The Expression And Dynamic Changes Of Notch Signaling Pathway In Ovarian Epithelial Where Enrich Ovarian Germline Stem Cells And Ovarian Germline Stem Cells

Objective:To clarify the effects of Notch signaling pathway on the proliferation,differentiation and senescence of ovarian germline stem cells through investigatingthe dynamic changes in the expression of Notch-related molecules in ovarianepithelial where enrich ovarian germline stem cells and germline stem cells fromnewborn, adult and aged mice, and to provide a theoretical basis for the preventionand treatment of premature ovarian failure and the related diseases.Methods:1. Dynamic changes in the expression of germ cell marker MVH and Notchpathway molecules (Notch1, Jagged1and Hes1) were determined using immunoh-istochemistry and real-time PCR in ovarian epithelial from3-day-,2-month-and20-month-old mice.2. Dynamic changes in the co-expression of ovarian germ cell marker MVH andNotch pathway molecules (Notch1, Jagged1and Hes1) were detected usingdouble-label immunofluorescence histochemistry in ovarian epithelial from3-day-,2-month-and20-month-old mice.3. Ovarian germline stem cells were isolated from ovaries of3-day-and2-month-old mice and were dispersed using two-step enzymatic digestion method.Cells were cultured in stem cell culture medium supplemented with leukemiainhibitory factor (LIF), epidermal growth factor (EGF) and basic fibroblast growthfactor (bFGF), and were maintained on feeder layers of STO cellsat37℃in5%CO2.4. Dynamic changes in the co-expression of ovarian germ cell marker MVH andNotch pathway molecule notch1were detected using double-label immunofluor-escence histochemistry in3-day-and2-month-old mice. Results:1. Immunohistochemistry showed the protein expression of MVH, Notch1,Jagged1and Hes1in ovarian epithelial was highest in3-day-old mice, medium in2-month-old mice and lowest in20-month-old mice.2. Real-time PCR showed that the mRNA expression of MVH, Notch1, Jagged1and Hes1in ovarian tissue was highest in3-day-old mice, medium in2-month-old mice and lowest in20-month-old mice.3. Double-label immunofluorescence histochemistry showed that ovarian germcell marker MVH and Notch pathway molecule Notch1, Jagged1and Hes1wereco-expressed in ovarian epithelial. The co-expression of MVH/Notch1, MVH/Jagged1and MVH/Hes1was highest in3-day-old mice, medium in2-month-old mice and lowest in20-month-old mice.4. RT-PCR showed that germ cell marker MVH, early germ cell marker Fragilisand pluripotent stem cell marker Oct-4were expressed in the ovarian cells isolatedby two-step enzymatic digestion. However, the expression of oocyte marker Zp3wasnot observed in these cells. These results indicated that the isolated cells areundifferentiated germ stem cells.5. Double-label immunofluorescence histochemistry showed that germ cellmarker MVH and Notch pathway receptor Notch1were co expressed in ovariangermline stem cells.Conclusion:Notch-related molecule Notch1, Jagged1and Hes1were expressed in ovariangermline stem cells, and the expression levels decreased with reproductive age inmice. Furthermore, MVH/Notch1, MVH/Jagged1and MVH/Hes1wereco-expressed in ovarian epithelial, and the co-expression levels decreased withreproductive age in mice. Moreover, MVH and Notch1were co-expressed in ovariangermline stem cells. There results suggested that Notch signaling pathway wasinvolved in the regulation of proliferation, differentiation and senescence of ovariangermline stem cells.