Beyond apoptosis: evidence of other regulated cell death pathways in the ovary throughout development and life

1Regulated cell death plays an integral role in tissue and organ differentiation during foetal development, and in the maintenance of cell turnover throughout subsequent stages of life. These cell death pathways are critical for responding to toxic insults or microbial infection, while dysfunctional cell death signalling can be implicated in the development of some disease states in humans. Accordingly, some cell death regulators represent viable therapeutic targets. The ovary houses the female germline in the form of oocytes, which are encapsulated by somatic granulosa and theca cells to form ovarian follicles. Women are born with their lifetime supply of follicles termed the ‘ovarian reserve’. Therefore, maintaining the quality of the reserve of long-lived oocytes is crucial to ensure the generation of healthy, viable children.

2Across the reproductive lifespan, regulated cell death plays fundamental roles during follicle formation, follicle development, ovulation, and the elimination of damaged oocytes. Therefore, understanding the specific pathways and key factors involved in the regulation of oocyte attrition (death of the oocyte specifically) and follicle atresia (death of the whole follicle, oocyte, and supporting somatic cells) has been the topic of intensive study over the past several decades. Animal models have contributed vital insights since the availability of human tissue for study is limited.

3During foetal development, there is a considerable oversupply of female gametes, which are initially generated by foetal germ cell proliferation before entry into meiosis. However, this supply is significantly depleted by extensive oogonial and oocyte loss during follicle formation. Oogonia and oocyte survival during ovarian development is dependent on the activation or suppression of cell death signals. These signals dictate the size and quality of the follicle pool that women will be endowed with, which will ultimately define their lifetime fertility. The process of follicle development (folliculogenesis) then commences, during which a subset of immature primordial follicles is activated to develop and mature through discrete stages of growth (pre-antral, antral, and pre-ovulatory), ultimately producing a single mature ovulatory oocyte with each menstrual cycle in women. During puberty, there is a surge in follicle atresia, which is thought to be predominately hormonally induced, though the exact reasons for this remain unknown. Folliculogenesis occurs throughout reproductive life and, like follicle formation, is subject to a significant amount of redundancy, since more follicles are activated than will actually be ovulated. Indeed, over 99% of activated follicles will undergo natural atresia.

4In addition to natural atresia, exposure to endogenous and exogenous insults that may be detrimental to oocyte quality can readily trigger regulated cell death pathways and follicle loss. Although, in some cases, this might be necessary in order to maintain the integrity of the female germline. For example, it is well-established that exposure to ionizing radiation, certain chemotherapies, and various environmental toxicants can induce DNA damage and significantly alter the balance of follicle atresia versus survival and maturation. Additionally—depending on the severity of damage—exposure to such agents can completely ablate the follicle pool, leading to permanent infertility and premature menopause. Thus, understanding the precise mechanisms of regulated cell death that occur within ovarian follicles in response to various exogenous insults has important implications for developing effective fertility preservation strategies.

5In this review, we provide a comprehensive overview of the most recent literature that identifies cell death pathway regulators in the ovary, highlighting apoptotic and non-apoptotic pathways, and their functions throughout the discrete stages of ovarian development and reproductive life.

6Comprehensive literature searches using PubMed and Google Scholar were conducted for this review to identify peer-reviewed English publications for human, animal, and cellular studies published until August 2022. The search included keywords in the following areas: oogenesis, follicle formation, follicle atresia, oocyte loss, oocyte apoptosis, regulated cell death in the ovary, non-apoptotic cell death in the ovary, premature ovarian insufficiency (POI), primordial follicles, oocyte quality control, granulosa cell death, autophagy in the ovary, autophagy in oocytes, necroptosis in the ovary, necroptosis in oocytes, pyroptosis in the ovary, pyroptosis in oocytes, parthanatos in the ovary, and parthanatos in oocytes.

7Regulated cell death refers to the programmed induction of cell death by specific molecular pathways. In the ovary, apoptosis has been the most widely investigated regulated cell death pathway to date, with new insights into the mechanisms of apoptotic cell death within the ovary published regularly (Table I). However, recent evidence indicates that other cell death pathways—including autophagy and autophagic cell death, necroptosis, pyroptosis, and parthanatos—may also play important roles in regulating ovarian function across the lifespan (Fig. 1; Table II). The classification of these various pathways differs in their gross cellular morphology, pathophysiological relevance, and by the specific signal transduction molecules that activate and execute the cell’s demise. However, it is important to appreciate that additional regulated cell death pathways exist in other tissues and cell types (reviewed in detail by ).

