Theriogenology xxx (2014) 1–12

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Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes Junchul D. Yoon a, Yubyeol Jeon a, Lian Cai a, Seon-Ung Hwang a, Eunhye Kim a, Eunsong Lee b, Dae Y. Kim c, **, Sang-Hwan Hyun a, * a

Laboratory of Veterinary Embryology and Biotechnology (VETEMBIO), College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea Laboratory of Theriogenology, College of Veterinary Medicine, Kangwon National University, Kangwon, Republic of Korea c Department of Life Science, College of BioNano Technology, Gachon University, Incheon, Republic of Korea b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 December 2013 Received in revised form 15 September 2014 Accepted 20 September 2014

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions between follicular mural somatic cells and COCs. To restore the microenvironment, a coculture system was established using cumulus-derived somatic cells (CSCs) for IVM. The CSCs were cultured in Dulbecco’s modified Eagle’s medium for 48 hours with varying numbers of CSCs (0.0, 2.5  104, 5.0  104, and 10.0  104) and then cultured in tissue culture medium 199 (TCM 199) for 4 hours before adding the oocytes. Cumulus-oocyte complexes from 3- to 6-mm follicles were matured in 500 mL of TCM 199 with eCG and hCG for 22 hours and then cultured in TCM 199 without hormones for 22 hours. After IVM, the group with 2.5  104 CSCs showed a significant increase in intracellular glutathione levels compared with the control group. In the evaluation of sperm penetration, efficient fertilization was increased in the groups with 2.5  104 and 5.0  104 CSCs compared with controls (44.9 and 46.5 vs. 32.1, respectively). The mRNA expression pattern analysis in matured COCs showed a significant upregulation of PCNA, COX-2, Has2, Ptx3, and Nrf2 in the 2.5  104 CSC group compared with controls. During COC maturation at 0, 11, 22, 33, and 44 hours, the 2.5  104 and 5.0  104 CSC groups showed a significantly altered mRNA expression of BMP15 and GDF9. The developmental competence of the matured oocytes in all groups was evaluated after IVF and parthenogenetic activation (PA). After IVF, the 2.5  104 CSC group showed significantly higher cleavage, blastocyst formation rate, and total cell numbers compared with controls (60.0%, 35.7%, and 127.3 vs. 43.2%, 21.1%, and 89.3, respectively). After PA, the 2.5  104 CSC group had significantly higher blastocyst formation rate and total cell number than the control group (52.0% and 120.4 vs. 35.4% and 90.9, respectively). In conclusion, these results suggest that the presence of a population of 2.5  104 CSCs during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by increasing the intracellular glutathione level via changing of a specific gene expression pattern during oocyte maturation. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Porcine Oocyte In vitro maturation Coculture Somatic cell

1. Introduction * Corresponding author. Tel.: þ82 43 261 3393; fax: þ82 43 267 3150. ** Alternate corresponding author. Tel.: þ82 32 820 4544; fax: þ82 32 821 2734. E-mail addresses: [email protected] (Dae Y. Kim), shhyun@cbu. ac.kr (S.-H. Hyun). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2014.09.025

Various transgenic animals have been generated through assisted reproductive technologies, including IVM and IVF, using gametes [1–3]. In particular, pigs have been used to develop some of the most important large animal models for biomedical research [4–6]. For translational

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research, the development of IVM, which can be attributed to improved culture conditions for the embryo, has improved fertilization efficiency in transgenic pigs. Nonetheless, compared with oogenesis in vivo, IVM conditions are still suboptimal for oocyte maturation, because numerous factors, including maturation time, temperature, and culture medium composition, affect oocyte maturation in vitro [7]. Therefore, a greater understanding of the in vivo environment will allow the modifications of culture conditions necessary to improve the developmental potential of oocytes matured in vitro [8]. The successful development of in vivo follicles requires bidirectional communication between oocytes and follicular somatic cells, such as granulosa cells and theca cells [9,10]. During the progression of follicular growth, the expressions of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) have been demonstrated to be positive regulatory paracrine factors in a variety of species, including rodents, humans, and ruminants [11–13]. Oocyte-secreted GDF9 and BMP15 regulate the oocyte microenvironment, for instance, by promoting granulosa cell proliferation and modulating FSH-dependent follicular function [14]. In response to GDF9 and BMP15, follicular somatic cells secrete their own paracrine factors, such as anti-Mullerian hormone, inhibin, activin, and BMP2, -5, -6, which affect oocyte intracellular maturation [15]. However, because cumulus-oocyte complexes (COCs) separate from the follicular microenvironment for maturation during typical IVM, they are not able to respond to factors secreted by follicular somatic cells. From the time of first trial of porcine IVM, to improve the IVM environment, the coculture system has been modified to contain diverse types of somatic cells, such as granulosa cells, theca cells, sections of the follicle wall, oviductal epithelial cells, and cumulus clumps, from various mammalian species [16–19]. In one case, denuded buffalo oocytes were embedded in cumulus cell clumps to restore the threedimensional environment and allow maturation. The embedded denuded oocytes had significantly higher maturation rates and blastocyst formation after parthenogenetic activation (PA) [17]. Analogously, ovine oocytes cocultured with somatic cells of a cumulus origin had altered expression levels of the Transforming Growth Factor-beta (TGF-b) ligand and receptor and higher maturation rates than controls [20]. In porcine models, prematured oocytes cocultured with oviductal epithelial cell monolayers during IVM resulted in increased oocyte cytoplasmic maturation and blastocyst development [19]. Another study revealed that cocultures with ovarian cortex cell monolayers during porcine oocyte maturation enhanced the maturation quality and rate of porcine oocytes as well as the blastocyst formation rate on subsequent embryonic development after IVF [21]. To restore the follicular microenvironment, many studies have applied coculture systems using various cell types during porcine IVM [19,21–24]. However, until now, no information has been available on the effect of cumulusderived somatic cells (CSCs) on porcine oocyte maturation, and the concentration of feeder cells has not yet been determined. In this study, the effects of coculture with CSCs were investigated on porcine oocyte IVM and subsequent embryonic development after IVF and PA. Oocyte nuclear

