Accepted Manuscript

Anti-oxidative effect of carboxyethylgermanium sesquioxide (Ge-132) on in vitromaturation of porcine oocytes and subsequent embryonic development afterparthenogenetic activation and in vitro fertilization

Eunhye Kim, Yubyeol Jeon, Dae Young Kim, Eunsong Lee, Sang-Hwan Hyun

PII: S0093-691X(15)00133-8

DOI: 10.1016/j.theriogenology.2015.03.006

Reference: THE 13116

To appear in: Theriogenology

Received Date: 6 October 2014

Revised Date: 25 February 2015

Accepted Date: 11 March 2015

Please cite this article as: Kim E, Jeon Y, Kim DY, Lee E, Hyun S-H, Anti-oxidative effect ofcarboxyethylgermanium sesquioxide (Ge-132) on in vitro maturation of porcine oocytes and subsequentembryonic development after parthenogenetic activation and in vitro fertilization, Theriogenology (2015),doi: 10.1016/j.theriogenology.2015.03.006.

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revised 1

Anti-oxidative effect of carboxyethylgermanium sesquioxide (Ge-132) on in vitro maturation of 2 porcine oocytes and subsequent embryonic development after parthenogenetic activation and in vitro 3

fertilization 4

5

Eunhye Kima, Yubyeol Jeona, Dae Young Kimb,* , Eunsong Leec and Sang-Hwan Hyuna,* 6

7 aLaboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk 8

National University, Cheongju, 362-763, Chungbuk, Republic of Korea. bDepartment of Life Science, 9

College of BioNano Technology, Gachon University, Incheon, 406-799, Republic of Korea. 10 cLaboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, 11

Chunchon 200-701, Kangwon, Republic of Korea. 12

13

*Corresponding authors 14

15

Sang-Hwan Hyun, DVM, Ph.D. 16

Vice Director, BK21 PLUS Creative Veterinary Research Group 17

Professor, Lab. of Veterinary Embryology and Biotechnology (VETEMBIO) 18

College of Veterinary Medicine, Chungbuk National University 19

1 Chungdae-ro, Seowon-gu, Cheongju 362-763, Republic of Korea 20

Email: shhyun@cbu.ac.kr Mobile: +82-10-6755-6088 21

Office: +82-43-261-3393 Fax: +82-43-267-3150 22

23

Dae Young Kim, DVM, PhD 24

Associate Professor, Department of Life Science, College of BioNano Technology, 25

Gachon University, Incheon, 406-799, Republic of Korea 26

E-mail: davekim@gachon.ac.kr 27

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Abstract 28

Carboxyethylgermaniumsesquioxide (Ge-132) is an organogermanium compound known to 29

exert biological activities, such as antioxidant and anticancer effects. In this study, we investigated the 30

effect of Ge-132 on the in vitro maturation (IVM) of porcine oocytes via analysis of nuclear 31

maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent 32

embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 40 33

h of IVM, no significant difference in nuclear maturation was observed in the 100-, 200- and 400-34

µg/ml Ge-132 treatment groups (89.9%, 91.3% and 90.4%, respectively) compared with the control 35

group (89.0%). However, intracellular GSH levels in oocytes treated with 200 µg/ml Ge-132 36

increased significantly (P< 0.05), and the 200- and 400-µg/ml Ge-132 treatment groups exhibited a 37

significant (P< 0.05) decrease in intracellular ROS levels compared with the control group. Oocytes 38

matured with 200 and 400µg/ml of Ge-132 during IVM displayed significantly higher cleavage rates 39

(78.7% and 82.7% vs. 67.5%, respectively), and the 200-µg/ml Ge-132 treatment group displayed 40

higher blastocyst formation rates and greater total cell numbers after PA (59.5% and 38.2 vs. 67.8 and 41

55.3, respectively) than the control group. Furthermore, oocytes matured with 200 µg/ml Ge-132 42

during IVM failed to display significantly higher blastocyst formation rates (31.6% vs. 36.7%) but 43

exhibited greater total cell numbers after IVF (71.5 vs. 101.3) than the control group. We also found 44

that the Ge-132-treated oocytes showed significantly higher mRNA expression levels of the oxidative-45

related gene Nrf-2 and lower mRNA expression levels of the pro-apoptotic gene Bax compared with 46

the control group (P< 0.05). In conclusion, our results suggest that treatment with Ge-132 during IVM 47

improves the developmental potential of PA and IVF porcine embryos by increasing the intracellular 48

GSH levels, thereby decreasing the intracellular ROS levels and reducing oxidative stress-induced 49

apoptosis, thereby regulating the mRNA expression of oocytes during oocyte maturation. 50

51

Keywords: Ge-132, porcine oocytes, GSH, ROS, pre-implantation embryonic development 52

