Sirtuin inhibition leads to autophagy and apoptosis in porcine preimplantation blastocysts

Min Gyeong Kim, Duk Hyoun Kim, Hye Ran Lee, Jun Sung Lee, Su Jin Jin, Hoon Taek Lee

Abstract

Sirtuins are nicotinamide adenine dinucleotide dependent class III histone deacetylase proteins that play a crucial role in several cellular processes, including DNA repair, apoptosis, and lifespan. Previous studies have shown that sirtuin inhibition leads to embryonic developmental arrest and oxidative stress in porcine and murine. However, sirtuin-mediated mechanisms have not been examined in porcine preimplantation blastocysts. We therefore investigated the relationship between sirtuins and autophagy. Embryos were cultured with 100 μM sirtinol (SIRT1/2 inhibitor) in NCSU-23 media after in vitro fertilization. Treatment with sirtinol significantly reduced the rates of morula (21.34 ± 1.84 vs. 11.89 ± 2.01), blastocyst development (17.18 ± 1.81 vs. 9.00 ± 2.02), and total cell number (50.80 ± 1.47 vs. 37.71 ± 1.79), compared to controls, with an associating decrease the levels of Sirt2 transcript. Sirtinol treatment induced autophagy through an increase in LC3 transcript and LC3 protein. BECLIN1 and ATG5 expression showed a slight increase in treated group. Finally, treatment with sirtinol dramatically increased TUNEL indices (6.55 ± 0.84 vs. 11.44 ± 0.81) and fragmentation indices (0.33 ± 0.05 vs. 1.40 ± 0.30). BCL2L1 expression was lower, while Caspase-3 expression was significantly elevated in the sirtinol-treated group. Therefore, these findings suggest that sirtuins may elicit their effects through modifying autophagy and apoptosis, leading to developmental arrest and reducing the quality of porcine preimplantation embryos.

Keywords: Apoptosis; Autophagy; In vitro fertilization; Pig blastocyst; Sirtinol; Sirtuin.

1. Introduction

The human sirtuin family consists of seven members that act as nicotinamide adenine dinucleotide (NAD+) dependent class III histone deacetylases. They interact with a number of transcriptional factors to regulate cellular processes, such as DNA repair, apoptosis, lifespan, and stress response [1]. In previous studies, levels of Sirt1 to 3 mRNA were detected in porcine embryos for the first time and levels are lower in blastocysts relative to matured oocytes [2]. Sirt1 plays an important role in determining the quality of aged pig oocytes and is also involved in the inflammatory response of bovine embryos [3,4]. Sirt2 protects against spindle defects, chromosomal misalignment, and influences the process of germinal vesicle break-down in mouse oocytes [5,6]. Sirt3 is related to folliculogenesis and mitochondrial function against oxidative stress [7,8]. SIRT1, SIRT2, and SIRT3 proteins have each been localized to the nuclei, cytoplasm, and mitochondria of mouse embryos, respectively [8]. Especially, sirtuins play an essential role in the transition from the morula to blastocyst stage in porcine [2]. Treatment using sirtuin inhibitors in mice, such as nicotinamide, sirtinol, and BML-210, induces developmental arrest and increases intracellular reactive oxygen species [8]. Nicotinamide or sirtinol treatment during in vitro maturation suppresses porcine oocyte meiosis through disrupting actin cap and spindle formation [9]. Conversely, resveratrol (RSV) treatment as sirtuin activator increases porcine embryonic development after in vitro fertilization (IVF) [10]. Other studies have reported important roles for SIRT1 and SIRT2 in the regulation of autophagy through the deacetylation of ATG proteins and Forkhead box O (FoxO) family in the nucleus and cytoplasm [11]. Treatment with sirtinol also leads to the accumulation of autophagy- and apoptosis-related proteins, resulting in an increase in cell death in human MCF-1 cancer cells [12]. SIRT1 inhibition by sirtinol reduces autophagy by decreasing LC3-II expression via the mammalian target of rapamycin (mTOR) pathway in human THP-1 cells [13]. In other words, sirtuins have been shown to both suppress and induce autophagy, depending on the specific cellular microenvironment.
Autophagy is the cellular process of self-digestion, acting to remove targeted proteins and organelles under various environmental stresses. Autophagy is initiated by activation of the ULK1 complex, leading to phosphorylation of the BECLIN1-Vps34 complex via mTOR inhibition [14]. Expansion of the autophagosome is mediated by two ubiquitin-like conjugation systems. ATG5 participates in the first conjugation system, leading to formation of ATG12-ATG5-ATG16L complexes that are involved in LC3/ATG8 lipidation. Conversion of LC3I-phosphatidylethanolamine to its conjugated form (termed LC3-II) occurs in the autophagosomal membranes. LC3-II is a key protein involved in autophagosome formation and is found bound to targeted proteins with ubiquitinated proteins, resulting in protein degradation by lysosomal proteases [14]. It has previously been shown that autophagy is an important role in fertilization, the regulation of pro-survival pathways, and the endoplasmic reticulum (ER) stress response [15–17]. Meanwhile, autophagy is also closely related to apoptosis and type II cell death, in addition to pro-survival pathways [18]. For example, mitochondrial dysfunction and hyperglycemia increases LC3 protein levels and apoptosis in porcine parthenotes [19,20]. Modulation of autophagy also influences apoptosis, mitochondrial contents, abnormal autophagosome formation, and maternal mRNA degradation [21, 22]. However, whether sirtuins regulate autophagy via the prosurvival or cell death pathways has not yet been investigated in porcine IVF embryos.
Therefore, we hypothesized that sirtuins may influence porcine embryonic development by regulating autophagy. We investigated the relationship between sirtuins and autophagy, examining the effects of sirtuin inhibition using sirtinol (SIRT1/2 inhibitor) as a non-competitive inhibitor. We assessed developmental competence, levels of Sirt1-3 transcript, and autophagic- and apoptotic-related pathways, in porcine embryos, with or without sirtinol-treatment. This revealed that sirtuin inhibition may lead to developmental arrest and reduced embryo quality, which is mediated by autophagy and apoptosis.

