S‐nitrosoglutathione reductase maintains mitochondrial homeostasis by promoting clearance of damaged mitochondria in porcine preimplantation embryos

Abstract Objectives S‐nitrosoglutathione reductase (GSNOR), a protein denitrosylase, protects the mitochondria from mitochondrial nitrosative stress. Mammalian preimplantation embryos are mitochondria‐rich, but the effects of GSNOR on mitochondrial function in preimplantation embryos are not well‐studied. In the present study, we investigate whether GSNOR plays a role in mitochondrial regulation during porcine preimplantation embryo development. Materials and Methods GSNOR dsRNA was employed to knock down the expression of GSNOR, and Nω‐Nitro‐L‐arginine methyl ester hydrochloride (L‐NAME), a pan‐NOS inhibitor, was used to prevent protein S‐nitrosylation. Mitochondrial amount and function in embryo development were assessed by performing immunofluorescence staining, Western blot, fluorescent probe and real‐time reverse transcription PCR. Results GSNOR knock‐down significantly impaired blastocyst formation and quality and markedly induced the increase in protein S‐nitrosylation. Notably, GSNOR knock‐down‐induced overproduction of S‐nitrosylation caused mitochondrial dysfunction, including mitochondrial membrane potential depolarization, mitochondria‐derived reactive oxygen species (ROS) increase and ATP deficiency. Interestingly, GSNOR knock‐down‐induced total mitochondrial amount increase, but the ratio of active mitochondria reduction, suggesting that the damaged mitochondria were accumulated and mitochondrial clearance was inhibited. In addition, damaged mitochondria produced more ROS, and caused DNA damage and apoptosis. Importantly, supplementation with L‐NAME reverses the increase in S‐nitrosylation, accumulation of damaged mitochondria, and oxidative stress‐induced cell death. Interestingly, autophagy was downregulated after GSNOR knock‐down, but reversed by L‐NAME treatment. Thus, GSNOR maintains mitochondrial homeostasis by promoting autophagy and the clearing of damaged mitochondria in porcine preimplantation embryos.


| INTRODUC TI ON
Mitochondria are key organelles in mammalian preimplantation embryos that supply ATP for most energy-requiring cellular activities via oxidative phosphorylation. In porcine preimplantation embryos, 91-97% of the ATP produced is derived from oxidative phosphorylation, with glycolysis making a small contribution (2.6-8.7% of the total ATP production). 33 Furthermore, mitochondria also play essential roles in cellular metabolic homeostasis and apoptosis. Numerous studies have indicated that excess production of reactive oxygen species (ROS) in sub-physical conditions induces mitochondrial dysfunction and compromises preimplantation embryo development. 11,20 Thus, mitochondrial quality control system is essential for protection from mitochondrial dysfunction and oxidative stress-induced impairment of embryo development. Damaged mitochondria are cleared via mitophagy, a specific process that degrades membrane potential depolarized mitochondria. 24 Previous studies have showed that overproduction of reactive nitrogen species, another important redox signal, causes nitrosative stress in mammals. 36,38 Introducing the nitric oxide group (-NO) to cysteine thiol in proteins easily forms S-nitrosothiol (SNO) and has been termed protein S-nitrosylation. 8 S-nitrosylation is catalysed by NO synthases (NOSs), and the enzymes termed S-nitrosylases. 32 Physiological S-nitrosylation modulates the activity of proteins involved in regulating various physiological and biochemical processes in mammalian cells. 9 Excessive S-nitrosylation during nitrosative stress has been linked to oocyte ageing, impaired mitochondrial respiratory function and impaired mitophagy.4,21 This can trigger protein misfolding, endoplasmic reticulum stress, mitochondrial quality control compromise and mitochondrial dysfunction. 8,16,27 Therefore, the NO-related redox signal may have an important role in the mitochondrial regulation of mammalian oocytes and preimplantation embryos. However, the mitochondrial nitrosative stress and NO-related redox signal in preimplantation embryos are not well understood.
In mammals, the S-nitrosoglutathione reductase (GSNOR) gene encodes a polypeptide of 385 amino acids with a molecular mass of approximately 40 kDa. Numerous studies have indicated that GSNOR regulates protein S-nitrosylation by functioning as a protein denitrosylase. 35 Denitrosylation is considered as an important route for nitrosative stress tolerance. GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. 28 GSNOR deficiency causes tumorigenesis 29 and extensively disrupts cellular homeostasis, including energy metabolism 30, DNA damage repair 39 and cardiovascular function. 2 Additionally, GSNOR deficiency causes excessive S-nitrosylation of dynamin-related protein 1 (Drp1) and Parkin, impairing mitochondrial dynamics and mitophagy. 28 Our previous studies have revealed that mitochondrial quality control has an important role in maintaining mitochondrial homeostasis and early embryo development in pigs. 18,20 However, involvement of GSNOR function in NO-related redox signalling and mitochondrial protection in preimplantation embryos is poorly understood. Therefore, in this study, we employed porcine preimplantation embryos to investigate the effects of GSNOR on mitochondrial amount and function and its underlying mechanisms.
First, GSNOR mRNA and protein expressions in porcine embryos from the parthenotes to blastocyst stages were assessed. Additionally, GSNOR function in porcine embryonic development was investigated using double-strand mRNA-mediated gene knock-down.

