Ethics statement

All procedures and use of pigs were approved by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee (Approval No. KRIBB-AEC-16075).

Chemicals

All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise indicated.

Oocyte collection and in vitro maturation (IVM)

Porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory in 0.9% saline supplemented with 75 μg/mL potassium penicillin G and 50 μg/mL streptomycin sulfate and maintained at 25–30°C. Intact cumulus oocyte complexes (COCs) were aspirated from follicles (3–8 mm in diameter) using a disposable 10-mL syringe with an 18-gauge needle. Aspirated COCs with at least three layers of compact cumulus cells and homogeneous cytoplasm were washed three times in low-carbonate tyrode’s albumin lactate pyruvate-HEPES (TL-HEPES) medium [36] containing 1 mg/mL polyvinyl alcohol (PVA). Approximately 70 oocytes were matured in 500 μL of the IVM medium in a four-well multi-dish (Nunc, Roskilde, Denmark) at 38.5°C in 5% CO₂ in air. Tissue culture medium 199 (TCM-199) was used during the 44-h maturation. During the first period of maturation, 0–22 h, the medium was supplemented with 10% porcine follicular fluid (PFF), 0.57 mM cysteine, 25 μM β-mercaptoethanol, 10 ng/mL epidermal growth factor (EGF), 10 IU/mL pregnant mare serum gonadotropin (PMSG) and 10 IU/mL human chorionic gonadotropin (hCG). During the second stage (22–44 h), the same media was used without hormones. After IVM, the COCs were exposed briefly to 0.1% hyaluronidase and gently aspirated using a small bore glass pipette to remove the cumulus cells. Only oocytes with a visible polar body, regular morphology and homogenous cytoplasm were used.

Parthenogenetic activation (PA)

Matured oocytes were placed on a 1-mm gab wire chamber (CUY5000P1, Nepagene, Chiba, Japan) overlaid with 10 μL 280 mM mannitol solution containing 0.1 mM MgSO₄∙7H₂O, 0.1 mM CaCl₂∙2H₂O, 0.5 mM HEPES, 0.01% PVA, as described previously [37]. Immediately, oocytes were activated with 110 V DC for 50 μsec using an electro cell fusion generator (LF101, Nepagene). Approximately activated oocytes were transferred into the activation medium, IVC medium [porcine zygote medium-3 (PZM-3) medium containing 0.4% BSA] supplemented with 5 μg/mL cytochalasin B (CB) and 2 mM 6-dimethylaminopurine (6-DMAP) for 4 h at 38.5°C in 5% CO₂ in air. After 4 h, the oocytes were washed in IVC medium and transferred to fresh IVC medium at 38.5°C in 5% CO₂ in air. Cleavage and blastocyst formation were evaluated at days 2 and 6, respectively.

In vitro fertilization (IVF)

In vitro fertilization (IVF) of porcine oocytes was performed as described previously [29]. IVF was performed in modified Tris-buffered medium (mTBM) consisting of 113.1 mM NaCl, 3 mM KCl, 7.5 mM CaCl₂∙2H₂O, 20 mM Tris (crystallized free base; Fisher Scientific, Waltham, MA, USA), 11 mM glucose and 5 mM sodium pyruvate. Ejaculated fresh semen from swine was washed three times by centrifugation (100 g for 3 min at room temperature) with Dulbecco’s phosphate-buffered saline (DPBS; Gibco-BRL, NY, USA) supplemented with 1 mg/mL BSA, 100 μg/mL penicillin G and 75 μg/mL streptomycin sulfate. After the washing procedure was complete, the spermatozoa were re-suspended in mTBM at pH 7.8. The oocytes were washed three times in mTBM with 2.5 mM caffeine sodium benzoate and 1 mg/mL BSA and placed into 48 μL mTBM under mineral oil. Next, 2 μL diluted spermatozoa was added to 48 μL mTBM containing 10–15 oocytes, yielding a final concentration of 1.5ⅹ10⁵ spermatozoa/mL. The oocytes were co-incubated with the spermatozoa for 6 h at 38.5°C in an atmosphere of 5% CO₂ in air. After 6 h, the oocytes were stripped by gentle pipetting and transferred to IVC medium. The IVF embryos were washed three times in fresh IVC medium, transferred into 40 μL IVC droplets under mineral oil and then stored at 38.5°C and 5% CO₂ in air for 6 days. Cleavage and blastocyst formation were evaluated at days 2 and 6, respectively.