2. Materials and methods

Unless otherwise specified, all chemicals were obtained from Sigma-Aldrich Co. (Sigma, St. Louis, MO, USA). Each experiment was repeated at least three times and embryos were distributed randomly among each group.

2.1. In vitro production of porcine embryos

Prepubertal ovaries were transferred from a local slaughterhouse maintaining the 32-36℃ of saline. Cumulus-oocyte complexes were aspirated from follicles and then matured in 500 µL of Tissue Culture Medium 199 with Earle’s salts (TCM-199; Gibco BRL, Grand Island, NY, USA) supplemented with 25 mM NaHCO3, 0.57 mM cysteine, 10% (v/v) porcine follicular fluid, 10 ng/mL epidermal growth factor, 0.5 µg/mL FSH (Folltropin V; Vetrepharm, Ontario, Canada), 1 mg/mL estradiol-17β and 0.22 µg/mL sodium pyruvate. They were then covered with mineral oil and incubated for 42-44 h at 39℃ in a humidified incubator with 5% CO2. Matured oocytes were partially denuded using 0.1% hyaluronidase and 20 oocytes were distributed into 50 µL droplets of fertilization medium (modified Tris-buffered medium (mTBM) supplemented with 1 mM caffeine sodium benzoate and 0.1% bovine serum albumin [BSA]). Ejaculated sperm were obtained from Darby Pig Breeding Co. (Anseong, Korea). Swim-up procedures were performed at 39℃ using Sp-TALP medium and injected to mTBM droplet at final sperm concentration of 5×105 cells/mL. Finally, oocytes were co-incubated with sperm for 6 h and zygotes were transferred to North Carolina State University-23 (NCSU-23) media containing 0.4% (w/v) essential fatty acid-free BSA for 7 days.

2.2. Isolation of mRNA and Real-time RT PCR

mRNA from embryos was isolated using a Dynabeads mRNA DIRECT Kit (Life technology, Oslo, Norway). Extracted mRNA was reverse transcribed into cDNA using a reverse transcription kit from Applied Biosystems (Applied Biosystems, Waltham, MA, USA). Primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/) or were obtained from previous studies. Real-time PCR was performed using a SYBR green premix Ex Taq (Takara, Otsu, Japan) containing TaKaRa Ex Taq HS, SYBR Green I, dNTP Mixture, Mg2+ and TliRNase H. PCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA). Relative quantification of gene expression was analyzed using 2-∆∆CT method. In total, 10-20 embryos were examined in each group, with four replicates per group. β-actin was used as an endogenous control and internal standard.

