- Email: [email protected]
Human female meiosis: what makes a good egg go bad?
Review
Human female meiosis: what makes a good egg go bad? Patricia A. Hunt and Terry J. Hassold School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164-4660, USA
Critical events of oogenesis occur during three distinct developmental stages: meiotic initiation in the fetal ovary, follicle formation in the perinatal period, and oocyte growth and maturation in the adult. Evidence from studies in humans and mice suggests that the genetic quality of the egg may be influenced by events at each of these stages. Recent experimental studies add additional complexity, suggesting that environmental influences might adversely affect all three stages. Thus, understanding the molecular control of oogenesis during these critical developmental windows will not only contribute to an understanding of human aneuploidy, but also provide a means of assessing potential effects of environmental exposures on human reproductive health. ‘We have met the enemy and (s)he is us’ – Pogo The high incidence of chromosomally abnormal pregnancies in humans has been an intractable genetic puzzle for decades. Approximately 7–10% of clinically recognized pregnancies are chromosomally abnormal [1], but this understates the problem for two reasons. First, the loss or gain of some human chromosomes is so lethal that the pregnancy does not survive long enough to come to clinical attention. Second, the incidence is strongly affected by age and, for women at the end of their reproductive lifespan, the risk of ovulating a chromosomally abnormal egg might be 50% or higher [2]. Studies of human trisomies have provided considerable insight. We know that the vast majority of errors occur during oogenesis (see Glossary), that the incidence of errors increases exponentially with advancing maternal age and that the number and placement of recombination events between homologous chromosomes influences the fidelity of their segregation at the first meiotic division [3–5]. Despite considerable research effort, the effect of maternal age remains the ‘black box’ of human aneuploidy. Virtually all hypotheses have attempted to explain the effect in simple terms of cause and effect [6], but no single hypothesis provides a satisfactory explanation. Indeed, the picture that has emerged in recent years suggests that female fertility is influenced by a complex series of events occurring at different stages of egg development. Thus, it seems increasingly likely that the genetic quality of the human egg reflects a complex interplay of events. In addition, experimental data, largely from studies in the mouse, have provided evidence that environmental effects – most notably Corresponding author: Hunt, P.A. ([email protected]).
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exposure to estrogenic substances – can negatively impact oogenesis. In this review, we outline three different ‘windows’ of vulnerability during mammalian oogenesis (Figure 1) and examine the evidence that events during each can influence female fertility. Glossary Aneuploidy: A chromosome number that is not a multiple of the haploid number. In humans, sperm and eggs should have 23 chromosomes (the haploid number), and fertilization should create a zygote with 46 chromosomes (the diploid number). However, because of errors in meiotic or early mitotic cleavage divisions, a surprisingly high number of human conceptions have 47 or 45 chromosomes; these two types of aneuploidy are termed trisomy and monosomy, respectively. Azoospermia: The absence of sperm cells in the ejaculate. Azoospermia and oligospermia (too few sperm, typically defined as <20 million sperm/ml in the ejaculate) are common causes of human male infertility. Endocrine disruptor: A synthetic chemical that disturbs the body’s normal endocrine function. Endocrine disruptors can mimic or block the actions of endogenous hormones (e.g. bisphenol A, discussed in this review, is weakly estrogenic). Follicle: An oocyte surrounded by a layer of somatic cells (granulosa cells). During follicle growth, the granulosa cells proliferate and the oocyte grows and becomes surrounded by a noncellular layer (the zona pellucida). Meiocyte: A cell undergoing meiosis. Meiosis: The cell division that produces haploid gametes from diploid progenitor cells. Meiosis involves one round of DNA replication followed by two cellular divisions, meiosis I (MI) and meiosis II (MII). MI, the reductional division, involves the segregation of homologous chromosomes. MII the equational division, involves the segregation of sister chromatids. Meiotic prophase: The first – and most complicated – phase of meiosis I, in which homologous chromosomes pair, become intimately associated (synapsis), and exchange genetic material (crossing over or meiotic recombination). The complex series of prophase events is subdivided into five stages (leptotene, zygotene, pachytene, diplotene and diakinesis) that reflect the progression of synapsis and recombination. In mammalian females, meiosis arrests at a modified diplotene stage (dictyate) and remains in this state for months, years or decades, depending on the species. Meiotic recombination: Exchanges between homologous chromosomes that occur during meiotic prophase. Recombination is initiated by programmed double-strand breaks at the onset of prophase. A proportion of these breaks are repaired to yield crossover events. The physical manifestations of recombination, chiasmata, perform an essential role in maintaining connections between homologous chromosomes before their segregation at anaphase of MI. Oogenesis: The process by which a premeiotic female germ cell develops into a mature egg. Pachytene: A substage of meiotic prophase; at this stage, synapsis is complete and the sites of meiotic recombination have been established. Sister chromatid cohesion: A complex of proteins (cohesin) that bind sister chromatids. During MI, cohesion below the sites of chiasmata is essential to maintain a physical connection between homologous chromosomes to facilitate their segregation at MI anaphase. These connections must be lost before homologous chromosome segregation at anaphase, but some cohesion at the centromere must be retained to maintain a connection between sister chromatids until their separation at MII anaphase. Synaptonemal complex: A tri-partite protein structure that forms between homologous chromosomes during meiotic prophase. Meiotic recombination occurs in the context of the synaptonemal complex. Trisomy: An aneuploid conception with one additional chromosome. Clinically, the most important is trisomy 21, which is the chromosome constitution associated with Down syndrome.
0168-9525/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.11.010 Available online 14 January 2008
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Figure 1. The three vulnerable stages of oogenesis: (i) the meiotic prophase events of synapsis and recombination, which occur in the fetal ovary; (ii) follicle formation, which occurs during the second trimester of fetal development and is associated with a dramatic loss of oocytes caused by atresia, and (iii) oocyte growth, which occurs in the adult ovary and culminates in the resumption and completion of meiosis I and ovulation of a metaphase II–arrested egg. Figure by Crystal Lawson.