8Apoptosis is the programmed, controlled death of a cell; which involves degradation and fragmentation of protein and DNA, and engulfment of the collapsed cell by neighbouring cells and/or phagocytes in a non-inflammatory manner. It occurs in all multicellular organisms throughout life from foetal development onwards and is an essential homeostatic mechanism to maintain healthy cell populations in tissues and organs. There are two major apoptotic pathways: the intrinsic and extrinsic pathways (Fig. 2). The intrinsic (mitochondrial) pathway is activated from within the cell and is predominately regulated by mitochondria. On the other hand, the extrinsic (death receptor) pathway is triggered from outside the cell, typically in response to conditions and factors within the extracellular environment.

9The intrinsic apoptosis pathway is initiated by a variety of non-receptor-mediated microenvironmental perturbations, including growth factor withdrawal, DNA damage, endoplasmic reticulum stress, reactive oxygen species (ROS) overload, replication stress, and microtubular alterations or mitotic defects, among others. These stimuli produce intracellular signals that cause disruptions to the mitochondrial membrane, which result in mitochondrial inner and outer membrane permeabilization, loss of mitochondrial transmembrane potential, and release of normally sequestered pro-apoptotic proteins from the intermembrane space into the cytosol. This release of pro-apoptotic proteins is considered the ‘point of no return’ in apoptosis, after which cytochrome c release, caspase activation (predominately caspase-3), formation of the apoptosome, and death of the cell will occur.

10The intrinsic apoptosis pathway is controlled by members of the B cell lymphoma 2 (BCL-2) family of proteins. These can be divided into three subgroups based on their structure and function: the anti-apoptotic BCL-2 proteins, including BCL-2, BCL-XL, BCL-W, MCL-1, and A1; the pro-apoptotic proteins, BAX, BAK, and BOK; and the BH3-only proteins PUMA, NOXA, BH3 interacting-domain death agonist (BID), BAD, BIM, BIK, HRK, and BCL-2 modifying factor (BMF). The BH3-only proteins are responsible for sensing apoptotic signals and transmitting them to other BCL-2 family members to ultimately trigger the apoptotic cascade. They do this by binding and inhibiting the core anti-apoptotic BCL-2 proteins, leading to conversion of BAX, BAK, and BOK from inert monomers into membrane-permeabilizing oligomers. Once permeabilized, apoptogenic factors, such as cytochrome c, are released from the mitochondrial intermembrane space and trigger caspase activation.

11The extrinsic apoptotic pathway can be activated by two types of plasma membrane receptors: death receptors, which are activated by cognate ligand binding; and dependence receptors, which are activated when specific ligands drop below a certain threshold. The most widely characterized death receptors include, but are not limited to, FAS cell surface death receptors and the tumour necrosis factor (TNF) receptor superfamily members. Briefly, binding of the death receptor ligand to the receptor allows the assembly of the death-inducing signalling complex that regulates the activation of pro-caspase-8 and -10.

12Although the mitochondrial pathway is strongly associated with intrinsic apoptosis; in certain cell types, the extrinsic pathway can crosstalk with the intrinsic pathway through caspase-8-mediated proteolytic cleavage of tBID to BID, which triggers the release of apoptogenic factors to activate BAX and induce apoptosis.

13Derived from the Greek language, meaning ‘self-eating’, autophagy is a tightly-regulated process whereby cells degrade and recycle their own cytosolic components inside lysosomes, which can lead to cell death. Unlike apoptosis, this form of regulated cell death occurs in the absence of chromatin condensation and phagocytes. Autophagic cell death manifests in the accumulation of large numbers of autophagic vesicles containing cytoplasmic material for degradation by lysosomes, and results in early degradation of organelles and late degradation of cytoskeleton, which is the reverse for apoptotic cells.

14Autophagy is mediated by dozens of autophagy-related (ATG) proteins that regulate expanding ‘isolation membranes’, which encapsulate and enclose proteins/organelles into a double-membrane structure called the autophagosome. These autophagosomes then fuse with liposomes to degrade the internal components. Once fused, acidic hydrolases in the lysosome can degrade the autophagic cargos, and salvaged nutrients are released to the cytoplasm to be recycled by cells. Genetic models have demonstrated that some autophagic machinery is essential for regulated cell death (e.g. ATG1, reviewed by ); however, it has been suggested that it might be more appropriate to name the process ‘autophagy-mediated cell death’. Although, more recent literature suggests that autophagy-dependent cell death, that is independent of apoptosis or other regulated cell death pathways, can occur (Fig. 2). Indeed, there is strong evidence that apoptotic and autophagic machineries are highly interconnected during developmental regulated cell death.

