Elsevier

Theriogenology

Volume 78, Issue 2, 15 July 2012, Pages 235-243
Theriogenology

Research article
L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression

https://doi.org/10.1016/j.theriogenology.2012.02.027Get rights and content

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.

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.

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