Reproductive senescence in the prime of life The relationship between maternal age and aneuploidy is striking. Among women in their early 20s, the risk of trisomy in a clinically recognized pregnancy is 2–3%. By contrast, among women in their 40s, the risk increases to 30–35% [7]. Although the basis of the age effect is unknown, studies of the origin of human trisomies make it clear that age influences the likelihood of errors at both meiosis I (MI) and meiosis II (MII) [8,9]. Because MI is initiated in the fetal ovary (Boxes 1 and 2) but major portions of oogenesis – including the cell divisions – occur in the adult ovary, it is likely that age-related changes acting at several different stages of oocyte development influence chromosome behavior. The high rate of aneuploidy is not the only curious aspect of human female reproduction. The fact that, on average, women are fertile for only two thirds of their adult life has long puzzled evolutionary biologists. Explanations range from adaptive theories that invoke the benefits of postmenopausal maternal or grandmaternal care [10] to theories that suggest menopause is an artifact of recent increases in human lifespan [11]. Recent human reproductive trends make the early onset of reproductive senescence a significant concern. In the United States, the average age at first pregnancy has risen by 3.6 years during the past 30 years [12], resulting in an increase in the percentage of live births occurring to women over the age of 35. Because this age group experiences the highest rate of pregnancy failure owing to chromosome abnormalities, it is likely that the live birth data grossly underestimate the number of women attempting to reproduce in their late 30s and early 40s. To quote the esteemed
geneticist Dorothy Warburton, ‘‘Our expectancy for productive and healthy life has increased by many years, so that a woman can look forward, if she wishes, to continuing in her chosen career beyond the age of 70. However, at least so far, we have not learned how to reprogram a woman’s biological clock, nor to turn off her desire to nurture her children’’ [13]. The risk of a chromosomally abnormal pregnancy is calculated on the basis of maternal age, but few would argue that chronological age per se is the culprit. The pool of ovarian follicles is progressively reduced during the reproductive lifespan of the female [14]. In human-assisted reproduction, serum follicle-stimulating hormone (FSH) levels provide an indicator of follicle reserve and an accurate predictor of fertility; the higher the FSH levels, the lower the likelihood of achieving a pregnancy. There is also evidence from research on aneuploidy that physiologic age is key, because menopause has been reported to occur at an earlier age among women with trisomic pregnancies than among women with chromosomally normal pregnancies [15]. Thus, we can make the assumption that factors that shorten reproductive lifespan will also accelerate the onset of ‘age-related’ aneuploidy. Meiotic prophase: the first window of vulnerability The historical description of female sexual differentiation as the ‘default pathway’ implies that making an ovary is a passive process when, in fact, it simply means that the controlling factors remain unknown. Recently, however, it has been recognized that retinoic acid provides the signal for meiotic entry in the fetal ovary [16]. Germ cells in the male escape this fate because the somatic cells of the 87
Review Box 1. A human oogenesis timeline Meiosis is initiated in the human fetal ovary at 11–12 weeks of gestation [18]. Oocytes enter prophase and homologous chromosomes undergo pairing, synapsis and the exchange of genetic material in a process known as recombination or crossing over. On completion of recombination, the oocyte progresses to diplotene of prophase and enters a protracted arrest stage known as dictyate. Around the time of arrest, oocytes become surrounded by somatic cells (pregranulosa cells), forming primordial follicles. Many oocytes are lost during follicle formation, and the newborn ovary contains only a fraction of the total oocytes that entered meiosis in the fetal ovary [42]. Primordial follicles remain quiescent for years until they are recruited to undergo the process of growth. In the sexually mature female, new follicles are recruited into the growing pool and supported in their development by the pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Follicle growth takes an estimated 85 days in humans, and most follicles die at some stage during the process [14]. In humans, one oocyte, on average, completes growth each month and is ovulated in response to a mid-cycle surge of LH. In response to the LH surge, the oocyte resumes meiosis; chromosomes condense, and the homologs (still connected by crossovers) orient on the metaphase I plate and segregate from each other at anaphase I. One group of chromosomes remains in the oocyte and the other is segregated to a small bleb of cytoplasm, the first polar body. Hence, the first division reduces the number of chromosomes in the oocyte by half. After meiosis I (MI) chromosome segregation, a second meiotic spindle forms immediately, the remaining chromosomes align at the spindle equator and the cell arrests. The metaphase II arrested cell is known as an egg, and it remains in arrest until it is fertilized or degenerates. Fusion of the sperm and egg plasma membranes at fertilization triggers the resumption and completion of MII. MII is essentially a mitotic division, albeit with one half the number of chromosomes and with sister chromatids that differ from one another as a result of crossing over; sister chromatids segregate, with one group of chromosomes remaining in the egg and the other segregated into a second polar body. After the division, separate nuclear envelopes form around the remaining egg chromosomes and the chromosomes contributed by the sperm, forming a zygote. The chromosomes in the male and female pronuclei undergo DNA replication and condense in preparation for the first mitotic cleavage division, and maternally and paternally derived chromosomes mix for the first time on the mitotic spindle.
differentiating testis produce a testis-specific enzyme (Cyp26b1) that degrades retinoic acid [17]. In the human female, the first germ cells initiate meiosis at 11–12 weeks of gestation [18], with subsequent groups of cells entering meiosis over the course of the next several weeks. Oocytes progress through meiotic prophase, undergoing the complex events of synapsis and recombination, and then enter a protracted arrest phase in late prophase (Box 1). The duration of arrest has made this a prominent component of human maternal age theories Box 2. A static or dynamic oocyte pool? The dogma in mammalian oogenesis is that the pool of oocytes in the ovary is established during fetal development, and all eggs ovulated by the adult female initiated meiosis in the fetal ovary. Johnson et al. [69,70] have recently challenged this belief, suggesting the existence of germline stem cells that give rise to new oocytes in the adult. Their data and interpretation have been challenged and hotly debated (e.g. [71–74]). However, there is not yet evidence that these ‘adult’ oocytes – if they exist – mature and are ovulated. Thus, we will operate under the assumption that, in both humans and mice, the oocytes present at the time of birth represent the female’s lifetime supply of oocytes, and this supply dictates the length of her reproductive lifespan.
