Autonomous follicle quality control mechanisms: Innate immune signaling capabilities of granulosa cells

Abstract

We synthesize current evidence that granulosa cells possess unique innate immune signaling capabilities. We suggest the novel concept that this serves as a quality control surveillance mechanism by integrating signals from the oocyte and ovarian microenvironment to prevent poor-quality follicles from producing gametes that contribute to the next generation.

Point of View

The follicle, comprised of an oocyte surrounded by granulosa and theca cells, is the functional unit of the ovary (Wang et al., 2023, Tomac et al., 2021). Individuals are born with a finite number of follicles, and only a small fraction develop to ovulate a mature egg. Tight regulatory mechanisms are needed to ensure only the highest quality gametes develop and contribute to the next generation. Folliculogenesis, the process of follicle growth and development, depends on sophisticated bi-directional communication between the oocyte and granulosa cells (GCs). This allows for nutrient and signaling molecule exchange which promotes oocyte growth and GC proliferation, necessary for maturation of a developmentally competent oocyte (Clarke, 2018, Doherty et al., 2022). Direct contact between the oocyte and GCs is mediated in part by physical structures, including transzonal projections and gap junctions, which facilitate material exchange (Clarke, 2018, Doherty et al., 2022).

The follicle develops within a complex ovarian microenvironment including fibroblasts, immune cells, epithelial cells, lymphatics, and vasculature (Kinnear et al., 2020). However, the oocyte and granulosa cells within a follicle are physically separated from the surrounding theca cells and broader ovarian microenvironment by a basement membrane, comprised of type IV collagen, laminin, and proteoglycans, which prevents passage of molecules larger than 100 kDa (Herath et al., 2007, Hummitzsch et al., 2015, Biswas et al., 2022, Rodgers and Irving-Rodgers, 2010). In healthy ovarian follicles, this basement membrane keeps the GCs and oocyte in a largely avascular environment that cannot be penetrated by immune cells (Herath et al., 2007, Hummitzsch et al., 2015), raising the question of how oocyte quality is sensed in preantral follicles.

In this Point of View, we synthesize current evidence that GCs possess innate immune signaling capabilities and propose the novel concept that this serves as a quality control surveillance mechanism by integrating signals from the oocyte and ovarian microenvironment. We posit that GCs mediate an inflammatory cascade when exposed to specific stimuli ensuring poor-quality follicles, due to damaged oocytes or an inhospitable ovarian microenvironment, do not contribute to the next generation (Figure 1A). Specifically, GCs acquire inflammatory signatures which can impact reproductive outcomes in response to specific inducers, including infection (Herath et al., 2007, Ibrahim et al., 2016, Tomac et al., 2021, Yan et al., 2017), environmental toxins (Wang et al., 2023, Fan et al., 2021), and reproductive aging (Duncan et al., 2017) (Figure 1B & C). These stimuli have the potential to compromise oocyte quality, and GCs respond by creating an inflamed microenvironment, preventing further development of their respective oocytes.

Figure 1.

Granulosa cells (GCs) have innate immune signaling capabilities. (A) Schematic highlighting the inducers of innate immune signatures in granulosa cells and the impact on bi-directional communication and oocyte development. (B) Summary table of studies included in this POV with information on species, experimental design, model system and the inducer utilized. Abbreviations utilized: VACV70 (synthetic vaccinia virus analog), MCMV (murine cytomegalovirus), LPS (lipopolysaccharide), MC-LR (microcystin-LR), DON (deoxynivalenol). (C) Heat map of immune-associated genes mined from Duncan et al. 2017 that are differentially expressed with age in early secondary follicles (Y = young, O = old).

Interestingly, when somatic cells in other tissues localize to “immune-privileged” areas not accessible to immune cells, they can adopt immune cell-like functions (Typiak and Zurawa-Janicka, 2024). For example, podocytes within the kidney, cochlear resident cells, and mesenchymal stem cells (MSCs) are phagocytic and release cytokines and chemokines, typical leukocyte functions (Typiak and Zurawa-Janicka, 2024). All three cell types express pattern recognition receptors (PRRs) expressed by many immune cells that detect pathogen-specific molecular features. Podocytes and MSCs also express costimulatory molecules CD80 and CD86, which activate T cells (Typiak and Zurawa-Janicka, 2024) and express major histocompatibility molecules (Class I&II) expressed by many immune cells to support recognition of foreign pathogens and are antigen-presenting cells (Typiak and Zurawa-Janicka, 2024). Thus, non-immune somatic cells in immune-privileged locations exhibit many immune-related features.

