L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression.
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.
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ABSTRACT: We have previously shown that fatty acid oxidation (FAO) is required for AMP-activated protein kinase (PRKA)-induced maturation in vitro. In the present study, we have further investigated the role of this metabolic pathway in hormone-induced meiotic maturation. Incorporating an assay with 3H-palmitic acid as substrate, we first examined the effect of PRKA activators on FAO levels. There was a significant stimulation of FAO in cumulus cell-enclosed oocytes (CEO) treated with AICAR and RSVA405. In denuded oocytes (DO), AICAR stimulated FAO only in the presence of carnitine, the molecule that facilitates fatty acyl CoA entry into mitochondria. The carnitine palmitoyltransferase 1 (CPT1B) activator C75 successfully stimulated FAO in CEO. All three of these activators trigger germinal vesicle breakdown. Meiotic resumption induced by follicle-stimulating hormone (FSH) or amphiregulin was completely inhibited by the FAO inhibitors etomoxir, mercaptoacetate, and malonyl CoA. Importantly, FAO was increased in CEO stimulated by FSH and epidermal growth factor (EGF), and this increase was blocked by FAO inhibitors. Moreover, compound C, a PRKA inhibitor, prevented the FSH-induced increase in FAO. Both carnitine and palmitic acid augmented hormonal induction of maturation. In a more physiological setting, etomoxir eliminated hCG-induced maturation in follicle-enclosed oocytes. In addition, CEO and DO from hCG-treated mice displayed an etomoxir-sensitive increase in FAO, indicating that this pathway was stimulated during in vivo meiotic resumption. Taken together, our data indicate that hormone-induced maturation in mice requires a PRKA-dependent increase in FAO.Biology of Reproduction 07/2013; · 4.03 Impact Factor
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ABSTRACT: Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of (3)H2O from [(3)H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid oxidation during in vitro maturation and resulted in poor embryo development points to the developmental importance of fatty acid oxidation and the need for it to be optimized during in vitro maturation to improve this reproductive technology.PLoS ONE 01/2014; 9(2):e87327. · 3.53 Impact Factor
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ABSTRACT: The objective of this study was to examine the developmental competence of IVM pig oocytes in relation to the pattern of morphologic changes after exposure to hyperosmotic medium to select oocytes of a higher quality. IVM oocytes were treated with a hyperosmotic (593 mOsm) medium containing NaCl, sorbitol, or sucrose. Oocytes that shrunk spherically (SSP oocytes) or in irregular shapes (SIR oocytes) were collected separately, and washed for 15 minutes in an isotonic (297 mOsm) medium for recovery. Irrespective of the chemicals used, hyperosmotic treatment of oocytes for 1 hour or 15 minutes did not alter embryonic development after parthenogenesis (PA) and SCNT. A significantly higher proportion of SSP oocytes developed to the blastocyst stage (34.0%) compared with SIR oocytes (15.8%) after PA. The intracellular glutathione content was significantly higher in SSP oocytes than in SIR oocytes. Conversely, the reactive oxygen species level was significantly higher in SIR oocytes than in SSP oocytes. The maturation promoting factor level as measured by p34(cdc2) kinase activity was not influenced by hyperosmotic treatment itself but was 1.3-fold higher (P < 0.05) in SSP oocytes than in SIR oocytes. When IVM oocytes were divided into two groups according to their diameters (large and small), and treated separately in hyperosmotic medium, significantly more SSP oocytes (71.4%) were found in the large oocytes than in the small oocytes (51.4%). Moreover, the proportion of metaphase II oocytes was significantly higher in SSP oocytes than in SIR oocytes in both groups (98.5% vs. 73.1% in large oocytes, and 92.2% vs. 48.0% in small oocytes). After SCNT, a significantly higher proportion of SSP oocytes displayed blastocyst formation (36.4%) than untreated (29.0%) and SIR oocytes (22.1%). Our results demonstrated that SSP oocytes were of a higher quality than SIR oocytes, which was shown by higher intracellular glutathione and maturation promoting factor levels, lower reactive oxygen species levels, and improved embryonic development to the blastocyst stage after PA and SCNT.Theriogenology 01/2014; · 2.08 Impact Factor