15Necrosis is morphologically distinct from apoptosis and characterized by a gain in cell volume, organelle swelling, plasma membrane rupture, and loss of intracellular contents. Unlike apoptosis, necrosis provokes an inflammatory response by spilling the cell’s cytosolic constituents into the extracellular space through the damaged plasma membrane. During apoptosis, however, these products are safely isolated by membranes and then consumed by phagocytes. Necroptosis is a programmed, regulated form of necrosis that is initiated by various extracellular and intracellular stressors, including viral infection, inflammation, and factors detected by specific death receptors (e.g. FAS, TNFR1) or pathogen recognition receptors (Fig. 2). Importantly, these death receptors can also activate the extrinsic apoptosis pathway. At the molecular level, necroptosis critically depends on activation of the receptor-interacting serine/threonine-protein kinase (RIPK) 1/3 necrosome and mixed lineage kinase domain-like pseudokinase (MLKL), in the absence of caspase-8 activation, to ultimately cause cell membrane rupture. Indeed, necroptosis is generally observed as a fall-back regulated cell death mechanism that is triggered when apoptosis is hindered, such as during pathogen infection.

16Exogenous insults also extend to infection, and regulated cell death is proposed to contribute to immune defence against infections. Pyroptosis is mediated by the cleavage of gasdermins, caspases (namely caspase-1), or granzymes; leading to the formation of pores in the cell membrane, lysis of the cell, and the release of inflammatory molecules (Fig. 2). This type of regulated cell death is primarily observed in inflammatory cells, such as macrophages, and occurs most frequently upon infection with intracellular pathogens. As such, it is likely to form part of the host response to control bacterial, viral, fungal, or protozoan pathogens. While the sterile inflammatory response is required for organ development and tissue repair, dysregulation of this process may lead to inflammatory disease, for example asthma, Type 2 diabetes, and inflammatory liver diseases. Indeed, there is accumulating evidence that pyroptosis and inflammasome dysregulation may contribute to sterile inflammatory diseases, gynaecological diseases, autoimmune diseases, neuronal diseases, and even cancer. Moreover, cytokine dysregulation resulting in a pre-inflammatory phenotype that occurs with age, known as ‘inflammageing’, has also been associated with pyroptosis.

17Parthanatos is a poly[ADP-ribose] polymerase 1 (PARP1)-dependent and apoptosis-inducing factor (AIF)-mediated, caspase-independent cell death pathway, which is distinct from apoptosis, necroptosis, or other known forms of regulated cell death (Fig. 2). Parthanatos is associated with various diseases including several retinal diseases, Parkinson’s disease, stroke, heart attack, and diabetes. Parthanatos is triggered by an excessive ROS response, which leads to an accumulation of poly[ADP-ribose] (PAR) polymer and translocation of AIF from mitochondria to the nucleus, resulting in chromatin condensation and nuclear fragmentation. Parthanatos does have shared characteristics with necroptosis, including loss of membrane integrity and depletion of cellular energy stores (NAD and ATP). However, cells undergoing parthanatos experience regulated chromatolysis without swelling and rupturing of cell membranes, as occurs during necroptosis. Key morphological features of parthanatos include shrunken, condensed nuclei, and membrane disintegration.

18Regulation of cell death pathways is critical to orchestrating numerous aspects of ovarian development and function across the lifespan (Fig. 1). More than 99% of mammalian follicles will not reach ovulation, instead undergoing atresia. It is well-established that follicle atresia is predominately mediated by apoptosis. In women, germ cell number peaks at ∼5 months gestation, with ∼6.8 million germ cells present in the ovary. This number falls to ∼1 million at birth, and by the onset of puberty, the follicle pool contains only 300 000 follicles. Despite this, over a woman’s reproductive life, only ∼400 oocytes will survive and undergo maturation to ovulation. At ∼50 years of age, ovarian senescence and menopause is triggered when a critical threshold of <1000 follicles remain in the ovary.

19Whilst there is continuous loss of germ cells throughout life, there are two distinct waves during which large numbers of oocytes and primordial follicles are lost in a short period of time (Fig. 3). The first occurs prior to birth in humans, during which primordial follicle formation is occurring, and a second occurs at the onset of puberty. It is now widely accepted that granulosa cell apoptosis is primarily responsible for the atresia of growing follicles, whereas primordial follicle atresia is largely initiated by oocyte apoptosis. However, alternative regulated cell death pathways can also trigger follicle atresia across the lifespan, especially following exposure to certain environmental stressors and toxicants.

