The Effects On Mouse Oocyte Meiosis And Preimplantation Embryo Development By Dot1l RNA Interference

In recent years, epigenetics research on the mechanism of histone covalent modifications become a research hotspot gradually. Histone covalent modifications regulate a variety of biological processes such as cell cycle, growth, development and disease. Different histone modifications involved in a variety of cellular function in cell cycle regulation, transcription and DNA breaks repair. The mouse histone H3K79 methyltransferase Dotll catalyzes mono-, di-, tri-methylation of H3K79. Dotll plays a role on DNA double-strand breaks repair, gene activation, transcription and cell cycle regulation. Methylation of histone H3K79 is essential on the telomere recruitment of Sir protein which maintains the heterochromatic gene silencing. Lack of H3K79 methylation, Sir protein is mislocated from silent areas of heterochromatin and unable to complete the telomere positioning. H3K79 methylation can regulate DNA repair course. Via its conserved tudor domain, checkpoint protein 53BP1 binds indirectly to methylated Lys79 and activates the checkpoint pathway to repair damaged DNA.Mouse GV stage oocytes blocked in the diplotene stage of meiosis, the homologous recombination of chromatin has been basically completed. Although DOT1 is the pachytene checkpoint in yeast, dotl mutations lead to an increase of inviable spores. Dotll role is unclear in oocytes of GV stage to the M-II stage in mice. This experiment is intended to suppress Dotll expression in oocytes by the method of cytoplasm injection of Dot11 siRNA and observe meiosis in oocytes.1. RNA interference of Dotll expression during oocytes meiosisWe design Dotll siRNA for mice by software. The GV stage oocytes were randomly divided into three groups:the first two groups were injected Dotll siRNA or scramble RNA, and the third group was control group without injection. After the injection, oocytes cultured in IVM solution containing IBMX for 24h and Dotll mRNA was detected by quantitative PCR, Dotll protein was detected by Westerablot and H3K79me2 by immunofluorescence. The results showed that:the injection of siRNA Dotll significantly reduced the Dotl 1 mRNA, protein level and H3K79me2 in the oocytes. Each of the groups of oocytes cultured in IVMI medium for 24h, then moved into the IVM medium to resume meiosis in oocytes, and observed the GVBD and MⅡ process. The results showed that: oocytes injected with Dot11 siRNA passed through GVBD smoothly, but arrested in the early MⅠ or mid MⅠ. Furthermore, immunofluorescence staining with the checkpoint protein BubRl antibody found that a large number of the BubR1 attached to oocytes telomeres in siRNA injected group suggest that Dot11 deficiency will result in the kinetochore and the spindle can not establish an appropriate attachment, and oocytes can not enter the M Ⅰ anaphase. The results of H3K79me2 and H3K79me3 immuno-fluorescence experiment showed that, in 10 month old mice and 3-week-old mice oocytes, H3K79me2 level was significantly lower in the aging mouse oocytes than in young mice. Finally, we examined the level of acetylation in oocytes after Dot11 siRNA injection. The results showed that H3K27 acetylation and H4K12 acetylation level rise significantly after siRNA injection, suggesting incomplete deacetylation.H3K79me2 was expressed in mitosis and not expressed in interphase in pre-implantation embryos of mouse. H3K79me3 is not expressed in preimplantation developmental stages. We also injected Dot11 siRNA into the prokaryotic embryos to interfere the expression of Dotll during pre-implantation. Based on the observation of embryonic development, transcription activity, karyotype and other indicators, we want to clarify Dot11 functions in mouse preimplantation embryos.2. The effects of Dot11 interference on the mouse preimplantation embryo developmentFive hours after in vitro fertilization (IVF 5h), the embryos containing two pronuclei were selected and divided into three groups randomly. The first two groups were injected with Dotll siRNA or scramble RNA, and the third group was control group without injection. Three groups of embryos were moved into KSOM medium and cultured in vitro. Two-cell embryos were collected at IVF 24h and detected Dot11 mRNA level by quantitative PCR; four-cell embryos were collected at IVF 45h and detected the protein Dot11 level by immunofluorescence; four-cell embryos in M phase were collected at IVF 50h and detected H3K79me2 level by immunofluorescence.The results showed that:the injection of siRNA can significantly reduce Dot11 mRNA level of the two-cell stage, Dot11 protein level of the four-cell embryos and H3K79me2 level of the four-cell embryos in M phase. The embryos of three treatment groups were cultured in vitro in KSOM medium respectively, and the embryos development stages were checked at IVF 24,48,72 and 96h. The results showed that the injection of siRNA can decrease the embryos development ratio significantly. The transcription activity of the three treatment groups embryos were detected at four, eight-cell embryo and morula stage. The results indicated that there were no significant differences among the three groups. The embryos of the three groups were cultured to the blastocyst in vitro, and submitted to TUNEL assay and karyotype analysis. The results showed that the injection of Dotll siRNA can significantly increase the cell apoptosis rate of blastocyst, and can significantly increase the blastocyst cell aneuploidy ratio.Our results reveal that in the Dotll defects mouse oocytes, kinetochores can not attach to the spidle appropriately and oocytes were arrested at M I pro-metaphase, and deacetylation incompletely. Defected Dotll in preimplantation embryo results in decreasing embryonic development rate, increasing the apoptosis rate and increasing aneuploidy rate.