Preparation of donor cells

Donor cells were obtained from cultured minced porcine kidney tissue. The kidney was obtained from a neonatal pig (2 days old, male) by surgical operation. Harvest kidney tissues were stored in DPBS washing buffer containing 10% (v/v) penicillin/streptomycin (Pen/Strep; Invitrogen, Carlsbad, CA, USA) on ice until isolation. Small pieces of kidney tissue were placed in 60-mm culture dishes and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; 16000–044, GIBCO, Carlsbad, CA, USA), 10 ng/mL basic fibroblast growth factor (bFGF) and 1% penicillin/streptomycin (Invitrogen) at 38.5°C in 5% CO₂ in air. These porcine kidney-derived fibroblasts were used at passages 4–6. The cells were synchronized at the G0-G1 phase of the cell cycle by an additional 3 days of culture, without changing the medium, after reaching confluency.

Somatic cell nuclear transfer (SCNT)

After IVM, matured oocytes with a visible first polar body were selected for somatic cell nuclear transfer (SCNT). Cumulus-free oocytes in DPBS supplemented with 4 mg/mL BSA, 75 μg/mL penicillin G, 50 μg/mL streptomycin sulfate and containing 7.5 μg/mL CB were penetrated by cutting the pipette to make a slit and then squeezing approximately 10% of the cytoplasm and the first polar body out of the oocytes under an automated inverted microscope (DMI 6000B, Leica, Wetzlar, Germany) equipped with a micromanipulator (NT-88-V3, Nikon-Narishige, Tokyo, Japan). Porcine kidney cells that were round, 15–20 μm in diameter and had good refractivity were selected and placed into the perivitelline space through the near slit in the zona pellucida made during enucleation. The oocyte-cell couplets were maintained in IVC medium at 38.5°C in 5% CO₂ in air until electrical fusion. A single cell-oocyte couplet was equilibrated in fusion medium consisting of 280 mM mannitol containing 0.1 mM MgSO₄∙7H₂O and 0.01% PVA [37], then sandwiched between two parallel electrodes (100-μm diameter; CUY 5100–100, Nepagene) attached to the micromanipulator, rotated to the contact surface between the cytoplasm and the donor cell perpendicular to the electrodes and activated by one direct current pulse of 23 V for 50 μsec using an electro cell fusion generator and incubated at 38.5°C in 5% CO₂ in air. After 2 h, oocyte–cell couplets that were completely fused, as observed under an automated inverted microscope, were selected, then transferred to a 1-mm gab wire chamber overlaid with 10 μL 280 mM mannitol solution containing 0.1 mM MgSO₄∙7H₂O, 0.1 mM CaCl₂∙2H₂O, 0.5 mM HEPES and 0.01% PVA and activated with 110 V DC for 50 μsec using an electro cell fusion generator. The electro-activated oocytes were transferred into the chemically assisted activation medium supplemented with 50 nM trichostatin A (TSA) for 4 h at 38.5°C in 5% CO₂ in air. After 4 h, activated embryos were transferred to IVC medium supplemented with 50 nM TSA and cultured for 20 h. After 20 h, SCNT embryos were washed in IVC medium and transferred to IVC medium at 38.5°C in 5% CO₂ in air. Cleavage and blastocyst formation were evaluated at days 2 and 6, respectively.