20Germ cell loss occurs throughout the process of primordial follicle formation, predominately via apoptosis. However, our analysis of studies performed in mice reveals some discrepancies in the exact timing of apoptosis. One of the earliest characterizations was by, who identified germ cell degeneration (death) by their condensed nuclei containing rounded clumps of densely stained chromatin. This study reported increased germ cell degeneration during the last mitotic divisions, as oocytes become arrested in the first meiotic prophase. At this time, germ cell nests begin to break down and facilitate primordial follicle assembly, culminating in loss of up to 98% of germ cells. Data examining germ cell loss during the early stages of meiosis (embryonic day 13.5–17.5 in mice; approximately mid-gestation in humans) are variable. Some reports highlight little to no germ cell loss and few TUNEL-positive-stained germ cells at this time. Meanwhile, others have identified a continuous decline and increased proportion of apoptotic germ cells during the first meiotic prophase compared to oogonia undergoing mitosis. Thus, it is still debated by the field as to whether there are specific windows of germ cell loss during foetal development, or if this loss occurs continuously in the lead up to nest breakdown and follicle formation. Irrespective of the timing of loss, it is well-established that the key anti-apoptotic proteins (e.g. BCL-XL and BCL-2) and pro-apoptotic proteins (e.g. BAX, BMF, and PUMA) are clearly required for this process.

21Extensive germ cell loss occurs during nest breakdown. It has been proposed that these dying cells could be acting as nurse-like cells that transfer important organelles (including centrioles, Golgi, and mitochondria) and the cytoplasm to a select few oogonia, before undergoing regulated cell death. The remaining oogonia that acquired these additional cellular components become progressively larger, mature, and form the primordial follicle pool. Apoptosis is fundamental to orchestrating nest breakdown and primordial follicle assembly. However, autophagy likely also plays a key role in this process (recently reviewed by ). Interestingly, inhibiting autophagy mediators using 3-methyladenine delays follicle formation and results in an increased number of cyst oocytes. In support, germ cell survival immediately prior to and during follicle formation is severely impaired in mice with genetic loss of autophagy-related genes Atg7 and Becn1. Lysosome amplification—a hallmark of autophagy—is also observed in oocytes upon birth and is most apparent in primordial follicle oocytes. Lysine-specific demethylase 1 (LSD1)—a critical repressor of autophagy—is highly expressed in mouse foetal ovaries, but sharply reduces during the period of follicle assembly. Expression data, together with functional studies in vitro, indicate that LSD1 is an indispensable regulator of oocyte death during ovarian reserve establishment, via regulation of autophagy. During the foetal-to-neonatal transition in mammals, which is associated with a transformation in nutrient supply from the maternal–foetal blood interface to lactation, there is a disruption in nutrient supply to the ovary. Interestingly, autophagy has been shown to play a critical role in nutrient stress adaptation to prevent excessive germ cell loss during this period in mice. Together, these studies imply key roles for autophagy mediators in regulating oocyte survival, particularly during nest breakdown and primordial follicle formation.

22Once established, primordial follicles enter meiotic arrest. Deemed ‘non-growing follicles’, the oocytes remain arrested at the diplotene stage of meiotic prophase 1, and the surrounding granulosa cells have low mitotic potential. These primordial follicles represent the stockpile of oocytes available to females for their reproductive life. As such, these primordial follicle oocytes are some of the longest living cells in the mammalian body, and may remain arrested for decades in humans. These follicles will ultimately leave this period of dormancy, either by activation to continue through stepwise maturation via follicle development, or to undergo follicle atresia and oocyte loss. Regulation of this process and a balance between growth, survival, and atretic factors are essential in maintenance of normal reproductive function.

23During puberty, rising gonadotrophin levels cause dynamic physiological changes in the ovary, including the development of antral follicles to the Graafian (pre-ovulatory) stage and the onset of ovulation. Strikingly, this window also coincides with a significant spike in the loss of the ovarian reserve of primordial follicles, by approximately half during the adolescent/young adult period (ages 13–25 years) in humans and roughly two-thirds in mice. This loss is thought to be gonadotrophin-mediated, though the precise mechanisms remain unknown. Notably, overexpression of LH in juvenile mice has been shown to trigger depletion of the primordial follicle reserve. Conversely, LH and FSH suppression prevented follicle loss in a more recent study. Importantly, this primordial follicle loss is regulated by apoptosis, as demonstrated by the essential requirement for the pro-apoptotic BH3-only protein, BMF, in this process.

