1. INTRODUCTION

The ovaries are major endocrine organs in females that, in mammals, serve two principal functions: (1) to produce a female germ cell (oocyte) that is capable of fertilization and successful embryonic development, yielding a viable, healthy offspring; and (2) to secrete a number of hormones that drive development of primary and secondary sex characteristics in the female and, during adulthood, prepare the uterus for establishment and maintenance of pregnancy.1,2 These functions are carried out by structures termed follicles,3 which are often referred to as the functional units of the ovaries. Each follicle is composed of an oocyte that is surrounded by one or more layers of somatic granulosa cells and, at more advanced stages of follicle development, theca cells. The granulosa and theca cells are responsible for much of the ovarian hormone production and support the maturation and growth of the enclosed oocyte. There are several different types of follicles present in the ovaries, which, depending on the size of the oocyte as well as the number of granulosa and theca cell layers, are classified as primordial (resting oocyte surrounded by a single layer of quiescent granulosa cells), primary (the first stage of immature follicles activated to initiate growth, characterized by oocyte enlargement and granulosa cell mitotic activity), secondary or preantral (larger maturing follicles with several layers of mitotically active granulosa cells, as well as some theca cells), and antral (mature follicles that have a fluid-filled cavity called an antrum and the ability to be ovulated in response to the pituitary gonadotropin, luteinizing hormone).1,2,3 The growth and maturation of a primordial follicle to the antral stage capable of ovulation can take weeks to months, depending on species,2,3,4 during which time the majority of follicles actually fail to mature and are

eliminated by a degenerative process involving apoptosis that is referred to as atresia.2,5,6,7,8,9

During embryonic development, oocytes are derived from primordial germ cells (PGCs), which, through extensive mitotic divisions, establish the initial germ cell pool.10,11,12,13 Generally speaking, approximately halfway through gestation, the mitotically active PGCs in the embryonic female gonads, termed oogonia, enter meiosis and arrest at prophase of meiosis I to form oocytes (oogenesis). At this time, the oocytes are in dictyate arrest and begin the process of follicle formation (folliculogenesis) by recruitment of surrounding somatic cells that will eventually become granulosa cells.10 Throughout development and most of postnatal life, large numbers of germ cells and follicles are lost via a process that has many hallmark features of apoptotic cell death.5,6,7,8,9,14,15,16 Indeed, of the peak number of germ cells produced in the human ovaries (roughly 7 × 106 at week 20 of gestation12), greater than 99.9% will undergo cell death at some point before complete exhaustion of the oocyte-containing follicle pool at menopause.8,17 Thus the “normal” fate of an oocyte is death, not survival. Because of this ongoing and extensive loss, the ovaries have provided an excellent model system for understanding the process of physiologic cell death. In fact, one of the first detailed documentations of apoptosis (long before the term was coined) was made with rabbit ovaries by Flemming in 1885,18 who detailed the microscopic features of dying granulosa cells in antral follicles undergoing atresia. Flemming termed this process chromatolysis on the basis of his observations of the condensation and ultimate disintegration of nuclear chromatin in the dying cells. In fact, Flemming’s descriptions of chromatolytic cell death in 1885 bear close parallels to the morphological features of apoptosis detailed by Kerr, Wyllie, and Currie when the term was first coined

in 1972.19 As limited methods were available in the late 1800s and early 1900s to study oocyte and granulosa cell death in detail, the apoptotic nature of these processes was not revisited until about 100 years later. Today, a wealth of morphological, biochemical, and genetic evidence is available demonstrating that controlled physiologic cell death occurs at relatively high levels in both

fetal and postnatal ovaries.7,8,9,14,15,16,20,21,22,23,56

As a consequence of the continuous depletion of female germ cells, natural cessation of female reproductive function occurs around mid-life (termed the menopause in humans),14,23 coincident with essentially complete depletion of the oocyte-containing follicle pool.13,14,15,16 This key event in the life of females has many ramifications. First, functional ovarian lifespan determines the maximum age at which women are able to conceive naturally and via assisted reproductive technologies.24,25,26 Because increasing numbers of women are electing to postpone their childbearing to ages of suboptimal fertility (i.e., late 30s and 40s), the decline in ovarian function with age has become an increasingly challenging issue for many women in Western societies.27,28 In addition to fertility issues, ovarian failure with age also results in other physiologic complications.14,23 For example, as a consequence of complete follicle depletion, the lack of cyclic ovarian hormone production results in widespread endocrine changes that are well-known risk factors for development of several age-related health issues in women, such as osteoporosis, heart disease, and cognitive decline. In turn, the development of methods to delay agerelated oocyte depletion29 could have enormous implications for improving the quality of life in aging women.

