The in vitro production (IVP) of porcine embryos is an important technology for agricultural and biomedical animal models, including development of advanced reproductive technologies (ARTs), embryos transfer, vitrification, and production of transgenic animals (Prather et al., 2003). However, the in vitro culture systems still have many problems, such as embryos development arrest and fragmentation (Chen et al., 2018). It is difficult to mimic the complex environment of mammalian uteri and oviducts in vitro. Furthermore, in vitro derived porcine blastocysts have low development rate and quality of blastocysts compared with in vivo derived porcine blastocysts (Rath et al., 1995; Macháty et al., 1998). In order to solve these problem, natural materials, nutrients, and various antioxidants were added to improve the quality of blastocysts (Lee et al., 2015; Yang et al., 2018).

During respiration and cellular metabolism in mammalian cells, reactive oxygen species (ROS) such as superoxide anion radical (O₂ㆍ^-), hydrogen peroxide (H₂O₂), and hydroxyl radical (ㆍOH) are produced. Maintaining homeostasis of ROS is critical for cell growth, proliferation, division, and survival (Covarrubias et al., 2008). Excessive ROS induced oxidative stress (OS) occurs due to an imbalance between the production of ROS and the antioxidant reaction that remove ROS. Many studies have demonstrated that oxidative damage to preimplantation embryos is harmful in various species, such as human (Yang et al., 1998), cow (Favetta et al., 2007), and mouse (Goto et al., 1993). Furthermore, our previous studies have reported increase in efficiency of IVP embryos using antioxidant and natural substances to suppress ROS (Lee et al., 2015; Yang et al., 2018). However, no reports have investigated this possible interaction between GD1a/GT1b and antioxidative effects response to ROS in porcine embryos.

Glycosphingolipids are divided into neutral glycosphingolipids and acidic glycosphingolipids. Acidic glycosphingolipids containing sialic acid residues in carbohydrate moiety are referred to as gangliosides. Gangliosides are found in the central nervous system and vertebrate plasma membranes (Kwak et al., 2011). Gangliosides are known to play a critical role in cellular functions, such as cell growth, differentiation, and signaling (Kim et al., 2008). Ganglioside GD1a and GT1b are synthesized in GM1a and GD1ab by the ST3GAL2 enzyme (Sturgill et al., 2012). GD1a and GT1b are known for being involved in cell survival and signaling pathways in cancer and neuron. In mice, GT1b was reported to be expressed during embryonic development (Kim et al., 2008). Also, after freezing and thawing, GT1b was expressed in surviving embryos, but GT1b was not expressed in dead embryos (Kim et al., 2008). In addition, GT1b was reported to protect sperm against H₂O₂-mediated cellular damage in human spermatozoa (Gavella et al., 2012; Gavella et al., 2013), and to reduce ROS during in vitro maturation (IVM) of porcine oocytes (Hwang et al., 2015). GD1a has been reported to increase the survival rate of PC12 cell and reduce ROS induced by LPS (Nikolaeva et al., 2015). However, there are no known effects of GD1a and GT1b on embryos development competence in porcine zygote. The aim of the present study was to determine whether the addition of GD1a and GT1b to culture medium affects embryonic developmental competence and quality of blastocysts through the reducing ROS production in porcine embryos.

Chemicals

Unless otherwise stated, all chemicals reagents used in this study were purchased from Sigma-Aldrich Korea (Sigma-Aldrich Korea, Yongin, Korea).

In vitro maturation (IVM)

Porcine ovaries obtained from a local slaughterhouse were transported to the laboratory in 0.9% saline supplement with 75 µg/mL potassium penicillin G at 30-35℃. Cumulus-oocyte complexes (COCs) were aspirated from 3-6 mm follicle using 10 mL syringe and 18 gauge needle. Obtained COCs were selected from thick surrounding cumulus cells and equality cytoplasm using mouth pipetting, then were washed three times in Tyrode’s lactat-N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid (TL-HEPES). Next, 50 immature COCs were matured in 500 µL of in vitro maturation medium in four-well dish (Nunc, Roskilde, Denmark) at 38.5℃ under 5% CO₂ and saturated humidity. BSA-free North Carolina State University (NCSU)-23 medium supplemented with 0.57 mM cysteine, 10 IU/mL pregnant mare’s serum gonadotropin (PMSG), 10 IU/mL human chorionic gonadotropin (hCG), 25 µM β-mercaptoethanol (β-me), 10 ng/mL EGF, 10% follicular fluid (v/v) were used for COCs maturation. After 22 h maturation, COCs were washed three times in PMSG and hCG-free maturation medium and then cultured in medium for 22 h.

