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L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression
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Theriogenology 78 (2012) 235–243 www.theriojournal.com
L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression Jinyoung Youa,1, Joohyeong Leea,1, Sang-Hwan Hyunb, Eunsong Leea,c,* b
a College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea c Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea
Received 22 September 2011; received in revised form 3 January 2012; accepted 15 February 2012
Abstract The objective of this study was to examine the effect of L-carnitine treatment during in vitro maturation (IVM) of immature pig (Sus scrofa) oocytes. Specifically, the effects of L-carnitine treatment on nuclear maturation and oocyte intracellular glutathione (GSH) levels, embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT pig embryos were determined. During IVM culture, immature oocytes were either treated or not treated with 10 mM L-carnitine. L-carnitine treatment did not improve the nuclear maturation of oocytes but significantly increased intracellular GSH levels, which led to a reduction of reactive oxygen species (ROS) levels in IVM oocytes. Oocytes treated with L-carnitine showed higher (P ⬍ 0.05) rates of blastocyst formation after PA (39.4% vs. 27.1%) and SCNT (23.2% vs. 14.9%) compared with untreated oocytes. SCNT embryos that were derived from L-carnitine-treated oocytes showed increased (P ⬍ 0.05) expression levels of DNMT1, PCNA, FGFR2, and POU5F1 mRNA compared with control embryos. Treatment of recipient oocytes with L-carnitine increased (P ⬍ 0.05) the expression of both BAX and p-Bcl-xl mRNA in SCNT blastocysts. However, the increase was more prominent in BAX than in p-Bcl-xl mRNA. Our results demonstrate that L-carnitine treatment during IVM improves the developmental competence of SCNT embryos. This effect is probably due to increased intracellular GSH synthesis in recipient ooplasts, which reduces ROS levels, and the stimulation of nuclear reprogramming via increased expression of POU5F1 and transcription factors. © 2012 Elsevier Inc. All rights reserved. Keywords: L-carnitine; Gene expression; Nuclear transfer; Oocyte maturation; Pig
1. Introduction Recent progress in reproductive technologies, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer (SCNT) 1
These authors contributed equally to this work.
* Corresponding author. Tel.: ⫹82 33 250 8670; fax: ⫹82 33 244 2367. E-mail address: [email protected] (E. Lee). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2012.02.027
has made it possible to produce specific animals successfully in a variety of species [1–3]. The efficiency of reproductive technologies, especially SCNT, is influenced by various factors, including the quality of oocytes, culture conditions, cell cycle stage of the donor cell, and oocyte activation methods [4,5]. Of those, the quality of oocytes is one of the most critical factors for determining the in vivo viability and in vitro developmental competence of in vitro-produced embryos. Oocytes or embryos are inevitably exposed to unde-
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sirable environments during in vitro production. Reactive oxygen species (ROS) can be generated by handling or culturing oocytes or embryos in a high-oxygen atmosphere, and artificial treatments, such as electric stimulus for cell fusion or activation of SCNT oocytes, are known to increase intracellular ROS levels [6,7]. It is well-understood that a high level of ROS causes cell membrane lipid peroxidation [8,9] and DNA fragmentation, and also influences RNA transcription and protein synthesis [10]. These activities lead to in vitro developmental blocks and early embryonic death [9,11]. Recent studies [12,13] on oocyte maturation have shown that modifications of a culture system for the in vitro maturation (IVM) of oocytes can improve cytoplasmic maturation. This stimulates embryonic development by increasing intracellular glutathione (GSH) levels in IVM oocytes, which reduces ROS activity during the culturing of embryos. Thus, it is crucial to establish a stable IVM system to produce mature oocytes of a higher quality to increase in vitro production efficiency. Previously, supplementation of IVM medium with antioxidants, such as -mercaptoethanol, cysteine, and cysteamine, has been demonstrated to stimulate the synthesis of intracellular GSH, which in turn plays an antioxidative role and enhances viability of IVF and ICSI embryos [14,15]. L-carnitine (-hydroxy-␥-trimethylammoniumbutyric acid), an antioxidative agent, is known to have a beneficial role in cellular metabolism and embryonic development in mammalian species. L-carnitine protects cell membranes and DNA from damage induced by oxygen free radicals [16]. When mouse metaphase II (MII) oocytes and 8-cell embryos were incubated in a medium supplemented with L-carnitine (0.6 mg/mL), a significant improvement in the integrity of microtubule and chromosome structural integrity and a decreased level of apoptosis were observed [17]. In addition, supplementation of culture medium with 0.3 mg/mL L-carnitine improved blastocyst formation in mice by reducing the blocking effects of actinomycin-D, hydrogen peroxide, and tumor necrosis factor-␣ on embryonic development and decreasing levels of DNA damage [18]. In bovine, L-carnitine has also been demonstrated to improve embryonic development in vitro by exhibiting an extensive relocation of active mitochondria to the inner oocyte cytoplasm [19]. Many studies have been performed in various species to determine the beneficial effect of L-carnitine on in vitro embryonic development, but there is limited information available in pigs on the effect of L-carnitine on oocyte maturation and subsequent embryonic development. In this study, we examined the effect of
L-carnitine treatment during the oocyte maturation process on the developmental competence of parthenogenetic and SCNT embryos. We did this by studying the nuclear maturation of oocytes, intracellular levels of GSH and ROS in IVM oocytes, embryonic cleavage, and blastocyst formation. In addition, expression levels of several genes (DNMT1, ERK2, PCNA, FGFR2, and POU5F1) and apoptosis-related genes (BAX and p-Bclxl) were analyzed in SCNT pig embryos. Our findings demonstrate that L-carnitine treatment during oocyte maturation improves SCNT embryonic development. This probably occurs through increasing the intracellular GSH level of oocytes, which leads to the inhibition of ROS activity and stimulates expression of POU5F1 and transcription factor genes during nuclear reprogramming in SCNT pig embryos. 