Effect of ganglioside GT1b on the in vitro maturation of porcine oocytes and embryonic development.

Ganglioside is an acidic glycosphingolipid with sialic acids residues. This study was performed to investigate the effect and mechanism of ganglioside GT1b in porcine oocytes in the process of in vitro maturation (IVM) and preimplantation development. Metaphase II (MII) rates were significantly (P < 0.05) different between the control group and the 5 nM GT1b treatment group. Intracellular glutathione (GSH) levels in oocytes matured with 5 nM and 20 nM and GT1b decreased significantly (P < 0.05). The 10 nM group showed a significant (P < 0.05) decrease in intracellular reactive oxygen species (ROS) levels compared with the control group. Subsequently, the level of intracellular Ca(2+) in oocytes treated with different concentrations of GT1b was measured. Intracellular Ca(2+) was significantly (P < 0.05) increased with a higher concentration of GT1b in a dose-dependent manner. Real-time PCR was performed and showed that the expression of bradykinin 2 receptor (B2R) and calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ) in cumulus cells was significantly (P < 0.05) decreased in the 20 nM GT1b treatment group. Treatment with 5 nM GT1b significantly (P < 0.05) decreased the expression of CaMKIIδ. In oocytes, treatment with 5 nM GT1b significantly (P < 0.05) decreased CaMKIIγ and POU5F1 (POU domain, class 5, transcription factor 1). However, treatment with 20 nM GT1b significantly (P < 0.05) increased the expression of POU5F1. Finally, embryonic developmental data showed no significant differences in the two experiments (parthenogenesis and in vitro fertilization). In conclusion, the results of the present study indicated that GT1b plays an important role in increasing the nuclear maturation rate and decreasing the intracellular ROS levels during IVM. However, GT1b inhibited maturation of the cytoplasm by maintaining intracellular Ca(2+) in the process of oocyte maturation regardless of the cell cycle stage. Therefore, GT1b is thought to act on another mechanism that controls intracellular Ca(2+).

Pigs are anatomically and physiologically similar to humans, and in the field of regenerative medicine, it is known that is suitable in comparison with mice and rats. However, the in vitro maturation system for pig oocytes is still inefficient, mainly due to the loss of the developmental capacity in the preimplantation stage [1]. This inefficiency might be associated with an insufficient understanding of the epigenetic mechanisms of porcine oocytes during in vitro maturation (IVM), macro- and micronutrients, endocrine status and oxidative stress compared with our understanding of other mammals [2, 3].

To date, porcine IVM conditions have been improved by treatment with various antioxidants, such as melatonin [4,5,6] and resveratrol [7,8,9], and growth factors, such as epidermal growth factor (EGF) [10,11,12], granulocyte-macrophage colony-stimulating factor (GM-CSF) [13] and vascular endothelial growth factor (VEGF) [14, 15]. Through these studies, the quality of porcine oocytes has shown some improvement. In a recent study, it was also confirmed that a glycosphingolipid called ganglioside is diversely expressed in the process of mouse embryonic development [16].

Ganglioside is an acidic glycosphingolipid that has sialic acids residues and is known to play an important role in biological processes, such as cell differentiation, adhesion, regulation of growth and signal transduction [17]. Specifically, it is distributed mainly in the central nervous system and is involved in the development of the brain and memory [18, 19]. The structure of gangliosides includes a sugar chain with one or more sialic acid (e.g., n-acetylneuraminic acid, NANA) residue. More than 60 gangliosides exist according to the number and the position of the NANA residues. They can be classified into a-, b- and c-series gangliosides according to the number of sialic acids bound to the galactose portion of the molecule [20]. The a-series includes GM3, GM2, GM1, GD1a and GT1a; the b-series includes GD3, GD2, GD1b, GT1b and GQ1b; and the c-series includes GT3, GT2, GT1c, GQ1c, and GP1c [21].

