CDCA8 regulates meiotic spindle assembly and chromosome segregation during human oocyte meiosis

Journal Pre-proofs Research paper CDCA8 regulates meiotic spindle assembly and chromosome segregation dur‐ ing human oocyte meiosis Changquan Zhang, Lei Zhao, Lizhi Leng, Qinwei Zhou, Shuoping Zhang, Fei Gong, Pingyuan Xie, Ge Lin PII: DOI: Reference:

S0378-1119(20)30164-5 https://doi.org/10.1016/j.gene.2020.144495 GENE 144495

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Gene Gene

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Please cite this article as: C. Zhang, L. Zhao, L. Leng, Q. Zhou, S. Zhang, F. Gong, P. Xie, G. Lin, CDCA8 regulates meiotic spindle assembly and chromosome segregation during human oocyte meiosis, Gene Gene (2020), doi: https://doi.org/10.1016/j.gene.2020.144495

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CDCA8 regulates meiotic spindle assembly and chromosome segregation during human oocyte meiosis Changquan Zhang1,2,#, Lei Zhao3,#, Lizhi Leng1,2,3, Qinwei Zhou3, Shuoping Zhang3, Fei Gong1,2,3,4, Pingyuan Xie4, Ge Lin1,2,3,4 1 Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha 410078, China 2 Key Laboratory of Reproductive and Stem Cells Engineering, Ministry of Health, Changsha 410078, China 3 Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha 410078, China 4 National Engineering and Research Center of Human Stem Cells, Changsha 410078, China # These authors contributed equally to the study. Corresponding author: Ge Lin, Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, 8 Luyun Road, Changsha, Hunan, China. E-mail: [email protected]

CDCA8 regulates meiotic spindle assembly and chromosome segregation during human oocyte meiosis Changquan Zhang1,2,#, Lei Zhao3,#, Lizhi Leng1,2,3, Qinwei Zhou3, Shuoping Zhang3, Fei Gong1,2,3,4, Pingyuan Xie4, Ge Lin1,2,3,4 1 Institute of Reproduction and Stem Cell Engineering, School of Basic Medical

Science, Central South University, Changsha 410078, China 2 Key Laboratory of Reproductive and Stem Cells Engineering, Ministry of Health, Changsha 410078, China 3 Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha 410078, China 4 National Engineering and Research Center of Human Stem Cells, Changsha 410078, China # These authors contributed equally to the study. Corresponding author: Ge Lin, Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, 8 Luyun Road, Changsha, Hunan, China. E-mail: [email protected]

Abstract

As a member of the chromosomal passenger complex, CDCA8 (cell division cycle associated 8) plays an important role in human mitosis, but its roles in human meiosis are unknown. Here, we show that CDCA8 expression is increased and its encoded protein has dynamic localization in human oocytes from germinal vesicle breakdown (GVBD) to metaphase Ⅱ (MⅡ), and that there are multipolar spindles, disordered chromosomes, and that microtubule assembly is affected after CDCA8 RNA interference (RNAi) in GV-stage oocytes. The GVBD and polar body extrusion (PBE) rates were not affected following CDCA8 depletion, but the PBE time was extended. There was no statistical difference between CDCA8 expression of oocytes from older and younger women, but the first polar body from older women was prone to chromosome

abnormalities, and oocytes with such abnormalities had lower CDCA8 expression than oocytes with normal polar bodies. These results indicate that CDCA8 is associated with bipolar spindle formation, chromosome segregation, PBE during human oocyte meiosis, and that it may affect the incidence of aneuploidy embryos in older women. Keywords: Chromosomal passenger complex; CDCA8; Meiosis; Human oocyte 1. Introduction During cell division, the chromosomal passenger complex (CPC) plays important roles in proper chromosome segregation and the execution of cytokinesis (van der Horst and Lens, 2014), and it is composed of the enzymatic core Aurora- B kinase (AURKB), the scaffold protein inner centromere protein (INCENP), Borealin (CDCA8, cell division cycle associated 8), and two other nonenzymatic subunits Survivin (also known as BIRC5). The CPC can localize correctly to the relevant destinations as different stages of mitosis and interact with its different substrates for various functions in the mitotic cell (Sampath et al., 2004; Vader et al., 2006). The CPC is involved in chromosome condensation, spindle assembly, chromosome alignment and the completion of cytokinesis (Vagnarelli and Earnshaw, 2004; Tanaka, 2005). The interactions within the CPC proteins support the stability of the individual CPC subunits. In mammalian cells, knockdown or depletion of each CPC subunit can result in very similar and severe outcomes, such as improper spindle formation and impaired mitotic checkpoint function, giving rise to chromosome congression and segregation defects (Honda et al., 2003; Klein et al., 2006; Santaguida et al., 2011). In mouse oocytes, AURKC replaces AURKB in the CPC as a critical regulator of chromosome segregation in meiosis (Yang et al., 2010; Balboula and Schindler, 2014). As a CPC component, Borealin is encoded by the human CDCA8 gene. Borealin was first

