Research advances in the regulation of the putative ovarian germline stem cell niche on female germline stem cells

ABSTRACT

Stem cells are ideal seeding cells, which have the potential for self-renewal and multiple differentiation, and they play a fundamental role in maintaining homeostasis and regenerating and repairing tissue. The discovery of female germline stem cells (FGSCs) brings much hope for the postnatal renewal of oocytes and solving some female infertility problems. Ovarian function declines with increasing female age. Moreover, ovarian germline stem cell niche-aging could be the main cause of ovarian senescence, which ultimately leads to decreased follicle generation, declining female fertility, and age-related diseases, such as osteoporosis and ovarian cancer. The ovarian germline stem cell niche is the surrounding microenvironment in which FGSCs live, and it helps control the biological characteristics of FGSCs in many ways, such as nutritional supply and immunological cytokine secretion. This paper reviews the knowledge about the ovarian germline stem cell niche and its probable regulatory mechanisms on FGSCs, which provides valuable scientific information and scope for the prevention and treatment of ovarian senescence.

Abbreviations: BMP: bone morphogenetic protein; Dpp: decapentaplegic; FGSC: female germline stem cell; IL, interleukin; OGSC: ovarian germline stem cells; ROS: reactive oxygen species; TGF, transforming growth factor; TNF, tumor necrosis factor

KEYWORDS:

• Niche
• ovarian germline stem cell niche
• female germline stem cells
• ovarian aging

Introduction

The environment in which stem cells live consists of niche cells, extracellular matrix, vessels, and cytokines that support stem cell survival. This stem cell microenvironment is also called as stem cell niche (Pan Z et al. 2016). The ovarian germline stem cell niche includes immune cells, cytokines, collagen, enzymes, and their regulation which involves the Hippo and Notch signaling pathways (Sato et al. 2011; Li et al. 2015; Van De Bor et al. 2015; Ye et al. 2016, 2017). Normally, signals under a steady-state as well as cell–cell and cell–matrix interactions are required for the maintenance of the function of stem cells (Ferraro et al. 2010; Rodgers and Rando 2012). The extracellular matrix is a major component of the microenvironment that regulates the polarity of stem cells via the cell–matrix interactions and keeps the balance between self-renewal and differentiation of the stem cells (Ahmed and Ffrench-Constant 2016). The non-stem cells in the ovarian germline stem cell niche, such as cap cells, play an important role in controlling the number, differentiation, and behavior of FGSCs through crosstalk (Song et al. 2007). Additionally, subtle variations in the niche, such as pH, oxygen level, and ion concentration, can also modulate the activity of stem cells (Inaba et al. 2016).

However, aging can lead to organ dysfunction and diseases. Several events that occur in the ovarian germline stem cell niche will influence the regenerative and proliferative abilities of the FGSCs, including DNA damage, mitochondrial damage, and epigenetic modification. The homeostasis of the ovarian microenvironment is consequently broken. Up to now, the research has been relatively detailed on structure and function of the intestinal, myocardial, neural, and hematopoietic stem cell niche. Once stem cell niches are broken, stem cells lose their self-renewal ability, and dysfunction of digestive, circulatory, and nervous systems will occur. However, the structure of ovarian germline stem cell niche and its regulatory mechanisms remains unclear. Maintaining the homeostasis of ovarian germline stem cell niche and delaying ovarian aging is beneficial for the health and quality of life of women.

The discovery of intestinal, myocardial, neural and hematopoietic stem cell niches and their regulatory effects on stem cells

