International Journal of

Molecular Sciences

Review
The Role of BDNF, YBX1, CENPF, ZSCAN4, TEAD4, GLIS1
and USF1 in the Activation of the Embryonic Genome in
Bovine Embryos
Bingnan Liu 1,† , Jiaxin Yan 1,† , Junjie Li 1,2 and Wei Xia 1,2, *

1 College of Animal Science and Technology, Hebei Agricultural University, Baoding 071000, China;
[email protected] (B.L.); [email protected] (J.Y.); [email protected] (J.L.)
2 Research Center of Cattle and Sheep Embryo Engineering Technique of Hebei Province,
Baoding 071000, China
* Correspondence: [email protected]
† These authors contributed equally to this work.

Abstract: Early embryonic development relies on the maternal RNAs and newly synthesized proteins
during oogenesis. Zygotic transcription is an important event occurring at a specific time after
fertilization. If no zygotic transcription occurs, the embryo will die because it is unable to meet the
needs of the embryo and continue to grow. During the early stages of embryonic development, the
correct transcription, translation, and expression of genes play a crucial role in blastocyst formation
and differentiation of cell lineage species formation among mammalian species, and any variation
may lead to developmental defects, arrest, or even death. Abnormal expression of some genes may
lead to failure of the embryonic zygote genome before activation, such as BDNF and YBX1; Decreased
expression of CENPF, ZSCAN4, TEAD4, GLIS1, and USF1 genes can lead to embryonic development
failure. This article reviews the results of studies on the timing and mechanism of gene expression of
these genes in bovine fertilized eggs/embryos.

Keywords: bovine; zygotes/embryos; genetic program; zygotic transcription

Citation: Liu, B.; Yan, J.; Li, J.; Xia, W.

The Role of BDNF, YBX1, CENPF,
ZSCAN4, TEAD4, GLIS1 and USF1 in 1. Introduction
the Activation of the Embryonic
The prerequisites for pregnancy require oocyte maturation, successful fertilization,
Genome in Bovine Embryos. Int. J.
and the acquisition of a high-quality embryo. Most infertility in both humans and animals
Mol. Sci. 2023, 24, 16019. https://
is caused by alterations in various developmental regulatory factors, such as mutations in
doi.org/10.3390/ijms242216019
multiple developmental regulators. These causes may result in impaired oocyte maturation,
Academic Editor: Alfonso failure of fertilization, or cessation of embryo development at an early stage. Spermatogonia
Gutiérrez-Adán undergo proliferation, growth, and maturation stages to develop into mature spermatozoa,
Received: 30 August 2023
but to achieve fertilization; they also need to undergo the “acrosome reaction” and the ener-
Revised: 31 October 2023
gization stage, while the development of the primordial oocyte into a “competent” egg cell
Accepted: 1 November 2023 is even more difficult. The maturation of the oocyte is not continuous; after undergoing the
Published: 7 November 2023 proliferative process of mitosis, it undergoes meiosis, but this does not allow it to develop to
maturity, and after undergoing the first meiosis, the follicle ruptures and is expelled. At this
point, the oocyte is not capable of fertilization and needs to undergo a second meiosis to
develop into a capable fertilization oocyte. After fertilization in mammals, the fertilized egg
Copyright: © 2023 by the authors. undergoes cleavage and division to develop into a blastocyst. During this period, there were
Licensee MDPI, Basel, Switzerland.
three important developmental events: chromosome group activation, zygotic genome acti-
This article is an open access article
vation (ZGA), polarization/compression, and the first specification of the cell lineage [1,2].
distributed under the terms and
Two distinct cell types emerge, the inner cell mass (ICM) and the trophoblastic ectoderm
conditions of the Creative Commons
(TE), which subsequently form the blastocyst and then continue to differentiate. Most
Attribution (CC BY) license (https://
animals, including humans, are influenced by maternal genes prior to ZGA, and embryonic
creativecommons.org/licenses/by/
development changes from maternal factor-dependent to embryonic factor-dependent
4.0/).

Int. J. Mol. Sci. 2023, 24, 16019. https://doi.org/10.3390/ijms242216019 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 16019 2 of 12