2. Materials and methods 2.1. Culture media All chemicals used in this study were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA), unless otherwise stated. The medium for IVM was Tissue Culture Medium-199 (M-199; Invitrogen, Grand Island, NY, USA) supplemented with 0.6 mM cysteine, 0.91 mM pyruvate, 10 ng/mL epidermal growth factor, 75 g/mL kanamycin, 1 g/mL insulin, and 10% (vol/vol) porcine follicular fluid. For the first 22 h of maturation culture, IVM media was supplemented with 10 IU/mL eCG (Intervet International BV, Boxmeer, Holland) and 10 IU/mL hCG (Intervet International BV). The in vitro culture (IVC) medium for embryo development was a porcine zygote medium (PZM-3) containing 0.3% (wt/ vol) bovine serum albumin (BSA) [20] which was modified by adding 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and 10 M -mercaptoetanol. 2.2. Oocyte collection and IVM Porcine ovaries were obtained from 6 to 7 mo old prepubertal gilts weighing 110 to 120 kg at a local abattoir and transported to the laboratory at 37 °C. In the ovaries, cumulus-oocyte complexes (COCs) were aspirated from 3- to 8-mm diameter follicles using an 18-gauge needle and a 10-mL syringe. COCs having multiple layers of compacted cumulus cells were selected and washed three times in HEPES-buffered Tyrode’s medium containing 0.05% (wt/vol) polyvinyl alcohol (TLH-PVA). COCs were placed into each well of a four-well multi-dish (Nunc, Roskilde, Denmark) that contained 500 L of
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IVM medium with hormones. The COCs were cultured at 39 °C with 5% CO2 under maximum humidity. After 22 h in the maturation culture, the COCs were washed three times in fresh hormone-free IVM medium. The COCs were then cultured in hormone-free IVM medium for an additional 18 and 22 h for SCNT and parthenogenetic activation (PA), respectively. During IVM, COCs were treated or not treated with 10 mM (1.98 mg/mL) L-carnitine according to the experimental design. 2.3. Experimental design In Experiment 1, the effect of L-carnitine in IVM medium on intracellular oocyte GSH and ROS levels was determined. The in vitro development of PA and SCNT embryos, which were derived from L-carnitinetreated oocytes, was examined in Experiments 2 and 3, respectively. The effects of L-carnitine treatment during IVM on the expression of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA in 4-cell cloned embryos and the apoptosis-related genes (BAX and p-Bcl-xl) in SCNT blastocysts were analyzed in Experiments 4 and 5, respectively. 2.4. Measurement of intracellular GSH and ROS levels IVM oocytes at the MII stage oocytes were sampled 44 h after IVM to determine the intracellular ROS and GSH levels. Reactive oxygen species and GSH levels were measured by methods previously described [8,21]. Briefly, H2DCFDA (2’, 7’-dichlorodihydrofluorescein diacetate; Invitrogen) and CellTracker Blue CMF2HC (4-chloromethyl-6.8-difluoro-7-hydroxycoumarin; Invitrogen) were used to detect intracellular ROS as green fluorescence and GSH level as blue fluorescence, respectively. Ten oocytes from each treatment group were incubated for 30 min in TLH-PVA supplemented with 10 M H2DCFDA and 10 M CellTracker in the dark. After incubation, oocytes were washed with D-PBS (Invitrogen) containing 0.1% (wt/vol) PVA, placed into 10-L droplets and fluorescence was observed under an epifluorescence microscope (TE300; Nikon, Tokyo, Japan) with UV filters (460 nm for ROS and 370 nm for GSH). Oocytes that were stained with H2DCFDA or CellTracker Blue were discarded after observation of GSH and ROS levels. Fluorescent images were saved as graphic files in tiff format. The fluorescence intensities of oocytes and embryos were analyzed by ImageJ software (version 1.41o; National Institutes of Health, Bethesda, MD, USA) and normalized to untreated control oocytes.
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2.5. Preparation of donor cells and nuclear transfer Skin fibroblasts from a miniature pig were seeded into four-well culture plates and grown in Dulbecco’s modified Eagle medium (DMEM) with the nutrient mixture F-12 (Invitrogen), which was supplemented with 15% (vol/vol) fetal bovine serum until a complete monolayer of cells had formed. Donor cells were synchronized at the G0/G1 stage of the cell cycle by contact inhibition for 48 to 72 h. Cells of the same passage (passage 5 to 8) were used in each replicate for SCNT. A suspension of single cells was prepared by trypsinization of the cultured cells, followed by resuspension in TLH containing 0.4% (wt/vol) BSA (TLHBSA) before the nuclear transfer. After 40 h of IVM, MII stage oocytes were incubated for 15 min in a manipulation medium (calciumfree TLH-BSA) containing 5 g/mL Hoechst 33342, and then washed twice with fresh manipulation medium. Oocytes were transferred into a drop of manipulation medium containing 5 g/mL cytochalasin B and were overlaid with mineral oil. Oocytes were enucleated by aspirating the first polar body and MII chromosomes using a 17-m beveled glass pipette (Humagen, Charlottesville, VA, USA). Enucleation was confirmed under an epifluorescence microscope (TE300; Nikon). After enucleation, a single cell was inserted into the perivitelline space of each oocyte. Cell-oocyte couplets were placed on a 1-mm fusion chamber overlaid with 1 mL of 280 mM mannitol solution containing 0.001 mM CaCl2 and 0.05 mM MgCl2 as previously described [22]. Membrane fusion was induced by applying an alternating current field of 2 V cycling at 1 MHz for 2 sec, followed by two pulses of 170 V/mm direct current (DC) for 25 sec using a cell fusion generator (LF101; NepaGene, Chiba, Japan). Immediately after fusion, the oocytes were incubated for 1 h in TLH-BSA and evaluated for membrane fusion under a stereomicroscope before activation. 2.6. Activation and in vitro culture of embryos Reconstructed oocytes were activated with two pulses of 120 V/mm DC for 60 sec in a 280 mM mannitol solution containing 0.01 mM CaCl2 and 0.05 mM MgCl2. For PA, the oocytes that reached MII stage at 44 h of IVM were activated using the same pulse sequence as used to activate SCNT oocytes. After electrical activation, the SCNT and PA embryos were treated with 0.4 g/mL demecolcine and 5 g/mL cytochalasin B in IVC medium for 4 h, respectively [22]. The SCNT and PA embryos were washed three times in fresh IVC medium, transferred into 30-L IVC droplets of medium under mineral oil, and then cultured at 39 °C in a humidified atmosphere of
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Table 1 Primers used for gene expression analysis. mRNA
Direction
Primer sequences
Product size (base pairs)
18s
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5=-CGGCTACCACATCCAAGGAA-3= 5=-CTCCAATGGATCCTCGTTAAAGG-3= 5=-CTGGACAAGGAGAAGCTGGAG-3= 5=-AAGAGAACCCCCAAAGTGAGC-3= 5=-AGGTGAGGACATGCAGCTTT-3= 5=-ATTCTGGTGCCGGATGAAGAC-3= 5=-ATTCTGGTGCCGGATGAAGAC-3= 5=-GGTGTTGGAGTTCATGGAGG-3= 5=-GTCGATGGACTTGGTGTAGCC-3= 5=-CTCAAACCTTCCAACCTGCTG-3= 5=-TAATGCAGACACCTTGGCACT-3= 5=-GCAAATTCACCAGAAGGCATC-3= 5=-GTTGACTTTCTCTCCTACAAGC-3= 5=-GGTACCTCAGTTCAAACTCATC-3= 5=-CTACTTTGCCAGTAAACTGG-3= 5=-TCCCAAAGTAGGAGAGGA-3=
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POU5F1 DNMT1 FGFR2 ERK2 PCNA p-Bcl-xl BAX
