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Theriogenology 220 (2024) 1–11
Available online 28 February 2024
0093-691X/© 2024 Elsevier Inc. All rights reserved.
Original Research Article
Neddylation inhibition affects early embryonic development by disrupting
maternal-to-zygotic transition and mitochondrial function in mice
Mingxiao Liu
a
,
1
, Zhiming Ding
c
,
d
,
1
, Peihao Sun
a
, Shuo Zhou
a
, Hanxiao Wu
a
, Lijun Huo
a
,
b
,
Liguo Yang
a
,
b
, John S. Davis
e
, Aixin Liang
a
,
b
,
*
a
Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong
Agricultural University, Wuhan, 430070, PR China
b
Frontiers Science Center for Animal Breeding and Sustainable Production (Huazhong Agricultural University), Ministry of Education, Wuhan, 430070, PR China
c
Department of Obstetrics and Gynecology, The First Afliated Hospital of Anhui Medical University, Hefei, 230022, PR China
d
NHC Key Laboratory of Study on Abnormal Gametes and Reproductive Tract, Anhui Medical University, Hefei, 230032, PR China
e
Olson Center for Women’s Health, Department of Obstetrics and Gynecology, University of Nebraska Medical Center, and Veterans Affairs Medical Center, Omaha, NE,
68198, USA
ARTICLE INFO
Keywords:
Neddylation
Embryonic development
MLN4924
Maternal-to-zygotic transition
Mitochondria
ABSTRACT
Post-translational modications (PTMs) are critical for early development in mice because early cleavage-stage
embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that
regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic
development remain largely unknown. In the present study, we found that inhibition of neddylation by specic
inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that
neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation in-
hibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-
zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant
mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further
analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and su-
peroxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this
study demonstrates that neddylation is vital for early embryonic development in mice. Our ndings suggest that
proper neddylation regulation is essential for the timely inter-stage transition during early embryonic
development.
1. Introduction
The early development of animal embryos is guided by maternal
gene products. Then, the developmental control is transferred from
maternally supplied gene products to the zygotic genome. The maternal-
to-zygotic transition (MZT) is required for the acquisition of develop-
mental competence by cleavage-stage embryos. The MZT involves the
degradation of maternal transcripts and proteins that are unnecessary
for subsequent embryonic development, and the activation of zygotic
genes [1]. Zygotic genome activation (ZGA) is regulated in a precisely
timed manner, which varies among mammals. It mainly occurs at the
2-cell stage in mice, and at the 4-cell and 8-cell stages in humans [2].
Totipotency and further development of early embryos require the
establishment of specic gene expression patterns during ZGA [3].
Recently, several key regulators, such as RNA polymerase II [4] and
PRD-like homeobox transcription factors (TPRXs) [5], have been iden-
tied. Functional disruption of these regulators results in failure of ZGA
and arrest of early embryonic development in 2-cell stage embryos [4,
5].
Since the zygotic genome is inactive during the initial stages of early
embryonic development, post-translational modications (PTMs) of
proteins would appear to be particularly crucial. Various PTMs, such as
phosphorylation, methylation and ubiquitination, have been identied
to participate in the MZT [6]. Neddylation is another important
* Corresponding author. Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, College of Animal Science and
Technology, Huazhong Agricultural University, No.1 Shizishan Street, Wuhan, 430070, PR China.
E-mail address: lax.pipi@mail.hzau.edu.cn (A. Liang).
1
These authors should be considered joint rst author.
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
https://doi.org/10.1016/j.theriogenology.2024.02.029
Received 1 October 2023; Received in revised form 27 February 2024; Accepted 27 February 2024
Theriogenology 220 (2024) 1–11
2
post-translational modication, involving the attachment of the
ubiquitin-like molecule NEDD8 to substrate proteins [7]. Like ubiquiti-
nation, neddylation is characterized by a series of enzymatic cascades
including NEDD8-activating enzyme (NAE) E1, NEDD8
ubiquitin-conjugating enzyme (UBC12) E2, and substrate-specic
NEDD8 ligase E3. Specically, NEDD8 is activated by NAE1 in an
ATP-dependent manner. Next, the activated NEDD8 is transferred to E2,
and then transferred to the targeted substrates by E3 [8]. Recent reports
indicate that neddylation is involved in the regulation of reproduction.
