| Backgrounds:Apoptosis can cause failure of oocyte fertilization, arrest of embryonic development, which resulting in failure of in vitro fertilization and embryo transfer (IVF-ET). Thus, antiapoptotic artificial interfering technique may be a new way of improving embryonic development quality and promoting clinical outcomes of IVF-ET. There are about75%embryos have different degrees of cell debris and asymmetry of human preimplantation embryos, and about50%of which are arrested during the first week in vitro. The reasons for this high attrition rate during early development are unclear, possibly including chromosomal abnormalities, poor culture conditions or oocytes maturation disability. It has been found that apoptosis was implicated in the arrested and fragmented embryos. The aim of this study was therefore to improve the embryo quanlity by the addition of anti-apoptotic factors.Sphingolipids are ubiquitous components of cell membranes and their metabolites sphingosine-1-phosphate (SIP) have important physiological functions, including regulation of cell survival, growth, proliferation and apoptosis. SIP is utilized as a regulator of cell function not only by binding to extracellular receptors but also work as an intracellular second messenger. SIP could protect the ovary, oocyte and male germ cells from apoptosis by radiation and heat shock, and so on.Most of the metabolites have important physiological functions. Ceramide and sphingosine inhibit cell growth and induce apoptosis, while SIP promotes cell growth and inhibits apoptosis. Stress stimuli activate sphingomyelinase produces Ceramide and promote cell apoptosis, survival factor activate sphingosine kinase1SphKl) produces S1P and inhibit the apoptosis induced by Ceramide. Therefore, the cellular balance of Cer and SIP form the "sphingolipid rheostat". Because Cer and SIP have opposite biological function, and these metabolites are interconvertible, thus the change of S1P/Cer ratio can lead to "sphingolipid rheostat" imbalance, and the dynamic balance of SIP and Cer is of crucial importance in regulating cell fate. Our previous study have found that25nM SIP could improve the blatosyst development rate, decrease2-4cell arrested rate and fragmented embryos rate of mouse early embryos in vitro. While50uM CED could significantly reduce the blatosyst development rate and increase the2-4cell arrested rate and fragmented embryos rate. Therefore, we study pre-implantation mouse embryo to evaluate the apoptotic factor C2-ceramide (CED) and anti-apoptotic factor S1P on early mouse embryo by adding50uM CED and25nMSlP into the embryo culture medium in vitro. And we use TUNEL and Caspase to detect the levels of apoptosis and proliferation of mouse embryos in order to see the mechanism of SIP inhibits C2-ceramide-induced apoptosis during murine preimplantation embryonic development.Global gene-expression profiling microarray technology become an effective tool for the study of development related genes, which can simultaneously monitor a large number of gene expression in embryos at different developmental stages. There are researches on gene expression of mouse preimplantation embryo development and cerebellar development using microarray technology. We perform global gene-expression profiling of two-cell mouse embryo using Affymetrix GeneChip(?) Mouse Genome4302.0Array as to study the effect of sphingosine-1-phosphate(S1P) on gene expression of the pre-implantation mouse embryo induced by C2-ceramide.The tumor suppressor gene p53plays an important role in several intracellular biological processes including regulation of cell cycle progression, DNA repair and apoptosis. p53can induce the expression of different proapoptosis genes through the activation of death receptor and mitochondrial apoptosis signal pathway. In early studies of tumor response to stresses, p53was treated as a downstream target. In addition, p53regulated the ratio of Bcl-2/bax and expression/activation of cysteinyl aspartate-specific protease in the apoptosis of the SKN-SH cell induced by ceramide. Therefore, we detect the expression of apoptotic gene in the pre-implantation mouse embryo by quantitative real-time PCR as well as to further investigate the anti-apoptotic mechanism of SIP. This can provide experimental evidence for application of anti-apoptotic artificial interfering technique in the clinical treatment of IVF-ET.Part I Effects of Sphingosine-1-phosphate on gene expression of two-cell mouse embryos induced by C2-ceramideObjective:To understand differentially expressed genes on two-cell mouse embryos treated by SIP and CED. To explore the apoptosis-related genes involving in the signal transduction pathways induced by CED and SIP.Methods and materials:1. Zygotes fertilized in vivo and cutured24h in vitro were randomly divided into four groups:the blank control group, CED group, CED plus methanol group and CED plus S1P group.2. Total RNA was isolated from these four groups, and performed global gene-expression profiling of two-cell mouse embryo using Affymetrix GeneChip(?) Mouse Genome4302.0Array. Affymetrix GeneChip(?) have4500probe, which can detect about34000identified genes in mice.3. The expression values of each gene were acquired using GeneChip(?) operating system. Data normalization and filtering were conducted with a DNA-ChiP analyzer. In present study, significant fold chages among genes were determined by taking the ratio of the expression between different groups. A differentially expressed gene was defined as a variation in gene expression