Inhibition of Fatty Acid Synthase Reduces Blastocyst Hatching through Regulation of the AKT Pathway in Pigs
Inhibition of Fatty Acid Synthase Reduces Blastocyst Hatching through Regulation of the AKT Pathway in Pigs
Jing Guo 0 1
Nam-Hyung Kim 0 1
Xiang-Shun Cui 0 1
0 Department of Animal Sciences, Chungbuk National University , Chungbuk, Cheongju , Republic of Korea
1 Editor: Qing-Yuan Sun, Institute of Zoology Chinese Academy of Sciences , CHINA
Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of longchain fatty acids. During oncogenesis, FASN plays a role in growth and survival rather than acting within the energy storage pathways. Here, the function of FASN during early embryonic development was studied using its specific inhibitor, C75. We found that the presence of the inhibitor reduced blastocyst hatching. FASN inhibition decreased Cpt1 expression, leading to a reduction in mitochondria numbers and ATP content. This inhibition of FASN resulted in the down-regulation of the AKT pathway, thereby triggering apoptosis through the activation of the p53 pathway. Activation of the apoptotic pathway also leads to increased accumulation of reactive oxygen species and autophagy. In addition, the FASN inhibitor impaired cell proliferation, a parameter of blastocyst quality for outgrowth. The level of OCT4, an important factor in embryonic development, decreased after treatment with the FASN inhibitor. These results show that FASN exerts an effect on early embryonic development by regulating both fatty acid oxidation and the AKT pathway in pigs.
Data Availability Statement; All relevant data are within the paper
Funding: This research was supported by Basic
Science Research Program through the National
Research Foundation of Korea (NRF) funded by the
Ministry of Education (No.
2015R1D1A1A01057629), and the
NextGeneration BioGreen 21 Program (PJ011126),
Rural Development Administration, Republic of
Korea. The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Fatty acid synthase (FASN) is a key enzyme catalyzing the de novo synthesis of long-chain fatty
acids from acetyl-CoA and malonyl-CoA. Fatty acids (FAs) are essential constituents of lipids
involved in membrane biogenesis and are critical substrates in energy metabolism. There are
two sources of FAs: exogenous FAs and endogenous FAs. The biosynthesis of endogenous FAs
is catalyzed by FASN[
]. The synthesis of FAs by FASN is initiated by the conversion of
acetyl-CoA to malonyl-CoA. Malonyl-CoA is then used for FA synthesis and is involved in
elongation. FAs are important constituents of sphingolipids, ceramides, and glycolipids and
are involved in many biological processes[
]. Under normal conditions, FASN-synthesized
FAs are stored as triacylglycerols and are catabolized through FA oxidation (FAO) when
]. De novo FA synthesis is very active during embryogenesis and plays a critical role in
In some cases, FASN contributes to growth and survival rather than the energy storage
pathway. FASN inhibition impairs DNA replication, causing cell cycle arrest before the G1
Competing Interests: The authors have declared
that no competing interests exist.
phase through mechanisms involving p21, p27, BRCA1, and NFκB[
]. Furthermore, FASN
inhibition induces tumor cell apoptosis through the down-regulation of AKT and suppression
of p53 function[
]. In addition, during the menstrual cycle, FASN and E2-ER signaling
control endometrial cell proliferation[
FASN studies primarily focus on its role in cancer biology. Thus, the function of FASN in
early embryonic development is poorly understood. In this study, C75, a pharmacological
inhibitor of FASN, was used to study the role of FASN in embryogenesis. C75 is a
ceruleninderived synthetic FASN inhibitor and has been used in many previous studies [
inhibits purified mammalian FASN by blocking its KS domain. Specific depletion of
FASN by RNAi leads to loss of sensitivity to C75, confirming that C75-induced damage is
dependent on inhibition of FASN activity[
]. Here, we hypothesized that FASN might be
involved in porcine embryonic development either through its action in lipid metabolism or
through other pathways. C75 was used to determine the function of FASN in embryogenesis
and to elucidate the mechanisms involved. Our results show that FASN plays critical roles
during embryonic development via its regulatory functions in FA synthesis and the AKT pathway.
