The Role And Mechanism Of Tumor Suppressor Gene Pten In Primordial Follicles Initiation And Growth

Objective:This topic to be of primordial follicles in vitro culture model to explore theregulation of primordial follicles initiation and growth of an important gene ptenexpression in primordial follicles, and used the carrying of pten-shRNA lentiviralvector to transfect vitro ovary, observed pten down impact the initiation and growthof primordial follicles, and then explore the pten in the role and mechanism ofprimordial follicles initiation and growth.Methods:1. Took2days old female SD rat ovaries under sterile conditions, appliedWaymouth culturing system to culture0,4,8days. Applied HE staining to observenormal0,4,8days expression and change of the primordial follicles in the ovary.Applied PCR method to observe pten mRNA expression in the primordial follicle.Applied immunohistochemistry to observe0,4,8days localization and expression ofPTEN protein in the primordial follicle.2. Selected effective interference fragment, pten-shRNA lentiviral packaged,choosed the best transfer time and best multiplicity of infection (MOI: infected virusand the ratio of the number of cells),applied RT-PCR and western blot method toverify pten-shRNA lentiviral gene silencing efficiency. Applied fluorescencemicroscope to observe GFP expression and change, meanwhile,the levels of hormonein the0,4,8days of culture solution were detected by radioimmunoassay.3. Applied PI3K inhibitor LY294002, the best interference fragment oflentiviral transduction pten-shRNA. Applied HE staining to observe growth anddevelopment of the primordial follicle, applied RT-PCR method to observe changesin mRNA expression of contents, applied western blot to observe PTEN protein andPI3K protein expression. The result:1. The number of primordial follicles in the proportion of the total number offollicles reduced with the culture days increasing; ptenmRNA expression in theprimordial follicle, with the development of follicles expression intensity wasgradually reduced;0,4,8days group of PTEN protein expressed in the primordialfollicle, expression site by oocyte cytoplasm turned granular cytoplasm, expressionamount reduced.2. Pten-shRNA lentiviral packaged, best time to transfer was the ovary removed36h, the best multiplicity of infection was MOI=1.0×10⁹×20×10^-3/1×10⁶=20. AppliedHE staining, RT-PCR and western blot, compared with the control group, primordialfollicle proportional of the total number of follicles reduced, the expression ofpten-shRNA group pten mRNA and PTEN protein decreased markedly. Estrogencontent in the media reduced, progesterone reduced significantly.3. Added the PI3K inhibitor LY294002, compared with the control group,ptenmRNA expression did not change significantly, but primordial follicleproportional of the total number of follicles increased, primordial follicle growthwas suppressed. After pten-shRNA lentiviral transduction, observed by western blotPTEN protein expression in normal8days group than normal0day group fell, thebest interference8days group than normal8days group fell. PI3K proteinsexpression in the best interference8days group than normal8days group fell.Conclusions:1. Pten mRNA express in the primordial follicles, and the amount of PTENprotein expression reduce with the primordial follicles initiation and growth.2. After pten-shRNA lentiviral transfection, the number of primordial folliclesreduce, promote the initiation and growth of primordial follicles.3. Tumor suppressor gene pten by PI3K signaling pathway on primordial follicledevelopment, its upstream and downstream relationship may be the ptenâ†'PI3K.Meanwhile, through progesterone secretion in the follicle cells in the regulationof primordial follicle development.