| Antioxidant of Bamboo Leaves (AOB) is a kind of flavonoid extracted from bamboo leaves. Many kinds of flavonoids have estrogen efficiency, which can cause dose-dependent effects on animal reproduction and embryonic development. Giant panda (Ailuropoda melanoleuca) is a rare and endangered species in China, known as "national treasure" and "living fossils", because of its’ low reproductive capacity, the giant panda population has become extinct. Bamboo is the main food source of the giant panda, whether the low reproductive capacity of the giant panda is affected by the accumulation of AOB in vivo is unknown. So we used mouse embryo fibroblasts (MEF) cells and embryoid bodies (mEB) which differentiated from mouse embryonic stem cells (mESC) as models, to explore the effects and mechanism of AOB on the expression of reproduction and embryonic development related genesis mice, to predict the effect of AOB on Giant pandas’fertility. Main results obtained in the present study are as follows:1. AOB’s toxicity and effects on genes expression of MEF CellsMTT test, flow cytometry and morphological observation were performed to detect the effects of different concentrations of AOB on cell proliferation of MEF. The results showed that low concentrations (0 to 10 μg/ml) of AOB promote cell growth of MEF, while high levels of AOB (>100 ug/ml) has significant inhibitory effect on cells proliferation, and enhanced the apoptosis rate of MEF cell. Morphological observation found that AOB can make the attached MEF cells shrivel, unable to maintain their spindle type states.According to gene expression profile test, a total of 792 differential expressed genes (DEGs) were identified, including 478 up-regulated genes and 314 down-regulated genes. GO term analysis found that 94 GO terms are related with mouse’s reproductive and embryonic development, such as sperm-egg binding, spermatogenesis, sperm motility, embryonic development and germ cell development. KEGG analysis indicated that many DEGs are involved in pathways like oocyte meiosis, MAPK signaling pathway, P53 signaling pathway, Hippo signaling pathway, Wnt signaling pathway which are related to mouse’s reproductive and embryonic development.20 DEGs(Cxxc4 Fzdl Sfrpl, Ntrk2, Fgf10, Kng2, Cpz, Dhrs9, Pla2g4e, Mapk12, Smc3, Lrp6, Cabyr, Pcsk4, My12, Pla2g4b, B23120H23Rik, Rapla,Pdgfb,Pdgfra) which participate in apoptosis(negative regulating cell proliferation, apoptosis, keto acid metabolism, metabolism, and organic acid metabolism, etc.) and reproductive regulating function(spermatogenesis, sperm-egg binding, embryonic development, etc.) were choosed for validation by qRT-PCR. It showed that the expression profiles of these genes were consistent with the result of gene chip.2. AOB impact the activation levels of ERK and Wnt signaling pathways in MEF cellsUsing Western blot experiments to detect the different expression of ERK and Wnt signaling pathways’key proteins in MEF cells that before and after AOB treated, and the results showed that the expression level of P-ERK and beta-catenin have significantly up-regulated, shows that AOB can activate the two signaling pathways. In addition, Western blot experiments results show that after AOB treated, the expression of proteins related to reproduction (CABYR, SOX17, P450scc) have significantly up-regulated. But after treated with ERK and Wnt pathways’inhibitors-three kinds of proteins expression were significantly reduced. So we speculate that AOB may influence the expression of proteins involved in development and reproductive functions through regulating the two classical pathways’activation,3. Impact of AOB on the toxicological effect on mEB and mES CellsALP staining and immunofluorescence of Oct4 were used to identify the state of mESC. qRT-PCR test was used to detect the relative expression level of the marker genes from EB’s three layers and mES’s pluripotency to study the effects of AOB on mEB and embryonic development. The results showed that:under treatment by 50 μg/ml and 100 μg/ml of AOB, Oct4 and Nanog, the marker genes of mES’s pluripotency are up-regulated, indicating that AOB may significantly inhibit the differential procession of mESC. After treated by 50 μg/ml of AOB, Gata6, the marker genes of primitive ectoderm of mEB is significantly decreased, Sox7 and Pdgfrb are significantly increased. The marker genes of ectoderm Nestin and Fgf5 are significantly down-regulated and the marker gene of Mesoderm Brachyuary is significantly down-regulated, whereas Flkl is significantly up-regulated. Meanwhile, endoderm marker genes Afp and Soxl 7 is significantly down-regulated.After treated by 100 μg/ml of AOB, the relative expression level of marker genes of primitive ectoderm in mEB:Gata6, Sox7, Pdgfrb are significantly up-regulated; the marker genes of ectoderm Nestin and Fgf5 are significantly down-regulated; the marker genes of mesoderm Brachyuary is significantly down-regulated, whereas Flkl is significantly up-regulated; the marker genes of endoderm Afp and Sox17 are significantly up-regulated. That suggest AOB may have significant effects on mouse’s early embryonic development.The expression microarray showed that the DEGs that choosed from mEB treated by 50 μg/ml of AOB were less than that treated by 100 μg/ml, indicating that the higher concentration of AOB, the greater effect on genes’expression related to mouse’s reproductive. Base on the results of GO and KEGG analysis, DEGs related to mouse’s reproductive and embryonic development were selected and use qRT-PCR to validate the results, and the results are basically consistent with the results from gene chips. Therefore, AOB might affect mouse’s reproductive and embryonic development through the regulation of the relative expression level of the genes participating in reproductive and embryonic developmentFurthermore, the AOB significantly influence the expression of the key genes in the signaling pathways, such as Wnt and MAPK that involved in mouse’s reproductive and embryonic development.Compared with the control group, Fgf10 and Pdgfra, the target genes of miRNA-294/295-294/295 and miRNA-449abc/34ab were up-regulated in the mEB treated with 50μg/ml of AOB and 100μg/ml of AOB. It indicated that miRNA regulation may also contribute to the impact of AOB on mouse’s reproductive and embryonic development.4. AOB impact the activation levels of ERK and Wnt signaling pathways in MEF cellsUsing Western blot experiments to detect the different expression of ERK and Wnt signaling pathways’ key proteins in mEB that before and after AOB treated, and the results showed that the expression level of P-ERK and beta-catenin have significantly up-regulated, shows that AOB can activate the two signaling pathways. In addition, Western blot experiments results show that after 100 μg/ml AOB treated, the expression of proteins related to reproduction (CABYR, SOX 17, P450scc) have significantly up-regulated. But after treated with ERK and Wnt pathways’inhibitors, three kinds of proteins expression were significantly reduced. So we speculate that AOB may influence the expression of proteins involved in development and reproductive functions through regulating the two classical pathways’ activation,5. Impact of AOB on biological function caused by the differential expression genesIn order to explain how AOB affect mouse’s reproductive and embryonic development, bioinformatics was used to predict the effect of the DEGs in signaling networks and the upstream and downstream nodes of the DEGs detected from the gene chip experiments. IPA analysis was used to detect the down-stream biological effects caused by DEGs, namely the disease and functional analysis. The results showed that DEGs caused by AOB treatment mainly related to the diseases like variety of tumors, neurological symptoms, bone myopathy, etc. Pathway analysis showed that the DEGs mainly participate in MAPK, Wnt, p53 and PI3K/AKT signaling pathways.In conclusion, our results showed that MEF cells and mESC are the ideal models for the study of the effects of exogenous factor on the embryonic development and reproduction. The certain concentration of AOB may have significant impacts on animals’reproductive system and embryonic development. That may provide new thoughts to further explore the reason and mechanism of the giant panda’s reduced fertility. |
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