Chemical treatment

To investigate the effects of FBS (GIBCO) on early embryogenesis and the related signaling pathways, the presumed zygotes were cultured in IVC medium supplemented with 10% FBS in the presence or absence of glutathione (GSH; 0.5 and 1 mM), hydrogen peroxide (H₂O₂; 0.1 and 0.5 mM), SB203580 [p38 mitogen-activated protein kinase (MAPK) inhibitor; 10 μM] or LY294002 (AKT inhibitor; 10 μM) for various culture periods (absence, day 0; entire period, days 0–6 of IVC; early phase, days 0–1 or 0–2 of IVC; late phase, days 4–6 or 5–6 of IVC). All chemicals were provided as lyophilized powders and dissolved in porcine zygote culture medium or dimethyl sulfoxide. The respective diluents were used as negative controls in each experiment.

Terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL) assay

To detect apoptotic cells in the blastocysts, the terminal deoxynucleotidyl transferase-mediated dUTP-digoxygenin nick end-labeling (TUNEL) assay was conducted using an In Situ Cell Death Detection Kit (Roche, Basel, Swiss). Blastocysts on day 6 were washed three times with 0.1% PVA in (PBS-PVA) and fixed in 4% (v/v) paraformaldehyde diluted in PBS for 1 h at room temperature. For membrane permeabilization, the fixed embryos were incubated in PBS containing 1% (v/v) Triton X-100 for 1 h at room temperature. Subsequently, the embryos were washed three times in PBS-PVA and stained with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase for 1 h at 38.5°C. As a positive control for the TUNEL reaction, a group of blastocysts was incubated in 1000 U/mL deoxyribonuclease at 38.5°C for 10 min, followed by TUNEL staining. Another group of blastocysts was incubated in fluorescein-dUTP in the absence of terminal deoxynucleotidyl transferase as a negative control. Subsequently, the blastocysts were washed three times in PBS-PVA and mounted on slides with a mounting solution containing 1.5 g/mL 4, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA). DAPI-labeled or TUNEL-positive nuclei were observed under a fluorescence microscope, and the number of apoptotic nuclei and total number of nuclei were counted. These experiments were performed in triplicate with 10–15 blastocysts per group for each experiment.

Differential staining

Blastocysts were fixed in 4% paraformaldehyde for 40 min and washed three times in PBS-PVA for 10 min each. For membrane permeabilization, the fixed blastocysts were incubated in PBS containing 0.2% (v/v) Triton X-100 for 40 min at room temperature. Subsequently, blastocysts were washed three times in PBS-PVA and stored in PBS-PVA supplemented with 1% BSA (PBS-PVA-BSA) at 4°C overnight. The blastocysts were blocked with 10% normal goat serum (NGS) for 45 min and then incubated overnight at 4°C with primary antibody, mouse monoclonal anti-Cdx2 (an undiluted solution; Biogenex Laboratories Inc., San Ramon, CA, USA). Subsequently, the blastocysts were washed three times in PBS-PVA-BSA for 10 min each and incubated for 40 min at room temperature with conjugated secondary antibodies, Alexa-Fluor-488-labeled goat anti-mouse IgG (1:200 in PBS-PVA-BSA). After the blastocysts were washed three times in PBS-PVA-BSA for 10 min each, the DNA was stained with 2 μg/mL DAPI. DAPI-labeled or Cdx2-positive nuclei were observed using a fluorescence microscope (Olympus, Tokyo, Japan).