53

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1. Introduction 54

Mammalian embryonic development is a complicated process regulated by the interaction between 55

inherent embryo characteristics and environmental factors. Changes in either component can modify 56

developmental and biological pathways. Trace elements, in minute amounts, are considered to play a 57

key role in these pathways by functioning in several physiological processes [1-2]. Recent studies 58

have shown the importance of numerous trace elements, such as zinc, copper and selenium, in the 59

support of female reproduction in many animal species (reviwed in [3-5]). It is thought that these trace 60

elements may help to improve current inefficient porcine in vitro maturation (IVM) protocols for 61

generating mature oocytes, which remain suboptimal compared with the in vivo oogenesis system. An 62

improved IVM system would provide important benefits for a range of applications including more 63

efficient embryo production, cloning and transgenic technologies and would represent a critical 64

research tool in mammalian reproductive and developmental biology [6-9]. 65

Germanium (Ge) is one of such trace elements, and compounds of Ge are classified into organic 66

and inorganic forms, which are present in certain plants, such as ginseng, shiitake mushroom, pearl 67

barley, waternut and garlic. Among the many organogermanium compounds, carboxyethylgermanium 68

sesquioxide (Ge-132), also known by a variety of other names including bis (2-69

carboxyethylgermanium) sesquioxide, propagermanium and 2-carboxyethyl-germasesquioxane, 70

exhibits low toxicity and elevated biological activity, thereby attracting the attention of researchers. 71

Ge-132 is synthesized via the hydrolysis of a trihalogenopropionic acid derivative formed by the 72

supplement of a trihalogenogermane to acrylic acid or its alkyl ester [10]. Some studies have 73

illustrated that Ge-132 exhibits anti-tumor [11-12] and anti-oxidative activity [13-15], thereby 74

representing a potentially useful compound in a wide variety of applications for the health care 75

industry [16]. 76

Although these studies have reported the biological effects of Ge-132 on a variety of cells, it remains 77

unclear whether Ge-132 exerts biological activities on the female reproductive system, including 78

oocytes and cumulus cells. In this study, we supplemented conventional medium used for maturing 79

porcine oocytes with Ge-132 and subsequently investigated its effect on nuclear maturation, 80

intracellular levels of glutathione (GSH) and reactive oxygen species (ROS), mRNA expression 81

(PCNA, POU5F1, Nrf-2, Bax, Bcl-2 andCaspase-3) and embryo developmental competence after PA 82

and IVF. 83

84

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2. Materials and Methods 85

2.1. Chemicals 86

All chemicals and reagents used in this study were purchased from Sigma-Aldrich Chemical 87

Company (St. Louis, MO, USA), unless otherwise stated. 88

89

2.2. Ooctye collection and in vitro maturation 90

Ovaries of prepubertal gilts were collected at a local abattoir and transported to the laboratory within 91

2 h in 0.9% (w/v) NaCl solution supplemented with 100 IU/L penicillin G and 100 mg/mL 92

streptomycin sulfate at 32 °C to 35 °C. The cumulus oocyte complexes (COCs) in the ovaries were 93

aspirated from 3- to 6- mm diameter superficial follicles using an 18-gauge needle attached to a 10-94

mL disposable syringe and allowed to settle in 15-mL conical tubes at 37 °C for 5 min. The 95

supernatant was discarded, and the precipitate was resuspended with HEPES-buffered Tyrode’s 96

medium (TLH) containing 0.05% (wt/vol) polyvinyl alcohol (TLH-PVA). Next, the precipitate was 97

examined using a stereomicroscope to recover the COCs. Only COCs with ≥ 3 uniform layers of 98

compact cumulus cells and homogenous cytoplasm were selected and washed three times in TLH-99

PVA. Approximately 60 COCs were placed into each well of a four-well Nunc dish (Nunc, Roskilde, 100

Denmark) containing 500 µL of culture medium (TCM199; Invitrogen Corporation, Carlsbad, CA, 101

USA) supplemented with 0.6 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth 102

factor, 75µg/mL kanamycin, 1 µg/mL insulin, 10% (vol/vol) porcine follicular fluid (pFF), 10 IU/mL 103

equine chronic gonadotropin (eCG), and 10 IU/mL hCG (Intervet, Boxmeer, Netherlands). The 104

selected COCs were incubated at 39 °C with 5% CO2 in a 95% humidified chamber for IVM. After 21 105

to 22 h of maturation with hormones, the COCs were washed twice in fresh hormone-free IVM 106

medium and then cultured in hormone-free IVM medium for an additional 21 to 22 h. The COCs 107

recovered during IVM were treated with or without Ge-132. The COCs were treated with or without 108

Ge-132 during the entire maturation time. 109

110

2.3. Evaluation of nuclear maturation 111

The oocytes at the Metaphase II (MII) stage, 42-44 h after IVM, were sampled to analyze nuclear 112

maturation. Samples of oocytes (a total of 967 oocytes were used for the nuclear maturation study) 113

were denuded by gently pipetting with 0.1% hyaluronidase in IVM medium and washed in TLH-PVA. 114

The denuded oocytes were fixed with fixative solution containing 2% formaldehyde and 0.25% 115

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glutaraldehyde and then stained with TLH-PVA containing 5µg/mL Hoechst 33342 for at least 5 min. 116

The stained oocytes were evaluated by use of a fluorescence microscope (Nikon Corp., Tokyo, Japan) 117

and classified as germinal vesicle (GV), metaphase I (MI), anaphase-telophase I (AT-I), or metaphase 118

II (MII) according to meiotic maturation stage. The oocytes at metaphase II were considered to have 119

matured. The experiment was repeated three times. The total number of oocytes used per treatment 120

group is presented in Table 2. 121

122

2.4. Measurement of intracellular GSH and ROS levels 123

The oocytes at 42-44 h after IVM were sampled to determine intracellular GSH and ROS levels. 124

GSH and ROS level assessment was carried out as previously described [17-18]. Briefly, 2’, 7’-125

dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen Corporation) and 4-chloromethyl-6.8-126

difluoro-7-hydroxycoumarin (CellTracker Blue; CMF2HC; Invitrogen Corporation) were used to 127

detect intracellular ROS levels (green fluorescence) and GSH levels(blue fluorescence), respectively. 128