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[19]. As discussed below, however, recent data have suggested that the prophase events that occur during fetal development are a potential source of vulnerability and contribute to human aneuploidy. Synapsis and recombination: failure to find and co-mingle with your partner jeopardizes your reproductive health The prophase events of synapsis and recombination that occur in the fetal ovary are essential for germ cell survival and meiotic progression. Early cytological studies in the mouse showed that translocations and other chromosomal aberrations that impede synapsis result in the loss of a significant number of meiocytes in both males and females [20]. Similarly, direct analysis of prophase in human males has revealed a correlation between synaptic and/or recombination defects and cases of azoospermia or oligospermia [21,22]. These cytological studies are consistent with the analyses of mice carrying targeted disruptions of meiotic genes. For example, null mutants for genes involved in synaptonemal complex formation invariably exhibit meiotic disruption or arrest (e.g. Sycp3 [23] and Syce2 [24]) in both males and females. Similarly, mutations in genes acting early (e.g. Spo11 [25]), midway (e.g. Msh4 [26]) or late (e.g. Mlh1 [27]) in the recombination pathway are associated with germ cell demise in both sexes. The demise of cells with synaptic or recombination defects has been ascribed to the actions of a checkpoint control mechanism acting at pachytene [28]. Although the mechanistic details remain to be fully elucidated, recent evidence has suggested that different control mechanisms act to detect synaptic and recombination defects. Specifically, in the mouse, chromosomal regions that remain unsynapsed at pachytene are transcriptionally silenced [29–32]. In addition, Li and Schimenti [33] have identified Trip13, the mouse ortholog of yeast PCH2, an essential component of the pachytene checkpoint. The mouse gene seems to be required for successful completion of recombination but not synapsis. Regardless of the molecular details of these checkpoint mechanisms, there are apparent sex-specific differences in their efficiency. Specifically, several meiotic mutations affecting synapsis and recombination (including most of those listed above) cause prophase arrest in the male while allowing at least a proportion of oocytes to progress through meiotic prophase. Possibly checkpoint mechanisms are more effective in spermatogenesis than in oogenesis [34,35]. However, some of the discrepancy between the sexes likely reflects differences in the ability to detect the consequences of synaptic or recombination errors. That is, in males, defects during pachytene that cause the elimination of a large number of spermatocytes result in a drop in sperm counts and infertility or reduced fertility. In the female, however, such loss would reduce the reproductive lifespan by decreasing the total follicle reserve. This effect could easily be missed if ‘fertility’ is defined as the ability to reproduce without reference to the duration of the fertile period. In the human, however, the consequences would be serious; premature ovarian failure and/or accelerated onset of human age-related aneuploidy.
Review Recombination sets the stage for chromosome segregation errors Germ cell death and infertility are not the only consequences of synaptic and/or recombinational defects. Studies of human trisomies have shown that the number and location of crossovers influence the likelihood of segregation errors at the first meiotic division [3]. Sites of recombination are established in the fetal oocyte, and some homologous chromosomes seem to be ‘at risk’ because they are tethered by exchanges at suboptimal locations (near telomeres or centromeres). Indeed, when homologs are segregated years later in the human oocyte, exchanges that are either too proximal or too distal are more likely to be associated with nondisjunctional events [3]. Thus, the establishment of recombination sites in the fetal ovary is an important window of vulnerability with respect to the genetic quality of the oocyte. Can environmental factors influence the number and placement of exchange events? Although considerable information exists about patterns of meiotic recombination, little is known regarding the mechanisms that control the number and placement of these exchanges. Most mammals exhibit sex-specific differences in recombination, and these are especially pronounced in the human. The rate of recombination is 1.6 times higher in human females than males [36], and the placement of exchanges differs, with more exchanges in distal regions in males. Furthermore, direct studies of prophase in humans provide evidence of considerable variation in recombination levels both within and among individuals of the same sex [21,37]. In the mouse, the availability of inbred strains can be exploited to examine the genetic control of recombination. The available data indicate that there is significant variation among inbred stains, that recombination levels are likely controlled by a few genes in the male, and that different control mechanisms likely operate in the female [38,39]. Although the strain-specific differences suggest that genetic factors are largely responsible for variation in recombination patterns, recent studies in mice provide evidence that environmental factors also can influence recombination. Exposure of pregnant females to low, environmentally relevant levels of the estrogenic chemical bisphenol A (BPA) (Box 3) disrupts prophase events in the fetal ovary [40]. Specifically, oocytes from treated females exhibit a high frequency of synaptic defects and increased levels and altered placement of crossovers. Importantly, these abnormalities herald subsequent errors in segregation, as a dramatic increase in meiotic aneuploidy is observed in oocytes from adult females exposed to BPA in utero [40]. This not only provides the first direct evidence of an environmental effect on recombination in mammals but raises concerns about the potential effects of environmental contaminants on human fertility. The follicle: developing a long-term relationship Before an oocyte can resume and complete the first meiotic division, it must undergo an extensive, hormone-dependent growth phase [41]. Oocyte growth and maturation
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Box 3. Bisphenol A Bisphenol A (BPA) is one of the highest volume chemicals produced, with >6 billion pounds manufactured annually. It is the major component of polycarbonate plastic, an additive in other forms of plastic, and a component of both resins used to line food and beverage containers and of some composites used in dentistry. Its presence in a wide variety of common household products (e.g. baby bottles, food storage containers, plastic wrap and canned foods and beverages) means that BPA is a part of everyday life. BPA has been shown to leach from food and beverage containers, and it is widely believed that the bulk of human exposure is through the ingestion of contaminated food and beverages. Recent studies, however, have shown that BPA contaminates our air, water and soil [66]. Human populations in many different countries in the developed world have now been screened for BPA, and detectable levels of the chemical have been found in human blood, urine, amniotic fluid, cord blood, breast milk and follicular fluid [66]. Although the levels of BPA vary widely among individuals within a population and among studies, analyses using the most sensitive detection methods suggest that the average level of BPA circulating in the blood of adults is in the 2- to 4-ng/ml range [66]. Unfortunately, these values provide no means of accurately determining current human exposure levels. A troubling aspect of the human BPA data are results of studies of pregnant women and their fetuses, which have reported high levels in some placental tissues and amniotic fluid samples. Indeed, in some cases, substantially higher levels were found in fetal tissues than in maternal serum. Studies in rodents suggest that the fetus is exquisitely sensitive to BPA; low-dose exposure has been reported to produce a variety of adverse reproductive effects, including morphological alterations of the reproductive tract, accelerated onset of puberty, disruption of estrus cycles and reduced sperm counts. These and other in vivo effects of BPA on laboratory rodents have recently been summarized in a review from an NIH-sponsored meeting [67].