after ZGA. However, all processes involved in cell differentiation, as well as in a range of
embryonic development, are closely linked to changes in gene expression and histones. In
mammals, the correct transcription, translation, and expression of genes play a pivotal role
in the formation of blastocysts and the differentiation of cell lineage species. During the
development of bovine embryos, different genes are expressed at different stages, and the
expression of these genes is regulated by many factors, including transcription factors and
epigenetic modifications. The study of these genes and their regulatory mechanisms will
lead to a better understanding of the genetic program of bovine embryonic development.
Many maternal genes are synthesized and accumulated in the oocyte and play a key role
in early embryonic development. Among them, the maternal effector genes (MEGs) refer
to the genes that play an important role in maintaining the survival and development of
mammalian embryos during the cleavage stage after fertilization. For example, knocking
out NOBOX in bovine embryos significantly reduced not only the blastocyst rate but also
the expression of pluripotent genes (POU5F1/OCT4 and NANOG) and the number of cells
in the inner cell mass within the blastocyst [3]. ZNFO has intrinsic transcriptional repressor
activity and is another maternally derived oocyte-specific nuclear factor essential for early
bovine embryonic development. Knockdown of ZNFO by siRNA significantly reduced
embryo development to the 8–16 cell stage and blastocyst stage [4]. Maternal genes can
support early embryonic development before the syn gene is activated and then begins
to degrade. Previous studies have found that nearly 90% of maternal genes are degraded
when cattle congenital gene activation occurs at the 8–16 cell stage [5]. Since the embryonic
genome is transcribed up to the ZGA stage, it is likely that the accumulation of maternal
factors (proteins and mRNAs) during oocyte development has led to the recording of this
phenomenon. In addition, the requirement for extensive epigenetic reorganization prior to
embryo implantation is associated with the pluripotency and activation of the embryonic
genome. Transcription of the zygotic genome is required for the normal development
and differentiation of early animal embryos, and ZGA-related factors are essential for the
transcriptional regulation of the zygotic genome. For instance, OCT-4, a transcription factor
that plays a pivotal role in maintaining the pluripotency of embryonic stem cells (ESCs), has
been demonstrated to elicit a diminished rate of blastocyst development in bovine embryos
with targeted knockdown of OCT-4 via siRNA injection [6,7]. It has been confirmed that
the knockdown of SOX2, an additional transcription factor associated with embryo quality
and essential for maintaining embryonic pluripotency, leads to a reduction in the number
of blastocysts and blastomeres in cattle [8–10]. Knockdown of TSPY by microinjection in
bovine fertilized eggs had no effect on the female embryo population but caused male
embryo development to stop before the blastocyst stage [11].
In recent years, an increasing number of genes associated with oocyte maturation
and early embryonic development have been identified. In order to successfully develop
into healthy offspring, growing and maturing mammalian oocytes and embryos must
undergo matching gene expression and epistatic modifications before they can develop
into new life. Therefore, knowledge of key genes and epistatic modifications for normal
development before implantation is essential to ensure normal gametogenesis, normal
fertilization and maintenance of high fecundity rates, and normal early embryogenesis in
domestic animals. Compared with natural mating and artificial insemination, the success
rate of in vitro production (IVP) of bovine embryos to establish pregnancy is low, and
oocyte quality is a key factor limiting the success of animal pregnancy. Therefore, in order
to better understand the genetic regulation of early embryo development and improve the
efficiency of in vitro embryo production, we summarized the genetic factors that have been
found to inhibit oocyte maturation in recent years.

2. Adverse Effects of Abnormal Gene Expression on ZGA

2.1. BDNF (Brain-Derived Neurotrophic Factor) Is Associated with Egg Maturation
BDNF is detected in numerous mammals, including ovaries and oocytes, and holds
significance in the progression of mammalian follicles, ovulation, proliferation of granulosa
Int. J. Mol. Sci. 2023, 24, 16019 3 of 12

cells, fertilization, and subsequent embryonic development [12–16]. A variety of animals

(such as rodents, sheep, cattle, etc.) can secrete high-affinity neurotrophins (NTs) in the
ovaries, and there are p75NTR receptors, which play an important role in signal transduc-
tion. High-affinity receptors for neurotrophin-4/5 (NT-4/5) and BDNF are important in
early follicular growth and oocyte survival, and BDNF is secreted by cumulus and granu-
losa cells, so it is speculated that they can affect oocyte maturation and early embryonic
development in many species [17–19].
Zhao et al. first used immunofluorescence staining to detect the expression pattern of
BDNF and its receptors during early buffalo embryonic development and found that BDNF
expression first increased and peaked at the 4-cell stage and then gradually decreased
(Figure 1) [20]. The mRNA manifestation profile of BDNF was akin to that of its receptor
NTRK2, and the manifestation of an alternative receptor, p75, was significantly limited
in comparison to NTRK2. The high synchronization of the two suggests that both are
involved in follicular development. When buffalo oocytes were treated with K-252a and
p75 inhibitor (pep5) for in vitro maturation, the reaction rate was significantly reduced
when BDNF and receptor inhibitor K-252a were added simultaneously, demonstrating that
K-252a can eliminate the effect of BDNF on oocyte maturation, receptor NTRK2 works
with BDNF during maturation of buffalo oocytes The receptor NTRK2 acts with BDNF
during oocyte maturation, while the receptor p75 does not seem to play a role in oocyte
maturation. Therefore, it is concluded that p75, as a low-factor receptor, may not function
primarily in buffalo granulosa cells.
The high synchronization of BDNF with its receptor NTRK2 suggests that both are
involved in follicle development, so it is speculated that the enhancement of the devel-
opmental capacity of buffalo embryos may be related to BDNF. A discovery was made
that the utilization of 10 ng/mL BDNF greatly enhanced the rate of oocyte maturation and
blastocyst formation in buffalo embryos using in vitro fertilization. Nevertheless, as the
concentration increased to 100 ng/mL, there was a decline in the blastocyst rate, indicating
a two-way effect of BDNF [20]. To further study the mechanism of BDNF on cumulus
cells, the mRNA expression changes of apoptosis-related genes and BDNF receptor genes
during IVM and the selection of developmental genes in cumulus cells were analyzed by
RT-qPCR. Further study on the mechanism of BDNF’s action on cumulus cells during IVM
finally found that BDNF can down-regulate apoptosis-related genes CASP9 and FAS. The
expression levels of receptor-related genes NTRK2 and cumulus cell development-related
genes CCNB1, PCNA, GJA4, GJA1, HAS2, PTX3, and TNFAIP6 were up-regulated, which
enhanced the proliferation of cumulus cells, promoted the expansion of cumulus cells, and
thus improved the maturation rate of buffalo oocytes. It can also promote the proliferation
of cumulus cells.