maturation, intracellular levels of glutathione (GSH) and reactive oxygen species (ROS), embryonic cleavage, blastocyst formation, and blastocyst cell numbers were analyzed. In addition, the expression patterns of GDF9 and BMP15 during oocyte maturation and embryonic developmental marker and apoptosis-related genes, proliferating cell nuclear antigen (PCNA), POU domain, class 5, transcription factor 1 (POU5F1), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak), caspase-3 (cas3), and B-cell lymphoma 2 (Bcl-2), in IVF- and PA-derived blastocysts were measured. 2. Materials and methods 2.1. Chemicals Unless otherwise indicated, all chemicals and reagents used in the present study were purchased from SigmaAldrich Corporation (St. Louis, MO, USA). 2.2. Oocyte collection and IVM Porcine ovaries (mixed Yorkshire, Landrace, and Duroc breeds, predominantly from 6-month-old gilts) were collected from a local abattoir and transported to the laboratory in physiological saline supplemented with 100 IU/L of penicillin G and 100 mg/mL of streptomycin sulfate at 32  C to 39  C within 2 hours. The ovaries were washed twice with physiological saline. The COCs were aspirated from follicles 3 to 6 mm in size using an 18-gauge needle attached to a 10 mL syringe, collected in 15 mL conical centrifuge tubes, and allowed to settle to the bottom of the tube. After 10 minutes, the supernatant was removed, a conical tube was filled with HEPES-buffered Tyrode’s medium containing 0.05% (wt/vol) polyvinyl alcohol (TLH-PVA), and the COCs were observed under a stereomicroscope. More than three layers of COCs comprising compact cumulus cells and homogenous cytoplasm were selected from the collected fluid for IVM and washed three times in TLH-PVA. The 50 to 60 randomly selected COCs were transferred into a four-well dish (Nunc, Roskilde, Denmark) containing 500 mL of maturation medium (tissue culture medium 199; Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 0.6 mM of cysteine, 0.91 mM of sodium pyruvate, 10 ng/mL of epidermal growth factor, 75 mg/mL of kanamycin, 1 mg/mL of insulin, and 10% (v:v) porcine follicular fluid. The porcine follicular fluid was extracted from 3- to 6-mm follicles of prepubertal gilt ovaries, prepared according to Hyun et al. [25] and stored at 70  C until used. The transferred COCs were cultured with 10 IU/mL of eCG and 10 IU/mL of hCG for 22 hours at 39  C in a humidified atmosphere of 5% CO2 and 95% air. After 22 hours of maturation with the hormones, the COCs were washed twice in a hormone-free maturation medium and subsequently cultured in that medium for an additional 20 hours. During IVM, COCs were incubated with varying CSC numbers of 0, 2.5  104, 5.0  104, and 10.0  104 CSCs. 2.3. Isolation of CSCs The isolation of CSCs for coculture with COCs was performed according to Kyasari et al. [20] with a few

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modifications. Briefly, the collected COCs were transferred to TLH-PVA with 0.1% hyaluronidase, and the cumulus cells were mechanically segregated by pipetting. Detached cumulus cells were centrifuged at 2000 g for 3 minutes, and the cell pellets were suspended in cell culture medium (Dulbecco’s modified Eagle’s medium–high; Gibco Ltd., Carlsbad, USA) supplemented with 10% Fetal bovine serum (FBS; Gibco Ltd.) (Gibco Ltd.), 100 U/mL of penicillin, and 100 mg/mL of streptomycin. The suspended media were transferred to 100-mm cell culture dishes (SPL, Anyang, Gyeonggi, South Korea) and incubated in a humidified atmosphere of 5% CO2 and 95% air at 39  C. The cell culture media were replaced every 2 days until CSCs reached 80% to 90% confluence, and the attached CSCs were washed twice with PBS and detached from the plate using trypsin-EDTA (Gibco Ltd.) for 2 minutes. The detached CSCs were washed with cell culture media to inactivate the trypsin and reseed (passage 1, P1) in new cell culture dishes for further expansion. Once the cultured CSCs reached passages 3 (P3) to 6 (P6), the cells were reseeded at varying concentrations (0, 2.5  104, 5  104, and 10  104 CSCs per milliliter) in four-well dishes as feeder cells.

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2.6. Parthenogenetic activation and IVC of porcine embryos Parthenogenetic activation was performed according to Kwak et al. [29]. Briefly, after IVM, the MII-stage oocytes of each group were denuded as described previously and washed twice with activation medium (280 mM mannitol solution containing 0.01 mM of CaCl2 and 0.05 mM of MgCl2). The oocytes were transferred between electrodes covered with activation medium and activated with two pulses of 120 V/mm direct current for 60 microseconds. The produced embryos by electrical activation (henceforth named as PA embryos) were treated with 5 mg/mL of cytochalasin B in the IVC medium for 5 hours at 39  C in a humidified atmosphere of 5% CO2 and 95% air. After treatment, PA embryos were washed three times in fresh IVC medium (porcine zygote medium 3) [30] and transferred into 30 mL fresh IVC droplets (10 activated oocytes per drop) with mineral oil. Then, PA embryos were cultured at 39  C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 7 days. In all experiments, the zygote culture media were renewed after 48 hours (Day 2) and 96 hours (Day 4). 2.7. In vitro fertilization

