Effect Of Transcription Factor TFⅡB On Mouse Oocyte Maturation And Early Embryo Development

In mammals, oocyte maturation is a process of cell cycle transition induced and stimulated by a series of external signals. During this period, two automatic cessations will take place, which can be resumed by outside stimulations, and finally induced gene transcription and protein synthesis. Early embryo development starts at the time of zygotic genome activation (ZGA) after fertilization. During ZGA process, most of the masked genes stored in the cytoplasm gradually express and are modified. Transcription factors (TFs) are necessary for RNA polymerase to catalyze gene transcription. TFIIB mainly involves in assembling of the pre-initiation complex (PIC) and mediates the connection between RNA polymerase II and gene promoter region. TFIIB combines with TFIID to regulate the activity of catalyzing center in RNA polymerase. In addition, TFIIB is also considered as a target protein associated with many transcription regulators and modulates cellular transcription. Alfa-tubulin is one of the basic cytoskeletal component proteins for assessment of spindles and directly affects the behavior of chromosomes. Meanwhile, as a cell skeleton structure, it also participates in the interaction among proteins. In the present study, the dynamic changes of transcription factors including BAF155, Brg-1, HDAC2, HP1β,TAFII, TFIIB, TFIID, TopoIIa and TopoⅡβwere investigated during mouse oocyte maturation. We especially focuse to investigate the TFIIB in the regulation of mouse and bovine early embryo development, which may provide some beneficial clues to understand functions and mechanisms of transcription factors in the regulation of meiosis and early embryo development. The major results are as followings:1. The distributions of transcription factors during mouse oocyte maturation By using immunofluorescence staining protocol, we analyzed the dynamics of nine transcription factors, BAF155, Brg-1, HDAC2, HP1β, TAFⅡ, TFⅡB, TFⅡD, TopoIIa and TopoⅡβ, in mouse maturing oocytes at different stages. The results showed that transcription factors always concentrated in GV nucleus in germinal vesicle stage. After meiotic resumption, transcription factors were with two different distributions:HDAC2, HP1β, TAFⅡ, TFⅡB, and TopoIIa always distributed along with chromosomes and co-located with spindles; however, BAF155, Brg-1, TFIID, and TopoⅡβgradually evenly distributed in cytoplasm. These results suggest transcription factors associated with chromosomes directly involve in the assembling of PIC and modify chromosomal structure. The factors evenly distributed in cytoplasm regulate the activity of transcription and re-gather to chromosomes when the binding sites are exposed.2. The dynamic relationship between TFIIB andα-tubulin in meiosis and the effects of TFIIB on mouse oocyte maturationAfter treatment of oocytes with colcemid, a specific inhibitor of microtubule assembling, the spindles were seriously twisted and destroyed, but the TFIIB distribution was not changed. Two bimolecular fluorescence complementation vectors pHA-tf2b and pFlag-tubala were constructed and both of the two vectors were co-transfected to mouse embryonic fibroblast cells (MEF). After examination of the transfected cells, TFIIB were found tightly interacted withα-tubulin. To further investigate the effect of TFIIB on oocyte meiosis and early embryo development, RNA interference of TFIIB was performed. Around 6h after siRNA injection, the mRNA level of tf2b began to decrease, and TFIIB expression was significantly lower in siRNA injection groups than the controls. However, TFIIB decline did not significantly affected oocyte maturation (P> 0.05).3. TFIIB affected mouse early embryo development After injection of tf2b siRNA into GV-stage oocytes, the injected oocytes were incubated in vitro for maturation and then the matured oocytes were activated by SrCl2. The results showed that the interference of TFIIB expression caused a significantly lower blastocyst development (P<0.01), most of the embryos were with obvious developmental blocki at 2-cell stage. When Mil stage oocytes were interference by tf2b siRNA, the tf2b mRNA level decreased but did not influence the blastocyst development. We also found the absent of transcription factors during pronuclear to 2-cell period induced abnormal transition of maternal to zygote genome activation, which subsequently led to embryo development arrest. Once the transcription of zygotic genome was fully activated, the interference of transcription factors did not significantly affect embryo development, but affected the embryo quality with lower blastocyst cell number.4. Abnormal cloned embryos were with irregular transcription factors distribution patternWe compared the distribution patterns of nine transcription factors (BAF155,Brg-1 HDAC2,HP1β,TAFⅡ,TFⅡB,TFⅡD,TopoⅡαand TopoⅡβ) in morphological normal and abnormal bovine cloned embryos by immunofluorescence staining. The results showed that the target transcription factors could be detected in normal cloned embryos, while in abnormal embryos, the chromosomes arranged disorderly, and the level and distribution of some transcription factors were obviously irregular. This result suggested that the defects of TFs in cloned embyros may be one of the factors which caused development abnormal.