2.2. Y-Box Binding Protein 1 (YBX1) Reduces Attenuation of Damaging Maternal Genes
YBX1 has a significant function in the stabilization of RNA and regulation of tran-
scription. This particular gene encodes a protein with a cold shock domain that is highly
conserved and possesses broad binding properties toward nucleic acids. The encoded
protein serves as a multifaceted nucleic acid-binding macromolecule, demonstrating its
involvement in manifold cellular endeavors encompassing the orchestration of transcrip-
tion and translation, intricate pre-mRNA splicing, intricate DNA reparation, and delicate
mRNA packaging. Additionally, this protein is an integral constituent within messenger
ribonucleoprotein (mRNP) formations, suggesting potential implications in the intricate
realm of microRNA processing [21]; Y-Box Binding Protein is reported to be enriched in
oocytes [22,23] and recognized as primary constituents of cytoplasmic messenger ribonu-
cleoproteins (mRNPs) with a diverse array of RNA-binding capacities [24]. Deng et al.
used the public RNA-seq dataset to reanalyze the transcription level of YBX1 in bovine
embryos. Bovine YBX1 was progressively up-regulated from oocyte to blastula, and during
the early development of bovine embryos, YBX1 expression was significantly increased in
8-cell embryos [25] (Figure 1). YBX1 is highly expressed in bovine mature oocytes, and its
 ribonucleoproteins (mRNPs) with a diverse array of RNA-binding capacities [24]. Deng
et al. used the public RNA-seq dataset to reanalyze the transcription level of YBX1 in bo-
vine embryos. Bovine YBX1 was progressively up-regulated from oocyte to blastula, and
Int. J. Mol. Sci. 2023, 24, 16019 during the early development of bovine embryos, YBX1 expression was significantly 4 of 12
in-
creased in 8-cell embryos [25] (Figure 1). YBX1 is highly expressed in bovine mature oo-
cytes, and its expression is further increased after fertilization, especially during MZT, as
confirmed
expressionby is Grurgia et al. [26].after fertilization, especially during MZT, as confirmed by
further increased
In order
Grurgia et al.to[26].
further study the effect of YBX1 on embryonic development ability, siRNA
injection was performed
In order to knock
to further study out the
the effect of expression of YBX1 and
YBX1 on embryonic observe the
development expression
ability, siRNA
changes
injectionofwas
YBX1. A total of
performed to 5154
knockdifferentially expressed
out the expression genesand
of YBX1 (DEGs) were
observe obtained
the expressionby
using
changesDESeq2.
of YBX1.It was found
A total of that
5154in the embryosexpressed
differentially with low genes
YBX1 (DEGs)
knockdown,
were the number
obtained by
of 2-cell
using and 4-cell
DESeq2. stage
It was blocked
found embryos
that in increased,
the embryos andYBX1
with low the percentage
knockdown, of the
blastocysts
number
of 2-cell and
decreased 4-cell stageAmong
significantly. blocked embryos
them, 1623 increased, and the percentage
and 3531 up-regulated of blastocysts
and down-regulated
decreased
genes weresignificantly.
enriched in the Among
regionsthem,
that 1623 and RNA
regulate 3531 up-regulated
splicing and RNAand down-regulated
stability. More-
genesawere
over, largeenriched
numberinofthe regions
genes that regulate
related RNA were
to Z-decline splicing and RNA
changed, andstability. Moreover,
these results all
a large number
indicated that YBX1 of genes related to
knockdown Z-decline
would lead towere changed, and
the impairment ofthese resultssplicing
alternative all indicated
(AS)
thatRNA
and YBX1stability
knockdown would
during ZGA, lead to theasimpairment
as well of alternative
the attenuation splicing
of maternal mRNA (AS) and RNA
[25].
stability during ZGA, as well as the attenuation of maternal mRNA [25].

