Redox signaling as a modulator of germline stem cell behavior: Implications for regenerative medicine

Abstract

Germline stem cells (GSCs) are crucial for the generation of gametes and propagation of the species. Both intrinsic signaling pathways and environmental cues are employed in order to tightly control GSC behavior, including mitotic divisions, the choice between self-renewal or onset of differentiation, and survival. Recently, oxidation-reduction (redox) signaling has emerged as an important regulator of GSC and gamete behavior across species. In this review, we will highlight the primary mechanisms through which redox signaling acts to influence GSC behavior in different model organisms (Caenorhabditis elegans, Drosophila melanogaster and Mus musculus). In addition, we will summarize the latest research on the use of antioxidants to support mammalian spermatogenesis and discuss potential strategies for regenerative medicine in humans to enhance reproductive fitness.

Graphical Abstract

1. Introduction

Germline stem cells (GSCs) are a group of unipotent cells that play an important role in the development of the gonad and are essential for generation of gametes, the specialized cells that are required for reproduction from generation to generation (ie., eggs and sperm). As such, germ cells are occasionally described to be immortal. Upon cell division, GSCs can divide to self-renew, in order to maintain a reservoir of GSCs, or generate progenitor cells that eventually differentiate into mature, functional gametes. GSCs reside in a specialized microenvironment composed of somatic, stromal cells, referred to as the stem cell “niche”, that influences the decision between self-renewal and differentiation through signaling events. A detailed review on the generation of gametes, referred to as gametogenesis, and the different gonadal components can be seen in [1]. In most cases, the differentiating daughter cells will undergo a series of mitotic divisions, prior to undergoing germline-specific meiotic divisions and eventually maturing into either oocytes or sperm. Several important themes regarding GSC behavior are conserved across species. Signals from the GSC niche influence stem cell maintenance and self-renewal by promoting the repression of differentiation signals [1, 2]. In addition, signals from the somatic stromal cells can influence germline differentiation and maturation [3–7]. In this review, we will summarize the main findings regarding the role of redox signaling in influencing GSC behavior and discuss potential strategies for regenerative medicine in humans to enhance reproductive fitness.

2. Redox signaling in GSC niches

2.1. GSCs in Caenorhabditis elegans (C. elegans)

The gonad in the roundworm C. elegans is a U-shaped tube that contains a population of GSCs anchored at the very distal tip. In hermaphrodites, GSCs will give rise to sperm in late stage larvae and sustain the life-long production of oocytes in adults. Meanwhile, in males, GSCs only sustain sperm production [8]. During development, the migration of the pair of somatic distal tip cells (DTCs), which are located on opposing sides of an oval-shaped gonad primordium, is required for proper gonad elongation and morphology [9]. The DTCs are also a crucial niche component, controlling the proliferation and self-renewal of GSCs during development and adulthood, primarily through Notch signaling [2]. Proximally, the C. elegans germline is encapsulated by somatic gonadal sheath cells, which actively participate in the control of germline maturation (Figure 1A). For instance, the superoxide-generating globin GBL-12 was found to be anchored in the plasma membrane of gonadal sheath cells to generate superoxide (O₂⁻). GBL-12-generated O₂⁻ is then converted into a hydrogen peroxide (H₂O₂) gradient by both intra- and extracellular superoxidase dismutases (SODs) throughout the gonadal sheath cells, influencing fecundity, gonad morphology and germline apoptosis through the modulation of p38/JNK MAPK signaling [10] (Figure 1A, inset). Consistent with a causative role for redox signaling in germline differentiation, H₂O₂ levels increase in the nematode gonad from GSCs to later germline stages, peaking in oocytes [11]. Multiple mechanisms that generate reactive oxygen species (ROS) appear to be at play during germline differentiation, as an ERKMAPKdependent mitochondrial maturation process has been recently reported to drive GSC differentiation, in part, through production of ATP and ROS [12]. Additionally, redox signaling has been shown to modulate the activity of the Rho GTPase and influence tissue contractility of the spermatheca, a muscle-rich region of the gonad where the nematode sperm is stored [13]. It is tempting to speculate that the DTC may have a more active role in preventing redox signaling in GSCs, perhaps by expressing high levels of antioxidants that could dampen ROS levels in the niche, yet it remains to be seen if genetic or pharmacologic increases in levels of redox signaling specifically in GSCs would promote differentiation.