24It remains unclear why such vast numbers of primordial follicles are eliminated at the time of sexual maturation. One explanation for this may be the fact that there are two separate populations of primordial follicles in the ovary that each have distinct functional roles. Class 1 primordial follicles, which are localized to the ovarian medulla, will activate and grow during pre-pubertal life but never be ovulated. These are fast growing, taking 19–21 days to reach maturity in mice. They will contribute to the first wave of follicle activation, which is in turn likely required for the establishment of the hypothalamic–pituitary–ovarian axis, and puberty onset. Class 2 primordial follicles, located at the ovarian cortex, are slower growing (∼47 days) and are thought to represent the source of all mature ovulatory oocytes for fertilization. Therefore, it is possible that the spike in oocyte loss observed at the transition of puberty involves the clearance of any remaining Class 1 primordial follicles in the medulla. While the two classes of follicles have not been clearly identified in human ovaries, the growth pattern of the first wave of activated follicles during foetal development is conserved in humans. Moreover, in human ovaries, nearly 20%, 5%, and 0% of the primordial follicles in pre-pubertal, pubertal, and adult ovaries, respectively, are classed as morphologically abnormal. Cultures of human cortical tissue containing primordial follicles and isolated pre-antral follicles from pre-pubertal and pubertal girls exhibited low activation rates and compromized oocyte growth respectively, compared to adult samples. Thus, it can be speculated that these abnormal primordial follicles are either eliminated or preferentially activated before puberty onset and the commencement of ovulation in humans. This provides an intriguing parallel to the two distinct types of primordial follicles identified by in mice.

25Atresia is a complex process that naturally occurs to regulate the follicle pool across the lifespan. It affects all stages of follicular development and involves multiple forms of regulated cell death. The highest incidence of follicular degeneration is observed when follicles become dependent on FSH, at the early antral follicle stage. Atresia is essential for maintaining ovarian homeostasis, and the dysregulation of atresia contributes to reproductive disorders, including PCOS and POI.

26The regulation of intrinsic apoptosis ensures the maintenance of the number and the quality of the long-lived primordial follicle pool. This pathway is responsible for eliminating defective oocytes, which is paramount to sustain fertility and generate healthy offspring. Genotoxic stress (i.e. DNA damage) within oocytes—particularly in the form of double-stranded breaks—is extremely harmful to chromosome structure and overall DNA integrity. DNA damage can readily accumulate within primordial follicle oocytes as a consequence of normal cellular metabolism and increased levels of oxidative stress during the ageing process. Additionally, DNA damage can be induced exogenously following exposure to various exogenous insults, including ionizing radiation, environmental toxicants, and certain chemotherapies causing extensive primordial oocyte apoptosis.

27In oocytes, intrinsic apoptosis is predominately regulated by TAp63α—an isoform of p63, which is the major p53 family member present. TAp63α, and its downstream effector PUMA, are primarily responsible for initiating oocyte apoptosis in response to genotoxic stress in vivo. Building on this work, a recent report showed that the ovaries in neonatal mice with a Trp63 exon 13 deletion (which leads to selective silencing of the TAp63α, but not the β isoform) were almost completely devoid of oocytes. This phenotype was a consequence of increased transcription of Puma and Noxa expression cause by the constitutively active TAp63β isoform. These data reveal that control of p63 signalling, and the intrinsic apoptosis pathway, is fundamentally important for oocyte maintenance.

28Antral follicular degeneration is predominantly initiated by granulosa cell apoptosis. Activation of the death ligand–receptor system is the most common trigger of granulosa cell apoptosis, via the extrinsic apoptosis pathway specifically. The FAS–FAS ligand (FAS-L) system has been localized to the human and rodent ovary, primarily in the granulosa and theca cells of unhealthy pre-antral and antral follicles, and in luteal cells of the corpus luteum, indicating a role in ovarian follicular atresia and luteolysis. Indeed, co-culture of interferon-γ pre-treated granulosa cells and denuded oocytes resulted in granulosa cell apoptosis, which could be blocked by an inhibitor of the FAS–FAS-L interaction. Additionally, new evidence suggests that the extrinsic apoptosis pathway may also regulate atresia of primordial follicles. A recent study examining the impact of checkpoint inhibitor immunotherapy on ovarian function in a mouse model revealed that TNF-α can directly induce primordial follicle loss via BID, which is a key member of the extrinsic apoptosis pathway.