occur in the ovary of the sexually mature female and require the coordinated actions of the oocyte and its somatic cell companions. Two distinct somatic cell compartments are required. Granulosa cells surround and are in direct contact with the oocyte and, together with the oocyte, comprise the follicle. The other key somatic cells in the ovary are the theca cells, which reside in the stromal tissue between follicles and, in conjunction with the granulosa cells, create an appropriate endocrine environment for oocyte growth and maturation. Forming follicles: a fetal event with direct relevance to reproductive lifespan Follicle formation occurs during the second trimester of human fetal development [42] and, in the mouse, immediately after birth [43]. The signal that triggers the formation of follicles is unknown. A sudden drop in estrogen levels (i.e. the loss of maternal estrogens at birth in the mouse or decreased availability of estrogens as a result of increased a fetal protein production in the human [44]) has been postulated to initiate the process. However, given the timing difference between humans and mice and the fact that the oocyte appears to regulate its own fate during follicle growth [45], it seems more likely that the signal comes from the oocyte and is linked to entry into dictyate arrest (Box 1). Despite our ignorance of the initiating event, the molecular control of follicle formation is beginning to be elucidated [46]. Although the specifics are beyond the scope of 89
Review this review, recent studies suggest that the packaging of oocytes into follicles can be perturbed by both genetic and environmental factors and that these perturbations might profoundly impact reproductive success. Before meiotic entry, primordial germ cells in the fetal ovary undergo a period of mitotic proliferation characterized by incomplete cytokinesis. This process creates groups of interconnected cells, called germ cell cysts, and these connections persist as oocytes enter meiosis [43]. Thus, the formation of a follicle requires two events: breakdown of intracellular bridges between oocytes and enclosure of individual oocytes by somatic cells (the precursors of granulosa cells). Intriguingly, recent data from studies of wildlife and experimental animals indicate that environmental contaminants can disrupt the process, resulting in the formation of follicles containing two or more oocytes (multioocyte or polyovular follicles) [47]. In alligators, the occurrence of multioocyte follicles in adult females exposed to environmental contaminants during fetal development is correlated with high levels of embryonic mortality [47]. In mice, neonatal exposure to both manmade and naturally occurring estrogenic substances increases the frequency of multioocyte follicles [44] and, as in alligators, a corresponding increase in embryo mortality has been reported [48]. Furthermore, there is evidence that multioocyte follicles might be selected against during oocyte growth [49]. Thus, the induction of multioocyte follicles might have reproductive repercussions by shortening the reproductive lifespan of the female and/or diminishing the quality of the oocytes she produces. Multioocyte follicles have been reported in a variety of species, including humans, [47,49], and available evidence suggests that environmental exposures are not the only precipitating event. In the mouse, defects in activin expression or signaling [50] and a variety of single gene mutations, including the growth factor genes bone morphogenetic protein 15 (Bmp15) [51] and growth differentiation factor-9 (Gdf9) [51] and the DM-domain encoding gene Dmrt4 [52], increase the frequency of multioocyte follicles. Recent studies of changes in mRNA and protein levels in response to neonatal estrogenic exposures showed suppression of the transforming growth factor b (TGF-b) superfamily member, activin [50]. Thus, although we are in the early stages of unraveling the molecular control of follicle development, understanding both activin and estrogen signaling and how these pathways are interrelated is key. In the interim, it is clear that follicle formation is a critical aspect of female fertility, and experimental studies raise concerns about the potential reproductive impact of environmental exposures during this critical developmental window. Oocyte atresia: programmed loss or a quality control mechanism? Around the time of follicle formation, a dramatic loss of oocytes, termed atresia, occurs in both humans and mice. In humans, it has been estimated that this loss reduces the pool of oocytes by >80% [42]. Because the pool of oocytes in the newborn ovary represents the reproductive resources of that female (Box 2), the amount of loss incurred influences the length of reproductive lifespan (i.e. the age at 90
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which reproductive senescence occurs in the adult). Because the period of atresia coincides with follicle formation, it has been likened to the programmed cell death that occurs in Drosophila melanogaster females with the breakdown of germline cysts [43]. However, in flies, only one of the 16 cells in each cyst is destined to become an oocyte, with the others serving as nurse cells. Because a separate somatic cell compartment – the granulosa cells – serve the comparable nurse cell function in the mammalian ovary, it is difficult to know how far the analogy can be stretched. Furthermore, as detailed above, there is compelling evidence that at least a portion of the cells is lost because of the actions of prophase quality control mechanisms. Nevertheless, because the reduction in oocyte pool during this period directly influences the reproductive lifespan of the female, it is clear that we need to better understand oocyte atresia. Oocyte growth and meiotic maturation: the final frontier The complex events of prophase set the stage for chromosome segregation when meiosis resumes in the adult ovary. The completion of the first meiotic division and ovulation of the mature egg is preceded by oocyte growth, a process that rivals prophase in complexity. The trigger that recruits an arrested oocyte into the pool of growing follicles remains unknown. Two things, however, are clear: first, most follicles that initiate growth die before ovulation. Second, growth is protracted. There is a tendency to think of oocyte maturation as the culmination of a single ovarian cycle, but the entire process – from initiation of growth to ovulation – takes an estimated 85 days in humans and 2 weeks in mice [14]. The first group of oocytes initiates growth in the sexually immature female but, in the absence of appropriate endocrine support, these follicles cannot complete growth and die. On sexual maturation, pituitary gonadotropin levels become sufficient to support the late stages of follicle development and ovulation. The intraovarian control of oocyte growth involves both the interplay of signals between the steroidogenic cells (the granulosa and theca cells) and intricate bi-directional communication between the oocyte and the granulosa cells. The process is complex, but the pivotal roles of both estrogen and members of the TGF-b superfamily are beginning to be elucidated [53,54]. From a meiotic standpoint, the late stages of oocyte growth are crucial, because the ability to resume and complete the first meiotic division is acquired by the oocyte in a stepwise fashion [41]. Although this implies that the oocyte is passively nursed along by its granulosa cells, recently it has become evident that, through the production of growth factors, the oocyte influences both the proliferation and differentiation of the granulosa cells. Thus, although communication is bi-directional, the oocyte clearly participates in the regulation of its own development [45]. The endocrine environment, maternal age and human aneuploidy The decade preceding menopause is marked by hormonal changes that influence the length of the menstrual cycle.
