Study On RDNA Activity And Nucleoli Changing In Mouse Nuclear Transfer Embryos

Objective To study the impact of donor cell’s rDNA status on rDNA activity aswell as rRNA synthesis and processing of nuclear transfer (NT) embryos. MethodsWe firstly established mouse embryonic stem cells (ESCs), cumulus cells (CCs) andembryonic fibroblasts (MEFs) cell lines of B6D2F1strain and examined the activeNORs numbers, rDNA methylation levels and nucleoar related genes expression ofthese three cell lines. Then the three kinds of cells were used as donor cells toconstruct NT embryos with one-step NT technique while intracytoplasmic sperminjection (ICSI) embryos were used as control. We examined the pronuclei formation,nucleolar protein distribution, active NORs numbers, rDNA methylation levels andnucleolar related genes expressionof these embryos. Results1) Nucleolar precursorbodies (NPBs) started to form5h after sperm injection/activation in mouse ICSI andNT embryos. The number of NPBs reduced until8hpa.Then the NPBs number keptunchanging until pronuclei fused at16-17hpa.2) ESNT embryos only formed onepronucleus at pronuclear stage, whiching containing7.20NPBs in average. Cumuluscell nuclear trasnfer (CCNT) rmbryos, MEFs nuclear transfer (MEFNT) embryos,ICSI embryos and parthenogenetic activated (PA) embryos all formed two pronuclei/pseudo-pronuclei and the average NPBs numbers per pronuclei/pseudo-pronucleiwere3.71,4,27,1.58and1.59, respectively.3) The reticular structure started toappear around the nucleoli in ICSI, ESNT and CCNT embryos at late-2cell stage(27hpa). The nucleoli appeard as compeletely reticular structure in ICSI and ESNTembryos at4-cell stage, but nucleoli in CCNT embryos only showed partial reticularstructure at the same stage.4) The CCs lost rRNA synthesis and processing activities after treated with0.8μg/ml actinomycin D for1h. The NT embryos reconstructedfrom AD treated CCs had no significant difference with un-treated group inpreimplantation development competence.5) ESCs had the most active NORs number(7.66) and the lowest rDNA methylation level (6.76%) and its UBF (upstream bindingfactor), FBL (fibrillarin) and B23(nucleophosmin) expression levels weresignificantly higher than CCs and MEFs. MEFs had the least active NORs numbers(4.70) and the highest rDNA methylation levels (22.57%) while CCs had the middleof both (6.45and13.59%).6) At4-cell stage, ESNT embryos had almost the sameactive NORs numbers as ICSI embryos (7.19vs.7.44) and which was significantlyhigher than that in CCNT (6.68) and MEFNT embryos (5.77). MEFNT embryos hadthe highest rDNA methylation level than the other3groups （15.52%vs.9.39%/ICSIvs.6.36%/ESNT vs.9.67%/CCNT）.7) The B23and UBF signals vanished in20minin the nuclei of CCs and MEFs after entering oocyte cytoplasm. But the B23/UBFsignals of ESCs nuclei can also be detected ever at1h after NT.8) Thepreimplantation developmental competence of MEFNT embryos was significantlylower than ICSI, ESNT and CCNT embryos. MEFNT embryos at morula stage hadsignificantly lower FBL and18S rRNA expression levels than the other3groups andtheir UBF and47S rRNA expression levels were significantly lower than ICSIembryos. Conclusion We demonstrated the distribution and changing of nucleolarprotein in donor cells and NT embryos at different preimplantation developnmentalstages. We found these donor cells owned different rDNA activities. And thisdiffenrence determined the different rDNA activities in NT embryos to a large extent,even affected the preimplantation developmental competence of NT embryos.