5% CO2, 5% O2, and 90% N2 for 7 days. Embryo cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively (the day of SCNT or PA was designated as Day 0). The total blastocyst cell count was performed using Hoechst 33342 staining under an epifluorescence microscope. 2.7. Gene expression analysis by real-time polymerase chain reaction (RT-PCR) The transcript abundance of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA in 4-cell cloned embryos and BAX and p-Bcl-xl mRNA in SCNT blastocysts was analyzed by RT-PCR as previously described [23]. Total RNA was isolated from 4-cell cloned embryos and blastocysts developed on Days 2 and 7, respectively, of IVC using AccuZol (Bioneer, Alameda, CA, USA) according to the manufacturer’s instructions. Complementary DNAs were synthesized from total RNA using the Reverse Transcription system (Promega, Madison, WI, USA). Then, the expression levels of specific genes in embryos were quantified by RT-PCR (Rotor-Gene 3000 system; Corbett, Mortlake, Australia) using the PrimeScript RT-PCR kit (Takara, Shiga, Japan). The melting curve data were collected to check PCR specificity. The Primary3 software (Whitehead Institute, MIT Center for Genome Research, Cambridge, MA, USA) was used for primer design in this study (Table 1). All data were normalized to 18 sec mRNA. The relative mRNA level was presented as 2-⌬⌬Ct where Ct ⫽ threshold cycle for target amplification, ⌬Ct ⫽ Cttarget gene – Ctinternal reference (18 sec), and ⌬⌬Ct ⫽ ⌬Ctsample – Ctcalibrator [24,25]. The experiment was repeated three times. Forty four-cell cloned embryos per sample were used for the analysis of DNMT1, ERK2,
155 213 121 183 153 277 158
PCNA, FGFR2, and POU5F1 mRNA levels and 10 cloned blastocysts per sample were analyzed for BAX and p-Bcl-xl mRNA levels. 2.8. Statistical analysis Statistical analyses were performed using the Statistical Analysis System (version 9.1; SAS Institute, Cary, NC, USA). Data were analyzed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P ⬍ 0.05. Percentage data were arcsinetransformed before analysis to maintain the homogeneity of variances. The results are expressed as mean ⫾ standard error of the mean (SEM). 3. Results 3.1. Effect of L-carnitine on oocyte maturation and intracellular levels of GSH and ROS (Experiment 1) During IVM, the proportion of oocytes that reached the MII stage (91.6% vs. 90.5% for control and L-carnitinetreated oocytes, respectively) was not influenced by the L-carnitine treatment. However, L-carnitine increased (P ⬍ 0.05) intracellular GSH levels and decreased (P ⬍ 0.05) ROS generation in MII oocytes after IVM (Table 2). 3.2. Effect of L-carnitine on embryonic development after PA (Experiment 2) In vitro development of PA embryos to the blastocyst stage (39.4%) was increased (P ⬍ 0.05) by the L-carnitine treatment compared with control (27.1%). However, embryonic cleavage (84.4% to 87.9%) and
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Table 2 Effect of L-carnitine treatment on nuclear maturation and intracellular glutathione (GSH) and reactive oxygen species (ROS) levels of oocytes during in vitro maturation (IVM). L-carnitine treatment during IVM No (control) Yes
Number of oocytes cultured for maturation*
% of oocytes that reached MII
244 210
91.6 ⫾ 1.1 90.5 ⫾ 0.2
Relative level (pixels/oocytes) GSH (N ⫽ 30†)
ROS (N ⫽ 30†)
1.0 ⫾ 0.03a 1.4 ⫾ 0.05b
1.0 ⫾ 0.13a 0.4 ⫾ 0.04b
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates. † Number of oocytes examined. Four replicates.
a,b
cell number in blastocyst (32.3 to 34.6 cells per blastocyst) were not influenced by the treatment (Table 3).
relative transcript abundance of BAX to p-Bcl-xl mRNA was significantly increased by the L-carnitine treatment (Fig. 2).
3.3. Effect of L-carnitine on embryonic development after SCNT (Experiment 3)
4. Discussion
As shown in Table 4, SCNT embryos that were derived from L-carnitine-treated oocytes showed a higher (P ⬍ 0.05) rate (23.2%) of blastocyst formation than control embryos (14.9%). However, oocyte-cell fusion (71.7% and 72.4% for control and treated oocytes, respectively), embryo cleavage (83.3% to 87.7%), and cell number in the blastocyst (33.2 to 34.2 cells per blastocyst) after SCNT were not altered by the L-carnitine treatment during IVM. 3.4. Effect of L-carnitine on the expression of DNMT1, FGFR2, PCNA, and POU5F1 mRNA (Experiment 4) L-carnitine treatment of recipient oocytes during IVM increased (P ⬍ 0.05) the transcript abundance of DNMT1, FGFR2, PCNA, and POU5F1 in four-cell SCNT embryos compared with the control. However, the expression of the housekeeping gene 18S was not influenced (Fig. 1). 3.5. Effect of L-carnitine on the expression of BAX and p-Bcl-xl mRNA (Experiment 5) During IVM of recipient oocytes, L-carnitine treatment increased (P ⬍ 0.05) the expression of the BAX gene in SCNT blastocysts compared with control. In addition, the
The culture conditions for oocyte maturation and embryonic development are critical determinants for the successful development of in vitro-produced embryos in a wide variety of mammalian species. In this study, the effects of an antioxidant (L-carnitine) treatment on oocyte maturation during IVM, embryonic development after PA and SCNT, and intracellular levels of GSH and ROS in oocytes were examined through a series of experiments. In addition, expression levels of several transcription factors and apoptosis-related genes were analyzed in SCNT embryos that were derived from L-carnitine-treated oocytes. Our findings demonstrated that treatment of pig oocytes with L-carnitine during IVM effectively increased the intracellular level of GSH, scavenged ROS in oocytes, and stimulated embryonic development after PA and SCNT. In addition, L-carnitine increased the expression of POU5F1 and transcription factors, such as DNMT1, PCNA, and FGFR2 in SCNT pig embryos but showed no effect on the inhibition of apoptosis at the gene expression level. As in other studies reporting the beneficial effects of L-carnitine on embryonic development in mice, bovine, and porcine [18,19,26], the treatment of immature oocytes with L-carnitine during IVM resulted in im-
Table 3 Effect of L-carnitine treatment on embryonic development during in vitro maturation (IVM) after parthenogenetic activation. L-carnitine treatment during IVM No (control) Yes
No. of embryos cultured* 240 198
Embryo development (%) ⱖ Two-cell
Blastocyst
84.4 ⫾ 3.4 87.9 ⫾ 2.2
27.1 ⫾ 2.7a 39.4 ⫾ 1.6b
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates.
a,b
Cells in blastocyst (N) 32.3 ⫾ 1.6 34.6 ⫾ 1.7
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Table 4 Effect of L-carnitine treatment on embryonic development during in vitro maturation (IVM) after somatic cell nuclear transfer. L-carnitine treatment during IVM No (control) Yes
No. of embryos cultured* 120 110
Embryo development (%) ⱖ Two-cell
Blastocyst
83.3 ⫾ 2.1 87.7 ⫾ 4.3
14.9 ⫾ 2.1a 23.2 ⫾ 1.9b
No. cells in blastocyst 34.2 ⫾ 4.2 33.2 ⫾ 2.8
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates.
