cenp-a regulates chromosome segregation during the first meiosis of mouse...

J Huazhong Univ Sci Technol［Med Sci］ 37(3):2017 313

CENP-A Regulates Chromosome Segregation during the First Meiosis of Mouse Oocytes* Li LI (李莉)1, Shu-tao QI (戚树涛)2, Qing-yuan SUN (孙青原)2, Shi-ling CHEN (陈士岭)1# 1Center for Reproductive Medicine, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China 2State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2017

Summary: Proper chromosome separation in both mitosis and meiosis depends on the correct connec-tion between kinetochores of chromosomes and spindle microtubules. Kinetochore dysfunction can lead to unequal distribution of chromosomes during cell division and result in aneuploidy, thus kinetochores are critical for faithful segregation of chromosomes. Centromere protein A (CENP-A) is an important component of the inner kinetochore plate. Multiple studies in mitosis have found that deficiencies in CENP-A could result in structural and functional changes of kinetochores, leading to abnormal chro-mosome segregation, aneuploidy and apoptosis in cells. Here we report the expression and function of CENP-A during mouse oocyte meiosis. Our study found that microinjection of CENP-A blocking anti-body resulted in errors of homologous chromosome segregation and caused aneuploidy in eggs. Thus, our findings provide evidence that CENP-A is critical for the faithful chromosome segregation during mammalian oocyte meiosis. Key words: oocytes; meiosis, CENP-A; chromosome segregation

Before metaphase of mitosis and meiosis, chromo-somes are pulled by spindle microtubules to be aligned in the equator of the cell, which is essential for proper chromosome segregation[1, 2]. Centromeres are structural components on chromosomes that attach to spindle microtubules. They are comprised of multiple regions, including a region that maintains cohesion of the two sister chromatids, a central area with special centromere DNA, and the kinetochores[3]. The kinetochore is a DNA/protein complex that provides the site where spin-dle microtubules and chromosomes connect, thus allow-ing chromosomes to move toward the two opposite spin-dle poles during cell division progression[4]. During mi-tosis or meiosis, kinetochore dysfunctions can lead to unequal chromatid distribution, resulting in aneuploidy and cell division abnormalities[5, 6].

In most organisms, the centromere consists of a large number of repeating satellite DNA sequences (α-satellite sequence in human) and these DNA se-quences directly recruit kinetochore proteins, which are highly conserved in humans and various model organ-isms[4]. Satellite DNAs in mammalian centromeres form special chromatin-containing specific histones, in which the canonical histone H3 is replaced by a histone H3 variant, centromere protein A (CENP-A, CID in fruit flies, Cse4 in buding yeast, Cnp1 in fission yeast), which is involved in recruiting other proteins to form a func-tional kinetochore[7–9]. Defects of CENP-A may lead to abnormal kinetochore protein recruitment and dislocation of other kinetochore proteins[10, 11]. Loss-of-function analyses of CENP-A were per-

Li LI, E-mail: [email protected] #Corresponding author, E-mail: [email protected] *This study was supported by the National Natural Science Foundation of China (No. 30930065 and No. 31271605).

formed in different ways, including microinjection of anti-CENP-A antibody into the nucleus of HeLa cells[12], RNA interference (RNAi) deficiency of CENP-A in HeLa cells[13] , and creation of Cenpa null mice[14] . The results of these analyses led us to conclude that CENP-A is absolutely essential for viability in all organisms. CENP-A deletion or mutation can cause abnormal mito-sis and early studies have found that CENP-A may also play a role in meiosis[15]. In Drosophila, CENP-A is found to accumulate in prophase of meiosisⅠof oocytes and spermatocytes[16, 17], and CENP-A deficiency can increase the error rate of chromosome segregation during both meiosisⅠand Ⅱ of spermatocytes[17]. At present, the function of CENP-A in mammalian meiosis has not been investigated. Our purpose was to study the role of CENP-A in mouse oocyte meiosis. We found that microinjection of CENP-A antibody led to homologous chromosome segregation errors and ane-uploidy in mouse oocytes. Thus, CENP-A is critical for correct alignment of chromosomes and homologous chromosome separation during the first meiosis of mouse oocytes. 1 MATERIALS AND METHODS 1.1 Oocyte Collection and In Vitro Culture