Ten oocytes from each treatment group were incubated (in the dark) for 30 min in TLH-PVA 129

supplemented with 10 µM H2DCFDA and 10 µM CellTracker Blue. After incubation, the oocytes 130

were washed with Dulbecco’s phosphate buffered saline (DPBS) (Invitrogen Corporation) containing 131

0.1% (wt/vol) polyvinyl alcohol (PVA), placed into 10-µL droplets, and fluorescence was evaluated 132

using an epifluorescence microscope (TE300; Nikon, Tokyo, Japan) with UV filters (460 nm for ROS 133

and 370 nm for GSH). These fluorescent images were saved as graphic files in TIFF format. The 134

fluorescence intensity of the oocytes was analyzed using Image J software (Version 1.41; National 135

Institutes of Health, Bethesda, MD, USA) and normalized to control oocytes. The experiment was 136

repeated three times (GSH samples, N=30; ROS samples, N =30). 137

138

2.5. Parthenogenetic activation of oocytes 139

For parthenogenetic activation (PA), the COCs 42-44h after IVM were denuded by gently pipetting 140

with 0.1% hyaluronidase, washed three times in TLH-PVA and then rinsed twice in activation medium 141

(280 mM mannitol solution containing 0.01 mM CaCl2 and 0.05 mM MgCl2). For activation, the MII 142

stage oocytes were placed between electrodes covered with activation medium in a chamber 143

connected to an electrical pulsing machine (LF101; Nepa Gene, Chiba, Japan). Oocytes were 144

activated with two direct-current (DC) pulses of 120 V/mm for 60 µsec. After electrical activation, 145

oocytes were immediately placed in IVC medium supplemented with 5 µg/mL cytochalasin B for 6 h. 146

The PA embryos were washed three times in fresh IVC medium, placed in 30 µL IVC droplets (10 147

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gametes per drop) covered with pre-warmed mineral oil, and then cultured at 39 °C in a humidified 148

atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. The experiment was repeated four times. The 149

total number of oocytes used per treatment group is presented in Table 3. 150

151

2.6. In vitro fertilization and culture 152

For in vitro fertilization (IVF), the COCs 42-44h after IVM were denuded by gently pipetting with 153

0.1% hyaluronidase and washed three times in TLH-PVA. Groups of 15 oocytes at the MII stage were 154

randomly placed in 40µL droplets of modified Tris-buffered medium (mTBM) in a 35 × 10 mm Petri 155

dish (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ, USA) covered with pre-warmed 156

mineral oil. Next, liquid semen supplied weekly from the Veterinary Service Laboratory (Department 157

of Livestock Research, Yong-in city, Gyeonggi-do, Republic of Korea) was stored at 17 °C for 5 days 158

before use. The semen sample was washed two times with Dulbecco’s phosphate buffered saline 159

(DPBS) supplemented with 0.1% BSA via centrifugation at 2000g for 2 min. After washing, the 160

sperm pellet was resuspended in mTBM [19], which had been pre-equilibrated for 18 h at 39 °C in 5% 161

CO2. After appropriate dilution, 5 µL of the sperm suspension was added to a 40-µL drop of 162

fertilization medium (mTBM) to set the final sperm concentration at 1 ×10 6 sperm/mL. Just before 163

fertilization, sperm motility was assessed, and more than 80% motile sperm were used in every 164

experiment. To use stored liquid semen, a modified two-step culture system [20]was applied. The 165

oocytes were co-incubated with the sperm for 20 min at 39 °C in a humidified atmosphere of 5% CO2 166

and 95% air. After 20 min of co-incubation with the sperm, the loosely attached sperm cells were 167

removed from the zona pellucid (ZP) via gentle pipetting. Next, the oocytes were washed three times 168

in mTBM and incubated in mTBM without sperm for 5 to 6 h at 39 °C in a humidified atmosphere of 169

5% CO2 and 95% air. Thereafter, the gametes were washed three times with embryo culture medium 170

and cultured in 25 µL microdrops (10 gametes/drop) of porcine zygote medium 3 (PZM3) [21] with 171

pre-warmed mineral oil. The embryos with cultured drops were incubated at 39 °C for 168 h under a 172

humidified atmosphere of 5% O2, 5% CO2, and 90% N2. For all experiments, the culture media were 173

renewed at 48 h (Day 2) and 96 h (Day 4) after IVF. The experiment was repeated four times. The 174

total number of oocytes used per treatment group is presented in Table 3. 175

176

2.7. Embryo evaluation and total cell count of blastocysts 177

The day of PA or IVF was considered Day 0. The embryos were evaluated for cleavage using a 178

stereomicroscope on Day 2 (48 h). Evenly cleaved embryos were classified into three groups (2 to 3, 4 179

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to 5, and 6 to 8 cells). Blastocyst formation was assessed on Day 7 (168 h) after IVF, and blastocysts 180

were classified according to degree of expansion and hatching status [22] as follows: early blastocyst 181

(small blastocyst with a blastocoel equal to or less than half of the embryo volume), expanded 182

blastocyst (a large blastocyst with a blastocoel greater than half of the embryo volume or a blastocyst 183

with a blastocoel completely filling the embryo), and hatched blastocyst (hatching or already hatched 184

blastocyst). To quantify the total cell number of blastocysts, at Day 7, the blastocysts were collected 185

and washed in 1% (wt/vol) PBS-BSA and stained with 5µg/mL Hoechst-33342 (bisbenzimide) for 5 186

min. After final wash in PBS-BSA, the embryos were fixed briefly in 4% paraformaldehyde in PBS. 187