In vitro fertilization and culture (IVF & IVC)

In current study, embryo development experiment was performed by evaluating the in vitro culture system, according to the methods described by Kim (2016). At the end of maturation period, matured COCs were performed in vitro fertilization. The IVF medium, modified Tris-buffered medium (mTBM), consisted of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl₂, 5 mM sodium pyruvate, 11 mM glucose, 20 mM Tris, 2.5 mM caffeine sodium benzoate, and 1 mg/mL BSA. Cumulus cells in matured COCs were removed using 0.1% hyaluronidase. Denuded oocytes were washed three times by mTBM and 15-20 oocytes were transferred into 48 µL drop of mTBM under mineral oil. Porcine semen was supplied twice a week by AI (Darby pig AI Center, Anseong, Korea) and kept at 17℃ for 2-3 days. Semen was washed three times by centrifugation in PBS supplemented with 1 mg/mL BSA (w/v), 100 mg/mL penicillin G, and 75 mg/mL streptomycin sulfate. Then, spermatozoa were made to swim up in mTBM. Then 2 µL of spermatozoa were added to 48 µL drop give to a final concentration of 1.5 × 10⁵ sperms/mL. Oocytes were co-incubated with spermatozoa for 6 h at 38.5℃ under 5% CO₂. Approximately 6 h after in semination, embryos were cultured in 50 µL drops of PZM-3 medium with 3 mg/mL BSA at 38.5℃ under 5% CO₂. After 48 h of culture, 25-30 cleaved embryos were further cultured in 50 µL drops of PZM-3 medium with 3 mg/mL BSA at 38.5℃ under 5% CO₂ for 4 days. Blastocyst formation was evaluated after 6 days of culture. Ganglioside GD1a and GT1b were added in different concentration in culture medium.

Assessment of apoptosis in blastocysts

Cellular apoptosis analysis was performed by TUNEL assay, according to the methods described by Kim (2016). Apoptotic blastocysts were detected using an In Situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. On day 6, the blastocysts from IVF were washed with 0.1% PVA in PBS and then fixed in 4% PFA in PBS for 1 h at room temperature. Next, blastocysts were permeabilized using 0.1% (v/v) Triton X-100 for 30 min at 4℃. The fixed embryos were incubated in TUNEL reaction medium for 1 h at 38.5℃, then washed and mounted on slides. Whole-mount embryos were examined under an epifluorescence microscope (Olympus, Tokyo, Japan) following TUNEL assay and DAPI staining, and the number of apoptotic nuclei and total number of nuclei were counted.

Measurement of ROS levels

ROS levels was performed by DCF-DA staining, according to the experimental methods described by Kim (2016). The levels of H₂O₂ in each embryo were measured using the difluorodihydrofluorescein diacetate method (H2DCFDA; Molecular Probes, Eugene, OR, USA) as described previously (Choi et al., 2008). H2DCFDA produces an intermediate difluorodihydrofluorescein (DCF) after reaction with ROS. Oxidation DCF produces difluorofluorescein, which can be monitored in the fluorescence microscope (Crow et al., 1997). At days 6 after IVF, IVP blastocysts were recovered and used for the experiment. After three times of washing in IVC medium, blastocysts were transferred into IVC medium containing 5 mM H2DCFDA for 20 min at 38.5℃. A stock solution of H2DCFDA dissolved in dimethylsulfoxide (DMSO) was then diluted in IVC medium, after which the permeabilized blastocysts in H2DCFDA were washed three times with 0.1% polyvinylalcohol (PVA)-PBS and placed into a 50 µL drop covered with mineral oil. The fluorescent emissions from the embryos were recorded with a fluorescent microscope (IX 51; Olympus, Tokyo, Japan) equipped with a cooled charge coupled device (CCD) camera which filters at 488 nm and 520 nm were used for excitation and emission, respectively. The recorded fluorescent images were analyzed by subtracting background and measuring integrated density with Image J software Version 1.38 (National Institutes of Health, Bethesda, MD, USA). Overall, blastocysts in each treatment groups were examined.

Statistical analysis

All percentage data obtained in this study are presented as the mean ± standard deviation (SD). Moreover, Western blot experiments were performed in triplicate and all values were presented as the mean ± standard error of the mean (SEM). The results were analyzed using either a one-way ANOVA followed by Bonferroni’s Multiple Comparison Test or by performing a t-test. All data were analyzed using the GraphPad Prism 5.0 software package (San Diego, CA, USA). Differences were considered significant at *p < 0.05, **< 0.01, and ***< 0.001.

