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ABSTRACT: We examined the expression patterns of porcine sirtuin 1 to 3 (Sirt1-3) genes in preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the effects of sirtuin inhibitors (5 mM nicotinamide [NAM] and 100 μM sirtinol) on embryonic development of PA and IVF embryos under in vitro culture (IVC). The expression patterns of Sirt1-3 mRNA in preimplantation embryos of PA, IVF, and SCNT were significantly (P < 0.05) decreased from metaphase stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1-3 in SCNT blastocysts were significantly (P < 0.05) lower and Sirt2 in PA blastocyst was significantly higher compared with the IVF blastocysts. Treatment with sirtuin inhibitors during IVC resulted in significantly (P < 0.05) decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate in both PA and IVF embryos. The early and expanded blastocyst formations at Day 7 were significantly lower in the sirtuin inhibitors-treated groups than the control. It was demonstrated that sirtuin inhibitor (NAM) influenced the percentage of blastocyst formation and total cell number of PA derived blastocyst when NAM was added during Day 4 to 7 (22.1% and 32.4) or Day 0 to 7 (23.1% and 31.6) of IVC compared with the control (41.8% and 41.5). No significant difference in cleavage rates appeared among the groups. The blastocysts derived from PA embryos treated with sirtuin inhibitors showed lower (P < 0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly decreased in both NAM and sirtinol treated groups compared with the control. In conclusion, these results suggest that sirtuins may have a physiological and important role in embryonic development of porcine preimplantation embryos by regulating essential gene expressions of developing embryos. These findings could have implications for understanding the role of sirtuins during embryo development and for improving SCNT and related techniques.
Theriogenology 10/2012; 78(7):1597-610. · 1.96 Impact Factor
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ABSTRACT: We examined the effects of porcine granulocyte-macrophage colony-stimulating factor (pGM-CSF) on the in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) for the first time. We evaluated the effects of pGM-CSF on SCNT-derived blastocyst formation and investigated gene expression. A total of 522 cloned embryos in 6 replicates were treated with 10 ng/ml pGM-CSF during in vitro culture (IVC). This treatment significantly (P<0.05) increased blastocyst formation and total cell number in blastocysts compared with the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no effect on cleavage rate. The numbers of cells in the inner cell mass and trophectoderm were significantly higher in the pGM-CSF treatment group (6.0 and 43.0, respectively) compared with the control (4.4 and 31.9, respectively). Treatment with 10 ng/ml pGM-CSF significantly increased POU5F1 and Cdx2 mRNA expression in blastocysts. In addition, Bcl-2, Dnmt1 and proliferating cell nuclear antigen (PCNA) mRNA expression were upregulated in blastocysts in the pGM-CSF supplemented group compared with the control. These results suggest that pGM-CSF improves the quality and developmental viability of porcine SCNT embryos by regulating transcription factor expression.
Journal of Veterinary Medical Science 04/2012; 74(9):1095-102. · 0.85 Impact Factor
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ABSTRACT: We investigated the effects of resveratrol, a phytoalexin with various pharmacologic activities, on in vitro maturation (IVM) of porcine oocytes. We investigated intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, as well as gene expression in mature oocytes, cumulus cells, and in vitro fertilization (IVF)-derived blastocysts, and subsequent embryonic development after parthenogenetic activation (PA) and IVF. After 44 h of IVM, no significant difference was observed in maturation of the 0.1, 0.5, and 2.0 μM resveratrol groups (83.0%, 84.1%, and 88.3%, respectively) compared with the control (84.1%), but the 10.0 μM resveratrol group showed significantly decreased nuclear maturation (75.0%) (P < 0.05). The 0.5- and 2.0-μm groups showed a significant (P < 0.05) increase in intracellular GSH levels compared with the control and 10.0 μM group. Intracellular ROS levels in oocytes matured with 2.0 μM resveratrol decreased significantly (P < 0.05) compared with those in the other groups. Oocytes treated with 2.0 μM resveratrol during IVM had significantly higher blastocyst formation rates and total cell numbers after PA (62.1% and 49.1 vs. 48.8%, and 41.4, respectively) and IVF (20.5% and 54.0 vs. 11.0% and 43.4, respectively) than the control group. Cumulus-oocytes complex treated with 2.0 μM resveratrol showed lower expression of apoptosis-related genes compared with mature oocytes and cumulus cells. Cumulus cells treated with 2.0 μM resveratrol showed higher (P < 0.05) expression of proliferating cell nuclear antigen than the control group. IVF-derived blastocysts derived from 2.0 μM resveratrol-treated oocytes also had less (P < 0.05) Bak expression than control IVF-derived blastocysts. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF porcine embryos by increasing the intracellular GSH level, decreasing ROS level, and regulating gene expression during oocyte maturation.
Theriogenology 03/2012; 78(1):86-101. · 1.96 Impact Factor