Measurement of intracellular ROS

Measurement of ROS levels in embryos was performed as described previously [38]. 5-(and-6)-chloromethyl-2ʹ,7ʹ-dichlorodihydro-fluorescein diacetate, acetyl ester (CM-H₂DCFDA; Invitrogen) was used to detect intracellular ROS as green fluorescence. At least 10 embryos from each treatment group were incubated for 20 min in a solution of TL-HEPES-PVA mixed with 5 μM CM-H₂DCFDA. After incubation, embryos were washed with DPBS containing 0.1% (w/v) PVA and aliquoted into 10 μL droplets; fluorescence was observed under a fluorescence microscope (DMI 4000B; Leica) with ultraviolet filters (460 nm for ROS). Fluorescent images were saved as graphic files in tagged image file format. The fluorescence intensities of the embryos were analyzed using Image J software (version 1.47; National Institute of Health, Bethesda, MD, USA) and normalized to those of control embryos.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Poly(A) mRNAs were extracted from 10 blastocysts using the Dynabeads mRNA Direct kit (Invitrogen Dynal AS, Oslo, Norway) according to the manufacturer’s protocol. Briefly, after thawing, samples were lysed in 300 μL lysis/binding buffer at room temperature for 10 min, and 10 μL Dynabeads oligo(dT)₂₅ were added to each sample. The beads were hybridized for 5 min and then separated from the binding buffer using a Dynal magnetic bar (Invitrogen). Bound poly(A) mRNAs and beads were washed with buffers A and B and then separated by adding 10 μL Tris buffer. The resulting poly(A) mRNAs were reverse-transcribed in 20 μL reactions containing oligo(dT)₂₀, 5ⅹRT buffer (containing 25 mM Mg²⁺), 10 U of the RNase inhibitor ReverTra Ace (Toyobo, Osaka, Japan) and a 10 mM mixture of dNTPs. Secondary RNA structure was denatured by incubating at 42°C for 60 min to facilitate cDNA production. The reaction was terminated by incubation at 99°C for 5 min. The resulting cDNA was used as a template for PCR amplification. The following PCR conditions were used: 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, followed by extension at 72°C for 5 min. The Mx3000P QPCR system (Agilent, Santa Clara, CA, USA) and SYBR premix Ex Taq (Takara Bio Inc., Shiga, Japan) were used for quantitative real-time polymerase chain reaction (qRT-PCR). The threshold cycle (Ct) is defined as the fractional cycle number at which the fluorescence passes a fixed threshold above baseline. For the comparative analyses, mRNA expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are expressed as the fold change. The sample delta Ct (^SΔCT) value was calculated from the difference between the Ct values of GAPDH and the target genes. The relative gene expression levels between the samples and the controls were determined using the formula 2⁻(^{SΔCT−CΔCT}). The primers used in the current study are listed in S1 Table.

Western blot analysis

Twenty embryos in each group were washed twice with PBS and lysed in 20 μl lysis buffer (20 mM HEPES, 150 mM NaCl, 2 mM EGTA, 1 mM EDTA, 20 mM glycerol phosphate, 1% Triton X-100 and 10% glycerol) containing protease inhibitor cocktail for 1 h subsequently boiled at 100°C for 5 min. The protein samples were separated on a 10% SDS-polyacrylamide gel electrophoresis gel (iNtRON Biotechnology, Gyeonggi-do, Korea) and transferred to a Polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, Canada). The membrane was blocked in Tris-buffered saline (TBS) containing 0.25% Tween 20 (TBST) and 5% BSA for 1 h, rinsed in TBST, and probed with an anti-phospho-p38-mitogen-activated protein kinase (MAPK) [phospho-p38 (Thr180/Tyr182), 1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA], anti-p38-MAPK (1:1000 dilution; Cell Signaling Technology), anti-phospho-AKT [p-AKT (ser473) 1:1000 dilution; Cell Signaling Technology] and AKT (1:1000 dilution; Cell Signaling Technology), anti- antibodies at 4°C overnight. The blot was washed with TBST and subsequently incubated with horseradish peroxidase-conjugated secondary antibody. The membranes were visualized using enhanced chemiluminescence detection reagent (Thermo Fisher scientific, Walltham, MA, USA) according to the manufacturer’s instructions.

Statistical analyses

All experiments were repeated at least three times, and data are presented as the means ± standard error (SE). Differences in the mean percentages of blastocysts and hatching blastocysts among the treatments were compared using factorial ANOVA, followed by Duncan’s multiple range tests using sigma stat software (Systat Software Inc., San Jose, CA, USA). P-values less than 0.05 were considered statistically significant.