Figure 1. Schematics of GSC niches and the redox signaling at play.

A) One arm of the hermaphrodite C. elegans gonad is represented, with a distal tip cell (DTC) acting as the major somatic niche component for GSCs, and somatic sheath cells influencing germline differentiation. Superoxide generated by membrane-bound globins in sheath cells are converted into hydrogen peroxide by superoxide dismutases both intra- and extra-cellularly, influencing germline differentiation through the activation of p38/JNK. B) In the Drosophila testis, ROS generated in early germ cells by the mitochondria (and possibly other sources) can activate the EGFR pathway non-autonomously in somatic cyst cells. EGFR activation promotes the cell-autonomous differentiation of cyst cells, and the non-autonomous differentiation of the germline. C) In the Drosophila ovary, Wnt signaling promotes the expression of glutathione S-transferase genes (Gst), which results in changes in the redox status of escort cells (ECs) that ultimately influence the differentiation of the germline. D) In mouse testis, Sertoli cell-derived GNDF and FGF2 signaling maintains basal ROS levels in SSCs, which are crucial for the maintenance of these cells through the activation of JNK and p38.

2.2. GSCs in the Drosophila melanogaster (D. melanogaster) testis

Two resident stem cell populations — GSCs and somatic cyst stem cells (CySCs) — are present at the tip of the testis in the fruit fly D. melanogaster. GSCs and CySCs surround a group of quiescent somatic cells that form an organizing structure called the hub that secretes factors important for the maintenance of both stem cell populations [14–16]. GSCs divide to self-renew and give rise to a gonialblast (GB), which in turn will undergo four rounds of mitotic, transit amplification divisions, giving rise to cysts of 16 interconnected spermatogonia. The spermatogonia initiate a tissue specific gene expression program to differentiate further into spermatocytes and undergo meiosis, yielding 64 haploid spermatids that ultimately mature into sperm. Meanwhile, CySCs divide to repopulate the niche and to give rise to a cyst cell which, along with another cyst cell, will encapsulate the GB and ensure differentiation of the germ cells [14] (Figure 1B). Crosstalk between soma and germ line is required for proper differentiation. For example, germ cells secrete the epidermal growth factor (EGF) ligand Spitz that binds to the EGF receptor (EGFR) on cyst cells, activating the MAPK cascade, which is required for the co-differentiation of germ and cyst cells [17–19]. In addition, this pathway controls the balance between Rac and Rho GTPase activation, which is important for cyst cell encapsulation of the germ line [20]. Interestingly, cyst cells also provide a permeability barrier to reinforce an environment conducive to differentiation, similar to the blood-testis barrier in mammals [21]. Hence, in many ways, cyst cells act in an analogous fashion as mammalian Sertoli cells [22].

Recently, changes in ROS levels in the testis have been shown to influence GSC maintenance. Treating flies with the oxidant paraquat, or depletion of the NF-E2-related factor 2 (Nrf2) homolog cap’n’colar C (CncC) or overexpression of its inhibitor Kelch-like ECH-associated protein 1 (Keap1) in GSCs, led to elevated ROS and a reduction in stem cell number [23]. Interestingly, levels of the EGF-ligand Spitz were high under conditions of elevated ROS, and GSC loss could be suppressed if EGFR signaling was decreased in cyst cells [23]. Similarly, when the activity of the one protein responsible for directing mitochondrial fission, Dynamin related protein-1 (Drp1), was blocked, mitochondria were hyper-fused, and ROS levels increased dramatically [24]. Subsequently, GSCs were lost due to EGFR activation in the somatic cyst cells. Treatment with potent antioxidants suppressed the increase in Spitz expression in germ cells, EGFR activation in cyst cells and consequent GSC loss, further validating the concept that ROS levels must be kept low in the testis in order to prevent GSC loss due to EGFR activation in the soma [24]. As mitochondria in germ cells increase in complexity with differentiation, it is tempting to speculate that these mitochondria could be an important source of ROS that regulates germline development in Drosophila, similar to what was described in C. elegans [12],.

