Inhibition of Fatty Acid Synthase Reduces Blastocyst Hatching through Regulation of the AKT Pathway in Pigs

Fatty acid synthase (FASN) is an enzyme responsible for the de novo synthesis of long-chain 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.


Introduction
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 [1,2]. 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 [3]. FAs are important constituents of sphingolipids, ceramides, and glycolipids and are involved in many biological processes [4]. Under normal conditions, FASN-synthesized FAs are stored as triacylglycerols and are catabolized through FA oxidation (FAO) when necessary [5]. De novo FA synthesis is very active during embryogenesis and plays a critical role in embryonic development [6].
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 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 phase through mechanisms involving p21, p27, BRCA1, and NFκB [7,8]. Furthermore, FASN inhibition induces tumor cell apoptosis through the down-regulation of AKT and suppression of p53 function [9,10]. In addition, during the menstrual cycle, FASN and E 2 -ER signaling control endometrial cell proliferation [11].
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 [12,13]. C75 inhibits purified mammalian FASN by blocking its KS domain [14]. 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 [9,10]. 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.

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 37˚C 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 for culture [15]. 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 38.5˚C for 44 h in a humidified atmosphere of 5% CO 2 . 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 38.5˚C in a humidified atmosphere with 5% CO 2 . On the 5 th 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 formation.

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 (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.

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 37˚C in a humidified atmosphere of 5% CO 2 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) [16]. 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 [17]. The reactions were performed as follows: 95˚C for 3 min followed by 40 cycles of 95˚C for 15 s, 60˚C for 20 s, and 72˚C for 20 s, and a final extension at 72˚C 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 experiment.

Reactive Oxygen Species (ROS) staining
Blastocysts were incubated for 15 min in IVC medium containing 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H 2 DCF-DA) at 37˚C. 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.

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 37˚C. 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 37˚C. 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).

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 37˚C 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 37˚C for 1 h and then with anti-BrdU at 4˚C overnight. After washing in PBS/PVA 5 times, blastocysts were incubated at 37˚C 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.

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: 95˚C for 3 min followed by 40 cycles of 95˚C for 15 s, 60˚C for 30 s, and 72˚C for 20 s, and a final extension at 72˚C 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 H 2 O, 0.5 μL of 10× RT Buffer, 5-plex stem-loop RPs, and 0.065 μL of RNase inhibitor, followed by incubation at 95˚C 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: 16˚C for 30 min, followed by 60 cycles of 20˚C for 30 s, 42˚C for 30 s, and 50˚C for 1 s, and a final step at 85˚C 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 H 2 O. Amplification parameters used for quantitative RT-PCR were consistent with the 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.

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.

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.

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  Fig 2E). Therefore, FASN can affect FAO through the regulation of Cpt1.

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.

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 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).

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.

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.

FASN inhibition decreases cell proliferation
AKT-mediated phosphorylation may affect the expression of Oct4, an important gene in embryonic development [18]. 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 inhibition may influence OCT4 expression via the AKT pathway, and may also affect pluripotency. 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 pathway.
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).

Discussion
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 through FAO [5]. In this study, FASN inhibition caused a decrease in ATP content through Cpt1 down-regulation during embryonic development. ATP content is paramount for embryonic development through its regulation of mitosis, blastocyst formation, and hatching [19][20][21]. The inhibition of FASN leads to the accumulation of malonyl-CoA, which suppresses Cpt1 during FAO [22]. Cpt1 is the rate-limiting enzyme in FAO, and enables the transport of long-chain FA into the mitochondria [23]. In addition, Cpt1 has been shown to have an antiapoptotic function [24].
Silencing FASN causes a decrease in palmitic acid synthesis leading to the induction of apoptosis and formation of ROS [25]. In addition, the inhibition of FASN also induces the accumulation of NADPH, which in turn promotes the activation of ROS-generating enzymes such as NOX [26]. The excessive production of ROS results in DNA and protein damage, thereby affecting the oocytes and embryos [27]. 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 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 [12,28]. The inhibition of the AKT pathway causes a decrease in blastocyst hatching [29]. 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 [9,30]. 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 [31]. 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 [32]. Our results demonstrate that FASN inhibition induces apoptosis, leading to the initiation of autophagy.
Many studies have described the relationship between FASN and proliferation. FASNdependent FA synthesis is required for cell proliferation [26,33]. FASN inhibition causes NADPH accumulation, which plays an important role in sustaining cell growth and proliferation [34]. Extracellular ATP-induced proliferation also requires the AKT pathway [35]. During 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 pluripotency [18,36]. AKT-mediated phosphorylation of Oct4 is related to the proliferation of stem-like cancer cells [37]. 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 expression [38].
miRNAs are non-coding RNAs that regulate many biological processes, including embryonic development [39][40][41][42]. miRNAs play an important role in the maternal-conceptus interface [43]. FASN inhibition causes changes in the expression of two miRNAs, miR-19 and miR-let7a. Both miR-92 and miR-let7a show decreased expression in blastocysts from male factor infertility or polycystic ovaries compared with blastocysts obtained from fertile women [44]. Additionally, let-7a can regulate the implantation potential of activated blastocysts [45].
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).