Expression of ST3GAL2 protein in embryos during IVC

We measured the expression of ST3GAL2 (Table 1) as a synthesizing enzyme for GD1a and GT1b production in porcine embryos during IVC process (2-cells, 4-cells, 8-cells, and blastocysts) by immunofluorescence (IF). As shown in Fig. 1, the expression of ST3GAL2 was significantly increased in 4-8 cells stage than in 2 cells and blastocysts stage. This result demonstrated that the GD1a and GT1b synthesizing enzyme ST3GAL2 was expressed in embryos during development stage.

Table 1. Antibodies used for immunofluorescence.

Target protein	Dilution	Catalog number	Source*
ST3GAL2	1:3000	sc-292044	Santa Cruz Biotechnology

^*Santa Cruz Biotechnology, Dallas, TX, USA..

Figure 1.Fluorescence expression patterns of ST3GAL2 in porcine embryos during IVC. (A) ST3GAL2 expression was examined in embryonic development during IVC by immunofluorescence. (B) Quantitative analysis of ST3GAL2 protein levels in embryonic development during IVC. Data in the bar graph represent the means ± SEM of three independent experiments. Scale bar = 100 µm. Differences were considered significant at **p < 0.01 and ***< 0.001.

Effects of ganglioside GD1a and GT1b on embryonic developmental competence in pigs

To find the appropriate treatment concentration of GD1a and GT1b, we supplied exogenous ganglioside GD1a and GT1b with culture medium for porcine embryos development. After fertilization, porcine zygotes were cultured in IVC medium supplemented with 0.1, and 0.2 µM GD1a or GT1b for 6 days, respectively. As shown in Fig. 2 and 3, the rates of blastocysts formation and expanded blastocysts production in the presence of only 0.1 µM GD1a and GT1b were significantly higher than those of control (Table 2; Non-treatment: 27.0 ± 5.0% vs GD1a 0.1 µM: 37.5 ± 9.5%, Table 3; Non-treatment: 27.5 ± 6.0% vs GT1b 0.1 µM: 40.1 ± 9.0%). These results showed the positive roles of GD1a and GT1b on development confidence in porcine embryos.

Table 2. Effect of various concentrations of GD1a on preimplantation development of porcine embryos in vitro.

Treatment	No. of embryos examined	% of embryos cleaved (n)	% of blastocysts (n)
Non-treated	217	86.4 ± 12.0 (182)	27.0 ± 5.0 (56)a
GD1a 0.1 µM	217	80.8 ± 9.5 (172)	37.5 ± 9.5 (78)b
GD1a 0.2 µM	217	82.3 ± 6.8 (175)	21.9 ± 9.0 (46)a

Data are expressed as means ± SD of three independent experiments. Different superscript letters denote significant differences (p < 0.05)..

Table 3. Effect of various concentrations of GT1b on preimplantation development of porcine embryos in vitro.

Treatment	No. of embryos examined	% of embryos cleaved (n)	% of blastocysts (n)
Non-treated	197	89.1 ± 7.8 (175)	27.5 ± 6.0 (54)a
GT1b 0.1 µM	197	89.8 ± 8.2 (178)	40.1 ± 9.0 (79)b
GT1b 0.2 µM	197	84.2 ± 11.9 (164)	32.8 ± 8.1 (63)a

Data are expressed as means ± SD of three independent experiments. Different superscript letters denote significant differences (p < 0.05)..

Figure 2.Effects of ganglioside GD1a during IVC on development of porcine embryos. (A) Representative photographs of blastocysts according to treatment concentrations (0.1 and 0.2 µM) of GD1a. Scale bar = 100 µm. Rates of cleavage stage (B) and blastocyst stage (C) in the absence or presence of different concentrations of GD1a. Data in the bar graph represent the means ± SEM of three independent experiments. Differences were considered significant at *p < 0.05.

Cellular apoptosis in blastocysts derived from ganglioside GD1a and GT1b treated embryos

To investigate the effects of exogenous GD1a and GT1b treatment on quality of blastocysts during IVC, we confirmed apoptotic pattern of porcine blastocysts using TUNEL assay (Fig. 4). As shown Table 4, the number of total nuclei increased in blastocysts derived from GD1a and GT1b treated embryos than in the control group. However, no difference was observed in the number of TUNEL positive nuclei between GD1a or GT1b treated groups and control groups.

Table 4. Effect of ganglioside GD1a and GT1b on total cell number and apoptosis in blastocysts.

