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ISSN : 2671-4639(Print)
ISSN : 2671-4663(Online)
Journal of Animal Reproduciton and Biotechnology Vol.32 No.1 pp.17-24
DOI : https://doi.org/10.12750/JET.2017.32.1.17

Ganglioside GD1a Activates the Phosphorylation of EGFR in Porcine Oocytes Maturation in vitro

Hyo-Jin Park, Jin-Woo Kim, Jae-Young Park, Seul-Gi Yang, Jae-Min Jung, Min-Ji Kim, Deog-Bon Koo†
Department of Biotechnology, College of Engineering, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk 38453, Republic of Korea
Correspondence: Deog-Bon Koo Tel: +82 53 850 6557, Fax: +82 53 850 6559dbkoo@daegu.ac.kr
November 21, 2016 December 6, 2016 March 15, 2017

Abstract

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.


초록


    Daegu University

    INTRODUCTION

    Epidermal growth factor (EGF) and EGF-receptor (EGFR) signaling pathways have important roles in normal ovarian steroidogenesis and oocyte maturation both in vivo and in vitro (Jamnongjit et al., 2005). Activated EGFR signaling in cumulus cells is required for resumption of meiosis via inhibition of Gap junction and decreasing of cAMP during oocyte maturation progression (Edry et al., 2006; Sela- Abramovich et al., 2005; Bornslaeger and Schultz, 1985). Furthermore, EGFR-related signaling has an important role in cumulus cell expansion of cumulus-oocyte-complex (COC) and in ovulation by inducing specific cumulus-secreted genes (Cox-2; hyaluronan synthase 2, HAS-2; tumor necrosis factors- α induced protein 6, TNFAIP6, and others) (Hsieh et al., 2007). Previous researchers have reported on the addition of EGF to in vitro maturation media to improve the meiotic maturation and blastocyst rates in pigs (Illera et al., 1998), rats (Ben-Yosef et al., 1992), mice (De La Fuente et al., 1999), humans (Goud et al., 1998), and cattle (Lonergan et al., 1996).

    Gangliosides (GDs) are present in the plasma membrane of vertebrate tissues and in the central nervous system and are biosynthesized in the Golgi apparatus (Sandhoff and Harzer, 2013). GDs regulate biological processes such as cell differentiation, cell adhesion, and regulation of signal transduction (Kim et al., 2008). Based on structure, GDs can be divided into a-(GM3, GM2, GM1a GD1a, GT1a), b-(GD3, GD2, GD1b, GT1b, GQ1b), and c-(GT3, GT2, GT1c, GQ1c, GP1c) series. The GDs that contain sialic acid residues can be biosynthesized by sialyltransferases (ST3GAL5, ST3GAL2, ST8SIA1 and ST8SIA5) (Lloyd and Furukawa., 1998). In particular, GDs regulate the activity and phosphorylation of various receptors including epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGF), and fibroblast growth factor receptor (FGFR) (Julien et al., 2013). Previous researchers have reported on expressions of GM3 and GM1 in spermatogenesis, GM3, GM1, GD1a, GD1b, GT1b, GM2, and GD3 in ovary maturation, and GM3 and GT1b in the development of preimplantation embryos (Kwak et al., 2011).

    Synthesis of GD1a from ganglioside GM1a occurs via a specific synthetic enzyme (ST3 β-galactoside α-2, 3-sialyltransferase 2; ST3GAL2) (Yang et al., 2011). Exposure to exogenous ganglioside GD1a expression has been associated with enhancement of dimerization and phosphorylation of EGFR (Liu et al., 2004). In addition, GD1a treatment increases EGF-induced proliferation and enhances EGFR-mediated activation of the mitogen-activated protein kinase (MAPK) signaling pathways (Li et al., 2001). Recently, our research has shown that GD1a promotes preimplantation development of embryo blastocysts and blastocyst quality in addition to improving oocyte maturation and COC cumulus cell expansion (Kim et al., 2016). However, relationships between EGFR activation and exogenous ganglioside GD1a have not yet been thoroughly described in porcine oocyte maturation.

    Based on this absence, we speculated that GD1a can regulate EGFR activity by controlling EGFR phosphorylation during the porcine in vitro maturation process. Therefore, in this study, we investigated the expression patterns associated with phosphorylation of EGFR and ST3GAL2, a GD1a synthesizing enzyme, during porcine oocyte maturation and under different EGF and GD1a concentrations. Our aim was to identify the functional relationship between EGFR activity and the expression pattern of GD1a during maturation in pigs in vitro.

