Ovarian Germline Stem Cells (OGSCs) and the Hippo Signaling Pathway Association with Physiological and Pathological Ovarian Aging in Mice

Background/Aims: The isolation and establishment of female germline stem cells (FGSCs) is controversial because of questions regarding the reliability and stability of the isolation method using antibody targeting mouse vasa homologue (MVH), and the molecular mechanism of FGSCs self-renewal remains unclear. Thus, there needs to be a simple and reliable method for sorting FGSCs to study them. Methods: We applied the differential adhesion method to enrich FGSCs (DA-FGSCs) from mouse ovaries. Through four rounds of purification and 7-9 subsequent passages, DA-FGSC lines were established. In addition, we assessed the role of the phosphoinositide-3 kinase (PI3K)-AKT pathway in regulating FGSC self-renewal. Results: The obtained DA-FGSCs spontaneously differentiated into oocyte-like cells in vitro and formed functional eggs in vivo that were fertilized and produced healthy offspring. AKT was rapidly phosphorylated when the proliferation rate of FGSCs increased after 10 passages, and the addition of a chemical PI3K inhibitor prevented FGSCs self-renewal. Furthermore, over-expression of AKT-induced proliferation and differentiation of FGSCs, c-Myc, Oct-4 and Gdf-9 levels were increased. Conclusions: The differential adhesion method provides a more feasible approach and is an easier procedure to establish FGSC lines than traditional methods. The AKT pathway plays an important role in regulation of the proliferation and maintenance of FGSCs. These findings could help promote stem cell studies and provide a better understanding of causes of ovarian infertility, thereby providing potential treatments for infertility.

Section: Discussionmentioning

confidence: 99%

Enrichment of Female Germline Stem Cells from Mouse Ovaries Using the Differential Adhesion Method

Xiong

et al. 2018

“…The whole protocol was performed according to previously described methods [24]. Total RNA (1 µg in a 25-µL reaction) was reverse-transcribed using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, RR047A).…”

Section: Methodsmentioning

confidence: 99%

“…For immunofluorescence, the procedure was performed according to previously published protocols [24]. The primary antibodies used in this study were anti-LATS2 (1: 100, ab54073), anti-MST1 (1: 100, ab51134), anti-YAP1 (1: 100, ab56701), anti-S127 phosphorylated-YAP1 (1: 100, ab76252), anti-OCT4 (1: 100, ab18976) and anti-MVH (1: 100, ab27591).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the OSE layer has cell-cell contacts and local signaling that may be associated with GSC growth and development [22]. Importantly, evidence suggests that the Hippo signaling pathway is spatiotemporally correlated with the size of the primordial follicle pool [23] and that the existence of OGSCs in the mouse OSE and the expression of OGSCs markers are correlated with both the age of the mouse and with Hippo signaling molecules [24]. …”

Section: Introductionmentioning

confidence: 99%

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The Hippo Signaling Pathway Regulates Ovarian Function via the Proliferation of Ovarian Germline Stem Cells

Zheng

et al. 2017

Self Cite

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. Conclusion: 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.

“…Additionally, they can express MVH, OCT4, Nango, Fragilis, Stella, C-kit and many other markers of reproductive cells and stem cells as determined via PCR. After obtaining training in Professor Ji Wu’s laboratory of Shanghai Jiao Tong University and improving our culture techniques, our group has been able to expertly separate, purify and culture female ovarian stem cells, resulting in a solid foundation for female reproductive stem cells research [20-22]. …”

Section: Research Background and Characteristics Of Ovarian Germ Stemmentioning

confidence: 99%

Ovarian Stem Cell Nests in Reproduction and Ovarian Aging

Zheng

et al. 2017

Self Cite

The fixed primordial follicles pool theory, which monopolized reproductive medicine for more than one hundred years, has been broken by the discovery, successful isolation and establishment of ovarian stem cells. It has brought more hope than ever of increasing the size of primordial follicle pool, improving ovarian function and delaying ovarian consenescence. Traditional view holds that stem cell aging contributes to the senility of body and organs. However, in the process of ovarian aging, the main factor leading to the decline of the reproductive function is the aging and degradation of ovarian stem cell nests, rather than the senescence of ovarian germ cells themselves. Recent studies have found that the immune system and circulatory system are involved in the formation of ovarian germline stem cell niches, as well as regulating the proliferation and differentiation of ovarian germline stem cells through cellular and hormonal signals. Therefore, we can improve ovarian function and delay ovarian aging by improving the immune system and circulatory system, which will provide an updated program for the treatment of premature ovarian failure (POF) and infertility.