However, a ROS-mediated increase in Spitz levels is not likely to be the sole mechanism through which EGFR signaling is upregulated in cyst cells. In fact, ROS may affect other aspects of EGFR signaling, including ligand-independent phosphorylation and activation of EGFR and MAPK cascade components [25] (Figure 1B, inset). Moreover, gene expression changes were observed in testes with high ROS levels, including downregulation of the sole fly homolog of small Maf (Maf-S), which participates in the Keap1/Nrf2 response [26]. It will be interesting to understand precisely if and how other mechanism(s) may play a role in regulating germline differentiation in response to redox signaling and what the primary source of ROS is under homeostatic conditions in this system (i.e., environmental vs mitochondrial vs NADPH-derived). Given that other signaling pathways important for regulating cell fate decisions in the testis, such as JAK-STAT and Insulin Receptor pathways, are also composed of receptor tyrosine kinases and have phosphorylation-based mechanisms of activation, similar to EGFR signaling, it is tempting to speculate that ROS could also modulate the activity of additional pathways and, consequently, impact GSCs and/or CySC behavior.

2.3. GSCs in the D. melanogaster ovary

In contrast to mammals, ovaries from the fruit fly D. melanogaster contain GSCs that support the production of oocytes throughout the lifetime of the fly. GSCs are found at the tip of a structure called a germarium surrounded by somatic cells that support GSC maintenance and subsequent germline differentiation [1]. The adjacent terminal filament (TF) and cap cells (CC) secrete self-renewal signals, and CCs attach directly to GSCs, facilitating asymmetric outcomes to GSC divisions. Additionally, a group of somatic escort cells (also known as inner germarial sheath cells) wrap around GSCs and the differentiating germ cells to physically separate developing germline cysts. At roughly the 16-cell stage, a group of specialized epithelial cells generated from follicle stem cells (FSCs) take over the role of encapsulating the developing germline [1] (Figure 1C).

In the germarium, GSCs and FSCs are exposed to signals that promote maintenance, self-renewal, and differentiation [1, 6, 7, 27–29]. Many pathways, including EGFR, Rho and ecdysone signaling restrict self-renewal signals from diffusing beyond the GSC niche, thereby promoting germline differentiation [30–33]. In addition, Wnt4-modulated redox signaling was found to be an important component of the niche that supports differentiation in the ovary. Wnt4 signaling downregulates E-cadherin in escort cells as a way to promote germ cell differentiation [34]. However, Wnt2/4 signaling was also found to regulate the expression of glutathione-S-transferase (Gst) genes, which are required for the maintenance and proliferation of escort cells, as well as to prevent germline differentiation. Disrupting Wnt signaling or depletion of Gst and other oxidative genes increased ROS levels in both escort cells and germ cells and promoted germ cell differentiation [7]. Hence, reduced redox signaling is a crucial factor in maintaining GSCs in the ovary (Figure 1C, inset). Given the dual role of Wnt-signaling (i.e., promoting both maintenance and differentiation), it is tempting to speculate that ROS levels and cellular redox state must be precisely controlled in order to govern cell fate decisions. In addition, redox signaling may also affect other pathways, such as the EGFR pathway, that are known regulators of germline differentiation [32, 35, 36].

