6-Gingerol Improves In Vitro Porcine Embryo Development by Reducing Oxidative Stress

Simple Summary Excellent quality of early embryonic development contributes to a successful pregnancy. At present, most in vitro cultured embryos can only develop to the blastocyst stage at most, because an in vitro culture (IVC) system cannot replace the physiological environment in vivo. During IVC, excessive accumulated reactive oxygen species in embryos cannot be easily metabolized, which will cause oxidative stress and suppress embryo development. In this study, we found that anti-oxidation capacity of early embryo was improved by adding 6-gingerol to IVC. Moreover, 6-gingerol can also improve blastocyst rate, cell proliferation, mitochondrial function, inhibit cell apoptosis, autophagy, and regulate functional genes expression in blastocyst. These results are helpful to optimize the early embryo culture system, and thus provide a theoretical basis for improving the early embryo quality and the efficiency of subsequent pregnancy. Abstract 6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 μM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro.


Introduction
In vitro embryo culture systems, including procedures for in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro embryo culture (IVC), are currently widely used for embryonic developmental mechanism research, in vitro embryo production, and human assisted reproduction [1]. However, it is difficult to simulate the optimal embryonic growth

Parthenogenetic Activation and In Vitro Embryo Culture
Mature oocytes were parthenogenetically activated. Next, the oocytes were cultured in IVC medium with cytochalasin B (#C6762) for 3 h. Then, the oocytes were transferred into IVC medium with/without 0 µM, 5 µM, 10 µM, and 20 µM 6-G (#S3836, Selleck Chemicals, Shanghai, China). The rate of blastocyst formation was calculated as the ratio of the number of blastocysts to the number of cleavages. The details are indicated in the Supplementary Materials.

Cell Proliferation Analysis
Briefly, embryonic cell proliferation capacity was analyzed by a 5-ethynyl-2 -deoxyuridine (EdU) assay with a BeyoClick™ EdU Cell Proliferation Kit (#C0075; Beyotime, Shanghai, China). The cell proliferation rate was calculated as the number of EdU-positive cells to the total number of cells in blastocysts. The details are indicated in the Supplementary Materials.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End-Labeling (TUNEL) Assays
Briefly, the cell apoptosis level was analyzed by an In Situ Cell Death Detection Kit (#11684795910; Roche, Mannheim, Germany). The ratio of the number of TUNEL-positive nuclei to the total number of nuclei was calculated as the apoptosis rate. The details are indicated in the Supplementary Materials.

Determination of ATP Levels
Briefly, the ATP levels in 4-cell-stage embryos were measured using an ATP Determination Kit (#A22066; Invitrogen). The details are indicated in the Supplementary Materials.

Immunofluorescence
Briefly, embryos were fixed in 4% paraformaldehyde and permeabilized with 0.3% Triton X-100. Then, the embryos were blocked in 5% BSA and incubated with a primary antibody against microtubule-associated protein 1 light chain 3 beta (LC3B; #ab63817; Abcam, Cambridge, MA, USA). Then, the embryos were incubated with a secondary antibody (#ab150073; Abcam) for 1 h. The nuclei were stained with Hoechst 33342. The details are indicated in the Supplementary Materials.

RNA Extraction and Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis
Briefly, mRNA was extracted using the Dynabeads mRNA DIRECT Purification Kit (#61011; Invitrogen). A TIANScript First Strand cDNA Synthesis Kit (#KR118; Tiangen Biotech Co., Beijing, China) was used to synthesize cDNA. Gene expression was quantified using the 2 −∆∆Ct method with 18S rRNA as the standard. The details are indicated in the Supplementary Materials and all the primers are listed in Supplementary Table S1.

Western Blot Analysis
Briefly, a RIPA lysis buffer (#R0010; Solarbio, Beijing, China) was used for total protein extraction. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (0.45 µm; #IPVH00010; Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA and incubated with a primary antibody. Then, the membranes were incubated with secondary antibodies. The details are indicated in the Supplementary Materials and the antibody information is shown in Supplementary Table S2.

Statistical Analysis
All calculations were performed using SPSS software v.22.0 (SPSS, Inc., Chicago, IL, USA). Data from two groups were compared using the Student's t-test. Tests with three or more means were analyzed using a one-way ANOVA (Tukey-Kramer). All data are presented as mean ± SD. The total numbers of embryos used (N) in each group and replicates (R) in each experiment are shown in the results and figure legends. p < 0.05 and p < 0.01 were considered to indicate significant differences.