The fully germinal vesicle (GV) stage oocytes were isolated from 6–8 week-old female ICR mice. Animal care and handling were conducted in accordance with policies regarding the care and use of animals issued by the ethical committee of the Institute of Zoology, Chi-nese Academy of Sciences. For microinjection, oocytes were placed in M2 culture medium (Sigma, USA) con-taining 200 μmol/L IBMX (Sigma, USA) to arrest oo-cytes at the germinal vesicle (GV) stage. For in vitro culture, oocytes were placed in M16 culture medium

37(3):313-318,2017 J Huazhong Univ Sci Technol［Med Sci］ DOI 10.1007/s11596-017-1733-9

ORIGINAL ARTICLE

314 J Huazhong Univ Sci Technol［Med Sci］ 37(3):2017

(Sigma, USA) covered with paraffin oil droplets in a 37°C, 5% CO2 incubator. 1.2 Microinjection of siRNA and Antibody Narishige microforge was used for the microinjec-tion procedure that was completed within 30 min at room temperature. For knockdown experiments, Cenpa siRNA[18] (GenePharma, China) was microinjected into the cytoplasm of GV-stage oocytes at 30 µmol/L concen-tration. The injected oocytes were transferred into M16 culture medium containing 200 μmol/L IBMX to arrest oocytes at the GV stage for 24 h for depletion of the tar-get protein. The same amount of negative control siRNA was microinjected as control. The sequences of the siRNAs used in this study were shown as follows, Cenpa siRNA1: 5'-UCUCAGACACUGCGCAGAATT-3'; Cenpa siRNA2: 5'-CUACCUCCUCUCCUUACAUTT-3'. CENP-A antibody was microinjected into the cyto-plasm of oocytes. After microinjection, cells were cul-tured in M16 medium with IBMX for 3 h, in order to allow complete interaction of CENP-A antibody and CENP-A. Mouse oocytes of the control group were in-jected with nuclease-free water. 1.3 Immunofluorescence and Chromosomal Spread Antibody against CENP-A was purchased from Abcam Inc. (Cambridge, USA). Antibody against α-tubulin was purchased from Cell Signaling Technology (Beverly, USA). Immunofluorescent analysis and chro-mosomal spreads were performed using standard proto-cols[18]. For immunofluorescent analysis, oocytes were fixed in 4% paraformaldehyde in PBS with 0.5% Triton X-100 for 30 min and blocked in 1% BSA for 1 h at room temperature. Then oocytes were stained with anti-α-tubulin antibody (1:100) at room temperature for 2 h. After washing four times in PBS with 0.05% Tween 20, oocytes were incubated with propidium iodide (PI) in PBS containing 0.05% Tween 20 for 10 min. Finally, oocytes were mounted on glass slides and examined with a confocal laser scanning microscope (Zeiss LSM 780, Germany). For chromosomal spread, oocytes were incubated in acid Tyrode’s solution (Sigma, USA) to remove the zona pellucida. After washing three times in M2 medium, oo-cytes were transferred onto a glass slide and fixed with 1% paraformaldehyde, 0.15% Triton X-100 and 3 mmolL dithiothreitol in distilled water at room tempera-ture. After drying in air, the slides were blocked in PBS containing 1% BSA at room temperature for 1 h, then incubated with primary antibodies (1:100) overnight at 4°C, and then washed three times and incubated with secondary antibody at room temperature for 2 h. Finally, chromosomes were stained with PI and the slides were viewed with immunofluorescence microscopy. 1.4 Western Blot Analysis Mouse oocytes at different developmental stages of meiosis were collected for Western blot analysis[19]. Two hundred oocytes at each stage were collected and trans-ferred to SDS sample buffer, heated for 5 min at 100°C. The samples were loaded onto SDS-PAGE to separate proteins, and then electrically transferred to PVDF membranes. After washing with TBST, the PVDF mem-branes were blocked in TBST containing 5% BSA for 1 h at room temperature, and then incubated with anti-CENP-A antibody (1: 500) or anti-β-actin antibody (1:1000, Zhongshan Golden Bridge Biotechnology Co.