a,b
proved preimplantation development of PA and SCNT embryos in pigs in this study. L-carnitine did not improve nuclear maturation but was effective at increasing GSH levels in oocytes after IVM. This result indicated that the beneficial effect of L-carnitine was prominent on cytoplasmic maturation rather than nuclear maturation. This might have further contributed to the improvement of embryonic development after PA and SCNT. These results were similar with previous findings in porcine that L-carnitine treatment during IVM reduced ROS level in IVM oocytes and improved embryonic cleavage or development to the blastocyst stage of porcine PA and IVF embryos [26,27]. On the other hand, L-carnitine has been shown to improve the meiotic competence of porcine oocytes by holding back the apoptosis of granulose cells and improving mitochondrial activity [27,28]. In contrast, another study [26] reported that L-carnitine supplementation in 2 mg/mL concentration during IVM inhibited nuclear maturation of oocytes in pigs. In our study, neither improvement nor inhibition in nuclear maturation was observed after treatment with L-carnitine during IVM. The proportion of MII oocytes from untreated control was 91.6%. Considering the proportion of MII oocytes improved by L-carnitine was 82.2% to 82.5% in a
previous study [27], 91.6% of nuclear maturation in control oocytes in this study might be too high to be improved by L-carnitine. Mitochondria are the major generators of ROS and, therefore, need to be continuously protected from oxidative stress. Although oocytes and embryos possess multiple enzymatic and nonenzymatic defense systems against ROS [29,30], excessive amounts of ROS that cannot be detoxified by the defense system, especially when the GSH levels of oocytes drop after zygotic genome activation [31], may be harmful to mitochondrial integrity and decrease embryonic development. In this study, it was not clear whether the mitochondrial integrity and functionality were protected from oxidative stress by the action of a high level of GSH. However, our result of decreased ROS levels in L-carnitine-treated oocytes indicated that the increased GSH level due to the L-carnitine treatment improved embryonic development. This effect was probably due to ROS scavenging, which led to the protection of micro-organelles, including mitochondria. The concentration of L-carnitine (10 mM; 1.98 mg/mL) used in this study was adopted from the previous results of bovine and porcine oocyte maturation and embryonic development [19,27]. This concentration of L-carnitine showed significant effect on the intracellular
Fig. 1. Mean ⫾ SEM expression of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA levels in four-cell somatic cell nucleus transfer embryos treated with L-carnitine during in vitro maturation. Within the same mRNA, means without a common superscript (a, b) differed (P ⬍ 0.05).
Fig. 2. Mean ⫾ SEM expression of BAX, p-Bcl-xl, and BAX/p-Bcl-xl mRNA levels in somatic cell nucleus transfer blastocysts that were treated with L-carnitine during in vitro maturation of recipient oocytes. Within the same mRNA, means without a common superscript (a, b) differed (P ⬍ 0.05).
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GSH and ROS levels and embryonic development. Our result was consistent with the result of previous study [27] that significant improvement was shown in pig oocyte maturation and embryo cleavage after IVF when L-carnitine was supplemented in 0.6 to 5 mg/mL concentration during IVM. In this study, intracellular GSH level in IVM oocytes was assayed by fluorescence staining method using CellTracker blue that was a specific fluorescent dye to detect reduced GSH. Compared with enzymatic assay, this fluorescent assay does not quantify total GSH content, including oxidized GSH but is easy to perform, can measure GSH level in small number of cells or oocytes, and cultures can be sequentially used for additional assays [32,33]. Modifications in culture environment can modulate gene expression in mammalian cells and embryos [34,35]. Especially in SCNT oocytes that are reconstructed with somatic cell nuclei, the timely and apposite expression of transcription factor genes during the nuclear reprogramming process of introduced somatic cell nuclei is essential for obtaining a normal developmental competence of reconstructed embryos. It has been reported that DNMT1 is involved in DNA methylation and cell proliferation [36]. PCNA is an essential component of the DNA replication and repair machinery [37], and expression of FGFR2 and POU5F1 is essential for the early development of mouse and human embryos [38,39]. Thus, the expression of mRNAs of several transcription factors in SCNT embryos has been analyzed to monitor nuclear reprogramming and embryonic development [40,41]. When the mRNA expression levels of several developmentally important genes in SCNT embryos were analyzed to assess whether improved embryonic development was correlated with gene expression profiles, the treatment of recipient oocytes with L-carnitine during IVM significantly increased the DNMT1, PCNA, FGFR2, and POU5F1 mRNA levels in this study. It was not clear how L-carnitine treatment increased mRNA levels, but it could be hypothesized that cytoplasmic modification, including increased GSH level and reduced ROS activity that was induced by L-carnitine treatment created beneficial microenvironments for nuclear reprogramming of donor nuclei and stimulated gene expression in cloned embryos [40,41]. mRNA level in PA embryos was not analyzed in this study because PA embryos may have different nuclear reprogramming than SCNT embryos and therefore the gene expression pattern in PA embryos may not be a good reference for SCNT embryos. Apoptosis is considered a normal process that
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eliminates cells with DNA or chromosomal abnormalities [42]. However, an excessive number of apoptotic cells during embryonic development may lead to a loss of viability and developmental failure [43,44]. It has been reported that apoptosis can be induced via oxidative stress derived from ROS generation when oocytes or embryos are cultured in vitro and L-carnitine acts as an antioxidant in animal cells or embryos and reduces apoptosis induced by oxidative stress by increasing intracellular GSH level and affecting mitochondrial functions [18,26,45]. Thus, we examined the expression of the anti- and proapoptotic genes p-Bcl-xl and BAX, respectively, to determine whether apoptosis would be decreased in SCNT blastocysts after treatment of recipient oocytes with an antioxidant, L-carnitine. Although L-carnitine enhanced the expression of both anti- and proapoptotic genes compared with no treatment, the enhancement of expression was more prominent with the proapoptotic gene (BAX) than with that antiapoptotic gene. This was in contrast to the previous result [26] that L-carnitine treatment during IVM reduced incidence of apoptotic nuclei in parthenogenetic blastocysts in pigs. In this study, the reason for increased expression of the proapoptotic gene despite the reduced ROS levels in IVM oocytes and improved embryonic development due to L-carnitine treatment was not known. Recently, it has been reported that apoptosis appears more frequently in competent oocytes from cycling gilts than in less competent oocytes from prepubertal gilts [46]. The results of previous and present study suggest that incidence of apoptosis may not be a good indicator to predict developmental competence of oocytes and embryos. An additional analysis of anti- and proapoptotic gene expression in IVM oocytes would be helpful to determine the effect of L-carnitine on oocyte apoptosis and later embryonic development after PA or IVF. In summary, our results suggest that IVM treatment of oocytes with L-carnitine improves the preimplantation development of PA and SCNT embryos by increasing intracellular GSH levels, reducing ROS toxicity, and regulating expression of POU5F1 and transcription factor(s) expression, which are necessary for the normal development of SCNT embryos.
Acknowledgments The authors thank Gyeonggi Veterinary Service for the generous donation of pig ovaries. This work was sup-
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ported by a grant (# PJ007113022011) from the BioGreen21 Program, Rural Development Administration, Republic of Korea, and by the Institute of Veterinary Science, Kangwon National University.