    MATERIALS and METHODS

    Chemicals

    Unless noted otherwise, all chemicals used in the present study were purchased from Sigma Aldrich Korea (St. Louis, MO, USA).

    In vitro maturation (IVM)

    We were performed in vitro maturation using slight modified as described by Lee et al. (2015). Porcine ovaries were collected from at a local slaughterhouse and transported to the laboratory in 0.9% saline supplemented 75 μg/ml potassium penicillin G maintained at 30-35°C. Immature cumulus-oocyte complexes (COCs) were aspirated from follicles between 3 - 6 mm in diameter using an 18-gauge needle. After, undamaged COCs with the same quality cytoplasm and surrounded by cumulus cells were selected using mouth pipettes and then washed three times in Tyrode’s lactate-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (TLHEPES) medium supplemented with 0.3% BSA (w/v), approximately 50 COCs were matured in 500 μL of IVM medium at 38.5°C and under 5% CO2 in air. The IVM medium used for oocyte maturation was BSA-free North Carolina State University (NCSU)-23 medium supplemented with 10% follicular fluid (v/v), 0.57 mM cysteine, 10 ng/ml β -mercaptoethanol, 10 IU/ml pregnant mare’s serum gonadotropin (PMSG) and 10 IU/ml human chorionic gonadotropin (hCG). After culturing for 22 h, COCs were washed three times and then further cultured in PMSG and hCG-free maturation medium for 22 h. During the maturation periods, epidermal growth factor (EGF, 10 and 50 mg/ml) and exogenous ganglioside GD1a (10 μM) were added to the maturation medium.

    Immunofluorescence (IF) staining

    Maturated porcine denuded oocytes (DO) and COCs in IVM progression (22 h and/or 44 h) were washed with 0.3% PVA-PBS (w/v) and fixed in 4% (v/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde solution for 1 h at room temperature. Next, DO and COCs were transferred to permeabilization solution (0.2% Triton X-100) for 1 h. After blocking for overnight at 4°C in 0.1% PVA-PBS (w/v) containing 1% BSA (w/v), they incubated with ST3GAL2 antibody (SC-292044; Santa Cruz Biotechnology, CA, USA) diluted 1:2500 for overnight at 4 °C. After incubation, the COCs and DO were reacted with the secondary antibody, FITC-conjugated goat anti-rabbit IgG (Santa Cruz), diluted 1:1000 in 0.3% PVA-PBS (w/v) for 2h at room temperature. DAPI reagent (2 mg/ml) was used to stain the nuclei. Finally, we observed immune-reactivity under an epifluorescence microscope (IX 51, Olympus, Tokyo, Japan).

    Protein extraction and Western blot analysis

    Matured COCs (25-30 COCs per group) lysates were prepared in PRO-PREP protein lysis buffer (iNtRON, Daejeon, Korea) centrifuging at 10,000 × g for 20 min at 4°C. Proteins of COCs were separated by sodium dodecyl sulphate (SDS) polyacrylamaide gel electrophoresis (PAGE) in 8 or 10% gels. After electrophoresis, the separated proteins were transferred onto nitrocellulose (NC) membranes (Pall Corporation, Port Washington, NY, USA). The membrane was blocked by incubation with 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) for overnight at 4°C and then the membrane was incubated with the appropriate primary antibody; anti-ST3GAL2, anti-β-actin (SC-292044, SC-47778; Santa Cruz, CA, USA), anti-EGFR (SC-03, Santa Cruz) and anti-phospho-EGFR (SC-12351, Santa Cruz) antibodies diluted 1:1000 for overnight at 4°C. The membranes were then incubated with a secondary antibodies horseradish peroxidaseconjugated anti-polyclonal-rabbit /monoclonal-mouse IgG (# 31463, #31439; Thermo, Rockford, IL) diluted 1:5000 for 1 h at room temperature. Antibody binding was detected with a chemiluminescent system (BightTM ECL Kit, Advansta Inc., CA, USA). Band intensities were quantified with Image J software (NIH, MD, USA).

    Statistical analysis

    All 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 and ** < 0.01.

