Gene 741 (2020) 144495

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Gene
journal homepage: www.elsevier.com/locate/gene

Research paper

CDCA8 regulates meiotic spindle assembly and chromosome segregation T

during human oocyte meiosis
Changquan Zhanga,b,1, Lei Zhaoc,1, Lizhi Lenga,b,c, Qinwei Zhouc, Shuoping Zhangc,
⁎
Fei Gonga,b,c,d, Pingyuan Xied, Ge Lina,b,c,d,
a
Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha 410078, China
b
Key Laboratory of Reproductive and Stem Cells Engineering, Ministry of Health, Changsha 410078, China
c
Reproductive & Genetic Hospital of CITIC-Xiangya, Changsha 410078, China
d
National Engineering and Research Center of Human Stem Cells, Changsha 410078, China

A R T I C LE I N FO A B S T R A C T

Keywords: As a member of the chromosomal passenger complex, CDCA8 (cell division cycle associated 8) plays an im-
Chromosomal passenger complex portant role in human mitosis, but its roles in human meiosis are unknown. Here, we show that CDCA8 ex-
CDCA8 pression is increased and its encoded protein has dynamic localization in human oocytes from germinal vesicle
Meiosis breakdown (GVBD) to metaphase Ⅱ (MⅡ), and that there are multipolar spindles, disordered chromosomes, and
Human oocyte
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 ab-
normalities 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.

1. Introduction
During cell division, the chromosomal passenger complex (CPC)
plays important roles in proper chromosome segregation and the ex-
ecution of cytokinesis (van der Horst and Lens, 2014), and it is com-
posed of the enzymatic core Aurora- B kinase (AURKB), the scaffold
protein inner centromere protein (INCENP), Borealin (CDCA8, cell di-
vision cycle associated 8), and two other non-enzymatic subunits Sur-
vivin (also known as BIRC5). The CPC can localize correctly to the re-
levant 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 com-
pletion 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 de-
fects (Honda et al., 2003; Klein et al., 2006; Santaguida et al., 2011). In
mouse oocytes, AURKC replaces AURKB in the CPC as a critical reg-
ulator 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 struc-
tural features, Borealin is conserved across Animalia and Fungi
(Jeyaprakash et al., 2007; Nakajima et al., 2009). It is regulated by

Abbreviations: CDCA8, cell division cycle associated 8; CPC, chromosomal passenger complex; AURKB, Aurora-B 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
⁎
Corresponding author at: Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, 8 Luyun Road,
Changsha, Hunan, China.
E-mail address: linggf@hotmail.com (G. Lin).
1
These authors contributed equally to the study.

https://doi.org/10.1016/j.gene.2020.144495
Received 27 September 2019; Received in revised form 19 February 2020; Accepted 19 February 2020
Available online 20 February 2020
0378-1119/ © 2020 Published by Elsevier B.V.
C. Zhang, et al.
phosphorylation at multiple sites, including phosphorylation by CDK1
(cyclin-dependent kinase 1) to promote targeting of the CPC to cen-
tromeres (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 me-
taphase–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 mis-
attachments and ectopic spindle poles formation (Gassmann et al.,
2004), and also leads to defective cell proliferation, p53 accumulation
and mouse early embryonic lethality by 5.5 days post coitus (Yamanaka
et al., 2008). In general, in human mitotic cells, Borealin corrects ki-
netochore–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 re-
localizes 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 er-
rors 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 an-
imal 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 ab-
normalities 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 fi-
nancial payment.
We enrolled women undergoing intracytoplasmic sperm injection
(ICSI) treatment at the Reproductive & Genetic Hospital of CITIC-
2
Gene 741 (2020) 144495
XIANGYA, and divided them into two groups according to age (≥40
and ≤30 years). The women had tubal-factor infertility, and we ex-
cluded other factors, including premature ovarian failure, ovarian
dysfunction, ovarian radiotherapy or chemotherapy, thyroid dysfunc-
tion 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, 1 μg/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 ap-
pearance 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′-dTdTCCU
UUAUGCUUAGUUCGUU-5′, Ribobio, Guangzhou, China) were dis-
solved 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 in-
jection needle (Femtotips Ⅱ, Eppendorf, Hamburg, Germany) connected
to another micromanipulator (FemtoJet, Eppendorf). Non-injected oo-
cytes 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 4 μL lysis buffer
(YK-PGSTM Embryo Biopsy Sample Collection Kit, Yikon Genomics,
Shanghai, China) by a glass micropipette with a 20 μm- 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
C. Zhang, et al.
performed to generate the micrograms of DNA required for next-gen-
eration sequencing. Using an Illumina HiSeq 2000 platform (illumina,
San Diego, CA, USA), the amplified genome of each single PB was se-
quenced 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 eval-
uated 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 re-
leased 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 com-
pared 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–α-tubulin 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, A-
11012, Invitrogen) for 1 h at room temperature in the dark. The nuclei
were stained for 10 min with 4, 6-diamidino-2-phenylindole (DAPI,
1 μg/mL, Invitrogen). Finally, fluorescent images were analyzed by
Table 1
Primer details.
Primer Sequences Annealing Length (bp)
temperature
(℃)
β-actin 5′- CATCCTGCGTCTGGACCTGG-3′ 58 116
5′-TAATGTCACGCACGATTTCC-3′
AURKC 5′-TCTACAACACCCCAATATCCTGC-3′ 58 142
5′-GGCTGTGCGCTGTTCATCTA-3′
CDCA8 5′-GGACCCACAAATGAGACACC-3′ 58 202
5′-ATGGGAGGGTGAACAGACAG-3′
INCENP 5′-GTGCAGAGGAACCAGATGCT-3′ 58 118
5′-CCTTCTCGACGAAGCTGCAC-3′
BIRC5 5′-TGACGACCCCATAGAGGAACA-3′ 58 186
5′-CGCACTTTCTCCGCAGTTTC-3′
3
Gene 741 (2020) 144495
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 Mann-
Whitney-Wilcoxon tests if they did not. Data are reported as the
mean ± SEM, and differences at P < 0.05 were considered statisti-
cally 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 ana-
phase Ⅰ (AI), the chromosomes segregated away from the equatorial
plate, and Borealin separated from the chromosome site and con-
centrated 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 chromo-
somes 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,
C. Zhang, et al. Gene 741 (2020) 144495