Next, the blastocysts were mounted on glass slides in a drop of 100% glycerol, covered gently with a 188

cover slip, and observed using a fluorescence microscope (Nickon Corp., Tokyo, Japan) at ×400 189

magnification. The experiment was repeated four times. The total number of oocytes used per 190

treatment group is presented in Table 3. 191

192

2.8. Gene expression analysis via quantitative real-time polymerase chain reaction (RT-PCR) 193

For the gene expression study, 160 mature oocytes and cumulus cells per each group (control, 100-, 194

200- and 400-µg/ml Ge-132) were separately sampled using a stereomicroscope. All samples were 195

stored at −80 °C until analyzed. The expression levels of proliferating cell nuclear (PCNA), 196

POU5F1, Nrf-2, Bax, Bcl-2andCaspase-3 mRNA in the oocytes and cumulus cells were analyzed via 197

quantitative RT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen Corporation) 198

according to the manufacturer’s protocol, and the total RNA concentration was determined by 199

measuring the absorbance at 260 nm. First-strand complementary DNA (cDNA) was prepared by 200

subjecting 1 µg of total RNA to reversetranscription using Moloney murine leukemia virus (MMLV) 201

reverse transcriptase (Invitrogen Corporation) and random primers (9-mers; Takara Bio, Inc., Otsu, 202

Shiga, Japan). To determine the conditions for the logarithmic phase PCR amplification of target 203

mRNA, 1-µg aliquots were amplified using differing numbers of cycles. The housekeeping gene 204

GAPDH was PCR-amplified to rule out the possibility of RNA degradation and to control for the 205

variation in mRNA concentrations in the reverse transcription (RT) reaction. A linear relationship 206

between PCR product band visibility and the number of amplification cycles was observed for the 207

target mRNAs. GAPDH and the target genes were quantified using 40 cycles. The cDNA was 208

amplified in a 20-µL PCR reaction containing 1 U Taq polymerase (Intron Bio Technologies, Co., Ltd., 209

Seongnam, Korea), 2mM deoxyribonucleoside triphosphates (dNTPs) mix and 10 pM of each gene-210

specific primer. The quantitative RT-PCR was performed with 1 µL cDNA template added to 10 µL 2 211

× SYBR Premix Ex Taq (Takara Bio, Inc.) containing specific primers at a concentration of 10 pM 212

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each. The reactions were carried out at 40 cycles and the cycling parameters were as follows: 213

denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec. All 214

oligonucleotide primer sequences are presented in Table 1.The fluorescence intensity was measured at 215

the end of the extension phase of each cycle. The threshold value for the fluorescence intensity of all 216

samples was set manually. The reaction cycle at which the PCR products exceeded this fluorescence 217

intensity threshold was deemed the threshold cycle (Ct) in the exponential phase of the PCR 218

amplification. The expression of each target gene was quantified relative to that of the internal control 219

gene (GAPDH). The relative quantification was based on a comparison of Cts at constant 220

fluorescence intensity. The amount of transcript present was inversely related to the observed Ct and, 221

for every two-fold dilution in the amount of transcript, Ct was expected to increase by one. The 222

relative expression (R) was calculated using the following equation: R = 2-[∆Ct sample -∆Ct control]. To 223

determine a normalized arbitrary value for each gene, every obtained value was normalized to that of 224

GAPDH. The experiments were repeated at least three times. 225

226

2.9. Experimental design 227

In experiment 1, the effect of treatment with various concentrations (0, 100, 200 and 400µg/mL) of 228

Ge-132 during IVM on nuclear maturation was examined to identify the optimal concentration. In 229

experiment 2, the effect of Ge-132 treatment during IVM on intracellular levels of GSH and ROS was 230

investigated. In experiment 3, the effect of Ge-132 treatment during IVM on subsequent embryonic 231

development of PA and IVF embryos was examined. In experiment 4, the effects of Ge-132 treatment 232

on the mRNA expression of PCNA, POU5F1, Nrf-2, Bax, Bcl-2 and Caspase-3 in matured oocytes 233

and cumulus cells were analyzed. 234

235

2.10. Statistical analysis 236

The statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). 237

Percentage data (e.g., rates of maturation, cleavage, blastocyst formation, and number of nuclei) were 238

compared using one-way ANOVA followed by Duncan’s multiple range tests. All results are 239

expressed as the mean ± SEM.P values < 0.05 were considered to be statistically significant, unless 240

otherwise stated. 241

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3. Results 243

3.1. Effect of Ge-132 on nuclear and cytoplasmic maturation during IVM 244

We evaluated the Metaphase II (MII) stage to analyze the nuclear maturation rates. No significant 245

difference in nuclear maturation (MII) between the control group and Ge-132 treatment groups was 246

observed (Table 2). To assess cytoplasmic maturation, we examined intracellular GSH and ROS levels 247

in MII oocytes derived from the maturation medium supplemented with Ge-132 after IVM (Figure 1). 248

The 200 µg/mL Ge-132 treatment group showed significantly increased (P< 0.05) intracellular GSH 249

levels compared with the control group. The ROS generation levels tended to decrease (P< 0.05) as 250