The intestinal stem cell niche consists of substances, such as intestinal nerve cells, lymphocytes, endothelial cells, and vessels, which play a role in regulating the function of intestinal stem cells (Ferraro et al. 2010). During aging, changes in the intestine microenvironment damage the intestinal stem cells, leading to dysfunction in digestion and absorption. Equally, the proliferation and differentiation of the myocardial stem cells are regulated by the myocardial stem cell niche. The myocardial stem cell niche consists of supporting cells, such as myocardial cells, fibroblastic cells, and endothelial cells, the majority of which are distributed in the atrium, and the tip of the heart (Leri et al. 2014). An aged and damaged myocardial microenvironment will weaken the crosstalk of myocardial stem cells and the myocardial stem cell niche. This greatly damages the regenerative ability of the myocardial stem cells (Hariharan and Sussman 2015), which will increase susceptibility to myocardial injury, disrupt the homeostasis and may finally result in heart failure. The neural stem cells are located in the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus (Sottocornola and Lo Celso 2012). The neural stem cell niche is the site where neural stem cells proliferate, differentiate, migrate, and mature, and it consists of astrocytes, microglial cells, vessels, extracellular matrix, and the axon endings (Fitzsimons et al. 2014). Senescence and disorder of the microenvironment, caused by the reduction of the brain–blood flow, oxygen content, and nerve nutritional factors, will lead to the decrease of neuron regeneration and accelerate the apoptosis of neural stem cells, and thus may result in neurodegenerative diseases such as Alzheimer’s disease (Leenders et al. 1990; Ajmani et al. 2000; Bentourkia et al. 2000; Drummond-Barbosa 2008; Maldonado-Soto et al. 2014). In addition, the number of neurons in the hippocampus gradually decreases in aged individuals, which is a major cause of the decline of cognitive, learning, and memory abilities (Bishop et al. 2010; Lazarov et al. 2010; Lugert et al. 2010; Artegiani and Calegari 2012). Hematopoietic stem cells exist in the peripheral blood and bone marrow with self-renewal and pluripotent differentiation properties. They play an important role in hematopoiesis, immunology, tissue oxygen uptake, and coagulation (Drummond-Barbosa 2008; Yang et al. 2015). The hematopoietic stem cell niche is composed of supporting cells, extracellular matrix, and various hematopoietic growth factors, which provide support and protection for the hematopoietic stem cells (Park et al. 2012). The dysfunction of the hematopoietic stem cells is also relevant to the disturbance of their microenvironment. In a decaying hematopoietic microenvironment, the increased level of the inflammatory factors [such as interleukin-6 (IL-6)], along with the increase of the reactive oxygen species (ROS), and epigenetic modifications, such as DNA methylation, histone modification, and chromatin structural changes, will lead to deviation of the hematopoietic stem cells from their normal differentiated orientation. This will further induce diseases, such as myelodysplastic syndrome, myeloid tumor, and thrombus (Drummond-Barbosa 2008; de Mora and Juan 2013; Kohli and Passegue 2014; Choudry and Frontini 2016). Since the stem cell niche plays a decisive role in the fate of the stem cells, aberration of the intestinal, myocardial, neural, and hematopoietic stem cell microenvironments needs to be repaired in time. This will prevent further damage of stem cells, which is helpful for maintaining the function of stem cells and the treatment of diseases.

The discovery of the FGSCs and the regulatory effect of the ovarian germline stem cell niche

Female germline stem cells exist in invertebrates such as Drosophila and other lower vertebrates. The traditional view holds that women are born with a finite number of oocytes. Zou et al. 2009 have successfully isolated and purified FGSCs from mice and humans, identified FGSCs in postnatal mammalian ovaries, which challenged the prevailing view that female mammals lose the capacity for oocyte generation prior to birth; but these results still remain controversial (Zou et al. 2009; Celik et al. 2012; Woods and Tilly 2013; Dunlop et al. 2014). FGSCs exist in the ovarian cortical surface underneath epithelial cells, which can undergo self-renewal and differentiation and express germline cell marker MVH and can be labeled with the cell proliferation marker 5-bromodeoxyuridine. They serve as a source of germ cells and can differentiate into primary oocytes (Celik et al. 2012). As the discovery of FGSCs and the study of their functions were under laboratory conditions, whether transplanting FGSCs into infertile female ovaries could solve some infertility problems is also controversial, and it has become a current research hotspot.

The main function of the ovarian stem cell niche is to regulate FGSCs to divide into new stem cells symmetrically and to differentiate into germ cells. This will help to maintain the continuous proliferation of postnatal follicles and oocytes (Du and Taylor 2009). The crosstalk between the FGSCs and their niche depends on the cell adhesion, whereas cadherin plays an important role in this adhesion junction. FGSCs with a high level of cadherin expression are much more competitive than the neighboring cells in forming the stem cell niche (Eliazer and Buszczak 2011).

The regulatory effect of the ovarian germline stem cell niche on FGSCs

The ovary appears to age faster than that of other organs. Ovarian recession occurs with menopause, when women have decreased quantity and quality of oocytes and further lose fertility (Garg and Sinclair 2015). Some studies indicate that the ovarian recession is caused by destruction of the ovarian germline stem cell niche (Gheorghisan-Galateanu et al. 2014).

Other studies suggest that the reduced regeneration of the oocytes in the decaying ovary is a result of the disruption of the niche for FGSCs, rather than being caused by any primary dysfunction of germ cells. When the ovarian germline stem cell niche among menopausal women recedes, it cannot support the maturation and regeneration of the oocytes (Banerjee et al. 2014; Ozakpinar et al. 2015). Therefore, the stem cell niche is much more relevant to organ aging compared to stem cells themselves (Massasa et al. 2010).