2.4. Fluorescent staining for assessment of nuclear status To investigate nuclear maturation, the oocytes were sampled 42 to 43 hours after IVM and mechanically denuded from cumulus cells in TLH-PVA medium containing 0.1% hyaluronidase. The denuded oocytes were washed three times in TLH-PVA, fixed for 5 minutes in Dulbecco’s PBS containing 1% paraformaldehyde, and stained in TLH-PVA containing 10 mg/mL of Hoechst 33342 for 5 minutes. The stained oocytes were examined under an epifluorescence microscope (Nikon Corp., Tokyo, Japan) and classified as germina vesicles (GV), metaphase I (MI), anaphase-telophase I (AT), or metaphase II (MII) in accordance with the meiotic maturation stage [26]. 2.5. Measurement of intracellular ROS and GSH levels The MII-stage oocytes of each group were sampled after IVM to determine the levels of intracellular GSH and ROS, which were measured using methods described previously [27,28]. Briefly, 20 ,70 -dichlorodihydrofluorescein diacetate (Invitrogen) and CellTracker Blue 4-chloromethyl-6.8difluoro-7-hydroxycoumarin (Invitrogen) were used to detect intracellular ROS as green fluorescence and GSH level as blue fluorescence, respectively. Six to eight oocytes from each group were incubated (in the dark) for 30 minutes in TLH-PVA supplemented with 10 mM of 20 ,70 dichlorodihydrofluorescein diacetate or 10 mM of CellTracker. After incubation, the oocytes were washed three times with TLH-PVA, aliquoted into 10 mL droplets, and the fluorescence was observed under an epifluorescence microscope (TE300; Nikon Corp.) with UV filters (460 nm for ROS and 370 nm for GSH). Fluorescent images were saved as graphic files in tagged image file format. The fluorescence intensities of oocytes were analyzed using ImageJ software (Version 1.47; National Institutes of Health, Bethesda, MD, USA) and normalized to those of the control oocytes. The experiment was replicated five times.

In vitro fertilization was performed according to Biswas and Hyun [31]. Briefly, the oocytes were coincubated with sperm at a final concentration of 5.0  105 sperm/mL for 20 minutes at 39  C in a humidified atmosphere of 5% CO2 and 95% air. After 20 minutes of coincubation, the attached sperm were removed from the zona pellucida (ZP) by gentle pipetting. The oocytes were then washed three times in a modified Tris-buffered medium and incubated in the modified Trisbuffered medium without sperm for 5 hours at 39  C in a humidified atmosphere of 5% CO2 and 95% air. Thereafter, oocytes were cultured as described in the previous section. 2.8. Assessment of fertilization parameters The cells were stained to detect sperm penetration, which located inside the oocyte, and pronuclear formation according to Koo et al. [32] with a few modifications. Briefly, 10 hours after insemination, the ZP of oocytes was dissolved with 0.5% protease. The zona-free zygotes were washed three times in TLH-PVA medium and fixed in 1% formaldehyde in PBS for 5 minutes at room temperature. The fixed embryos were placed in a drop of mounting medium (25% [v:v] glycerol in PBS containing 2.5 mg/mL of sodium azide and 2.5 mg/mL of Hoechst stain) on a slide with a cover slide overlaying the embryos. The number of spermatozoa penetration, the sperm ratio, and male pronucleus (MPN) formation were examined under a fluorescence microscope. 2.9. Embryo evaluation and total cell counts Day 0 was considered the day at which PA and IVF were commenced. The embryos were assessed for cleavage under a stereomicroscope on Day 2 (48 hours). Blastocyst formation was evaluated on Day 7 after PA and IVF, and blastocysts were recognized as a blastocyst according to a degree of more than expansion grade [33]. To determine the total cell numbers comprising blastocysts at Day 7, blastocysts from

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each group were collected and the ZP (if not hatched) was dissolved with 0.5% proteases. The zona-free blastocysts were washed in PBS containing 1% (wt/vol) BSA and stained with 10 mg/mL of Hoechst 33342 for 5 minutes. After a final wash in PBS-BSA, the blastocysts were fixed briefly in 4% paraformaldehyde in PBS. The blastocysts were mounted on glass slides with a drop of 100% glycerol, gently pressed under a cover slip, and observed using a fluorescence microscope (Nikon Corp.) at 400 magnification. 2.10. Real-time polymerase chain reaction and gene expression analysis The expression patterns of two TGF-b superfamily genes, namely, BMP15 and GDF9, were assessed in COCs at 0, 11, 22, 33, and 44 hours during IVM. To perform real-time polymerase chain reaction (RT-PCR) for detecting expression patterns of two TGF-b superfamily genes during maturation time, each group of 120 COCs was collected by following time. The mRNA levels of cyclooxygenase 2 (COX-2; also as well known prostaglandin-endoperoxide synthase 2), PCNA, hyaluronan synthase 2 (Has2), pentraxin-related protein (Ptx3), nuclear factor erythroid-derived factor 2–related factor 2 (Nrf2), and Cas3 were analyzed in COCs after maturation. To detect the specific gene expression level in COCs after maturation, each group of 120 COCs was sampled after maturation. The mRNA expression of PCNA, POU5F1, Cas3, Bak, Bax, and Bcl-2 in IVF- and PA-derived blastocysts was also analyzed. To analyze specific mRNA levels in blastocysts, each group of 5 to 6 IVF- and PA-derived hatched blastocysts were collected under stereomicroscope on Day 7. All the samples

were stored at 70  C until analysis. The transcript abundance was analyzed by RT-PCR as described previously [34]. Briefly, total RNA was extracted from samples using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Complementary DNA was prepared by subjecting 1 mg of total RNA to reverse transcription using Moloney murine leukemia virus reverse transcriptase (Invitrogen) and random primers (9-mers; Takara Bio. Inc., Otsu, Shiga, Japan). Quantitative RT-PCR was performed on the Mx3000P qPCR System (Agilent Technologies, Santa Clara, USA) using 1 mL complementary DNA template plus 10 mL 2 SYBR Premix Ex Taq (Takara Bio Inc.) containing specific primers. The reactions were performed over 40 cycles with the following parameters: denaturation at 95  C for 30 seconds, annealing at 55  C for 30 seconds, and extension at 72  C for 30 seconds. Each primer pair was designed using Primer Express software (Primer 3; Whitehead Institute for Biomedical Research, Cambridge, MA, USA). The primer sequences are listed in Table 1. The expression of each target gene was quantified relative to glyceraldehyde 3-phosphate dehydrogenase as the internal control gene. The relative quantification was based on a comparison with the threshold cycle (Ct) at constant fluorescence intensity. The relative mRNA expression (R) was calculated using the equation R ¼ 2(DCt sample  DCt control). Each expression value was normalized to that of glyceraldehyde 3-phosphate dehydrogenase. 2.11. Statistical analysis Each experiment consisted of at least three replicates. Statistical analyses were performed using statistical

