Studies Related To The Expression Pattern And Actions Of ASF1A In Mouse And Buffalo Early Embryogenesis

The physiological activities of the early stages of preimplantation embryos are mainly regulated by maternal factors stored in oocytes.The developmental regulation gradually transforms from maternal-dependent to zygotic-dependent manner when the embryonic genome activation happens.Then,both the oocyte-derived and embryonic new-synthesized factors are involved in the developmental process of early embryos.Anti-silencing factor 1A（ASF1A）identified as a maternal factor abundantly expresses in metaphaseⅡ（MⅡ）oocytes.This study aims to profile the expression pattern of ASF1A throughout the mouse and buffalo oogenesis and early embryo development and explore the effect of ASF1A on the development of early embryos in mouse and buffalo.The major results of the study are listed:1.The expression pattern of ASF1A throughout mouse oogenesis and early embryo development was explored.（1）The immunohistochemical results of the mouse ovary showed that ASF1A expressed in oocyte nuclei from primordial to Graafian follicles.Moreover,the obvious ASF1A signal was observed in the nucleolus-like bodies of oocytes from the secondary to Graafian follicles.（2）The immunofluorescence results of mouse oocytes and early embryos showed that ASF1A immunofluorescence signal appeared in the germinal vesicle（GV）,especially on the nucleolus-like bodies,and dispersed in the cytoplasm of MII oocytes.ASF1A immunofluorescence signal was mainly observed on the nucleolus precursor bodies（NPBs）in 1-to 4-cell embryos,especially appeared on the NPBs of male pronuclei,and then ASF1A immunofluorescence signal was observed to distribute in the nuclei regions outside NPBs in the 8-cell embryos and morulae,and homogeneously dispersed in the nuclei in blastocysts.（3）Real-time PCR results showed that the expression level of ASF1A m RNA displayed no significant difference between the GV and MⅡoocytes.ASF1A m RNA displayed the highest expression level in the 1-cell embryos,and then obviously decreased in the 2-cell embryos,followed by a significant increase of ASF1A m RNA in 4-cell embryos.ASF1A m RNA gradually decreased from 4-cell emrbyos to blastocysts.（4）Western blot results showed that the expression level of ASF1A protein was similar between the GV and MⅡoocytes.ASF1A protein displayed the highest expression level in the 1-cell embryos,followed by a decrease in the 2-cell embryos and arrived at the lowest level from the 4-cell embryos to blastocysts.2.The regulatory mechanism of ASF1A accumulating onto the NPBs of mouse early embryos was investigated.（1）The location analysis of the different ASF1A domains labelled by enhanced green fluorescent protein（e GFP）showed that the e GFP signal in both ASF1＿hist＿chap domain（1-154 AA）and ASF1A group appeared on the NPBs of male pronuclei and 2-cell embryos.Further,the location analysis of the 36^thE37^thD or 94^thV（within ASF1＿hist＿chap domain）point mutations labelled by e GFP showed that no obvious e GFP signal was observed on NPBs and a decreased intensity of e GFP was observed in the nuclei in zygotes and 2-cell embryos.（2）When these mutations were connected to the sv40 nucleus location sequence,the e GFP signal in the 36^thE37^thD point mutation group appeared on NPBs,and the e GFP signal in the 94^thV point mutation group dispersed in the nuclei in zygotes and 2-cell embryos.（3）When injecting the RNAs of e GFP-labelling ASF1A and m Cherry-labelling histone H3 variants,which binding to ASF1A through a 94^thV-centered groove,the m Cherry signal of H3.3 co-located on NPBs with the e GFP signal of ASF1A,while the m Cherry signal of H3.1 and the e GFP signal of ASF1A dispersed in nuclei in mouse early embryos.（4）When deleting the ASF1A-binding 122^nd-135^th region of histone H3variants,the m Cherry signal of H3.3 was still observed to appear on NPBs.（5）When the 56^thK（ASF1A specifically promotes the acetylation modified in this site of H3）point mutations RNAs of histone H3 variants were injected in zygotes,the developmental rate of mouse embryos in the H3.3K56R group significantly decreased in comparison to the H3.3K56 group（P<0.05）,while the similar developmental rates were observed between the H3.1K56R and H3.1K56 groups,or between the H3.2K56R and H3.1K56 groups（P>0.05）.（6）Finally,the generative number of NPBs of 2-cell embryos in the ASF1A and ASF＿hist＿chap groups significantly increased in comparison to the control group（P<0.05）,while the number of NPBs in the 36^thE37^thD or 94^thV point mutation groups were similar to the control group（P>0.05）.3.The effect of ASF1A on the development of mouse early embryos was explored.（1）Inhibition of ASF1A expression by morpholino oligo significantly decreased the embryonic developmental rate and cell number in blastocysts,and remarkably increased the proportion ofγH2AX-positive nuclei in blastocysts.（2）Overexpression of ASF1A by m RNA microinjection significantly increased the embryonic developmental rate and cell number in blastocysts,and remarkably decreased the proportion ofγH2AX-positive nuclei in blastocysts.（3）ASF1A overexpression promoted the repair of DNA double-strand breaks by enhancing homologous recombination and inhibiting non-homologous end joining manner（P<0.01）.4.The expression pattern of ASF1A during buffalo oocyte and early embryo development and its effect on the development of buffalo early embryos were explored.（1）The full length of coding sequence of buffalo ASF1A acquired by PCR amplification was 615 bp,encoded 204 amino acids and contained a typical ASF1＿hist＿chap domain.（2）Real-time PCR results showed that the expression level of ASF1A m RNA displayed no significant difference between buffalo GV and MⅡoocytes.ASF1A m RNA displayed the highest expression level in the1-cell embryos,obviously decreased in the 2-cell embryos,and arrived at its lowest level from the 4-cell embryos to blastocysts.（3）Injection of ASF1A RNA significantly increased the development rate of buffalo early embryos.（4）Bimolecular fluorescence compensation experiment showed a direct interaction of buffalo ASF1A with OCT4 and SOX2.The above observations indicated:（1）ASF1A expressed throughout mouse oocyte development,maturation and early embryonic development and mainly accumulated onto NPBs of early embryos,specifically onto NPBs of male pronuclei.（2）The ASF1A accumulation onto NPBs was regulated by histone H3.3,and ASF1A may promote the acetylation of H3.3 56^thK,which be involved in the early embryonic development and the generation of NPBs.（3）Moreover,ASF1A can maintain the genome stability in early embryos through enhancing homologous recombination.（4）The coding sequence of buffalo ASF1A was 615bp,encoded 204 AA,and expressed in oocytes and early embryos.Injection of ASF1A RNA can promote the development of buffalo embryo,and the reason may be the interaction of ASF1A with OCT4 and SOX2,which may maintain the pluripotency of early embryos.