The decisive effect of the stem cell niche on stem cells is demonstrated by the fact that oxidative damage breaks the adhesive bond of FGSCs to the stem cell niche. Oxidative stress is caused by an imbalance between the production and elimination of the ROS in the body. ROS play an important role in modifying female reproductive function. Studies have shown that during the female perimenopausal period, the accumulation of ROS damages the ovarian germline stem cell niche, and leads to follicular atresia (Banerjee et al. 2014). Carbone’s experiment also confirmed that the clearance efficiency of ROS decreases with ovarian decay, which is caused by the decline of the body’s anti-oxidative function, thus disturbing the ovarian germline stem cell niche (Carbone et al. 2003). Experiments on the ovaries of sand flies suggest that maintenance of the activity of the superoxide dismutase could delay ovarian aging by eliminating ROS. Therefore, it is beneficial for survival of the FGSCs (Diaz-Albiter et al. 2011). However, the enzymatic activity of the stem cell niche should be strictly regulated. Over-expression of matrix metalloproteinase can damage the extracellular matrix and degrade the collagen, thus damaging the ovarian germline stem cell niche and leading to the abnormal proliferation of the FGSCs, which eventually causes ovarian cancer.

The deficiency of nutritional factors in the stem cell niche will also indirectly accelerate decay of FGSCs (Jasper and Kennedy 2012). This is compounded by mitochondria that exists in the cytoplasmic matrix provide energy for the development of the oocytes that with age in females, the copies of oocyte mitochondrial DNA decrease and the ATP level declines. In this manner, the energy of FGSCs provided by the ovarian germline stem cell niche greatly decreases increasing the risk of birth defects and infertility (Garg and Sinclair 2015).

The role of immunity in the niche

During ovarian development, the maintenance of the steady-state of the ovarian germline stem cell niche is inseparable from the function of immune cells. The stem cell niche consists of a perivascular compartment, which connects the stem cells to the immune and vascular system (Bukovsky 2011). The mature ovarian germline stem cell niche contains, e.g., both primary and activated monocyte-derived cells, T cells. The ovarian germline stem cells are also regulated by vascular pericytes, immunoglobulins, and autonomic innervation of the immune system, both of which are essential for the asymmetric division of the ovarian germline stem cells into new germline cells (Ye et al. 2016). Immune cells, such as macrophages, also play an important role in keeping the steady-state of the stem cell niche. Macrophages can secrete many cytokines, including IL-10 and tumor necrosis factor-alpha (TNF-α), which accelerate the removal of aging erythrocytes and necrotic tissues that may be the cause of the aging of the stem cell niche (Casanova-Acebes et al. 2014). In addition, cytokines, TNF-α, and IL-1, can facilitate the process of ovulation, which is closely correlated with angiogenesis. Animal model studies have demonstrated that for the developing corpus luteum, TNF can promote angiogenesis via enhancing the expression of vascular endothelial growth factor, but for the mature corpus luteum, TNF inhibits angiogenesis; thus, the dual-regulatory mechanism of TNF remains elusive (Galvao et al. 2013). However, the inflammatory microenvironment will greatly damage the stem cell niche, ablating the survival, and normal function of the stem cells (Dooley et al. 2014). Researchers also discovered that with increasing age, the immune system decays, characterized by thymic dysfunction and ovarian recession (Bukovsky and Caudle 2012). Ultimately, ovarian follicle renewal ceases (Bukovsky 2011).

The function of the immune system is also regulated by the hypothalamus and the level of estrogens is crucial for the function of immune cells. Studies have indicated that multiple immune cells exist in the hypothalamic-pituitary-ovary axis (Sato et al. 2011). The cytokines secreted by the immune cells can regulate estrogen secretion and follicular formation and further influence the maintenance of homeostasis of the stem cell niche (Sato et al. 2011). The appropriate application of immunopotentiators will enhance the immunity and reproductive function at the same time.