For example, neddylation deciency leads to defects in spermatogenesis
and sterility in male mice [9]. Disruption of neddylation in female mice
results in meiotic arrest in oocytes accompanied by the activation of the
spindle assembly checkpoint [10]. Importantly, inhibition of neddyla-
tion during the 4-cell to late 8-cell stage transition results in delayed
development of bovine embryos [11]. Further, Yang and colleagues
demonstrated that neddylation inhibition led to diminished embryo
quality in mice due to increased oxidative stress and reduced IL-1β levels
[12]. Despite these ndings, the precise role of neddylation in regulating
early embryonic development, especially regulating the MZT process,
remains unclear.
In this study, we used the specic neddylation inhibitor MLN4924 to
investigate the effects of neddylation on early murine embryonic
development. We also performed transcriptome analysis to uncover the
underlying mechanisms. The results showed that inhibition of neddy-
lation led to severe early arrest of embryonic development and MZT
defects. Moreover, the arrested embryos exhibited abnormal mito-
chondrial function, oxidative stress, and DNA damage.
2. Materials and methods
2.1. Animals and ethics statement
All SPF Kunming (KM) mice used in the experiments were obtained
from the Laboratory Animal Center of Huazhong Agricultural University
and housed in the SPF animal facility. All animal experiments were
approved by the Animal Ethics Committee of Huazhong Agricultural
University (HZAUMO-2022-0105).
2.2. Collection and culture of embryos
Female SPF KM mice (4–6 weeks old) were injected (i.p.) with 10 IU
of PMSG. After 48 h they were injected with 10 IU of hCG and mated
with adult males at a ratio of 1:1. Twenty hours later, the zygotes were
collected from the oviducts. Cumulus cells were disaggregated from
zygotes with 0.1% hyaluronidase (10176, Vitrolife, Sweden). Zygotes
were then cultured in G1 medium (10128, Vitrolife, Sweden) with or
without 0.1, 1, 10
μ
M of MLN4924 (S7109, Selleck, Houston, TX, USA)
dissolved by DMSO at 37 ◦C in humidied atmosphere of 5% CO
2
.
2.3. Immunouorescence and confocal microscopy assay
Embryos were briey xed with PBS containing 4% para-
formaldehyde and 0.5% Triton X-100 for 50 min. After blocking in PBS
containing 2% BSA, 0.1% Tween-20, and 0.01% Triton X-100 for 1 h at
room temperature, xed embryos were then incubated with rabbit anti-
NEDD8 (1:100, ab81264, Abcam, Cambridge, UK) or rabbit anti-Phos-
pho-Histone H2AX antibody (1:100, 9718, CST, Boston, MA, USA)
overnight at 4 ◦C. After washing, embryos were incubated with Cy3-
labelled goat anti-rabbit antibody (1:500, AS007, Abclonal, Wuhan,
China) at 37 ◦C for 1 h. DNA of embryos was labelled with DAPI (C0065,
Solarbio, Beijing, China) for 10 min at room temperature. Finally, em-
bryos were transferred onto glass slides, cover slipped with Antifade
Mounting Medium (P0128S, Beyotime, Shanghai, China), and observed
under a Zeiss confocal laser scanning microscope (LSM 800, Germany).
2.4. Low input RNA sequencing
For RNA sequencing library preparation, three sets of samples
(twenty embryos per sample) were collected in the MLN4924 treatment
and control groups, respectively. The RNA of embryos was amplied by
the Smart-Seq 2 method [13] and reverse transcribed using an Oligo-dT
primer to synthesize rst-strand cDNA. The resulting cDNA was ampli-
ed by PCR, and the PCR products were puried with magnetic beads
before analysis with Qubit® 3.0 Flurometer and Agilent 2100 Bio-
analyzer to ensure the length of cDNA to be around 1~2 kb prior to
shearing ultrasonically. The cDNA was then sheared randomly by ul-
trasonic wave. The cDNA library was constructed by Illumina protocols
including DNA fragmentation, end repair, 3
′
ends A-tailing, adapter
ligation, PCR amplication, and library validation. Afterwards, the
quality of constructed cDNA library was determined using PerkinElmer
LabChip® GX Touch and Step OnePlus™ Real-Time PCR System.
Qualied libraries were then loaded onto Illumina Hiseq platform for
PE150 sequencing. The differentially expressed genes (DEGs) in the
comparison of treatment group and control group were identied using
DESeq2 according to the thresholds of |log2Ratio|≥ 1 and a q-value
<0.05.
