Study On The Developmental Disorder Of Primordial Germ Cells In Fance-deficient Mice

Objective:Premature ovarian insufficiency（POI）is a common cause of female infertility,with an incidence of about 1-3.7%.The patient’s ovarian function declines before the age of 40,which seriously affects the reproductive health of women.About 70%of POI cases are idiopathic with unknown etiology,and up to 40%of these cases have a family history,suggesting that genetic causes predominate in the pathogenesis of POI.The study found that the pathogenic genes of POI were mainly concentrated in DNA damage repair,homologous recombination and meiosis.Fanconi anemia（FA）pathway is one of the most closely related to germ cell development in the DNA damage repair pathway.Mutations in the FA genes Fanca,-c,-d1,-g,-m,-s,-u are all related to the occurrence of POI.Fanconi anemia group E（FANCE）protein is an important substrate molecule necessary for the activation of FA pathway,and its gene defect seriously affects the fertility and development of mouse germ cells.Our previous study showed that the number of germ cells in Fance^-/-female mice was significantly reduced at the embryonic day 13.5（E13.5）,so we speculated that the germ cell developmental disorder of the Fance-deficient mice might occur at the developmental stage of Primordial germ cells（PGCs）before E13.5.This study aims to explore the potential mechanism of PGCs developmental disorders in Fance-deficient mice,and to provide new theoretical and experimental foundation for the development of PGCs and the pathogenesis and prevention of POI.Methods:（1）In the established Fance-deficient mouse model,sexually mature heterozygous(Fance^+/-)female and male mice were mated in a 1:1 ratio to obtain E9.5,E10.5,E11.5 and E12.5 stage embryos,respectively.Agarose gel electrophoresis was used to identify the gender and Fance genotype of the sample.（2）The embryo specimens were fixed,embedded,and serially sectioned.Fance^+/+,Fance^+/-and Fance^-/-PGCs were labeled and counted by immunofluorescence using germ cell-specific indicators（SSEA1,OCT4,MVH,STELLAR,DAZL）.（3）Immunofluorescence double staining was used to detect proliferation indicators（Ki-67,PH3）,apoptosis indicators（Cleaved Caspase3,Cleaved PARP）,DNA damage and repair indicators（γH2AX,53BP1）,as well as epigenetic programming indicators（H3K9me2,H3K27me3）in PGCs.（4）E9.5,E12.5 female,E12.5 male Fance^+/+and Fance^-/-PGCs were obtained respectively by flow cytometry.（5）Using Smart-seq2 technology in single-cell transcriptome sequencing,the sorted PGCs samples were sequenced,and the changes in the transcriptional profile of PGCs caused by Fance gene deficiency were described.（6）Perform functional enrichment analysis of differentially expressed genes in Fance-deficient PGCs,using R language,GO,KEGG database,etc,to predict and analyze PGCs development-related signaling pathways and regulatory genes.Results:（1）The ratio of Fance^+/+,Fance^+/-and Fance^-/-genotypes in embryonic mice was about 1:2:1,which conformed to Mendelian genetics,indicating that Fance gene defect has no obvious embryonic lethality.（2）The expression patterns of a series of germ cell-specific markers SSEA1,OCT4,MVH,STELLAR,and DAZL in Fance^-/-PGCs were consistent with those in Fance^+/+and Fance^+/-PGCs,suggesting that Fance gene deficiency did not affect the characteristics of mouse PGCs.SSEA1 was expressed on the membrane of E9.5-E12.5 PGCs,OCT4 and STELLAR were expressed in the nucleus of E9.5-E12.5 PGCs,while MVH and DAZL were expressed in the cytoplasm of E11.5-E12.5 PGCs.However,the number of PGCs in Fance^-/-embryonic mice was significantly decreased as early as E9.5,which was about 59%of that in Fance^+/+and Fance^+/-embryonic mice,and then the difference became more obvious.Until E12.5,the number of Fance^-/-PGCs was only 10%of that of Fance^+/+and Fance^+/-PGCs.Fance gene defect severely affects the development of PGCs.