more than two-fold. All the expressed genes were carried out by Cluster3.0and the cluster data were visualized by the Java Treeview software. The differentially expressed genes were mapped to Gene Ontology (GO) terms and the Kyoto Encyclopedia of Gene and Genomes (KEGG) pathways.Results:1. Using Clutster3.0to compare the gene expression profile changes of the two-cell mouse embryos between four groups, results showed that, compared with the blank control group, gene expression of the CED, CED plus methanol and CED plus S1P group had been changed, and the trend were similar.2. Analysis of Microarray (SAM) software was used to determine statistically significant genes. Compared with the blank group,1466differentially expressed genes were found24hours after treated by CED or CED plus methanol,408up-regulated,1058down-regulated. Of which,63apoptosis-related genes were included,29up-regulated,34down-regulated. Inadditonally, there were12anti-apoptosis genes,6up-regulated,6 down-regulated. Pathway anlysis showed the differentially expressed genes participate in the mitogen-activated protein kinase, cell cycle, p53signal pathway, oxidative phosphorylation and apoptosis signal pathway by KEGG.3. Compared with CED and CED plus methanol goup,51differentially expressed genes were found24hours after treated by both CED and S1P,26up-regulated,25down-regulated. There were three cell proliferation-related genes:Cdkn1b, Fgfr2were up-regulated, Scarbl were down-regulated. One cell differentiation-related gene:Navl was down-regulated. Three apoptosis-related genes:Tgm2was up-related:Traf3and Atm were down-regulated. Pathway anlysis showed the differentially expressed genes participate in the mitogen-activated protein kinase, cell cycle, p53signal pathway and apoptosis signal pathway by KEGG. The differentially expressed genes involved in those pathways were Cdkn1b. Atm, Fgfr2(Cdkn1b,Fgfr2were up-regulated, Atm was down-regulated).Conclusion:1. Compared with the blank control group, gene expression of the CED, CED plus methanol and CED plus SIP group have been changed, and the trend was similar.2. The gene expression of two-cell mouse embryos has been changed after treatment of CED, apoptosis-related genes, anti-apoptosis genes and genes involved in apoptosis-related signal pathways have been changed, some were up-regulated,some were down-regulated.3. SIP may inhibit embryonic cell apoptosis by down-regulating Atm and promote cell proliferation by up-regulating Cdkn1b, Fgfr2. SIP may inhibit embryonic cell apoptosis through cell cycle, p53signal pathway, mitogen-activated protein kinase and apoptosis signal pathway. Part II The effect of Sphingosine-1-phosphate and C2-ceramide during murine preimplantation embryonic developmentObjective:To investigate the effect of antiapoptotic factor S1P on suppressing apoptosis of preimplatation mouse embryos induced by C2-ceramid. As to understand the mechanism of antiapoptotic factor in inhibiting apoptosis and promoting development during murine preimplantation embryonic development. At the same time, to explore the roles of apoptotic-related genes p53during the development of mouse embryos.Methods and materials:1. Zygotes obtained from female mice were cultured in vitro for120hours in different groups. The number and morphous of proliferating embryonic cells were observed and counted regularly as well as blastocyst development rates were calculated.2. The levels of apoptosis and proliferation of mouse embryos were detected by the apoptotic assays for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Caspase in situ fluorescence methods.3. The expression of p53mRNA at morula stage was detected by quantitative real-time PCR.4. The experiment was divided into four groups:the blank group, the CED group, the CED+methanol group and the CED+S1P group.Results:1. The results showed that81.3%embryos of the control group and65.3%of the CED+S1P group developed to the blastocyst stage, whereas only24.1%of the CED group and27.9%of the CED+methanol group developed to the blastocyst stage (P<0.01). The results of the embryos cultured in vitro suggested that66.34%embryos from the CED group and61.33%embryos from the CED+methanol group arrested at morula stage.2. The green fluorescent signal active CASPASE and TUNEL signal were higher in the blastocysts derived from the CED group and the CED+methano group than the blank group and CED+S1P group.3. The apoptotic cell number of blastocysts from the CED group (9.3±0.7) and the CED+methanol group (8.7±0.5) were significantly higher than that from the control group(1.3±0.3) and the CED+S1P group (3.2±0.6)(P<0.05). The total cell number of the embryos cultured in the CED group (81.3±5.1, n=26) or the CED+methanol group at the blastocyst stage (83.9±5.4, n=26) was not significantly different from that of the control group (89.3±4.5, n=27) and the CED+S1P group (84.1±5.2, n=26).4. The expression level of p53mRNA in the CED group and CED+methanol group was increased compared with the control group and the CED+S1P group (P<0.05).Conclusion:1. CED decreased the mouse blastocyst formation rate, increased the expression level of p53at morla stage and the apoptosis of the blastomeres at blastocyst stage.2. S1P can partly inhibit the apoptosis of the blastomeres at blastocyst stage, increase the mouse blastocyst formation rate and decrease the expression level of p53at morla stage induced by CED.3. SIP may inhibit the murine embryonic apoptosis by reducing the expression of p53. |
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