In the mouse, the role of ganglioside in early embryonic development has been investigated. One of the b-series gangliosides GT1b was reported to suppress mitochondrial DNA (mtDNA) damage resulting from ROS in the mouse brain [22]. Moreover, it was shown that GT1b decreases DNA fragmentation and apoptotic changes by reducing ROS by blocking the diffusion of H2O2 into the sperm membrane in humans [23, 24]. GT1b expression was observed during the embryonic developmental process of mice using cryopreservation and a thawing method at different developmental stages [25]. It was also observed that the addition of GT1b into the culture media for rat hippocampal cells or neuroblastoma–glioma hybridoma (NG108-15) cells temporarily activated CaMKll [26]. In addition, it was recently reported that the B2R is part of the signal transduction pathway that is related to the maturation and differentiation of neurons induced by GT1b B2R activated by binding with Gq/11, which results in activation of phospholipase C (PLC), resulting in the production of inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 is able to induce Ca2+ release from the endoplasmic reticulum, and DAG stimulates protein kinase (PK) C [27]. Increased Ca2+ in the cumulus cells during this process is thought to diffuse into the egg through the gap junction, which would cause activation of CaMKII and likely to play an important role in the maturation of oocytes (Fig. 2) [28].

Fig. 2.

Identification of B2R gene expression in matured cumulus cells and oocytes. Genomic DNA (gDNA) and complementary DNA (cDNA) polymerase chain reaction analysis of GAPDH (gDNA, 374 bp and cDNA, 207 bp) and B2R (gDNA and cDNA, 196 bp). a, c and e: GAPDH. b, d and f: B2R. GAPDH was used a control.

However, GT1b is thought to contribute to the maturation of oocytes and the survival of embryonic development. So far, a number of studies have been conducted to identify the physiological functions and biological activities of GT1b in rodents; however, limited information is available regarding the effects of GT1b on the maturation of porcine oocytes. In the present study, the effect of GT1b treatment on porcine oocyte maturation and the subsequent premplantation embryonic development was investigated.

Materials and Methods

Chemicals

Trisialoganglioside GT1b (NH4+ salt) (Matreya LLC, 1063) was dissolved in deionized (DI) water to make a 1 mM stock solution. Stock solutions were stored at −20 C until required, at which time they were added to the oocyte maturation medium in specific amounts according to the experimental protocol. All of the other chemicals were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).

Oocyte collection and IVM

Porcine ovaries were collected from a slaughterhouse. Porcine follicular fluid (pFF) and cumulus-oocyte complexes (COCs) were recovered from 3–6 mm ovarian follicles using an aspiration method. The medium used during IVM was composed of TCM199 (Gibco, Life Technologies, Melbourne, Australia), 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/m epidermal growth factor, 75 μg/ml kanamycin, 1 μg/ml insulin, 10% (v/v) and pFF. GT1b was used to treat the oocytes during IVM at concentrations of 0 μM, 5 μM, 10 μM and 20 μM. Porcine COCs were co-cultured at 50 to 60 cells per well using a 4-well dish (Nunc, Roskilde, Denmark) with 500 μl of IVM medium (0 μM group). 20 μl of DI water was added to all groups. IVM was performed at 39 C in 5% CO2 using a humid incubator (Astec, Fukuoka, Japan). Maturation was performed in IVM media with 10 IU/ml equine chorionic gonadotropin (eCG) and 10 IU/ml human chorionic gonadotropin (hCG) for 22 h. The cells were then moved into hormone-free IVM medium and cultured for 18 h. Matured COCs were denuded by gentle pipetting with 0.1% hyaluronidase and TLH-PVS medium. Cumulus cells and denuded oocytes were obtained through this process and then used for subsequent experiments.

Assessment of nuclear maturation

Nuclear maturation of denuded oocytes was evaluated using 10 µg/ml Hoechst 33342 dye. Fluorescence was observed under an inverted fluorescence microscope (TE300, Nikon, Tokyo, Japan) with UV filters (330–385 nm) at 400 × magnification. Nuclear maturity was assessed by classifying cells into four stages (germinal vesicle, metaphase I, anaphase and telophase I and metaphase II).

Measurement of intracellular GSH and ROS levels

The intracellular GSH and ROS levels in matured oocytes were measured using 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CellTracker Blue CMF2HC, Invitrogen, Carlsbad, CA, USA) and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA, Invitrogen), respectively. Details of the protocol were described previously [7, 29, 30]. In short, the matured oocytes were added to 10 μM H2DCFDA or 10 μM CellTracker Blue with TLH-PVA medium and stained for 30 min in the dark. The stained oocytes were washed with TLH-PVA medium, and then, 10 oocytes were transferred to one drop of TLH-PVA medium. Fluorescence was observed under an inverted fluorescence microscope with UV filters (460 nm for ROS and 370 nm for GSH) at 200 × magnification. Fluorescent images were saved as picture files in jpeg format. Using the Adobe Photoshop software (version 13.0, Adobe Systems, San Jose, CA, USA), fluorescence intensity was measured only in the cytoplasmic portion of the oocytes.