Group	No. of embryos examined	No. of cells		TUNEL-positive cells (%)
		Total	TUNEL-positive
Con	14	33.4 ± 11.3	0.1 ± 0.3	0.8 ± 0.3
GD1a 0.1 µM	16	47.6 ± 16.2	0.2 ± 0.4	0.3 ± 0.6
GD1a 0.2 µM	16	34.6 ± 12.0	0.1 ± 0.3	0.3 ± 0.9
GT1b 0.1 µM	16	44.6 ± 13.0	0.2 ± 0.4	0.4 ± 0.9
GT1b 0.2 µM	10	37.9 ± 14.7	0.2 ± 0.4	0.4 ± 0.8

This experiment was replicated three times. Data are the mean ± SD..

Figure 4.Apoptotic patterns in blastocysts derived from GD1a and GT1b treated embryos. Total chromatin content were determined by staining with DAPI (blue) and fragmented DNA was labeled via terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; green, white arrows). Merged images show TUNEL and DAPI signals. Scale bar = 100 µm. Data in the bar graph represent the means ± SEM of three independent experiments. Differences were considered significant at *p < 0.05 and **< 0.01.

Effects of ganglioside GD1a and GT1b on intracellular ROS levels of porcine embryos

We investigated the intracellular levels of ROS in ganglioside GD1a and GT1b treated embryo at 4-cells, 8-cells, and blastocyst stages. As shown in Fig. 5, the intracellular level of ROS was significantly decreased in blastocysts derived from 0.1 µM GD1a and GT1b treated embryos compared with the control group. However, there was no significant difference at 4-cells and 8-cells.

Figure 5.Detection of ROS levels in blastocysts derived from ganglioside GD1a and GT1b treated embryos by H2DCFDA staining. (A) Fluorescence imaging of ROS production in blastocysts by H2DCFDA. (B) Quantification of ROS levels in blastocysts. Scale bar = 100 µm. Data in the bar graph represent the means ± SEM of three independent experiments. Differences were considered significant at ***p < 0.001.

In present study, we demonstrated that exogenous ganglioside GD1a and GT1b enhanced embryonic development competence and blastocysts quality in porcine embryo. In addition, intracellular ROS levels reduced in GD1a and GT1b treated blastocyst stage. Therefore, we first identified possible roles of ganglioside GD1a and GT1b in an antioxidative effect during in vitro culture (IVC) of porcine embryos. Interestingly, the results above showed that ganglioside GD1a and GT1b play major role as ROS scavenger in early embryo development of pigs.

Ganglioside GD1a and GT1b are known to be synthesized by the ST3GAL2 enzyme (Sturgill et al., 2012). In previous studies, in ST3GAL2 knockout(K/O) mice, the synthesis of GD1a and GT1b was decreased and the expression of GM1a and GD1b was increased (Sturgill et al., 2012). Fig. 1 shows expression patterns of ST3GAL2. To infer the expression of GD1a and GT1b in embryos development stages, we measured the expression of ST3GAL2 using IF staining. The red fluorescence expression of ST3GAL2 increased at the 4-cells and 8 cells stage of porcine embryos after IVF. Therefore, we speculated that there is a close correlation between GD1a and early embryonic development. Based on these results, we performed subsequent experiments using GD1a and GT1b treated culture medium during IVC.

Ganglioside GD1a and GT1b regulate cellular function, such as cell differentiation and growth control in neuron (Magistretti et al., 2019) and cancer cells (Groux-Degroote et al., 2018). Our previous study shows that GD1a can improve porcine oocyte quality, maturation capacity, and subsequent embryonic development through its various biological activities such as EGFR activation or ERK signaling (Kim et al., 2016; Park et al., 2017). In previous study, we confirmed that GT1b was expressed during the embryonic development in mice (Kim et al., 2008). However, effects of GD1a and GT1b treatment on embryos development in mammalian embryos has not investigated. Therefore, we examined the effects of GD1a and GT1b on embryos development in porcine embryos during IVC. We confirmed that the development rate of porcine embryos was significantly increased in the 0.1 µM GD1a and GT1b treated culture medium (Fig. 2 and 3). We first proved that GD1a and GT1b enhance porcine embryos development.

Figure 3.Effects of exogenous ganglioside GT1b during IVC on development of porcine embryos. (A) Representative photographs of blastocysts according to treated concentrations (0.1 and 0.2 µM) of GT1b. Scale bar = 100 µm. Rates of cleavage (B) and blastocyst stage (C) in the absence or presence of different concentrations of GT1b. Data in the bar graph represent the means ± SEM of three independent experiments. Differences were considered significant at *p < 0.05.