2.4. Spermatogonial stem cells in the mouse testis

The only GSCs present in mammals are the male spermatogonial stem cells (SSCs), as ovaries lack GSCs and, instead, harbor oocytes arrested in the diplotene stage of meiosis until maturation [37]. SSCs are found in the testis, immediately adjacent to somatic Sertoli cells, as well as peritubular myoid cells and interstitial Leydig cells. In addition, vascular tissue and immune cells in the interstitial tissue can contribute indirectly to the SSC niche (Figure 1D). Though all of these somatic cell types influence SSC behavior, Sertoli cells exert their function directly on the germline, providing SSCs and the differentiating spermatogonia with structural, nutritional and signaling components [38–40]. Estimated to be very few in number, SSCs divide to maintain the stem cell pool as well as to differentiate into later stages of spermatogenesis (spermatogonia, spermatocytes, spermatids and spermatozoa) [40, 41].

In the seminiferous tubules of humans and rodents, SSCs are located between the basement membrane and Sertoli cells. As described for other organisms, spermatogonia undergo several rounds of mitotic divisions prior to differentiating into spermatocytes, the latter of which transverse the Sertoli cell barrier and move to a physically separated compartment within the testis [40, 42]. This blood-testis barrier is important for the proper maturation of spermatocytes and overall fertility [43]. Interestingly, as the primary stromal component of the seminiferous tubule, Sertoli cells play a role in both the maintenance of SSCs and in spermatocyte differentiation. Signals such as glial cell derived-neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2) are secreted from Sertoli cells and are important for SSC self-renewal, while TGFβ ligands such as activin A and BMP4, and other factors such as stem cell factor (SCF) and retinoic acid (RA), support differentiation [38, 40, 41].

As discussed above for worms and flies, redox signaling also plays a role in controlling SSC behavior. It has been known for some time that elevated ROS levels cause oxidative stress and impact male fertility due, in part, to negative effects on spermatogonial survival, maintenance and activity [44–46]. However, basal levels of ROS are required for SSC self-renewal [47]. Both GDNF and FGF2 stimulation activate Ras, a critical factor in SSC self-renewal, via the PI3K-AKT and MAPK pathways [48–51]. Interestingly, Ras-overexpressing SSCs or wild-type SSCs stimulated with self-renewal factors generated detectable ROS in vitro. In addition, ROS was shown to be required and sufficient for the proliferation of SSCs in vitro and in vivo, as both genetic or pharmacologic manipulations in which ROS levels were decreased showed reduced spermatogonial proliferation, while H₂O₂ treatment increased SSC self-renewal. Both PI3K-AKT and MAPK pathways generate ROS through NADPH oxidase 1 (Nox1), activating p38/MAPK to control self-renewal through yet unidentified ways [47] (Figure 1D, inset).

The overall trend identified across species is that GSCs/SSCs tend to reside in niches where ROS levels are tightly controlled. While elevated ROS levels tend to be detrimental to stem cell maintenance, for instance, through the activation of cell-death pathways or the differentiation program, too little ROS may also negatively impact GSC/SSC behavior in many species. Although other stem cell populations, such as hematopoietic stem cells (HSCs) and neural stem cells (NSCs) reside in hypoxic niches where very little to no ROS is present [52], oxygen levels in the SSC niche remain uncertain and, as such, could influence the behavior of SSCs [53]. Moreover, the specific species created by reactive oxygen, as well as their sources, may influence cellular response in different ways. Further studies should aim on better understanding how redox signaling precisely controls GSC/SSC behavior, as well as determining how ROS levels are generated and/or maintained in the GSC/SSC niche.

3. Strategies for Regenerative Medicine

Common causes for the loss of fertility in men include side-effects of chemotherapy, as well as age-related decline in gamete number and quality. Though successful strategies have been developed in order to cryopreserve semen, procedures that aim to either preserve early spermatogonia in young/healthy individuals or to generate SSCs through iPS-mediated differentiation have not yet been successful [54]. Importantly, donor characteristics, such as age and any patho-physiological conditions, influence the ultimate efficiency of spermatogonial preservation and/or generation [53–56]. One major bottleneck in preservation of spermatogonia is that the optimal culture conditions for these cells is not known. For instance, as discussed above, the concentration of oxygen required for culturing SSCs in vitro remains debatable [53]. Given the role of redox signaling in GSC niches, finding the optimal concentration of oxygen that contributes to appropriate levels of ROS to maintain SSCs will be of extreme importance. As such, the search for methods that could improve SSC preservation and activity in mammals have been the focus of several recent studies [53, 55, 56].