Table 1 Primers used for gene expression analysis. mRNA

Primer sequences

Product size (base pairs)

GenBank accession number

PCNA

F: 50 -CCTGTGCAAAAGATGGAGTG-30 R: 30 -GGAGAGAGTGGAGTGGCTTTT-50 F: 50 -GCGGAAAACGCCTATGAGTA-30 R: 30 -GCAGTGATGCAGCATGAAGT-50 F: 50 -TGCCTCAGGATGCATCTACC-30 R: 30 -AAGTAGAAAAGCGCGACCAC-50 F: 50 -GCGGACAAGTATCGAGAACC-30 R: 30 -CCTCAAAATCCTCTCGTTGC-50 F: 50 -AGGGCATTCAGTGACCTGAC-30 R: 30 -CGATCCGACTCACCAATACC-50 F: 50 -CGTGCTTCTAAGCCATGGTG-30 R: 30 -GTCCCACTGTCCGTCTCAAT-50 F: 50 -GGCTGCGGGAACATAATAGA-30 R: 30 -GCAGCTCTGGGTCAAACTTC-50 F: 50 -TTACAATCCTCCTGGGTGGT-30 R: 30 -TCAAGCACCATGTCGTACTG-50 F: 50 -AGACTTTATGCCATGGTGCT-30 R: 30 -TGACAGTGAGCAATGAACAA-50 F: 50 -CCCATTCACAAAAGACAAACATTC-30 R: 30 -GCTTTTGCCCTTAGCTCATCTC-50 F: 50 -GTTGGGATCATTGGATCATT-30 R: 30 -TCAGGAGGATGCTAATAGGC-50 F: 50 -CTGAAGTGGGACAACTGGAT-30 R: 30 -ACTCAAAGGGCTGTACTTGG-50 F: 50 -GTCGGTTGTGGATCTGACCT-30 R: 30 -TTGACGAAGTGGTCGTTGAG-50

187

XM_003359883

189

XM_001928147

199

XM_003127290

200

NM_001113060

193

NM_214285

186

NM_214131

183

NM_214321

199

NM_214053

195

NM_001244783.1

Bak Bax POU5F1 Bcl-2 Caspase-3 COX-2 Has2 Ptx3 Nrf2 BMP15 GDF9 GAPDH

71

[52]

198

NM_001005155.1

192

NM_001001909.1

207

NM_001206359

Abbreviations: BMP15, bone morphogenetic protein 15; Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; COX-2, cyclooxygenase 2; CSC, cumulus-derived somatic cell; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GDF9, growth differentiation factor 9; Has2, hyaluronan synthase 2; Nrf2, nuclear factor erythroid-derived factor 2–related factor 2; PCNA, proliferating cell nuclear antigen; POU5F1, POU domain, class 5, transcription factor 1; Ptx3, pentraxin-related protein.

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package for the social sciences 17.0 software (SPSS Inc., Chicago, IL, USA). The levels of GSH and ROS, embryonic development (e.g., rates of cleavage, blastocyst formation, and number of nuclei), and the relative gene expression levels were compared for all groups using one-way ANOVA, followed by Duncan’s multiple range test. Data are presented as the mean or the mean  SEM. Differences were considered significant at P < 0.05.

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different (P < 0.05) among the 0, 2.5  104, 5.0  104, and 10.0  104 CSC groups (Fig. 1B). In this study, the coculture with CSCs during maturation has not affected the intracellular ROS level. However, the coculture system has affected the intracellular GSH level significantly on matured oocytes when cocultured with 2.5  104 CSCs. 3.3. Effects of coculture with CSCs after IVM on the development of PA and in vitro fertilized embryos (experiment 4)

3. Results 3.1. Effects of coculture with CSCs during porcine oocyte IVM (experiment 1) In the first set of experiments, the effects of CSCs on oocyte nuclear maturation were evaluated using a range of cell numbers (0, 2.5  104, 5.0  104, and 10.0  104). Under an epifluorescence microscope the oocytes were classified as GV, MI, AT, or MII, and only MII-stage oocytes were determined as nuclear matured oocytes. In the present study, all the coculture groups showed no significance in all nuclear maturation stages among 0, 2.5  104, 5.0  104, and 10.0  104 CSC groups such as GV (0.8  0.5%, 0.0  0.0%, 0.4  0.4%, and 0.0  0.0%, respectively), MI (11.8  0.6%, 8.4  1.6%, 9.4  1.2%, and 8.3  1.1%, respectively), and AT (5.9  2.1%, 4.6  1.5%, 4.3  1.1%, and 3.7  1.7%, respectively). Although the coculture groups showed to increase MII-stage oocytes after maturation, no significant differences (P < 0.05) in the nuclear maturation rate were observed among 0, 2.5  104, 5.0  104, and 10.0  104 CSC groups (81.5  2.4%, 87.0  2.2%, 86.0  2.0% and 88.1  1.9%, respectively) (Table 2). 3.2. Effects of coculture with CSCs on intracellular GSH and ROS levels in porcine oocytes matured in vitro (experiment 2) To evaluate intracellular GSH and ROS level, each group of matured oocytes was stained after IVM. In the stained oocytes, intracellular GSH level, which has been established as a developmental competence marker, was shown as blue fluorescence. The intracellular ROS level, which is well known for negative factor on matured oocytes, was shown as green fluorescence (Fig. 1A). Mature oocytes cocultured with 2.5  104 CSCs showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control group. Although matured oocytes derived from 5.0  104 and 10.0  104 CSC groups tended to increase the intracellular GSH levels, no significant differences were observed compared with the control group. Intracellular ROS levels in mature oocytes were not significantly