29Emerging evidence suggests that autophagic cell death plays a role in follicle atresia (recently reviewed by ). Granulosa cell apoptosis may also be triggered by the accumulation of autophagic vacuoles (autophagosomes) leading to the down-regulation of BCL-2 expression, suggesting that autophagy is closely related to apoptosis induction in granulosa cells. Interestingly, recent investigations show that different regulated cell death pathways beyond apoptosis play active roles in mediating follicle atresia, depending on the stage of follicle development. Pre-antral follicle atresia occurs largely via enhanced granulosa cell autophagy, meanwhile antral follicle atresia arises due to granulosa cell apoptosis. The oocyte residing within the atretic follicle may then be eliminated by mechanisms involving mediators common to both apoptosis and autophagy pathways. Unlike standard morphological features of apoptosis, oocytes within atretic follicles do not display normal chromatin compaction; however, they do display DNA fragmentation. Therefore, oocyte death may begin with autophagic degradation of cytoplasmic components, including mitochondria, which activates caspases that lead to DNA fragmentation without compaction; thus, triggering a non-conventional route of cell death.

30There is, in fact, evidence that autophagy contributes to the regulation of oocyte death and follicle atresia at all stages of development, though its role in cell survival versus cell death appears to be complex. The expression of autophagy-related genes (e.g. those encoding ATG proteins, microtubule-associated proteins 1A/1B light chain 3 A and B [LC3A/B], beclin-1 [BECN1] and [LAMPs]) have been detected in follicles at all stages of development in rodents and pigs. In mice, the relative mRNA expression of Becn1 is highest in primordial follicle oocytes compared with oocytes from primary, pre-antral, small antral, and large antral follicles, and protein is present in follicles (theca and granulosa cells) and oocytes of all stages including atretic follicles, but is absent from ovary epithelium. In the rat, the expression pattern of LC3A appears to be restricted to granulosa cells with weak staining in thecal cells, but no staining in oocytes. LC3B protein is localized to the cytoplasm of oocytes and granulosa cells of all follicle stages as well as in steroidogenic cells of the corpus luteum. Importantly, LC3B levels are significantly higher in ovaries of heat-stressed mice and after exposure to FSH. Less is known about the expression of these genes in humans; however, BECN1 and LC3A are present in the KGN immortalized human granulosa cell line. Importantly, a recent in vitro study demonstrated that human granulosa cells can remove apoptotic oocytes by unconventional autophagy-assisted phagocytosis, which may explain the expression of autophagy degradation machinery in these cells. LC3A is also expressed in cumulus cells from human cumulus–oocyte completes (COCs), with higher expression in cumulus cells classed as dysmature. Interestingly, oocytes from COCs with dysmature cumulus cells had a much lower fertilization rate, highlighting LC3A as a possible biomarker for lower quality human cumulus cells.

31The involvement of autophagy, with or without cell death, has been reported in the establishment and maintenance of the primordial follicle reserve, follicle development and atresia, luteinization of granulosa cells and formation of corpus luteum, and corpus luteum regression. Expression or activation of autophagy-related proteins is most evident in high stress conditions (such as starvation, heat, and hypoxia) and can suppress apoptotic signalling in the oocyte and/or granulosa cells in the ovary, promoting the survival of oocytes and follicles, and thereby ensuring fertility. Consistent with the concept that autophagy plays important survival roles in the ovary, deletion of the autophagy induction gene Atg7 in female mice leads to excessive germ cell loss during follicle formation, reduced ovarian reserve, and subfertility. It has been proposed that autophagy protects immature oocytes from elimination by apoptosis, under starvation conditions. Moreover, loss of function mutations in ATG7 and ATG9A are associated with autophagy impairment and ovarian failure in women. On the other hand, elevated expression of autophagy-related genes within granulosa cells appears to be important for the normal and insult-induced atresia of antral and pre-ovulatory follicles, but excessive or unregulated autophagy may be pathogenic. For example, increased autophagy has been observed in ovaries from women with PCOS, which may indicate an important role for autophagy in ovarian homeostasis.

32As a final note, in mature oocytes, BECN1—a key regulator of autophagosome formation and membrane trafficking—may also regulate chromosome segregation and cytokinesis during the last stages of meiosis, independent of its role in the autophagy pathway. Thus, it is important to consider that expression of proteins associated with autophagy does not necessarily imply autophagy is occurring.