Figure 1. Description of expression patterns of BDNF and YBX1mRNA. The horizontal coordinate
Figure 1. Description
represents of embryonic
the stages of expressiondevelopment;
patterns of BDNF and YBX1mRNA.
The ordinate The horizontal
only represents coordinate
the rise and fall; there
represents the stages of embryonic development; The ordinate only represents the rise and fall; there
is no actual value.
is no actual value.
3. Adverse Effects of Abnormal Gene Expression on Zygotic Genome Activation
3.3.1.
Adverse Effects of Centromeric
Down-Regulation Abnormal Gene Expression
Protein F (CENPF)on Zygotic
May Cause Genome Activation
the Embryo to Stagnate at the
8-Cell Stage Formatting of Mathematical Components
3.1. Down-Regulation Centromeric Protein F (CENPF) May Cause the Embryo to Stagnate at
the 8-Cell
CENPFStage is Formatting
a component of Mathematical Components
of the centromere-centromeric complex, and the expression of
this CENPF
gene results in the production of a protein that is
is a component of the centromere-centromeric complex,linked to this complex.
and theThis protein
expression
plays a critical role in the differentiation of somatic cells. It was found
of this gene results in the production of a protein that is linked to this complex. This pro-that the number of
CENPF
tein plays mRNA decreased
a critical gradually
role in the from 2 to
differentiation of8somatic
cells stage,
cells.and all of
It was themthat
found originated
the number from
the mother line. The transcription of CENPF in embryo begins at
of CENPF mRNA decreased gradually from 2 to 8 cells stage, and all of them originated the late stage of 8 cells,
so it can be assumed that after the appearance of EGA, its gene expression
from the mother line. The transcription of CENPF in embryo begins at the late stage of begins to rise
8
again and remains basically unchanged until the blastocyst stage (Figure
cells, so it can be assumed that after the appearance of EGA, its gene expression begins to 2) [27]. To investi-
gateagain
rise the impact of CENPF
and remains on embryonic
basically unchanged development potential,stage
until the blastocyst Tereza et al. 2)
(Figure employed
[27]. To
CENPF-specific double-stranded RNA (dsRNA) to suppress
investigate the impact of CENPF on embryonic development potential, Tereza et al. the corresponding mRNAem-
expression and found that the embryos showed obvious developmental abnormalities until
ployed CENPF-specific double-stranded RNA (dsRNA) to suppress the corresponding
the EGA stage and found that the most common defects in the embryos inoculated with
mRNA expression and found that the embryos showed obvious developmental abnor-
CENPF dsRNA were: The size varies, the edge of the blastomere is blurred, the part of
malities until the EGA stage and found that the most common defects in the embryos
the blastomere becomes transparent, there are obvious nuclear fragments in the embryo,
inoculated with CENPF dsRNA were: The size varies, the edge of the blastomere is
even there is no nucleus in the blastomere, only less than 1/3 of the embryos developed to
blurred, the part of the blastomere becomes transparent, there are obvious nuclear frag-
8 cells [28]. CENPF plays an important role in the aggregation, arrangement, and separation
ments in the embryo, even there is no nucleus in the blastomere, only less than 1/3 of the
of chromosomes because CENPF plays an important role in the interaction between cen-
embryos developed to 8 cells [28]. CENPF plays an important role in the aggregation, ar-
tromere and microtubule in somatic cells [29]. The protein functions as a vital component
rangement, and separation of chromosomes because CENPF plays an important role in
of the nuclear framework during the G2 stage of interphase. As G2 nears its conclusion,
the interaction between centromere and microtubule in somatic cells [29]. The protein
the protein establishes connections with the kinetochore and maintains this interaction
functions as a vital component of the nuclear framework during the G2 stage of inter-
until early anaphase. It is found in the spindle midzone during late anaphase and the
phase. As G2 nears
intracellular bridgeitsduring
conclusion, the protein
telophase establishes
and is thought connections
to undergo with the degradation.
subsequent kinetochore
The precise pattern of localization for this protein suggests its potential role in promot-
ing chromosome segregation during mitosis. Studies in humans and other animals have
shown that CENPF silencing affects cell division by disrupting the chromosome division
process [30,31]. The research results of cattle also showed that blastomere abnormalities, to
a certain extent, can also be understood as related to the process of chromosome division,
 and maintains this interaction until early anaphase. It is found in the spindle midzone
during late anaphase and the intracellular bridge during telophase and is thought to un-
dergo subsequent degradation. The precise pattern of localization for this protein suggests
its potential role in promoting chromosome segregation during mitosis. Studies in hu-
Int. J. Mol. Sci. 2023, 24, 16019 mans and other animals have shown that CENPF silencing affects cell division by disrupt-
5 of 12
ing the chromosome division process [30,31]. The research results of cattle also showed
that blastomere abnormalities, to a certain extent, can also be understood as related to the
process
but of chromosome
the specific division,
mechanism but the through
or pathway specific mechanism or pathway
which the impact through
on bovine which
embryos
the impact on bovine
remains to be explored.embryos remains to be explored.

Figure
Figure 2. Description of
2. Description of expression
expression patterns
patterns of
of CENPF,
CENPF, ZSCAN4,
ZSCAN4, TEAD4,
TEAD4, GLIS1,
GLIS1, and
and USF1mRNA.
USF1mRNA.
The horizontal
horizontal coordinate
coordinate represents
representsthe
thestage
stageofofembryonic
embryonicdevelopment;
development;The
Theordinate
ordinateonly
onlyrepre-
rep-
resents
sents thethe rise
rise and
and fallfall
andand has
has nono actual
actual value.
value.