6-G Enhanced Cell Proliferation
Subsequently, we examined the effect of 6-G on proliferation in early embryos by the EdU assay. As shown in Figure 2, compared with the NC group, the 6-G treatment group exhibited a significantly higher proportion of proliferating cells among the total number of cells (48.87% ± 12.46% (N = 70) versus 40.66% ± 12.57% (N = 71), p < 0.01). Based on the above results, 6-G can enhance cell proliferation during early embryo development.

6-G Reduced Apoptosis of Porcine Embryos
As shown in Figure 3, the proportion of TUNEL-positive nuclei in the 6-G-treated group was significantly lower than that in the NC group (3.94 ± 1.87% (N = 61) versus 5.70 ± 2.36% (N = 62), p < 0.01). This suggests that treatment with 6-G reduces apoptosis in blastomeres of porcine embryos.

6-G Enhanced Antioxidant Capacity in Porcine Embryos
To investigate whether 6-G can inhibit ROS accumulation, we used DCFH fluorescent probes to detect ROS levels in 4-cell-stage embryos. As shown in Figure 4, the fluorescence intensities of DCFH in blastomeres were significantly lower (0.79 ± 0.13-fold; N NC = 163, N 6-G = 168; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4A). In addition, we analyzed the relative CMF 2 HC level to detect GSH levels in 4-cell-stage embryos and found that they were significantly higher (1.26 ± 0.12-fold; N NC = 140, N 6-G = 155; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4B).

6-G Enhanced Cell Proliferation
Subsequently, we examined the effect of 6-G on proliferation in early embryos by EdU assay. As shown in Figure 2, compared with the NC group, the 6-G treatment gro exhibited a significantly higher proportion of proliferating cells among the total num of cells (48.87% ± 12.46% (N = 70) versus 40.66% ± 12.57% (N = 71), p < 0.01). Based on above results, 6-G can enhance cell proliferation during early embryo development.

6-G Enhanced Cell Proliferation
Subsequently, we examined the effect of 6-G on proliferation in early embryos by the EdU assay. As shown in Figure 2, compared with the NC group, the 6-G treatment group exhibited a significantly higher proportion of proliferating cells among the total number of cells (48.87% ± 12.46% (N = 70) versus 40.66% ± 12.57% (N = 71), p < 0.01). Based on the above results, 6-G can enhance cell proliferation during early embryo development.

6-G Reduced Apoptosis of Porcine Embryos
As shown in Figure 3, the proportion of TUNEL-positive nuclei in the 6-G-treated group was significantly lower than that in the NC group (3.94 ± 1.87% (N = 61) versus 5.70 ± 2.36% (N = 62), p < 0.01). This suggests that treatment with 6-G reduces apoptosis in blastomeres of porcine embryos.

6-G Enhanced Antioxidant Capacity in Porcine Embryos
To investigate whether 6-G can inhibit ROS accumulation, we used DCFH fluorescent probes to detect ROS levels in 4-cell-stage embryos. As shown in Figure 4, the fluorescence intensities of DCFH in blastomeres were significantly lower (0.79 ± 0.13-fold; NNC = 163, N6-G = 168; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4A). In addition, we analyzed the relative CMF2HC level to detect GSH levels in 4-cell-stage embryos and found that they were significantly higher (1.26 ± 0.12-fold; NNC = 140, N6-G = 155; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4B).

6-G Enhanced Antioxidant Capacity in Porcine Embryos
To investigate whether 6-G can inhibit ROS accumulation, we used DCFH fluorescent probes to detect ROS levels in 4-cell-stage embryos. As shown in Figure 4, the fluorescence intensities of DCFH in blastomeres were significantly lower (0.79 ± 0.13-fold; NNC = 163, N6-G = 168; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4A). In addition, we analyzed the relative CMF2HC level to detect GSH levels in 4-cell-stage embryos and found that they were significantly higher (1.26 ± 0.12-fold; NNC = 140, N6-G = 155; p < 0.01) in the 6-G treatment group than in the NC group ( Figure 4B).