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Accordingly, it has been suggested that the changing endocrine environment underlies the age-related increase in human aneuploidy. Perhaps it would be easier to understand age-related aneuploidy if we understood menopause. Although increasing FSH levels are the first indicator of reproductive senescence, it remains unclear whether FSH increases in response to declining follicle numbers in the ovary or whether rising FSH levels actively diminish the follicle pool. Indeed, there is some support for both ideas [55]. Recent studies of transgenic mice expressing pituitary-independent human FSH through the rat insulin II promoter, however, provided evidence that rising FSH levels alone might accelerate female reproductive failure [55]. Regardless of whether the brain or the ovary initiates menopause, oocyte quality suffers. Direct studies of human oocytes from both infertility patients [56] and normal donors [57] have provided evidence of age-related changes in spindle formation and chromosome alignment. Furthermore, in the assisted reproductive setting, increasing maternal age is the most important impediment to success [58], because it is associated with both a decline in the rate of fertilization and an increase in the frequency of aneuploidy.
and must be maintained until anaphase. In addition, sister centromeres must be constrained to act in tandem at metaphase I. During the past several years, meiosisspecific cohesins that mediate these events have been identified [59]. We remain ignorant, however, of a critical piece of the puzzle: cohesion is established during DNA replication and presumably modified during the repair of DNA double-stand breaks that produce crossovers [60]. Thus, the protein complex essential for maintaining connections between chromosomes until the onset of anaphase is established in utero. Does this mean that chromosome segregation in the oocyte of a 45-year-old woman is reliant on 45-year-old proteins? Attempts were made to address this question when an age effect was discovered in females homozygous for a targeted disruption of the meiosisspecific cohesin, Smc1b [61]. These studies revealed transcription of meiotic cohesins during oocyte growth; however, whether new proteins are synthesized and become incorporated into the cohesin complex remains unknown. The answer is pivotal. Human age-related aneuploidy could reflect the degeneration of components of the cohesin complex caused by the lack of protein turnover, or it could reflect inadequate synthesis or incorporation of essential replacement proteins during oocyte growth.
Can genetic quality be influenced by the growth environment? How compelling are the data that changes in oocyte growth influence the segregation of chromosomes in meiosis? At the beginning of oocyte growth, homologous chromosomes are joined together, but the cell is not yet capable of undergoing metaphase I. Thus, the above question is best considered in two parts: how might growth affect the connections between chromosomes and how might growth influence the metaphase machinery?
Oocyte growth and the meiotic cell cycle During the final stages of growth, changes occur in both the organization of the chromatin and the microtubule network in the egg, and these changes coincide with the acquisition of meiotic competence [41]. In addition, both spindle assembly and the formation of functional kinetochores occur at the time of nuclear envelope breakdown. Is there compelling evidence that disturbances in growth can affect these processes and lead to malsegregation at either meiotic division? Studies designed to test the effects of altered growth on meiotic chromosome segregation in the mouse provided evidence of disturbances in spindle formation and chromosome alignment. Importantly, effects were evident in several different mouse mutants, and defects observed at MI were correlated with increases in aneuploidy in metaphase II arrested eggs [62].
Oocyte growth and chromosomal connections In contrast to mitosis, meiosis requires highly specialized chromosomal connections (Figure 2). The connections between homologs (chiasmata) forged during prophase in the fetal ovary are essential for homolog segregation during MI
Figure 2. Meiosis necessitates specialized chromosome connections. The unique features of meiotic chromosomes are shown in these images of mouse (a) mitotic, (b) meiosis I (MI) and (c) MII chromosomes. Chromosomes have been immunostained with an antibody to CENP-E to highlight the kinetochores (red) and counterstained with DAPI (blue) [68]. During mitotic division (a), kinetochores form at the centromeres of both sister chromatids (arrows), and cohesion at the centromere and along the chromosome arms keeps sister chromatids physically associated until anaphase. Degradation of the cohesin complex at anaphase allows the sisters to move to opposite poles. During MI metaphase (b), meiosis-specific cohesins perform two distinct functions: (i) they constrain the sister chromatids of an individual chromosome (arrows) to act in unison at the first division, so that their kinetochores are captured by microtubules from the same spindle pole, and (ii) they maintain a physical connection between homologs distal to the site of a crossover (arrowhead). At anaphase I, loss of cohesion at the sites of exchanges allows homologs to segregate. At metaphase II (c), the tight association between the kinetochores of sister chromatids (arrows) and cohesion along the chromosome arms have been lost. The retention of some cohesion at the centromere, however, is essential to maintain a connection between sister chromatids until anaphase II. Figure adapted, with permission, from Ref. [68]. Left and center images reprinted with permission from Chromosoma.
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Review Perhaps the most worrying evidence that changes during the final stages of oocyte growth can impact the genetic quality of the oocyte comes from studies of mice accidentally exposed to the estrogenic chemical, BPA. In the course of meiotic studies, a sudden change in meiotic chromosome alignment and segregation was observed in control animals. These effects proved to be the result of BPA exposure owing to inadvertent damage to the polycarbonate caging materials, and subsequent studies showed that low-dose exposures during the final stages of oocyte growth were sufficient to elicit the meiotic defects [63]. Although the data linking BPA to reproductive defects in humans remain equivocal [64,65], it is clear that current human exposure falls within levels that elicit effects in mice [66,67]. Concluding remarks Aneuploidy is the leading cause of reproductive failure and congenital birth defects in humans. The strong association between maternal age and trisomy, coupled with changing reproductive trends that have steadily increased the maternal age at first birth, suggests that the aneuploidy problem is not going to diminish. In this review, we tried to dispel the notion that there is a single cause of human nondisjunction or a single stage of oocyte development when chromosomes become destined to nondisjoin. Instead, we argue that events during at least three distinct stages of mammalian oogenesis have the potential to influence the genetic quality of the egg. We suggest that the interplay of these factors accounts for the complex etiology of human aneuploidy and explains why no single hypothesis has been able to adequately explain the effect of maternal age. We included a discussion of the potential impact of environmental factors on each of the three stages. The endocrine disrupting chemical bisphenol A has been used as an example because studies in mice suggest that it can adversely affect events at each stage. Our intent in summarizing these bisphenol A studies is not to focus attention on this chemical, but rather to use bisphenol A as a ‘poster child’ to make the point that environmental factors that contribute to human aneuploidy might well exist. Given the complexity of oogenesis, providing definitive evidence that environmental effects contribute to human chromosome abnormalities will be difficult indeed. Nevertheless, the clinical importance of any such association makes this an essential consideration in future studies. Acknowledgements We thank Smith Lutu and Crystal Lawson for help with the figures. This work was supported by National Institutes of Health Grants HD37502 and ES013527 (P.A.H.) and HD21341 (T.J.H.).