2.3. Immunocytochemistry

Embryos were fixed in 10% formaldehyde after removal of the zona pellucida with 0.5% pronase. Embryos were then permeabilized for 1 h with 1.0% Triton X-100 in DPBS at 39°C and blocked at 39°C for 1 h using 1% BSA in DPBS. Blocked embryos were incubated at 4°C with a polyclonal antiLC3 antibody (MBL international, Woburn, USA). FITC-labeled anti-rabbit IgG was used as the secondary antibody (Jackson Immunoresearch, West Grove, PA, USA). Nuclei were stained with TOPRO-3 iodide (Life technology, Grand Island, NY, USA). Images were collected using a confocal laser microscope (Carl Zeiss, Oberkochen, Germany) from at least three independent experiments. The intensity of LC3 staining was measured using ZEN software. At least 10 embryos were investigated, with 10 different positions analyzed for each embryo.

2.4. TUNEL assay

Fixed embryos were permeabilized for 1 h using 1.0% Triton X-100 and 0.1% sodium citrate in DPBS. Apoptotic cells were stained with terminal deoxynucleotidyl transferase and fluorescein dUTP for 1 h at 39°C using an in situ Cell Death Detection Kit (Roche, Indianapolis, IN). Nuclei were stained with 12.5 µg/mL Hoechst 33342. Embryos were then examined under fluorescence microscopy (Nikon
Corporation, Tokyo, Japan) and nuclei were characterized according to three morphological categories [16]. Briefly, these were (i) healthy nuclei stained with Hoechst 33342 (F-), but no TUNEL signal (T-), (ii) fragmented nuclei (F+) without TUNEL signal (T-), or (iii) fragmented nuclei (F+) with TUNEL signal (T+). Fragmentation, TUNEL, and apoptosis indices were calculated for each embryo using the formulae fragmented index = (number of F+T- nuclei/ total number of nuclei) x 100, TUNEL index = (number of F+T+ and F-T+ nuclei/total number of nuclei) x 100, and total apoptotic index = (number of F+T+, F+T- and F-T+ nuclei/total number of nuclei) x 100. Experiments were repeated four times using 15-25 embryos per group.

2.5. Treatment of chemicals

Sirtinol (Sellekchem, USA) was dissolved in dimethyl sulfoxide (DMSO) to make a 50 mM stock solution (×2000 of final concentration) and kept frozen at -20°C until added to the NCSU-23 medium containing 0.4% BSA. DMSO of equal volume used as a control. Sirtinol concentration of 100 µM was used for all experiments, based on data from previous studies [2].

2.6. Statistical analysis

SPSS statistics (SPSS, Chicago, IL, USA) was used to analyze data using t-tests. A significant difference was indicated by p values less than 0.05 (p<0.05). The mean ± standard error of the mean (SEM) are provided for all data.

3. Results

3.1. Effect of sirtuin inhibition on developmental ability after IVF

We first performed a parthenogenetic activation to confirm the effective concentration of sirtinol suggested by previous study [2]. The rate of blastocyst expansion was significantly lower in sirtinoltreated group compared to controls (data not shown). We next evaluated the effect of sirtuin inhibition after IVF. This revealed a significant decrease in the rates of morula (21.34 ± 1.84 vs. 11.89 ± 2.01) and total blastocyst (17.17 ± 1.81 vs. 9.00 ± 2.02) in sirtinol-treated group compared to controls (Fig. 1). Notably, expanded blastocysts (9.90 ± 1.56 vs. 2.92 ± 0.94) were almost completely absent in sirtinol-treated group (p<0.05). However, there were no significant differences between the rate of cleavage in treated or control groups (72.66 ± 1.08 vs. 69.22 ± 1.29).