    RESULTS

    Changes of ST3GAL2 gene expression in porcine COCs according to EGF treatment

    We investigated the fluorescence expression patterns of ST3GAL2 as GD1a synthesizing enzyme by IF staining analysis in DO after IVM (44 h) supplemented with EGF (0, 10, 50 ng/ml), respectively (Fig. 1). The fluorescence expression of ST3GAL2 was significantly decreased (p<0.01) in cytoplasm (Fig. 1A) and surface (Fig. 1B) of porcine DO according to the EGF treatment (10 and 50 ng/ml) than in EGF non-treatment group. And, to investigate the changes of ST3GAL2 expression in cumulus cells of COCs according to EGF treatment, we performed the IF staining analysis. As shown in Fig. 2, ST3GAL2 fluorescence expression significantly decreased (p<0.01) in COC of EGF treated (10 and 50 ng/ml) groups compared to non-treated group. As well as, the expression of ST3GAL2 enzyme was significantly decreased (p<0.01) in DO and cumulus cells of COCs at the IVM II (44 h) compared to IVM I (22 h) phase. These results demonstrated that fluorescence and protein expression of ST3GAL2 decreased as EGF treatment in porcine DO and COCs during IVM progression.

    Expression of EGFR and p-EGFR protein levels in porcine COCs of IVM after EGF and/or GD1a treatment.

    We investigated the protein expression of ST3GAL2 using Western blotting analysis in maturated porcine COCs under the different EGF (0, 10, 50 ng/ml) concentration. As shown in Fig. 3A, the protein expression of ST3GAL2 was dramatically decreased in EGF high concentration (50 ng/ml) treatment (p<0.01), whereas expression of p-EGFR protein was significantly increased. To confirm the effect of exogenous GD1a on porcine COCs and oocyte maturation, we evaluated under the following three condition; (i) non-treatment group; control, (ii) only EGF (10 ng/ml) treatment group and (iii) only GD1a (10 μM) treatment groups. We investigated the protein levels of ST3GAL2 and EGFR activation related genes (EGFR, p-EGFR) in maturated porcine COCs. The protein expression of p-EGFR was significantly higher in porcine COCs of EGF (10 ng/ml) and GD1a (10 μM) treated group than control group. Total EGFR protein level was no changes in three condition groups (Fig. 3B). Also, we investigated that change of ST3GAL2 protein expression in COCs of IVM II phase according to the GD1a (10 or 50 μM) treatment after EGF (10 and 50 ng/ml) pre-treatment. As shown in Fig. 3C, ST3GAL2 expression of COCs was specific decreased in GD1a high concentration (50 μM) treated group regardless of EGF pre-treatment condition. These results demonstrated that expression of ST3GAL2 protein levels was influence to EGF or GD1a treatment. Moreover, EGF and GD1a treatment enhanced the p-EGFR expression in maturation of porcine COCs.

    DISCUSSION

    Ganglioside GD1a is an a-series GDs with a structure that contains two sialic acid residues. The synthesizing enzyme ST3GAL2 synthesizes GD1a from GM1a, and ST3GAL2 knock-down decreases the expression of GD1a (Zhang et al., 2011; Yang et al., 2011). Previous studies have investigated the expression patterns of gangliosides GD1a and GT1b (Kim et al., 2016; Hwang et al., 2015) and a variety of GDs in mouse during oocyte maturation and preimplantation development stages in vivo (Choo et al., 1995; Kim et al., 2006). In addition, it has been shown that GD1a is expressed in mouse oocyte, interstitial cells, and theca cells (Choo et al., 1995). Therefore, we investigated the expression of the ST3GAL2 enzyme protein levels in maturing porcine COCs after GD1a or EGF treatments.

    In present study, we showed, through IF staining and Western blotting analysis that the ST3GAL2 expression pattern decreased according to treatment by EGF in a concentrationdependent manner during porcine oocyte maturation in vitro. Fig. 1 and 2 show the expressions of ST3GAL2 enzyme protein in porcine DO or COCs at IVM II (44 h) after EGF treatment (10 or 50 ng/mL). Similarly, the fluorescence expression pattern of ST3GAL2 was higher in cytoplasm of DO and COCs at IVM II (44 h) than it was on the oocyte membrane surface. Based on these results, we conclude that a reduction of ST3GAL2 expression is related to the amount of GD1a in porcine COCs at the IVM II stage.

    Porcine oocyte meiotic maturation is a well-orchestrated process that includes binding of GDs to membrane receptors and phosphorylation or dimerization of specific receptor such as EGFR (Liu et al., 2004). In addition, disruption of ST3GAL2 gene expression has been shown to have an important role in reducing EGFR activity in cancer (Zhang et al., 2011). In particular, inhibition of ST3GAL2 suppresses the GD1a and EGFR signaling pathways (Sturgill et al., 2012). However, GD1a is unable to interact directly with EGFR activation, suggesting an alternate means of improving oocyte maturation. However, EGFR activation following a change in ST3GAL2 or GD1a expression in porcine oocyte maturation is not yet fully described.