Given that GCs are immune-privileged in basement membrane-enclosed follicles, it is not surprising they can assume immune cell-like characteristics. In fact, GCs from pre-ovulatory follicles initiate and execute inflammatory reactions without involvement of the innate immune system (Poulsen et al., 2019). Ovulation is the process whereby a follicle responds to the luteinizing hormone surge and ruptures to release a mature gamete. Ovulation is a highly inflammatory process characterized by TLR5 and IL-1 receptor activation, cytokine signaling, and MAPK and JAK/STAT pathway activation (Poulsen et al., 2019). Specifically, GCs isolated from women 36-hours after hyperstimulation but before ovulation exhibit an inflammatory signature with top upregulated pathways including those downstream of interleukin signaling events and general cytokine signaling (Poulsen et al., 2019). GC preparations were without immune cell contamination confirmed by absence of CD45 expression, indicating GCs can acquire an immune signature without pathogen exposure. As ovulation is associated with basement membrane degradation, allowing immune cell penetration (Hummitzsch et al., 2015), it is unclear whether GCs from preantral follicles also have similar immune-like capabilities.

Exogenous stimuli, including viral and bacterial infections, can impact fertility by inducing an inflammatory response in GCs to prevent poor-quality oocytes from maturing (Figure 1 A & B). GCs in mouse (Yan et al., 2017), bovine (Herath et al., 2007), and human (Ibrahim et al., 2016) express PRRs similar to immune cells. Mouse GCs also express two cytosolic DNA sensors, p204 and cGAS, which converge on a common signaling adaptor STING, a mediator of the innate immune response (Yan et al., 2017). STING initiates type I interferons (IFNα/β) (Yan et al., 2017), which activate an antiviral state across cells to reduce the spread of infection and recruit immune cells (Typiak and Zurawa-Janicka, 2024, Yan et al., 2017). Transfection of mouse cumulus cells with VACV70, a synthetic viral DNA analog, induces secretion of type I interferons and pro-inflammatory cytokines (IL-6 and TNF⍺) and suppresses steroidogenesis in vitro and in vivo (Yan et al., 2017). Estradiol is a steroid hormone produced by GCs essential for follicle growth and dominant follicle selection, and its suppression negatively impacts follicle and reproductive outcomes (Herath et al., 2007). Another study treated pregnant mice with murine cytomegalovirus (MCMV) and demonstrated viral penetration in corpora lutea (CL) and stroma but not follicles. Infected ovaries showed an innate immune response with upregulation of proinflammatory cytokines compared to controls, accompanied by early pregnancy loss. GCs in these animals demonstrated a type I interferon response (Tomac et al., 2021), demonstrating without MCMV penetration into the follicle, GCs adopt an immune profile and contribute to the inflammatory cascade potentially through bi-directional communication with the inflammatory microenvironment (Tomac et al., 2021).

The immune-like response of GCs is also observed when bovine cumulus cells (Herath et al., 2007) or human luteal GCs are exposed to lipopolysaccharide (LPS), a pathogenic component of E. coli (Herath et al., 2007, Ibrahim et al., 2016). GCs from growing and antral follicles express the TLR4 complex required for the LPS response (Herath et al., 2007, Ibrahim et al., 2016). LPS exposure in GCs results in IL-1β and IL-6 secretion and a significant reduction in estradiol and progesterone production, likely contributing to adverse reproductive outcomes (Herath et al., 2007, Ibrahim et al., 2016). In other tissues, LPS can alternatively activate the NLRP3 inflammasome through TLR4 signaling, stimulating caspase-1 to eventually release IL-1β and IL-18 (Wang et al., 2025). LPS exposure may also induce activation of this complex in granulosa cells, and the secretion of cytokine signaling may contribute to the known decrease in hormone production upon LPS exposure. Interestingly, knockout models of Asc, the adaptor protein of NLRP3, exhibit decreased serum and ovarian levels or pro-inflammatory cytokines (up to 18 months of age) and knockout models of Asc and NLRP3 both had an increase in follicular reserve (up to 12 months of age) relative to controls (Lliberos et al., 2020). These studies highlight that GCs possess the machinery necessary to detect and respond to pathogenic signals; this switch to immune signaling is accompanied by decreased hormone production which negatively impact follicle development (Figure 1A).

Along with infectious agents, environmental exposures can induce inflammatory gene expression in GCs (Figure 1B). Microcystin-LR (MC-LR) is a harmful toxin released by cyanobacterial algal blooms (Wang et al., 2023). In vivo, MC-LR exposure in mice results in toxin penetrance into the ovarian stroma and GCs with a decrease in the number of oocytes retrieved and reduced CL number post-stimulation relative to controls (Wang et al., 2023). When isolated follicles are cultured ex vivo with MC-LR, they exhibit increased expression of inflammation-related pathways, including upregulated cytokine and chemokine signaling, IL-10 signaling, and cytokine-cytokine receptor pathways. This inflammatory signature is accompanied by suppression of follicle maturation genes and functional defects in ovulation and progesterone secretion (Wang et al., 2023).