| MATERIAL S AND ME THODS
Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich Co., Inc (St. Louis, MO, USA) and all manipulations were performed on a heated stage adjusted to 38.5°C.

| Collection and in vitro maturation of porcine oocytes
All experimental protocols were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Chungbuk National University Laboratory Animal Center, Cheongju, South Korea.
Ovaries from pre-pubertal gilts were obtained from a local slaughterhouse (Farm Story Dodarm B&F, Umsung, Chungbuk, South Korea) and transported to the laboratory at 38.5°C in saline supplemented with 75 mg/mL penicillin G and 50 mg/mL streptomycin sulphate.
Follicles 3-6 mm in diameter were aspirated using an 18-gauge needle connected to a 10 mL disposable syringe. Cumulus-oocyte complexes were selected based on their morphologic characteristics, that is those showing at least three layers of compact cumulus cells and an evenly granulated ooplasm. After three rinses with in vitro maturation medium TCM-199 [(11150-059; Gibco, Grand Island, NY, USA) supplemented with 0.1 g/L sodium pyruvate, 0.6 mmol/L L-cysteine, 10 ng/ mL epidermal growth factor, 10% (v/v) porcine follicular fluid, 10 IU/ mL luteinizing hormone and 10 IU/mL follicle-stimulating hormone], approximately 50 cumulus-oocyte complexes were transferred to 4-well dishes (SPL life sciences, Seoul, South Korea) containing 500 μL of the maturation medium. The medium was covered with mineral oil, and the plates were incubated at 38.5°C in a humidified atmosphere containing 5% CO 2 for 44 hours.

| Parthenogenetic activation and in vitro culture
Parthenogenetic activation and in vitro culture were performed as described earlier. 42 After removing the cumulus cells by repeated pipetting in 1 mg/mL hyaluronidase, denuded oocytes were parthenogenetically activated using 2 direct-current pulses of 120 V for 60 µs in 297 mmol/L mannitol (pH 7.2) containing 0.1 mmol/L CaCl 2 , 0.05 mmol/L MgSO 4 , 0.01% polyvinyl alcohol (PVA, w/v) and 0.5 mmol/L HEPES. These oocytes were then cultured 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 hours to suppress extrusion of pseudo-second polar bodies. Next, the activated oocytes were thoroughly washed and cultured in bicarbonate-buffered PZM-5 supplemented with 4 mg/mL BSA in 4-well plates for 6 days at 38.5°C and 5% CO 2 . Blastocyst formation was examined on day 6 after activation. To determine the total cell number, day 6 blastocysts were randomly collected and stained with 10 mg/mL Hoechst 33342 prepared in PBS for 5 minutes.

| GSNOR double-stranded RNA (dsRNA) preparation
To prepare GSNOR dsRNA, GSNOR was amplified using a pair of primers Table 1 containing the T7 promoter sequence. The purified PCR products were then used to synthesize dsRNA with a MEGAscript T7 Kit (Ambion, AM1333, Huntingdon, UK) according to the manufacturer's instructions. After in vitro transcription, dsRNA was treated with DNase I and Rnase A to remove the DNA template and any single-stranded RNA was then purified by phenol-chloroform extraction and isopropyl alcohol precipitation. The purified dsRNA was dissolved in Rnase-free water and stored at −80°C until use.

| Inhibitor preparation and treatment
N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME, Sigma, N5751) was dissolved in H 2 O to prepare stock solution. To determine whether excess protein S-nitrosylation was involved in mediating the harmful effects of GSNOR knock-down in preimplantation embryos, the embryos in PZM-5 were treated with L-NAME at 250 µmol/L concentrations. The same amount of H 2 O was added to control oocytes to score the effect of the solvent on the outcome.

| Biotin-switched assay for detection of S-nitrosylated proteins
The biotin-switch method for detecting S-nitrosylated proteins was used as described previously. 7,13 In brief, blastocysts from the different experimental groups were fixed in 3.7% paraformaldehyde at room

| Immunofluorescence and confocal microscopy
After washing three times with PBS/PVA, embryos were fixed

| Quantitative Reverse Transcription PCR (qRT-PCR)
Blastocysts were collected, and total RNA was extracted from a pool

| Western blot analysis
A total of 100 porcine blastocysts per group were lysed with 1 × so-

| Statistical analysis
Each experiment was repeated at least three times, and representative images are shown in the figures. The GSNOR mRNA and protein expression at different stages were subjected to one-way analysis of variance. Differences among stages were examined using the Duncan multiple range test. Other data were subjected to the Student's t test. Further, all percentage data were subjected to arcsine transformation prior to statistical analysis and then presented as the mean ± standard error (SEM). Significance was set at P < .05.