Materials and Methods
All chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA),
unless otherwise indicated.
2.1. Oocyte collection, in vitro maturation, and embryo culture
All animal studies were performed in strict accordance with institutional guidelines and prior
approval was obtained from the Institutional Animal Care and Use Committee (IACUC) of
Chungbuk National University.
Ovaries from prepubertal gilts were obtained from a local slaughterhouse and transported
in saline at 37ÊC to the laboratory. Follicles 3±6 mm in diameter were aspirated.
Cumulusoocyte complexes (COCs) surrounded by more than three layers of cumulus cells were selected
]. COCs were isolated from follicles and washed three times in TL-HEPES.
COCs were cultured in tissue culture medium 199 (TCM 199) supplemented with 10% porcine
follicular fluid, 0.1 g/L sodium pyruvate, 0.6 mM L-cysteine, 10 ng/mL epidermal growth
factor, 10 IU/mL luteinizing hormone, and 10 IU/mL follicle stimulating hormone at 38.5ÊC for
44 h in a humidified atmosphere of 5% CO2. After maturation, cumulus cells were removed by
treatment with 0.1% hyaluronidase and repeated pipetting. For activation of parthenogenesis,
oocytes with polar bodies were selected, activated by two direct current pulses of 1.1 kV/cm
for 60 μs, and then incubated in porcine zygote medium (PZM-5) containing 7.5 μg/mL of
cytochalasin B for 3 h. Finally, embryos were cultured in PZM-5 for 8 d at 38.5ÊC in a
humidified atmosphere with 5% CO2. On the 5th day, fetal bovine serum was added to the medium
for a final concentration of 10%. To determine the effect of FASN on early porcine embryonic
development after embryo activation, the FASN inhibitor C75 was added to the medium at
final concentrations of 10 or 20 μM. The 10-μM concentration was used in the following
experiments as it represents the minimum concentration inducing an effect on blastocyst
2.2. ATP content assay
The ATP contents from 30 blastocysts per treatment group were measured using an ATP
Determination Kit (Invitrogen, Carlsbad, CA, USA). Briefly, samples were washed three times
with PBS and then transferred individually into 1.5-mL tubes. Media were removed and
blastocysts were frozen and thawed for lysis. Approximately 100 μL of ice-cold somatic cell reagent
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(FL-SAR) was added to each tube, and samples were incubated in an ice-water bath for 5 min.
Thereafter, 100 μL of ice-cold assay buffer (diluted 1:25 with ATP assay buffer, FL-AAB) was
added and the tubes were maintained at room temperature for 5 min under limited light
conditions. The ATP concentration was measured using a luminometer (Berthold, Wildbad,
Germany) with a sensitivity of 0.01 pmol. The ATP concentration in the control group was
arbitrarily set at 1. Three separate experiments were performed.
2.3. Membrane potential assay and mitochondrial copy number analysis
Day-8 blastocysts were washed three times with phosphate buffered saline/polyvinyl alcohol
(PBS/PVA) and incubated in culture medium containing 1 mM
5,5',6,6'-tetrachloro-1,1',3,3'tetraethyl-imidacarbocyanine iodide (JC-1) (Invitrogen) at 37ÊC in a humidified atmosphere
of 5% CO2 for 30 min. Membrane potential was calculated as the ratio of red florescence,
which corresponds to activated mitochondria (J-aggregates), to green fluorescence, which
corresponds to less-activated mitochondria (J-monomers)[
]. Fluorescence was visualized with
an epifluorescence microscope (Nikon Corp., Tokyo, Japan). The fluorescence intensity in the
control group was arbitrarily set at 1, and the fluorescence intensities in the treatment groups
were measured and expressed as relative values with respect to the control group. Three
separate experiments were performed, and 10 blastocysts were examined per experiment.