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54 Knight, P.G. and Glister, C. (2006) TGF-beta superfamily members and ovarian follicle development. Reproduction 132, 191–206 55 McTavish, K.J. et al. (2007) Rising follicle-stimulating hormone levels with age accelerate female reproductive failure. Endocrinology 148, 4432–4439 56 Battaglia, D.E. et al. (1996) Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum. Reprod. 11, 2217–2222 57 Volarcik, K. et al. (1998) The meiotic competence of in-vitro matured human oocytes is influenced by donor age: evidence that folliculogenesis is compromised in the reproductively aged ovary. Hum. Reprod. 13, 154–160 58 Van Voorhis, B.J. (2006) Outcomes from assisted reproductive technology. Obstet. Gynecol. 107, 183–200 59 Revenkova, E. and Jessberger, R. (2006) Shaping meiotic prophase chromosomes: cohesins and synaptonemal complex proteins. Chromosoma 115, 235–240 60 Revenkova, E. and Jessberger, R. (2005) Keeping sister chromatids together: cohesins in meiosis. Reproduction 130, 783–790 61 Hodges, C.A. et al. (2005) SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction. Nat. Genet. 37, 1351–1355 62 Hodges, C.A. et al. (2002) Experimental evidence that changes in oocyte growth influence meiotic chromosome segregation. Hum. Reprod. 17, 1171–1180 63 Hunt, P.A. et al. (2003) Bisphenol a exposure causes meiotic aneuploidy in the female mouse. Curr. Biol. 13, 546–553 64 Sugiura-Ogasawara, M. et al. (2005) Exposure to bisphenol A is associated with recurrent miscarriage. Hum. Reprod. 20, 2325–2329 65 Takeuchi, T. et al. (2004) Positive relationship between androgen and the endocrine disruptor, bisphenol A, in normal women and women with ovarian dysfunction. Endocr. J. 51, 165–169 66 Vandenberg, L.N. et al. (2007) Human exposure to bisphenol A (BPA). Reprod. Toxicol. 24, 139–177 67 Richter, C.A. et al. (2007) In vivo effects of bisphenol A in laboratory rodent studies. Reprod. Toxicol. 24, 199–224 68 Hodges, C.A. and Hunt, P.A. (2002) Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos. Chromosoma 111, 165–169 69 Johnson, J. et al. (2004) Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145–150 70 Johnson, J. et al. (2005) Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell 122, 303–315 71 Byskov, A.G. et al. (2005) Eggs forever? Differentiation 73, 438–446 72 Telfer, E.E. et al. (2005) On regenerating the ovary and generating controversy. Cell 122, 821–822 73 Bristol-Gould, S.K. et al. (2006) Fate of the initial follicle pool: empirical and mathematical evidence supporting its sufficiency for adult fertility. Dev. Biol. 298, 149–154 74 Liu, Y. et al. (2007) Germline stem cells and neo-oogenesis in the adult human ovary. Dev. Biol. 306, 112–120
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Human female meiosis: what makes a good egg go bad? Patricia A. Hunt and Terry J. Hassold School of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164-4660, USA
Critical events of oogenesis occur during three distinct developmental stages: meiotic initiation in the fetal ovary, follicle formation in the perinatal period, and oocyte growth and maturation in the adult. Evidence from studies in humans and mice suggests that the genetic quality of the egg may be influenced by events at each of these stages. Recent experimental studies add additional complexity, suggesting that environmental influences might adversely affect all three stages. Thus, understanding the molecular control of oogenesis during these critical developmental windows will not only contribute to an understanding of human aneuploidy, but also provide a means of assessing potential effects of environmental exposures on human reproductive health. ‘We have met the enemy and (s)he is us’ – Pogo The high incidence of chromosomally abnormal pregnancies in humans has been an intractable genetic puzzle for decades. Approximately 7–10% of clinically recognized pregnancies are chromosomally abnormal [1], but this understates the problem for two reasons. First, the loss or gain of some human chromosomes is so lethal that the pregnancy does not survive long enough to come to clinical attention. Second, the incidence is strongly affected by age and, for women at the end of their reproductive lifespan, the risk of ovulating a chromosomally abnormal egg might be 50% or higher [2]. Studies of human trisomies have provided considerable insight. We know that the vast majority of errors occur during oogenesis (see Glossary), that the incidence of errors increases exponentially with advancing maternal age and that the number and placement of recombination events between homologous chromosomes influences the fidelity of their segregation at the first meiotic division [3–5]. Despite considerable research effort, the effect of maternal age remains the ‘black box’ of human aneuploidy. Virtually all hypotheses have attempted to explain the effect in simple terms of cause and effect [6], but no single hypothesis provides a satisfactory explanation. Indeed, the picture that has emerged in recent years suggests that female fertility is influenced by a complex series of events occurring at different stages of egg development. Thus, it seems increasingly likely that the genetic quality of the human egg reflects a complex interplay of events. In addition, experimental data, largely from studies in the mouse, have provided evidence that environmental effects – most notably Corresponding author: Hunt, P.A. ([email protected]).