| GSNOR mRNA and protein expression during porcine preimplantation embryo development
First, GSNOR mRNA expression was detected using qRT-PCR. As shown in Figure 1A, the mRNA expression of GSNOR was decreased from 1-cell to 4-cell embryos and then slightly but significantly increased at the blastocyst stage. We next evaluated the expression and subcellular localization of GSNOR during porcine preimplantation embryo development by immunostaining. GSNOR protein was expressed at all stages in both cytoplasmic and nuclear of preimplantation embryos, but gradually reduced during embryo development Figure 1B,C.

| Effects of GSNOR knock-down on porcine preimplantation embryonic development
To examine why GSNOR was expressed at all stages during embryonic development, GSNOR double-stranded RNA (dsGSNOR) was injected into porcine parthenotes, which were then cultured in vitro for 6 days. The knock-down efficacy on GSNOR mRNA was evaluated at the 4-cell and blastocyst stages. EGFP dsRNA was injected into porcine parthenotes as the control group (dsControl). As shown in Figure 2A, compared with the dsControl, GSNOR mRNA was effectively downregulated by 76.1% and 79.3% at the 4-cell and blastocyst stages, respectively (P < .001). GSONR protein knock-down was confirmed by and Western blots Figure 2B,C, P< .05, Efficiency: 29.1%) and immunostaining Figure 2D,E, P < .001, Efficiency: 33.1%) at blastocyst stages. To detect whether knock-down of GSNOR caused increased protein S-nitrosylation, the biotin-switch assay for detection of S-nitrosylated proteins was performed. 10 S-nitrosylated proteins increased after GSNOR knock-down compared with control group Figure 2F,G, P< .05

| GSNOR knock-down induces damaged mitochondrial accumulation and mitochondrial ROS production during porcine preimplantation embryonic development
Overproduction of S-nitrosylation causes mitochondrial nitrosative stress and mitochondrial dysfunction. We therefore investigated whether mitochondrial function was compromised in porcine embryos after GSNOR knock-down. The active and total mitochondria were labelled with MitoTracker Red CMXRos and TOM20, respectively. As shown in Figure 3A-C, although the fluorescence intensity of TOM20 was significantly increased after GSNOR knock-down (P < .05), the ratio of fluorescence intensity (MitoTracker Red CMXRos/TOM20) was decreased (P < .05), indicating total mitochondrial amount increased, but active mitochondria decreased. Moreover, the increase in mitochondrial amount was confirmed by detecting mitochondrial DNA (mtDNA) copy number Figure  As expected, compared with dsControl blastocysts, mitochondria-derived ROS levels in GSNOR knock-down blastocysts were significantly upregulated by 118% Figure 3G,H, P < .001). Finally, the ATP level was significantly reduced in GSNOR knock-down embryos Figure 3I, P < .05). Taken together, in GSNOR deficiency embryos, damaged mitochondria accumulated and caused an increase in ROS, thereby further inducing the mitochondrial membrane potential depolarization and ATP deficiency.

| GSNOR knock-down induces oxidative stressderived apoptosis and DNA damage, as well as inhibits autophagic process
Mitochondrial dysfunction was closely linked to excessive intracellular ROS generation, apoptosis, DNA damage and autophagy.
Accordingly, total ROS was detected using the H 2 DCF-DA fluorescent reaction and active caspase 3 was quantified as an apoptosis biomarker. The total ROS and apoptosis were significantly increased by 41% (P < .001) and 35% (P < .01), respectively, in shown in Figure 4G, the number of BECLIN1 and LC3 dots decreased rather than increased in GSNOR knock-down blastocysts compared with control blastocysts. Quantified data also revealed that the fluorescence intensity of BECLIN1 and LC3 was significantly decreased after GSNOR knock-down by 44% (P < .01) and 55% (P < .01), respectively.

| L-NAME prevents GSNOR knock-downinduced excessive protein S-nitrosylation and embryo development impairment
To verify whether the high level of protein S-nitrosylation induced by GSNOR knock-down was the main reason for preimplantation embryo development impairments, the pan-NOS inhibitor, L-NAME, was added to the medium during in vitro embryo culture.
The results indicated that 250 µmol/L L-NAME could attenuate the vs. 187.0 ± 6.1 μm, P < .05) in the dsGSNOR + L-NAME group was significantly higher, indicating L-NAME could prevent GSNOR knock-down-induced embryo development impairment Figure 5A-D. In addition, the protein S-nitrosylation level was evidently decreased after L-NAME treatment when compared with GSNOR knock-down group Figure 5E,F, P < .05).