It is important to emphasize that in addition to the normal physiologic loss of female germ cells, many women experience an accelerated loss of oocytes resulting from exposure to pathological insults.30,31,32,33,34,35 For the purposes of discussion, these insults can be categorized as either those associated with clinical care for various diseases (e.g., chemotherapy and radiotherapy)30,31,32 or those arising from the environment (e.g., man-made chemicals and industrial by-products).33,34,35 Interestingly, a wealth of information from both animal and human studies has demonstrated that, contrary to what one might expect of the response to pathological insults, gene-regulated death of germ cells and follicles appears to play a primary role in the premature ovarian failure that is often observed.7,8,9,36,37,38,39,40,41 As will be addressed in more detail in the following sections, this information has provided a solid basis to explore ways to circumvent oocyte loss resulting from these types of

insults as a novel means to sustain ovarian function and fertility in women treated for cancer.

2. DETECTING CELL DEATH IN THE FEMALE GONADS

Initially, the occurrence of physiologic cell death in mammalian ovaries was derived from morphological characterization of dying oocytes and granulosa cells, followed by biochemical identification of fragmented chromosomal DNA.5,6,14,15,18,20,21,22 At present, morphological evaluation of ovarian tissue sections remains a standard, if not preferred, method for the detection of cell death in oocytes and granulosa cells. Typical morphological features of dying germ cells in embryonic and postnatal ovaries include chromatin condensation, cytoplasmic shrinkage and vacuolization, crowding of organelles, surface protuberances, cytoplasmic and nuclear condensation and fragmentation, and nucleolar segregation. It bears mentioning that the application of various biochemical and molecular biological techniques to detect apoptosis, including the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling (TUNEL) assay, immunostaining with active caspase-specific antibodies, and phosphatidyl serine exposure on the outer leaflet of plasma membrane, have produced conflicting results with respect to their detection in dying female germ cells.42,43,44 This likely stems from differences in the models employed by different investigators to trigger oocyte death, the maturational stage of the oocytes being studied, and the developmental window (i.e., fetal vs. postnatal life) under investigation. Nevertheless, there is agreement, and genetic evidence, that many genes and signaling pathways classically associated with apoptosis, such as the sphingomyelin pathway, death receptors (most notably Fas/CD95), Bcl-2 family members, and

caspases,7,8,9,16,23,36,37,38,39,40,41,45,46,47,48,49,50 play instru-

mental roles in the regulation of germ cell and follicle depletion under most situations. Given this and the morphological information available, for this chapter we use the term apoptosis when discussing instances of female germ cell death known to be regulated by these genedriven pathways, rather than attempting to use multiple terms (e.g., programmed cell death, autophagy, necroptosis) to refer to these various examples of controlled cell death.

3. OCCURRENCE AND REGULATION OF CELL

DEATH IN THE OVARIES

Cell death has been detected in nearly all cell types within the mammalian ovaries, although the vast