Parthenogenetic activation (PA) and in vitro culture (IVC)

Oocytes at the stage of metaphase II were chosen for PA, and they were activated with two pulses of 120 V/mm DC for 60 μsec in a 260 mM mannitol solution containing 0.1 mM CaCl2 and 0.05 mM MgCl2. After electrical activation, the PA embryos were incubated in 5 μg/ml cytochalasin B (CB) in drops of porcine zygote medium (PZM) for 4 h. Embryos were transferred to a PZM drop for IVC. On the second day after activation, embryo cleavage was evaluated (1 cell, 2 cell, 4 cell, 8 cell, fragment) and transferred to a new PZM drop. On the 7th day after activation, blastocyst (BL) formation was evaluated quantitatively (early BL, expanded BL, hatched BL) and qualitatively (total cell number per BL) using 10 µg/ml Hoechst 33342.

In vitro fertilization (IVF)

Among denuded oocytes, MII-stage, oocytes were selected and washed two times using mTBM, and 15 oocytes were then transferred to an mTBM drop. The motility and concentration of sperm in liquid semen was confirmed using a hemocytometer. The final concentration of sperm was diluted to 5 × 105/ml and co-cultured with oocytes at 39 C in a 5% CO2 humid incubator for 20 min. Next, sperm on the surface of oocytes were detached by gentle pipetting and moved to an mTBM drop. Embryos were cultured at 39 C in a 5% CO2 humid incubator for 5 h. They were then transferred to PZM drops for IVC, and embryonic development was evaluated using the same method as used for PA.

Reverse transcription and quantitative real-time PCR

TRizol reagent (Invitrogen) was used to extract total RNA from matured cumulus cells. To synthesize cDNA from the extracted total RNA, Moloney murine leukemia virus (MMLV) reverse transcriptase and random primers (Invitrogen) were used. All procedures were carried out according to the manufacturer’s instructions. A SuperPrep™ Cell Lysis & RT Kit for qPCR (Toyobo, Osaka, Japan) was used for total RNA extraction and the synthesis of cDNA in matured oocytes according to the manufacturer’s instructions. Quantitative real-time PCR (Mx3000P qPCR, Agilent Technologies, Santa Clara, CA, USA) analysis was conducted using the synthesized cDNA and 2×SYBR Premix Ex Taq (Takara Bio, Otsu, Shiga, Japan). All of the primer sequences are presented in Table 1. The reactions were performed for 40 cycles, and the cycling parameters were as follows: denaturation at 95 C for 30 sec, annealing at 57 C for 30 sec, and extension at 72 C for 30 sec. Relative quantification was based on a comparison of the threshold cycle (Ct) at constant fluorescence intensity. The relative mRNA expression (R) was calculated using the equation R = 2-[ΔCt sample - ΔCt control]. To determine a normalized arbitrary value for each gene, every value was normalized to that of GAPDH [31].

Table 1.

Primer sequences for analysis of mRNA gene expression

mRNA	Primer sequences	Tm	Product size (bp)	GenBank accession number
GAPDH	F: 5’-GTCGGTTGTGGATCTGACCT-3’	60.5	207	NM_001206359
	R: 5’-TTGACGAAGTGGTCGTTGAG-3’	58.4
PCNA	F: 5’-CCTGTGCAAAAGATGGAGTG-3’	57.3	187	XM_003359883
	R: 5’-GGAGAGAGTGGAGTGGCTTT-3’	59.8
POU5F1 (Oct4)	F: 5’-GCGGACAAGTATCGAGAACC-3’	59.3	200	NM_001113060
	R: 5’-CCTCAAAATCCTCTCGTTGC-3’	57.3
Bax	F: 5’-TGCCTCAGGATGCATCTACC-3’	59.3	199	XM_003127290
	R: 5’-AAGTAGAAAAGCGCGACCAC-3’	57.3
Bcl-2	F: 5’-AATGACCACCTAGAGCCTTG-3’	58.4	182	NM_214285
	R: 5’-GGTCATTTCCGACTGAAGAG-3’	58.4
Caspase-3	F: 5’-CGTGCTTCTAAGCCATGGTG-3’	59.3	186	NM_214131
	R: 5’-GTCCCACTGTCCGTCTCAAT-3’	59.3
B2R	F: 5’-GCTCTACAGCCTGGTGATCT-3’	57.1	206	NM_214146
	R: 5’-TGCAGTAGGTGATGATGCTC-3’	57.3
CaMKIIδ	F: 5’-ACAGTACCCATCAAGCCATC-3’	57.5	199	NM_214381
	R: 5’-ATGCATGAAGAGGAGGAGAG-3’	57.0
CaMKIIγ	F: 5’-CTTATCCAAGAACAGCAAGC-3’	55.3	202	NM_214193
	R: 5’-GCAGTGGTAGTGGACATTGA-3’	57.1

F: forward, R: reverse.