| L-NAME prevents GSNOR knockdown-induced accumulation of damaged mitochondria, oxidative stress-derived cell death and downregulation of autophagy in porcine preimplantation embryos
To further prove whether the high levels of protein S-nitrosylation induced by GSNOR knock-down were the main reason for accumulation of damaged mitochondria, oxidative stress-derived cell death and downregulation of autophagy, L-NAME was added to the medium during in vitro embryo culture. GSNOR knock-downinduced reduction in active mitochondria Figure 6A,B, P < .05) and increase in DNA copy number Figure 6C, P < .01) were abated after L-NAME treatment, indicating that the accumulation of damaged mitochondria was rescued. Furthermore, GSNOR knock-downinduced ROS production Figure 6D,E, p < .05), active caspase 3 increase Figure 6F,G, p < .05) and γH2A.X expression Figure 6H,I, p < .05) were all rescued by adding L-NAME, suggesting that oxidative stress-derived cell death in GSNOR knock-down group could be rescued by addition of L-NAME. However, GSNOR knock-downinduced autophagy prevention was attenuated after L-NAME treatment Figure 6J-L, P < .05).

| D ISCUSS I ON
Mammalian oocytes and preimplantation embryos contain a particularly high number of mitochondria. The ROS-related redox signal has been linked to the mitochondrial dysfunction, and deficiency in ageing oocytes and early embryos exposed to a toxic  previous reports in cells. 26 The main reason for this might be that the function of key proteins in the autophagy process is prevented because the protein was S-nitrosylated in nitrosative stress conditions. 15 However, the precise molecular pathways that are involved in autophagy inhibition after GSNOR knock-down require investigation in future studies.

F I G U R E 7
Schematic diagram showing that GSNOR maintains mitochondrial homeostasis by promoting damaged mitochondrial clearance in porcine preimplantation embryos. Normally, mitophagy and mitochondrial biogenesis maintain mitochondrial function and amount via promoting damaged mitochondrial clearance and production of new and healthy mitochondria. Furthermore, autophagy degrades unnecessary proteins and dysfunctional organelles. However, decrease in GSNOR protein levels by knock-down of GSNOR mRNA induces an increase in protein SNOs and prevents mitophagy and autophagy. Thus, GSNOR knock-down further induces accumulation of damaged mitochondria, oxidative stress and cell death. These harmful effects could be reversed via treatment with L-NAME. ∆Ψm: mitochondrial membrane potential; ROS: reactive oxygen species. Black arrow indicates the balance between mitophagy and mitochondrial biogenesis and autophagy-derived protect function. Red arrow indicates the GSNOR knock-down-induced increase in protein SNOs, accumulation of damaged mitochondria, downregulation of autophagy and subsequent processes NOSs use L-arginine as a substrate for the formation of NO.
L-NAME is a highly effective pan-NOSs inhibitor and as an analogue of L-arginine can compete with L-arginine for NOSs. 40 Therefore, to investigate whether the increase in protein S-nitrosylation, and its-induced accumulation of damaged mitochondria, oxidative stress-derived cell death, and embryo development impairments were due to denitrosylation downregulation in GSNOR knock-down embryos, L-NAME was used to inhibit the S-nitrosylation process.
The results indicated that impairment of blastocyst formation and quality in the GSNOR knock-down group were rescued via L-NAME treatment. Moreover, the number of active mitochondria increased and total mitochondrial amount was decreased, indicating that accumulation of damaged mitochondria was also abated with L-NAME compared with GSNOR knock-down,NOSs inhibition attenuated GSNOR knock-down-induced oxidative stress, DNA damage, and apoptosis. GSNOR knock-down-induced downregulation of autophagy was also recued with L-NAME addition, suggesting that GSNOR could denitrosylate the autophagy related proteins to control the autophagy process.
A schematic diagram Figure 7 demonstrates that when environmental factors or toxins induce mitochondrial dysfunction, depolarized mitochondria can be cleared by mitophagy.
Mitochondrial biogenesis can produce new and healthy mitochondria that compensate the deficiency incurred by this. However, GSNOR knock-down prevents mitophagy and damaged mitochondria keep accumulating in preimplantation embryos. Furthermore, these damaged mitochondria produce more ROS and induce oxidative stress, DNA damage and apoptosis. In addition, GSNOR knock-down downregulates autophagy and induces a loss of cell adaptive response in the embryos. In conclusion, GSNOR is stably expressed at all stages during porcine embryo development.
Protein S-nitrosylation because of GSNOR knock-down causes the loss of two important cell protection functions, mitophagy and autophagy, to the embryos.

ACK N OWLED G EM ENTS
This work was supported by the National Research Foundation

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.