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Theriogenology 78 (2012) 235–243 www.theriojournal.com
L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression Jinyoung Youa,1, Joohyeong Leea,1, Sang-Hwan Hyunb, Eunsong Leea,c,* b
a College of Veterinary Medicine, Kangwon National University, Chuncheon, Korea Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea c Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea
Received 22 September 2011; received in revised form 3 January 2012; accepted 15 February 2012
Abstract The objective of this study was to examine the effect of L-carnitine treatment during in vitro maturation (IVM) of immature pig (Sus scrofa) oocytes. Specifically, the effects of L-carnitine treatment on nuclear maturation and oocyte intracellular glutathione (GSH) levels, embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT pig embryos were determined. During IVM culture, immature oocytes were either treated or not treated with 10 mM L-carnitine. L-carnitine treatment did not improve the nuclear maturation of oocytes but significantly increased intracellular GSH levels, which led to a reduction of reactive oxygen species (ROS) levels in IVM oocytes. Oocytes treated with L-carnitine showed higher (P ⬍ 0.05) rates of blastocyst formation after PA (39.4% vs. 27.1%) and SCNT (23.2% vs. 14.9%) compared with untreated oocytes. SCNT embryos that were derived from L-carnitine-treated oocytes showed increased (P ⬍ 0.05) expression levels of DNMT1, PCNA, FGFR2, and POU5F1 mRNA compared with control embryos. Treatment of recipient oocytes with L-carnitine increased (P ⬍ 0.05) the expression of both BAX and p-Bcl-xl mRNA in SCNT blastocysts. However, the increase was more prominent in BAX than in p-Bcl-xl mRNA. Our results demonstrate that L-carnitine treatment during IVM improves the developmental competence of SCNT embryos. This effect is probably due to increased intracellular GSH synthesis in recipient ooplasts, which reduces ROS levels, and the stimulation of nuclear reprogramming via increased expression of POU5F1 and transcription factors. © 2012 Elsevier Inc. All rights reserved. Keywords: L-carnitine; Gene expression; Nuclear transfer; Oocyte maturation; Pig
1. Introduction Recent progress in reproductive technologies, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer (SCNT) 1
These authors contributed equally to this work.
* Corresponding author. Tel.: ⫹82 33 250 8670; fax: ⫹82 33 244 2367. E-mail address: [email protected] (E. Lee). 0093-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2012.02.027
has made it possible to produce specific animals successfully in a variety of species [1–3]. The efficiency of reproductive technologies, especially SCNT, is influenced by various factors, including the quality of oocytes, culture conditions, cell cycle stage of the donor cell, and oocyte activation methods [4,5]. Of those, the quality of oocytes is one of the most critical factors for determining the in vivo viability and in vitro developmental competence of in vitro-produced embryos. Oocytes or embryos are inevitably exposed to unde-
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sirable environments during in vitro production. Reactive oxygen species (ROS) can be generated by handling or culturing oocytes or embryos in a high-oxygen atmosphere, and artificial treatments, such as electric stimulus for cell fusion or activation of SCNT oocytes, are known to increase intracellular ROS levels [6,7]. It is well-understood that a high level of ROS causes cell membrane lipid peroxidation [8,9] and DNA fragmentation, and also influences RNA transcription and protein synthesis [10]. These activities lead to in vitro developmental blocks and early embryonic death [9,11]. Recent studies [12,13] on oocyte maturation have shown that modifications of a culture system for the in vitro maturation (IVM) of oocytes can improve cytoplasmic maturation. This stimulates embryonic development by increasing intracellular glutathione (GSH) levels in IVM oocytes, which reduces ROS activity during the culturing of embryos. Thus, it is crucial to establish a stable IVM system to produce mature oocytes of a higher quality to increase in vitro production efficiency. Previously, supplementation of IVM medium with antioxidants, such as -mercaptoethanol, cysteine, and cysteamine, has been demonstrated to stimulate the synthesis of intracellular GSH, which in turn plays an antioxidative role and enhances viability of IVF and ICSI embryos [14,15]. L-carnitine (-hydroxy-␥-trimethylammoniumbutyric acid), an antioxidative agent, is known to have a beneficial role in cellular metabolism and embryonic development in mammalian species. L-carnitine protects cell membranes and DNA from damage induced by oxygen free radicals [16]. When mouse metaphase II (MII) oocytes and 8-cell embryos were incubated in a medium supplemented with L-carnitine (0.6 mg/mL), a significant improvement in the integrity of microtubule and chromosome structural integrity and a decreased level of apoptosis were observed [17]. In addition, supplementation of culture medium with 0.3 mg/mL L-carnitine improved blastocyst formation in mice by reducing the blocking effects of actinomycin-D, hydrogen peroxide, and tumor necrosis factor-␣ on embryonic development and decreasing levels of DNA damage [18]. In bovine, L-carnitine has also been demonstrated to improve embryonic development in vitro by exhibiting an extensive relocation of active mitochondria to the inner oocyte cytoplasm [19]. Many studies have been performed in various species to determine the beneficial effect of L-carnitine on in vitro embryonic development, but there is limited information available in pigs on the effect of L-carnitine on oocyte maturation and subsequent embryonic development. In this study, we examined the effect of
L-carnitine treatment during the oocyte maturation process on the developmental competence of parthenogenetic and SCNT embryos. We did this by studying the nuclear maturation of oocytes, intracellular levels of GSH and ROS in IVM oocytes, embryonic cleavage, and blastocyst formation. In addition, expression levels of several genes (DNMT1, ERK2, PCNA, FGFR2, and POU5F1) and apoptosis-related genes (BAX and p-Bclxl) were analyzed in SCNT pig embryos. Our findings demonstrate that L-carnitine treatment during oocyte maturation improves SCNT embryonic development. This probably occurs through increasing the intracellular GSH level of oocytes, which leads to the inhibition of ROS activity and stimulates expression of POU5F1 and transcription factor genes during nuclear reprogramming in SCNT pig embryos. 