2.5. RNA extraction and quantitative real-time PCR (qRT-PCR)
The RNA was extracted with RNAprep Pure Micro Kit according to
the manufacturer’s protocol (DP420, TIANGEN, Beijing, China), and
cDNA was then synthesized using the HiScript II Q RT SuperMix (R222-
01,Vazyme, Nanjing, China). The qRT-PCR was performed with ChamQ
Universal SYBR qPCR Master Mix (Q711-02/03, Vazyme, Nanjing,
China) by using Bio-Rad uorescence quantitative PCR instrument
(CFX96, Bio-Rad, USA). Specic primers were designed by Primer Pre-
mier 5.0 and listed in Supplementary Table S1. The qRT-PCR conditions
were as follows: 95 ◦C for 1 min, followed by 40 cycles of 95 ◦C for 10 s
and 60 ◦C for 30 s. Relative gene expression was calculated by 2
−ΔΔCt
method and normalized to Gapdh.
2.6. Ethynyl uridine (EU) detection
The EU assay Kit (C10316-1, RiboBio, Guangzhou, China) was used
to examine RNA transcriptional activity. Embryos were incubated with
EU (500
μ
M) in G1 medium for 2 h at 37 ◦C. After washing with PBS,
embryos were xed in PBS containing 4% paraformaldehyde and 0.5%
Triton X-100 for 30 min. Then, embryos were stained with Apollo for 30
min at room temperature, and protected from light. Afterwards, em-
bryos were washed with PBS containing 0.1% Tween-20 and 0.01%
Triton X-100 three times for 10 min each. DNA was labelled with DAPI
for 10 min at room temperature. Finally, embryos were transferred onto
glass slides containing Antifade Mounting Medium (P0128S, Beyotime,
Shanghai, China) and observed with a Zeiss confocal laser scanning
microscope (LSM 800, Germany).
2.7. Mitochondrion staining
Mito-Tracker Red (C1049B, Beyotime, Shanghai, China) was applied
to evaluate mitochondrial localization in the embryos. After washing
with PBS, embryos were incubated in G1 medium containing 200 nM
Mito-Tracker at 37 ◦C incubator for 30 min. After washing, the embryos
were transferred to a laser confocal Petri dish and observed under Zeiss
confocal laser scanning microscope (LSM 800, Germany).
2.8. ATP content assay
The levels of ATP were measured using an ATP assay Kit (S0026,
Beyotime, Shanghai, China) following the manufacturer’s instructions.
In brief, embryos were placed into black 96-well culture plates (Lysis
buffer, 50
μ
L/well). Next, 50
μ
L of ATP working solution was added.
M. Liu et al.
Theriogenology 220 (2024) 1–11
3
Finally, ATP concentration in embryos was detected using an EnSpire®
Multimode Reader (PerkinElmer, USA) in luminescence mode.
2.9. Mitochondrial membrane potential detection
The mitochondrial membrane potential (MMP) assay Kit (C2006,
Beyotime, Shanghai, China) with JC-1 was used to examine MMP. After
washing with PBS, embryos were cultured in G1 medium containing JC-
1 working solution for 20 min at 37 ◦C. After washing, the embryos were
transferred to a laser confocal Petri dish and observed with uorescence
inverted microscope (IX73, Olympus, Japan).
2.10. Superoxide anion and ROS detection
A Dihydroethidium (DHE) Kit (S0063, Beyotime, Shanghai, China)
and a ROS Assay Kit (S0033S, Beyotime, Shanghai, China) were used to
determinate superoxide anion and ROS levels, respectively, in 2-cell
embryos according to the manufacturer protocols. Embryos per group
were incubated in G1 medium containing dihydroethidium (10
μ
M) or
DCFH-DA (10
μ
M) for 30 min at 37 ◦C. After washing in PBS, the em-
bryos were transferred to a suitable vessel for observation under uo-
rescence inverted microscope (IX73, Olympus, Japan).
2.11. Statistical analysis
All statistical data from at least three independent experiments are
presented as mean ±SEM and the numbers of analyzed embryos are
labelled in gure legends as (n). All uorescence signal images were
quantied by ImageJ software. ANOVA and Student’s t-test were per-
formed to examine the difference among groups. P <0.05 was consid-
ered as statistically signicant.