Intracellular Ca2+ staining

The denuded oocytes were stained using TCM199 with 5 μM Fluo-4, AM, at 39 C in a 5% CO2 humid incubator for 40 min. Next, oocyte nuclei were stained using TLH-PVA solution with 10 μg/ml Hoechst 33342 dye for 5 min. Stained oocytes were washed with TLH-PVA solution and placed in a confocal dish (Invitrogen) before being covered with a cover glass to immobilize them. Next, the distribution of intracellular Ca2+ in the oocytes was investigated using confocal laser microscopy (Carl Zeiss, Thornwood, NY, USA). All of the images were analyzed with the ZEN 2009 Light Edition software.

Statistical analysis

All of the experiments were replicated more than three times. All data were analyzed by one-way ANOVA followed by Duncan’s test using SPSS (Statistical Package for the Social Sciences) and are reported as the mean ± SEM. Differences were considered to be significant if the P-value was less than 0.05.

Results

Enhancement of nuclear maturation on oocytes by GT1b

The nuclear maturation of oocytes at each stage, germinal vesicle (GV), metaphase I (MI), anaphase and telophase I (Ana & Telo I) and metaphase II (MII), was evaluated after IVM for 40 h. The experiment was repeated 4 times. The results showed that the percentages of immature stage cells in the treated groups, including cells at the GV, MI and Ana & Telo I stages, were not significantly (P < 0.05) different compared with the control (GV, MI and Ana & Telo I stages: 2.0, 6.6 and 12.5% for the control; 0.6, 6.4 and 6.4% for the 5 nM GT1b group; 0.6, 5.1 and 11.4% for the 10 nM GT1b group; and 1.3, 10 and 8.8% for the 20 nM GT1b group, respectively). The rate of matured MII-stage cells after treatment with 5 nM GT1b was 86.6%, which was significantly (P < 0.05) higher than that of the control group (78.9%). However, the other treatment (82.9 and 80.0% for the 10 nM and 20 nM GT1b groups, respectively) did now show a significant (P < 0.05) differences compared with the control (Table 2).

Table 2.

Effect of GT1b treatment on nuclear maturation during IVM

GT1b concentration (nM)	Oocytes cultured for maturation, N*	Number of oocytes at the stage of
		Germinal vesicle (%)		Metaphase I (%)		Anaphase and telophase I (%)		Metaphase II (%)
0	152	3	(2.0 ± 0.7)	10	(6.6 ± 1.5)	19	(12.5 ± 2.0)	120	(78.9 ± 1.3) a
5	157	1	(0.6 ± 0.7)	10	(6.4 ± 2.0)	10	(6.4 ± 1.5)	136	(86.6 ± 1.1) b
10	158	1	(0.6 ± 0.5)	8	(5.1 ± 1.3)	18	(11.4 ± 1.3)	131	(82.9 ± 0.7) a,b
20	160	2	(1.3 ± 0.7)	16	(10.0 ± 4.4)	14	(8.8 ± 2.9)	128	(80.0 ± 2.5) a,b

Values with different superscript letters (a, b) within a column differ significantly (P < 0.05). The data represent the means ± SEM. * The experiment was replicated four time.

GSH and ROS levels were decreased by GT1b

MII-stage oocytes were selected after IVM for 40 h, and GSH and ROS were measured. The GSH levels in the 5 nM and 20 nM treatment groups were significantly lower than that of the control group. The 10 nM treatment group did not show a significant difference compared with the control. The ROS level in the 10 nM treatment group was significantly lower than that in the control group. The other treatment groups (5 nM and 20 nM treatment groups) did not demonstrate a significant differences compared with the control group (Fig. 1).

Fig. 1.

Measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in matured oocytes. (A) Fluorescent photomicrographic images of in vitro matured porcine oocytes. Oocytes were stained with CellTracker Blue (a–d) and 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA) (e–h). Metaphase II (MII) oocytes derived from the maturation medium supplemented 0 μM (a and e), 5 μM (b and f), 10 μM (c and g) and 20 μM GT1b (d and h). (B) Effect of the ganglioside GT1b in the maturation medium on intracellular GSH and ROS levels in in vitro matured porcine oocytes. Within each group (GSH and ROS) at the end point, bars with different letters (a–b) are significantly (P < 0.05) different. GSH samples, N = 50; ROS samples, N = 65. Experiments were replicated three times.

Expression of B2R was observed in cumulus cells but not in oocytes

B2R expression was confirmed by reverse transcription PCR. According to a previous study, bradykinin 2 is known to activate CaMKII in response to GT1b stimulation [32]. The PCR results revealed the B2R gene (gDNA and cDNA, 196 bp) in the porcine genomic DNA (gDNA). Expression of B2R mRNA was observed in cDNA of cumulus cells but not in oocytes. Expression of the GAPDH gene (gDNA, 374 bp and cDNA, 207 bp) was used as a control in all groups (Fig. 2).

Matured cumulus cells and oocytes showed completely different patterns of mRNA expression

To analyze the mechanism of effect of GT1b on IVM, we examined the expression of apoptosis-associated genes (Bax, Bcl-2, Caspase-3) and PCNA, POU5F1, B2R and CaMKII subunit genes (CaMKIIγ and CaMKIIδ) in matured cumulus cells and oocytes. In cumulus cells, the 20 nM treatment group showed significantly (P < 0.05) decreased the expression of B2R. The expression of CaMKIIδ was significantly decreased in all treatment groups (P < 0.05) (Fig. 3A). In oocytes, the expression of POU5F1 was significantly decreased in the 5 nM treatment group (P < 0.05), whereas its expression was significantly increased in the 20 nM treatment group (P < 0.05). The 5 nM treatment group exhibited significantly (P < 0.05) increased expression of CaMKIIγ (Fig. 3B).

Fig. 3.

Expression levels of genes relative to apoptosis and calcium signaling were determined by quantitative RT-PCR. Mean ± SEM expression of proliferating cell nuclear antigen (PCNA), pluripotency-associated gene (POU5F1), apoptosis-associated genes (Bax, Bcl-2, Caspase-3), bradykinin 2 receptor (B2R) and Ca2+/calmodulin-dependent protein kinase type II subunit (CaMKIIγ and CaMKIIδ) mRNA in matured (A) cumulus cells and (B) oocytes treated with the ganglioside GT1b during in vitro maturation (IVM). Within the same mRNA, values with different superscript letters differ significantly (P < 0.05). The experiment was replicated three times.

The intracellular Ca2+ concentration was maintained irrespective of the cell cycle in oocytes in the GT1b treatment groups

Oocytes were classified by stage using Hoechst staining to confirm the distribution of Ca2+ according to the degree of nuclear maturation, and intracellular Ca2+ in oocytes was stained using Fluo-4, AM, and observed based on the fluorescence intensity. The results showed that the intracellular Ca2+ level was significantly increased from the GV stage (16.41 ± 0.31) to the GVBD stage (29.30 ± 0.23), but oocytes in the MI stage (31.88 ± 3.94) did not show a significant (P < 0.05) difference. However, in the MII stage (10.48 ± 2.10), the intracellular Ca2+ level was significantly decreased in the MII stage (10.48 ± 2.10) and reached the level of the GV stage (P < 0.05) (Fig. 4).

Fig. 4.

Measurements of the Ca2+ concentration in oocytes according to the cell cycle. (A) Ca2+ distribution in porcine oocytes during different phases of in vitro maturation. a) Germinal vesicle (GV) stage, b) germinal vesicle breakdown (GVBD) stage, c) MI stage, d–f) anaphase & telophase stage, g) early MII stage, h) MII stage. (B) Comparison of Ca2+ fluorescence intensity in matured porcine oocytes treated with the ganglioside GT1b during in vitro maturation (IVM). Within each end point, bars with different letters (a, b) are significantly (P < 0.05) different for different concentrations of GT1b treatment.

Next, the intracellular Ca2+ level was evaluated in oocytes at 18 h and 40 h after IVM initiation, which are thought to be mainly in the MI stage, to identify the change in intracellular Ca2+ level in oocytes after GT1b treatment [33]. The results showed that the fluorescence intensity of cells that underwent 18 h of IVM and were treated with 5 nM GT1b was significantly decreased compared with the control and that other groups did not show a significant differences (P < 0.05). However, the Ca2+ fluorescence intensity of cells that underwent 40 h of IVM and were treated with 5 nM GT1b was significantly (P < 0.05) increased compared with the control. The Ca2+ fluorescence intensity of cells treated with 20 nM GT1b was significantly increased compared with that of the cells in the 5 nM treatment group (Fig. 5).