LTD , USA) at 4°C overnight. After TBST washes for three times, the membranes were further incubated with secondary antibody for 2 h at room temperature. Finally, the membranes were processed using the enhanced chemiluminescence detection system (Bio-Rad, USA). 1.5 Real-time PCR RNeasy micro purification kit (Qiagen, Germany) was used to extract total RNAs from 50 oocytes and M-MLV first strand cDNA synthesis kit (Invitrogen, USA) was used to synthesize the first-strand cDNA[18]. CENP-A fragment was amplified with the following primers, forward: 5'-CTCCAGTGTAGGCTCTCAGA-C-3'; reverse: 5'-CTGAAAGGCTTCTTCCTGAACA-3'; GAPDH gene was used as an internal control gene. SYBR Premix Ex TagTM kit (Takara, Japan) and ABI prism 7500 quantitative PCR instrument were used for the tests. The results were analyzed by the 2–ΔΔCt method. 1.6 Statistical Analysis Each experiment was repeated at least three times and the experimental data obtained were expressed as

±s . The total number of cells is shown as n. The ex-perimental results were analyzed using SPSS 16.0 statis-tical software. The independent sample t test was used for statistical analysis. P<0.05 was considered statisti-cally significant. 2 RESULTS 2.1 Expression and Centromeric Localization of CENP-A during Mouse Oocyte Meiosis To detect the expression level of CENP-A during mouse oocyte maturation, we collected mouse oocytes at 0, 8 and 12 h of in vitro culture (200 oocytes at each time-point), which corresponded to GV, meiosisⅠ andⅡ stage, respectively. These oocytes were analyzed by West-ern blotting. As shown in fig. 1, CENP-A was expressed in all the three meiotic stages of mouse oocytes: CENP-A expression was lowest in the GV stage oocytes, and reached the highest level at the meiosisⅡstage (fig.1A).

To investigate the localization of CENP-A on chro-mosomes, we performed immunofluorescent analysis on spread chromosomes, and found that CENP-A was lo-cated on chromosome centromeres at meiosisⅠand Ⅱ stages (fig. 1B). 2.2 CENP-A RNAi Fails to Efficiently Reduce the Expression of CENP-A RNA interference was used to study CENP-A func-tion in oocyte meiosis. The two small interfering RNAs (siRNAs) were microinjected into mouse oocytes at the GV stage and oocytes were arrested at this stage for 24 h by 200 µmol/L IBMX. Real-time PCR results showed that the Cenpa mRNA level was significantly reduced after siRNA microinjection (7.6%±1.7% for siRNA1 and 42.0%±1.5% for siRNA2, P<0.05, fig. 2A). Accordingly, we used Cenpa siRNA1 to perform further experiments. To detect the CENP-A protein expression level after RNAi treatment, we harvested 200 GV-stage oocytes after 24-h IBMX inhibition in the experimental group (Cenpa siRNA1 microinjection) and the control group (control siRNA microinjection). The results of Western blot analysis showed that the CENP-A protein level was only reduced to 51.3%±9.6% of the control group (P<0.05, fig. 2B and 2C), which is due to the long half-life of this protein.