3. Results
3.1. Inhibition of neddylation leads to early embryonic development arrest
NEDD8 localization was examined at different stages during early
embryonic development. We collected murine embryos at 42, 66, 80,
104, and 128 h post injection of hCG, which represent 2-cell, 4-cell, 8-
cell, morula, and blastocyst stages, respectively. Immunouorescence
staining showed that NEDD8 was mainly localized in the nucleus of 2-
cell and 4-cell stage embryos, and nuclear localization disappeared in
the later stages (Fig. 1A). MLN4924, a specic inhibitor of E1, which
suppresses the neddylation modication of proteins by forming an
adduct with NEDD8 [14], was employed to explore the roles of neddy-
lation during early embryonic development. We cultured zygotes with
different concentrations of MLN4924, and investigated embryonic
development at each stage. As shown in Fig. 1B–C and S1, MLN4924
treatment signicantly inhibited early embryonic development. Treat-
ment of embryos with 1
μ
M or 10
μ
M MLN4924 blocked progression
from the 2-cell stage by approximately 50% in both treatment groups.
No signicant difference was observed in response to 0.1
μ
M MLN4924
compared to control. Notably, the developmental progress at the 8-cell
stage was signicantly arrested with both 1
μ
M and 10
μ
M MLN4924,
resulting in no morula and blastocyst formation. Considering the similar
effects of 1
μ
M and 10
μ
M MLN4924 on early embryonic development,
we selected 1
μ
M MLN4924 as the working concentration for the sub-
sequent analysis.
Importantly, the nuclear-specic localization of NEDD8 at the 2-cell
stage was lost after MLN4924 treatment (Fig. 1D), and the proportion of
embryos with abnormal NEDD8 localization was signicantly higher in
the MLN4924 treatment group than the control group (90.95 ±1.32%
vs 6.52 ±1.55%, P <0.001, Fig. 1E), suggesting that neddylation might
play an important role at the 2-cell stage.
3.2. Neddylation inhibition alters the transcriptome proles of 2-cell
embryos
To explore the underlying mechanism of neddylation inhibition on
early embryonic developmental arrest, we compared the transcriptome
of the MLN4924-treated 2-cell embryos with that of the control group.
Principal component analysis (PCA) showed a good inter-group sample
repeatability (Fig. 2A), which was supported by the sample-to-sample
Heatmap results (Fig. 2B). A total of 3959 DEGs were identied be-
tween the MLN4924 treatment and control groups, of which 1597 DEGs
were up-regulated (Fig. 2C, yellow dots), and 2362 DEGs were down-
regulated (Fig. 2C, blue dots). DEGs were found to be enriched in 3
categories of Gene Ontology (GO) terms related to cellular component,
biological process, and molecular function. Specically, the signicantly
enriched GO terms in each category mainly included RNA metabolic
process, mitochondrion organization, antioxidant activity, and tran-
scription regulator activity (Fig. 2D and Fig. S2). Kyoto Encyclopedia of
Genes and Genomes (KEGG) analysis further showed that the DEGs were
mainly enriched in pathways such as RNA transport, RNA polymerase,
Ribosome biogenesis in eukaryotes, mRNA surveillance, and Protea-
some (Fig. 2E). Furthermore, we observed that a total of 953 differen-
tially expressed transcription factors were identied between the
MLN4924 treatment and control groups, of which 338 transcription
factors were up-regulated and 615 transcription factors were down-
regulated (Fig. 2F). The protein families related to these differentially
expressed transcription factors are shown in Fig. 2G. Collectively, our
data strongly suggest that neddylation inhibition changes the gene
expression patterns associated with mRNA transcription in 2-cell
embryos.