Total DNA was isolated from 10 blastocysts using the Puregene DNA Isolation Kit
(Invitrogen) according to the manufacturer's instructions. Blastocyst DNA samples were then used for
real-time polymerase chain reaction (PCR) experiments. The primers Ndufaf3 and Gapdh are
described in a previous study[
]. The reactions were performed as follows: 95ÊC for 3 min
followed by 40 cycles of 95ÊC for 15 s, 60ÊC for 20 s, and 72ÊC for 20 s, and a final extension at
72ÊC for 5 min. The relative quantification of the mitochondrial copy numbers was performed
using the 2-ΔΔCt method. Three separate experiments were performed, with three replicates per
2.4. Reactive Oxygen Species (ROS) staining
Blastocysts were incubated for 15 min in IVC medium containing 10 μM
2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) at 37ÊC. After incubation, 30 blastocysts per group
were washed three times in IVC medium and transferred to PBS drops covered with paraffin
oil in a polystyrene culture dish. The fluorescent signal was captured using an epifluorescence
microscope. The fluorescence intensity in the control group was arbitrarily set at 1, and the
fluorescence intensity in the treatment group was measured and expressed as a relative value
with respect to the control group.
2.5. Terminal deoxynucleotidyltransferase-mediated 2'-deoxyuridine 5'triphosphate (dUTP) nick-end labeling (TUNEL) assay
The blastocysts were collected after C75 treatment, fixed in 3.7% paraformaldehyde for 15 min
at room temperature, and permeabilized in 0.5% Triton X-100 for 1 h at 37ÊC. The embryos
were incubated with fluorescein-conjugated dUTP and the terminal
deoxynucleotidyltransferase enzyme (In Situ Cell Death Detection Kit, Roche; Mannheim, Germany) for 1 h at 37ÊC.
The embryos were washed three times in PBS/PVA, treated with Hoechst 33342 for 5 min,
washed three times in PBS/PVA, and mounted onto glass slides. Images were captured using a
confocal microscope (Zeiss LSM 710 META, Jena, Germany).
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2.6. Immunofluorescence and confocal microscopy
Embryos were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized
with PBS/PVA containing 0.5% Triton X-100 at 37ÊC for 1 h, and then incubated in PBS/PVA
containing 3.0% bovine serum albumin at 37ÊC for 1 h. Subsequently, the embryos were
incubated overnight at 4ÊC with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK),
anticytochrome C (ab110325, 1:100; Abcam, Cambridge, UK), anti-pAKT (9271, 1:100; Cell
Signaling Technology, Danvers, MA, USA), anti-p53 (sc6243, 1:100; Santa Cruz, CA, USA), or
anti-OCT4 (sc8628, 1:100; Santa Cruz, CA, USA) antibodies. After washing three times in
PBS/PVA, the oocytes and embryos were incubated at 37ÊC for 1 h with either goat anti-rabbit
IgG or rabbit anti-goat IgG. The oocytes and embryos were then stained with Hoechst 33342
for 5 min, washed three times in PBS/PVA, mounted onto slides, and examined using a
confocal microscope (Zeiss LSM 710 META, Jena, Germany). Images were processed using Zen
software (version 8.0, Zeiss, Jena, Germany).
2.7. 5-Bromo-deoxyuridine analysis
The rate of cell proliferation was analyzed using 5-bromo-deoxyuridine (BrdU). Blastocysts
were incubated in 100 μM BrdU for 6 h and then washed 3 times in PBS/PVA. Embryos were
fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/
PVA containing 0.5% Triton X-100 at 37ÊC for 30 min, and treated with 1N HCl at room
temperature for 30 min. Blastocysts were incubated in PBS/PVA containing 3.0% bovine serum
albumin at 37ÊC for 1 h and then with anti-BrdU at 4ÊC overnight. After washing in PBS/PVA
5 times, blastocysts were incubated at 37ÊC for 1 h with goat anti-mouse antibody. Finally,
blastocysts were stained with Hoechst 33342 for 5 min, mounted onto slides, and examined
with a confocal microscope.
2.8. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
Day-8 blastocysts were collected. mRNA was extracted from 10 blastocysts per group using a
DynaBeads mRNA Direct Kit (Dynal Asa, Oslo, Norway) according to the manufacturer's
instructions. cDNA was obtained by reverse transcription of mRNA using the Oligo(dT)12-18
primer and SuperScript III Reverse Transcriptase (Invitrogen Co., Grand Island, NY, USA).
