Alpha-lipoic acid attenuates heat stress-induced apoptosis via upregulating the heat shock response in porcine parthenotes

Heat stress (HS) is a long-standing hurdle that animals face in the living environment. Alpha-lipoic acid (ALA) is a strong antioxidant synthesized by plants and animals. The present study evaluated the mechanism of ALA action in HS-induced early porcine parthenotes development. Parthenogenetically activated porcine oocytes were divided into three groups: control, high temperature (HT) (42 °C for 10 h), and HT + ALA (with 10 µM ALA). The results show that HT treatment significantly reduced the blastocyst formation rate compared to the control. The addition of ALA partially restored the development and improved the quality of blastocysts. Moreover, supplementation with ALA not only induced lower levels of reactive oxygen species and higher glutathione levels but also markedly reduced the expression of glucose regulatory protein 78. The protein levels of heat shock factor 1 and heat shock protein 40 were higher in the HT + ALA group, which suggests activation of the heat shock response. The addition of ALA reduced the expression of caspase 3 and increased the expression of B-cell lymphoma-extra-large protein. Collectively, this study revealed that ALA supplementation ameliorated HS-induced apoptosis by suppressing oxidative and endoplasmic reticulum stresses via activating the heat shock response, which improved the quality of HS-exposed porcine parthenotes.


HS disrupted parthenotes development in pigs.
To confirm the harmful effects of high temperature (HT) on porcine parthenotes development, we first investigated the development of parthenotes exposed to HT at 42 °C for 10 h. After exposure to HT, the developmental rate from four-cell to morular stage was gradually decreased compared to that in the control group (four-cell stage: control: 87.56 ± 1.09% vs. HT: 71.99 ± 1.53%, p < 0.05; five to eight-cell stage: control: 78.85 ± 1.29% vs. HT: 54.73 ± 1.83%, p < 0.05; morular stage: control: 63.76 ± 1.07% vs. HT: 37.31 ± 1.1%, p < 0.001). Finally, the rate of blastocyst formation was significantly decreased (29.27 ± 2.99%, p < 0.01) compared to that in the control group (58.23 ± 4.61%, Fig. 1A,B), indicating that HT disrupts cleavage and consecutive progress during parthenotes development. These results show that HS reduced the potential for the development of porcine parthenotes.

ALA rescued HS-induced impairment of porcine parthenotes development.
To explore the protective effects of ALA on HT-induced parthenotes, we first assessed the rate of parthenotes development following the addition of ALA in a dose-dependent manner. The results showed that the addition of 10 µM ALA partially restored the rate of blastocyst formation compared to the HT group (control: 40.38 ± 0.99% vs. HT: 25.35 ± 1.13%, p < 0.01; HT: 25.35 ± 1.13% vs. HT + 10 µM ALA: 35.54 ± 0.95%, p < 0.05; HT + 15 µM ALA: 30.33 ± 1.32%; HT + 20 µM ALA: 28.76 ± 1.15%, Fig. 2A). In addition, we evaluated the quality of blastocysts using two methods: assessment of blastocyst diameter and TUNEL assays, which detect the number of dead nuclei. As shown in Fig. 2B, although HT exposure markedly reduced the diameter of blastocysts, treatment with 10 µM ALA partially restored its full diameter. However, addition of 20 µM ALA was diminished the quality in blastocysts (control: 1.00 ± 0.05 vs. HT: 0.83 ± 0.05, p < 0.001; HT: 0.83 ± 0.05 vs. HT + 10 µM ALA: 0.94 ± 0.06, p < 0.05). Therefore, 10 µM of ALA was used in subsequent studies. Moreover, as shown in Fig. 2C, low blastocyst quality was observed in the HT group, indicating an increase in TUNEL-positive cells and a decrease in the total number of cells. In contrast, ALA addition reduced the number of TUNEL-positive cells and increased the total number of cells, suggesting that it improves the quality of blastocysts under HT (total number of cells: control: 46.38 ± 0.68 vs. HT: 32.43 ± 0.89, p < 0.01; HT: 32.43 ± 0.89 vs. HT + ALA: 43.22 ± 0.87, p < 0.05; Apoptosis index: control: 5.65 ± 0.39% vs. HT: 10.66 ± 0.73%, p < 0.01; HT: 10.66 ± 0.73% vs. HT + ALA: 6.89 ± 0.57%, p < 0.05, Figure 1. Effect of high temperature (HT) on parthenotes development in pigs. (A) Representative images of the parthenotes development in the control group and groups exposed to HT for 10 h. (B) The developmental rate from four-cell to blastocyst stage in the control and HT group. *p < 0.05; ***p < 0.001.  Fig. 3A,C). These results suggest that ALA addition partially ameliorated the oxidative stress induced by HT in porcine parthenotes.