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exposure to estrogenic substances – can negatively impact oogenesis. In this review, we outline three different ‘windows’ of vulnerability during mammalian oogenesis (Figure 1) and examine the evidence that events during each can influence female fertility. Glossary Aneuploidy: A chromosome number that is not a multiple of the haploid number. In humans, sperm and eggs should have 23 chromosomes (the haploid number), and fertilization should create a zygote with 46 chromosomes (the diploid number). However, because of errors in meiotic or early mitotic cleavage divisions, a surprisingly high number of human conceptions have 47 or 45 chromosomes; these two types of aneuploidy are termed trisomy and monosomy, respectively. Azoospermia: The absence of sperm cells in the ejaculate. Azoospermia and oligospermia (too few sperm, typically defined as <20 million sperm/ml in the ejaculate) are common causes of human male infertility. Endocrine disruptor: A synthetic chemical that disturbs the body’s normal endocrine function. Endocrine disruptors can mimic or block the actions of endogenous hormones (e.g. bisphenol A, discussed in this review, is weakly estrogenic). Follicle: An oocyte surrounded by a layer of somatic cells (granulosa cells). During follicle growth, the granulosa cells proliferate and the oocyte grows and becomes surrounded by a noncellular layer (the zona pellucida). Meiocyte: A cell undergoing meiosis. Meiosis: The cell division that produces haploid gametes from diploid progenitor cells. Meiosis involves one round of DNA replication followed by two cellular divisions, meiosis I (MI) and meiosis II (MII). MI, the reductional division, involves the segregation of homologous chromosomes. MII the equational division, involves the segregation of sister chromatids. Meiotic prophase: The first – and most complicated – phase of meiosis I, in which homologous chromosomes pair, become intimately associated (synapsis), and exchange genetic material (crossing over or meiotic recombination). The complex series of prophase events is subdivided into five stages (leptotene, zygotene, pachytene, diplotene and diakinesis) that reflect the progression of synapsis and recombination. In mammalian females, meiosis arrests at a modified diplotene stage (dictyate) and remains in this state for months, years or decades, depending on the species. Meiotic recombination: Exchanges between homologous chromosomes that occur during meiotic prophase. Recombination is initiated by programmed double-strand breaks at the onset of prophase. A proportion of these breaks are repaired to yield crossover events. The physical manifestations of recombination, chiasmata, perform an essential role in maintaining connections between homologous chromosomes before their segregation at anaphase of MI. Oogenesis: The process by which a premeiotic female germ cell develops into a mature egg. Pachytene: A substage of meiotic prophase; at this stage, synapsis is complete and the sites of meiotic recombination have been established. Sister chromatid cohesion: A complex of proteins (cohesin) that bind sister chromatids. During MI, cohesion below the sites of chiasmata is essential to maintain a physical connection between homologous chromosomes to facilitate their segregation at MI anaphase. These connections must be lost before homologous chromosome segregation at anaphase, but some cohesion at the centromere must be retained to maintain a connection between sister chromatids until their separation at MII anaphase. Synaptonemal complex: A tri-partite protein structure that forms between homologous chromosomes during meiotic prophase. Meiotic recombination occurs in the context of the synaptonemal complex. Trisomy: An aneuploid conception with one additional chromosome. Clinically, the most important is trisomy 21, which is the chromosome constitution associated with Down syndrome.
0168-9525/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.11.010 Available online 14 January 2008
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Figure 1. The three vulnerable stages of oogenesis: (i) the meiotic prophase events of synapsis and recombination, which occur in the fetal ovary; (ii) follicle formation, which occurs during the second trimester of fetal development and is associated with a dramatic loss of oocytes caused by atresia, and (iii) oocyte growth, which occurs in the adult ovary and culminates in the resumption and completion of meiosis I and ovulation of a metaphase II–arrested egg. Figure by Crystal Lawson.
Reproductive senescence in the prime of life The relationship between maternal age and aneuploidy is striking. Among women in their early 20s, the risk of trisomy in a clinically recognized pregnancy is 2–3%. By contrast, among women in their 40s, the risk increases to 30–35% [7]. Although the basis of the age effect is unknown, studies of the origin of human trisomies make it clear that age influences the likelihood of errors at both meiosis I (MI) and meiosis II (MII) [8,9]. Because MI is initiated in the fetal ovary (Boxes 1 and 2) but major portions of oogenesis – including the cell divisions – occur in the adult ovary, it is likely that age-related changes acting at several different stages of oocyte development influence chromosome behavior. The high rate of aneuploidy is not the only curious aspect of human female reproduction. The fact that, on average, women are fertile for only two thirds of their adult life has long puzzled evolutionary biologists. Explanations range from adaptive theories that invoke the benefits of postmenopausal maternal or grandmaternal care [10] to theories that suggest menopause is an artifact of recent increases in human lifespan [11]. Recent human reproductive trends make the early onset of reproductive senescence a significant concern. In the United States, the average age at first pregnancy has risen by 3.6 years during the past 30 years [12], resulting in an increase in the percentage of live births occurring to women over the age of 35. Because this age group experiences the highest rate of pregnancy failure owing to chromosome abnormalities, it is likely that the live birth data grossly underestimate the number of women attempting to reproduce in their late 30s and early 40s. To quote the esteemed
geneticist Dorothy Warburton, ‘‘Our expectancy for productive and healthy life has increased by many years, so that a woman can look forward, if she wishes, to continuing in her chosen career beyond the age of 70. However, at least so far, we have not learned how to reprogram a woman’s biological clock, nor to turn off her desire to nurture her children’’ [13]. The risk of a chromosomally abnormal pregnancy is calculated on the basis of maternal age, but few would argue that chronological age per se is the culprit. The pool of ovarian follicles is progressively reduced during the reproductive lifespan of the female [14]. In human-assisted reproduction, serum follicle-stimulating hormone (FSH) levels provide an indicator of follicle reserve and an accurate predictor of fertility; the higher the FSH levels, the lower the likelihood of achieving a pregnancy. There is also evidence from research on aneuploidy that physiologic age is key, because menopause has been reported to occur at an earlier age among women with trisomic pregnancies than among women with chromosomally normal pregnancies [15]. Thus, we can make the assumption that factors that shorten reproductive lifespan will also accelerate the onset of ‘age-related’ aneuploidy. Meiotic prophase: the first window of vulnerability The historical description of female sexual differentiation as the ‘default pathway’ implies that making an ovary is a passive process when, in fact, it simply means that the controlling factors remain unknown. Recently, however, it has been recognized that retinoic acid provides the signal for meiotic entry in the fetal ovary [16]. Germ cells in the male escape this fate because the somatic cells of the 87