3.3. Neddylation inhibition affects the ZGA process of 2-cell embryos
The MZT is one of the most important events in mouse 2-cell stage
embryos, comprising both degradation of maternal effect genes and
ZGA. As described above, the nuclear-specic localization of NEDD8 at
the 2-cell stage was lost after inhibition of neddylation (Fig. 1D), indi-
cating that nuclear gene expression might have been affected. KEGG
analysis showed that RNA polymerase, an enzyme involved in ZGA, was
enriched after neddylation inhibition (Fig. 2E). Notably, homeobox
transcription factors, as the key regulators of ZGA, were downregulated
after MLN4924 treatment (Fig. 2G). Thus, we speculated neddylation
inhibition adversely affects ZGA. To test our speculation, 2279 ZGA-
related genes were rst screened from different stages of mouse embryos
according to published RNA-seq data [15]. Venn analysis showed that
719 out of the 3959 DEGs identied in this study belonged to
ZGA-related genes (Fig. 3A), 31.5% of which were regulated by ned-
dylation. Furthermore, cluster analysis based on RNA-seq data showed
that the expression of typical ZGA transcripts was signicantly
decreased, except for the Taf9b gene (Fig. 3B). In addition, qRT-PCR
validation of decreased ZGA transcripts by qRT-PCR revealed results
consistent with transcriptomic results, suggesting the reliability of
transcriptomic data (Fig. 3C). Subsequently, EU staining was performed
to evaluate the effects of neddylation inhibition on transcriptional ac-
tivity. The results showed that the EU uorescence intensity was
signicantly lower in the MLN4924 treatment group than that in the
control group (116.7 ±6.88 vs 146.3 ±6.56, P <0.01, Fig. 3D and E).
Taken together, these results indicate that neddylation inhibition in-
duces developmental arrest in 2-cell embryos by disrupting transcrip-
tional activity.
3.4. Neddylation inhibition impairs degradation of maternal effect genes
in 2-cell embryos
To examine the effect of neddylation inhibition on the degradation of
maternal effect genes, 7661 maternal genes were screened from
different stages of mouse early embryonic development according to the
M. Liu et al.
Theriogenology 220 (2024) 1–11
4
Fig. 1. Inhibition of neddylation led to early embryonic development arrest. (A) NEDD8 localization in 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae and
blastocysts. NEDD8, red; DNA, blue. Scale bar =20
μ
m. (B) Embryo morphologies after 1
μ
M MLN4924 treatment at the 2-cell stage. Scale bar =100
μ
m. (C) Rates of
different stages in early embryonic development after MLN4924 treatment (n =120 embryos per group). **P <0.01, ***P <0.001. (D) NEDD8 localization in 2-cell
embryos after MLN4924 treatment. NEDD8, red; DNA, blue. Scale bar =20
μ
m. (E) The proportion of embryos with abnormal NEDD8 localization in the control (n =
79 embryos) and MLN4924-treated (n =89 embryos) groups. ***P <0.001. (For interpretation of the references to color in this gure legend, the reader is referred to
the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
5
Fig. 2. Neddylation inhibition changed the transcriptome proles of 2-cell embryos. (A) The principle component analysis (PCA) analysis of 2-cell embryo RNA-seq
data comparing control with MLN4924-treated embryos. (B) The heatmap of differentially expressed genes (DEGs) comparing control with MLN4924-treated em-
bryos. (C) The volcano plot of DEGs comparing control with MLN4924-treated embryos. Down-regulated, blue; up-regulated, yellow. (D) GO analysis of DEGs
comparing control with MLN4924-treated embryos. (E) KEGG analysis of DEGs comparing control with MLN4924-treated embryos, the dot size represents the
enriched gene number, the larger the value, the greater the enrichment, and the dot color represents the adjusted p-value. (F) Differentially expressed transcription
factors comparing control with MLN4924-treated embryos. Down-regulated, yellow; up-regulated, dark blue. (G) The enriched protein family of differentially
expressed transcription factors. "C" represents "Control" and "T" represents "1
μ
M MLN4924 treatment". (For interpretation of the references to color in this gure
legend, the reader is referred to the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
6
Fig. 3. Neddylation inhibition affected the zygotic genome activation (ZGA) process of 2-cell embryos. (A) Venn analysis of the ZGA genes and DEGs. ZGA genes,
yellow; DEGs, blue. (B) The cluster analysis of DEGs related to ZGA. (C) The relative mRNA levels of some ZGA genes, Gapdh was set as the internal reference gene.