Scientific Reports
ALA rescued HS-induced ER stress in porcine parthenotes. Given the previous results on oxidative stress, we determined whether ALA had mitigating effects on ER stress after HT exposure. First, we measured the expression levels of glucose regulatory protein 78 (GRP78), an ER stress marker, among the groups. In the HT-treated group, the fluorescence intensity of GRP78 was significantly higher than that in the control group.   (Fig. 4A,B). The relative band intensity of GRP78 was also higher in the HT-treated group than in the control group. ALA addition remarkably reduced its band intensity compared to the HT group (control: 1 vs. HT: 1.24 ± 0.04, p < 0.05; HT: 1.24 ± 0.04 vs. HT + ALA: 1.02 ± 0.08, p < 0.05, Fig. 4C,D). We also investigated the mRNA expression of other marker genes, such as activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). The results showed that expression of these marker genes was higher in the HT-treated group, whereas the ALA group exhibited lower expression levels compared to the HT group ALA induced HSR in HS-treated parthenotes in pigs. HT exposure induces ER stress and results in an increase in damaged proteins. Based on the observed effect of ALA treatment on ER stress, we explored the effect of ALA on the HSR under HT conditions. As shown in Fig. 5A,B, the fluorescence intensity of heat shock factor 1 (HSF1) was significantly increased in the ALA treatment group compared to the other groups (control: 1 vs. HT + ALA: 1.26 ± 0.12, p < 0.05; HT: 1.05 ± 0.2 vs. HT + ALA: 1.26 ± 0.12, p < 0.05). The relative band intensity of HSP40 was also higher in the ALA group than in the other groups (control: 1 vs. HT + ALA: 1.13 ± 0.14, p < 0.05; HT: 1.01 ± 0.03 vs. HT + ALA: 1.13 ± 0.14, p < 0.05, Fig. 5C,D). Therefore, these results suggest that ALA addition induces an HSR to restore unfolded proteins under HT exposure in porcine parthenotes. www.nature.com/scientificreports/ ALA attenuated apoptosis induced by HS in porcine parthenotes. Considering the effects of oxidative and ER stresses, we evaluated the effect of ALA on apoptosis after HT exposure. We examined the expression of apoptotic genes such as caspase 3 and B-cell lymphoma-extra-large (Bcl-xL), which are involved in pro-apoptosis and anti-apoptosis, respectively. As shown in Fig. 6A,B, the fluorescence intensity of caspase 3 was significantly increased in the HT-treated group compared to that in the control group, whereas treatment with ALA restored its expression compared to the HT groups (control: 16.21 ± 0.48 vs. HT:19.65 ± 0.59, p < 0.05; HT: 19.65 ± 0.59 vs. HT + ALA: 15.30 ± 0.47, p < 0.01). In addition, the relative intensity of Bcl-xL was markedly increased in the ALA-treated group compared to that in the control group, suggesting that ALA suppressed apoptosis after HT exposure (control: 1 vs. HT + ALA: 1.18 ± 0.15, p < 0.05, Fig. 6C,D). Thus, these results indicate that ALA treatment attenuated apoptosis induced by HT exposure in porcine parthenotes.