majority of studies have focused on either germ cells (PGCs, oogonia, or oocytes) or granulosa cells.7,8,9 With respect to the germline, apoptosis occurs in this cell lineage very early in embryonic development.7,8,9,14,15 For example, PGCs can first be identified as alkaline phosphatase-positive cells posterior to the primitive streak region of the gastrula.51,52 From this region, PGCs migrate through the hindgut and dorsal midline region and then laterally into the genital ridges of the embryo.52,53,54 Extensive evidence, primarily from rodent studies, indicates that PGCs that fail to reach the developing gonads during this migration are eliminated through Bax-dependent apoptosis in extragonadal sites because of a lack of tropic factors (primarily Steel factor or stem cell factor) that support their survival.55,56,57,58,59 Those PGCs that successfully reach and colonize the genital ridges undergo successive mitotic divisions as oogonia that give rise to peak numbers of germ cells, some of which enter meiosis and arrest in the first meiotic prophase (oocytes).1,2,10,11,12,13 However, vast numbers of oogonia and oocytes also die during this time, such that only about one-third of the peak number of germ cells produced by the female survives shortly after birth as follicle-enclosed oocytes.7,8,9 Experimental evidence indicates that at least two principal pathways trigger this developmental germ cell death in the female.8 One pathway is activated as a response to an inadequate supply of external cytokine survival signals and involves the sphingomyelin pathway, the Bcl-2 family of proteins and caspases (primarily caspase-9 and caspase-2).37,39,57,58,60,61,62 The second pathway appears to be cytokineand Bax-independent and is triggered by defects in meiotic initiation or arrest.62 It should be mentioned that in Drosophila, incomplete cytokinesis during germ cell mitosis leads to the formation of germ cell cysts – structures in which several germ cells remain connected to each other by intercellular bridges.63 During germ cell differentiation, every cell in the cyst but one undergoes apoptosis, with the dying germ cells serving to provide the sole remaining germ cell with nutrients, macromolecules, and organelles necessary for further development. Although recent studies have identified the formation of germ cell cysts in rodent ovaries during development, only a few germ cells in the cyst appear to activate apoptosis.64

Oocytes continue to die throughout postnatal life as well. Of the approximately 1 to 2 × 106 oocytes present in humans at birth,12 the pool of oocyte-containing follicles declines to less than 3 × 105 by puberty,13 and fewer than 1 × 103 oocytes remain just before menopause.17 Because only one egg on average reaches the appropriate maturity to be ovulated each month, at most an

adult woman can ovulate 400 oocytes in total during her entire reproductive years. The vast majority (>99.9%) of oocytes are lost through follicle atresia. Atresia can apparently occur at any point during the maturation of follicles from the resting (primordial) to mature (antral) stage, although histological evidence indicates that most follicles die during transition from the primary to preantral, and from the preantral to antral, stages of development.65 Interestingly, histological data from studies of human and rodent ovaries indicate that immature follicle atresia (primary, early preantral) is probably triggered by initial death of the oocyte, whereas atresia during latter stages of follicle maturation (late preantral, antral) is a consequence of granulosa cell apoptosis being activated before discernible changes in the oocyte.7,8,9,65 Regardless, the end result is the same – irreversible loss of that oocyte and follicle from the available pool.

The signals and genes that serve as determinants of germ cell and granulosa cell fate in the ovaries have been a subject of considerable research interest since the early 1990s. Through a combination of in vitro cell and organ culture models, coupled with extensive validation in vivo (primarily in rats and mice), a complex network of converging receptor-mediated survival and death signals, coupled to activation of a variety of intracellular signaling cascades ranging from ceramide generation to phosphatidylinositol-3-kinase/Akt, have been described in detail.8,9,39,60 Further, the availability of mutant mouse lines lacking critical regulators of apoptosis – most notably, Bcl-2 family members and caspases – has had a tremendous impact on the field of ovarian cell death research, as correlative gene expression studies could be tested for their functional relevance to various models of oocyte and follicle loss.7,8,9,23 As a result, a number of pathways and genes have been identified as absolutely critical for apoptosis to occur in oocytes and granulosa cells, and cell lineage-specificity for the necessity of certain proteins, such as caspase-3, has been also been demonstrated.7,8,9,23,46 However, bcause this information has been recently reviewed in detail elsewhere,7,8,9,23 we devote the remainder of this chapter to a discussion of two specific examples in which the ability to manipulate apoptosis has considerable clinical potential for improving women’s health.

4. APOPTOSIS AND FEMALE REPRODUCTIVE AGING

As discussed previously, in human females, less than 5% of the peak germ cell population produced during development survives at puberty, and this number continues to decline during adulthood to the point of exhaustion at approximately age 50 years, driving the menopause.17