reported to stabilize the bipolar mitotic spindle in human mitosis in 2004 (Gassmann et al., 2004). Based on its structural features, Borealin is conserved across Animalia and Fungi (Jeyaprakash et al., 2007; Nakajima et al., 2009). It is regulated by phosphorylation at multiple sites, including phosphorylation by CDK1 (cyclin-dependent kinase 1) to promote targeting of the CPC to centromeres (Tsukahara et al., 2010), and

by MPS1 (monopolar spindle 1) on Thr230 to modulate

its dimerization and Aurora B activity (Bourhis et al., 2007). During the cell cycle in mitotic cells, Borealin has dynamic localization together with the CPC. In interphase, Borealin is visualized on the pericentromeric heterochromatin. Maximal concentration of the CPC occurs at the inner centromere by prometaphase, and during metaphase–anaphase transition, Borealin leaves the inner centromeres and transfers to the central spindle microtubules, thereafter localizing at the equatorial cortex.

Eventually during telophase and cytokinesis, Borealin localizes at the midbody (Carmena

et al., 2012). Depletion of Borealin delays mitotic progression and results in kinetochore–spindle misattachments and ectopic spindle poles formation (Gassmann et al., 2004), and also leads to defective cell proliferation, p53 accumulation and mouse early embryonic lethality by 5.5days post coitus (Yamanaka et al., 2008). In general, in human mitotic cells, Borealin corrects kinetochore– spindle misattachments, and stabilizes bipolar spindle, while the Borealin dimerization domain suppresses dynamic exchange at the centromere to allow optimal CPC function (Bekier et al., 2015). Different from mitosis, meiosis includes two cell divisions: meiosis Ⅰ and meiosis Ⅱ. In mammals, oocytes arrest at the diplotene stage of meiosis Ⅰ. Oocyte meiotic resumption is mainly triggered by follicle-stimulating hormone and luteinizing hormone (FSH). During mouse oocyte meiosis, Borealin accumulates near the chromosomes after germinal vesicle (GV) breakdown, and localizes at the spindle poles in metaphase Ⅰ and anaphase Ⅰ, at the midbody in telophase, and

relocalizes at the spindle poles during metaphase Ⅱ. Disruption of Borealin results in severe spindle assembly defects, but does not affect polar body extrusion (PBE) (Sun et al., 2010). In human oocytes, meiosis is more prone to chromosome–mediated spindle assembly errors than mitosis; in other organisms, this is true for female meiosis, which result in chromosome segregation defects (Danylevska et al., 2014; Holubcova et al., 2015). However, how CDCA8/Borealin affects cytokinesis during human cell meiosis, especially in meiosis Ⅰ, is not well known. CDCA8 not only plays an important role in mammalian mitotic cells, but is also an indispensable element in the meiosis of some model animal oocytes (Carmena et al., 2012; Date et al., 2012). However, due to the limitations of experimental materials, there is no direct evidence for the role of the CDCA8 gene or Borealin in human oocyte meiosis to date (Gorbsky, 2015). The first meiotic process in particular, which features homologous chromosome segregation rather than sister chromatid segregation similar to mitosis, has more different characteristics than mitosis. More importantly, most human embryo chromosomal abnormalities occur in oocyte meiosis I (Fragouli et al., 2013). Here, we investigated CDCA8 expression, and the location and functions of Borealin during human oocytes meiosis, and explored the relationship between CDCA8 expression and advanced maternal age women with aneuploid embryos. Together, our data show that CDCA8 regulates spindle assembly, bipolar spindle formation, and may affect the first polar body extrusion, and may be related to chromosome abnormality in the oocytes of advanced age women. 2. Materials and methods 2.1 Ethics statement and patient information This study was approved and guided by the Ethics Committee of the Reproductive & Genetic Hospital of CITIC-XIANGYA (LL-SC-2016-014). The enrolled patients were informed consent

