| Objective:Clarifying the molecular mechanisms by which the initiation of primordial follicles is regulated is crucial for the prevention and treatment of female infertility and premature ovarian failure.Our previous work had verified the expression of HIPPO signaling pathway in mouse ovaries and it has been proven to have spatio-temporal correlation with the size of primordial follicle pool.However,the role and underlying mechanism were poorly understood.In this study,we further explored the expression,effects and regulatory mechanisms of HIPPO signaling pathway in primordial follicle activation,new information could be accumulated to the molecular basis of initiation and maintenance of primordial follicles.Methods:1.Using immunofluorescence techniques and Western blot,combined with the culture system of mouse primordial follicles in vitro,the localization and expression of HIPPO pathway family genes were detected in 3 d mouse ovaries.2.Constructing lentiviral vectors of YAP1,the key effector of HIPPO signaling pathway:(1)YAP1 overexpressing lentiviral vector(Vector-YAP1)and negative controlling group(Vector).(2)YAP1 interfering lentiviral vector(sh YAP1-1、sh YAP1-2、sh YAP1-3)and negative controlling group(sh Control).3.Using immunofluorescence techniques and Western blot,the best effective YAP1 interference fragment was selected.4.By transfecting Vector-YAP1 and sh YAP1 in 3 d mouse ovaries respectively,the effects of YAP1 upregulation or downregulation on primordial follicle activation and growth,as well as the change of AKT expression,were observed by using HE staining and Western blot assay.5.Adding MK2206-2HCl,an AKT specific inhibitor to the culture system ofmouse primordial follicles in vitro,the effect of AKT on the primordial follicle activation was observed,and the expression of HIPPO pathway family genes including MST1,LATS2 and YAP1 were detected as well.6.By adding Vector-YAP1 to the MK2206-2HCl cultured model of mouse primordial follicles in vitro,the role of YAP1 in the process of primordial follicle activation was further verified.Results:1.Immunofluorescence results showed that: HIPPO signaling pathway family genes included MST1,P-MST,LATS2,YAP1 and P-YAP1 were expressed in 3 d and cultured 8 d mouse ovaries.Further more,MST1 co-expressed with LATS2.MST1 expressed in cytoplasm of oocytes in primordial follicles,and localized in cytoplasm of oocytes,nucleus of oocytes and granulosa cells in primary follicles.The expression of P-MST1 was distributed in cytoplasm of oocytes,nucleus of oocytes and granulosa cells in primordial and primary follicles.LATS2 expressed in cytoplasm of oocytes and nucleus of oocytes in primordial follicles,and localized in cytoplasm of oocytes and granulosa cells in primary follicles.YAP1 and P-YAP1co-expressed in cytoplasm of oocytes in primordial follicles,and co-expressed in cytoplasm of oocytes,nucleus of oocytes and granulosa cells in primary follicles.The results of Western blot showed that,the expression of MST1,P-MST and LATS2 was decreased significantly in cultured 8 d ovaries(p< 0.05,p< 0.01,p< 0.001),the expression of YAP1 and P-YAP1 was increased significantly(p< 0.01,p< 0.01),the ratio of P-MST/MST1 and P-YAP1/YAP1 was declined significantly(p< 0.05,p<0.05).It was showed that the expression of the core components of HIPPO pathway changed significantly with the initiation of primordial follicles in mice,which implied that primordial follicle activation related closely to the attenuation of HIPPO signaling pathway.2.Constructing the key effector of HIPPO signaling pathway,YAP1 gene lentiviral vectors.The results of Western blot and immunofluorescence showed that mouse ovaries could be transfected by YAP1 overexpressing,interfering and negative control lentiviral vector effectively.3.Selecting the best effective interference fragment: Compared with Vector group,the expression of YAP1 in Vector-YAP1 group increased significantly(p<0.05).Compared with sh Control group,the expression of YAP1 in sh YAP1-1,sh YAP1-2 and sh YAP1-3 groups decreased significantly(p< 0.05,p< 0.01,p< 0.001),with the best effective interference in sh YAP1-3 group,named as sh YAP1 group.4.Transfecting 3 d mouse ovaries with Vector-YAP1 and sh YAP1 lentiviral vector respectively.The results of HE showed that: Compared with Vector group,the number of primordial follicles decreased significantly(p< 0.05)and the number of primary follicles increased significantly(p< 0.05)in Vector-YAP1 group.Compared with sh Control group,the number of primordial follicles increased significantly(p<0.05)and the number of primary follicles decreased significantly(p< 0.05)in sh YAP1 group.It was shown that the primordial follicle activation could be influenced by the activity of YAP1,the key effector of HIPPO pathway.5.The results of Western blot showed that: YAP1 overexpression or downexpression had no influence on the phosphorylation of AKT(p> 0.05).After adding AKT specific inhibitor-MK2206-2HCl in the culture system of primordial follicles in vitro,there were no significant difference in the total protein expression of AKT,MST1 and YAP1 in MK2206 group compared with Control group.6.The results of HE showed that: Compared with Control group,the number of primordial follicles increased significantly(p< 0.01)and the number of primary follicles decreased significantly in MK2206 group(p< 0.05).In MK2206 +Vector-YAP1 group(MK2206 group cultured with YAP1 overexpressing lentiviral vector),the number of primordial follicles increased significantly compared with Control group(p< 0.05)and decreased significantly with MK2206 group(p< 0.05),while the number of primary follicles showed the opposite trend.It was shown that AKT upregulation could suppress the initiation of primordial follicles,which could be partially rescued by YAP upregulation.Conclusions:1.HIPPO signaling pathway is crucial to regulating the quiescence and activation of primordial follicles in mice.2.HIPPO signaling pathway is involved in AKT regulation of primordial follicle activation. |
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