3.2. Zinc Finger

3.2. Zinc Finger and and SCAN
SCAN Domain
Domain Containing
Containing 44 (ZSCAN4)
(ZSCAN4) May May Result
Result in in 16-Cell
16-Cell Phase
Phase
Growth Arrest
Growth Arrest
ZSCAN4 is an extraordinary gene responsible for encoding a protein that participates
ZSCAN4 is an extraordinary gene responsible for encoding a protein that participates
in the upkeep of telomeres. This remarkable gene plays a pivotal role in a crucial attribute
in the upkeep of telomeres. This remarkable gene plays a pivotal role in a crucial attribute
of mouse ESs, namely, defying cellular senescence and maintaining normal karyotypes
of mouse ESs, namely, defying cellular senescence and maintaining normal karyotypes
for many cell divisions in culture. Initially, its presence was discovered to be exclusive to
for many cell divisions in culture. Initially, its presence was discovered to be exclusive to
the late 2-cell stage of the pre-implantation embryo in mice [32,33]. Knockout of ZSCAN4
the late
with 2-cell stage
interfering of the pre-implantation
RNA(siRNA) results in delayed embryo in mice [32,33].
progression at the 2–4Knockout of ZSCAN4
cell stages, leading
to the failure of embryo implantation [32]. However, the expression status andleading
with interfering RNA(siRNA) results in delayed progression at the 2–4 cell stages, role of
to the failure
ZSCAN4 in theofpre-implantation
embryo implantation [32]. However,
development of bovinethe expression
embryos remains status and Kazuki
unclear. role of
ZSCAN4 in the
TAKAHASHI et al.pre-implantation
investigated thedevelopment
necessity of the of development
bovine embryos of the remains
ZSCAN4 unclear.
gene
Kazuki TAKAHASHI et al. investigated the necessity
before implantation in bovine embryos. They found that the expression of ZSCAN4of the development of the ZSCAN4
gene before
remained in aimplantation
low pattern until in bovine embryos.
the 4th cell stage They found thatafter
but increased the the
expression of ZSCAN4
4th cell stage, where
remained in a low pattern until the 4th cell stage but increased
the expression level was significantly increased at the 4–8 cell stage until the embryonic after the 4th cell stage,
where the expression
transcription level peaked level atwas
thesignificantly
16th cell stage increased
(Figure at 2).the 4–8 cell stage
In addition, it wasuntil the that
found em-
bryonic transcription
ZSCAN4 level peaked
increased significantly at theat4–8thecell
16th celldue
stage stage
to de(Figure 2). In addition,
novo synthesis of ZSCAN4 it wasin
found that
zygotes [34].ZSCAN4 increased
Additionally, they significantly
cultured bovine at the 4–8 cellthat
embryos stage
weredue to de novo
injected synthesis
with ZSCAN4-
of ZSCAN4
siRNA in zygotes
in vitro. [34]. Additionally,
The results demonstratedthey thatcultured
all embryosbovine embryos that
encountered werearrest
growth injected at
with ZSCAN4-siRNA in vitro. The results demonstrated that
the 16-cell stage, with only a limited number progressing to the blastocyst stage. Moreover, all embryos encountered
growth
the arrest atassessed
researchers the 16-cell thestage,
mRNA with onlyofa developmental
levels limited numberpluripotency-associated
progressing to the blastocyst gene
2stage. Moreover,
(DPPA2) the researchers
and Piwi-like assessedgene
RNA-mediated the mRNA
silencing levels of developmental
2 (PIWIL2) pluripo-
in 16-cell embryos
tency-associated
to evaluate the impact gene 2 of (DPPA2)
reducing andZSCAN4
Piwi-likeexpression
RNA-mediated gene silencing 2 (PIWIL2)
on reprogramming-associated
in 16-cell
gene embryosIttowas
transcripts. evaluate
foundthe impact
that PIWIL2 of reducing
expression ZSCAN4 expression
levels were reduced on inreprogram-
embryos
ming-associated
injected gene transcripts.
with ZSCAN4-siRNA. It washas
Research found that PIWIL2
indicated that the expression
Piwi proteinlevels and its were
linkedre-
duced in embryos injected with ZSCAN4-siRNA. Research
small RNAs, known as Piwi interacting RNAs (piRNAs), impede the transcription of late has indicated that the Piwi
protein and itsfactors
transposition linkedinsmall RNAs,
animal germ known
cells, as Piwi interacting
resulting in a notable RNAs (piRNAs), impede
upregulation of long
the transcription
terminal of late transposition
repeat retrotransposons factors
[35,36], among in animal germ cells,
them, pi-RNAs resulting
have in a notable
been shown to be
essential
upregulationfor targeted
of longelimination
terminal repeatof mRNA transcripts during
retrotransposons [35,36],theamong
[37]. Inthem,
addition, more
pi-RNAs
biological
have been reactions
shown to occur duringfor
be essential thetargeted
transcription of ZSCAN4,
elimination of mRNA including
transcripts instantaneous
during the
expression of other
[37]. In addition, moreZGA-specific groups, occur
biological reactions rapid during
expansion of telomere 5,
the transcription ofand blocking
ZSCAN4, in-
of translation of the entire protein [38,39]. It can be speculated
cluding instantaneous expression of other ZGA-specific groups, rapid expansion of that the down-regulation
of PIWIL2 expression will cause the dysfunction of other retrotransposons (including
transcriptional transposons) in bovine embryos, and thus halt the early development of
ZGA. However, the mechanism of the interaction between ZSCAN4 and PIWI-piRNA in
bovine embryos is unclear and remains to be investigated.
Int. J. Mol. Sci. 2023, 24, 16019 6 of 12