Homeostasis of ovarian germline stem cell niche is also dependent on the ovarian hematocyte. ‘Plasmatocytes’ can secrete collagen IV, which is a major component of the basement membrane of the ovarian germline stem cell niche. Reducing the level of collagen IV will significantly destroy the integrity of stem cell niche (Van De Bor et al. 2015). In addition, Drosophila ovarian experiments have proven that collagen IV can ensure a high level of bone morphogenetic protein (BMP) signaling by binding decapentaplegic (Dpp), thus increasing the identity of stem cells (Inaba et al. 2016). Therefore, we can regulate the ovarian germline stem cell niche by increasing the level of collagen secreted by the ‘plasmatocytes.’ Cap cells, which play a role in controlling the number of FGSCs, are also an important component of the germline stem cell niche (Song et al. 2007). It has previously been demonstrated that, in the Drosophila ovarian stem cell niche, cap cells were in direct contact with the germline stem cells, and disrupting this connection would bring about failure in the asymmetry of the stem cell division (Deng and Lin 1997). As the close adhesion between the FGSCs and the cap cells mediated by the E-cadherin is important for keeping the integrity of the ovarian germline stem cell niche, the decrease of cadherin expression will also lead to premature failure of the stem cell niche (Pearson et al. 2016). Generally, the discovery of the contribution of DE-cadherin and beta-catenin to this connection, and their requirement for maintaining the germline stem cells, supports the central role of cap cells in the niche (Deng and Lin 1997).

Signaling pathways are also a part of the niche

In addition to the extracellular matrix components, the cell–cell adhesion mechanism and the immune system related factors, signaling pathways are also required to maintain germline stem cells. Their exact mechanism of action remains to be investigated. For example, the transforming growth factor-beta (TGF-β) signaling pathway also plays an important role in regulating cell proliferation, differentiation, and apoptosis (Shull and Doetschman 1994; Godkin and Dore 1998; Ingman and Robertson 2002). As shown in an ovarian mouse model TGF-β signaling plays a physiological role in regulating the pool of primordial follicles by inhibiting the growth of primordial follicles. This is achieved by activation of S6K1-rpS6 involved in oocyte growth (Wang et al. 2014). Activating the BMP and insulin signaling pathway can increase nutritional ingestion of the stem cell niche, which provides energy support for the proliferation of the FGSCs (Gancz and Gilboa 2013). Additionally, it has been shown that, in Drosophila ovaries, insulin signaling could influence the level of E-cadherin at the germline stem cell–cap cell junction. Insulin has also been indicated to be crucial for maintaining the integrity of the niche and controlling the number of cap cells (Hsu and Drummond-Barbosa 2009; Kao et al. 2015). An increasing number of studies have indicated that the Hippo and Notch signaling pathways are crucial for controlling the process of stem cell proliferation and differentiation. Our recent studies suggested that both the Hippo signaling factor and the Notch signaling pathway could also help the self-renewal and differentiation of FGSCs and thus promote development of primordial follicles (Li et al. 2015; Pan Z et al. 2015; Ye et al. 2017). There has been emerging evidence that the Hippo signaling pathway could regulate organ size and tissue homeostasis in Drosophila and mammals (Pan D 2010). Three key molecules of the Hippo signaling pathway (LATS2, MST1, and YAP1) were both expressed in ovarian germline stem cells (OGSCs) and closely correlated with the size of the primordial follicle pool. The over-expression of YAP1, the main effect molecule in the Hippo signaling pathway could contribute to the proliferation of primordial follicles and increase the thickness of the ovarian surface epithelium. This finding provides evidence that the Hippo effector molecule, YAP1, plays an essential role in reconstructing ovarian function (Ye et al. 2017). In general, Hippo signaling can inhibit growth, mediate apoptosis, and regulate cell fate determination (Halder and Johnson 2011). However, the Hippo signaling pathway works together with multiple signaling pathways, such as Notch, TGF-beta, and P13K/Akt, at different levels (Xiang et al. 2015). It can be concluded that disrupting the Hippo signal, activating the Akt pathway, and increasing YAP nuclear localization could promote follicle growth (Kawamura et al. 2013). In addition, our recent study suggests that when the key components of the Notch pathway (Notch1, Hes1, and Hes5) were inhibited by DAPT, the expression of germline stem cell markers, especially MVH and OCT4, were also down-regulated. Moreover, the results were the same when ovarian aging occurred (Li et al. 2015; Pan Z et al. 2015; Ye et al. 2017). Experiments on Drosophila showed that decreased Notch signaling significantly reduced the number of cap cells and niche size in parallel with the number of FGSCs (Song et al. 2007). Disruption of Notch signaling will result in premature ovarian failure, accompanied by reduction of the number of follicles (Vanorny et al. 2014). It has been shown that replacing the aging ovary of mice with ovarian germline stem cell niche of young mice could restore the reproductive ability of the oocyte in the future (Pan Z et al. 2016). As summarized in Figure 1, this may help to resolve some cases of female infertility.