Discussion
The therapeutic properties of ALA against cellular stress have been previously described 10,16 . In the present study, we demonstrated that ALA significantly ameliorated the adverse effects of HS by decreasing cellular stress during porcine parthenotes development. Our results show that exposure to HT at 42 °C for 10 h reduced the rate of parthenotes development and its quality by decreasing the total cell number of blastocysts. In addition, HS conditions induced oxidative and ER stresses following an increase in unfolded proteins, which led to apoptosis caused by the increased expression of caspase3. In contrast, treatment with ALA partially ameliorated the oxidative and ER stresses induced by HS and also upregulated the HSR through higher expression levels of HSF1 and HSP40, which increased the refolding of unfolded proteins. Finally, it inhibited apoptosis under HT exposure by increasing Bcl-xL expression (Fig. 7).
In the current study, HT exposure markedly reduced the rate of parthenotes development in pigs, thereby decreasing the cleavage rate throughout the developmental process. These results indicate that HS reduces the developmental potential of porcine parthenotes. In the 4-cell stage of porcine embryos, important conversions from maternal to zygote gradually occur 19 , called major zygotic genome activation. Epigenetic reprogramming can be influenced by increased ambient temperature, and maternal HS leads to global DNA methylation, which reduces antioxidant competence 20 . In addition, early embryos are extremely vulnerable to HS because they develop thermotolerance from the 2-cell stage to the morular stage 21 . HS in the 2-cell stage compromises the development of bovine embryos 22 . To assess the possible protective effect of ALA on heat-stressed porcine www.nature.com/scientificreports/ parthenotes, we compared three groups: control, HT 10 h, and HT + ALA. The results show that treatment with ALA partially restored the rate of parthenotes development and its quality compared to the HT group, resulting in an increase in the diameter of blastocysts, higher total cell numbers, and fewer TUNEL-positive cells. ALA addition increases the ratio of inner cell mass cells to total cells in blastocysts, suggesting that it may improve the quality of goat embryos 23 . In addition, to confirm whether ALA inhibited apoptosis in blastocysts, we detected the expression of the pro-apoptotic and anti-apoptotic genes caspase 3 and Bcl-xL, respectively. The results show that ALA treatment not only reduced the expression of caspase 3 compared to the HT group but also increased the expression of Bcl-xL compared to the other groups. ALA supplementation improves the quality of blastocysts exposed to ethanol, resulting in fewer TUNEL-positive cells, and decreases expression of apoptotic genes in ovine oocytes 24 . Given the previous results showing a decrease in oxidative and ER stresses, ALA treatment markedly inhibited apoptosis under HS conditions. Additionally, the present results showed a decrease in DNA damage followed by fewer TUNEL-positive cells in the HT group in the presence of ALA. In previous reports, relieving ER stress and promoting HSR have been regarded as potential therapeutic responses against several chronic diseases 25,26 . In addition, HSF1 induces the expression of HSP40 and HSP70 and stabilizes Bcl-xL as a protective response 27 . Therefore, these results reveal the potential benefits of ALA as a therapeutic agent in heat-stressed porcine parthenotes by inhibiting apoptosis. Oxidative damage induced by HS leads to apoptosis with excess production of ROS 5 . To evaluate whether ALA can attenuate oxidative stress induced by HS in porcine parthenotes, we first detected ROS levels among the three groups. Although HT exposure induced ROS overproduction compared to the control, ALA treatment markedly reduced ROS levels compared to HT treatment. Moreover, higher GSH levels were observed in the ALA group than in the HT group, suggesting that it has strong antioxidant ability. GSH is mainly involved in scavenging ROS during the oocyte stage as a primary antioxidant component 28 . Several antioxidants have been shown to increase GSH levels and diminish ROS levels in oocytes exposed to HS. This suggests that the modulation of antioxidants under HS conditions may relieve thermal-oxidative stress in oocytes and enhance fertility 5 . In addition, ALA dissolves in both water and lipids, which enables it to freely pass through biological membranes, and generally serves as an antioxidant in the cytosol, extracellular spaces, and plasma, thereby effectively protecting cells against ROS injury 