signed by the donor couples. The informed consent confirmed that the couple donors were voluntarily donating immature oocytes for the research with no financial payment. We enrolled women undergoing intracytoplasmic sperm injection (ICSI) treatment at the Reproductive & Genetic Hospital of CITIC-XIANGYA, and divided them into two groups according to age (≥ 40 and ≤ 30 years). The women had tubal-factor infertility, and we excluded other factors, including premature ovarian failure, ovarian dysfunction, ovarian radiotherapy or chemotherapy, thyroid dysfunction or polycystic ovary syndrome. 2.2 Oocytes collection and in vitro maturation (IVM) Immature oocytes with a GV and normal morphological appearance were collected, and cultured at 37.5°C, in a humidified atmosphere of 6% CO2, 5% O2, and 89% N2. In total, 87 immature oocytes were collected and included in this study. The GV oocytes were placed in an IVM medium (Vitrolife, Göteborg, Sweden) supplemented with 0.075 IU/mL FSH, 0.5 IU/mL human chorionic gonadotropin, 1μg/mL estradiol, and 0.5% human serum albumin. After 12–18 h and 30 h culture for MⅠ and MⅡ analysis, respectively. MⅠ oocytes were identified by the lack of a GV and the absence of a PB; and mature MⅡ oocytes had a clear extruded PB and no GV. 2.3 Time-lapse recording Oocytes at the GV stage from patients in different age groups or that had undergone RNA interference (RNAi) were moved to wells of pre-equilibrated EmbryoSlide (Vitrolife) and cultured in IVM medium. After all the oocytes located had been placed correctly, the slides were placed in the embryoscope chamber immediately and cultured in a 6% CO2, 5% O2, and 89% N2 atmosphere at 37.5°C. Images of each oocyte were recorded every 10 min for at least 30 h. The time taken for the appearance of the first polar body (PB1) was noted.

2.4 Microinjection of small interfering RNA (siRNA) in GV oocytes To determine the possible role of CDCA8 in human oocyte meiosis, we performed microinjection of siRNA in the GV oocytes. Scrambled RNA (as a negative control or control siRNA) and human CDCA8 siRNA (sense: 5′-GGAAAUACGAAUCAAGCAAdTdT-3′, antisense: 3′-dTdTCCUUUAUGCUUAGUUCGUU-5′, Ribobio, Guangzhou, China) were dissolved in RNase-free water. A GV oocyte in G-MOPS PLUS (Vitrolife) covered with mineral oil was held with a holding pipette connected to a micromanipulator on an inverted microscope, and injected into the cytoplasm with approximately 10pL scrambled RNA solution (1 pg/pL) or CDCA8 siRNA solution(1 pg/pL) (Supplemental Fig. 1) with an injection needle (Femtotips Ⅱ, Eppendorf, Hamburg, Germany) connected to another micromanipulator (FemtoJet, Eppendorf). Non-injected oocytes were used as a blank control. Injected oocytes with normal shapes immediately after injection were cultured as described above, and were collected and analyzed after 30 h. A total of 43 oocytes were injected with siRNAs, of which 28 were CDCA8- siRNA. 2.5 Polar body biopsy and chromosome analysis After 30 h IVM, some oocytes reached meiosis Ⅱ with a visible PB. A total of 19 PBs were biopsied and underwent in chromosomes analysis, and comprised that from the younger group (CDCA8- siRNA group, n = 5; negative group, n = 4; blank group, n = 5) and older group (n = 5). Briefly, the PB was removed from perivitelline space of the oocyte with a PB biopsy needle after laser dissection opening of the zona pellucida, and then placed into a PCR tube with 4μL lysis buffer (YK-PGSTM Embryo Biopsy Sample Collection Kit, Yikon Genomics, Shanghai, China) by a glass micropipette with a 20μm- inner diameter. PB could be easily identified in the medium droplets using a high-contrast stereomicroscope and were aspirated for immediate transfer. The capillary was