*P <0.05, **P <0.01. (D) Fluorescence image of EU staining in the 2-cell embryos after MLN4924 treatment. EU, red; DNA, blue. Scale bar =20
μ
m. (E) Fluo-
rescence intensity of EU in the control (n =81 embryos) and MLN4924 treatment (n =81 embryos) groups, **P <0.01. (For interpretation of the references to color
in this gure legend, the reader is referred to the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
7
published RNA-seq data [15]. Venn analysis results showed that 1442
out of the 3959 DEGs identied in our study are maternal-related genes
(Fig. 4A). Maternal genes fall into three main categories according to the
degradation period: genes whose degradation mainly occurs during the
transition of GV oocytes to the zygote stage (Cluster 1), genes whose
degradation mainly occurs from the zygote to the 2-cell stage (Cluster 2),
genes whose degradation mainly occurs from GV oocytes to the 2-cell
stage (Cluster 3) [15]. Venn analysis results showed that Clusters 1, 2,
and 3 exhibited 27, 483, and 49 overlapping genes, respectively, with
783 up-regulated maternal effect genes (Fig. 4B). According to the above
maternal gene classication, the 483 genes identied in Cluster 2
overlapped with the 783 up-regulated maternal effect genes that should
be degraded in 2-cell stage embryos. However, RNA-seq data showed
that the expression of the representative genes from Cluster 2 (483
genes) were increased after neddylation inhibition, indicating that these
genes were not degraded at the 2-cell stage embryos (Fig. 4C). Our
qRT-PCR results were consistent with transcriptomic data (Fig. 4D),
further conrming these ndings. Overall, our results suggest that in-
hibition of neddylation leads to developmental arrest in 2-cell stage
embryos by disrupting the degradation of maternal effect genes.
3.5. Neddylation inhibition causes mitochondrial dysfunction in 2-cell
embryos
Transcriptome results showed that neddylation inhibition induced
the downregulation of several mitochondrial-related genes (Fig. 5A),
except for the Dnm1l gene, thus potentially interfering with mitochon-
drial function. Therefore, we investigated the distribution of mito-
chondria in 2-cell embryos, and found that in the control embryos,
mitochondria were evenly distributed in the cytoplasm, while mito-
chondria were aggregated and clustered in the MLN4924-treated em-
bryos (Fig. 5B). The rate of abnormal distribution was signicantly
higher in the MLN4924-treated embryos than that in the control em-
bryos (71.6 ±6.17% vs 9.68 ±1.87%, P <0.001, Fig. 5C). Next, we
investigated the changes of mitochondrial membrane potential (MMP)
by JC-1 staining. As shown in Fig. 5D–E, MMP was signicantly lower in
the MLN4924-treated embryos than that in the control embryos (P <
0.001). Moreover, the ATP content was signicantly lower in the
MLN4924-treated embryos compared to the control embryos (Fig. 5F, P
<0.05). These ndings suggest that neddylation inhibition results in
mitochondrial dysfunction.
Fig. 4. Neddylation inhibition impaired the degradation of maternal effect genes in 2-cell embryos. (A) Venn analysis of the DEGs and maternal genes. Maternal
genes, yellow; DEGs, blue. (B) Venn analysis of up-regulated maternal genes of DEGs and cluster 1–3 genes. (C) The cluster analysis of DEGs related to maternal
genes. (D) The relative mRNA levels of several maternal genes, Gapdh was set as the internal reference gene. *P <0.05, **P <0.01, ***P <0.001. (For interpretation
of the references to color in this gure legend, the reader is referred to the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
8
Fig. 5. Neddylation inhibition resulted in mitochondrial dysfunction in 2-cell embryos. (A) The cluster analysis of DEGs related to mitochondrial function. (B)
Fluorescence image of mitochondria staining after MLN4924 treatment. Mitochondria, red. Scale bar =20
μ
m. (C) The proportion of embryos with abnormal
mitochondria in the control (n =93 embryos) and MLN4924 treatment (n =89 embryos) groups. ***P <0.001. (D) Fluorescence image of JC-1 staining after
MLN4924 treatment. Scale bar =100
μ
m. (E) The uorescence intensity of Red/Green in the control (n =95 embryos) and MLN4924 treatment (n =114 embryos)
groups. ***P <0.001. (F) The changes of ATP content (fold change) in the control (n =90 embryos) and MLN4924 treatment (n =90 embryos) groups. *P <0.05.
(For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
9
3.6. Neddylation inhibition induces oxidative stress and DNA damage in
2-cell embryos
Mitochondrial dysfunction has been reported to induce ROS accu-
mulation. RNA-seq data showed that the expression of several genes
required for the oxidation-reduction process and oxidoreductase activity
was altered in the MLN4924 treatment group (Fig. 6A). Considering this,
we investigated whether ROS was elevated in 2-cell embryos upon
treatment with the neddylation inhibitor. As expected, uorescence
staining results showed that ROS levels were globally increased (P <
0.001, Fig. 6B and D). Moreover, our results indicated that superoxide
anion was also signicantly stronger in the MLN4924-treated embryos
than that in the control embryos (P <0.001, Fig. 6C and E). These results
suggest that neddylation inhibition induces oxidative stress in 2-cell
embryos.