Supplementation with FBS during late IVC improves developmental competence in porcine PA embryos

To study the effect of FBS on the early development of porcine embryos, PA embryos were cultured in IVC medium supplemented with 10% FBS for various culture periods (absence, day 0; entire period, days 0–6 of IVC; early phase, days 0–1 or 0–2 of IVC; late phase, days 4–6 or 5–6 of IVC), and the blastocyst development rate and total cell number were assessed after 6 days of IVC. In particular, blastocyst development rate and total cell number were the lowest in the entire culture period treatment group, whereas both parameters were most notably increased in the 4–6 day treatment group (Fig 1 and S2 Table). Consistent with these results, the proportion of hatched or hatching blastocysts was markedly increased by treatment with FBS during the late phase, especially for 4–6 days, compared to the other groups (S1 Fig and S3 Table).

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Fig 1. Effect of FBS supplementation timing on the developmental competence of porcine PA embryos.

(A) Representative photographs of blastocysts (white asterisks) developed in the presence or absence of FBS during the entire culture period (0–6 days), early phase (0–1 and 0–2 days) and late phase (4–6 and 5–6 days) of IVC. Bar = 50 μm. (B) Quantification of the blastocyst developmental rate in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (C and D) DAPI staining for scoring the number of cells in blastocysts of the indicated groups (C) and quantification of the total cell number (D). Bar = 50 μm (C). The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05; D).

https://doi.org/10.1371/journal.pone.0175427.g001

The effect of ROS levels in early phase treatment of FBS during early embryogenesis of porcine PA embryos

To investigate ROS generation during whole embryo developmental period, PA embryos were cultured in IVC medium. ROS levels were continuously increased during early embryogenesis, whereas the gradual decreases in antioxidant enzyme transcripts, including SOD1, GPx1, Prdx2, and Catalase, were found (S2 Fig), indicating possible role of the antioxidant enzymes as embryonic ROS scavengers. To investigate the relationship between FBS supplementation and ROS levels during early stage embryo development, PA embryos were cultured in the presence or absence of FBS for 0–2 days of IVC, and ROS reduction was determined using CM-H₂DCFDA and subjected to fluorescence microscopy (Fig 2A). As observed in the FBS treatment group, the blastocyst developmental rate and total cell number were significantly decreased relative to the control group (Fig 2B and S4 Table). Consistent with this, the embryonic ROS level was successfully decreased by adding 1 mM GSH, which led to significant decreases in cleavage rate, blastocyst development rate, TE cell number, and cellular survival compared with controls (S3 Fig, S5 and S6 Tables). However, little or no changes concerning ROS levels and blastocyst development rate were found following treatment with low GSH concentrations, such as 0.1, 0.2 and 0.5 mM (data not shown). In contrast, the decreases in cleavage and blastocyst developmental rates were significantly restored by the addition of hydrogen peroxide (S4 Fig and S7 Table). Consistent with, elevation of ROS levels by the addition of hydrogen peroxide in the FBS treatment group restored the blastocyst formation and proportion of TE cells (Fig 2C–2G, S8 and S9 Tables). Similarly, TUNEL analysis of blastocyst showed that the proportion of apoptotic cells significantly decreased in the presence of FBS and hydrogen peroxide to compare the control and only FBS groups (Fig 2H and 2I and S9 Table).

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Fig 2. Involvement of ROS in FBS-associated phenotypes during early IVC phase of PA embryos.