33ROS contribute to the physiological functions of follicles and human granulosa cells. Indeed, ROS are needed for various processes within the ovary, including ovulation. However, accumulation of ROS from physiological stress (i.e. release of cortisol) or exposure to environmental and/or endogenous toxins can trigger various regulated cell death pathways and follicle atresia. Besides apoptosis and autophagy, necroptosis of granulosa cells and oocytes has been reported in the ovary in response to ROS accumulation. In studies performed in vitro, serum starving human granulosa cells causes generation of ROS and induces both necroptosis and apoptosis. Indeed, mediators of necroptosis, such as phosphorylated MLKL, RIPK1, and RIPK3 proteins, are readily detected in the granulosa cells of pre-antral and antral follicles in macaque ovaries and human corpora lutea. Moreover, a recent publication identified elevated gene expression of RIPK1 and RIPK3 in atretic, but not healthy, bovine follicles. Collectively, these data suggest that necroptosis, in concert with apoptosis and autophagy, may play a role in regulating late-stage follicle atresia, but limited mechanistic information is available.

34In the ovary, granulosa cells of antral follicles are producers and targets of acetylcholine (ACh). ACh is an important neurotransmitter that has been implicated in the regulation of cell viability, proliferation, gap junctional communication and intracellular calcium levels, as well as expression of transcription factors. Two esterases cleave and inactivate ACh—butyrylcholinesterase and acetylcholinesterase (AChE), with several splice variants of AChE (e.g. AChE-E, -S, and -R)—which results in isoforms that differ in subcellular localization and enzyme activity. Importantly, the expression of splice variant AChE-R increases in response to oxidative stress and circulating levels also increase with age in humans. AChE-R can induce RIPK1-/MLKL-dependent necroptosis of granulosa cells, which can be blocked using key inhibitors of necroptosis (e.g. the RIPK1 inhibitor necrostatin-1 and MLKL-blocker necrosulfonamide). Locally inhibiting AChE using Huperzine A, via intrabursal injection, in vivo for 4 weeks in rats significantly increased the number of pre-antral follicles, corpora lutea, and pup numbers, highlighting an important role for ACh in follicular development and ovulation. Blocking the breakdown of ACh by inhibiting AChE (using Huperzine A) or interfering with necroptosis (necrostatin-1) did not improve follicle survival, but did promote oocyte development and growth of macaque follicles from the pre-antral to the small antral stage, and increased follicle granulosa cell number in vitro. These studies strongly implicate granulosa cell necroptosis as an additional regulated cell death pathway that can be utilized during follicle atresia. However, the mechanism by which AChE-R induces necroptosis in granulosa cells remains to be determined.

35Interestingly, oxidative stress can also prime the NLR family pyrin domain containing 3 (NLRP3) inflammasome, resulting in caspase-1 activation and pyroptosis. A recent study showed that polystyrene microplastics, which are transported to the ovary and taken in by the granulosa cells, result in the induction of NLRP3/caspase-1 signalling and pyroptosis activation in the ovary. In addition, a recent study in dairy cows demonstrated that culturing granulosa cells in the presence of non-esterified fatty acids (NEFAs) induces oxidative stress, pyroptosis, and inflammation. Specifically, NEFAs activate the toll-like receptor 4 (TLR4)/NF-κB pathway, increase the production of NLRP3 and caspase-1, and trigger granulosa cells to release inflammatory cytokines interleukin (IL)-1β and IL-6. Importantly, these effects were reversed when the granulosa cells were pre-treated with antioxidant N-acetylcysteine, validating the role of oxidative stress during NEFA-induced pyroptosis. The involvement of NEFAs in this process is particularly interesting as high levels of NEFAs are a hallmark of various metabolic diseases, including obesity, Type 2 diabetes, and ketosis in humans and animals. These circulating NEFAs can enter the ovary and the follicular fluid, and negatively impact on the steroidogenic functions of granulosa cells and oocyte quality.