29 . Thus, these results demonstrate that ALA ameliorates ROS production by regenerating GSH, resulting in a decrease in oxidative stress under HS conditions. www.nature.com/scientificreports/ A few pathological signal transduction pathways, as well as ER stress and apoptosis, are promoted by oxidative stress 30 . Accumulation of unfolded or aggregated proteins activates ER stress, which induces many chaperones to restore these proteins, regarded as the UPR pathway 31 . Although the UPR protects cells from several stresses and exerts its effects on protein homeostasis, prolonged ER stress can induce cell death 30 . Based on our results regarding oxidative stress after HT exposure, we further examined ER stress and evaluated the curative effect of  www.nature.com/scientificreports/ ALA under HT exposure. Our results revealed that HT exposure resulted in higher protein expression of GRP78 and mRNA levels of GRP78, ATF4, and CHOP, which are ER stress markers, indicating the occurrence of ER stress. ALA supplementation markedly reduced the expression of all marker genes and GRP78 protein levels in porcine parthenotes, suggesting that it inhibited ER stress under HS conditions. In previous studies, thermal stress stimulated the formation of protein aggregates in the ER, resulting in oxidative stress-related damage 31 . ALA has the potential to regenerate endogenous antioxidants such as vitamin C, vitamin E, and GSH and is attracting attention as a therapeutic support to ameliorate ER stress 32 . ALA protects against cadmium-induced ER stress in rats and attenuates heat-damaged injury by inhibiting ER stress in chicken testes 10,33 . Taken together, these results suggest that ALA attenuates ER stress in HS-induced porcine parthenotes. Interestingly, we found that HT exposure in the presence of ALA induced the HSR in porcine parthenotes, indicating an increase in the expression of HSF1 and HSP40. The HSR is induced by HS or oxidative damage, and HSPs serve as molecular chaperones and protect against misfolded or aggregated proteins 34 . Therefore, heat damage immediately activates HSFs, which leads to the synthesis of HSP70 and HSP40 as cell survival signaling molecules 35 . However, in the present study, there were no differences in the expression of HSF1 and HSP40 between the control and HT groups. ER stress induced by thermal stress suppresses HSR via translational blockade in rats 35 . ALA has a beneficial effect on modulating diverse components of the HSR including HSP25, HSP72, and HSF1, which results in the attenuation of ER stress and improvement in insulin sensitivity 36 . In addition, ALA prevents heat stroke-induced myocardial damage by acting as an antioxidative and anti-inflammatory agent with the induction of HSP70 37 . Thus, based on the previous results on ER stress recovery by ALA treatment, these results suggest that ALA supplementation induces HSR in parallel with suppression of ER stress in heat-stressed porcine parthenotes.
In conclusion, ALA ameliorated HT-induced apoptosis by suppressing oxidative and ER stresses by inducing the HSR in porcine parthenotes. Moreover, these results revealed that ALA had a strong protective function against HS in pigs and demonstrate its therapeutic role in protein stabilization via activation of the HSR.

Materials and methods
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated, and all animal studies were conducted following the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University, South Korea. In present study, parthenogenetic diploids were used due to the relatively high occurrence of polyspermy with in vitro fertilization of porcine embryos. However, the development of porcine parthenogenetic diploids to the blastocyst stage is comparable to the normal development of embryos 38-40 . Oocyte collection and in vitro maturation. Porcine ovaries were acquired from a local slaughterhouse (Farm Story Dodram B&F, Umsung, Chungbuk, South Korea) and transported to the laboratory at 38.5 °C in saline. Cumulus-oocyte complexes (COCs) were aspirated from ovarian follicles (3-6 mm in diameter), and oocytes enclosed by at least three layers of cumulus cells were collected for further experiments. After washing thrice with Tyrode lactate HEPES (TL-HEPES) buffer, and the COCs were transferred to an in vitro maturation medium containing TCM-199 (Invitrogen, Carlsbad, CA, USA), supplemented with 10% (v/v) porcine follicular fluid, 0.91 mM sodium pyruvate, 0.6 mM l-cysteine, 10 ng/mL epidermal growth factor, 10 μg/mL luteinizing hormone, and 0.5 μg/mL follicle-stimulating hormone, and were cultured for 44 h at 38.5 °C in a humidified 5% CO 2 incubator.