(A) Fluorescence microscopy of 4- to 8-cell embryos treated with CM-H₂DCFDA after treatment with FBS for 48 h (left panel) and quantification of ROS levels (right panel) in the indicated groups. Bar = 100 μm. (B) Representative photographs of the blastocysts (left panel; white asterisks) and quantification of the blastocyst developmental rate (center panel) and total cell number (right panel) within the indicated groups. Bar = 100 μm. (C) Fluorescence microscopy of 1-cell embryos treated with CM-H₂DCFDA after 6 h of cultivation in the presence or absence of either FBS and/or 0.5 mM hydrogen peroxide (left panel) and quantification of ROS levels (right panel) within the indicated groups. Bar = 100 μm. (D) Representative photographs of the developed blastocysts (left panel; white asterisks) and quantification of the blastocyst developmental rate within the indicated groups. Bar = 100 μm. (E–G) Immunocytochemical analysis of Cdx2/DAPI using blastocysts developed under the indicated IVC conditions for 0–2 days of IVC (E) and quantification of the total cell number (F) and ICM/TE proportions (G) in the indicated groups. Merged images (light green) between Cdx2 (green) and DAPI (blue) signals are shown (E). Bar = 100 μm (E). The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05; F and G). (H and I) Apoptosis detection analysis using blastocysts developed under the indicated IVC conditions for 0–2 days (H), and quantification of the number and proportion of apoptotic cells in the indicated groups (I). Merged images (light green) between TUNEL (green, white arrow) and DAPI (blue) signals are shown (H). Bar = 50 μm (H). The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05; I).

https://doi.org/10.1371/journal.pone.0175427.g002

The effect of ROS levels in late phase treatment of FBS during early embryogenesis of porcine PA embryos

To investigate the effect of FBS on the late phase development of porcine embryos, PA embryos were cultured in IVC medium supplemented with 10% FBS for 4–6 day. In particular, blastocyst development rate and total cell number were most notably increased in the 4–6 day FBS treatment group (Fig 3A and 3B and S10 Table) Based on qRT-PCR analysis, the transcript levels of antioxidant enzymes, including SOD1, GPx1, Catalase and Prdx2, were significantly upregulated by the addition of FBS during the late IVC phase compared to control (Fig 3C).

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Fig 3. Effects of FBS supplementation during late IVC phase on the developmental competence of PA embryos.

(A) Bright field (top of left panel) and fluorescence (bottom of left panel) images of blastocysts treated with CM-H₂DCFDA in the indicated groups (left panel) and quantification of ROS levels in the indicated groups (right panel). Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (B) Representative photographs of the developed blastocysts (white asterisks) and quantification of the blastocyst development rate (top of right panel) and total cell number (bottom of right panel) in the indicated groups. Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (C) qRT-PCR analysis of the relative abundances of antioxidant genes SOD1, GPx1, Catalase and Prdx2 in blastocysts from the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (D) Immunocytochemical analysis of Cdx2 using blastocysts cultured in the presence or absence of FBS for 4–6 days of IVC. Merged images (light green) between Cdx2 (green) and DAPI (blue) signals are shown. Bar = 50 μm. (E) Quantification of the total cell number (top panel) and TE/ICM proportions (bottom panel) in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (F) qRT-PCR analysis of the relative abundances of Oct4 and Cdx2 transcript levels in blastocysts from the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (G) Apoptosis detection analysis in blastocysts from the indicated groups. Merged images (light green) between TUNEL (green, white arrow) and DAPI (blue) signals are shown. Bar = 50 μm. (H) Quantification of the number (top panel) and proportion (bottom panel) of apoptotic cells in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (I) qRT-PCR analysis of the relative abundance of Bax and Bcl-xL transcript levels in blastocysts from the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05).

https://doi.org/10.1371/journal.pone.0175427.g003

To clarify the beneficial role of FBS during the late IVC phase of early embryogenesis, porcine PA embryos were cultured in the presence or absence of 10% FBS for 4–6 days of IVC, and the ICM/TE proportion and apoptotic cell number were scored based on immunocytochemical analyses of Cdx2 and TUNEL, respectively, using blastocysts from each group. Compared to control, the ICM proportion was decreased markedly in the FBS treatment group, whereas the TE proportion was increased markedly (Fig 3D and 3E and S11 Table). Similarly, the Cdx2 transcript level was significantly upregulated with FBS treatment compared to control (Fig 3F). Moreover, the number and proportion of apoptotic cells were notably decreased in the FBS treatment group (Fig 3G and 3H and S11 Table). Similarly, reduction of the pro-apoptotic Bax transcript level and elevation of the anti-apoptotic Bcl-xL transcript level were detected in the FBS treatment group compared to control (Fig 3I).