Figure 1. The ideograph of the supposed ovarian germline stem cell niche. Germline stem cells obtain nutrients from the vessels, which exist both inside and outside of the niche. Superoxide dismutase in the niche helps clear reactive oxygen species (ROS). The immune cells in the niche, such as macrophages, can secrete many cytokines, such as IL-10 and TNF-α, which help to slow the aging of stem cell niche. E-cadherin connects the cap cells and FGSCs and plays an important role in keeping the integrity of the niche.

Future directions and remaining questions

Premature ovarian failure causes loss of normal function of the ovary, which can be treated with hormones, but there is no way to completely solve it at this time. The discovery of ovarian germline stem cells fills a gap and has generated great attention in reproductive medicine offering the prospect of stem cell-based treatment. Germline stem cells may not only supply a feasible model for the production, development, and maturation of germ cells but also play a role in fertility rescue (Dunlop et al. 2014). However, before using ovarian germline stem cells to treat female infertility, the developmental mechanism is required.

The ovarian germline stem cell niche is composed of a body fluid perivascular compartment, which supports stem cell survival. The ovarian germline stem cell niche is formed during early development when the coelomic epithelium acquires the ability to differentiate into secondary germ cells in embryos (Bukovsky 2011). The ovarian stem cell niche harbors the function to regulate FGSCs to divide into new stem cells and to differentiate into germ cells, which helps to maintain continuous proliferation of postnatal follicles and oocytes (Du and Taylor 2009). Several issues need to be addressed and clarified before the application of FGSC niche for female infertility reaches clinical practice. These include: (1) Definition of the molecular mechanisms of action (Pan et al. 2016). (2) It has yet to be shown how to establish, stabilize, and prevent other cells from contaminating germline stem cell lines and how to freeze and thaw these germline stem cells without damage. (3) It is not yet known how the ovarian germline stem cell niche regulates ovarian germline stem cells. (4) It also remains unknown whether there is also a similar germline stem cell niche that exists in the human ovary, what role these cells play in the normal human body, and how they can be tested in the normal human body in the future. (5) Methods of germline stem cell isolation that can induce differentiation and in vitro maturation and the ethical issues of transplantation of the germline stem cells remain to be solved. The potential use of ovarian germline stem cells may help to recover the fertility of women with premature menopause or undergoing cancer treatment. However, the controversy about whether germline stem cell niche exists is far from over.

Aimed at the above problems, on the one hand, we could develop regulators to improve the nutritional status of the niche, increase energy supply to the FGSCs from the niche, and modulate immune responses to delay or reverse FGSC aging. On the other hand, we could further explore the mechanism of how FGSCs are regulated by ovarian germline stem cell niche, keep germline stem cells alive, maintain female fertility, and prolong life. For example, for women with premature ovarian failure, could we enhance the immune function via dietary therapy and exercise? This approach may provide the decaying ovary with a suitable microenvironment, to promote ovarian germline stem cell activation and differentiation into germ cells, filling the primordial follicle pool, delaying female reproductive aging, and further improving the outcome of perimenopausal syndrome and other age-related diseases caused by ovarian aging. For patients receiving chemotherapy before puberty, before they receive treatment, ovarian tissue could be partly resected and digested into tissue suspension and then cryopreserved. Following full recovery, the cryopreserved tissue could then be transplanted back into the remaining ovary. After puberty, the reproductive function could recover.

Up to now, the decline of female reproductive function caused by organism aging and perimenopausal-related diseases has been of great concern. As research of ovarian germline stem cell niche is a new field, there are many problems that remain to be solved. Thus, efforts to develop new laboratory research programs of potential clinical application should be continued. The opportunities for this research are unpredictable; the understanding and application of ovarian germline stem cell niche has the potential to make a great difference in regenerative medicine.

Acknowledgments

The authors are thankful for the guidance of Professor YueHuiZheng and all of the authors’ help. Special thanks to Professor JuQiu from Institute of Health Sciences, Shanghai Academy of Life Sciences.

Disclosure statement

The authors have no conflicts of interest. The authors alone are responsible for the content and writing of this paper. All authors approved revisions and the final paper. Views expressed in the submitted article are our own and not an official position of the institution or funder.

Additional information

Funding

This work was supported by the National Nature Science Foundation of China (Nos. 81671455, 81360100, 81260098, 81660245) and the Natural Science Foundation of Jiangxi province (Nos. 20152ACB20023, 20161BAB205207, 20161BAB205213, 20153BCB23031).

Notes on contributors

Yangchun Liu

Wrote the manuscript: YCL; Revising the manuscript: JX, FYZ, HFY, CH, JH, YHZ.