Parthenogenetic activation and in vitro culture. Parthenogenetic activation and in vitro culture were conducted as previously reported 41 . After removing the cumulus cells by repeated pipetting in 1 mg/mL hyaluronidase, denuded oocytes were parthenogenetically activated by two direct-current pulses of 120 V for 60 µs in 297 mM mannitol (pH 7.2) containing 0.1 mM CaCl 2 , 0.05 mM MgSO 4 , 0.01% polyvinyl alcohol (PVA, w/v), and 0.5 mM HEPES. Then, these oocytes were incubated in bicarbonate-buffered porcine zygote medium 5 (PZM-5) containing 4 mg/mL bovine serum albumin (BSA) and 7.5 µg/mL cytochalasin B for 3 h to inhibit extrusion of the pseudo-second polar body. Next, the oocytes were thoroughly washed and incubated in bicarbonate-buffered PZM-5 supplemented with 4 mg/mL BSA in 4-well plates for 7 days at 38.5 °C in humidified atmosphere containing 5% CO 2 . Four-cell cleavage rate and morula and blastocyst formation rates were recorded at 48, 96, and 144 h after activation, respectively. Blastocyst diameter was examined in parthenotes at the blastocyst stage on day 7 using ImageJ v.l.44 g software (National Institutes of Health, Bethesda, MD, USA). TUNEL assay. The intracellular apoptosis standard of blastocysts was determined with TUNEL assay using an In Situ Cell Death Detection Kit (11684795910; Roche, Basel, Switzerland), as described earlier 42 . Blastocysts were fixed in 3.7% formaldehyde for 30 min at room temperature (RT) and permeabilized by incubation in 0.5% Triton X-100 for 30 min at RT. Next, the blastocysts were cultured with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase enzyme for 1 h at 38.5 °C. After washing thrice with phosphatebuffered saline/polyvinyl alcohol (PBS/PVA), the blastocysts were treated with 10 µg/mL Hoechst 33,342 for 10 min and mounted onto glass slides. Images were obtained using a confocal microscope (LSM 710 Meta; Zeiss, Oberkochen, Germany). The apoptosis index was calculated by dividing the TUNEL-positive cell number by the total cell number.

Experimental design.
To ensure parthenotes of steady quality, the 2-cell cleavage rate was confirmed after 24 h of parthenogenetic activation. Parthenotes of the one-cell and two-cell stages and the same quality were separated evenly into three groups: control, HT, and HT + ALA (10 µM). ALA was diluted in ethanol 0.1% (the toxicity of which has been previously evaluated 44 and added at 10, 15, or 20 µM to porcine parthenotes cultured in vitro. The concentration of ALA was selected based on the results. The control group was cultured at 38.5 °C. HT and HT + ALA groups were cultured at 42 °C for 10 h and then returned to 38.5 °C 45 and continuously cultured for 6 days. Statistical analysis. Each experiment was performed three times, each sample in triplicates. Data were analyzed using one-way analysis of variance (ANOVA) or the Student's t-test. All percentage data were subjected to arcsine transformation before statistical analysis and are presented as mean ± SEM. Differences were consid- www.nature.com/scientificreports/ ered statistically significant at p < 0.05. All calculations were performed using the GraphPad Prism 6 software (GraphPad, San Diego, CA, USA).

Data availability
The datasets used or analyzed during the current study are available from the corresponding author upon reasonable request.