To examine the effect of oxidant treatment during the late IVC phase on early embryogenesis. PA embryos were cultured in the presence or absence of FBS or hydrogen peroxide for 4–6 days. Unlike the early IVC phase, elevation of ROS levels due to the addition of hydrogen peroxide for 4–6 days of IVC significantly decreased the blastocyst development rate in a dose-dependent manner (Fig 4A and 4B and S12 Table). In addition, the detrimental effects of hydrogen peroxide treatment for 4–6 days of IVC on blastocyst development rate, TE proportion and cellular survival were greatly ameliorated by co-treatment with FBS (Fig 4C–4G, S13 and S14 Tables). Interestingly, the transcript levels of antioxidant enzymes, including SOD1, GPx1 and Catalase, were upregulated by FBS treatment (Fig 4H).

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Fig 4. Involvement of ROS in FBS-associated phenotypes during late IVC phase of PA embryos.

(A) Fluorescence microscopy of blastocyst treated with CM-H₂DCFDA after cultivation in the presence or absence of hydrogen peroxide (0.1 and 0.5 mM) for 4–6 days of IVC (bottom of left panel) and bright field (BF; top of left panel) and quantification of ROS generation (right panel) within the indicated groups. (B) Representative photographs of the developed blastocysts (white asterisks; left panel) and quantification of blastocyst developmental rate (right panel) within the indicated groups. Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (C) Fluorescence microscopy of blastocysts treated with CM-H₂DCFDA after cultivation in the presence or absence of FBS and/or 0.5 mM hydrogen peroxide for 4–6 days of IVC (top of left panel) and bright field (BF; top of left panel) and quantification of ROS generation (right panel) within the indicated groups. (D) Representative photographs of the developed blastocysts (white asterisks; left panel) and quantification of blastocyst rate (right panel). The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (E-G) Immunocytochemical analysis of Cdx2 (left panel) and TUNEL staining (right panel). Bar = 50 μm. Quantification of the total cell number (F; left panel) ICM/TE proportion (F; right panel) and proportion of apoptotic cells (G) and in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (H) qRT-PCR analysis of the relative abundance of antioxidant genes SOD1, GPx1, Catalase and Prdx2 in blastocysts from the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05).

https://doi.org/10.1371/journal.pone.0175427.g004

Beneficial role of FBS in late IVC phase involves p38 MAPK and AKT cascades

Based on the previous evidences concerning the close association of developmental competence with activation of p38 MAPK and AKT signaling pathways [39, 40], we investigated whether FBS supplementation in late IVC phase modulated p-p38 MAPK and p-AKT levels in PA blastocysts. Compared to control, the levels of p-p38 MAPK and p-AKT in PA blastocysts were markedly increased in FBS treatment group (Fig 5A). Interestingly, addition of either p38 MAPK inhibitor SB203580 (10 μM) or AKT inhibitor LY294002 (10 μM) significantly decreased the blastocyst development rate and TE proportion (Fig 5B and 5C, S15 and S16 Tables). However, no changes in proportion of apoptotic cells were found in the inhibitor treatment groups compared to FBS treatment group (Fig 5D and S16 Table).

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Fig 5. Involvement of p-p38 MAPK and p-AKT cascades in FBS-associated phenotypes during late IVC phase.

