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Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 μM rapamycin/24 h, 47.52±5.68) or control oocytes (44 h IVM; 42.14±4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

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a b s t r a c tWe generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-L-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile DamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, DamiC mutant cells were unable to establish symbiotic association. But, DamiC mutant complemented with amiC gene restored in vivo symbiotic association. DamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When DamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont.

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Abstract. Embryonic stem (ES) cells are expanded versions of the inner cell mass cells that compose the early mammalian blastocyst. Components derived from ES cells may contain various bioactive materials (BM) helpful for early preimplantation embryo growth. In this study, we examined the effect of human ES cell derived BM (hES-BM) on in vitro culture of bovine embryos. When bovine parthenogenetic day 2 embryos were cultured in 10% hES-BM, a significantly higher embryo development rate (44.3%) and increased cell numbers were observed relative to control medium containing 3 mg/ml BSA (19.5%; P<0.01). Among the various concentrations (5, 10 and 15%) and days of treatment (2 or 4 days) tested, 10% hES-BM treatment for 4 days provided the best culture environment to support the growth of bovine embryos in vitro (P<0.05). Little difference was observed between 10% hES-BM and 10% FBS treatment in the examined parthenogenetic or in vitro fertilized embryos, although the hES-BM group developed at a slightly better rate. However, the ICM cell numbers were significantly higher in the hES-BM group in irrespective of embryo origin (P<0.05). In addition, the relative levels of pluripotency (Oct4, × 1.8 fold; Nanog. × 3.3 fold), embryogenesis (Stat3, × 2.8 fold; FGF4, × 18.8 fold; E-cad, × 2.0 fold) and growth (Glut5, × 2.6 fold) genes were significantly higher in the 10% hES-BM group than in the 10% FBS group (P<0.05), while the levels of other genes (Bax, Bcl2, MnSOD and Connexin43) were not different. This is the first report examining the positive effects of hES-BM on bovine embryo development in vitro. Based on our results, we conclude that hES-BM can be used as a new protein supplement for bovine preimplantation embryo development.

ObjectiveTo investigate the effect of pulsed radiofrequency (PRF) applied proximal to the injured peripheral nerve on the expression of tumor necrosis factor-α (TNF-α) in a neuropathic pain rat model.MethodsNineteen male Sprague-Dawley rats were used in the study. All rats underwent chronic constriction injury (CCI) procedure. After 7 days of CCI, withdrawal frequency of affected hind paw to mechanical stimuli and withdrawal latency of affected hind paw to heat stimulus were measured. They were randomly divided into two groups: group A, CCI group (n=9) and group B, CCI treated with PRF group (n=10). Rats of group B underwent PRF procedure on the sciatic nerve. Withdrawal frequency and withdrawal latency were measured at 12 hours, and 7 days after PRF. Immunohistochemistry and Western blot analysis were performed using a TNF-α antibody.ResultsBefore PRF, withdrawal frequency and withdrawal latency were not different in both groups. After PRF, withdrawal frequency decreased and withdrawal latency prolonged over time in group B. There was significant interaction between time and group for each withdrawal frequency and withdrawal latency. Group B showed decreased TNF-α immunoreactivity of the spinal cord and sciatic nerve at 7 days.ConclusionPRF applied proximal to the peripheral nerve injury is potentially helpful for the reduction of neuropathic pain by neuromodulation of inflammatory markers.