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, α-tubulin; blue, chromatin. Scale bar: 10 μm.

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 chromo-
somes). In the control siRNA group, 86% and 67% of MI and MII oo-
cytes, 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, respec-
tively, 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 ab-
normality (partial chromosome deletion and partial chromosome

4
C. Zhang, et al. Gene 741 (2020) 144495

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.

monosomy), and the other three were normal. In the CDCA8- siRNA monosomy. Clearly, CDCA8- siRNA group had a significantly higher
group, chromosome copy number abnormalities were found in all five chromosome abnormality than the other two groups (P < 0.05)
PBs, including partial chromosome deletion, partial chromosome tet- (Fig. 3D).
rasomy, partial chromosome trisomy, and partial chromosome

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C. Zhang, et al. Gene 741 (2020) 144495

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 ab-
normal 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).

3.4. Decreased CDCA8 possibly induced chromosome abnormalities in the younger group (≤30 years), and examined oocyte CDCA8 expression.
oocytes of older women There was no significant difference between the CDCA8 expression
from GV, MI and MII oocytes of the two groups. As AURKC plays es-
We divided the patients into two age groups: older (≥40 years) and sential roles in female meiosis, we detected the expression of other CPC

6
C. Zhang, et al.
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 ex-
pression in the GV oocytes was not significant and was near the chro-
mosomes 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
7
Gene 741 (2020) 144495
Fig. 4. Expression of Borealin and the CPC compo-
nents in oocytes from older women. (A–C) RT-qPCR
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 ex-
pression was not significantly different between the
two groups. There was no obvious Borealin expres-
sion 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.
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 con-
sistently 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 diffi-
culty in obtaining a large number of samples, we did not detect Borealin
protein expression levels during human oocyte meiosis. However, im-
munoblot analysis of mouse oocyte has shown that Borealin expression
increases gradually from the GV stage to MⅡ (Sun et al., 2010).
C. Zhang, et al. Gene 741 (2020) 144495

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).

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 immuno-
fluorescence 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), dif-
fering from that of mice. As in mitosis, Borealin has dynamic localiza-
tion during human meiosis, and is related to spindle formation, chro-
mosome 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

8
C. Zhang, et al.
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 compen-
sation 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 posi-
tion.
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 ab-
normal 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 in-
tegrity (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.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
9
Gene 741 (2020) 144495
Acknowledgments
Not applicable.
CRediT authorship contribution statement
Changquan Zhang: Funding acquisition, Investigation, Validation,
Writing-original draft, Writing-review & editing. Lei Zhao:
Investigation, Formal analysis, Software, Visualization. Lizhi Leng:
Data curation, Methodology, Project administration, Visualization.
Qinwei Zhou: Investigation, Validation. Shuoping Zhang: Resources.
Fei Gong: Conceptualization, Resources. Pingyuan Xie: Investigation,
Visualization. Ge Lin: Conceptualization, Funding acquisition, Project
administration, Supervision.
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).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.gene.2020.144495.
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