Research articleL-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression
Introduction
Recent progress in reproductive technologies, including in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer (SCNT) has made it possible to produce specific animals successfully in a variety of species [1], [2], [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 undesirable 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 β-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 (β-hydroxy-γ-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-α 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-Bcl-xl) 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.
Section snippets
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 μg/mL kanamycin, 1 μg/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
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-carnitine-treated 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).
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,
Discussion
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
Acknowledgments
The authors thank Gyeonggi Veterinary Service for the generous donation of pig ovaries. This work was supported by a grant (# PJ007113022011) from the BioGreen21 Program, Rural Development Administration, Republic of Korea, and by the Institute of Veterinary Science, Kangwon National University.
References (46)
- et al.
Electrical activation induces reactive oxygen species in porcine embryos
Theriogenology
(2008) - et al.
Effect of oxidative stress on development and DNA damage in in-vitro cultured bovine embryos by comet assay
Theriogenology
(2000) - et al.
Increased generation of reactive oxygen species in embryos cultured in vitro
Free Radic Biol Med
(1993) - et al.
A combination of FSH and dibutyryl cyclic AMP promote growth and acquisition of meiotic competence of oocytes from early porcine antral follicles
Theriogenology
(2011) - et al.
Presence of beta-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization
Theriogenology
(1998) - et al.
The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development effect of beta-mercaptoethanol, cysteine and cystine
Theriogenology
(2000) - et al.
L-carnitine supplementation reduces oocyte cytoskeleton damage and embryo apoptosis induced by incubation in peritoneal fluid from patients with endometriosis
Fertil Steril
(2009) - et al.
L-carnitine decreases DNA damage and improves the in vitro blastocyst development rate in mouse embryos
Fertil Steril
(2009) - et al.
Tyrosine phosphorylation of STAT3 by leptin through leptin receptor in mouse metaphase 2 stage oocyte
Biochem Biophys Res Commun
(1999) - et al.
L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs
Theriogenology
(2011)
A new method of quantifying glutathione levels in freshly isolated single superfused rat cardiomyocytes
J Pharmacol Toxicol Methods
Analysis of nuclear reprogramming in cloned miniature pig embryos by expression of Oct-4 and Oct-4 related genes
Biochem Biophys Res Commun
L-carnitine attenuates oxidant injury in HK-2 cells via ROS-mitochondria pathway
Regul Pept
The quality of porcine oocytes is affected by sexual maturity of the donor gilt
Reprod Biol
Viable offspring derived from fetal and adult mammalian cells
Nature
Cloned pigs produced by nuclear transfer from adult somatic cells
Nature
Development of porcine embryos and offspring after intracytoplasmic sperm injection with liposome transfected or non-transfected sperm into in vitro matured oocytes
Zygote
Developmental ability of cloned embryos from neural stem cells
Reproduction
Method of oocyte activation affects cloning efficiency in pigs
Mol Reprod Dev
Effect of oxygen concentration and free radicals on in vitro development of in vitro-produced bovine embryos
J Anim Sci
Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo
Development
Involvement of superoxide radicals in the mouse two-cell block
Mol Reprod Dev
Insulin-transferrin-selenium (ITS) improves maturation of porcine oocytes in vitro
Zygote
Cited by (74)
L-carnitine: Nutrition, pathology, and health benefits
2023, Saudi Journal of Biological SciencesEffects of cobalamin on meiotic resumption and developmental competence of growing porcine oocytes
2020, TheriogenologyCitation Excerpt :Despite many advancements in the in vitro production of porcine embryos in the past few years [8,9], the number of transferable embryos developed from in vitro-matured porcine oocytes [10,11] remains lower than that of transferable embryos developed from in vivo-matured porcine oocytes [12]. Moreover, oocyte quality is the limiting factor for in vivo viability and developmental competence of embryos produced in vitro [13]. A possible reason for this low developmental competence of in vitro-matured porcine oocytes is improper cytoplasmic maturation despite complete nuclear maturation [14].
- 1
These authors contributed equally to this work.