In the PA experiment, 448 oocytes were used for three independent replicate experiments to evaluate the effects of coculture with CSCs during IVM and subsequent embryonic development after PA. The results revealed a significant increase in the rate of blastocyst formation in the 2.5  104 CSC group (52.0  4.4%) compared with the control group (35.4  4.4%) and 10.0  104 CSC group (32.0  6.6%), whereas no differences in cleavage rate were observed in all groups. Furthermore, total blastocyst cell numbers were significantly higher in the 2.5  104 coculture group (120.4  8.6) compared with the control group (90.9  6.1) respectively (Table 3). In the IVF experiment, MPN formation was significantly higher in the 2.5  104 CSC group (93.7  2.9%) than the control group (83.9  2.9%). The monospermy rate and the efficiency of fertilization were higher, and the number of penetrating sperm per penetrated oocyte was significantly lower in both 2.5  104 and 5.0  104 CSC groups (55.7  4.4% and 56.1  6.9%, 44.9  2.1%, and 46.5  4.5% and 1.6  1.0 and 1.6  1.5, respectively) than the control group (36.6  4.5%, 32.1  3.1%, and 2.2  2.3, respectively) (Table 4). However, no significant differences in sperm penetration or polyspermy rates were observed among the control and coculture groups. As shown in Table 5, after IVF the cleavage rates were significantly higher in 2.5  104 and 5.0  104 coculture groups (60.0  4.7% and 64.5  5.9%) than the control group (43.2  5.0%). Moreover, blastocyst rate and total blastocyst cell number, indicators of embryonic developmental competence, after IVF were significantly higher in the 2.5  104 coculture group (35.7  2.9% and 127.3  7.7, respectively) than the control group (21.1  3.8% and 89.3  4.0, respectively) (Table 5). 3.4. Effects of coculture with CSCs during IVM on gene expression in maturing COCs, matured COCs, and PA- and IVFderived blastocysts (experiment 5) The expression patterns of the TGF-b superfamily members, BMP15 and GDF9, in COCs at 0, 11, 22, 33, and 44 hours of IVM were measured. As shown in Figure 2, the

Table 2 Effects of coculture with cumulus-derived somatic cells on nuclear maturation during IVM. Feeder cell concentration (104)

No. of oocytes cultured for maturationa

No. of oocytes at the stage of Germinal vesicle (%)

0 2.5 5.0 10.0 a

Five times replicated.

238 239 235 218

2 0 1 0

(0.8 (0.0 (0.4 (0.0

   

0.5) 0.0) 0.4) 0.0)

Metaphase I (%) 28 20 22 18

(11.8  0.6) (8.37  1.6) (9.4  1.2) (8.3  1.1)

Anaphase and telophase I (%) 14 11 10 8

(5.9 (4.6 (4.3 (3.7

   

2.1) 1.5) 1.1) 1.7)

Metaphase II (%) 194 208 202 192

(81.5 (87.0 (86.0 (88.1

   

2.4) 2.2) 2.0) 1.9)

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Fig. 1. Epifluorescent photomicrographic images of in vitro matured porcine oocytes. (A) Oocytes were stained with CellTracker Blue (a–d) and 20 ,70 -dichlorodihydrofluorescein diacetate (e–h) to detect intracellular levels of GSH and ROS, respectively. Metaphase II oocytes derived from cocultures with 0 (a, e), 2.5  104 (b, f), 5.0  104 (c, g), or 10.0  104 (d, h) CSCs. (B) The effect of coculture with CSCs on intracellular GSH and ROS levels in in vitro matured porcine oocytes. Bars with different letters (a, b) represent significant differences within the respective end point (GSH or ROS) (P < 0.05). Number of GSH samples ¼ 30, ROS ¼ 30. The experiment was replicated five times. CSC, cumulus-derived somatic cell; GSH, glutathione; ROS, reactive oxygen species. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

levels of BMP15 mRNA in the control group were higher than those of the coculture groups at 11 and 22 hours during maturation. However, after 33 hours, the 2.5  104 coculture group showed higher mRNA levels than the control and 10.0  104 coculture groups; furthermore, both the 2.5  104 and 5.0  104 coculture groups had higher BMP15 expression at 33 hours than at 22 hours. Although the 10.0  104 coculture group showed the lowest mRNA levels among all groups, the 2.5  104 coculture group maintained higher levels than the control and 5.0  104 coculture groups at 44 hours. Similar to the BMP15 mRNA pattern, GDF9 mRNA levels were greater in the control

group than the coculture groups at 11 and 22 hours of maturation; yet the 2.5  104 and 5.0  104 coculture groups showed higher mRNA levels than the control at 33 and 44 hours. To examine the expression of genes related to developmental competence and apoptosis, PCNA, COX-2, Has2, Ptx3, Nrf2, and Cas3 mRNA levels were evaluated in matured COCs of each coculture group (Fig. 3). The mRNA transcript levels of PCNA, COX-2, Has2, and Nrf2 were significantly increased in matured COCs of the 2.5  104 coculture group compared with those of the control group. The transcription levels of PCNA, COX-2, and Has2 were

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Table 3 Effects of the coculture system on embryonic development after parthenogenetic activation. Treatment group (104)