rinsed after transfer to verify that the PB had been placed the reaction tube. Then, multiple annealing and looping-based amplification cycles (MALBAC) (MALBACTM single cell WGA Kit, Yikon Genomics) were performed to generate the micrograms of DNA required for next-generation sequencing. Using an Illumina HiSeq 2000 platform (illumina, San Diego, CA, USA), the amplified genome of each single PB was sequenced at approximately 0.04 × genome depth, with approximately 1-Mb resolution to detect the variation. 2.6 Single oocyte quantitative reverse transcriptase PCR (RT-qPCR) At 30 h after injection, CDCA8 mRNA expression levels were evaluated using RT-qPCR. We tested a total of 56 oocytes (CDCA8- siRNA group, n = 18; negative group, n = 15; blank group, n = 23). cDNA libraries were generated using the Smart-seq2 protocol as described previously (Leng et al., 2019). Briefly, the zona pellucida was removed using acidic Tyrode’s solution (Sigma, St. Louis, MO, USA) and then the oocytes were placed in PCR tubes. Following cell lysis, mRNA was released and Oligo-dT primer was added at 72℃ for 3 min. For first-strand cDNA synthesis, the RT reaction was carried out using SuperScript II reverse transcriptase (Invitrogen, Gaithersburg, MD, USA). Then, the cDNA was amplified by PCR (18 cycles) using KAPA HiFi Hot-Start ReadyMix (KAPA Biosystems, Boston, USA) and purified using Ampure XP beads (Beckman Coulter, Brea, CA, USA). The cDNA of single oocyte equivalent was used as templates for real-time PCR analysis in a Roche LightCycler 480 II System (Roche, Basal, Switzerland) using FastStart Universal SYBR Green Master (Roche). Samples were run in triplicate to ensure amplification integrity. The PCR conditions were as follows: 95℃ for 5 min, followed by 45 cycles of 95℃ for 15 s, 58℃ for 10 s and 72℃ for 10 s. The relative expression levels of each target gene compared with β-actin were calculated using the comparative threshold cycle (2-ΔΔCT) method. Specific PCR primer pairs are

summarized in Table 1. 2.7 Immunofluorescence analysis The zona pellucida was removed by incubation with acidic Tyrode’s solution for an instant, and then zona-free oocytes were washed three times with phosphate-buffered saline (PBS) and fixed in microtubule stabilizing buffer (0.1 M PIPES, pH 6.9, 2 mM MgCl2.6H2O, 2.5 mM EGTA

[ethyleneglycoltetraacetic acid], 2% paraformaldehyde, 0.5% Triton X-100,10 μM taxol) at 37℃ for 30 min. Then, the oocytes were blocked in 1× PBS containing 5% donkey serum, 1% BSA, 0.1 M glycine, and 0.01% Triton X-100 at room temperature for 1 h, followed by incubation with rabbit polyclonal anti-Borealin antibody (1:50, SAB1300184, Sigma) and mouse monoclonal anti–αtubulin antibody (1:200, T6199, Sigma) at 4℃ overnight. After rinsing three times in washing buffer (0.1% Tween 20 and 0.01% Triton X-100 in PBS), the oocytes were incubated with the appropriate secondary antibodies, i.e., Alexa 488 (1:1000, A-11001, Invitrogen) and Alexa 594 (1:1000, A11012, Invitrogen) for 1 h at room temperature in the dark. The nuclei were stained for 10 min with 4, 6-diamidino-2-phenylindole (DAPI, 1μg/mL, Invitrogen). Finally, fluorescent images were analyzed by Olympus FV1000 laser confocal fluorescence microscope (Olympus, Tokyo, Japan). Images in the same dataset were acquired at the same laser power intensity. Negative control with no primary antibodies were also established. 2.8 Statistical analysis Data were analyzed using the Statistical Package for Social Sciences (SPSS, version 18.0). Categorical variables were analyzed by chi-squared tests or Fisher’s exact tests; continuous variables were analyzed by Student’s t-tests if they followed a normal distribution or by MannWhitney-Wilcoxon tests if they did not. Data are reported as the mean ± SEM, and differences at P

< 0.05 were considered statistically significant. 3. Results 3.1 CDCA8 expression and protein localization from GV to MⅡ stage We cultured immature oocytes in IVM medium and examined the expression of CDCA8 during human oocyte meiotic maturation. The results showed that CDCA8 was expressed at every stage of meiosis, and its expression increased significantly from MⅠ to MⅡ, which was similar to the transcriptome data from oocytes matured in vivo (Hendrickson et al., 2017) (Fig. 1A). Immunofluorescence analysis of the subcellular localization of Borealin at the different stages of human oocyte meiosis showed that Borealin expression was not significant in the GV oocytes, but gradually strengthened from GV breakdown (GVBD) to MⅡ and had dynamic localization pattern during meiosis (Fig. 1B). After GVBD, the localization of Borealin overlapped with the chromosome region, and microtubules accumulated and assembled near chromosomes. By anaphase Ⅰ (AI), the chromosomes segregated away from the equatorial plate, and Borealin separated from the chromosome site and concentrated in the middle of the spindle. By telophase Ⅰ (TI), the division furrow formed, the contractile ring was contracted, and the spindle was constricted in the midbody, and Borealin accumulated mainly at the division furrow and midbody. When the PB1 extruded and oocytes soon reached metaphase Ⅱ, Borealin again overlapped with the chromosome site and localized at the equatorial plate. 3.2 RNAi caused inaccurate spindle assembly and disordered chromosomes To observe the effect of CDCA8 depletion, CDCA8 was silenced by specific siRNA in the GV oocytes. We monitored the development of these oocytes and found that, after 30 h in vitro culture, some oocytes arrested at the GV stage and some oocytes developed to MⅠ or MⅡ both in the CDCA8