Ge-132 concentrations increased. 251

252

3.2. Effect of Ge-132 supplemented to IVM media on subsequent embryonic development after 253

PA and IVF 254

Mature oocytes from each IVM group were subjected to parthenogenetic activation (PA) or in vitro 255

fertilization (IVF). As shown in Table 3, the cleavage rates of the PA embryos tended to increase (P< 256

0.05) as Ge-132 concentrations increased. Therefore, PA embryos from the 400 µg/mL Ge-132 group 257

displayed the highest (P< 0.05) cleavage rates (82.7%) compared with the other groups. However, 258

embryonic developmental competence to the blastocyst stage and total cell number in blastocysts after 259

PA were significantly (P< 0.05) higher in the 200 µg/mL Ge-132 group (59.5% and 67.8) than in the 260

control group (38.2% and 55.3), respectively. On day 2, there were significantly higher 6- to 8-cell PA 261

embryos in the 400 µg/mL Ge-132 group compared with the 100-µg/mL Ge-132 group, and no 262

significant difference was observed in the cleavage patterns of 2- to 3-cell PA embryos and 4- to 5-cell 263

PA embryos (Figure 2, A). Hatched blastocyst formation on day 7 was significantly higher in the 200-264

µg/mL Ge-132 group than the other groups (Figure 2, B). However, no significant difference was 265

observed in early expanded PA blastocyst formation rates between the groups. 266

In the IVF experiment, the cleavage rate of the IVF embryos was significantly (P< 0.05) higher in 267

the 200 µg/mL Ge-132 treatment group (73.1%) compared with the control group (61.3%) (Table 4). 268

Regarding the cleavage pattern, significantly more 2- to 3-cell IVF embryos were observed in the 200 269

µg/mL Ge-132 group than the control group (Figure 3, A). However, no significant differences were 270

observed in the cleavage pattern of 4- to 5-cell and 6- to 8-cell IVF embryos. No significant difference 271

in blastocyst formation rates was observed between the control group and Ge-132 treated groups 272

(Table 4). However, the 200 µg/mL Ge-132 group displayed the significantly greatest (P< 0.05) total 273

cell numbers (101.3) compared with the other groups. When comparing blastocyst formation patterns 274

on day 7, expanded IVF blastocyst numbers were significantly (P< 0.05) higher in the 100 µg/mL Ge-275

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132group than in the other groups (Figure 3, B). Monospermy and efficiency of fertilization were 276

significantly greater in all Ge-132 treated groups (45.3%, 47.7% and 48.9%, 40.7, 41.4 and 38.8, 277

respectively) compared with the control group (25.1% and 21.7) (Table 5). However, no significant 278

differences in sperm penetration, MPN formation rate or polyspermy rates were observed between the 279

control and Ge-132 treated groups. 280

281

3.3. Effect of Ge-132 treatment during IVM on gene expression in oocytes and cumulus cells 282

To examine the effect of Ge-132 on the expression of DNA repair-related, anti-oxidative-related and 283

apoptosis-related genes, we evaluated the mRNA expression levels of PCNA, POU5F1, Nrf-2, Bax, 284

Bcl-2 and Caspase-3 in the oocytes and the cumulus cells of each group. As shown in Figure 4, Nrf-2 285

transcript levels were significantly higher in oocytes treated with 100 and 200 µg/mL of Ge-132 286

compared with the control group (P< 0.05). Bax transcript levels showed a tendency to decrease when 287

Ge-132 concentrations increased. The 200- and 400-µg/mL Ge-132 group expressed significantly 288

higher Bax transcript levels than control oocytes (P< 0.05). No significant difference in PCNA, 289

POU5F1, Bcl-2 and Caspase-3 transcript levels was observed in the Ge-132-treated oocytes 290

compared with the control group. Furthermore, PCNA transcript levels were significantly higher in 291

cumulus cells treated with 100 µg/mL of Ge-132, although no difference was observed in the other 292

Ge-132 treatment groups compared with the control. Nrf-2 transcript levels showed a tendency to 293

increase when Ge-132 concentrations increased. The 400 µg/mL Ge-132 group expressed the 294

significantly highest Nrf-2 transcript levels (P< 0.062). No significant difference in Bax, Bcl-2 and 295

Caspase-3 transcript levels was observed in the Ge-132treatmentgroups compared with the control 296

group. 297

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4. Discussion 298

The goal of the present study was to investigate the effect of Ge-132 supplementation during IVM. 299

Importantly, we found that Ge-132 treatment was helpful to improve cytoplasmic maturation of 300

porcine oocytes by upregulating anti-oxidative-related genes and downregulating pro-apoptotic-301

associated genes. Ge-132 also improved the developmental potential of PA and IVF porcine embryos. 302

Exposure to environmental stressors, such as ultraviolet light, ionizing radiation, chemotherapeutics 303

and inflammatory cytokines, can cause oxidizing imbalances in the cellular redox state, thereby 304

resulting in the production of reactive oxygen species (ROS), a condition termed “oxidative stress” 305

[23]. The occurrence of oxidative stress in oocytes has been considered one of the most important 306

parameters for evaluation of oocytes health [24-25]. Oocytes appear to be particularly sensitive to 307

elevated ROS levels during maturation, and the levels of glutathione (GSH) is a critical factor that 308

influence oocyte IVM [17, 26]. Our initial results of GSH and ROS level analysis are highly 309

indicative that Ge-132treatment during IVM relieves oxidative stress and cellular damage. 310