No. of embryos cultureda

0 2.5 5 10

198 196 190 197

Total cell number (n)b

No. (%) of embryos developed to 2 cells 151 163 143 141

(76.3 (83.2 (75.3 (71.6

Blastocyst    

3.7) 3.8) 4.7) 5.8)

70 102 89 63

(35.4 (52.0 (46.8 (32.0

   

4.4)A 4.4)B 4.5)A,B 6.6)A

90.9 120.4 105.7 108.4

   

6.1 8.6 8.7 8.4

(36)A (34)B (34)A,B (34)A,B

Values with different superscripts (A, B) within a column differ significantly (P < 0.05). a Five times replicated. b Number of examined blastocysts.

significantly higher and of Cas3 were significantly lower in matured COCs derived from 5.0  104 cocultured oocytes in comparison with those from the control group. The gene expression levels did not differ between the control and 10.0  104 coculture groups. We examined the relative abundance of PCNA, POU5F1, Bax, Bak, Cas3, and Bcl-2 transcripts in IVF- and PA-derived blastocysts in each group of cocultured mature oocytes (Fig. 4). The transcript levels of PCNA and POU5F1 were significantly higher in IVF blastocysts derived from the 2.5  104 and 5.0  104 cocultured oocytes than those from the control group. Blastocysts derived from 10.0  104 cocultured oocytes after IVF had significantly higher Cas3 and Bak transcript levels than the other groups. The PA blastocysts derived from 2.5  104 and 5.0  104 cocultured oocytes showed significantly higher PCNA transcript levels than the control group. The transcript levels of Cas3 and Bak were significantly higher in PA blastocysts derived from the 10.0  104 cocultured oocytes than the control group. The expression levels of POU5F1, Bax, and Bcl-2 were not different among the control and coculture groups. 4. Discussion We demonstrated that coculture with CSCs during IVM had beneficial effects on oocyte maturation and the subsequent development of PA and in vitro fertilized embryos. Coculture with porcine oocytes and 2.5  104 CSCs during IVM effectively increased the GSH concentration in mature

oocytes and altered TGF-b superfamily mRNA expression patterns during maturation of COCs. This led to enhanced in vitro development of PA and in vitro fertilized embryos, increased developmental competence–related gene expression, and reduced apoptosis-related gene expression in blastocysts. In general, IVM oocytes have lower developmental competence compared with oocytes matured in vivo, and this is caused by inappropriate maturation conditions and insufficient markers for cytoplasmic maturation [35]. When oocytes mature in vivo, the oocyte is surrounded by immediately adjacent cumulus cells that play an essential role in oocyte growth and development; mural somatic cells attach to the surrounding follicular wall and perform endocrine functions during follicular growth [36]. In contrast to in vivo oocyte maturation, during IVM the COCs are removed from the follicular microenvironment and from the specific endocrine factors secreted by mural somatic cells. These microenvironmental differences lead to decreased nuclear maturation and inadequate cytoplasmic maturation in IVM. Therefore, the CSCs that show similar characteristics with mural somatic cells were isolated and cultured to better simulate the basal microenvironment for oocyte maturation. With respect to simulation of the in vivo microenvironment for IVM, many studies have been conducted on IVM using various somatic cells during porcine IVM [19,37]. Moreover, Abeydeera et al. [23] reported increases in the nuclear maturation rate and relative

Table 4 Effects of the coculture system on sperm penetration of in vitro matured porcine oocytes 10 hours after insemination. Parameter

Feeder cell number (104) 0

2.5

5

10

No. of oocytes No. of penetration (%)a No. of MPN formed (%)b No. of monospermy (%)b No. of polyspermy (%)b No. of 3PN (%) No. of 4PN (%) No. of 5PN (%) Sperm per penetrated Efficiency of fertilizationc

106 93 (87.7  2.7) 78 (83.9  2.9)A 34 (36.6  4.5)A 44 (47.3  7.4) 16 (17.2  2.7) 14 (15.1  2.0) 14 (15.1  3.6) 2.2  2.3A 32.1  3.1A

98 79 (80.6  2.5) 74 (93.7  2.9)B 44 (55.7  4.4)B 30 (38.0  6.0) 14 (17.7  0.5) 10 (12.7  3.1) 6 (7.6  2.5) 1.6  1.0B 44.9  2.1B

99 82 (82.8  3.9) 74 (90.2  3.2)A,B 46 (56.1  6.9)B 28 (34.2  4.6) 15 (18.3  6.4) 9 (11.0  1.2) 4 (4.9  2.5) 1.6  1.5B 46.5  4.5B

100 81 (81.0  2.4) 71 (87.7  2.0)A,B 38 (46.9  6.0)A,B 33 (40.7  6.1) 16 (19.8  3.0) 9 (11.1  0.9) 8 (9.9  1.6) 2.0  1.4A,B 38.0  3.8A,B

Data are given as the mean  SEM from four times replicated experiment. Values with different superscripts (A, B) within a column differ significantly (P < 0.05). Abbreviation: MPN: male pronucleus. a Percentage of the number of oocytes examined. b Percentage of the number of oocytes penetrated. c Efficiency of fertilization was the percentage of monospermic oocytes from total examined.