The amplification cycles were as follows: 95ÊC for 3 min followed by 40 cycles of 95ÊC for 15 s,
60ÊC for 30 s, and 72ÊC for 20 s, and a final extension at 72ÊC for 5 min. The primers used in
the study were listed in Table 1. Relative gene expression was normalized to internal porcine
Gapdh mRNA level using the 2-ΔΔCt method.
2.9. Quantitative RT-PCR for microRNA analysis
microRNA (miRNA) was obtained from day-8 blastocysts using the High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Ten blastocysts per
group were lysed in RT master mix containing 3.61 μL of H2O, 0.5 μL of 10× RT Buffer, 5-plex
stem-loop RPs, and 0.065 μL of RNase inhibitor, followed by incubation at 95ÊC for 5 min.
The RT mixture contained 4.3 μL of RNA, 0.065 μL of RNase inhibitor, 0.335 μL of MMLV
RT, and 0.25 μL of dNTP RT-PCR. The amplification was performed using the following
steps: 16ÊC for 30 min, followed by 60 cycles of 20ÊC for 30 s, 42ÊC for 30 s, and 50ÊC for 1 s,
and a final step at 85ÊC for 5 min. Quantitative RT-PCR for each miRNA was carried out in
20-μL reaction mixtures that included 1 μL of RT product, 2× SYBR mix, 1 μL of primers, and
8 μL of H2O. Amplification parameters used for quantitative RT-PCR were consistent with the
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Product size (bp)
manufacturer's protocol (KAPA Biosystems, Korea). The primers used in this study were listed
in Table 2. miRNA expression was normalized to internal U6 level using the 2-ΔΔCt method.
2.10. Statistical analysis
All data were analyzed using the one-way analysis of variance and Chi-square test embedded
in Statistical Package for the Social Sciences (SPSS). Each experiment was performed in
triplicate and differences were considered significant if P < 0.05.
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Fig 1. Effect of FASN inhibition on porcine early embryonic development. (A) Effect of increasing FASN
inhibitor concentrations on embryonic development. (B) Blastocyst formation and hatching rates. (C) mRNA
expression of hatching-related genes is shown in the presence or absence of the FASN inhibitor. *P < 0.05,
**P < 0.01, ***P < 0.001. BL:Blastocyst; HB: Hatching Blastocyst. Scale bar: 200μm.
3.1 Depletion of FASN impairs porcine embryonic development
To understand the role of FASN during porcine early embryonic development, we used its
inhibitor, C75. Parthenotes were treated with C75 at different doses. As shown in Fig 1A and
1B, there was no significant difference in blastocyst development rates between the control
and the 10-μM treatment group (59.55 ± 7.47% vs. 47.52 ± 12.40%). However, blastocyst
development decreased significantly in the 20-μM C75 treatment group (25.90 ± 6.23%). In
addition, the rate of blastocyst hatching was significantly lower in the presence of 10 μM and
20 μM C75 than in the control group (, 12.48 ± 0.46%,0 vs 22.90 ± 0.08%). Therefore, FASN
was confirmed as a critical component in early embryonic development. The expressions of
hatching-related genes were also reduced in the treatment groups compared with those in the
control (Fig 1C). A FASN inhibitor concentration of 10 μM was used for further experiments.
3.2 Inhibition of FASN affects FAO
Because of the important role of FASN in FA synthesis, the ATP content was assessed after the
inhibition of FASN. First, the mitochondrial membrane potential and copy number were
examined. No significant differences in membrane potential were observed following the
addition of the FASN inhibitor (Fig 2A and 2B). However, the mitochondrial copy number was
significantly lower after treatment with the FASN inhibitor (Fig 2C). We also detected a lower
ATP content in the control group than in the FASN-inhibitor group (P < 0.05), as shown in
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Fig 2. Effect of the FASN inhibitor on fatty acid oxidation. (A) Red fluorescence corresponds to activated mitochondria and green fluorescence
corresponds to less-activated mitochondria. (B) Membrane potential was measured as the ratio of red fluorescence to total fluorescence. (C) The
relative mtDNA copy number is shown. (D) The ATP content was measured in both the control and treatment groups. (E) The mRNA expression level
of Cpt1 is shown in the presence or absence of C75. *P < 0.05, **P < 0.01. Scale bar: 200μm.