siRNA and control siRNA groups. CDCA8 mRNA was examined by RT-qPCR in single oocytes. Compared with the control siRNA group, CDCA8 expression levels were decreased by >75% in the developmental oocytes (MⅠ and MⅡ) of the CDCA8- siRNA group, and there was no significant difference in the arrested GV oocytes (Fig. 2A). Immunofluorescence examination of Borealin expression in these oocytes showed that Borealin signal was not found in the oocytes of the two groups arrested at the GV stage (Fig. 2B), and the chromosomes were condensed and microtubules mainly localized under the oocyte cytomembrane, with only a small amount of enrichment around the chromosomes. By metaphase Ⅰ, Borealin in the control siRNA group occurred mainly in the middle of the spindle, basically coincident with the position of the chromosome. In the CDCA8- siRNA group, Borealin expression was very weak, chromosomes were disordered, and there was multipolar spindle formation (Fig. 2B MⅠ-a), or even a complete absence of the Borealin signal(Fig. 2B MⅠ-b), with no microtubules accumulating near the chromosomes. By metaphase Ⅱ, the chromosomes in the control siRNA group were arranged in a line at the equatorial plate, and Borealin localized in the middle of the spindle, overlapping with the chromosomes site. In the CDCA8- siRNA group, Borealin expression was weak, the chromosomes were disordered, and the microtubules were scattered around the chromosomes (Fig. 2B MⅡ-a), or there was even a complete absence of Borealin and microtubules signals around the chromosomes (Fig. 2B MⅡ-b). Based on whether microtubules occurred near the chromosomes, we divided the above abnormal performance into two types: microtubules signals type 1 (microtubules expressed around chromosomes), and microtubules signals type 2 (no microtubules signals around chromosomes). In the control siRNA group, 86% and 67% of MI and MII oocytes, respectively, showed normal

chromosome and microtubule morphology, and 14% and 33% were type 1, respectively. In the CDCA8- siRNA group, 88% and 85% of MI and MII oocytes, respectively, showed abnormal expression, and the proportion of type 1 was slightly higher than that of type 2 (Fig. 2C). 3.3 Prolonged PBE time and PB1 chromosome abnormality after RNAi To observe oocyte maturation after siRNA microinjection, we monitored oocytes development via a time-lapse system, recorded the time of GVBD and PBE, and analyzed the GVBD and PBE rates. Compared with the blank and negative control groups, the CDCA8- siRNA group had significantly prolonged time from GVBD to PBE (Fig.3A, 3B), and there was no significant difference between the GVBD and PBE rates the CDCA8- siRNA group and the blank and negative control groups. The chromosome composition of PB1 can reflect the chromosomal condition of oocyte, so we examined the PB1 chromosomes (Fig. 3C). In the blank control group, one of five oocytes had PB chromosomal copy numbers abnormality (partial monosomy), and the others were diploid. The negative control group had one chromosome copy number abnormality (partial chromosome deletion and partial chromosome monosomy), and the other three were normal. In the CDCA8siRNA group, chromosome copy number abnormalities were found in all five PBs, including partial chromosome deletion, partial chromosome tetrasomy, partial chromosome trisomy, and partial chromosome monosomy. Clearly, CDCA8- siRNA group had a significantly higher chromosome abnormality than the other two groups (P < 0.05) (Fig. 3D). 3.4 Decreased CDCA8 possibly induced chromosome abnormalities in the oocytes of older women We divided the patients into two age groups: older (≥ 40 years) and younger group (≤ 30 years),