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Table 5 Effects of the coculture system on embryonic development after IVF. Treatment group (104)

No. of embryos cultureda

0 2.5 5 10

190 185 186 184

Total cell number (n)b

No. (%) of embryos developed to 2 cells 82 111 120 99

Blastocyst

(43.2 (60.0 (64.5 (53.8

   

5.0)A 4.7)B 5.9)B 5.0)A,B

40 66 63 59

(21.1 (35.7 (33.9 (32.1

   

3.8)A 2.9)B 3.6)A,B 2.6)A,B

89.3 127.3 121.7 92.7

   

4.0 7.7 5.5 3.7

(18)A (21)B (19)A,B (17)A

Values with different superscripts (A, B) within a column differ significantly (P < 0.05). a Five times replicated. b Number of examined blastocysts.

intracellular GSH level during IVM using follicular shell pieces in porcine models. In the present study, coculturing COCs with CSCs during IVM did not significantly improve nuclear maturation, although it was increased in

all CSC coculture groups. On completion of IVM, intracellular GSH levels can be used as a marker for cytoplasmic maturation [38]. In matured oocytes, the increased intracellular GSH concentration stimulated MPN formation

Fig. 2. Messenger RNA expression patterns of TGF-b family members, (A) BMP15 and (B) GDF9, in porcine COCs cocultured with CSCs during IVM. The experiment was replicated four times. Values represented by different superscripts (a–c) differ significantly for the respective gene (P < 0.05). BMP15, bone morphogenetic protein 15; COC, cumulus-oocyte complex; CSC, cumulus-derived somatic cell; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; GDF9, growth differentiation factor 9; transforming growth factor-beta (TGF-b). (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

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Fig. 3. The mRNA expression of PCNA, COX-2, Has2, Ptx3, Nrf2, and Cas3 in matured COCs cocultured with CSCs during IVM. The values represent the mean  SEM. Values represented by different superscripts (a, b) differ significantly for the respective gene (P < 0.05). The experiment was replicated four times. Cas3, caspase-3; COC, cumulus-oocyte complex; CSC, cumulus-derived somatic cell; COX-2, cyclooxygenase 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; Has2, hyaluronan synthase 2; Nrf2, nuclear factor erythroid-derived factor 2–related factor 2; PCNA, proliferating cell nuclear antigen; Ptx3, pentraxinrelated protein. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

and improved developmental competence by protecting against oxidative stress [39,40]. Among the different CSC numbers used in the study groups, coculture with 2.5  104 CSCs during IVM significantly increased intracellular GSH levels and may play a role in both cytoplasmic and nuclear maturation.

Although IVF techniques have progressed over several decades, the polyspermy rate is still higher compared with in vivo fertilization. Cytoplasmic maturation is critical for preventing polyspermic penetration in mammals [31]. In this study, the monospermy rate and fertilization efficiency were dramatically increased in cocultures with 2.5  104

Fig. 4. Mean  SEM expression values of PCNA, POU5F1, Caspase-3, Bax, Bak, and Bcl-2 mRNA in (A) IVF blastocysts and (B) PA blastocysts incubated with CSCs during IVM. Values represented by different superscripts (a–c) differ significantly for the respective gene (P < 0.05). The experiment was replicated three times. Bak, Bcl-2 homologous antagonist/killer; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CSC, cumulus-derived somatic cell; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; PCNA, proliferating cell nuclear antigen; POU5F1, POU domain, class 5, transcription factor 1. (For interpretation of the references to color in this Figure, the reader is referred to the web version of this article.)

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and 5.0  104 CSCs compared with the control group. Furthermore, the MPN formation rate was significantly higher in cocultures with 2.5  104 CSCs. As described previously, intracellular GSH levels in matured oocytes influence MPN formation after fertilization [41]. The rates of MPN formation and monospermy were significantly improved; accordingly, the rates of sperm penetration per oocyte were meaningfully reduced in cocultures with 2.5  104 CSCs, potentially affecting additional embryo development. Thus, our findings suggest that coculture with 2.5  104 CSCs during IVM plays a beneficial effect in matured oocytes, which could enhance monospermy and the MPN formation rate. It has been reported that coculture during IVM has a beneficial effect on embryonic development. Chen et al. [37] found that when porcine oocytes were cocultured with porcine ovarian cortex cell monolayers, the percentage of blastocysts formed after fertilization was significantly higher compared with the control group. In the present study, the 2.5  104 CSC group showed not only an increased blastocyst formation rate but also higher cleavage rate and average total blastocyst cell number after IVF. Although there were no significant differences in cleavage rate during the development of PA embryos, the 2.5  104 CSC group showed a significantly higher rate of blastocyst formation and total cell number compared with the control group. The changes in the development and viability of the embryos were highly associated with the mRNA expression patterns of early development and apoptotic genes [42,43]. The present study compared the transcription of six genes, PCNA, POU5F1, Cas3, Bak, Bax, and Bcl-2, in porcine blastocysts derived from IVF and PA. It has been reported that PCNA is an essential component of DNA replication, cell cycle regulation [44], and developmental potential in bovine embryos [45]. The expression of POU5F1 is also an essential marker for early development in humans [46]. The other genes evaluated are involved in environmental stress, which is highly related to apoptosis and lower embryonic viability [47–49]. The mRNA levels of PCNA and POU5F1 were highly increased in IVF blastocysts from the 2.5  104 and 5.0  104 coculture groups. The upregulation of both genes may lead to increase in cleavage and blastocyst formation rate, total cell numbers, and consequently embryonic viability. In PA-derived blastocysts, the mRNA level of PCNA was highly increased from the 2.5  104 coculture group than the control group. Although in difference with IVF experiment the POU5F1 mRNA expression seemed higher in PA-derived blastocysts from the 2.5  104 coculture group, we could not find significance compared with the control group. The upregulation of PCNA gene would lead to correct early embryonic development. In contrast with the control group, 10.0  104 coculture group showed significantly higher mRNA levels of Cas3 and BAK in both IVF- and PA-derived blastocysts. The high expression of proapoptotic genes in the blastocyst could lead to abnormal development and lower embryonic viability [50], despite there being no change in the expression of PCNA and POU5F1. We speculate that the gene expression patterns suggestive of environmental stress and unplanned apoptosis were caused by inappropriate administration of feeder cells and their secreted metabolites during