Fig 2D. Cpt1 is the limiting factor in FAO and is a target of FASN. The mRNA expression level
of Cpt1 also decreased when embryos were incubated with the FASN inhibitor, which further
confirmed the role of FASN in FAO (Fig 2E). Therefore, FASN can affect FAO through the
regulation of Cpt1.
3.3 Inhibition of FASN increases ROS
The inhibition of FASN has a negative effect on FAO, and FAO can induce ROS formation.
Embryos were treated with H2DCF-DA to determine the impact of FASN inhibition on ROS
production. As shown in Fig 3A and 3B, the fluorescence intensity significantly increased after
treatment with C75 (P < 0.001), indicating an increase in oxidation activity. The mRNA
expression levels of ROS-related genes were examined. Mnsod, Gpx1, and Tfam showed
significantly lower expression in treated blastocysts than in control blastocysts (Fig 3C), which
confirmed the impact of FASN inhibition on ROS production.
3.4 Inhibition of FASN induces cell apoptosis
The embryonic development is influenced by apoptosis; therefore, we checked whether FASN
could affect the apoptosis process. The rate of apoptosis was calculated as the ratio between the
number of TUNEL-positive nuclei and the total cell number of nuclei. The rate of apoptosis
increased significantly in the FASN-inhibitor treated group (Fig 4A and 4B). We also observed
decreased expression of the anti-apoptotic genes Bcl2 and Bcl-xl, and the increase of the
apoptotic gene Casp3 (Fig 4C), suggesting that FASN may have a role in modulating apoptosis
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Fig 3. FASN inhibition causes an increase in ROS. (A) ROS staining in porcine blastocysts for various
concentrations of FASN inhibitor. (B) Green fluorescence intensity was measured. (C) mRNA expression of
Gpx1, Mnsod, and Tfam in blastocysts with or without FASN inhibitor. *P < 0.05, ***P < 0.001. Scale bar:
during embryogenesis. Furthermore, apoptosis can induce cell autophagy. The expression
level of LC3 was examined after treatment with the FASN inhibitor. Immunofluorescence
staining showed a significant decrease in fluorescence intensity and the mRNA expression
level of LC3 was also significantly reduced (Fig 4D and 4E).
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Fig 4. Effect of FASN inhibition on apoptosis and autophagy. (A) TUNEL-positive cells were detected in the presence or
absence of the FASN inhibitor. (B) The apoptotic rate is shown. (C) mRNA expression of Bcl2, Bcl-xl, and Casp3 in blastocysts
with or without FASN inhibitor. (D) Laser scanning confocal microscopy images of immunostaining for the LC3 protein in porcine
blastocysts after treatment with the FASN inhibitor. (E) Relative fluorescence intensity of LC3 is shown. *P < 0.05, **P < 0.01.
Scale bar: 50μm.
3.5 FASN inhibition causes release of mitochondrial cytochrome C
Cytochrome C is a component of the electron transport chain in mitochondria. The release of
cytochrome C from the mitochondria into the cytoplasm is involved in the initiation of
apoptosis. The increased release of cytochrome C into the cytoplasm following C75 treatment was
assessed by colocalization of cytochrome C with the mitochondria and is shown in Fig 5.
Taken together, these data show that the inhibition of FASN causes the release of cytochrome
C, which induces the initiation of apoptosis.
3.6 FASN influences apoptosis through the AKT pathway
AKT is an important target of FASN, and FASN regulates a number of biological processes
through the AKT pathway. We hypothesized that the inhibition of FASN induces apoptosis
through the AKT pathway. Immunofluorescence staining showed that pAKT protein levels
decreased after the parthenotes had been treated with the FASN inhibitor (Fig 6A and 6B). p53
is a downstream product of the AKT pathway and an increased expression of p53 results in
poor developmental potential. We observed that p53 protein levels increased significantly in
the FASN inhibitor-treated group (Fig 6C and 6D). Therefore, these results suggest that the
inhibition of FASN induces apoptosis through the AKT pathway.
