Effects Of Inhibition Of Fanconi Anemia Signaling Pathway On Follicle Development And Oocyte Maturation In Female Mice

The process of mammalian follicle development is regulated by endocrine,paracrine,and genes.This process is affected by the DNA damage produced by the cells in the tissue and the effect of DNA damage caused by the external environment.It has been reported that the Fanconi anemia(FA)gene plays a role in DNA cross-linking damage repair during follicular maturation.Fanconi anemia(Fanconi,FA)is a rare autosomal or X chromosome linked recessive genetic disease,derived from the mutation of the FA genes.It mainly plays the role in homologous recombination repair in DNA damages.The patients with FA pathogenic genes are often accompanied by bone marrow failure,congenital dysplasia and high carcinogenicity.The female patients are often accompanied by premature ovarian failure(premature ovarian insufficiency,POI)in the symptoms,in the progress of sperm meiosis,FA proteins accumulate in male sex chromosome,the deletion of FA genes lead to male infertility,and in meiotic stage serious sex chromosome regulation defects.The clinical symptoms of FA mutations in patients with diversity,heterogeneity,and complexity are not clear.It has been reported that the role of FA gene in the process of follicle development is mainly shown in the following aspects: promoting PGCs proliferation,inhibiting the formation of meiosis,promoting homologous recombination repair,and maintaining the stability of the chromosome structure.The maturation process of oocytes is regulated by many factors and undergo a series of morphological and ecological changes.However,the role of FA pathway in the maturation of oocytes is not yet reported.Whether FA gene affects follicle maturation is still worth further exploring.In order to explore the effect of FA pathway abnormality in the development of female mouse oocyte maturation,we use the USP1 de ubiquitinase inhibitor SJB3-019 A to inhibit the FANCD2-Ub de generalization in repair process of DNA damage in vitro.The experimental results suggest that the oocyte maturation is not affected by the addition of USP1 inhibitor in the oocyte culture medium,but the expression level of the transcriptional transposon in oocyte is up to rise.The abnormal upregulation of these transcriptional transposons will cause genomic instability,mutation and abnormal recombination.Using immunofluorescence to detect the morphological changes and chromosomal changes of the spindle of the oocyte after USP1 inhibitor treatment,we found that the spindle of various morphologic abnormalities and disorganized chromosomes appeared in the inhibitor treatment group.Abnormal spindles are mainly polar bodies,including polar bodies,unipolar bodies,multipolar bodies,etc.Other spindles are unassembled and elongated or shortened spindles.Chromosomal abnormalities are arranged in disorder.Therefore,we speculate that the abnormality of FA repair pathway by using USP1 inhibitors will affect the spindle assembly and spindle stability of the meiotic maturation of mouse oocytes during meiosis maturation.The instability of these genomes affects the ability of oocytes to be fertilized in vitro,and the results of IVF after fertilization in vitro show that abnormal FA pathway leads to a decrease in fertilization rate in mouse oocytes,which is consistent with our inference.In addition to in vitro experiments,we tried to explain the functions of important genes in FA pathway through in vivo experiments.In the experiment in vivo,we first analyzed the expression of some important genes in the oocyte and early embryo in the FA pathway by BIOGPS software.After comparison,we found that Ube2 t is a E2 ubiquitin binding enzyme in the FA pathway,which is more specific in oocytes.After retrieval,we also found that UBE2 T protein is in human and in human.The homology of mice is relatively high.It is of some reference significance to study the role of UBE2 T protein in human beings by using mouse model as a research tool.The realtime-PCR results of multiple tissues in mice showed that Ube2 t mRNA was highly expressed in GV oocytes,MII oocytes and fertilized eggs,and the level of expression decreased after cleavage.The UBE2 T protein immunofluorescence results suggest that the protein is expressed in mouse GV and MII oocytes,and that the expression and expression level of the fertilized egg and early embryo have not been changed significantly.It suggests that UBE2 T may play an important role in the development of oocyte and embryo embryo as a potential mother source effect factor.Therefore,in the experiment in vivo,we constructed and designed the Ube2 t single guide RNA(Single-guide RNA,sgRNA)using the CRISPR-Cas9 method.By co-microinjection with Cas9 mRNA,we obtained two gene inactivation lines with the mutation of the code.The results of gene sequencing analysis in F0 generation mice showed that the Ube2 t gene shift mutation gene knockout mice were successfully constructed,and these mutations could be steadily inherited to the offspring.In the process of breeding and breeding,we found that the number of progeny homozygous of knockout mice was very low,the percentage of homozygosity was about 3.9%(9/228),and it was seriously inconsistent with Mendel’s law of inheritance.We speculated that partial homozygous mice died in the embryonic period,and we analyzed the results of 4.5dpc,6.5dpc and 10.5dpc in mice.It is preliminarily speculated that the lethal time of homozygous mouse embryos may be between 4.5dpc-6.5dpc.The test of fertility test showed that both the Ube2 t gene knockout mice and the control mice showed a lack of fertility in both the female and the male mice,and the abnormal development of the eyelid,rodents and limbs appeared so on.Further studies on UBE2 T protein knockout mice showed that female homozygous mice had different degrees of follicle number loss and reproductive system atrophy,and male homozygous mice showed small testicular volume and no sperm.Through exogenous injection of PMSG and HCG,it was found that female homozygous mice had no oocyte excretion.It was speculated that UBE2 T protein knockout might lead to lower ovarian response to hormones.We detected a large number of DNA damage sites in the ovaries of female homozygous mice by detecting the DNA damage(double-strand damage,DSB)fluorescent marker-H2 AX signal.We speculate that the deletion of UBE2 T protein causes the dysfunction of FA pathway repair,which makes the oocytes retained in the ovaries of mice susceptible to the effects of endogenous / exogenous genotoxic substances,causing an increase in the accumulation of DSB an d accelerating the aging or atrophy of the ovaries.