3.3. Interaction of TEA Domain Transcription Factor 4 (TEAD4) and CCN2

During embryonic development, TEAD4 plays an important role in organ formation
and development by regulating gene transcription. Studies have shown that TEAD4
deficiency leads to early embryo death and developmental deformities [40,41]. TEAD4
is a regulator of blastomere TE properties in mouse models and plays a key role in TE
differentiation [40,42], participating in a variety of life processes such as cell proliferation,
cell survival, tissue regeneration, and stem cell maintenance; TEAD4 potentially plays
a role in the development of porcine embryo blastocysts by modulating the activity of
SOX2, thereby influencing the conversion of morula into blastocyst [43]; bovine TEAD4
has a unique function to activate the specific pregnancy recognition factor interferon tau
in ruminants [44]. More studies have shown that TE is a single layer of epithelioid cells
surrounding the outer layer of the blastula, and the expression level of transcription factor
caudal homo box 2(CDX2) determines its development direction. During embryonic
development, CCN2 is involved in the regulation of many growth factors and extracellular
matrix interactions and has different expression patterns and effects in different organs and
tissues [42,45]. Therefore, CDX2 and CCN2 play important roles in blastocyst development.
In addition, CCN family 2(CCN2) is an important downstream gene of TEAD4, which is
widely used in mammalian somatic cells, and TEAD4 regulates the proliferation of CCN2 by
regulating its expression. Bovine TEAD4 was studied by Hiroki Akizawa et al. It was found
that TEAD4 mRNA was expressed in TE and ICM, and Tead4 mRNA content was higher
in TE than in ICM, and Tead4 was mainly expressed in TE nucleus. The initial amount of
TEAD4mRNA was low and increased from the 8-cell stage, and TEAD4 reached its peak
after the morula stage (Figure 2). By using short hairpin RNA (shRNA) to interfere with
the TEAD4 gene, TEAD4 was knocked out KD, and it was found that CDX2, GATA2, and
CCN2 genes were significantly reduced, and the change of CCN2 expression was the most
prominent [46]. When CCN2KD was performed in bovine embryos, TEAD4 expression
levels were significantly reduced in CCN2KD blastocysts. It is worth noting that the
expression level of TEAD4 in CCN2KD blastula also showed a significantly decreased trend.
Interestingly, neither TEAD4KD nor CCN2KD had an effect on cleavage development rate
or blastocyst formation in vitro, but CCN2KD led to a significant reduction in the ratio of TE
to ICM cell numbers without changing the total number of TE and ICM cells. Regulation of
cell composition in bovine blastula is related to the expression of CCN2 in TE; since bovine
CCN2 is expressed in endometrial epithelial cells, it is speculated that maternal CCN2
may influence pre-implantation development. These results indicate that TEAD4 and
CCN2 regulate and influence each other, and TEAD4 directly regulates the transcriptional
activation of CCN2, leading to changes in cell characteristics [47,48]. The expression of
CCN2 is also affected by TEAD4. The interaction between TEAD4 and CCN2 is important
for normal cell differentiation during pre-implantation development.

3.4. Deletion of GLI-Similar 1 (GLIS1) May Lead to ZGA Failure

GLIS1 is a transcription factor 15 residue closely related to the Gli family [49–51].
GLIS1 plays an important role in the formation and development of organs such as the
cardiovascular system, kidney, eye, thyroid, and pancreas and is considered to be a direct
recombinant coding factor that can promote the production of pluripotent stem cells and
is richly expressed in both unfertilized mouse oocytes and 1-cell stage embryos [52,53].
In addition, Glis1 also has the function of temporal and spatial regulation, so it can be
speculated that Glis1 regulates the embryonic development process in a certain period
of time [50,54]. Kazuki Takahashi et al. studied GLIS1 in bovine oocytes in vitro and
found that a large amount of the GLIS1 gene could be detected in both bovine oocytes and
embryos at stage 1 to 4 cells, and the GLIS1 gene decreased from the stage 1 cell to the
stage of 8 cells and beyond (Figure 2) [55]. By injecting Glis1-siRNA into bovine embryos
to investigate the relationship between the effects of bovine embryo development and the
downregulation of the GLIS1 gene, it was found that the injected embryos had no effect
on 16-cell stage development, but the rate of 32-cell stage development was significantly
Int. J. Mol. Sci. 2023, 24, 16019 7 of 12

reduced. In order to further explore the effect of GLIS1 downregulation on gene transcripts,
mRNA expressions of PGK1, PDHA1, heat shock homologous protein 70 (HSPA8), and
X non-live specific transcripts (XIST-) at cell stage 8–16 were detected. The expression of
PDHA1 and HSPA8 decreased significantly. PDHA1 is involved in glucose metabolism and
plays a key role in embryonic development, especially in cattle and mice [56,57]. HSPA8
encodes HSC70, which is involved in the pretreatment and selective autophagy of intron
RNA, so HSC70 knockdown will lead to a large number of cell death of various types.
In conclusion, GLIS1 down-regulated embryos may lead to the failure of ZGA initiation,
suggesting that GLIS1 may be an important factor in the pre-implantation development of
bovine embryos.