maturation. In this study, coculture with 2.5  104 CSCs during IVM had an advantageous effect on early embryonic development after IVF and PA by upregulating the expression of PCNA and POU5F1. To understand their association with embryonic development, we measured the mRNA expression of PCNA, COX-2, Has2, Ptx3, Nrf2, and Cas3 in matured COCs using quantitative RT-PCR. Coculture with 2.5  104 and 5.0  104 CSCs showed a nearly twofold upregulation of PCNA in matured COCs, which could be a good parameter for evaluating porcine oocyte quality as described earlier. Only the 2.5 104 coculture group showed significantly higher mRNA expression of Nrf2 in matured COCs compared with the control; Nrf2 is a transcription factor that regulates the expression of several antioxidant enzymes and is involved in cellular protection against oxidative stress [51,52]. Upregulation of Nrf2 in oocytes and cumulus cells might affect the GSH level in matured COCs. On the basis of these findings, Nrf2 expression in oocytes and cumulus cells could be used as a biomarker of intracellular maturation. The expression level of Cas3 was significantly downregulated in 2.5  104 and 5.0  104 coculture groups. The lower expression of such proapoptotic genes in the matured COCs may help to reduce unplanned apoptosis during subsequent embryonic development. The expression levels of COX-2, Has2, and Ptx3 were significantly upregulated in matured COCs derived from 2.5  104 and 5.0  104 coculture groups compared with the control group. The well-known molecular markers of oocyte developmental competence involved in cumulus cell expansion and oocyte maturation initiation are COX-2, Has2, and Ptx3 [53–56]. These findings further suggest that coculture with CSCs has positive effects on the quality of matured COCs through the activation of genes related to cumulus expansion and protection from oxidative stress, which can increase oocyte quality and reduce the expression of genes involved in apoptosis during IVM. The BMP15 and GDF9 genes, which are expressed in oocytes, are downstream of TGF-b superfamily receptor signaling and intracellular cascades activated by SMAD (homologs of both the Drosophila protein, mothers againstdecapentaplegic [MAD] and the Caenorhabditis elegans protein SMA) [57,58], which allow communication between cumulus cells and oocytes and regulate the gene expression during IVM. In the previous study, we analyzed the expression patterns of BMP15 and GDF9 at 0, 11, 22, 33, and 44 hours after the start of maturation, and their mRNA levels showed significant differences at every time point analyzed compared with the control group. Moreover, 33 hours after maturation commenced, BMP15 mRNA levels dramatically recovered and those of GDF9 were significantly higher compared with the control group. During late timing of IVM recovery of BMP15, the mRNA expression level shows a different pattern with general porcine IVM. When mRNA levels of BMP15 and GDF9 were analyzed temporally in porcine COCs during IVM by Li et al. [59], the mRNA levels of BMP15 and GDF9 were correlated with their translated protein levels. Consequently, increased BMP15 and GDF9 mRNA levels on late maturation time could indicate more abundant amount of BMP15 and GDF9 protein expressions than at the same stage of general IVM [60]. The increased mRNA levels of BMP15 and GDF9 in COCs may lead to well

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expansion of the late timming of IVM through induced expression of cumulus expansion-related genes such as COX-1, Has2, and Ptx3 [59,61]. In comparing with the general mRNA expression pattern of BMP15 and GDF9 during IVM, 2.5  104 CSC coculture group partially restored the BMP15 and GDF9 mRNA expression level at 33 hours. A subsequent study by Diaz et al. [55] in mice showed that the increases in COX-2, Has2, and Ptx3 expressions in oocytes required activation of SMAD2/3. Therefore, taking into consideration the mRNA expression patterns observed, we suggest that the SMAD2/3 signaling pathway, which increases maturation-related gene expression and the quality of the induced oocytes, is highly activated during maturation via changes in BMP15 and GDF9 expressions. Over the past decade, many researchers have reported significant insights into oocyte-somatic cell communication. The most convincing model is that the matured oocyte is a fundamental regulator of ovarian somatic cell differentiation and follicular development [13]. In this study, oocyte-somatic cell communication and subsequent embryonic development were shown after IVF and PA using a CSC coculture model of IVM. In conclusion, this coculture system was advantageous for cytoplasmic maturation of porcine oocytes by increasing intracellular GSH levels, upregulating genes involved in developmental competence, and downregulating proapoptotic factors. Furthermore, porcine oocytes from this coculture system showed greater developmental competence, significantly improved monospermic fertilization and fertilization efficiency in in vitro fertilized embryos, and higher developmental competence–related gene expression, blastocyst formation rate, and cell numbers of IVF- and PA-derived blastocysts. These results may have been affected significantly by the upregulation of cumulus cell expansion–related genes such as COX-2, Has2, and Ptx3 during maturation, and these positive effects are predicted by the activation of SMAD2/3, as suggested by changes in the expression of BMP15 and GDF9. Although further molecular studies are required to verify this model, these findings will benefit the early development of porcine embryos by increasing the quality of cytoplasmic maturation in oocytes. Therefore, these results suggest that coculture with optimized number of feeder cells during IVM improves the quality of porcine oocytes and subsequent early embryonic development. Acknowledgments This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (no. PJ0095692014), Rural Development Administration, and the National Research Foundation of Korea grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1 A2A2A04008751), Republic of Korea. References [1] Yu Y, Wang Y, Tong Q, Liu X, Su F, Quan F, et al. A site-specific recombinase-based method to produce antibiotic selectable marker free transgenic cattle. PLoS One 2013;8:e62457. [2] Zhang P, Liu P, Dou H, Chen L, Lin L, Tan P, et al. Handmade cloned transgenic sheep rich in omega-3 fatty acids. PLoS One 2013;8: e55941.

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Effects of coculture with cumulus-derived somatic cells on in vitro maturation of porcine oocytes.

In the process of IVM, cumulus-oocyte complexes (COCs) separate from the follicular microenvironment, leading to the loss of endocrine interactions be...
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