Fig. 1 Expression of CENP-A A: expression of CENP-A in mouse oocyte meiotic maturation. Each sample included 200 mouse oocytes which were cultured in vitro for 0, 8 or 12 h, corresponding to GV, meiosis Ⅰ(MⅠ) and Ⅱ(MⅡ) stages, respectively. B: Immunofluorescent staining shows the localization of CENP-A (white) on the centromere of each chromosome (red). Scale bars: 10 μm

Fig. 2 CENP-A RNAi can not efficiently knock down CENP-A. A: the Cenpa mRNA expression after Cenpa siRNA1 and siRNA2 microinjection. Data are presented as ±s , *P<0.05 vs. control; #P<0.05 vs. siRNA1. B: Western blot analysis of CENP-A. Cenpa siRNA1 was microinjected into the oocytes of the RNAi group, and control siRNA into the oocytes of the control group. C: CENP-A expression was reduced by half after CENP-A RNAi. Data are presented as ±s . *P<0.05 vs. control

2.3 CENP-A RNAi Does Not Affect Oocyte Meiotic Progression and Chromosome Separation in Meiosis Ⅰ

We then tried to investigate whether partial knock-down of CENP-A could affect the mouse oocyte meiotic process and chromosome arrangement. The oocytes in the experimental group and the control group were ar-rested at the GV stage by 200 µmol/L IBMX for 24 h, then washed thoroughly and cultured in fresh M16 me-dium to observe the progress of meiosis. After culturing in vitro for 14 h, we found that the germinal vesicle breakdown (GVBD) rates were 92.0%±2.9% (n=191) and 87.3%±5.8% (n=159), respectively, in the CENP-A RNAi group and the control group. There was no statis-tically significant difference between the two groups (P>0.05, fig. 3A), indicating that CENP-A RNAi did not affect the resumption of mouse oocyte meiosis. Statisti-cal analysis of the first polar body extrusion (PBE) rates in the RNAi group and the control group also did not show a significant difference (78.3%±9.9% in CENP-A RNAi group, n=175; 81.6%±4.8% in control group, n=136; P>0.05, fig. 3B).

We then investigated the chromosome alignment in meiosisⅡ stage oocytes after RNAi; the proportions of oocytes with disorganized chromosomes in the CENP-A RNAi group and the control group were 16.8%±2.4% (n=114) and 14.9%±1.2% (n=129), respectively, and no significant difference was observed between the two groups (P>0.05, fig. 3C and 3D). Thus partial knock-down of CENP-A did not significantly affect mouse oo-cyte meiosis and metaphase chromosome alignment.

Chromosome segregation after CENP-A RNAi for meio-sis Ⅰ was also investigated. We collected meiosis Ⅱ oocytes after CENP-A RNAi and chromosome spread was performed to display the pattern and number of chromosomes. As known for meiosisⅡ-stage mouse oocytes, the number of univalents is 20. However, oo-cytes in both CENP-A RNAi and control group showed abnormal chromosome number (11.8%±1.4% in CENP-A RNAi group, n=85; 10.3%±2.7% in control group, n=79; P>0.05, fig. 3E and 3F), indicating that CENP-A RNAi had little effect on homologous chromo-somes separation at meiosis Ⅰ. 2.4 CENP-A Blocking Causes Chromosome Mis-alignment and Oocyte Aneuploidy

Because CENP-A RNAi could only decrease about half of the protein level of CENP-A, this unexpected phenotype was probably due to the low efficiency of CENP-A knockdown. We then employed CENP-A anti-body microinjection to study the function of CENP-A in meiosis. Oocytes in the experimental group were micro-injected with CENP-A antibody, cultured in M16 me-dium containing 200 µmol/L IBMX for 3 h for antibody binding to CENP-A, and then released into fresh M16 medium for further culture. We found that the CENP-A antibody microinjection did not significantly affect the GVBD rate (CENP-A antibody group: 88.9%±4.8%, n=159; control group: 91.8%±6.7%, n=165; P>0.05, fig. 4A) and PBE rate (CENP-A antibody group: 81.6%±4.9%, n=142; control group: 78.3%±8.9%, n=152; P>0.05, fig. 4B). However, CENP-A antibody microin-