One of the critical cellular defense mechanisms against mammalian oxidative stress is the Nrf2-311

Keap1 system [27]. Under basal nonactivated conditions, Nuclear respiratory factor (Nrf-2) interacts 312

with Kelch-like erythroid cell-derived protein 1 (Keap-1), a cytoplasmic repressor protein, and limits 313

Nrf2-mediated gene expression [28-31]. Upon activation, the Keap-1-Nrf2 complex is dissociated and 314

Nrf-2 coordinates the expression of other antioxidant gene products and several dozen cytoprotective 315

genes that enhance cell survival. In the present study, Ge-132-treated oocytes and cumulus cells 316

displayed significantly increased expression levels of Nrf-2 mRNA. This finding is consistent with 317

previous results showing that Ge-132 promotes the production of GSH and represses ROS levels in 318

oocytes. So, it is likely to be exerted by increased activation of transcription factor Nrf-2 by Ge-132 319

along with its direct antioxidant effect to ameliorate the oxidative damage during IVM. Moreover, 320

oxidative stress triggers programmed cell death in response to DNA damage. It has been reported that 321

aging is the result of cellular damage caused by free radicals, primarily ROS-generated, as a 322

consequence of oxidative phosphorylation in the mitochondrial electron transport chain [32]. 323

Numerous studies published over the past decade have revealed that ROS is an important mediator of 324

the intrinsic (mitochondrion-initiated) pathway of oxidative stress-induced apoptosis [33]. ROS 325

commonly induces DNA breakdown and causes cellular damage leading to apoptosis [34-36]. During 326

intrinsic stress signaling, BAX which is the most well-characterized pro-apoptotic BCL-2 family 327

member, becomes activated and induces the release of other apoptogens from the mitochondria [37-328

38]. Therefore, the results of decreased expression of Bax mRNA provide further evidence that Ge-329

132 plays a role in the reduction of oxidative stress-induced apoptosis. 330

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Although these data have shown that Ge-132 may be a promising anti-oxidative agent during IVM, 331

the mechanism that causes this anti-oxidative effect remains unclear. Elucidating these mechanisms is 332

critical to understanding how anti-oxidative capacity is activated in oocytes through the IVM process. 333

Electron scavenging is the one of the major mechanisms that may provide insight. As shown in Figure 334

5, Ge-132 has a unique chemical structure with a Ge-C bond so that electron transfer tends to occur 335

between Ge and free radicals. Therefore, Ge-132 treatment in the form of ((HOOCCH2CH2Ge)2O3)n 336

during IVM protects the oocytes from oxidative damage via electron scavenging. We postulate that it 337

is beneficial to scavenge free radicals and then reduce oxidative damage from oxidative stressors 338

during IVM. The other potential mechanism involves cytoskeleton stabilization via the membrane 339

stabilizing effect of Ge-132, although more studies are necessary to further understand the mechanism. 340

In leukocyte function, a study has reported that higher concentrations of Ge-132 (50µg/mL) increased 341

membrane stability [14]. Therefore, the membrane stabilizing effect of Ge-132 might relieve the 342

cytoskeletal alterations caused by increased ROS production during IVM. Ge-132 may block ion-343

channels via immobilization of protein kinase-C, phospholipases and NADPH oxidase. 344

Among the trace elements, it is known that a deficiency in zinc, copper and selenium in female 345

results in infertility, and such supplements promote pregnancy and embryonic development in 346

mammals [39]. For example, a previous experiment has shown that the presence of zinc during 347

porcine IVM enhanced the developmental potential by regulating the intracellular GSH and ROS 348

levels and transcription factor expression [40]. Contrary to the beneficial function of Ge-132 proposed 349

in the current study, copper supplementation displayed no significant differences in oocyte maturation 350

and cleavage rates compared with the control but rather increased the rates of morulae and blastocyst 351

formation by decreasing the number of apoptotic blastomeres [41]. The addition of selenium to IVM 352

media has also been described to improve the developmental competence of bovine oocytes; therefore, 353

selenium is routinely used in several IVM systems [42-43]. 354

Our study reveals that Ge-132 had a beneficial effect on the developmental competence of porcine 355

oocytes showing enhanced monospermic fertilization, blastocyst formation, and blastomere viability 356

in PA- and IVF-derived embryos. Interestingly, these data clearly indicate that the 400 µg/mL Ge-132 357

group displayed detrimental effects concerning oocyte maturation, while the 200 µg/mL Ge-132 358

group displayed the most beneficial effect on embryonic development after PA and IVF. Why does the 359

capacity decline with 400 µg/mL of Ge-132? We believe that the detrimental effect of 400 µg/mL of 360

Ge-132 is caused by the weak acidic pH levels in the IVM media due to the following reasons. Indeed, 361

little work has been done to examine the effects of external pH (pHe) on the oocyte during IVM. 362

However, several reports have repeatedly demonstrated that embryos were very susceptible to 363

disruption via reduction in pH [44]. In embryo, it is known that pH affects the distribution of 364