2. Materials and methods 2.1. Culture media All chemicals used in this study were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA), unless otherwise stated. The medium for IVM was Tissue Culture Medium-199 (M-199; Invitrogen, Grand Island, NY, USA) supplemented with 0.6 mM cysteine, 0.91 mM pyruvate, 10 ng/mL epidermal growth factor, 75 g/mL kanamycin, 1 g/mL insulin, and 10% (vol/vol) porcine follicular fluid. For the first 22 h of maturation culture, IVM media was supplemented with 10 IU/mL eCG (Intervet International BV, Boxmeer, Holland) and 10 IU/mL hCG (Intervet International BV). The in vitro culture (IVC) medium for embryo development was a porcine zygote medium (PZM-3) containing 0.3% (wt/ vol) bovine serum albumin (BSA) [20] which was modified by adding 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and 10 M -mercaptoetanol. 2.2. Oocyte collection and IVM Porcine ovaries were obtained from 6 to 7 mo old prepubertal gilts weighing 110 to 120 kg at a local abattoir and transported to the laboratory at 37 °C. In the ovaries, cumulus-oocyte complexes (COCs) were aspirated from 3- to 8-mm diameter follicles using an 18-gauge needle and a 10-mL syringe. COCs having multiple layers of compacted cumulus cells were selected and washed three times in HEPES-buffered Tyrode’s medium containing 0.05% (wt/vol) polyvinyl alcohol (TLH-PVA). COCs were placed into each well of a four-well multi-dish (Nunc, Roskilde, Denmark) that contained 500 L of
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IVM medium with hormones. The COCs were cultured at 39 °C with 5% CO2 under maximum humidity. After 22 h in the maturation culture, the COCs were washed three times in fresh hormone-free IVM medium. The COCs were then cultured in hormone-free IVM medium for an additional 18 and 22 h for SCNT and parthenogenetic activation (PA), respectively. During IVM, COCs were treated or not treated with 10 mM (1.98 mg/mL) L-carnitine according to the experimental design. 2.3. Experimental design In Experiment 1, the effect of L-carnitine in IVM medium on intracellular oocyte GSH and ROS levels was determined. The in vitro development of PA and SCNT embryos, which were derived from L-carnitinetreated oocytes, was examined in Experiments 2 and 3, respectively. The effects of L-carnitine treatment during IVM on the expression of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA in 4-cell cloned embryos and the apoptosis-related genes (BAX and p-Bcl-xl) in SCNT blastocysts were analyzed in Experiments 4 and 5, respectively. 2.4. Measurement of intracellular GSH and ROS levels IVM oocytes at the MII stage oocytes were sampled 44 h after IVM to determine the intracellular ROS and GSH levels. Reactive oxygen species and GSH levels were measured by methods previously described [8,21]. Briefly, H2DCFDA (2’, 7’-dichlorodihydrofluorescein diacetate; Invitrogen) and CellTracker Blue CMF2HC (4-chloromethyl-6.8-difluoro-7-hydroxycoumarin; Invitrogen) were used to detect intracellular ROS as green fluorescence and GSH level as blue fluorescence, respectively. Ten oocytes from each treatment group were incubated for 30 min in TLH-PVA supplemented with 10 M H2DCFDA and 10 M CellTracker in the dark. After incubation, oocytes were washed with D-PBS (Invitrogen) containing 0.1% (wt/vol) PVA, placed into 10-L droplets and fluorescence was observed under an epifluorescence microscope (TE300; Nikon, Tokyo, Japan) with UV filters (460 nm for ROS and 370 nm for GSH). Oocytes that were stained with H2DCFDA or CellTracker Blue were discarded after observation of GSH and ROS levels. Fluorescent images were saved as graphic files in tiff format. The fluorescence intensities of oocytes and embryos were analyzed by ImageJ software (version 1.41o; National Institutes of Health, Bethesda, MD, USA) and normalized to untreated control oocytes.
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2.5. Preparation of donor cells and nuclear transfer Skin fibroblasts from a miniature pig were seeded into four-well culture plates and grown in Dulbecco’s modified Eagle medium (DMEM) with the nutrient mixture F-12 (Invitrogen), which was supplemented with 15% (vol/vol) fetal bovine serum until a complete monolayer of cells had formed. Donor cells were synchronized at the G0/G1 stage of the cell cycle by contact inhibition for 48 to 72 h. Cells of the same passage (passage 5 to 8) were used in each replicate for SCNT. A suspension of single cells was prepared by trypsinization of the cultured cells, followed by resuspension in TLH containing 0.4% (wt/vol) BSA (TLHBSA) before the nuclear transfer. After 40 h of IVM, MII stage oocytes were incubated for 15 min in a manipulation medium (calciumfree TLH-BSA) containing 5 g/mL Hoechst 33342, and then washed twice with fresh manipulation medium. Oocytes were transferred into a drop of manipulation medium containing 5 g/mL cytochalasin B and were overlaid with mineral oil. Oocytes were enucleated by aspirating the first polar body and MII chromosomes using a 17-m beveled glass pipette (Humagen, Charlottesville, VA, USA). Enucleation was confirmed under an epifluorescence microscope (TE300; Nikon). After enucleation, a single cell was inserted into the perivitelline space of each oocyte. Cell-oocyte couplets were placed on a 1-mm fusion chamber overlaid with 1 mL of 280 mM mannitol solution containing 0.001 mM CaCl2 and 0.05 mM MgCl2 as previously described [22]. Membrane fusion was induced by applying an alternating current field of 2 V cycling at 1 MHz for 2 sec, followed by two pulses of 170 V/mm direct current (DC) for 25 sec using a cell fusion generator (LF101; NepaGene, Chiba, Japan). Immediately after fusion, the oocytes were incubated for 1 h in TLH-BSA and evaluated for membrane fusion under a stereomicroscope before activation. 2.6. Activation and in vitro culture of embryos Reconstructed oocytes were activated with two pulses of 120 V/mm DC for 60 sec in a 280 mM mannitol solution containing 0.01 mM CaCl2 and 0.05 mM MgCl2. For PA, the oocytes that reached MII stage at 44 h of IVM were activated using the same pulse sequence as used to activate SCNT oocytes. After electrical activation, the SCNT and PA embryos were treated with 0.4 g/mL demecolcine and 5 g/mL cytochalasin B in IVC medium for 4 h, respectively [22]. The SCNT and PA embryos were washed three times in fresh IVC medium, transferred into 30-L IVC droplets of medium under mineral oil, and then cultured at 39 °C in a humidified atmosphere of
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Table 1 Primers used for gene expression analysis. mRNA
Direction
Primer sequences
Product size (base pairs)
18s
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
5=-CGGCTACCACATCCAAGGAA-3= 5=-CTCCAATGGATCCTCGTTAAAGG-3= 5=-CTGGACAAGGAGAAGCTGGAG-3= 5=-AAGAGAACCCCCAAAGTGAGC-3= 5=-AGGTGAGGACATGCAGCTTT-3= 5=-ATTCTGGTGCCGGATGAAGAC-3= 5=-ATTCTGGTGCCGGATGAAGAC-3= 5=-GGTGTTGGAGTTCATGGAGG-3= 5=-GTCGATGGACTTGGTGTAGCC-3= 5=-CTCAAACCTTCCAACCTGCTG-3= 5=-TAATGCAGACACCTTGGCACT-3= 5=-GCAAATTCACCAGAAGGCATC-3= 5=-GTTGACTTTCTCTCCTACAAGC-3= 5=-GGTACCTCAGTTCAAACTCATC-3= 5=-CTACTTTGCCAGTAAACTGG-3= 5=-TCCCAAAGTAGGAGAGGA-3=
147
POU5F1 DNMT1 FGFR2 ERK2 PCNA p-Bcl-xl BAX