Hence the ovaries represent one of the first major organ systems to fail with advancing chronological age, and their failure marks a transitional period characterized by increased risk for development of a spectrum of debilitating health complications in the ensuing years.24 Similar reductions in oocyte numbers have been reported in female mice throughout postnatal life, with complete depletion of the oocyte pool also noted several months before death due to chronological age.7,8,9,66 The postnatal loss of oocytes and follicles through atresia is mediated in large part via apoptosis, a point most clearly established by studies of mutant female mice lacking expression of the proapoptotic Bax protein.67 In these investigations, it was shown that oocytes of Bax-deficient female mice are resistant to developmental cues responsible for triggering apoptosis, thus leading to a reduced incidence of immature follicle atresia and a dramatic extension of functional ovarian lifespan into advanced chronological age.29 In addition to providing the first vertebrate animal model that fails to undergo its equivalent of menopause with age, Bax-deficient mice validated a critical concept in mammalian female reproductive biology – experimentally increasing the size of the immature follicle pool can extend the functional lifespan of the ovaries well past their normal time of senescence. Of further note, oocytes in the ovaries of very old Bax-deficient females remain fully capable of generating viable offspring if the aged ovarian tissue is transplanted into young adult recipient females.29

Subsequent characterization of aging Bax-deficient females revealed that sustaining ovarian function through maintenance of the follicle pool yielded a number of significant health benefits.68 For example, the age-related onset of bone and muscle loss, excess fat deposition, alopecia, cataracts, deafness, increased anxiety, and selective attention deficit observed in aging wild-type females was attenuated or absent in Bax-deficient female siblings. Further, maintaining ovarian function with age did not increase the incidence of cancer in any tissues, particularly those responsive to ovarian steroid hormones. Somewhat surprisingly, however, the reduced incidence or severity of age-related health complications noted in aged Bax-deficient females did not equate to increased longevity.68 Nevertheless, these findings support the idea that significant improvements in the overall quality of life in aging females can be achieved by approaches that sustain follicle numbers and, consequently, ovarian function. With that said, it is important to keep in mind that extrapolation to humans is difficult to foresee given that one cannot inactivate specific genes (e.g., Bax) in humans to achieve a similar outcome. Accordingly,

some consideration must be given to exploring and developing more practical methods of extending the fertile lifespan of females that could be reasonably viewed as amenable for translation and human application at some point in the future. Some progress has been made in this regard over the past few years.

The first example of this revolves around a series of recent findings challenging the dogma that, unlike males, mammalian females do not share the luxury of a renewable germ cell pool during postnatal life. For more than five decades, a central underpinning of mammalian reproductive biology has been that the reserve of oocytes set forth at birth cannot be replenished or replaced.69 However, experimental results from an increasing number of labs have challenged this belief, thus opening the door to a number of new possibilities to consider with respect to exploring the role that adult stem cells may play in oogenesis, fertility, and ovarian aging in females.70,71,72,73,74,75,76,77,78 In turn, efforts are now needed to determine whether and when apoptosis of these stem/progenitor cells, or the cells comprising the microenvironments that support them, occurs in the lifespan of the female and how this process plays into ovarian failure with age. Some insights into this may come from transplantation models, as exemplified by very recent studies with mice showing that infusion of bone marrow–derived stem cells from young donor females into aging recipients remarkably extends their reproductive lifespan.76 Although the mechanisms underlying this outcome remain to be identified, it is possible that the “young” stem cells have replaced missing stem cells lost through apoptosis as a result of replicative arrest associated with advancing age.

The second example is related to the impact of dietary caloric intake on fertility in females with age. Although there are several historical reports documenting a positive effect of caloric restriction (CR) on reproductive performance in rodents, much of the past work evaluating the effect of CR on long-term fertility in female mice initiated CR at or before weaning, which has obvious limitations in the context of human application.79,80,81 However, recent studies have shown that female mice placed on a moderate CR protocol during adulthood have increased numbers of oocytes later in life compared with age-matched ad libitum (AL)–fed controls, and this in turn is associated with a dramatic extension of reproductive lifespan.82 Although the impact of CR on the incidence of germ cell apoptosis was not assessed in this study, it is logical to assume that the expanded oocyte reserve seen in CR females during adulthood reflects a reduced rate of postnatal loss via atresia. Further, although the feasibility of applying adult-onset CR

protocols in humans for the sole purpose of extending ovarian lifespan is debatable, current efforts to develop small-molecule CR mimetics83,84,85,86,87 may provide an alternative strategy to achieve this outcome in aging females.

