536 The unique distributions of phosphorylated Plk1 imply the relevance of Plk1 activity in some essential cellular events controlling chromosome division during meiotic progression. We then explored the roles of Plk1 activity by using the Plk1 inhibitor BI2536. Firstly, we took an overview of the effects of BI2536 on oocyte maturation rate. GV oocytes were matured in culture medium supplemented with either 100 nM BI2536 or same volume of DMSO, and the number of oocytes developed to MI as well as MII stage was assessed by spindle morphology and chromosome alignment at 8, 10, 12, 14 and 17 h of culture. As showed in Figure 5, when checked at 8 and 10 h of culture, the majority of oocytes were matured to MI stage, there was no significant difference in the number of MI oocytes between BI2536 treatment group and control, indicating BI2536 exacted no obvious effects Figure 3. Unique distribution pattern of pPlk1Ser137 on chromosomes. (A) pPlk1Ser137 was accumulated peron the restart of meiosis and the sistently on centeromeres and dynamically between chromosome arms. Chromosome spreads from promeiotic progression to MI. With MI, MI, and MII oocytes were processed for immunofluorescence staining with pPlk1Ser137 antibodies and the increase in incubation time, the CREST serum. Chromosome was labeled in blue, pPlk1Ser137 in red and CREST in green. Scale bar D 10 mm. portion of MI oocytes continupPlk1Ser137 was accumulated between chromosome arms from pro-MI (a-d) to MI (e-h), and only focused at local space between sister centromeres at MII stage (i-l). pPlk1Ser137 was persistently concentrated on cenously decreased while that of MII tromeres from pro-MI to MII (i-l, arrow). (B) Quantitative analysis demonstrated that higher level of oocytes, characterized by the extrupPlk1Ser137 between chromosome arms in MI oocytes than that in pro-MI cells (P