3.7 FASN inhibition decreases cell proliferation
AKT-mediated phosphorylation may affect the expression of Oct4, an important gene in
]. FASN inhibition led to a decrease in OCT4 expression (Fig 7A,
7B and 7C). The Oct4 gene is a marker of pluripotency. The expression levels of other
pluripotency-related genes were also evaluated. The inhibition of FASN caused a reduction in the
expression of Nanog, but had no effect on Sox2 (Fig 7C). These results indicate that FASN
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Fig 5. Effect of FASN inhibition on the co-localization of cytochrome C and mitochondria. (A) The localizations of
both cytochrome C and mitochondria detected by immunostaining. (B) The co-localization of cytochrome C and
mitochondria is shown. (C) The level of co-localization was examined using Pearson's correlation. *P < 0.05. Scale bar:
inhibition may influence OCT4 expression via the AKT pathway, and may also affect
The AKT pathway also influences cell proliferation. Therefore, the effect of FASN on
proliferation was measured through BrdU staining (Fig 7D). The percentage of cells undergoing
DNA synthesis was calculated by dividing the number of BrdU positive nuclei by the total cell
number. As shown in Fig 7E, the percentage of proliferative cells was lower in the presence of
FASN inhibitor. Therefore, FASN inhibition can block cell proliferation through the AKT
To investigate the relationship between FASN and miRNA content, the expression of
implantation-related miRNAs was determined. The miR-19 level remained unchanged after
treatment with the FASN inhibitor, whereas the levels of both miR-92 and miR-let7a decreased
significantly (Fig 7F).
Our results showed that FASN inhibition blocks porcine embryonic development by causing a
decrease in ATP content and inducing apoptosis. Therefore, we have shown that FASN acts as
a critical regulator of blastocyst formation and hatching. We describe a molecular mechanism
by which the FASN inhibitor induces the decrease in FA through the down-regulation of Cpt1.
In addition, the regulation of apoptosis by FASN occurs via the AKT-p53 pathway.
FAs provide twice as much ATP as carbohydrates. FASN-synthesized FAs can be utilized
]. In this study, FASN inhibition caused a decrease in ATP content through
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Fig 6. FASN inhibition triggers the activation of the AKT-p53 pathway. (A) Staining of pAKT showed activation and expression of pAKT. (B) The
relative intensity of pAKT is shown as bars. (C) Laser scanning confocal microscopy images of immunostaining of p53. (D) Relative intensity of p53
was measured. *P < 0.05. Scale bar: 50μm.
Cpt1 down-regulation during embryonic development. ATP content is paramount for
embryonic development through its regulation of mitosis, blastocyst formation, and hatching [19±
21]. The inhibition of FASN leads to the accumulation of malonyl-CoA, which suppresses
Cpt1 during FAO[
]. Cpt1 is the rate-limiting enzyme in FAO, and enables the transport of
long-chain FA into the mitochondria[
]. In addition, Cpt1 has been shown to have an
Silencing FASN causes a decrease in palmitic acid synthesis leading to the induction of
apoptosis and formation of ROS[
]. In addition, the inhibition of FASN also induces the
accumulation of NADPH, which in turn promotes the activation of ROS-generating enzymes such
]. The excessive production of ROS results in DNA and protein damage, thereby
affecting the oocytes and embryos [
]. The analysis of ROS-related genes further confirmed
FASN increases ROS generation.
