Journal of Animal Reproduction and Biotechnology 2020; 35(4): 347-356
Published online December 31, 2020
https://doi.org/10.12750/JARB.35.4.347
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jin-Woo Kim1,2,# , Hyo-Jin Park1,2,#
, Seul-Gi Yang1,2
and Deog-Bon Koo1,2,*
1Department of Biotechnology, College of Engineering, Daegu University, Gyeongsan 38453, Korea
2Institute of Infertility, Daegu University, Gyeongsan 38453, Korea
Correspondence to: Deog-Bon Koo
E-mail: dbkoo@daegu.ac.kr
ORCID https://orcid.org/0000-0001-7825-9598
#The first two authors contributed equally to this work.
Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1µM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 µM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 µM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.
Keywords: GD1a, GT1b, In vitro culture (IVC), porcine embryo, reactive oxygen species (ROS)
The
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
Unless otherwise stated, all chemicals reagents used in this study were purchased from Sigma-Aldrich Korea (Sigma-Aldrich Korea, Yongin, Korea).
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 current study, embryo development experiment was performed by evaluating the
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.
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.
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.
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..
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
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 (
Table 3 . Effect of various concentrations of GT1b on preimplantation development of porcine embryos
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 (
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..
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.
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
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.
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
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 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
Journal of Animal Reproduction and Biotechnology 2020; 35(4): 347-356
Published online December 31, 2020 https://doi.org/10.12750/JARB.35.4.347
Copyright © The Korean Society of Animal Reproduction and Biotechnology.
Jin-Woo Kim1,2,# , Hyo-Jin Park1,2,#
, Seul-Gi Yang1,2
and Deog-Bon Koo1,2,*
1Department of Biotechnology, College of Engineering, Daegu University, Gyeongsan 38453, Korea
2Institute of Infertility, Daegu University, Gyeongsan 38453, Korea
Correspondence to:Deog-Bon Koo
E-mail: dbkoo@daegu.ac.kr
ORCID https://orcid.org/0000-0001-7825-9598
#The first two authors contributed equally to this work.
Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1µM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 µM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 µM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.
Keywords: GD1a, GT1b, In vitro culture (IVC), porcine embryo, reactive oxygen species (ROS)
The
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
Unless otherwise stated, all chemicals reagents used in this study were purchased from Sigma-Aldrich Korea (Sigma-Aldrich Korea, Yongin, Korea).
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 current study, embryo development experiment was performed by evaluating the
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.
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.
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.
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..
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
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 (
Table 3. Effect of various concentrations of GT1b on preimplantation development of porcine embryos
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 (
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..
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.
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
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.
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
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 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
Table 2 . Effect of various concentrations of GD1a on preimplantation development of porcine embryos
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 (
Table 3 . Effect of various concentrations of GT1b on preimplantation development of porcine embryos
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 (
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..
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