3.2. Effects of sirtinol on Sirt1 to 3 transcript levels in blastocysts

To investigate whether 100 µM sirtinol treatment led to reduced sirtuin mRNA levels, we examined sirtuins transcript abundance using real-time PCR (Fig. 2). We found that the abundance of Sirt2 mRNA was significantly lower in sirtinol-treated blastocysts compared to controls (p<0.05). However, the levels of Sirt1 and Sirt3 mRNA were similar in both treated and control groups.

3.3. Effects of sirtuin inhibition on autophagy-related genes (ATGs) and LC3 proteins in blastocysts

To identify whether sirtuin inhibition is involved in autophagy, we next investigated the levels of certain ATGs (LC3, BECLIN1, and ATG5) and LC3 protein in blastocysts treated with or without sirtinol. We found that sirtinol treatment significantly increased the levels of LC3 transcript (Fig. 3A, p<0.05). BECLIN1 and ATG5 mRNA levels were found to be elevated in sirtinol-treated blastocysts compared to controls, although this was not significant to a threshold of p<0.05. In addition, the relative fluorescence intensity of LC3 signal was also increased in sirtinol-treated blastocysts, compared to controls (Fig. 3B, p<0.05). Furthermore, LC3 was intracellularly distributed throughout the cytoplasm, with LC3 dots noticeably higher in the sirtinol-treated group (Fig. 3C).

3.4. Effect of sirtuin inhibition on embryo quality

Finally, to examine whether sirtuin inactivation affects embryo quality, we assessed the abundance of apoptosis-related genes, total cell number and apoptotic indices in sirtinol-treated with or without blastocysts. Total cell number (50.80 ± 1.47 vs. 37.71 ± 1.79) was significantly lower, while TUNEL indices (6.55 ± 0.84 vs. 11.44 ± 0.81) and fragmentation indices (0.33 ± 0.05 vs. 1.40 ± 0.30) were significantly higher in sirtinol-treated group (Table 2, Fig. 4A, p<0.05). In addition, the abundance of BCL2L1 transcript was lower in sirtinol-treated blastocysts compared to controls, while Caspase-3 transcript levels were higher (Fig. 4B, p<0.05). Furthermore, BAX mRNA levels showed a slight increase in sirtinol-treated blastocysts compared to controls.

