Effect Of H1foo Expression On Oocyte Meiotic Maturation And Nuclear Transfer In Bovine

Since the cloned sheep Dolly was generated using somatic cell nuclear transfer (SCNT), a number of mammalian species have been successfully cloned. However, the success rate is still very low and the relevant mechanisms involved in reprogramming are largely unknown. The main cause of these problems may be attributed to the uncompleted reprogramming of the donor genome. Therefore, trying to improve the quality of oocyte and promote the efficient reprogramming of donor nuclear will response well to these problems. As one of the linker histones, the expression of oocyte specific linker histone (H1foo) is restricted in oocytes and early embryos, which makes them have a specific chromosome structure and gene expression pattern. In mouse oocyte, H1foo is indispensable for meiotic maturation. During the process of SCNT either amphibians Xenopus or mammalians mouse, the rapid linker histone H1 transitions can be observed, which is considered to be essential for donor nuclear reprogramming.In our study, the bovine oocyte is used to determine the function of H1foo during the process of oocyte maturation and SCNT. To examine the effect of H1foo expression on oocyte maturation, we microinjected effective siRNA against H1foo or H1foo mRNA transcripted in vitro into the maturing bovine oocytes. Meanwhile, to determine whether the replacement occurs during bovine nuclear transfer, we performed the nuclear transfer, using RFP-taged somatic linker histone H1e stable transfected donor cells and MII oocytes expressing exogenous fusion protein H1foo-Venus. After fusion, the replacement of linker histones can be real-time monitored under fluorescence microscope. The results of the project are as follows:1. We successfully established an improved RT-PCR, which is efficient for gene cloning from trace oocytes. The complete coding sequence of H1foo was cloned from 5 bovine oocytes, which was totally identical with previous reports. After the recombinant plasmid pVenus-H1foo was transfected into Hela cells, H1foo was efficiently expressed identified by Fluorescence microscopy observation,RT-PCR and Western Blotting. H1foo was localized accurately on chromosomes of the oocyte and polar body after H1foo mRNA transcripted in vitro was microinjected into bovine oocytes. Then we co-transfected the pVenus-H1foo and the 4 siRNAs into Hela cells respectively to screen a valid candidate sequence. Fluorescence microscopy observation,RT-PCR and Western Blotting indicated that siRNA-3 had a higher efficiency, which decreased the H1foo expression 90% and 60% in mRNA and protein level, respectively. Finally, we microinjected H1foo mRNA or siRNA-3 into the maturing bovine oocytes, and the results showed that compared to un-injected control group (71.2%), the maturation rate of H1foo-Venus injected group and siRNA-3 injected group is 88.7% and 36.0%, respectively, which indicated that knowdown of H1foo significantly impaired oocyte maturation, and overexpression of H1foo can apparently promote oocyte maturation.2. Since there are no introns existing in linker histone H1e, we successfully amplified the complete coding sequences of H1e from genome DNA of the bovine fibroblast cells. Fluorescence microscopy observation and RT-PCR detection showed that the recombinant plasmid pdsred1-H1e was efficiently expressed in Hela cells. The plasmid pdsred1-H1e was transfected into bovine skin fibroblast cells, and the stable transfected cell lines were successfully established after 3-4 weeks of selection with neomycine (G418). RT-PCR and Western Blotting showed that H1e-RFP mRNA and fusion protein were expressed in these cell lines, and the fluorescence was localized accurately on nuclear. Finally, to determine whether the replacement occurs during bovine nuclear transfer, the different treated fibroblasts were injected into the perivitelline space of different treated enucleated MII-arrested oocytes. Under fluorescence microscope, release of H1e in the donor nucleus, acquisition of H1foo by donor chromosome and H1foo-to-H1e transition were observed. However, unlike that in mouse, the replacement in bovine was slower. Subdued but diffused red fluorescence (H1e) in donor nucleus was still detectable, while green fluorescence (H1foo) has incorporated into the donor chromosome at 5 h, and the complete replacement occurred until 8 h after fusion.Our results indicate that overexpression of H1foo can apparently promote bovine oocyte maturation, while knowdown of H1foo significantly impaired oocyte maturation, which is agreement with that in mouse. During the bovine SCNT, the linker histones transition was observed, which demonstrates that the phenomenon perhaps is sufficiently conserved among different species rather than specific just in rodent oocytes. Our findings provided further evidence for its important role in nuclear reprogramming.