Apart from its action on metabolism, FASN is also involved in a number of additional
processes. Although FASN is always considered as a key synthase in fatty acid synthesis,
sometimes FASN play a role rather than fatty acid synthesis process. The AKT was a known target
of the FASN, and AKT was involved in several biological signal pathways. In this study, we
checked the function of FASN in embryonic development. We found that the FASN affect the
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Fig 7. Effect of FASN inhibition on pluripotency. (A) Laser scanning confocal microscopy images of OCT4
protein immunostaining in porcine blastocysts with or without FASN inhibitor. (B) Relative intensity of p53 was
measured. (C) Relative Oct4, Nanog, and Sox2 mRNA expression levels are shown as bars. (D) BrdU
incorporation in blastocysts after treatment with the FASN inhibitor was examined. (E) Graph summarizing
relative proliferation rates. (F) Expression of microRNAs after treatment with the FASN inhibitor. Data are
presented as relative expression levels. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 50μm.
porcine early embryonic development mainly through the AKT-p53 pathway. Many studies
have demonstrated that FASN regulates AKT, which is a known target of FASN[
inhibition of the AKT pathway causes a decrease in blastocyst hatching. Therefore, we
hypothesize that FASN affects embryonic development via the AKT pathway. p53, which is
associated with apoptosis, is one of the downstream products of the AKT pathway. FASN
inhibition effectively initiates apoptosis by enabling p53. Many studies have also demonstrated
that p53 induces growth arrest and apoptosis after DNA damage[
]. TUNEL staining and
analysis of apoptosis-related gene expression confirmed the effect of FASN on apoptosis.
Furthermore, the co-localization of cytochrome C and the mitochondria showed that FASN
inhibition induces the release of cytochrome C from the mitochondria. Many apoptotic factors
stimulate the release of cytochrome C, which, in turn, initiates the apoptosis pathway.
Studies have provided insight into the crosstalk between apoptosis and autophagy. The
antiapoptotic proteins belonging to the BCL2 family bind Beclin-1 to suppress autophagy.
Moreover, the caspase-mediated cleavage of autophagy-related proteins inhibits autophagy[
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Fig 8. Schematic representation of the function of FASN during porcine early embryonic development. FASN is essential for the embryonic
development in pigs. FASN impairment causes the inhibition of FAO through the regulation of Cpt1. Cpt1 is a key factor in the FAO process via its
influence on the transport of FAs into the mitochondria. In addition, the inhibition of FASN can affect embryonic development through the AKT-p53
pathway, potentially inducing apoptosis. AKT can also influence cell proliferation through the regulation of pluripotency.
results demonstrate that FASN inhibition induces apoptosis, leading to the initiation of
Many studies have described the relationship between FASN and proliferation.
FASNdependent FA synthesis is required for cell proliferation[
]. FASN inhibition causes
NADPH accumulation, which plays an important role in sustaining cell growth and
proliferation. Extracellular ATP-induced proliferation also requires the AKT pathway[
embryonic development, FASN inhibition can also reduce cell proliferation. However, the
mechanism by which FASN influences cell proliferation needs further study.
AKT is not only an anti-apoptosis and cell survival factor, but also a key regulator of
]. AKT-mediated phosphorylation of Oct4 is related to the proliferation of
stem-like cancer cells. As expected, FASN inhibition yielded a faint staining for OCT4 and
a decrease in Oct4 mRNA levels. In addition, apoptosis also generates a decrease in OCT4
miRNAs are non-coding RNAs that regulate many biological processes, including
embryonic development [39±42]. miRNAs play an important role in the maternal-conceptus
]. FASN inhibition causes changes in the expression of two miRNAs, miR-19 and
miRlet7a. Both miR-92 and miR-let7a show decreased expression in blastocysts from male factor
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infertility or polycystic ovaries compared with blastocysts obtained from fertile women[
Additionally, let-7a can regulate the implantation potential of activated blastocysts[
In conclusion, FASN plays a critical role in porcine early embryonic development. FASN
exerts its embryogenesis function through two pathways. It provides ATP through
FASNdependent FAO and it affects apoptosis and survival through the AKT pathway (Fig 8).
This research was supported by Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education (No.
2015R1D1A1A01057629), and the Next-Generation BioGreen 21 Program (PJ011126), Rural
Development Administration, Republic of Korea.
Conceptualization: N-HK X-SC JG.
Data curation: JG.
Formal analysis: JG.
Funding acquisition: N-HK X-SC.
Project administration: JG.
Writing ± original draft: JG.
Writing ± review & editing: JG.
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