3.1. Antioxidant therapies

One strategy employed by a number of groups recently focuses on the use of molecules that produce an antioxidant response during SSC cryopreservation, including the use of antioxidants themselves. For instance, the pineal gland hormone N-acetyl-5-methoxy-tryptamine (melatonin) has been used in a few studies related to SSC behavior [57–59]. Melatonin positively impacts cell viability, in part, by reducing cellular ROS levels and DNA mutation rates [60]. Melatonin receptors are expressed in testes from many mammals [61–64], and melatonin has been shown to protect mammalian gametes in culture from oxidative damage [65]. Supplementation of melatonin to the culture media was shown to increase neonatal mouse SSC proliferation in vitro [59]. In addition, melatonin reduced the oxidative stress and SSC apoptosis caused by the chemotherapy alkylating agent busulfan in mouse testes. Melatonin stimulated the expression of MnSOD, to reduce ROS levels, and SIRT1, which led to the deacetylation and ultimately degradation of p53 [58]. Melatonin also reduced the busulfan-induced endoplasmic reticulum stress response that contributes to SSC cell death [57].

Alpha-Tocopherol (α-TCP), a vitamin E-related antioxidant, and catalase, an antioxidant enzyme normally present in peroxisomes, have also been used successfully to improve murine SSC cryopreservation conditions. The addition of these antioxidants to the cryopreservation media improved SSC viability and proliferation rates in vitro after freeze-thawing, a process known to be stressful and detrimental to cell survival. In addition, restoration of spermatogenesis was improved after transplantation into mice that were previously treated with busulfan. Antioxidant treatment in SSCs induced the expression of the anti-apoptotic gene Bcl2 and of the self-renewal genes Plzf and ID4, while reducing the expression of the pro-apoptotic gene Bax [66]. Another antioxidant used in a recent study was caffeic acid (CA). Though the precise mechanisms of action in cells are still elusive, the addition of CA to the media helped maintain a healthy pool of SSCs upon freeze-thawing by stimulating apoptosis in damaged SSCs [67] (Table 1).

Table 1:

Summary of molecules, conditions, and primary effects of antioxidants on mammalian spermatogenesis.

Antioxidant Molecules	Condition	Phenotypes	Reference
Melatonin	in culture	increased neonatal mouse SSC proliferation	59
	in culture	increased survival of mammalian gametes after freeze-thaw	65
	in respone to chemotherapy (busulfan) in vivo	reduced oxidative stress and murine SSC apoptosis	57
α-TCP and catalase	in culture	improved murine SSC viability and proliferation after freeze-thaw	66
	in respone to chemotherapy (busulfan) in vivo	improved restoration spermatogenesis in mice	66
Caffeic acid	in culture	stimulated damaged murine SSC apoptosis	67

In sum, studies suggest that the pharmacological decrease in ROS levels in cultured murine SSCs may positively impact stem cell maintenance and activity, at least in the short term. However, whether or not these findings will hold true for cultured, human SSCs remains to be seen. Given that there is a threshold beyond which ROS is detrimental and that antioxidants may work via different mechanisms, future approaches may involve titrating and/or combining antioxidants to find the perfect concentrations to support each stage of germ cell development. Additionally, it will be interesting to explore whether interventions that control the redox status in the SSC niche in vivo could also prime any remaining healthy SSCs to proliferate and restore spermatogenesis in older individuals or patients that have undergone procedures such as chemotherapy.

Highlights.

• GSCs often reside in niches with little to no ROS

• Increased ROS levels correlate with germline differentiation

• Differentiation pathways can be targeted and activated by ROS

• Regenerative strategies for SSC maintenance/activity focus on the use of antioxidants