Since the cluster analysis results indicated that the expression of
several DNA damage-related genes were changed (Fig. 6F), we specu-
lated that neddylation inhibition might cause DNA damage in 2-cell
embryos. To test this, we determined the levels of γH2AX immunou-
orescence as a measure of DNA damage in 2-cell embryos. As shown in
Fig. 6G–H, strong γH2AX uorescence signals were observed in the
nucleus of MLN4924-treated embryos compared to the control group (P
<0.001), suggesting that neddylation inhibition results in DNA damage
in 2-cell embryos.
4. Discussion
Preimplantation embryonic development is a sensitive period sus-
ceptible to the adverse effects of various internal and external stimuli,
which can trigger developmental arrest. Precise regulation of gene
transcription and translation in early mouse embryonic development is
essential. Post-translational modications (PTMs) are critical for early
embryonic development because transcription of the zygote genome is
non-existent during these stages [6,16,17]. Neddylation is an important
ubiquitin-like PTM, which appears to be a good candidate as an
important player during preimplantation mouse embryo development.
The present study documents that the intracellular location of Nedd8
changes during early stages of embryonic development, implying that
neddylation may play an important role in regulating early embryonic
development in mice. Treatment with MLN4924, a specic inhibitor of
neddylation, resulted in the arrest of embryonic development in vitro,
conrming the idea that neddylation may play an important role in
regulating early embryonic development. Furthermore, transcriptome
analysis revealed that MLN4924 treatment altered gene expression
proles in 2-cell stage embryos. Bioinformatics analysis using GO and
KEGG jointly showed that the DEGs were mainly involved in tran-
scription regulator activity, RNA transport, and RNA polymerase, which
are related to the maternal-to-zygotic transition.
The maternal-to-zygotic transition consists of two important molec-
ular processes, the degradation of maternal effect genes and zygotic
genome activation (ZGA) [1]. In our study, the inhibition of neddylation
resulted in the loss of nuclear-specic localization of NEDD8 at the 2-cell
stage. Notably, we found that transcription factor activity, RNA poly-
merase, and homeobox transcription factor family, which have been
recently reported to participate in ZGA [3,4], were enriched after ned-
dylation inhibition. Combing Venn analysis and EU staining, we
conrmed that inhibition of neddylation adversely affects ZGA. On the
other hand, clearance of maternal mRNA is important for early embry-
onic development and impairment of this clearance process can lead to
developmental defects or even death of the embryo [18]. Our results
revealed that neddylation inhibition disrupted the degradation of
maternal effect genes, especially degradation of a portion of
ZGA-dependent maternal effect genes. As far as we know, this is the rst
study to reveal that neddylation inhibition can delay the MZT in the
mouse. Previously, Yang et al. demonstrated that neddylation inhibition
resulted in poor embryo quality and failure of blastocyst hatching [12].
In bovine embryos, MLN4924 treatment delayed initiation of embryonic
genome activation (EGA) as evidenced by the reduction of EGA markers
[11], results which indirectly support our ndings. Besides neddylation,
previously reports indicate that other PTMs including phosphorylation,
methylation, and ubiquitination are involved in the MZT [6], suggesting
that the MZT is regulated by several PTMs.
Mitochondria are vital for numerous physiological processes, such as
energy production, metabolite synthesis, calcium signaling, as well as
cell proliferation and death [19]. A recent study demonstrated that
neddylation is involved in the regulation of morphology, trafcking, and
function of mitochondria [20]. Herein, cluster analysis based on
RNA-seq data indicated that MLN4924 treatment downregulated the
expression of mitochondrial function-related genes. Moreover,
MLN4924 treatment altered the intracellular localization of mitochon-
dria and decreased the membrane potential of mitochondria and ATP
content. All the above data suggest that neddylation inhibition induces
mitochondrial dysfunction of 2-cell stage embryos. Previous studies
suggest that mitochondrial dysfunction adversely affects embryonic
development and ZGA [21]. Notably, our ndings indicate that neddy-
lation inhibition-induced ZGA impairment may be associated with
mitochondrial dysfunction. However, the exact effects and related
mechanisms of neddylation inhibition on the mitochondrial activity are
complicated and remain to be further investigated.