5% CO2, 5% O2, and 90% N2 for 7 days. Embryo cleavage and blastocyst formation were evaluated on Days 2 and 7, respectively (the day of SCNT or PA was designated as Day 0). The total blastocyst cell count was performed using Hoechst 33342 staining under an epifluorescence microscope. 2.7. Gene expression analysis by real-time polymerase chain reaction (RT-PCR) The transcript abundance of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA in 4-cell cloned embryos and BAX and p-Bcl-xl mRNA in SCNT blastocysts was analyzed by RT-PCR as previously described [23]. Total RNA was isolated from 4-cell cloned embryos and blastocysts developed on Days 2 and 7, respectively, of IVC using AccuZol (Bioneer, Alameda, CA, USA) according to the manufacturer’s instructions. Complementary DNAs were synthesized from total RNA using the Reverse Transcription system (Promega, Madison, WI, USA). Then, the expression levels of specific genes in embryos were quantified by RT-PCR (Rotor-Gene 3000 system; Corbett, Mortlake, Australia) using the PrimeScript RT-PCR kit (Takara, Shiga, Japan). The melting curve data were collected to check PCR specificity. The Primary3 software (Whitehead Institute, MIT Center for Genome Research, Cambridge, MA, USA) was used for primer design in this study (Table 1). All data were normalized to 18 sec mRNA. The relative mRNA level was presented as 2-⌬⌬Ct where Ct ⫽ threshold cycle for target amplification, ⌬Ct ⫽ Cttarget gene – Ctinternal reference (18 sec), and ⌬⌬Ct ⫽ ⌬Ctsample – Ctcalibrator [24,25]. The experiment was repeated three times. Forty four-cell cloned embryos per sample were used for the analysis of DNMT1, ERK2,
155 213 121 183 153 277 158
PCNA, FGFR2, and POU5F1 mRNA levels and 10 cloned blastocysts per sample were analyzed for BAX and p-Bcl-xl mRNA levels. 2.8. Statistical analysis Statistical analyses were performed using the Statistical Analysis System (version 9.1; SAS Institute, Cary, NC, USA). Data were analyzed using a general linear model procedure followed by the least significant difference mean separation procedure when the treatments differed at P ⬍ 0.05. Percentage data were arcsinetransformed before analysis to maintain the homogeneity of variances. The results are expressed as mean ⫾ standard error of the mean (SEM). 3. Results 3.1. Effect of L-carnitine on oocyte maturation and intracellular levels of GSH and ROS (Experiment 1) During IVM, the proportion of oocytes that reached the MII stage (91.6% vs. 90.5% for control and L-carnitinetreated oocytes, respectively) was not influenced by the L-carnitine treatment. However, L-carnitine increased (P ⬍ 0.05) intracellular GSH levels and decreased (P ⬍ 0.05) ROS generation in MII oocytes after IVM (Table 2). 3.2. Effect of L-carnitine on embryonic development after PA (Experiment 2) In vitro development of PA embryos to the blastocyst stage (39.4%) was increased (P ⬍ 0.05) by the L-carnitine treatment compared with control (27.1%). However, embryonic cleavage (84.4% to 87.9%) and
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Table 2 Effect of L-carnitine treatment on nuclear maturation and intracellular glutathione (GSH) and reactive oxygen species (ROS) levels of oocytes during in vitro maturation (IVM). L-carnitine treatment during IVM No (control) Yes
Number of oocytes cultured for maturation*
% of oocytes that reached MII
244 210
91.6 ⫾ 1.1 90.5 ⫾ 0.2
Relative level (pixels/oocytes) GSH (N ⫽ 30†)
ROS (N ⫽ 30†)
1.0 ⫾ 0.03a 1.4 ⫾ 0.05b
1.0 ⫾ 0.13a 0.4 ⫾ 0.04b
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates. † Number of oocytes examined. Four replicates.
a,b
cell number in blastocyst (32.3 to 34.6 cells per blastocyst) were not influenced by the treatment (Table 3).
relative transcript abundance of BAX to p-Bcl-xl mRNA was significantly increased by the L-carnitine treatment (Fig. 2).
3.3. Effect of L-carnitine on embryonic development after SCNT (Experiment 3)
4. Discussion
As shown in Table 4, SCNT embryos that were derived from L-carnitine-treated oocytes showed a higher (P ⬍ 0.05) rate (23.2%) of blastocyst formation than control embryos (14.9%). However, oocyte-cell fusion (71.7% and 72.4% for control and treated oocytes, respectively), embryo cleavage (83.3% to 87.7%), and cell number in the blastocyst (33.2 to 34.2 cells per blastocyst) after SCNT were not altered by the L-carnitine treatment during IVM. 3.4. Effect of L-carnitine on the expression of DNMT1, FGFR2, PCNA, and POU5F1 mRNA (Experiment 4) L-carnitine treatment of recipient oocytes during IVM increased (P ⬍ 0.05) the transcript abundance of DNMT1, FGFR2, PCNA, and POU5F1 in four-cell SCNT embryos compared with the control. However, the expression of the housekeeping gene 18S was not influenced (Fig. 1). 3.5. Effect of L-carnitine on the expression of BAX and p-Bcl-xl mRNA (Experiment 5) During IVM of recipient oocytes, L-carnitine treatment increased (P ⬍ 0.05) the expression of the BAX gene in SCNT blastocysts compared with control. In addition, the
The culture conditions for oocyte maturation and embryonic development are critical determinants for the successful development of in vitro-produced embryos in a wide variety of mammalian species. In this study, the effects of an antioxidant (L-carnitine) treatment on oocyte maturation during IVM, embryonic development after PA and SCNT, and intracellular levels of GSH and ROS in oocytes were examined through a series of experiments. In addition, expression levels of several transcription factors and apoptosis-related genes were analyzed in SCNT embryos that were derived from L-carnitine-treated oocytes. Our findings demonstrated that treatment of pig oocytes with L-carnitine during IVM effectively increased the intracellular level of GSH, scavenged ROS in oocytes, and stimulated embryonic development after PA and SCNT. In addition, L-carnitine increased the expression of POU5F1 and transcription factors, such as DNMT1, PCNA, and FGFR2 in SCNT pig embryos but showed no effect on the inhibition of apoptosis at the gene expression level. As in other studies reporting the beneficial effects of L-carnitine on embryonic development in mice, bovine, and porcine [18,19,26], the treatment of immature oocytes with L-carnitine during IVM resulted in im-
Table 3 Effect of L-carnitine treatment on embryonic development during in vitro maturation (IVM) after parthenogenetic activation. L-carnitine treatment during IVM No (control) Yes
No. of embryos cultured* 240 198
Embryo development (%) ⱖ Two-cell
Blastocyst
84.4 ⫾ 3.4 87.9 ⫾ 2.2
27.1 ⫾ 2.7a 39.4 ⫾ 1.6b
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates.
a,b
Cells in blastocyst (N) 32.3 ⫾ 1.6 34.6 ⫾ 1.7
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Table 4 Effect of L-carnitine treatment on embryonic development during in vitro maturation (IVM) after somatic cell nuclear transfer. L-carnitine treatment during IVM No (control) Yes
No. of embryos cultured* 120 110
Embryo development (%) ⱖ Two-cell
Blastocyst
83.3 ⫾ 2.1 87.7 ⫾ 4.3
14.9 ⫾ 2.1a 23.2 ⫾ 1.9b
No. cells in blastocyst 34.2 ⫾ 4.2 33.2 ⫾ 2.8
Different superscript letters indicate a significant difference within a column (P ⬍ 0.05). * Three replicates.