6-G Improved Mitochondrial Function
Mitochondrial activity directly affects embryonic cell proliferation and developmental potential. To study whether 6-G can improve mitochondrial function in porcine early embryos, we measured MMP in 4-cell-stage embryos. As shown in Figure 5A, the JC-1 Red/Green fluorescence intensity ratio in 4-cell-stage embryos was significantly higher (1.40 ± 0.14-fold; N NC = 81, N 6-G = 70; p < 0.01) in the 6-G-treated group than in the NC group ( Figure 5B). In addition, the ATP level in 6-G treated embryos was also higher (1.30 ± 0.03-fold; N NC , N 6-G = 270; p < 0.01) than that in non-6-G-treated embryos ( Figure 5C). tal potential. To study whether 6-G can improve mitochondrial function in porcine early embryos, we measured MMP in 4-cell-stage embryos. As shown in Figure 5A, the JC-1Red/Green fluorescence intensity ratio in 4-cell-stage embryos was significantly higher (1.40 ± 0.14-fold; NNC = 81, N6-G = 70; p < 0.01) in the 6-G-treated group than in the NC group ( Figure 5B). In addition, the ATP level in 6-G treated embryos was also higher (1.30 ± 0.03fold; NNC, N6-G = 270; p < 0.01) than that in non-6-G-treated embryos ( Figure 5C).

6-G Inhibited Autophagy in Blastocysts
Excessive ROS production can induce autophagy in cells. Therefore, we analyzed the autophagy levels of embryos after 6-G treatment. As shown in Figure 6A, compared with the NC group, the 6-G group exhibited a significantly lower number of intracellular LC3Bpositive puncta (0.76 ± 0.33-fold; NNC = 57, N6-G = 64; p < 0.01). This finding indicates that 6-G may reduce intracellular autophagy levels.

6-G Inhibited Autophagy in Blastocysts
Excessive ROS production can induce autophagy in cells. Therefore, we analyzed the autophagy levels of embryos after 6-G treatment. As shown in Figure 6A, compared with the NC group, the 6-G group exhibited a significantly lower number of intracellular LC3Bpositive puncta (0.76 ± 0.33-fold; N NC = 57, N 6-G = 64; p < 0.01). This finding indicates that 6-G may reduce intracellular autophagy levels.

6-G Regulated MAPKs Activations in Blastocysts
The MAPK signaling pathway can regulate cell proliferation, differentiation, apoptosis, and autophagy. Therefore, we used Western blotting to detect the effects of 6-G treatment on the extracellular regulated protein kinase 1/2 (ERK1/2), JNK1/2/3, and p38 signaling pathways. As shown in Figure 8, the addition of 6-G significantly increased the level

Discussion
Studies have shown that 6-G alleviates oxidative damage and inflammation in the ovaries [20,21]. Porcine early embryos are more sensitive to oxidative stress during in vitro embryo production than in vivo due to their unique structures [22]. The results of this study indicate that the addition of 6-G can effectively improve the developmental potential of early porcine embryos.
In this study, 6-G treatment effectively reduced the accumulation of ROS in blastomeres at the 4-cell stage, likely because 6-G can effectively prevent -OH-induced DNA damage, in particular by scavenging various free radicals [23]. This effect was likely mediated by 6-G-induced high levels of GSH. Intracellular ROS levels are highly correlated with GSH levels. The findings show that when ROS levels in the body are increased, 6-Gmediated enhancement of the GSH defense system can increase the removal of excess oxygen free radicals and maintain dynamic redox balance. These results are consistent with previous suggestions that 6-G can alleviate ROS-induced diseases and conditions by increasing GSH levels [24]; such conditions include impaired ovarian follicle development and abnormal fertilization [25].
At the 4-cell stage, zygotic genome activation is essential for porcine early embryo development and pregnancy [26,27]. During IVC of embryos, activation of the zygotic ge- Figure 8. Effects of 6-gingerol (6-G) on the extracellular regulated protein kinase 1/2 (ERK1/2), c-Jun Nterminal kinase 1/2/3 (JNK1/2/3), and p38 pathways. (A) The level of p-ERK1/2 in the 6-G treatment group was higher than that of the NC group. R = 3. (B) The level of p-p38 in the 6-G treatment group was lower than that of the NC group. R = 3. (C) The level of p-JNK1/2/3 in the 6-G treatment group was lower than that of the NC group. R = 3. ** p < 0.01.