Another study investigated the impact of deoxynivalenol (DON), a mycotoxin produced by fungi, on mouse GCs. GCs isolated and cultured with DON for up to 72-hours exhibit a significant decrease in cell viability and estradiol and progesterone secretion, and increased apoptosis relative to controls (Fan et al., 2021). RNA-Seq analysis indicates DON exposure in GCs upregulates inflammation-related pathways, including NF-kB and MAPK signaling, potentially contributing to the negative reproductive outcomes associated with DON (Fan et al., 2021). Taken together, these studies demonstrate that exogenous stimuli can induce an inflammatory immune response in GCs and elicit altered follicular microenvironments with reduced steroidogenesis. Toxicants, which are man-made substances or heavy metals, can also negatively impact reproductive and fertility outcomes (Yao et al., 2023a, Priya et al., 2021). However, whether exposure to various toxicants induces innate immune signaling in granulosa cells has not been directly investigated.

Physiologic processes, such as ovarian aging, are also associated with an inflammatory signature in GCs. Advanced reproductive age is associated with a decline in gamete quantity and quality, with increased genomic instability in oocytes (Sturm and Vellai, 2022, Wasserzug-Pash et al., 2022), and the aging ovarian microenvironment becomes fibro-inflammatory with features of chronic, sterile inflammation (Duncan et al., 2017, Briley et al., 2016). RNA-Seq of individual early secondary follicles from reproductively young and old mice demonstrate an age-dependent increase in innate immune function and chemokine signaling gene expression specifically in the GC compartment (Duncan et al., 2017) (Figure 1C). Chemokine signaling is associated with immune cell migration suggesting potential leukocyte recruitment to the follicle. Thus, follicles may initiate crosstalk with the ovarian microenvironment to recruit immune cells to mediate removal of follicles containing poor-quality gametes. Additionally, inflammatory signaling in GCs may create an inhospitable environment that fails to support gamete development. In fact, there is an age-dependent increase of oocyte secreted factors which may be a compensatory response to enhance bi-directional communication due to poorly functioning GCs (Duncan et al., 2017) (Figure 1A). These findings highlight the dynamic communication of GCs both with their respective oocytes and the ovarian microenvironment.

How poor-quality oocytes are prevented from developing further and contributing to the next generation is not completely understood. However, mechanisms to maintain tissue homeostasis may become activated, including autophagy, apoptosis, and ferroptosis. Autophagy is the process of regulated self-degradation to preserve cellular homeostasis through recycling of cell components (Shao et al., 2022, Scudieri et al., 2024). Apoptosis is induced intrinsically through factors including DNA damage, oxidative stress, mitochondrial dysfunction or extrinsically through activation of death receptors or infectious or cytotoxic exposures, leading to a signaling cascade of caspase activation resulting in the removal of damaged or unwanted cells (Nossing and Ryan, 2023). Ferroptosis is initiated by the accumulation of intracellular ferrous (Fe2+) iron due to dysregulation of iron metabolism or inactivation of GPX4 which is responsible for repairing oxidative damage in cell membranes, triggering the accumulation of reactive oxygen species leading to membrane failure and cell death (Dou et al., 2022). The immune signaling capabilities of GCs may activate these mechanisms.

All three mechanisms to maintain tissue homeostasis are activated by viral and/or bacterial infection or environmental toxins (Choi et al., 2018, Wang et al., 2022, Kang et al., 2022), and both autophagy and ferroptosis are dysregulated with age (Barbosa et al., 2018, Yao et al., 2023b). For example, dying GCs activate autophagy in hypoxic conditions, and contribute to follicular atresia. Moreover, immune responses which produce cytokines have been shown to induce autophagy (Schmeisser et al., 2014, Ge et al., 2018), apoptosis (Demine et al., 2020), and ferroptosis (Dou et al., 2022). Thus, GC immune functions may activate these mechanisms to prevent continued development of poor-quality oocytes and maintain ovarian homeostasis.

In this Point of View, we demonstrated that in response to diverse stimuli, GCs acquire an innate immune signature and produce signaling molecules which impact folliculogenesis and oocyte outcomes (Figure 1). We propose this function, moderated by communication within the follicle and between the follicle and the ovarian microenvironment, may occur as a self-preservation mechanism. Promoting the development and fertilization of only high-quality oocytes, while intentionally preventing poor-quality ones, ensures survival and health of the next generation. Studies are ongoing to determine how follicle survival and growth is impacted after immune activation, whether GCs have altered sensitivity to stimuli with reproductive age as innate immunity becomes dysregulated, and if pathways that maintain tissue homeostasis are activated in response to GC immune signaling to remove poor-quality follicles and safeguard the next generation.