Mitochondria are the main sources of ROS in living cells. Appropriate
levels of ROS are required for oocyte maturation, fertilization, cell di-
vision and blastulation [22,23]. Oxidative stress occurs when there is an
imbalance between ROS production and consumption [24], and an in-
crease in ROS has been reported to impair preimplantation embryonic
development exhibiting development arrest at the 2-cell stage and a
signicant decrease in blastocyst formation [25]. Since neddylation in-
hibition led to abnormal mitochondrial function, we further examined
the intracellular oxidative stress. Our results showed that MLN4924
treatment caused signicantly higher levels of superoxide anion and
ROS, which were accompanied by altered expression levels of oxidative
stress-related genes. A recent study reports that inhibition of neddyla-
tion resulted in the activation of oxidative stress, poor mouse embryo
quality, and blastocyst hatching failure [12]. These results are consistent
with the possibility that neddylation inhibition impairs early embryonic
development through oxidative stress.
ROS can affect adversely genomic and mitochondrial DNA, thus
leading to various types of DNA damage. DNA damage in turn can affect
the integrity of the biological genome, activating complex DNA repair
mechanisms to avoid further DNA damage [26]. DNA double-strand
break (DSB) is one of the most severe form of DNA damage, capable
of causing genetic mutations and impairing embryo development. In this
study, elevated levels of γH2AX were observed in MLN4924-treated
embryos, suggesting neddylation inhibition resulted in DNA damage,
which could lead to arrest of early embryonic development. Jia et al.
reported that MLN4924 treatment triggered DNA damage response as
observed by the formation of γH2AX, ultimately resulting in senescence
in cancer cells [27]. Remarkably, ROS can induce intracellular DNA
damage, while DNA damage in return can elevate ROS levels. Therefore,
neddylation plays a complex, rather than a simple, regulatory role in
embryonic development.
5. Conclusion
Our ndings reveal multiple roles of neddylation in early embryonic
development. Specically, neddylation inhibition causes developmental
arrest of mouse preimplantation embryos by altering gene expression
and impairing the MZT. Moreover, neddylation inhibition results in
mitochondrial dysfunction, oxidative stress, and DNA damage. Further
studies should reveal how downstream target genes of Nedd8 are spe-
cically affected during the MZT.
M. Liu et al.
Theriogenology 220 (2024) 1–11
10
Fig. 6. Neddylation inhibition induced oxidative stress and DNA damage in 2-cell embryos. (A) The cluster analysis of DEGs related to oxidation-reduction process
and oxidoreductase activity. (B) Fluorescence image of ROS staining after MLN4924 treatment. ROS, green. Scale bar =50
μ
m. (C) Fluorescence image of superoxide
anion staining after MLN4924 treatment. Superoxide anion, red. Scale bar =100
μ
m. (D) The uorescence intensity of ROS in the control (n =81 embryos) and
MLN4924 treatment (n =85 embryos) groups. ***P <0.001. (E) The uorescence intensity of superoxide anion in the control (n =78 embryos) and MLN4924
treatment (n =80 embryos) groups. ***P <0.001. (F) The cluster analysis of DEGs related to DNA damage. (G) Fluorescence image of γH2AX staining after MLN4924
treatment. γH2AX, red; DNA, blue. Scale bar =20
μ
m. (H) The uorescence intensity of γH2AX in the control (n =68 embryos) and MLN4924 treatment (n =65
embryos) groups. ***P <0.001. (For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.)
M. Liu et al.
Theriogenology 220 (2024) 1–11
11
Funding information:
Fundamental Research Funds for the Central Universities
(2662023DKPY001) and National Natural Science Foundation of China
(32072729). JSD is supported by a Senior Research Career Scientist
Award from the Department of Veterans Affairs.
Data availability
Data will be made available on request.
CRediT authorship contribution statement
Mingxiao Liu: Writing – review & editing, Validation, Methodology,
Investigation, Formal analysis, Data curation. Zhiming Ding: Writing –
original draft, Supervision, Methodology, Investigation, Conceptualiza-
tion. Peihao Sun: Project administration, Investigation. Shuo Zhou:
Investigation. Hanxiao Wu: Investigation. Lijun Huo: Resources,
Conceptualization. Liguo Yang: Supervision, Conceptualization. John
S. Davis: Supervision, Conceptualization. Aixin Liang: Writing – review
& editing, Writing – original draft, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing nancial
interests or personal relationships that could have appeared to inuence
the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.theriogenology.2024.02.029.
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