5. ANTIAPOPTOTIC AGENTS AND FERTILITY

PRESERVATION FOR CANCER SURVIVORS

Recent estimates from the American Cancer Society indicate that 2% of all young girls and reproductiveage women in the United States are diagnosed with cancer annually.88 The majority of these patients will undergo some type of treatment protocol involving the administration of cytotoxic drugs and/or radiation therapy in an attempt to eradicate their cancer. Unfortunately, because it is currently not possible to target only the cancerous cells, side-effect damage to healthy tissues is an inevitable consequence of these treatments. In this regard, ovarian follicles are remarkably vulnerable to ionizing radiation and many chemotherapeutic drugs.8,9,30,31,32,89,90,91,92 Indeed, accelerated loss of oocytes, premature ovarian failure, and infertility are well-recognized, and undesired, outcomes in young girls and reproductive-age women treated for cancer. Yet, despite the significance of these complications to the quality of life of cancer survivors and the fact that cancer treatment–induced loss of ovarian function has been recognized since the 1950s, relatively little has been done to protect the ovaries from these insults. Hence female cancer patients are forced to rely on assisted reproductive technologies before and after their treatment for a chance at fertility.93,94 However, oocyte retrieval for either cryopreservation or in vitro fertilization requires extensive hormonal manipulation, which may not be ideal for patients combating cancers that are aggravated by exposure to estrogens (i.e., breast cancer) or requiring a rapid initiation of treatment.93,94 In addition, many assisted reproductive technologies are not suitable for prepubertal girls or single (unmarried) women and, perhaps more importantly, none prevent the premature loss of ovarian function and the ensuing health problems that arise from it.

However, progress toward preserving ovarian function and fertility in female cancer patients without the use of assisted reproductive technologies has been made in the recent years. This stems primarily from a pivotal study published in 1997, which showed that the mechanism of anticancer therapy–induced oocyte loss involves apoptotic cell death as opposed to uncontrolled necrosis.36 Thus the possibility of preventing follicle death caused by chemoor radiotherapy via a targeted

inhibition of apoptosis emerged as a testable hypothesis. Importantly, this work was built on a platform of substantial information already regarding the specific genes and pathways involved in the activation and execution of cell death in oocytes and ovarian follicles under normal physiologic conditions.7,8,9 Drawing parallels from this body of work, extensive thought was given to the many potential targets for therapeutic development. The most logical to test were the earliest steps leading to apoptosis commitment, as inhibiting events after mitochondrial destabilization delays apoptosis but the cells nonetheless eventually die via a process more akin to necrosis.

A key mediator of mitochondrial permeabilization in many types of cells is the proapoptotic Bax protein.95 Experiments with gene mutant mice also identified Bax as being absolutely required for oocyte loss under normal physiologic conditions, as well as under of host of pathological situations associated with premature ovarian failure.29,36,40,41 However, the lack of a small-molecule Bax inhibitor at that time necessitated consideration of other, perhaps earlier, steps in the signaling cascade leading to apoptosis commitment. In this regard, generation of ceramide via acid sphingomyelinase (ASMase)-mediated membrane hydrolysis was subsequently reported as an initiator of proapoptotic signaling in oocytes during development.36,39 Ceramide is sphingolipid produced in many cell types after exposure to stress.96 Ceramide can either signal for apoptosis, or it can, if the damage to the cell is not irreparable, be metabolized to sphingosine, which serves as a precursor for the generation of sphingosine-1-phosphate (S1P). In some cells, S1P can effectively counteract the proapoptotic effects of ceramide, leading to enhanced cell survival.97 In initial studies conducted to test whether S1P could protect oocytes from death caused by cancer treatments, it was shown that the majority of oocytes cultured in vitro in presence of S1P were insensitive to the proapoptotic effects of the chemotherapeutic drug, doxorubicin.36 Additional in vitro studies with oocytes harvested from female mice lacking expression of ASMase supported that the generation of ceramide from sphingomyelin was an early critical step in chemotherapy-induced oocyte death.39 In vivo studies with mice soon followed, confirming that delivery of S1P to the ovaries of adult female mice 2 hours before irradiation maintained their ovarian follicle reserves.39 Importantly, these radioprotected females were able to give birth to healthy and cytogenetically normal offspring,98 indicating that the targeted suppression of female germline cell death after exposure to a cytotoxic insult does not simply result in the accumulation of damaged or “undead” oocytes in the ovaries.