and examined oocyte CDCA8 expression. There was no significant difference between the CDCA8 expression from GV, MI and MII oocytes of the two groups. As AURKC plays essential roles in female meiosis, we detected the expression of other CPC components (AURKC, BIRC5, INCENP), and also found no difference (Fig. 4A–C). Immunofluorescence detection of Borealin expression at different stages of oocyte meiosis in the older group showed that Borealin expression in the GV oocytes was not significant and was near the chromosomes at the MI and MII stages (Fig. 4). In addition, the disordered microtubules arrangement resulted in obstructed spindle formation in some oocytes of the older group. The PBs of IVM oocytes from the older group were biopsied and the chromosomes analyzed. Three of five PBs had abnormal chromosomes, including partial chromosome trisomy and partial chromosome monosomy (Fig. 5A). RT-qPCR examination of CDCA8 expression in the corresponding oocytes showed significantly lower CDCA8 expression in the oocytes with abnormal chromosomes as compared with the oocytes with normal chromosomes (P < 0.05); AURKC, BIRC5 and INCENP expression were not significantly different (Fig. 5B). 4. Discussion Progress toward understanding the function of CDCA8 in human meiosis has so far been limited by the difficulty in obtaining human samples. Here, we show that CDCA8 expression increased at the MII stage in human oocytes; that the protein localized to the middle of the spindle at anaphase Ⅰ; that CDCA8 RNAi resulted in spindle formation defects, chromosome abnormalities, and prolonged PBE time; and that advanced maternal age oocytes with abnormal chromosomes in the PB1 had lower CDCA8 expression. We conclude that CDCA8 is required for spindle assembly, bipolar spindle formation, and PBE, and that its function may be influenced by age.

Human single oocyte show high CDCA8 expression level at the MⅠ stage, which decrease at the MⅡ stage (Avo Santos et al., 2011). In the present study, CDCA8 expression was low in GV and MⅠ oocytes and was increased significantly at the MⅡ stage. Our results trend consistently with the transcriptome data of Hendrickson et al. (Hendrickson et al., 2017). However, different from the above two studies, the oocytes in our study were matured in vitro. Generally, maternal mRNA transcription occurs during follicular growth but ceases as the GV undergoes breakdown during meiosis (Watson, 2007), so oocytes should be transcriptionally silent. However, IVM oocytes exhibit lower developmental competence than oocytes matured in vivo (Lonergan et al., 2003), which indicates that IVM oocytes do not mature completely. Active transcription in the fully grown oocytes suggests that they are still in the process of synthesizing substances required for meiotic maturation (Liu and Aoki, 2002). Meanwhile, due to the difficulty in obtaining a large number of samples, we did not detect Borealin protein expression levels during human oocyte meiosis. However, immunoblot analysis of mouse oocyte has shown that Borealin expression increases gradually from the GV stage to MⅡ (Sun et al., 2010). Therefore, we believed that CDCA8 expression at both mRNA and protein level increases during human oocyte meiotic maturation. The obvious increase in CDCA8 at the MⅡ stage could be related to oocyte maturation and preparation for subsequent fertilization and mitosis. In mouse meiosis, Borealin localized to the spindle poles at the anaphase of meiosis Ⅰ (Sun et al., 2010). However, our immunofluorescence observations showed that Borealin was localized to the middle of the spindle at anaphase Ⅰ, indicating that it has consistent localization changes in human mitosis (Gassmann et al., 2004), differing from that of mice. As in mitosis, Borealin has dynamic localization during human meiosis, and is related to spindle formation, chromosome

separation, and cytoplasmic division (Sun and Kim, 2012; van der Horst and Lens, 2014). Meanwhile, species differences between mice and humans mean that protein localization also differs, indicating that mouse and human CPC may have different mechanisms of action. Currently, there are no consistent results for the GVBD and PBE rates after disruption of Borealin. In mouse meiosis, disruption of Borealin function by antibody injection resulted in spindle assembly defects but did not affect PBE (Sun et al., 2010). Here, our study shows that inhibiting CDCA8 mRNA does not affect the GVBD and PBE rates, but significantly prolonged the time from GVBD to PBE after RNAi. This indicates that Borealin dysfunction can affect the progress of human oocyte meiosis and hamper PBE, leading to a time extension, but does not completely block oocyte development, which may not influence spindle assembly checkpoint activity and be related to some compensation mechanism in oocytes (Honda et al., 2003).Spindle rotation is indispensable for PBE, and microfilaments play a vital role in regulating meiotic spindle rotation (Zhu et al., 2003). Therefore, CDCA8 might also influence microfilaments assembly and regulate spindle the position. Borealin is required for proper chromosome segregation (Bourhis et al., 2009); Borealin mutant cells of Drosophila undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and polyploidy (Hanson et al., 2005). Here, the CDCA8 siRNA group had multipolar spindles and disordered chromosome. The results are also consistent with research on human mitosis (Gassmann et al., 2004) and mouse embryo (Zhang et al., 2009). Borealin may be indispensable for meiotic bipolar spindle stability. As the spindle is closely linked with chromosome separation, we examined PB1 chromosome copy numbers for reflecting the condition of the chromosome after CDCA8 RNAi. Our findings proved that CDCA8 RNAi resulted in spindle