3.5. Upstream Stimulating Factor 1 (USF1) Gene Knockout Affects Early Embryonic Development
in Cattle
USF1 is a transcription factor with a basic helix-loop-helix structure that selectively
attaches to E-box DNA motifs. It is recognized as a cis-element of crucial genes responsible
for oocyte expression, which is vital for early embryonic and oocyte development [58]. Datta
T et al., therefore, first examined the expression patterns of USF1 in bovine oocytes and
embryos at different times. USF1 mRNA was found to increase during meiosis, increase
significantly during 2–8 cells, and then decrease until it is almost undetectable at the
blastocyst stage, indicating that it may play a role in embryo genome activation, indicating
that the gene is maternal in origin and may be consumed or degraded during embryo
genome activation (Figure 2). In order to investigate the role of USF1 in bovine oocyte and
embryonic development, the siRNA program was used to mediate gene silencing in bovine
embryos, and the abundance of USF1 transcripts was significantly reduced after injection
of USF1 siRNA. The study found that the total cleavage rate in bovine embryos after USF1
knockout had no effect but reduced the number of development to the 8–16 cell stage, and
in particular, the blastocyst rate was significantly reduced. In addition, these genes TWIST 2,
JY-1, GDF 9, and FST were found to carry USF1-binding elements (e-boxes) in their promoter
region necessary for the ability of bovine oocytes to develop. The abundance of TWIST2
and JY1 mRNA increased, but the abundance of GDF9 and FST transcripts decreased in
USF1 siRNA oocytes collected at the MII stage. The abundance of GDF9 transcripts was
moderately decreased, suggesting that negative control siRNA had a moderate off-target
effect on GDF9 expression. The transcription factor TWIST2 functions as a molecular switch,
which can either activate or suppress target genes by directly binding to conserved E-box
sequences in promoter regions and enlisting coactivators or suppressors [59]. As such,
USF1 has the potential to regulate the transcriptional levels of GDF9, FST, TWIST2, and
JY-1 during oocyte maturation.

4. Conclusions
After fertilization, mammals may require a specific object or abundant eggs for mRNA
transcription and protein synthesis to give them the ability to develop fully. In mice,
blastocyst formation is dependent on the presence of maternal factor(s) as mRNA in the egg
and also on syncytial genetic information [60,61]. The normal expression of these genes is
inextricably linked to the normal development of the blastocyst and the normal attachment
of the early embryo. Moreover, abnormal oocyte mRNA level abundance and pattern
of oocyte–follicle axis of development may lead to oocyte development failure and thus
affect later development [62]. Large mammals similar to mice, such as cattle and sheep,
have similar mechanisms. Among them, transcripts of genes required for oocytes and
zygotes of some domestic animals have been identified, and different levels of transcript
deletion cause developmental arrest and other developmental disorders at different stages,
including BDNF, GLS1, YBX1, GENPF, ZSCAN4, and TEAD4, and at which stage they play
a key role, as shown in Figure 3. A general pattern emerges from various studies: During
the continuous division and maturation of the primordial follicle, a series of transcriptome
changes occur. Two attenuation occurs during the period; the first is M-attenuation, starting
 and zygotes of some domestic animals have been identified, and different levels of tran-
script deletion cause developmental arrest and other developmental disorders at different
stages, including BDNF, GLS1, YBX1, GENPF, ZSCAN4, and TEAD4, and at which stage
they play a key role, as shown in Figure 3. A general pattern emerges from various studies:
During the continuous division and maturation of the primordial follicle, a series of tran-
Int. J. Mol. Sci. 2023, 24, 16019 scriptome changes occur. Two attenuation occurs during the period; the first is M-attenu- 8 of 12
ation, starting from the GVBD stage. The second attenuation from the MII phase is called
Z-attenuation; after fertilization, maternal mRNA will be heavily degraded via a key de-
velopmental
from the GVBD process
stage.known as maternal
The second to zygotic
attenuation transition
from the MII phase(MZT), in which
is called develop-
Z-attenuation;
mental
after control is transferred
fertilization, from maternally
maternal mRNA supplied
will be heavily gene products
degraded to products
via a key synthe-
developmental
process known
sized from as maternal
the zygotic to zygotic
genome, transition
resulting (MZT),ofinthe
in activation which developmental
syngeneic control
genome [63–66].
is transferred
Thus, from maternally
oocyte-derived mRNAs supplied genemay
and proteins products
play to
anproducts
important synthesized
role in thisfrom the
process
zygotic
[67]. genome, resulting in activation of the syngeneic genome [63–66]. Thus, oocyte-
derived mRNAs and proteins may play an important role in this process [67].

