| Aims:The studies concerning mammal preimplantation embryo development, differentiation and embryonic genome activation have become the focus in the field of reproductive medicine. To study the regulation mechanism of early embryogenesis is essential for understanding the development of embryo preimplantation and improving the success rate of assisted reproduction technology. And it will facilitate to find out the pathogenesis of the infertility and sterility, birth defects, and hereditary diseases.The genome of mouse and human has extensive homology. Mouse embryo and human embryos also have many common features. Research on mouse embryo implantation helps people to understand the process of human embryo implantation. mir-290 cluster is highly expressed during mouse preimplantation embryo development, while specific effects of mir-290 cluster on mouse preimplantation embryos and related regulation mechanisms are still not fully clarified. Recent researches show that micro RNA involved in the process of autophagy by targeting autophagy-related genes, in response to intra- or extracellar stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Autophagy is a fundamental biological process that controls the quality andquantity of intracellular biomass. During the oocyte-to-embryo transition, autophagy leads to the degradation of maternal components for the dynamic remodeling of subcellular compartments, turnover and recycling of macromolecules, and regulation of cellular activity through the control of speci?c intracellular signaling pathways. The recent study shows that, mir-290 cluster suppresses autophagic cell death of mouse melanoma cells by targeting autophagy-related genes Atg7 and ULK1. However, the involvement of mir-290 cluster though autophagy pathway and its function during the development of mouse preimplantation embryo remain to be investigated. Thus, the present study will focus on mi R-291a/b-5p to explore its function on Atg5 and Becn1, its effects of autophagy, and observe its influence on the development of mouse preimplantation embryos.Methods:1. To collect mouse oocytes and embryos, female C57BL/6J mice, averaging 4 weeks of age, were superovulated to provide embryos. After superovulation, females were mated with C57BL/6J males and inspected the following morning for copulation plugs. Preimplantation embryos were collected at each cell stage. 2. To analyze the expression of target genes in mouse preimplantation embryos from different stages, total RNA was isolated from embryos at different developmental stages using Pico Pure® RNA Isolation Kit following the manufacturer’s protocol. c DNA was prepared by using SYBR® Prime ScriptTM mi RNA RT-PCR Kit for Real Time PCR detection. 3. Mouse embryo immune-fluorescence detection. oocytes and embryos were prepared by fixation and permeabilized in 1% PFA and 0.2% Triton? X-100 solution at room temperature for 1 h. They were then washed under the stereomicroscope and blocked. Oocytes and embryos were then double stained to visualize LC3 B and LAMP, and stained for DNA. Oocytes and embryos were observed in fluorescence microscope. 4. For mouse embryo TEM specimen preparation and testing, 200 embryos were collected under the stereomicroscope, and moved into the ampulla of mouse fallopian tube. The embedded embryos with fallopian tube were fixed in cold 3%inglutaraldehyde solution at 4℃ overnight. The samples were then prepared for subsequent routine and observed by transmission electron microscopy. 5. Dual luciferase-reporter assay. Vectors containing wild-type or mutated targeted sequences from Atg5 and Becn1 3’UTR were cotransfected with mi R-291a/b-5p into NIH 3T3(mouse embryonic fibroblast cell line) cells. Firefly and renilla luciferase activities were measured using a dual luciferase-reporter assay system. 6. mi R-291a/b-5p were overexpressed in NIH/3T3 cells for testing rapamycin-induced and autophagy-related LC3-â… to LC3-â…¡conversion. For EGFP-LC3 tests, mi RNA and EGFP-LC3 were co-transected into NIH/3T3 cells. Cells were treated with rapamycin for 24 h, fixed and analysed using an fluorescence microscope. 7. Microinjection and culture of mouse embryo in vitro. 1-cell embryos were collected for cytoplasm microinjection of 5’FAM modified mi R-291a-5p inhibitor. After injection, embryos were cultured in KSOM medium. The expression of injected mi R-291a-5p inhibitor in mouse embryos were observed using an fluorescence microscopy. The effect of mi R-291a-5p inhibitor on the expression of mi R-291a-5p, Atg5 m RNA and Becn1 m RNA in embryos was detected by Real Time PCR. The influence of mi R-291a-5p inhibitor on the early development of mouse embryos were observed daily and photographed.Results:1. mi R-291a/b-5p, Atg5 and Becn1 were expressed dynamically during mouse preimplantation embryos development. During the 2~4-cell stage, mi R-291a/b-5p expression increased while Atg5 and Becn1 m RNA expression decreased. An corresponding relationship in opposite direction between the expression of mi R-291a/b-5p and the m RNA of Atg5 and Becn1 were observed during this stage. 2. Immune fluorescence detection was used to monitor the formation of autophagosomes in mouse preimplantation embryos. After fertilization, the formation of autophagosomes and autolysosomes increased in 1-cell, decreased from 2-cell stage, and rapidly decreased during 4~8-cell stages.3. TEM was used to observe autophagosomes in the cytoplasm. It is found that there are autophagosomes in the cytoplasm of fertilized egg with a double-layer membrane structure, whereas such structure was not observed in unfertilized oocyte cytoplasm. 4. Dual luciferase-reporter assay showed that mi R-291a/b-5p targeted Atg5 and Becn1. mi R-291a/b-5p inhibited the expression of Atg5 and Becn1 m RNA and protein in NIH/3T3 cells. mi R-291a-5p inhibited rapamycin-induced autophagy-related LC3-I to LC3-II conversion, and inhibited the formation of EGFP-LC3 dots. 5. After microinjection of 5’FAM modified mi R-291a-5p inhibitor into the cytoplasm of mouse zygotes, the green fluorescence of mi R-291a-5p inhibitor were observed under fluorescence microscope. After the injection of mi R-291a-5p inhibitor, the expression of mi R-291a-5p was downregulated, while the expression of Becn1 m RNA and Atg5 m RNA was upregulated. Further, the embryo development rates of mi R-291a-5p inhibitor injection group in 1-cell stage to 2-cell stage were higher than those in other groups, and its blastocyst formation rate in 4.5dpc were higher than that of control group.Conclusion:1. mi R-291a/b-5p located in mir-290 clusters, and expressed dynamically during mouse preimplantation embryo development. The expression of mi R-291a/b-5p in mouse embryos increased during 2~4-cell stage, and was accompanied by the decreasing expression of Atg5 and Becn1 m RNA. 2. mi R-291a/b-5p regulates rapamycin-induced autophagy by targeting of autophagy related genes- Atg5 and Becn1 in NIH/3T3 cells. 3. Downregulation of endogenous mi R-291a-5p in mouse zygote can increase the expression of Atg5 and Becn1 m RNA in mouse embryos, and facilitate mouse embryos first cleavage and blastocyst formation. |
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