Correlation Study Between The Ovarian Aging And The Notch Signaling Pathway In Ovarian Germline Stem Cells

Objective:Exploring the regulation of Notch signaling pathway on the proliferation and differentiation of germline stem cells via detecting the expression and dynamic changes about the Notch signaling pathway in germline stem cells as well as in ovarian surface epithelium（OSE）of physiological and pathological aging mice,which will provide new directions for further research in the regulatory mechanisms of mammalian ovarian germline stem cells in vivo and bring more possibilities in clinical treatment of female infertility by ovarian germline stem cells.Methods:1.Isolating the ovarian germline stem cells from 3-5-day-old mice by primitive culture method.These ovarian germline stem cells were cultivated in 37℃along with 5%CO₂,while STO cells were taken for feeder layer and MEM-αwas as basal medium.2.We chose 20-month-old mice as physiological aging mice.Injected the combined preparation of cyclophosphamide/busulfan intraperitoneally into mice to construct infertility models as pathological aging mice.Transfected the high-expressed vector of lentivirus NICD into pathological aging mice in vitro.3.HE staining:Observed the changes of follicular development in ovaries of2-month-old mice,infertility models,20-month-old mice as well as infertility models with lentivirus NICD high-expressed vectors.4.Immunohistochemical staining:To observe the expressions and dynamic changes of marker molecules MVH,Oct4 from germ cells and stem cells as well as marker molecules Notch1,Hes1 of Notch signaling pathway among mice OSE.5.Dual immunofluorescence staining:To detect the co-expressions and dynamic changes of marker molecules MVH/Oct4,MVH/Notch1,MVH/Hes1 in mice OSE.6.Western Blot detection:Observed the expression and dynamic changes of marker molecules MVH,Oct4,Notch1,Hes1 in mice OSE.Results:1.Co-expressions of marker molecules MVH and Oct4,which from germ cells and stem cells are detected in ovarian germline stem cells by Dual immune-ofluorescence staining as well as the co-expressions of MVH and marker molecules Notch1,Hes1 of Notch signaling pathway.2.Abundant follicles can be seen in the 2-month-old mice ovaries while little follicles are observed in the ovaries of the infertility models and 20-month-old mice by HE staining.However,the amount of follicles increase among the infertile mice ovaries with high-expressed lentivirus NICD vectors.3.Immunochemical method reveals that the expressions of the markers MVH,Oct4,Notch1 and Hes1 in 2-month-old mice’s ovarian cortex are all in high quantity while these four decrease obviously in aging mice’s ovarian cortex.Among the ovarian cortex of mice with lentivirus NICD high-expressed vectors,all the expressions of these genes above increase to some extent.4.Dual immunofluorescence staining showed that the co-expressions of MVH/Oct4,MVH/Notch1 and MVH/Hes1 in 2-month-old mice’s ovarian cortex were high but low among aging and infertile mice’s ovarian cortex.However,the expressions of these genes above increase to some extent among the ovarian cortex of mice with lentivirus NICD high-expressed vectors.5.Western blot detection displayed the expressions of MVH,Oct4,Notch1 and Hes1 of the 2-month-old mice are higher than those in aging and infertile mice.In addition,the genes expressions are all increasing among the mice injected by lentivirus NICD high-expressed vectors.Conclusion:1.The co-expressions of the marker molecules from germ cells as well as the stem cells and Notch signaling pathway relevant molecules do exist in mice’s ovarian germline stem cells and among the OSE.One to mention,the expression quantity decrease obviously in physiological and pathological aging mice’s OSE.2.Activating the Notch signaling pathway in the ovaries of pathological aging mice can improve the expressions of MVH,Oct4,Notch1 and Hes1 in OSE greatly and increases the amount of follicles as well,which indicates that Notch signaling pathway can delay ovarian senility by regulating the ovarian germline stem cells.