Studies On Histone Acetylation In Oocyte Maturation In Vitro And Early Embryo Development In Pigs

In the past decades,the studies on epigenetic modifications flourished. Thefunction of epigenetic modification in cell oncogenesis,imprint gene expression,celltotipotency and pluripotency had been overdicussed. However, the effects of thereprogramming of the genome and changes in gene expression patterns onmammalian embryo development need to be researched further. How to promote theembryo development is one of the problems of assisted reproduction in vitro andcloned transgenic animal production to be solved urgently. In order to elucidate therole of histone acetylation in embryos and find the beneficial method for embryoproduction, the present study changed the level of the histone acetylation with histonedeacetylase inhibitor (HDACi)---sodium butyrate (NaBu) and shRNA interferingtechnique and investigated the effects of histone acetylation on porcine oocytesmaturation in vitro and the early development of the embryos.The role of NaBu on porcine oocytes maturation and subsequent embryonicdevelopmental competence were evaluated firstly. Cumulus oocyte complexes (COCs)collected from the ovaries obtained at a local slaughterhouse were divided intodifferent groups randomly and matured in vitro in maturation medium at39℃,5%CO2.After44h maturation in vitro (IVM), the oocytes with evenly dark ooplasmand visible first polar bodies were considered matured. The levels of histoneacetylation were examined with immunostaining.(1)To explore the role of NaBu onmeiotic resumption, the oocytes were cultured with1.0mMNaBu for a short time (2h)and a long time (24h) in maturation medium respectively. The results showed thatNaBu delayed porcine oocyte meiosis in the germinal vesicle stage (GV) and GVBDin proportion to the length of exposure.(2)To study the effects of NaBu on meioticprogression through metaphase I to metaphase II, oocytes were cultured for24h and subsequently treated with1.0mM NaBu for a short time (24h+NaBu2h) and a longtime (24h+NaBu20h). The results showed that the short treatment (2h) with1.0mMNaBu after GVBD (86.03±3.07) improved the meiotic competence significantly(P<0.05) compared with the control (73.95±5.27).(3)The matured oocytes fromeach group were collected and incubated with spermatozoa. The rate of malepronuclear formation and in vitro fertilization (IVF) blastocyst were determined underan inverted fluorescence microscope after staining with10μg/ml Hoechst33342.However, there were no positive effects of NaBu on levels of glutathione (GSH) andsubsequent embryonic development following IVF. These results indicated that thetransient exposure to NaBu after GVBD improved meiotic competence but notsubsequent developmental competence in porcine oocytes.The next, to promote the reprogramming of the donor cell nuclei in ooplasm aftersomatic cell nuclear transfer (SCNT), we evaluated the effects of NaBu onpreimplantation development, histone acetylation and the gene expression of theporcine SCNT embryos. The results showed that the blastocyst rate (24.88±2.09) ofcloned embryos treated with1.0mM NaBu for12h after activation was significantlyhigher (P<0.05) than that of untreated cloned embryos (13.15±3.07). In addition,treated embryos displayed a global acetylated histone H3at lysine14profile similarto that of IVF embryos during preimplantation development. Lower levels of Pou5f1and Bcl-2, but higher levels of Hdac1, in SCNT embryos at the2-cell and blastocyststages were observed, compared with those in the IVF counterparts. The4-cellembryos showed no differences in the levels of these genes among IVF embryos orSCNT embryos treated with or without NaBu; however, the levels of Dnmt3b weresignificantly different. NaBu-treated SCNT embryos showed similar levels of Pou5f1,Bcl-2, and Dnmt3b as in IVF blastocysts. These results indicated that NaBu treatmentin SCNT embryos alters their histone acetylation pattern to provide beneficial effectson in vitro developmental competence and gene expression.At last, we evaluated the effect of downregulation of P300on the development ofporcine embryos.(1) Before the transfection, we detected the expresstion and locationof P300during oocyte maturation and embryo development with real-time PCR and immunofluorescence staining. The results showed P300mRNA expression washighest in4-cell stage and lowest in pronuclear stage, P300protein was expressedmainly on the nuclear in GV oocyte,8-cell stage and blastocyst and in cytoplasma inother stages respectively, which indicated P300was required in the development ofporcine embryos.(2)We designed three shRNA sequences corresponding to P300mRNA. The shRNA vectors were transfected into the porcine fibroblast cell firstly toidentify which was the most efficient shRNA in P300downregulation, with the resultsof real-time PCR and Western blotting, shRNA-1876was determined. Followed thedownregulation of P300protein, the level of H3K14acetylation decreased in porcinefibroblast cell.(3)The lentiviral vector with shRNA-1876was constructed andtransfected into the embryos by microinjecting into the perivitelline space of theembryos. Results of real-time PCR indicated that the P300mRNA weredownregulated in8-cell embryo, morula and blastocyst after transfection inpronuclear stage. But green fluorescent protein (GFP) expressed by lentiviral vectorwas observed only in blastocyst stage, which accompanied with decrease of H3K14histone acetylation level. The transfection of sh-1876of P300in pronuclear and4-cellembryos inhibit the embryos development significantly, especially in the pronuclearembryos.In conclusion, we investigated the effects of HDACi sodium butyrate andshRNA-1876on the development of porcine oocytes and embryos, which indicatedthat Histone acetylation is closely related to oocyte maturation and embryodevelopment, especially in the pronuclear embryo. This work may be useful for theresearch on the mechanism of epigenetic modifications in embryonic developmentand embryo production.