Discussion
Studies have shown that 6-G alleviates oxidative damage and inflammation in the ovaries [20,21]. Porcine early embryos are more sensitive to oxidative stress during in vitro embryo production than in vivo due to their unique structures [22]. The results of this study indicate that the addition of 6-G can effectively improve the developmental potential of early porcine embryos.
In this study, 6-G treatment effectively reduced the accumulation of ROS in blastomeres at the 4-cell stage, likely because 6-G can effectively prevent -OH-induced DNA damage, in particular by scavenging various free radicals [23]. This effect was likely mediated by 6-G-induced high levels of GSH. Intracellular ROS levels are highly correlated with GSH levels. The findings show that when ROS levels in the body are increased, 6-G-mediated enhancement of the GSH defense system can increase the removal of excess oxygen free radicals and maintain dynamic redox balance. These results are consistent with previous suggestions that 6-G can alleviate ROS-induced diseases and conditions by increasing GSH levels [24]; such conditions include impaired ovarian follicle development and abnormal fertilization [25].
At the 4-cell stage, zygotic genome activation is essential for porcine early embryo development and pregnancy [26,27]. During IVC of embryos, activation of the zygotic genome generally occurs 24-48 h after parthenogenetic activation or IVF. This stage is more sensitive than other stages to negative conditions in the external environment, especially the accumulation of ROS, which leads to the failure of zygotic genome activation and leads to the suppression of embryo development [28]. The results indicate that 6-G may exert biological effects at least as early as at this stage.
Mitochondria play essential roles during embryo development both before and after zygotic genome activation [29]. Excessive accumulation of ROS causes oxidative damage, MMP depolarization [30], aging, and apoptosis [31]. In this study, we found that 6-G supplementation significantly increased the MMP and ATP level in blastomeres at the 4-cell stage, improved the ability of embryos to proliferate and effectively reduced the number of apoptotic cells in the embryos. This result is consistent with previous findings that 6-G inhibits disruption of MMP [32] and is probably related to the roles of 6-G in increasing the activity of the mitochondrial enzymes NADH oxidase, succinate dehydrogenase, and Sirtuin 3 [33]. Previous studies have also shown that 6-G can reduce intracellular ROS levels by regulating nuclear factor kappa B translocation [34]. In addition, 6-G upregulated antioxidant enzymes, such as glyoxylate carboligase and heme oxygenase 1, and further protects against cytotoxicity and apoptotic cell death resulting from processes such as ROS-induced DNA fragmentation, disruption of MMP, and autophagy. These effects seem to be mediated by regulation of nuclear factor 2 [35], p38 MAPK, JNK [36], and phosphatidylinositol 3-kinase/Akt [37]. In this study, we found that 6-G increased p-ERK levels and decreased p-JNK and p-p38 levels. This indicates that 6-G exerts a regulatory effect on cell survival or apoptosis by affecting the dynamic balance between the growth factor-activated ERK pathway and the stress-activated JNK-p38 pathway [38]. On the one hand, this regulation depends on ERK pathway-mediated direct targeting and regulation of the cell cycle and indirect regulation of RNA metabolism and transport [39]. On the other hand, it also depends on the regulation of the JNK pathway by various cellular stress and growth factors [40]. 6-G may enable the effects mediated by these pathways to regulate biological processes, such as cell morphology changes, immune responses, and apoptosis [41,42].
To explore the potential mechanism by which 6-G promotes embryo development, we also examined functional gene expression changes. The results showed that the addition of 6-G significantly upregulated the anti-apoptotic gene RICTOR and downregulated the pro-apoptotic gene CASP3. These factors help stabilize the intracellular environments of blastomeres. The results are consistent with the findings that 6-G can improve embryo development and reduce apoptosis. Early embryos with stable cell states and enhanced mitochondrial function show high proliferation ability and reduced nuclear apoptosis rates [43,44]. Notably, the quality of blastomeres is an important basis for embryo development before implantation [45]. Combined with the result that 6-G improved the proliferation-related gene mTOR [46], these findings suggest that 6-G can stabilize or even optimize the intracellular environment of blastomeres, creating a relatively stable internal environment and promoting cell proliferation and development. This study also revealed that the addition of 6-G can significantly increase the cell proliferation capacity in blastocysts. Interestingly, 6-G significantly decreased the expression level of the apoptosis-related gene BIRC5. BIRC5 is widely known as an anti-apoptosis gene. However, a previous study has found that overexpression of BIRC5 significantly inhibits cell survival [47]. In addition, compared with those