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cytoskeletal elements and mitochondrial localization which are associated with developmental 365

competence in oocytes [45- 47]. Thus, we propose that similar effects may be occurring within the 366

oocytes. Furthermore, oocyte metabolism is likely affected by pH and has been correlated with 367

cytoplasmic maturation and developmental competence [48-50]. Overall, it is likely that even small 368

decreases in pHe by Ge-132 perturb oocyte maturation, which can profoundly affect subsequent 369

development after PA and IVF (see data supplement Figure 1). 370

Finally, although this study presents a beneficial effect of Ge-132 associated with oocyte cytoplasmic 371

maturation, some limitations should be taken into account when interpreting the results. It is worth 372

noting that some in vivo studies in this area have suggested that high doses of Ge-132 can exert a 373

toxic effect similar to that of inorganic Ge, GeO2 [51-52]. Based on this study, although the 374

mechanism by which this occurs remains unclear, accumulating evidence suggests that there were no 375

toxic effects of Ge-132 in the optimal range during IVM. Furthermore, concentration experiments 376

may need to be employed to further dissociate these effects. Currently, the potential biological 377

activities of Ge-132 have been investigated in the form of a novel complex with other materials [53-378

54]. 379

In summary, we focused on the function of Nrf-2 and apoptotic-associated genes under the oxidative 380

stress. These results strongly support the notion that Ge-132 improves the quality of porcine oocytes 381

and hence subsequent in vitro development when used at an optimal concentration of 200 µg/mL. The 382

current study provides insight into the role that organogermanium, especially Ge-132, plays in the 383

IVM system via anti-oxidation and anti-apoptosis. This knowledge will enable IVM preconditioning 384

for more successful use in IVP therapies mimicking that of in vivo conditions for better efficiency. 385

386

5. Acknowledgement 387

This research was supported, in part, by a grant from the National Research Foundation of Korea 388

Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), and a grant 389

from the Next-Generation BioGreen 21 Program (No. PJ0095692014), Rural Development 390

Administration, Republic of Korea. 391

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References 393

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germanium dioxide. Biological trace element research. 1991;29:267-80. 503 [53] Jiang J, Yao S, Cai H-H, Yang P-H, Cai J. Synthesis and synergetic effects of chrysin–organogermanium 504 (IV) complex as potential anti-oxidant. Bioorganic & medicinal chemistry letters. 2013;23:5727-32. 505 [54] Yao S, Jiang J, Yang P, Cai J. Synthesis, Characterization and Antioxidant Activity of a Novel 506 Organogermanium Sesquioxide with Resveratrol. BULLETIN OF THE KOREAN CHEMICAL SOCIETY. 507 2012;33:1121-2. 508

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FIGURE LEGENDS 509

Fig 1. Epifluorescent photomicrographic images of in vitro matured porcine oocytes. (A) Oocytes 510 were stained with CellTracker Blue (a–d) and 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) 511 (e–h) to detect the intracellular levels of glutathione (GSH) and reactive oxygen species (ROS), 512 respectively. Metaphase II (MII) oocytes derived from the maturation medium supplemented with 0 513 µg/mL Ge-132 (A and E), 100 µg/mL Ge-132 (B and F), 200 µg/mL Ge-132 (C and G) and 400 514 µg/mL Ge-132 (D and H). (B) The relative levels of intracellular GSH and ROS in in vitro-matured 515 porcine oocytes among the four groups (0 µg/mL, 100 µg/m, 200 µg/mL and 400 µg/mL Ge-132). 516 Within each group end point (GSH and ROS), the bars with different letters (a-d) are significantly (P 517 < 0.05) different. Total number of examined oocytes: GSH samples, N= 30; ROS samples, N =30. 518 The experiment was replicated three times. The experiment was replicated three times. 519 520

Fig 2. Effect of different concentrations of Ge-132 during IVM on the cleavage pattern of 521 parthenogenetic activation (PA) embryos at day 2 (A), and the percentage of PA embryos that 522 developed to the blastocyst stage at day 7 (B). Within each end point, bars with different letters (a and 523 b) are significantly (P < 0.05) different for different concentrations of Ge-132. EarBL, early 524 blastocyst; ExpBL, expanded blastocyst; HatBL, hatched blastocyst. The experiment was replicated 525 four times. 526 527

Fig 3. Effect of different concentrations of Ge-132 during IVM on the cleavage pattern of in vitro 528 fertilization (IVF) embryos at day 2 (A), and the percentage of IVF embryos that developed to the 529 blastocyst stage at day 7 (B). Within each end point, bars with different letters (a and b) are 530 significantly (P < 0.05) different for different concentrations of Ge-132. EarBL, early blastocyst; 531 ExpBL, expanded blastocyst; HatBL, hatched blastocyst. The experiment was replicated four times. 532 533

Fig 4. mRNA expression levels (mean ± SEM) of PCNA, POU5F1, Nrf-2, Bax, Bcl-2, and Caspase-3 534 relative to GAPDH in oocytes and cumulus cells treated with Ge-132 during in vitro maturation 535 (IVM). Within the same target mRNA, values with different superscript letters are significantly (P < 536 0.05) different. The experiment was replicated three times. 537

538

Fig 5. Proposed antioxidant mechanism of Ge-132. 539

540

Supplement Figure 1. The pH level of IVM media treated with various concentrations of Ge-132 541 with or without hormones. eCG; equine chronic gonadotropin ; hCG ; human chronic gonadotropin. 542

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Table 1. 544

Primers used for gene expression analysis. 545

mRNA Primer sequences Product size (base pairs)

Gene Bank accession number

GAPDH F: 5'-GTCGGTTGTGGATCTGACCT-3' R: 3'-TTGACGAAGTGGTCGTTGAG-5'

207 NM_001206359

PCNA F: 5'-CCTGTGCAAAAGATGGAGTG-3' R: 3'-GGAGAGAGTGGAGTGGCTTTT-5'