Apoptosis has been used as an important indicator for embryonic development and embryonic loss following the stress response of cells in IVP embryos. Previous studies have reported that the rate of apoptosis was increased by harmful substances such as H₂O₂ during embryos development (Lee et al., 2015). On the other hand, the rate of apoptosis was decreased in the melatonin and antioxidants treated groups (Do et al., 2015; Lee et al., 2015). Therefore, apoptosis in blastocysts is an important indicator for evaluation the quality of blastocysts and in vitro environments. In addition, we investigated that the ganglioside GM3 is involved the apoptotic stress in porcine oocyte and embryo during in vitro production (IVP) progression (Chae et al., 2015; Kim et al., 2018). However, no reports have identified this crosstalk between GD1a or GT1b and blastocyst development capacity in porcine embryos. In our results, there was no significant difference in the number of total cells and ratio of apoptosis rate. However, we confirmed that the total cell number was increased in 0.1 µM GD1a and GT1b treated groups (Table 4). This result support that the GD1a and GT1b treatment improves blastocyst quality by protecting DNA damage mediated cellular apoptosis.

ROS are produced during normal cell activation and are involved in a variety of biological processes, including cell differentiation and gene expression. Therefore, maintaining homeostasis of ROS is very important for cell growth, survival (Covarrubias et al., 2008), embryo development, and cloned embryos production (Fang et al., 2018). Oxidative stress is a phenomenon in which the ROS in the cell increases due to an imbalance between the production of ROS and the antioxidant reactions that remove it, causing it to react with and damage DNA, proteins, and lipids (Tarín, 1996). This damage is known to cause many cellular problems on embryos development during IVP of cow (Favetta et al., 2007) and mice (Goto et al., 1993). Our previous results have reported increase in the efficiency of IVF embryos production and development potential using natural substances or antioxidants to suppress ROS or oxidative stress in pigs (Lee et al., 2015; Yang et al., 2018). According to previous studies, Ganglioside GT1b protected spermatozoa against H₂O₂ damage in human (Gavella et al., 2012; Gavella et al., 2013), and reduced ROS during in vitro maturation of porcine oocytes (Hwang et al., 2015). Also, Ganglioside GD1a reduced ROS induced by LPS and increased survival rate in PC12 cells (Nikolaeva et al., 2015). In present study, there was no significant difference in intracellular ROS levels between GD1a and GT1b treated groups and control group at the cleavage stage (4-8 cells). However, in the blastocyst stage, intracellular ROS levels were significantly decreased in the GD1a and GT1b treated groups compared with control group (Fig. 5). These results suggest that ganglioside GD1a and GT1b decrease intracellular ROS levels of porcine blastocysts in vitro.

In conclusion, the results of the present study suggest that treatment of ganglioside GD1a and GT1b in porcine preimplantation embryos enhanced rate of blastocyst development. Although there was no significant difference in the rate of apoptosis, number of total cell was increased. Furthermore, we confirmed the intracellular ROS levels decreased in the blastocyst stage. Therefore, we suggest that treatment of ganglioside GD1a and GT1b during IVC may assist in the preimplantation development of porcine embryos.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2018R1C1B002922 and NRF-2019R1A2C1085199) funded by the Ministry of Education and the Ministry of Science and ICT, Republic of Korea.

No potential conflict of interest relevant to this article was reported.

Conceptualization: Jin-Woo Kim, Hyo-Jin Park and Deog-Bon Koo

Data curation: Jin-Woo Kim and Seul-Gi Yang

Formal analysis: Jin-Woo Kim, Seul-Gi Yang, Hyo-Jin Park

Supervision and funding acquisition: Deog-Bon Koo

Investigation: Jin-Woo Kim and Seul-Gi Yang

Methodology: Jin-Woo Kim, Seul-Gi Yang and Hyo-Jin Park

Resources, Project administration: Jin-Woo Kim, Seul-Gi Yang and Deog-Bon Koo

Validation: Jin-Woo Kim and Seul-Gi Yang

Writing - original draft: Jin-Woo Kim and Hyo-Jin Park

Writing - review & editing: Seul-Gi Yang, Hyo-Jin Park and Deog-Bon Koo

JW Kim, PhD, https://orcid.org/0000-0002-0089-680X

HJ Park, PhD, https://orcid.org/0000-0003-3287-2914

SG Yang, PhD Candidate, https://orcid.org/0000-0003-3247-2443

DB Koo, Professor, https://orcid.org/0000-0001-7825-9598