in unfrozen bovine blastocysts, the levels of apoptosis and the expression of apoptosis-related genes BIRC5 and CASP3 were also significantly increased in frozen embryos [48], which was similar to our results that 6-G reduced the level of apoptosis and downregulated the expression levels of BIRC5 and CASP3 in porcine blastocysts. We speculate that this may be because the addition of 6-G improves the embryonic development environment while the high level of anti-apoptotic factors is not needed. In addition, embryo pluripotency-related genes (NANOG and SOX2) were significantly upregulated in this study, which indicates that 6-G has the potential to promote establishment of the epiblast and hypoblast [49], improve the quality of inner cell masses [50,51], and stabilize the basic functions of embryonic stem cells [52,53]. However, there are some reports showing that there are differences in the expression of NANOG and SOX2 during embryo development not only between embryos produced in vivo and in vitro, but also between parthenogenetic and IVF embryos [54]. Additional research about the roles of 6-G in IVF and somatic cell nuclear transfer embryos is still needed. The upregulation of COX2 observed in this study suggests that 6-G has potential effects on early embryo implantation and decidualization [55,56]. Our results showed that the expression levels of autophagy-related genes ATG12 and BECN1 with 6-G treatment. ATG12 can target the elongation of the autophagosome membrane. A previous study showed that inhibition of ATG12 significantly decreased the autophagy level [57]. BECN1 also plays important roles in autophagy [58]. However, mTOR plays opposite roles in the regulation of autophagy [59]. The early stages of the autophagic process are inhibited by mTOR, and mTOR can also regulate the lysosomal degradative capacity by preventing the transactivation of genes encoding catalytic, regulatory, and structural factors [60]. These results were related to the inhibition of autophagosome formation with 6-G treatment, indicating that 6-G can significantly inhibit the autophagy level in porcine early embryos. Moreover, autophagy is one of the most important factors in mediating apoptosis [61], which indicates that the decreased level of autophagy with 6-G treatment may also be one of the reasons for the inhibition of apoptosis.
Conversely, some studies have shown that 6-G can play roles in inducing ROS production, reducing mitochondrial function, and promoting autophagy and apoptosis. However, such studies have mostly focused on cancer research [62]. In addition, we believe that different effects are caused by different concentrations of 6-G. 6-G (20 µM) inhibited blastocyst formation. This is similar to the results showing that an appropriate concentration of antioxidant supplements could improve early embryo development by reducing oxidative stress, while they would inhibit early embryo development at a high concentration [63][64][65]. This may be because oxidative stress signaling is also required during early embryonic development. A large amount of 6-G will reduce the ROS level too much. Reductive stress is just as dangerous as oxidative stress [66]. At the same time, the concentration of 6-G in the culture medium may decrease with time. However, during the 7 days of in vitro culture, the corresponding concentration of 6-G still affected the embryonic development. Furthermore, many studies have shown that biological substances can induce ROS production and promote apoptosis in cancer cells while exerting beneficial effects on normal cells, promoting cell proliferation and enhancing embryo development [67]. These different effects are related mainly to the abnormal physiological statuses of cancer cells, such as their abnormal gene expression (including that of insulin-like growth factor-1, DNA methyltransferase 1, and histone deacetylases) [68], enzyme activity [69], and activation of signaling pathways including the AKT, MAPK, and nuclear factor kappa B pathways [70]. In this study, 6-G was found to regulate the p38, JNK, and ERK pathways. However, how 6-G regulates key downstream genes through these pathways during early embryo development still needs further research.
In summary, our results indicate that the addition of 6-G to IVC systems increases GSH and regulates functional gene expression and the ERK, JNK, and p38 signaling pathways, thereby reducing intracellular ROS accumulation, autophagy, and apoptosis, and enhancing mitochondrial activity and cell proliferation to improve porcine preimplantation embryo development and competence. Our findings will suggest new methods and provide a theoretical basis for improving the quality of embryo development in vivo and in vitro.

Conclusions
6-G improved blastocyst rate, cell proliferation, mitochondrial function, and inhibited cell apoptosis and autophagy by reducing oxidative stress. Moreover, 6-G can regulate related gene expressions in blastocyst.
Funding: This study was supported by the Science and Technology Project of Jilin Province (SXGJSF2017-6, Jiabao Zhang; and 20200703013ZP, Hao Jiang).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available in the article or supplementary material.