    As shown in Fig. 3, expression of ST3GAL2 dramatically decreased following EGF treatment in a concentrationdependent manner in porcine COCs at the M II stage (44 h). Although there was no direct observation of the expression pattern of GD1a in porcine oocyte, we speculate that GD1a or ST3GAL2 is involved in EGFR activity. Previous reports have shown that phosphorylated EGFR to EGF and EGF-like factors such as amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), which are generated in granulosa cells and are activated by EGFR in cumulus cells (Conti et al., 2005). In addition, exposure to EGF and exogenous GD1a can significantly increase the protein levels of phosphorylated EGFR (p-EGFR) (Liu et al., 2004; Kim et al., 2008). As expected, expression of p-EGFR increased in COCs at IVM II after EGF treatment in a concentration-dependent manner, whereas expression of ST3GAL2 rapidly decreased under those conditions (Fig. 3A). In addition, p-EGFR protein expression was increased in maturing COCs in the EGF- or GD1a-treated groups (Fig. 3B). These results suggest that exogenous GD1a induced an increase in p-EGFR expression in a manner similar to that of EGF. Interestingly, the maturing COCs in the high concentration (50 μM) GD1a treatment group induced a reduction of ST3GAL2 expression regardless of the EGF pre-treatment condition.

    The GD1a ganglioside regulates the activity of EGFR. In particular, it has a role in increasing EGFR phosphorylation and inducing EGFR activity. In the current study, we investigated, for the first time, the activity of EGFR in extracorporeal cumulus cells of COCs and in oocyte maturation. Interestingly, our results showed that treatment with exogenous GD1a increased the activity of EGFR in COCs during IVM II (44 h). In a previous paper, we confirmed that meiotic maturation of porcine oocyte increased when only GD1a was treated, except for EGF. Therefore, we speculated that exogenous GD1a treatment was the cause of increased in vitro maturation of porcine oocytes and that it had a role in regulating the activity of EGFR.

    In summary, the present study provides the first evidence that EGF and/or GD1a can influence the synthesis of ST3GAL2, a GD1a-synthesizing enzyme, during porcine oocyte maturation (Fig. 4; Graphical summary). Furthermore, we have shown that exposure to exogenous GD1a enhances EGFR activity through p-EGFR expression during porcine oocytes maturation. Therefore, EGF and exogenous GD1a perform similar roles in regulating of EGFR activity during porcine IVM progression. These results suggest that GD1a may have an important role in enhancing porcine oocyte maturation via EGFR signaling.

    ACKNOWLEDGMENTS

    This research was supported by a Daegu University Research Grant, 2014.

    Figure

    JET-32-17_F1.gif

    Immunofluorescence staining of ST3GAL2 as GD1a synthesizing enzyme in porcine DO. Fluorescent confocal image of ST3GAL2 (Red) enzyme in porcine oocyte cytoplasm (A) and surface (B) at IVM II stage (44 h maturation) under the different EGF concentration (0, 10, 50 ng/ml). EGF non-treatment group is control. Scale bar = 20 μm.

    JET-32-17_F2.gif

    Immunofluorescence staining of ST3GAL2 enzyme in porcine COCs. Fluorescent expression patterns of ST3GAL2 enzyme in porcine maturing COCs at 22 h (IVM I, A) and 44 h (IVM II, B) after the EGF treatment (10, 50 ng/ml). Red, ST3GAL2; bright field, BF; blue, DAPI. Scale bar = 50 μm.

    JET-32-17_F3.gif

    Western blot analysis of ST3GAL2 and EGFR (EGFR, p-EGFR) protein levels in matured (44 h) porcine COCs. (A) Changes of ST3GAL2, EGFR and p-EGFR protein expression levels in various concentration of EGF treated porcine COCs at 44 h. (B) Changes of EGFR and p-EGFR protein expression levels in EGF (10 ng/ml) and GD1a (10 μM) treated porcine COCs at 44 h. (C) Changes of ST3GAL2 protein expression levels in EGF (10 and 50 ng/ml) and GD1a (1 and 10 μM) treated porcine COCs at 44 h. The protein expression of ST3GAL2 and EGFR was normalized to β-actin expression. The p-EGFR protein was normalized to total EGFR protein expression. Bar graph data represent as means ± SEM. Differences were considered significant at * P < 0.05; ** < 0.01.

    JET-32-17_F4.gif

    Graphical summary; effects of exogenous GD1a on porcine oocyte of IVM progression related to the expression of EGFR and p-EGFR. Both EGF and GD1a exposure induced the decreasing of ST3GAL2 enzyme and increasing of EGFR activity in porcine oocyte maturation. Based on these results, it was speculated that GD1a had the same role as EGF in the in vitro maturation of oocytes.

    Table

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