Fig. 5.

Measurements of the Ca2+ concentration in matured porcine oocytes treated with the ganglioside GT1b during in vitro maturation (IVM). (A) Ca2+ distribution in matured porcine oocytes treated with the ganglioside GT1b. (a and e) 0 nM (control), (b and f) 5 nM, (c and g) 10 nM, (d and h) 20 nM. (B) Comparison of Ca2+ fluorescence intensity in matured porcine oocytes treated with the ganglioside GT1b. Within each end point, bars with different letters (a, b, c) are significantly (P < 0.05) different for different concentrations of GT1b treatment. * P < 0.05.

GT1b tended to improve development of preimplantation embryo

The cleavage and blastocyst formation rates of parthenogenetic embryo were evaluated 2 days and 7 days after the electrical activation. There were no significant differences between the control group and treatment groups. However, there was a tendency for a dose-dependent increase in both cleavage ratio and blastocyst development rate. Furthermore, the blastocyst formation rate was significantly different between the 5 nM and 20 nM treatment groups (Fig. 6).

Fig. 6.

Effect of GT1b treatment during IVM on embryonic development after parthenogenetic activation (PA) in terms of (A) the cleavage pattern and (B) the blastocyst formation pattern of the PA embryo. Within each end point, bars with different letters (a, b) are significantly (P < 0.05) different for different concentrations of GT1b treatment. CL, cleavage; BL, blastocyst. (C) Summary of embryonic development after PA. The cleavage rate was measured on day 2, and the blastocyst formation rate was evaluated on day 7 of culture.

The cleavage and blastocyst formation rates of fertilized embryo were evaluated 2 days and 7 days after fertilization. There were no significant differences between the control group and treatment groups (Fig. 7).

Fig. 7.

Effect of GT1b treatment during IVM on embryonic development after in vitro fertilization (IVF) in terms of (A) the cleavage pattern and (B) the blastocyst formation pattern of IVF embryo. Within each end point, bars with different letters (a, b) are significantly (P < 0.05) different for different concentrations of GT1b treatment. CL, cleavage; BL, blastocyst. (C) Summary of embryonic development after IVF. The cleavage rate was measured on day 2, and the blastocyst formation rate was evaluated on day 7 of culture.

Discussion

Ganglioside is abundant in the nervous system and is thought to play an important role [18, 19]. However, in a recent study using a mouse model, it was confirmed that various types of gangliosides are dynamically expressed during embryonic development [25]. Gangliosides can be classified by the number of sialic acid residues. Among them, the b-series ganglioside GT1b is thought to stimulate B2R during neuronal differentiation in mice, which results in changes in the Ca2+ concentration [26]. In addition, some studies using mouse and human germ cells revealed that GT1b reduced ROS and DNA damage [22,23,24,25, 34]. Therefore, this study was performed to examine the effect of ganglioside on porcine germ cells.

GT1b forms micelles when it is dissolved in an aqueous solution [35]. Attachment of these micelles to the cell surface increases the diffusion barrier [36]. Micelles of GT1b also reduce mtDNA damage by an antioxidant effect in neurons. [22, 34] and protect spermatozoa from hydrogen peroxidase-induced DNA and membrane damage [23].