jection caused increased incidence of chromosome mis-alignment (antibody injection group: 77.1%±4.5%, n=84; control group: 12.4%±1.1%, n=82; P<0.05, fig. 4C and 4D). To know whether these chromosomal misalign-ments can lead to aneuploidy in oocytes at the meiosisⅡ stage, we employed chromosome spreading to analyze the chromosome number in meiosisⅡ oocytes. In the control group, the chromosome number in most oocytes

was 20 pairs, while in the CENP-A antibody group, the chromosome number was more or less than 20 pairs, causing increased incidences of aneuploidy (antibody group: 79.2%±4.2%, n=77; control group: 8.9%±1.5%, n=78; P<0.05, fig. 4E and 4F). Therefore, CENP-A an-tibody injection resulted in chromosomal disorder and aneuploidy in meiosisⅡ oocytes.

Fig. 3 CENP-A RNAi had little effect on mouse oocyte meiosis and chromosome arrangement. A: rates of GVBD in Cenpa and control siRNA injected group. Oocytes after microinjection were arrested in IBMX for 24 h, and then released and in vitro cultured. Data are presented as ±s . B: Percentages of PBE after siRNA microinjection. Data are presented as ±s . C, D: There is no significant difference in the percentage of misarranged chromosomes between the two groups. CENP-A RNAi did not affect the arrangement of chromosomes. Green indicates spindle and red DNA. Scale bars: 10 μm. Data are presented as ±s . E, F: No significant difference is shown in the percentages of aneuploidy between the two groups. Choromosme separation in meiosis Ⅰ was not affected by CENP-A RNAi. DNA was stained with PI (red). Scale bars: 10 μm

3 DISCUSSION In mitotic cells, incorrect CENP-A assembly causes improper cell division, aneuploidy and cell death[12–14]. But the role of CENP-A in mammalian meiosis has not been well explored. In this study, we investigated the expression and subcellular localization of CENP-A dur-ing mouse oocyte meiosis, and its role in meiosis. CENP-A blocking demonstrated that CENP-A influences the faithful chromosome separation in meiosis Ⅰ. Chromosome spread showed CENP-A was faith-fully localized to the kinetochores in meiosis Ⅰ and Ⅱ oocytes. The expression of CENP-A was detected at GV phase, the prophase of meiosis I, indicating CENP-A assembly occurs prior to chromosome segregation. In addition, we observed a significant increase in CENP-A expression at the metaphase of meiosis Ⅰ. These results are consistent with previous observations in human cells[20–22] and the timing and dynamics of CID assembly in male and female meiosis in Drosophila

melanogaster[17]. Due to the segregation of sister chro-matids, CID intensity per oocyte nucleus drops at meta-phase of male meiosis Ⅱ[17]. However, we detected a higher expression level of CENP-A in meiosisⅡ than meiosisⅠ, as expected due to the residual first polar bodies and the stability of CENP-A.

At first, we microinjected Cenpa siRNAs into mouse oocytes and expected to knockdown the expres-sion of CENP-A. Most of Cenpa mRNAs were degraded in mouse oocytes after 24-h CENP-A RNAi, implying that there might be no new protein synthesis of CENP-A. However, the total CENP-A expression was only de-creased partially (about a half). Like other core histones, CENP-A is quite stable and its half-time is significantly greater than the cell time[20]. Previous studies suggested that adequate degradation of CENP-A after RNAi re-quired longer incubation. In HeLa cells, CENP-A is knocked down after 68-h RNAi, while at 24 and 48 h of RNAi, CENP-A is still found on the centromeres[13]. In mouse embryonic fibroblasts, adequate knockdown re-


quires 72-h RNAi incubation to suppress CENP-A[23]. However, mouse oocytes could only tolerate RNAi treatment for 24 h; otherwise the oocytes would undergo

aging and loose their meiotic maturation capability. Thus the 24-h CENP-A inhibition by RNAi in this study could only partially knock down CENP-A protein.