36Reproductive ageing coincides with a decline in ovarian follicle number, leading to loss of fertility and endocrine function, and eventually menopause. Notably, emerging evidence indicates that the ovary naturally transitions to a low-level inflammatory microenvironment with advancing maternal age, termed ‘inflammageing’. The NLRP3/apoptosis-associated speck-like protein (ASC) inflammasome, which activates caspase-1, appears to be central to this process, raising the possibility that pyroptosis might contribute to age-associated follicle loss. Although pyroptosis has not been directly studied during ovarian ageing, this hypothesis is supported by recent reports demonstrating that genetic loss or pharmacological inhibition of NLRP3 or ASC reduces caspase-1 levels in the ovary, increases oocyte number, and delays ovarian ageing. Furthermore, the granulosa cells of women with POI exhibit elevated NLRP3, caspase-1, and IL-1α levels. Further investigations of how pyroptosis is regulated in the ovary will provide valuable therapeutic targets to potentially delay natural ovarian ageing.

37Some new evidence suggests that parthanatos may also contribute to premature ovarian ageing. Cumulus granulosa cells collected from women with diminished ovarian reserve showed increased levels of nuclear purified PAR and AIF, suggesting a role for PARP-dependent cell death in diminished ovarian reserve pathophysiology. Therefore, inhibition of parthanatos, amongst other regulated cell death pathways, may prove useful for patients with diminished ovarian reserve.

38Granulosa cells are specialized somatic cells in the ovary, vital for oocyte survival and female fertility. When follicles are formed, oocytes not surrounded by granulosa cells are eliminated. Impaired granulosa cell function also dysregulates oocyte growth and causes POI, characterized by early loss of fertility and hormone production. Recent endeavours to derive human or mouse oocytes from induced pluripotent stem cells rely exclusively on mouse granulosa cells to support the germ cell-like cells. Collectively, these observations reveal that granulosa cells have unique and essential functional properties that cannot be replaced by other somatic cell types.

39In response to activation signals, primordial follicles give rise to large, hormone-producing follicles, and mature ovulatory oocytes. The founding population of ∼5–8 granulosa cells present in a primordial follicle undergoes clonal divisions to eventually produce >2000 granulosa cells that support a mature oocyte. After primordial follicle formation, there is no evidence of new granulosa cell formation from other somatic cell types. Thus, all mature granulosa cells appear to be clones of primordial follicle granulosa cells. As oocytes cannot survive without these essential granulosa cells, granulosa cell apoptosis invariably causes follicle atresia. However, the possibility that primordial follicle granulosa cells are susceptible to ageing and exogenous insults has not been investigated. Understanding the regulated cell death pathways utilized by each cell type comprising ovarian follicles is important to better understand the effects of maternal ageing, and to develop appropriate fertility preservation strategies for female cancer patients.

40Studies of the ovarian environment, including the stroma and other supporting somatic cell types, have emerged as a recent area of interest. Some studies have established that fibrosis is one early hallmark of the aging ovarian stroma. It has thus been proposed that this altered microenvironment could contribute to the age-associated decline in oocyte number and/or quality. However, a direct link between the ovarian stroma and regulated cell death in ovarian follicles or other somatic cell types is lacking, and should be the focus of further investigation.

41Inhibiting key mediators of oocyte death is a promising strategy for protecting primordial follicles from anti-cancer treatment and age-mediated depletion. Such strategies require characterization of the pathways and specific mediators involved in order to identify suitable targets. Of relevance to cancer treatment, Bax–/– mice have prolonged fertility after chemotherapy. Similarly, irradiated TAp63–/–, Puma–/–, and checkpoint kinase 2 (Chk2)–/– mice remain fertile after treatment with radiation or chemotherapy, unlike their wild-type counterparts, demonstrating the potential of this strategy for fertility preservation. Translation of this information is currently limited by the availability of effective small-molecule inhibitors to these proteins. However, an inhibitor of CHK2 does exist, and its transient use reduced irradiation-mediated primordial follicle loss in mice, leading to the generation of healthy offspring.

42Targeting other regulated cell death pathways, independently or in concert with apoptosis, may also provide novel fertility preservation strategies. Indeed, investigations on the use of AChE and necroptosis inhibitors to improve folliculogenesis, or inflammasome inhibitors to delay ovarian ageing, are exciting potential avenues for fertility preservation. However, more studies are needed to understand when and how these pathways are activated in the ovary, in order to determine the best timing and suitability of these therapies.

43The contribution of apoptosis to the processes of follicle formation during foetal development and around birth, as well as throughout other life stages, is well defined. However, here, we highlight substantial gaps in knowledge of the localization, activation, and contribution of key players from other types of regulated cell death pathways in ovarian follicles. Understanding the relative importance of all the different regulated cell death pathways, and how they are activated in both granulosa cells and oocytes, is vital to identify other potential therapeutic targets for fertility preservation strategies.