In the present study, we first evaluated the effect of GT1b on oocyte maturation by examining, nuclear maturation and cytoplasmic maturation were examined. Although the nuclear maturation rate of MII-stage oocytes in the 5 nM treatment group was significantly increased compared with that of the control, the GSH levels in the 5 nM and 20 nM treatment groups were lower than that in the control group. Interestingly, the ROS level in the 10 nM treatment group was significantly lower than those in the control and 5 nM treatment groups. In previous reports of porcine IVM, various antioxidants, including resveratrol [7,8,9], cysteine [34] and β-mercaptoethanol [37], increased intracellular GSH levels and reduced ROS levels in matured oocytes. These antioxidants showed positive effects on oocyte maturation and embryonic development by improving cytoplasmic maturation. ROS are generated in the form of superoxide radicals, hydrogen peroxide and hydroxyl radicals as a necessity in the process of aerobic respiration to produce energy in cells [38,39,40,41]. Due to their high reactivity, they are thought to trigger cell damage due to cell membrane degeneration, protein degeneration, lipid oxidation, DNA damage and the inhibition of DNA synthesis [42, 43]. Oxidative stress due to ROS formation can be suppressed by endogenous antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase [44, 45]. However, the partial pressure of oxygen in vitro is higher than that in vivo, leading to the accumulation of ROS, which in turn generates oxidative stress. Oxidative stress in vitro causes a lower maturation rate during IVM and a lower embryonic development rate [46,47,48,49]. In this study, treatment with 10 nM GT1b did not result in a significant difference in GSH antioxidant expression compared with the control. However, 5 nM and 10 nM of GT1b result in rather lower expression of GSH. Therefore, other antioxidative mechanisms, but not GSH, might help reduce ROS during porcine IVM.

As one of the ROS reduction mechanisms, the intracellular Ca2+ level was evaluated in this study. According to the report of Jiao et al., oxidative stress reduces intracellular Ca2+, which in turn leads to polyspermy by affecting cortical granule redistribution and causing the disorder of activation and damage to the microfilament network during the fertilization process [50]. Intracellular Ca2+ regulates a major event of mammalian cells and also regulates the cell cycle. In Xenopus eggs, CaMKII regulates the conversion of metaphase into anaphase in the meiotic cell cycle, and targeting Ca2+/CaM and CaMKII stimulates the escape from metaphase arrest twice as much as continuous InsP3-dependent Ca2+ oscillations from MII to interphase in mouse oocytes [51]. Hence, we evaluated the intracellular Ca2+ level at the different nuclear stages during porcine IVM. In the non-treated groups, the intracellular Ca2+ level of GVBD- and MI-stage oocytes were higher than that of GV- and MII-stage cells. These results are consistent with previous reports that intracellular Ca2+ regulates the cell cycle [52,53,54]. Indeed, most matured oocytes arrested at the MII stage, and their intracellular Ca2+ levels were very low. In contrast, in the GT1b-treated groups, the intracellular Ca2+ levels at 40 hours were similar to those of the 18 hour maturation groups. These results indicated that GT1b could regulate intracellular Ca2+ during oocyte maturation, regardless of the cell cycle stage. Based on these results, the reason for ROS reduction without an increase in GSH might be the maintenance of intracellular Ca2+ levels by GT1b treatment.

Recently, it was confirmed that GT1b stimulates B2R signals in yeast cells that express mammalian B2R [55]. However, other studies using nonneuronal cells that also express B2R did not obtain a similar result [56]. An aim of the present study was to confirm whether B2R is expressed in matured porcine cumulus cells and oocytes. The expression of B2R was observed in cumulus cells but not in oocytes. B2R expression did not significantly differ from the zygote to blastocyst stage at different concentrations of GT1b treatment in the process of IVC in embryos that had been subjected to PA after the general IVM process (data not shown). Indeed, porcine PA/IVF was performed after the removal of cumulus cells, and zygotes produced from in vitro fertilization were cultured to the blastocyst stage without cumulus cells. According to a previous hypothesis [32], GT1b is thought to directly affect cumulus cells and then increase intracellular Ca2+ in oocytes.

GT1b is distributed throughout in the granular layers [57], and it activates CaMKII distributed in synapses by activating cdc42 [32]. CaMKII belongs to the serine/threonine protein kinase family and has 28 isoforms originating from alpha (α), beta (β), gamma (γ), and delta (δ) genes [58, 59]. It has been reported that CaMKII can interact with molecules that control Ca2+ signaling and the cell cycle in mouse oocytes [60]. In porcine oocytes, CaMKII is diversely involved in meiotic resumption and activation-like regulation of other proteins [61]. In a recent study using mouse oocytes, CaMKIIα was found to be neuron specific, and CaMKIIβ was found primarily on neurons; neither of these isoforms are expressed in mouse oocytes [62, 63]. However, CaMKIIγ and CaMKIIδ are reportedly expressed in various tissues [63]. In this study, the expression of CaMKIIγ and CaMKIIδ was confirmed in porcine cumulus cells and oocytes. Backs et al. reported that oocytes from CaMKIIγ-knockout mice could not exit metaphase II arrest in a meiotic resumption experiment [64]. However, the expression of exogenous CaMKIIγ cDNA in the oocytes resulted in the resumption of meiosis, and this rescue might be possible with the expression of CaMKIIδ because it is not isoform specific. Similar to this finding, CaMKIIγ signaling is required to activate oocytes in vivo, downstream of Ca2+ oscillations [64]. Another previous study revealed that Ca2+ influx is required to activate downstream signaling molecules, including CaMKIIγ, during meiotic resumption induced by sperm and during embryonic development. In particular, it was reported that CaMKIIγ signaling is crucial in spindle rotation and polar body emission [65].