4. Discussion

Recently, poly(ADP-ribosyl)ation (PARylation) has been shown to regulate pro-survival autophagy via mTORC1 signalling in porcine blastocysts [16, 23]. Sirtuins interact with poly(ADP-ribose) polymerases (PARPs) in various biological processes through common NAD+ substrate and regulation of gene expression [24]. Sirtuins regulate autophagy through deacetylation of ATG proteins and FoxO family (FoxO3a and FoxO1), affecting pro-survival or cell death pathways in nuclei (via SIRT1) and cytoplasm (via SIRT2) [11]. Therefore, we hypothesized that sirtuin inhibition may influence the regulation of autophagy in porcine embryos, such as PARylation. However, it has not been studied about sirtuin-mediated autophagy in porcine preimplantation blastocysts. We therefore investigated the relationship between sirtuins and autophagy in porcine embryos. We demonstrate that sirtuin inhibition may protect against autophagic cell death and apoptosis during porcine embryonic development.
We found that sirtinol treatment decreased the developmental ability and embryo quality via autophagy and apoptosis. Notably, the expanded blastocysts were hardly observed in sirtinol-treated group. These results are consist with previous reports showing that sirtuins play an important role in the development from morula to blastocysts [2]. Nicotinamide or sirtinol treatment have also been shown to decrease embryo quality by reducing POU5f1 and Cdx2 expression [2]. In contrast, RSV treatment improves the embryo quality by reducing BAX and Caspase-3 levels in porcine parthenotes [10]. SIRT1 activation was found to increase bovine embryonic development by protecting cells from apoptosis, oxidative stress and inflammation through decreasing nuclear factor (NF)-κB and COX-2 protein levels [4]. Sirtuins can act as co-factor that directly deacetylate the transcriptional factors linked to cell death and apoptosis, such as p53 and NF-kB [1]. SIRT2 inhibition induces acetylation of p53 in NSCL cancer cells, leading to the activation of p53 targets downstream as the apoptosisrelated genes [25]. Sirt3-knockdown mouse embryos induces p53-mediated developmental arrest, and mitochondrial reactive oxygen species production [8]. Considering previous research, our results indicate that sirtuins participate in porcine preimplantation development and apoptosis pathway. We next examined whether sirtinol treatment affects the level of sirtuin transcript in IVF blastocysts. We found that the levels of Sirt2 were decreased in treated blastocysts, while Sirt1 and Sirt3 were unaffected. However, a previous report suggested that sirtinol treatment downregulates Sirt2 and Sirt3 expression, but not Sirt1, in porcine parthenotes [2]. This discrepancy in Sirt3 may be due to sirtinol being specific to SIRT1/2, and particularly more specific to SIRT2 in IC50 value of sirtinol [26].
A relationship between SIRT2 and autophagy regulation has been demonstrated in several prior studies. For example, Sirt2-knockdown increases autophagy through LC3-II accumulation and SQSTM1/p62 degradation as ubiqitinated protein prior to lysosomal degradation in HCT116 cancer cells [27]. In addition, SIRT2 as FoxO1 deacetylase induces autophagic cell death rather than cell survival through acetylation of FoxO1 and subsequent dissociation of FoxO1 from SIRT2-FoxO1 complex in HCT116 cells [28]. SIRT2 in cytoplasm also interacts with HDAC6 that plays a role in lysosome-autophagosome fusion [29,30]. Sirtinol treatment induces cytotoxicity by increasing the LC3-II proteins, apoptosis-related proteins and acetylation of p53 in human MCF cancer cell [12]. p53 can also induce autophagy through transactivation of autophagy-inducing genes [31]. These observations are similar to our present results that sirtinol treatment induces autophagic cell death and apoptosis by increasing the levels of LC3 mRNA, LC3 protein and expression of apoptosisrelated genes. Considering previous results, sirtinol treatment may influence acetylation of FoxO1 and p53 or may affect SIRT2-mediated interaction with other molecules, such as HDAC6. This would lead to autophagic cell death and apoptosis during preimplantation development. Therefore, our data suggest that inhibition of Sirt2 transcripts in treated group may strongly influence autophagic cell death and apoptosis pathways, rather than pro-survival pathways. However, SIRT2-mediated mechanism remains unclear in present study, it is necessary to investigate about sirtuin protein analysis under sirtinol treatment.
Furthermore, autophagy and apoptosis may be closely connected by common regulators [32]. Autophagic cell death show excessive levels of autophagy and ATG5 can directly affect pro-apoptotic factors to initiate apoptosis pathway [33]. Mitochondrial dysfunction and hyperglycemia decrease the rate of embryonic development through increasing the levels of LC3 protein and apoptosis in porcine parthenotes [19, 20]. Induction of autophagy by rapamycin induces mitochondrial DNA reduction, abnormal autolysosome formation, maternal mRNA degradation and apoptosis in blastocysts [21, 22]. Other studies have shown that ATG5 regulates apoptosis by translocating to the mitochondria and inducing the release of cytochrome c and caspase activation [34]. The BCL family can also inhibit the autophagic pathway through interaction with BECLIN1 as well as BAX blocking [33]. Importantly, we found that Caspase-3 transcript was elevated and BCL2L1 transcript lower in sirtinol-treated blastocysts relative to controls. Considering previous studies, we suggest that a rapid reduction in BCL2L1 mRNA may contribute to autophagy through interactions with BECLIN1. The slight increase in ATG5 may also affect caspase activation. Therefore, sirtuin inhibition is closely involved in the regulation of autophagy and apoptosis, thereby controlling embryo quality during porcine preimplantation development.
In summary, our study demonstrated that sirtuin inhibition may play an important role during porcine preimplantation development by modulating the autophagic and apoptotic pathways. Sirtinol treatment led to lower levels of Sirt2 transcript in blastocysts, reduced developmental ability, and embryo quality through the regulation of ATGs, LC3 proteins and apoptosis-related genes. However, further experiments are necessary to fully ascertain whether upstream signaling factors, such as FoxO family and p53, are involved in autophagy under sirtuin inhibition during porcine preimplantation development. Conclusively, our study represents an important advance in elucidating the links between sirtuins and basal autophagy in porcine blastocyst-related mechanism.

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