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

ArticleinTheriogenology 78(2):235-43 · May 2012with31 Reads
DOI: 10.1016/j.theriogenology.2012.02.027 · Source: PubMed
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.
    • "De plus, des études menées également chez la souris montrent que la supplémentation in vitro de L-carnitine (un co-facteur de CTP1) dans le milieu de culture des follicules améliore la bêta-oxydation mais aussi le taux de développement des embryons au stade blastocyste (Dunning et al 2011). De même chez le porc, la supplémentation du milieu de maturation en L-carnitine augmente la concentration en glutathion (un antioxydant) et favorise le développement des ovocytes (You et al 2012). L'importance de la bêta-oxydation des acides gras a également été démontrée dans la maturation nucléaire des ovocytes de souris, de vache et de porc (Paczkowski et al 2013, Dunning et al 2014 "
    [Show abstract] [Hide abstract] ABSTRACT: Abstract The influence of weight and energy intake on fertility animals and humans has been known for a long time. Animals in poor condition, or losing weight, have generally poor reproductive performance. Economic losses associated with infertility can be very important and for example in cattle, they can considerably exceed the cost of artificial insemination. Some evidence suggests that the effects of nutrition on reproductive function are mediated through dietary components such as lipids, glucose, amino acids, and minerals acting on the hypothalamic-pituitary axis but also directly on the gonads. These nutritional effects may also be mediated through changes in metabolic hormones such as insulin, insulin-like growth factor 1, growth hormone, ghrelin, thyroid hormones or hormones produced and secreted by the white adipose tissue. Here, we will review the known effects of these nutritional metabolites, nutrients and metabolic hormones on the follicular development, the activity of ovarian cells, oocyte quality and on early embryonic development in different mammalian species. Abstract (French) L’influence du poids et des apports énergétiques sur la fertilité chez les animaux, mais aussi chez l’Homme est reconnue depuis très longtemps. Les animaux ou individus en mauvaise condition, ou perdant du poids, ont généralement des performances reproductives décevantes. Les pertes économiques associées à l’infertilité sont parfois importantes, et dépassent chez le bovin largement le coût de l’insémination et de la semence. De nombreux arguments suggèrent que l’influence de la nutrition sur la reproduction s’exerce par l’intermédiaire des composants du régime alimentaire comme les lipides, le glucose, les acides aminés et les minéraux au niveau de l’axe hypothalamo-hypophysaire et aussi directement au niveau des gonades. Ces effets nutritionnels peuvent aussi s’exercer par une modulation des hormones du métabolisme comme l’insuline, l’insulin-like growth factor 1, l’hormone de croissance, la ghréline, les hormones thyroïdiennes ou encore les hormones produites et secrétées par le tissu adipeux blanc. Dans cette revue nous rapportons les effets connus de ces nutriments et hormones métaboliques sur le développement folliculaire, les cellules ovariennes, la qualité ovocytaire ainsi que sur le développement embryonnaire précoce en prenant l’exemple de différentes espèces de mammifères.
    Full-text · Article · Jul 2016 · Theriogenology
    • "IVM medium was supplemented with 80 g/mL FSH (Antrin R-10; Kyoritsu Seiyaku, Japan) and 10 IU/mL hCG (Intervet International BV, the Netherland) for the first 22 h of IVM. Porcine zygote medium (PZM)-3 containing 0.3% (w/v) bovine serum albumin (BSA) was used as the in vitro culture (IVC) medium for embryonic development, which was modified by adding 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and 10 M -mercaptoetanol as previously described [39]. "
    [Show abstract] [Hide abstract] ABSTRACT: The objective of this study was to investigate the effect of rapamycin treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Morphologically good (MGCOCs) and poor oocytes (MPCOCs) were untreated or treated with 1 nM rapamycin during 0-22 h, 22-42 h, or 0-42 h of IVM. No significant effect of rapamycin was observed in nuclear maturation and blastocyst formation after PA of MGCOCs. Notwithstanding no stimulating effect on nuclear maturation, blastocyst formation after PA was significantly increased by the rapamycin treatment during 22-42 h and 0-42 h (46.6% and 46.5%, respectively) compared to control (33.3%) and 0-22 h groups (38.6%) in MPCOCs. In SCNT, blastocyst formation tended to increase in MPCOCs treated with rapamycin during 0-42 h of IVM than in untreated oocytes (20.3% vs. 14.3%, 0.05 < P < 0.1) while no improving effect was observed in MGCOCs. Gene expression analysis revealed that transcript abundance of Beclin 1 and microtubule-associated protein 1 light chain 3 mRNAs was significantly increased in MPCOCs by rapamycin compared to control. Our results demonstrated that autophagy induction by rapamycin during IVM improved developmental competence of oocytes derived from MPCOCs.
    Full-text · Article · Mar 2015
    • "In supplementing IVM culture media, it has been reported that addition of superoxide dismutase or catalase decreases ROS and improves oocyte maturation and embryonic development in mice and cattle [43,46]. In porcine IVM, L-carnitine, anthocyanin, and resveratrol improved embryonic development in vitro by decreasing intracellular ROS levels [26,27,44]. Resveratrol is an efficient scavenger of ROS radicals such as OH, NO 2 , NO , and Cl 3 OO [47,48] and consequently suppresses lipid peroxidation of cell membranes. "
    [Show abstract] [Hide abstract] ABSTRACT: We investigated the effect of resveratrol supplementation during IVM and/or trolox during IVC on the development of porcine embryos derived from parthenogenetic activation (PA) and SCNT. In this study, we evaluated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in blastocysts, and embryonic development after PA and SCNT. To determine the combined effects of resveratrol during IVM and trolox during IVC on PA embryos, we selected optimal concentrations (2 μM of resveratrol and 200 μM of trolox) and designed four groups: (1) control, (2) resveratrol, (3) trolox, and (4) combined. All treatment groups showed significantly increased intracellular GSH levels and decreased ROS levels. Resveratrol supported significantly higher cleavage and blastocyst formation rates than the control (80.3% and 38.0% vs. 71.1% and 22.4%, respectively) by downregulating Bax/Bcl-2, Caspase-3, and Bak. Trolox showed significantly increased blastocyst formation rates (36.7%) compared with the control (22.4%) by downregulating only Caspase-3. The combined group had significantly higher cleavage and blastocyst formation rates and greater total cell numbers than the control (81.7%, 36.3%, and 67.1 vs. 71.1%, 22.4%, and 47.8, respectively) by downregulating Bax/Bcl-2, Caspase-3, and Bak. On the basis of these results, we applied sequential treatments with resveratrol and trolox to SCNT, and blastocyst formation rates and total cell numbers were significantly increased compared with the control (17.2% and 52.1 vs. 11.8% and 36.6, respectively), with increased GSH, decreased ROS levels, upregulated proliferating cell nuclear antigen, and downregulated Bax/Bcl-2 and Caspase-3. These results indicate that sequential treatment with resveratrol during IVM and trolox during IVC improved the development of PA and SCNT porcine embryos by regulating intracellular GSH, ROS levels, and gene expression. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015
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