title: The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells.
abstract: OBJECTIVE
  To improve the separation, identification and cultivation of ovarian germline stem cells (OGSCs), to clarify the relationship between the Hippo signaling pathway effector YAP1 and the proliferation and differentiation of OGSCs in vitro and to identify the major contribution of Hippo signaling to ovarian function.
  
  
  METHODS
  Two-step enzymatic separation processes and magnetic separation were used to isolate and identify OGSCs by determining the expression of Mvh, Oct4, Nanog, Fragilis and Stella markers. Then, YAP1, as the main effector molecule in the Hippo signaling pathway, was chosen as the target gene of the study. Lentivirus containing overexpressed YAP1 or a YAP1-targeted shRNA was transduced into OGSCs. The effects of modulating the Hippo signaling pathway on the proliferation, differentiation, reproduction and endocrine function of ovaries were observed by microinjecting the lentiviral vectors with overexpressed YAP1 or YAP1 shRNA into infertile mouse models or natural mice of reproductive age.
  
  
  RESULTS
  (1) The specific expression of Mvh, Oct4, Nanog, Fragilis and Stella markers was observed in isolated stem cells. Thus, the isolated cells were preliminarily identified as OGSCs. (2) The co-expression of LATS2, MST1, YAP1 and MVH was observed in isolated OGSCs. Mvh and Oct4 expression levels were significantly increased in OGSCs overexpressing YAP1 compared to GFP controls. Consistently, Mvh and Oct4 levels were significantly decreased in cells expressing YAP1-targeted shRNA. (3) After 14-75 days of YAP1 overexpression in infertile mouse models, we detected follicle regeneration in ovaries, the activation of primordial follicles and increased birth rate, accompanied by increasing levels of E2 and FSH. (4) However, we detected decreasing follicles in ovaries, lower birth rate, and decreasing E2 and FSH in serum from healthy mice of reproductive age following YAP1 shRNA expression.
  
  
  CONCLUSIONS
  Methods for the isolation, identification and culture of OGSCs were successfully established. Further results indicate that isolated OGSCs can specifically recognize Hippo signaling molecules and that manipulation of YAP1 expression can be used to regulate the proliferation and differentiation of OGSCs, as well as ovarian function in mice. This study suggests that the Hippo signaling pathway may represent a new molecular target for the regulation of mouse ovarian functional remodeling.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Ye
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.forename: Tuochen
authors.initials: T
authors.lastname: Zheng
authors.forename: Chuan
authors.initials: C
authors.lastname: Hu
authors.forename: Zezheng
authors.initials: Z
authors.lastname: Pan
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.forename: Yuehui
authors.initials: Y
authors.lastname: Zheng
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002868:Chromosomal Proteins, Non-Histone; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D059346:Magnets; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D000071317:Nanog Homeobox Protein / Q000235:genetics / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D000072977:Oogonial Stem Cells / Q000166:cytology / Q000378:metabolism*; D006080:Ovarian Follicle / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C462180:Dppa3 protein, mouse; D008565:Membrane Proteins; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; D010750:Phosphoproteins; C494174:Pou5f1 protein, mouse; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000091102:YAP-Signaling Proteins; C486223:Yap1 protein, mouse; C462181:fragilis protein, mouse; D049452:Green Fluorescent Proteins; C090864:Ddx4 protein, mouse; D053487:DEAD-box RNA Helicases
keywords: Hippo signaling pathway; Mice; Ovarian function; Ovarian germline stem cells; YAP1