a,b
proved preimplantation development of PA and SCNT embryos in pigs in this study. L-carnitine did not improve nuclear maturation but was effective at increasing GSH levels in oocytes after IVM. This result indicated that the beneficial effect of L-carnitine was prominent on cytoplasmic maturation rather than nuclear maturation. This might have further contributed to the improvement of embryonic development after PA and SCNT. These results were similar with previous findings in porcine that L-carnitine treatment during IVM reduced ROS level in IVM oocytes and improved embryonic cleavage or development to the blastocyst stage of porcine PA and IVF embryos [26,27]. On the other hand, L-carnitine has been shown to improve the meiotic competence of porcine oocytes by holding back the apoptosis of granulose cells and improving mitochondrial activity [27,28]. In contrast, another study [26] reported that L-carnitine supplementation in 2 mg/mL concentration during IVM inhibited nuclear maturation of oocytes in pigs. In our study, neither improvement nor inhibition in nuclear maturation was observed after treatment with L-carnitine during IVM. The proportion of MII oocytes from untreated control was 91.6%. Considering the proportion of MII oocytes improved by L-carnitine was 82.2% to 82.5% in a
previous study [27], 91.6% of nuclear maturation in control oocytes in this study might be too high to be improved by L-carnitine. Mitochondria are the major generators of ROS and, therefore, need to be continuously protected from oxidative stress. Although oocytes and embryos possess multiple enzymatic and nonenzymatic defense systems against ROS [29,30], excessive amounts of ROS that cannot be detoxified by the defense system, especially when the GSH levels of oocytes drop after zygotic genome activation [31], may be harmful to mitochondrial integrity and decrease embryonic development. In this study, it was not clear whether the mitochondrial integrity and functionality were protected from oxidative stress by the action of a high level of GSH. However, our result of decreased ROS levels in L-carnitine-treated oocytes indicated that the increased GSH level due to the L-carnitine treatment improved embryonic development. This effect was probably due to ROS scavenging, which led to the protection of micro-organelles, including mitochondria. The concentration of L-carnitine (10 mM; 1.98 mg/mL) used in this study was adopted from the previous results of bovine and porcine oocyte maturation and embryonic development [19,27]. This concentration of L-carnitine showed significant effect on the intracellular
Fig. 1. Mean ⫾ SEM expression of DNMT1, ERK2, PCNA, FGFR2, and POU5F1 mRNA levels in four-cell somatic cell nucleus transfer embryos treated with L-carnitine during in vitro maturation. Within the same mRNA, means without a common superscript (a, b) differed (P ⬍ 0.05).
Fig. 2. Mean ⫾ SEM expression of BAX, p-Bcl-xl, and BAX/p-Bcl-xl mRNA levels in somatic cell nucleus transfer blastocysts that were treated with L-carnitine during in vitro maturation of recipient oocytes. Within the same mRNA, means without a common superscript (a, b) differed (P ⬍ 0.05).
J. You et al. / Theriogenology 78 (2012) 235–243
GSH and ROS levels and embryonic development. Our result was consistent with the result of previous study [27] that significant improvement was shown in pig oocyte maturation and embryo cleavage after IVF when L-carnitine was supplemented in 0.6 to 5 mg/mL concentration during IVM. In this study, intracellular GSH level in IVM oocytes was assayed by fluorescence staining method using CellTracker blue that was a specific fluorescent dye to detect reduced GSH. Compared with enzymatic assay, this fluorescent assay does not quantify total GSH content, including oxidized GSH but is easy to perform, can measure GSH level in small number of cells or oocytes, and cultures can be sequentially used for additional assays [32,33]. Modifications in culture environment can modulate gene expression in mammalian cells and embryos [34,35]. Especially in SCNT oocytes that are reconstructed with somatic cell nuclei, the timely and apposite expression of transcription factor genes during the nuclear reprogramming process of introduced somatic cell nuclei is essential for obtaining a normal developmental competence of reconstructed embryos. It has been reported that DNMT1 is involved in DNA methylation and cell proliferation [36]. PCNA is an essential component of the DNA replication and repair machinery [37], and expression of FGFR2 and POU5F1 is essential for the early development of mouse and human embryos [38,39]. Thus, the expression of mRNAs of several transcription factors in SCNT embryos has been analyzed to monitor nuclear reprogramming and embryonic development [40,41]. When the mRNA expression levels of several developmentally important genes in SCNT embryos were analyzed to assess whether improved embryonic development was correlated with gene expression profiles, the treatment of recipient oocytes with L-carnitine during IVM significantly increased the DNMT1, PCNA, FGFR2, and POU5F1 mRNA levels in this study. It was not clear how L-carnitine treatment increased mRNA levels, but it could be hypothesized that cytoplasmic modification, including increased GSH level and reduced ROS activity that was induced by L-carnitine treatment created beneficial microenvironments for nuclear reprogramming of donor nuclei and stimulated gene expression in cloned embryos [40,41]. mRNA level in PA embryos was not analyzed in this study because PA embryos may have different nuclear reprogramming than SCNT embryos and therefore the gene expression pattern in PA embryos may not be a good reference for SCNT embryos. Apoptosis is considered a normal process that
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eliminates cells with DNA or chromosomal abnormalities [42]. However, an excessive number of apoptotic cells during embryonic development may lead to a loss of viability and developmental failure [43,44]. It has been reported that apoptosis can be induced via oxidative stress derived from ROS generation when oocytes or embryos are cultured in vitro and L-carnitine acts as an antioxidant in animal cells or embryos and reduces apoptosis induced by oxidative stress by increasing intracellular GSH level and affecting mitochondrial functions [18,26,45]. Thus, we examined the expression of the anti- and proapoptotic genes p-Bcl-xl and BAX, respectively, to determine whether apoptosis would be decreased in SCNT blastocysts after treatment of recipient oocytes with an antioxidant, L-carnitine. Although L-carnitine enhanced the expression of both anti- and proapoptotic genes compared with no treatment, the enhancement of expression was more prominent with the proapoptotic gene (BAX) than with that antiapoptotic gene. This was in contrast to the previous result [26] that L-carnitine treatment during IVM reduced incidence of apoptotic nuclei in parthenogenetic blastocysts in pigs. In this study, the reason for increased expression of the proapoptotic gene despite the reduced ROS levels in IVM oocytes and improved embryonic development due to L-carnitine treatment was not known. Recently, it has been reported that apoptosis appears more frequently in competent oocytes from cycling gilts than in less competent oocytes from prepubertal gilts [46]. The results of previous and present study suggest that incidence of apoptosis may not be a good indicator to predict developmental competence of oocytes and embryos. An additional analysis of anti- and proapoptotic gene expression in IVM oocytes would be helpful to determine the effect of L-carnitine on oocyte apoptosis and later embryonic development after PA or IVF. In summary, our results suggest that IVM treatment of oocytes with L-carnitine improves the preimplantation development of PA and SCNT embryos by increasing intracellular GSH levels, reducing ROS toxicity, and regulating expression of POU5F1 and transcription factor(s) expression, which are necessary for the normal development of SCNT embryos.
Acknowledgments The authors thank Gyeonggi Veterinary Service for the generous donation of pig ovaries. This work was sup-
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ported by a grant (# PJ007113022011) from the BioGreen21 Program, Rural Development Administration, Republic of Korea, and by the Institute of Veterinary Science, Kangwon National University.
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