defects and abnormal chromosomes, including partial chromosome deletion, partial chromosome monosomy, partial chromosome tetrasomy and partial chromosome trisomy. This indicates that CDCA8 may not only affect the correct distribution of homologous chromosomes in PB and oocytes, but may also affect centromere stability between sister chromatids in homologous chromosomes. As an important CPC component, Borealin is involved in regulating the spindle checkpoint in human oocyte meiosis. It is suggested that CDCA8 has a potential role in the genomic integrity of human oocyte meiosis, as its function in mitotic cells has been proved, where it contributes to mitotic fidelity and genomic integrity (Liu et al., 2012). We examined CDCA8 expression in the older women to investigate the relation between CDCA8 and the high incidence of aneuploidy embryos in older women. There was no significant difference in CDCA8 expression in the GV, MI, and MII oocytes between the older group and the younger, and three of five PBs in the older group had abnormal chromosomes, wherein CDCA8 expression was significantly lower than that of the normal in the corresponding oocytes, while there was no difference for the other CPC components. All of this suggests that CDCA8 may affect spindle assembly and chromosome segregation in human oocyte meiosis. Based on the high CDCA8 expression in the MII oocytes, and the chromosome distribution pattern and microtubule abnormalities at the MII stage in the RNAi group, it is reasonable to believe that CDCA8 may also play a role in MII, as it does in mitosis. However, as human oocyte material is precious, we did not continue with studying MII, and the sample size of the present study also should be increased. Future studies are needed to explore the depth the specific molecular mechanism of CDCA8 in meiosis to deepen the understanding of human oocyte meiosis.

Acknowledgments: Not applicable. Author Contribution: LG, GF, and LLZ. designed, conceived, and performed the experiments. ZSP collected the samples. ZL, ZQW, and ZCQ performed the experiments and analyzed the data. XPY performed the chromosome analysis. ZCQ wrote the manuscript.

Funding: This work was supported by the National Natural Science Foundation of China (grant number: 81471510) and the Innovation Funds for Postgraduate of Central South University (grant number: 2016zzts112). Conflict of Interest: The authors declare that there is no conflict of interest.

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Table 1 Primer details Primer

Sequences

Annealing temperature (℃)

Length(bp)

5′- CATCCTGCGTCTGGACCTGG-3′ β-actin

58

116

58

142

58

202

58

118

58

186

5′-TAATGTCACGCACGATTTCC-3′ 5′-TCTACAACACCCCAATATCCTGC-3′ AURKC 5′-GGCTGTGCGCTGTTCATCTA-3′ 5′-GGACCCACAAATGAGACACC-3′ CDCA8 5′-ATGGGAGGGTGAACAGACAG-3′ 5′-GTGCAGAGGAACCAGATGCT-3′ INCENP 5′-CCTTCTCGACGAAGCTGCAC-3′ 5′-TGACGACCCCATAGAGGAACA-3′ BIRC5 5′-CGCACTTTCTCCGCAGTTTC-3′

Figure legends

Fig. 1. CDCA8 expression and subcellular localization during human oocyte meiotic maturation. (A) RT-qPCR of CDCA8 mRNA expression (left) and CDCA8 transcriptome data of Hendrickson et al., 2017 (right; FPKM, fragments per kilobase of transcript per million mapped reads). Samples were collected when oocytes reached the GV (n = 8), MⅠ (n = 8), and MⅡ (n = 7) stages; CDCA8 expression increased significantly at MⅡ (**P < 0.01). (B) Immunofluorescence staining of Borealin subcellular localization. No Borealin was expressed at the GV stage. From GVBD to MⅠ, Borealin overlapped with the chromosome region. As the oocytes progressed to AⅠ, Borealin

migrated to the middle of the spindle. By TⅠ, Borealin accumulated at the midbody and the division furrow. At the MⅡ stage, Borealin again localized at the equatorial plate. Red, Borealin; green, αtubulin; blue, chromatin. Scale bar: 10 μm.