187 XM_003359883

POU5F1 F: 5'-GCGGACAAGTATCGAGAACC-3' R: 3'-CCTCAAAATCCTCTCGTTGC-5'

200 NM_001113060

Nrf2 F: 5'-CCCATTCACAAAAGACAAACATTC-3' R: 3'-GCTTTTGCCCTTAGCTCATCTC-5'

71 [31]

Bax F: 5'-TGCCTCAGGATGCATCTACC-3' R: 3'-AAGTAGAAAAGCGCGACCAC-5'

199 XM_003127290

Bcl-2 F: 5'-AGGGCATTCAGTGACCTGAC-3' R: 3'-CGATCCGACTCACCAATACC-5'

193 NM_214285

Caspase-3 F: 5'-CGTGCTTCTAAGCCATGGTG-3' R: 3'-GTCCCACTGTCCGTCTCAAT-5'

186 NM_214131

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Table 2. Effect of Ge-132 treatment during porcine IVM on nuclear maturation 548

Ge-132 concentration

(µg/mL)

Oocytes cultured

for maturation,

N*

Number of oocytes at the stage of

Germinal vesicle (%)

Metaphase I (%) Anaphase and Telophase I

(%) Metaphase II (%)

0 (control) 244 3 (1.2±0.5) 11 (4.5±0.7) 13 (5.3±1.0) 217 (89.0±1.2)

100 240 1 (0.4±0.4) 13 (5.4±0.8) 10 (4.2±0.7) 216 (89.9±1.3)

200 242 0 (0.0±0.0) 7 (2.9±0.6) 14 (5.8±0.8) 221 (91.3±0.9)

400 241 3 (1.3±0.5) 10 (4.1±1.1) 10 (4.2±0.8) 218 (90.4±1.2)

* Three times replicated. 549

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Table 3. Effect of Ge-132 treatment during IVM on embryonic development in porcine 551 parthenogenetic activation (PA) embryos 552

Ge-132 concentration

(µg/mL)

Embryo cultured, N

Embryos developed to (N, %) Total cell numbers in

Blastocyst (N) ≥ 2-cell Blastocyst

0 (control) 187 126 (67.5 ± 4.5) a 71 (38.2 ± 2.8) a 55.3 ± 3.4a (25)

100 193 138 (71.5 ± 7.0)a, b 103 (53.2 ± 8.2)a, b 77.0 ± 8.8a, b(21)

200 188 148 (78.7± 5.2)b, c 112 (59.5 ± 5.3) b 67.8 ± 4.7b (27)

400 193 160 (82.7± 6.2) c 77 (39.9 ± 4.9) a 57.8 ± 3.6a (24)

* Percentage of total cultured oocytes.† Number of examined blastocysts. a-c Values with different superscripts within a column differ significantly ( P< 0.05).

The experiment was repeated four times. The data represent the mean±SEM.

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Table 4. Effect of Ge-132 treatment during IVM on embryonic development after in vitro fertilization 556 (IVF) 557

Ge-132 concentration

(µg/mL)

Embryo cultured, N

Embryos developed to (N, %) Total cell numbers in

Blastocyst (N) ≥ 2-cell Blastocyst

0 (control) 181 111 (61.3 ± 2.9)a 57 (31.6 ± 3.0) 71.5 ± 7.8a (19)

100 178 118 (66.0 ± 3.7)a, b 72 (40.5 ± 3.5) 87.3 ± 7.8a, b(18)

200 182 133 (73.1± 1.2)b 67 (36.7 ± 1.3) 101.3 ± 10.6b (19)

400 186 130 (69.9± 0.9)a, b 72 (38.9 ± 4.0) 95.1 ± 10.1a, b(20)

* Percentage of total cultured oocytes.† Number of examined blastocysts. a-b Values with different superscripts within a column differ significantly (P< 0.05).

The experiment was repeated four times. The data represent the mean±SEM.

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Table 5. Effect of Ge-132 treatment on sperm penetration ofin vitro matured porcine oocytes at 10 h 559

post-insemination 560

Parameter Ge-132 concentration (µg/mL)

0 (control) 100 200 400

Number of oocytes

examined 104 106 111 127

Penetrated (%)* 87.7 ± 4.5 89.8 ± 3.8 87.2 ± 2.7 82.2 ± 7.4

MPN formed (%)† 88.9 ± 5.2 93.9 ± 3.8 97.5 ± 2.5 93.9 ± 1.9

Monospermy (%)† 25.1 ± 3.6a 45.3 ± 5.3b 47.7 ± 4.0b 48.9 ± 9.0b

Polyspermy (%)† 63.8 ± 5.6 48.6 ± 7.2 49.8 ± 4.8 45.0 ± 10.1

Efficiency of

fertilization‡ 21.7 ± 2.1a 40.7 ± 5.3b 41.4 ± 2.5b 38.8 ± 3.2b

Data are provided as the mean ± SEM. Values with different superscript letters within rows are 561 significantly different (P< 0.05). 562

The experiment was repeated three times. 563

MPN, male pronucleus. 564

*Percentage of the number of oocytes examined. 565

†Percentage of the number of oocytes penetrated. 566

‡Efficiency of fertilization was the percentage of monospermic oocytes from total examined. 567

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Figure 1. 569

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Figure 2. 572

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Figure 3. 575

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Figure 4. 578

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Figure 5. 583

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Supplement Figure 1. 585

586