(A) Western blot analysis of p-p38 MAPK and p-AKT in control and FBS supplementation during late phase in porcine PA embryos. (B) Representative photographs of the developed blastocysts (white asterisks; left panel) and quantification of blastocyst developmental rate (right panel) in the indicated groups. Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (*P < 0.05). (C) Immunocytochemical analysis of Cdx2/DAPI. Merged images (light green) between Cdx2 (green) and DAPI (blue) signals are shown (top panel) and quantification of the total cell number (bottom left panel) and ICM/TE proportion (bottom right panel) in the indicated groups. Bar = 50 μm. (D) Apoptosis detection analysis in blastocysts from the indicated groups. Merged images (light green) between TUNEL (green, white arrow) and DAPI (blue) signals are shown (top panel) and quantification of apoptotic cells (bottom panel). Bar = 50 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05).

https://doi.org/10.1371/journal.pone.0175427.g005

Treatment with FBS during the late IVC phase supports early embryogenesis of IVF and SCNT embryos

Next, we investigated whether FBS supplementation for 4–6 days of IVC continues to benefit early embryogenesis of IVF and SCNT embryos. ROS levels were greatly reduced by FBS treatment (Fig 6A), leading to improved blastocyst development and hatching rates (Fig 6B and S17 Table). In addition, the transcript levels of antioxidant enzymes, including SOD1, GPx1 and Catalase, were relatively high in the FBS treatment group compared to control (Fig 6C). Consistent with these results, the proportion of TE cells and cellular survival were significantly increased with FBS treatment (Fig 6D–6F and S18 Table).

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Fig 6. Beneficial effect of FBS during late IVC phase on developmental competence of porcine IVF embryos.

(A) Bright field (top panel) and fluorescence (bottom panel) images of blastocysts treated with CM-H₂DCFDA after cultivation in the presence or absence of FBS for 4–6 days of IVC (left panel) and quantification of ROS levels in the indicated groups (right panel). Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (B) Representative photographs of the blastocysts (white asterisks; left panel) developed in the presence or absence of FBS for 4–6 days of IVC after IVF and quantification of blastocyst developmental (center panel) and hatching (right panel) rates in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (C) qRT-PCR analysis of the relative abundances of antioxidant genes SOD1, GPx1, Catalase and Prdx2 in blastocysts from the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (D) Immunocytochemistry of Cdx2 (top panel) and TUNEL staining (bottom panel) in blastocysts from the indicated groups. Bar = 50 μm. Merged images (light green color) between green (Cdx2, top panel; TUNEL, bottom panel) and blue (DAPI; both panels) signals are shown. (E and F) Quantification of ICM/TE (E) and apoptotic cell (F) number and proportion in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05).

https://doi.org/10.1371/journal.pone.0175427.g006

To further examine the effect of FBS supplementation during the late phase of early embryogenesis, SCNT embryos were cultured in the presence or absence of FBS for 4–6 days of IVC. In agreement with other results of this study, FBS treatment led to reduced ROS levels (Fig 7A) as well as an increased rate of blastocyst development (Fig 7B and S19 Table). TE cell proportion and cellular survival in SCNT blastocysts were also improved by the addition of FBS during the late IVC phase (Fig 7C–7E and S20 Table).

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Fig 7. Beneficial effect of FBS during late IVC phase on developmental competence of porcine SCNT embryos.

(A) Bright field (top of left panel) and fluorescence (bottom of left panel) images of SCNT blastocysts treated with CM-H₂DCFDA after cultivation in the presence or absence of FBS for 4–6 days of IVC (left panel) and quantification of ROS levels in the indicated groups (right panel). Bar = 100 μm. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (B) Representative photographs of the blastocysts (white asterisks) developed in the presence or absence of FBS for 4–6 days of IVC after SCNT (left panel) and quantification of the blastocyst developmental rate in the indicated groups (right panel). The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05). (C) Immunocytochemical analysis of Cdx2 (top panel) and TUNEL staining (bottom panel) in SCNT blastocysts from the indicated groups. Merged images (light green color) between green (Cdx2, top panel; TUNEL, bottom panel) and blue (DAPI; both panels) signals are shown. Bar = 50 μm. (D and E) Quantification of ICM/TE (D) and apoptotic cell (E) number and proportion in the indicated groups. The data are from three independent experiments, and values represent the means ± SE (^*P < 0.05).

https://doi.org/10.1371/journal.pone.0175427.g007