Figure 3. The process of the action of each gene is summarized. BDNF and YBX1 played an important
role before
Figure ZGA.
3. The CENPF
process of ZSCAN4,
the actionTEAD4
of eachGLIS1,
gene isand USF1 affect BDNF
summarized. the growth of the late
and YBX1 ZGA.
played an im-
portant role before ZGA. CENPF ZSCAN4, TEAD4 GLIS1, and USF1 affect the growth of the late
ZGA.For example, if BDNF is defective during the maturation and early embryonic devel-
opment of buffalo follicles and oocytes, the expression levels of related genes and receptor
genesForareexample,
dysregulatedif BDNFso that cumulus
is defective cells and
during their receptor
the maturation andNTRK2 cannot promote
early embryonic devel-
oocyte
opmentmaturation. However,
of buffalo follicles and after YBX1
oocytes, thegene knockdown,
expression levels ofit related
affects genes
the stability of AS
and receptor
and RNA, thus leading to the development defects of pre-ZGA
genes are dysregulated so that cumulus cells and their receptor NTRK2 cannot promote embryos. However, the
difference is that studies on pigs, sheep, mice, zebrafish, and other
oocyte maturation. However, after YBX1 gene knockdown, it affects the stability of AS species have shown
that
and the
RNA,development
thus leading of embryos is affected bydefects
to the development m6A modification, while the mechanism
of pre-ZGA embryos. However, the of
the effect on cattle has not been reported [68–71]. CENPF, ZSCAN4,
difference is that studies on pigs, sheep, mice, zebrafish, and other species have shown GLIS1, TEAD4, and
CDX2
that thealldevelopment
inhibited the development
of embryos isofaffected
early ZGA to varying
by m6A degrees. while
modification, For example, CENPF-
the mechanism
specific knockdown resulted in mRNA and protein silencing
of the effect on cattle has not been reported [68–71]. CENPF, ZSCAN4, GLIS1, TEAD4, during pre-implantation
development, disrupting the morphology of blastomere and inhibiting the development
and CDX2 all inhibited the development of early ZGA to varying degrees. For example,
of 8 cells after implantation. ZSCAN4 knockdown affected PIWIL2 mRNA level, which
CENPF-specific knockdown resulted in mRNA and protein silencing during pre-implan-
may affect the normal function of transposons and lead to a decrease in the number of
tation development, disrupting the morphology of blastomere and inhibiting the devel-
16-cell stage embryos. Down-regulation of GLIS1 affected the expression levels of PDHA1
opment of 8 cells after implantation. ZSCAN4 knockdown affected PIWIL2 mRNA level,
and HSPA8, thus inhibiting the embryonic development from the 16–32 cell stage. TEAD
which may affect the normal function of transposons and lead to a decrease in the number
and CDX2 should interact with each other to ensure the stable expression of the TE gene;
of 16-cell stage embryos. Down-regulation of GLIS1 affected the expression levels of
otherwise, the transcriptional regulation of pluripotency-related genes in bovine blastula
PDHA1 and HSPA8, thus inhibiting the embryonic development from the 16–32 cell stage.
TE and ICM cell lines will be affected, leading to the failure of embryonic development.
TEAD and CDX2 should interact with each other to ensure the stable expression of the TE
USF1, on the other hand, alters the developmental capacity of oocytes by affecting the
gene; otherwise, the transcriptional regulation of pluripotency-related genes in bovine
promoter-binding element E-box. In addition, the time of the first division after embryo
blastula TE and
fertilization ICM
is also cell lines
closely willtobethe
related affected,
normalleading to the failure
development of the of embryonic
embryo. If thedevel-
time
opment. USF1, on the other hand, alters the developmental capacity
interval of the first division after embryo fertilization is too long, the igf-1 ligand of oocytes by affect-
may
ingreduced
be the promoter-binding
or even absent,element and theE-box.
mRNA In abundance
addition, themaytimebeof the firstindivision
changed responseafter
to
embryo fertilization is also closely related to the normal development
the unfavorable growth environment, thus adversely affecting the development of the of the embryo. If
embryo [72,73]. The role of the fallopian tube in the development of the early embryo
should not be ignored, and the mechanism of embryo-fallopian tube interaction also af-
fects changes in transcription levels [74,75]. We can see that there is up-regulation and
down-regulation of gene expression before embryo implantation. During this period, no
matter how large or small the gene is, once the expression disorder occurs, the embryo
development will encounter problems. Therefore, the research on important genes related
to genes before embryo implantation is of great significance and also poses great challenges.
However, as technology advances and we learn more about genes and the mechanisms by
which they work, the more comprehensive the study will be.
Int. J. Mol. Sci. 2023, 24, 16019 9 of 12

Author Contributions: W.X. and J.L. conceived and designed this review. B.L. and W.X. wrote the
manuscript. J.Y. contributed to the revisions. All authors have read and agreed to the published
version of the manuscript.
Funding: Supported by grants from the National Key R&D Program of China (2021YFD1200401),
Biological Agriculture Joint Fund of Hebei Natural Science Foundation (C2023204007), Key Project
of Educational Commission of Hebei Province of China (ZD2022005), Project for overseas talents
in Hebei Province (ZD20220513), the Special Project for Talents Enrollment of Hebei Agricultural
University (YJ2021013) and Basic Research Funds for Colleges of Hebei province (KY2021006).
Institutional Review Board Statement: These studies were approved by the Hebei Agriculture
University Animal Research Ethics Committee (Baoding, Hebei, China).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors gratefully acknowledge all the teachers and students in the Research
Center of Cattle and Sheep Embryo Engineering Technique of Hebei Province.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations

TE Trophoblastic ectoderm
ICM Inner cell mass
ESCs Embryonic stem cells
MEGs Maternal effector genes
NOBOX Newborn ovary homeobox
OCT4 Octamer-binding transcription factor 4
ZNFO Krüppel-associated box (KRAB) containing zinc finger transcription factor
SOX2 SRY (sex determining region Y)-box 2
K-252a NTRK2 inhibitor
pep5 p75 inhibitor
K-252a NTRK2 inhibitor
TSPY Testis specific protein Y-encoded
BDNF Brain-derived neurotrophic factor
IVM Oocytes in vitro maturation
NTs Neurotrophins
YBX1 Y-Box Binding Protein 1
MZT Maternal-to-zygotic transition
AS Alternative Splicing
m6A N6-methyladenosine
CENPF Centromeric protein F
ZSCAN4 Zinc Finger and SCAN Domain Containing 4
PIWIL2 Piwi-like RNA-mediated gene silencing 2
DPPA2 Developmental pluripotency-associated gene 2
TE Trophoblast ectoderm
PGK1 Phosphor glycolate 15 kinase 1
PDHA1 Pyruvate dehydrogenase 1α
HSC70 Heat shock homologous protein 70
GLIS1 GLI-Similar 1
USF1 Upstream stimulating factor 1

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