Fig. 4 CENP-A antibody injection had no effect on meiotic progression, but affected separation of homologous chromosomes. A: rates of GVBD after microinjection. Oocytes of the CENP-A antibody group were injected with CNEP-A antibody, and oo-cytes of the control group were injected with the same amount of water. All the oocytes were incubated in M16 with IBMX for 3 h, and then cultured in M16 in vitro. Data are presented as ±s . B: percentages of PBE after microinjection. *P<0.05 vs. control. Data are presented as ±s . C, D: CENP-A antibody injection caused increasing chromosome disorders in meiosisⅡ stage mouse oocytes. Green indicates spindle and red DNA. Scale bars: 10 μm. Data are presented as ±s . E, F: Chromosome spreads demonstrated aneuploidy at the meiosisⅡ stage in mouse oocytes of the antibody group. CENP-A antibody injection significantly increased the occurrence of aneuploidy. DNA was stained with PI (red). Scale bars: 10 μm.

Meanwhile, CENP-A replaces histone H3 to form a CENP-A nucleosome (which includes CENP-A, histone H2A, H2B, and H4), depositing centromeres as tetramer or octamer with dynamic and changing configurations during the cell cycle[24, 25]. Unbound CENP-A nu-cleosomes are in a very unstable state and are therefore degraded by the E3 ligase-ubiquitin-proteasome pathway, while bound CENP-A nucleosomes on the centromeres interact with specific proteins (such as Psh1, Ppa) that compete with E3 ligase for binding sites, thereby pro-tecting bound CENP-A nucleosomes from E3 li-gase-mediated degradation[26–28]. Considering the higher stability of the bound CENP-A nucleosomes on the cen-tromeres, we speculated that 24-h RNAi inhibition only degraded the unbound CENP-A, while the bound CENP-A on the centromeres was not degraded. Conse-quently, kinetochore structure and function was not af-fected after CENP-A RNAi inhibition, accompanied by normal meiosis progression and chromosome arrange-ments. Considering the stable of endogenous CENP-A and the limit of incubated time in mouse oocytes, we could not successfully knock down or knock out the endoge-

nous CENP-A in mouse oocytes. Therefore, CENP-A blocking antibody was injected into mouse oocytes dur-ing the GV stage. The CENP-A antibody is a synthetic peptide corresponding to the region within C-terminal amino acids 100 of CENP-A. The C-terminal region of CENP-A includes several important functional mo-tifs[29–31] and is indispensible for centromere localiza-tion[8]. Previous studies have confirmed that the func-tional disruption of CENP-A protein affected the re-cruitment of other kinetochore proteins and changes in kinetochore configuration[12, 13]. Indeed, we found that the blocking of CENP-A function may affect the kineto-chore function, leading to chromosome alignment and segregation errors during meiosis Ⅰ[32]. However, the progression of meiosis was not affected by antibody microinjection. Apparently the assembly of specific CENP-A centromeric nucleosomes, which are sensitive to antibody inhibition, have been completed by prophase of meiosis Ⅰ[12, 17]. In summary, our study revealed the important role of CENP-A in meiosis of mouse oocytes. As an important component of the inner kinetochore plate, immunoblocking of CENP-A by antibody injection affected meiotic chro-


mosome arrangement and segregation. Acknowledgements

We thank Shi-wen LI and Hua QIN for their technical help with confocal laser microscopy. We also thank the other mem-bers in Dr. Sun’s laboratory for their kind discussions and help. Conflict of Interest Statement

The authors declare no conflict of interest in this work. REFERENCES 1 Akiyoshi B, Biggins S. Reconstituting the kineto-

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(Received May 22, 2016; revised Sep. 9, 2016)

cenp-a regulates chromosome segregation during the first meiosis of mouse...

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