ABSTRACT
The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%) or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.
Subject(s)
Animals , Blastocyst/cytology , Cloning, Organism/veterinary , Culture Media/metabolism , Dogs/embryology , Embryo Culture Techniques/veterinary , Embryonic Development , Nuclear Transfer Techniques/veterinaryABSTRACT
The Rho GTPases are the sub-group of Ras super family and identified in all eukaryotes. The Rho GTPases effect different cellular signaling pathways involved in a number of diseases such as cancer, neurological and cardiovascular disorders. Members of Rho GTPases including RhoA, RhoC and Rac1 play a major role in regulation of apoptosis in different kind of stress conditions. Here we investigated the Rho GTPase activating protein 15 [ArhGAP15] gene knock-down effect on apoptosis induced by ethanol in bovine fibroblast cells. The bovine Fibroblast cells were treated and transfected with two different concentrations [50 and 100 nM] of ArhGAP15 siRNA for 48 h respectively. Both concentrations of siRNA were effective and the results of RT-PCR revealed an efficient knock-down of ArhGAP15 mRNA in fibroblast cells. Further, the normal cells exposed to a 100 mM ethanol concentration showed a reduction in cell viability and induced the ratio of apoptosis related Bax/Bcl-2 proteins compared with ArhGAP15 siRNA transfected ethanol treated cells. Ethanol also increased caspase-3 expression in normal fibroblast cells compared with transfected cells. The ArhGAP15 knock-down cells treated with ethanol decreased Bax/Bcl-2 ratio and lower caspase-3 protein levels in ArhGAP15 knocked-down cells. Our results suggest that apoptosis induced by ethanol involves the activation of Rho GTPase activating protein 15 and silencing of the said gene protects apoptosis
Subject(s)
Animals , RNA, Small Interfering/genetics , Transfection/methods , bcl-2-Associated X Protein/genetics , RNA, Messenger , Fibroblasts/drug effects , Ethanol/pharmacology , Cell Survival/genetics , Cells, Cultured , Apoptosis/genetics , Caspase 3/genetics , CattleABSTRACT
Fetal alcohol syndrome (FAS) is a developmental neuropathology resulting from in utero exposure to ethanol; many of ethanol's effects are likely to be mediated by the neurotransmitter gamma-aminobutyric acid (GABA). We studied modulation of the neurotransmitter receptor GABABR and its capacity for intracellular signal transduction under conditions of ethanol treatment (ET) and RNA interference to investigate a potential role for GABA signaling in FAS. ET increased GABAB1R protein levels, but decreased protein kinase A-alpha (PKA-alpha), calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylation of cAMP-response element binding protein (p-CREB), in cultured hippocampal neurons harvested at gestation day 17.5. To elucidate GABAB1R response to ethanol, we observed the effects of a GABABR agonist and antagonist in pharmacotherapy for ethanol abuse. Baclofen increased GABABR, CaMKII and p-CREB levels, whereas phaclofen decreased GABABR, CaMKII and p-CREB levels except PKA-alpha. Furthermore, when GABAB1R was knocked down by siRNA treatment, CaMKII and p-CREB levels were reduced upon ET. We speculate that stimulation of GABAB1R activity by ET can modulate CaMKII and p-CREB signaling to detrimental effect on fetal brain development.
Subject(s)
Animals , Pregnancy , Rats , Baclofen , Brain , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Carrier Proteins , Ethanol , Fetal Alcohol Spectrum Disorders , gamma-Aminobutyric Acid , Hippocampus , Neurons , Neurotransmitter Agents , Phosphorylation , Protein Kinases , Receptors, Neurotransmitter , RNA Interference , RNA, Small Interfering , Signal TransductionABSTRACT
To evaluate ethanol effects to induced activation of caspsae-3, and to observe the protective effects of Vitamin C [vit-C] on ethanol-induced apoptotic neurodegeneration in rat cortical area of brain. Administration of a single dose of ethanol in 7-d postnatal [P7] rats triggers activation of caspase-3 and widespread apoptotic neuronal death. Western blot analysis, cells counting and Nissl staining were used to elucidate possible protective effect of vit-C against ethanol-induced apoptotic neurodegeneration in brain. The results showed that ethanol significantly increased caspase-3 expression and neuronal apoptosis. Furthermore, the co-treatment of vit-C along with ethanol showed significantly decreased expression of caspase-3 as compare to control group. Our findings indicate that vit-C can prevent some of the deleterious effect of ethanol on developing rat brain when given after ethanol exposure and can be used as an effective protective agent for Fetal Alcohol Syndrome [FAS]