To understand the effect of GT1b in this study, oocytes were treated with different concentrations of GT1b during the process of IVM, and real-time PCR was carried out using cDNA obtained porcine cumulus cells and oocytes that had completed maturation. A previous study reported that B2R activates the PLC/PKC and cAMP/PKA pathways, and cAMP is known to inhibit cell growth [66]. Another study reported that cAMP/PKA pathway activation suppresses B2R and leads to the reduction of bradykinin-dependent inositol triphosphate and Ca2+ mobilization [67,68,69]. In the present study, the expression of B2R and CaMKIIδ in cumulus cells from the 20 nM GT1b treatment group was significantly decreased compared with the control. This result showed that the expression of B2R is suppressed by cAMP/PKA pathway stimulation with GT1b treatment. Reduced B2R expression can lead to the suppression of another pathway of B2R, that is, the PLC/PKC pathway. As a result, it is thought that inositol triphosphate and Ca2+ mobilization are reduced and that the expression of MAPK is also decreased.

PCNA is known to be a critical gene in many cellular processes, such as DNA replication, DNA repair, DNA damage avoidance, cell cycle control, and cell survival. Recently, PCNA was reported to have an impact on the development of ovarian follicles [70,71,72,73]. However, the concentration of Ca2+ in oocytes was increased in the present study, and this would have stimulated the cell cycle and increased the expression of POU5F1. Furthermore, the expression of CaMKIIγ and CaMKIIδ tended to increase compared with the control, but the difference was not significant. It could be concluded that there was no significant effect on oocyte maturation because the concentration of GT1b was not insufficient. In the 5 nM GT1b group, the expression of B2R in cumulus cells was slightly inhibited, but this inhibition was not significant. Like the other groups, the expression of CaMKIIδ was decreased. Moreover, the Ca2+ concentration was increased in oocytes, and the expression of CaMKIIγ was significantly increased. In a previous study, increased Ca2+ concentrations in mouse oocytes stimulated the activation of CaMKIIγ, which led to the reduced activity of MAPK [65]. In addition, it was observed that POU5F1 phosphorylation in human ES cells was dependent on MAPK [74], and POU5F1 is thought to be downstream of MAPK, as shown in a recent study [75]. Therefore, it can be concluded that the increased expression of CaMKIIγ decreases MAPK expression, which decreases POU5F1 expression in oocytes. The additional NH4+ contained in the GT1b used in this experiment was previously reported to be cytotoxic [76]. Thus, we examined the expression of Caspase-3, but there were no significant differences.

Then PA and IVF were also carried out using GT1b-treated matured oocytes, but there were no significant differences among the treatment groups with regard to the cleavage rate, blastocyst formation rate, and the cell number in the blastocysts. However, embryo development rates showed a tendency to increase in a dose-dependent manner, but this trend was not significant. Therefore, treatment with a sufficient concentration of GT1b is thought to result in good quality oocytes.

The present study suggests that GT1b stimulates nuclear maturation at some concentrations, reduces ROS during IVM and inhibits cytoplasmic maturation by maintaining consistent intracellular Ca2+ levels regardless of the cell cycle. However, low concentrations of intracellular Ca2+ due to ROS or an inappropriate composition of the in vitro culture medium could be compensated for treatment with GT1b to raise the intracellular Ca2+ level to within the range comparable to that in vivo. Increased intracellular Ca2+ levels in the oocytes stimulated the cell cycle so that POU5F1 expression also increased. It was reported in a previous study that the injection of 0.1 M CaCl2 in metaphase II-arrested porcine oocytes readily activated cells [77]. Treatment with excessive levels of GT1b might activate matured oocytes. GT1b is thought to regulate the level of Ca2+ in oocytes in a dose-dependent manner and to be associated with another mechanism that controls the level of intracellular Ca2+. Further studies are needed to confirm the changed level of intracellular Ca2+ in cumulus cells, and it is also necessary to determine the precise mechanism that maintains the intracellular Ca2+ level in oocytes.