Fig. 2. CDCA8 microinjection in human oocytes disrupted normal spindle organization and oocyte maturation. After 30 h culture, non-matured GV oocytes (without PB1) or MⅡ-arrested oocytes (with PB1) were analyzed. (A) RT-qPCR analysis of CDCA8 expression. Control siRNA group: GV oocytes, n = 4; MⅠ oocytes, n = 5; MⅡ oocytes, n = 6; CDCA8 siRNA group: GV oocytes, n = 5; MⅠ oocytes, n = 6; MⅡ oocytes, n = 7. (*P < 0.05). (B) Immunofluorescence staining of Borealin. GV, MⅠ, MⅡ: control siRNA group; GV-a, MⅠ-a, MII-a, MI-b, MII-b: CDCA8 siRNA group. Borealin expression was very weak at MⅠ-a and MⅡ-a; there was no signal at MⅠ-b and MⅡ-b. Red, Borealin; green, α-tubulin; blue, chromatin. Scale bar: 10 μm. (C) Constitution ratio of microtubule abnormal morphology. The CDCA8 siRNA group had a significantly higher abnormal ratio than the control siRNA group.

Fig. 3. Effects of CDCA8 siRNA microinjection on the cell cycle, PBE time, and chromosome composition. (A)There was no significant difference of the percentages of GVBD and PBE between the blank and negative control groups (control) and the CDCA8 siRNA group (CDCA8 siRNA). (B) CDCA8 siRNA group had significantly prolonged PBE time. (C) Normal PB1 had diploid chromosome copy numbers (Normal). The CDCA8 siRNA group had abnormal PB1 chromosome composition, including partial chromosome deletion, partial chromosome tetrasomy, partial chromosome trisomy, and partial chromosome monosomy. (D) Percentage of abnormal

chromosome copy numbers in the blank, negative control (control siRNA), and CDCA8 siRNA groups (n = 5, 4, 5, respectively). The CDCA8 siRNA group had a significantly higher chromosome abnormality rate than the other two groups. (*P < 0.05).

Fig. 4. Expression of Borealin and the CPC components in oocytes from older women. (A–C) RTqPCR analysis of AURKC, CDCA8, BIRC5, and INCENP mRNA expression in oocytes of the older and younger groups, and immunofluorescence staining of Borealin (a–c) in the (A) GV, (B) MⅠ, and (C) MⅡ oocytes of the older group. CPC components expression was not significantly different between the two groups. There was no obvious Borealin expression in the GV oocytes of the older patients (a). Borealin signals were at MⅠ and MⅡ stages (b, c); defective spindle and microtubules misalignment could be seen in the MⅡ oocytes (c). Red, Borealin; green, α-tubulin; blue, chromatin. Scale bar: 10 μm.

Fig. 5. Oocytes from older women were prone to have abnormal chromosomes and decreased CDCA8 expression. (A) Chromosome analysis of PB1 from older women. There were many abnormal chromosome conditions, including chromosome 11 trisomy, chromosome 9 monosomy, chromosome 21 monosomy, and chromosome 22 monosomy. (B) RT-qPCR Analysis of AURKC, CDCA8, BIRC5, and INCENP mRNA in oocytes from the older women. Oocytes with PB1 abnormal chromosome had significantly decreased CDCA8 expression (*P < 0.05); AURKC, BIRC5, and INCENP expression was not significantly different (P > 0.05).

Abbreviations CDCA8, cell division cycle associated 8; CPC, chromosomal passenger complex; AURKB, AuroraB kinase; INCENP, inner centromere protein; AURKC, Aurora-C kinase; GV, germinal vesicle; GVBD, germinal vesicle breakdown; MⅠ, metaphase Ⅰ; MⅡ, metaphase Ⅱ; PB, polar body; PBE, polar body extrusion; PBS, phosphate-buffered saline; DAPI, 4, 6-diamidino-2-phenylindole; NGS, next-generation sequencing; IVM, in vitro maturation; AI, anaphase Ⅰ; TI, telophase Ⅰ; FPKM, fragments per kilobase of transcript per million mapped reads;

LG, GF, and LLZ. designed, conceived, and performed the experiments. ZSP collected the samples. ZL, ZQW, and ZCQ performed the experiments and analyzed the data. XPY performed the chromosome analysis. ZCQ wrote the manuscript.

Declaration of interests

√ The authors declare that they have no known competing financial interests or personal □

relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Highlights CDCA8 protein has dynamic localization during human oocyte meiosis. Knock-down of CDCA8 affects spindle assembly and chromosomes arrangement in oocytes. Decreased CDCA8 in the oocytes of older women may lead to chromosome abnormalities.