（3）Consistent with Fance^+/+and Fance^+/-,E9.5 to E12.5 Fance^-/-PGCs all expressed the proliferation indicator Ki-67,indicating that Fance-deficient PGCs entered the mitotic cell cycle normally.However,the expression of G2/M phase marker PH3 was significantly increased in E12.5 Fance^-/-PGCs,suggesting that the G2/M phase of Fance-deficient PGCs was prolonged and the proliferation rate was slowed down.（4）More apoptosis indexes Cleaved Caspase3 and Cleaved PARP were detected in E9.5,E11.5,and E12.5 Fance^-/-PGCs.Increased apoptosis is a possible reason for the loss of PGCs in Fance-deficient mice.（5）Compared with Fance^+/+and Fance^+/-,the expression rate of DNA damage indicatorγH2AX in E12.5 Fance^-/-PGCs was significantly increased,but the formation of repair protein 53BP1 foci was not detected at this time,which suggested the accumulation of DNA damage and repair defects of Fance^-/-PGCs.（6）The expression patterns of epigenetic markers H3K9me2 and H3K27me3 in Fance^-/-PGCs were consistent with Fance^+/+and Fance^+/-embryonic mice.PGCs expressed H3K27me3 but not H3K9me2,suggesting that Fance gene deficiency may not affect the epigenetic reprogramming process of mouse PGCs.（7）The results of Smart-seq2 single-cell transcriptome sequencing showed that,compared with Fance^+/+,E9.5 Fance^-/-PGCs showed significant differences in the expression of 1586 genes,which were mainly enriched in biological processes such as cell fate determination,development-related and cell communication.KEGG analysis showed that Fance gene defect in E9.5 PGCs up-regulated MAPK signaling pathway,Notch signaling pathway,and down-regulated cell adhesion molecules.（8）Compared with Fance^+/+,E12.5 Fance^-/-female and male PGCs showed significant differences in the expression of 3026 and 1836 genes,respectively.These differential genes were mainly enriched in cell component such as extracellular vesicles,cell protrusions,and biological processes such as cellular metabolism.Fance gene deficiency in E12.5PGCs up-regulated the lysosomal pathway and down-regulated the expression of genes related to mitosis,meiotic cell cycle,and DNA damage repair.（9）During the developmental stage from E9.5 to E12.5,Fance^+/+PGCs mainly up-regulated genes related to meiosis,sex differentiation,p53 signaling pathway,and down-regulated genes related to organ morphogenesis,Wnt signaling pathway,PI3K/Akt signaling pathway,stem cell pluripotency regulation pathway,etc.However,Fance-deficient PGCs were more likely to upregulate genes related to lysosome,apoptosis,and autophagy,suggesting the possible cell death mode in Fance^-/-PGCs.（10）A number of genes known to be closely related to germ cell proliferation,migration,DNA methylation,DNA damage response and meiosis,are abnormally expressed in Fance-deficient PGCs,suggesting that the Fance gene gene plays an important role in multiple events in the development of PGCsConclusion:（1）The PGCs development of Fance-deficient mice was abnormal.The number of Fance^-/-PGCs was significantly reduced at E9.5,and there were detectable accumulated DNA damage,increased apoptosis and slowed proliferation.（2）Fance gene plays different biological roles at different developmental stages of PGCs,which was closely related to their main life activities at different developmental stages.At E9.5,Fance gene defect may affect the pluripotency maintenance and migration process of PGCs through various mechanisms such as up-regulation of MAPK signaling pathway,Notch signaling pathway,and down-regulation of cell adhesion molecules.At E12.5,Fance gene defects caused the accumulation of DNA damage,slowed proliferation and increase apoptosis in PGCs by down-regulating DNA damage repair,cell cycle,ribosome-related genes and up-regulating lysosome-related genes.（3）A number of known genes related to germ cell proliferation,migration,DNA methylation,DNA damage response and meiosis,are abnormally expressed in Fance-deficient PGCs.The cellular and molecular mechanism of Fance gene regulating PGCs development still needs to be further explored and verified.