Review Box 1. A human oogenesis timeline Meiosis is initiated in the human fetal ovary at 11–12 weeks of gestation [18]. Oocytes enter prophase and homologous chromosomes undergo pairing, synapsis and the exchange of genetic material in a process known as recombination or crossing over. On completion of recombination, the oocyte progresses to diplotene of prophase and enters a protracted arrest stage known as dictyate. Around the time of arrest, oocytes become surrounded by somatic cells (pregranulosa cells), forming primordial follicles. Many oocytes are lost during follicle formation, and the newborn ovary contains only a fraction of the total oocytes that entered meiosis in the fetal ovary [42]. Primordial follicles remain quiescent for years until they are recruited to undergo the process of growth. In the sexually mature female, new follicles are recruited into the growing pool and supported in their development by the pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Follicle growth takes an estimated 85 days in humans, and most follicles die at some stage during the process [14]. In humans, one oocyte, on average, completes growth each month and is ovulated in response to a mid-cycle surge of LH. In response to the LH surge, the oocyte resumes meiosis; chromosomes condense, and the homologs (still connected by crossovers) orient on the metaphase I plate and segregate from each other at anaphase I. One group of chromosomes remains in the oocyte and the other is segregated to a small bleb of cytoplasm, the first polar body. Hence, the first division reduces the number of chromosomes in the oocyte by half. After meiosis I (MI) chromosome segregation, a second meiotic spindle forms immediately, the remaining chromosomes align at the spindle equator and the cell arrests. The metaphase II arrested cell is known as an egg, and it remains in arrest until it is fertilized or degenerates. Fusion of the sperm and egg plasma membranes at fertilization triggers the resumption and completion of MII. MII is essentially a mitotic division, albeit with one half the number of chromosomes and with sister chromatids that differ from one another as a result of crossing over; sister chromatids segregate, with one group of chromosomes remaining in the egg and the other segregated into a second polar body. After the division, separate nuclear envelopes form around the remaining egg chromosomes and the chromosomes contributed by the sperm, forming a zygote. The chromosomes in the male and female pronuclei undergo DNA replication and condense in preparation for the first mitotic cleavage division, and maternally and paternally derived chromosomes mix for the first time on the mitotic spindle.
differentiating testis produce a testis-specific enzyme (Cyp26b1) that degrades retinoic acid [17]. In the human female, the first germ cells initiate meiosis at 11–12 weeks of gestation [18], with subsequent groups of cells entering meiosis over the course of the next several weeks. Oocytes progress through meiotic prophase, undergoing the complex events of synapsis and recombination, and then enter a protracted arrest phase in late prophase (Box 1). The duration of arrest has made this a prominent component of human maternal age theories Box 2. A static or dynamic oocyte pool? The dogma in mammalian oogenesis is that the pool of oocytes in the ovary is established during fetal development, and all eggs ovulated by the adult female initiated meiosis in the fetal ovary. Johnson et al. [69,70] have recently challenged this belief, suggesting the existence of germline stem cells that give rise to new oocytes in the adult. Their data and interpretation have been challenged and hotly debated (e.g. [71–74]). However, there is not yet evidence that these ‘adult’ oocytes – if they exist – mature and are ovulated. Thus, we will operate under the assumption that, in both humans and mice, the oocytes present at the time of birth represent the female’s lifetime supply of oocytes, and this supply dictates the length of her reproductive lifespan.
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[19]. As discussed below, however, recent data have suggested that the prophase events that occur during fetal development are a potential source of vulnerability and contribute to human aneuploidy. Synapsis and recombination: failure to find and co-mingle with your partner jeopardizes your reproductive health The prophase events of synapsis and recombination that occur in the fetal ovary are essential for germ cell survival and meiotic progression. Early cytological studies in the mouse showed that translocations and other chromosomal aberrations that impede synapsis result in the loss of a significant number of meiocytes in both males and females [20]. Similarly, direct analysis of prophase in human males has revealed a correlation between synaptic and/or recombination defects and cases of azoospermia or oligospermia [21,22]. These cytological studies are consistent with the analyses of mice carrying targeted disruptions of meiotic genes. For example, null mutants for genes involved in synaptonemal complex formation invariably exhibit meiotic disruption or arrest (e.g. Sycp3 [23] and Syce2 [24]) in both males and females. Similarly, mutations in genes acting early (e.g. Spo11 [25]), midway (e.g. Msh4 [26]) or late (e.g. Mlh1 [27]) in the recombination pathway are associated with germ cell demise in both sexes. The demise of cells with synaptic or recombination defects has been ascribed to the actions of a checkpoint control mechanism acting at pachytene [28]. Although the mechanistic details remain to be fully elucidated, recent evidence has suggested that different control mechanisms act to detect synaptic and recombination defects. Specifically, in the mouse, chromosomal regions that remain unsynapsed at pachytene are transcriptionally silenced [29–32]. In addition, Li and Schimenti [33] have identified Trip13, the mouse ortholog of yeast PCH2, an essential component of the pachytene checkpoint. The mouse gene seems to be required for successful completion of recombination but not synapsis. Regardless of the molecular details of these checkpoint mechanisms, there are apparent sex-specific differences in their efficiency. Specifically, several meiotic mutations affecting synapsis and recombination (including most of those listed above) cause prophase arrest in the male while allowing at least a proportion of oocytes to progress through meiotic prophase. Possibly checkpoint mechanisms are more effective in spermatogenesis than in oogenesis [34,35]. However, some of the discrepancy between the sexes likely reflects differences in the ability to detect the consequences of synaptic or recombination errors. That is, in males, defects during pachytene that cause the elimination of a large number of spermatocytes result in a drop in sperm counts and infertility or reduced fertility. In the female, however, such loss would reduce the reproductive lifespan by decreasing the total follicle reserve. This effect could easily be missed if ‘fertility’ is defined as the ability to reproduce without reference to the duration of the fertile period. In the human, however, the consequences would be serious; premature ovarian failure and/or accelerated onset of human age-related aneuploidy.
Review Recombination sets the stage for chromosome segregation errors Germ cell death and infertility are not the only consequences of synaptic and/or recombinational defects. Studies of human trisomies have shown that the number and location of crossovers influence the likelihood of segregation errors at the first meiotic division [3]. Sites of recombination are established in the fetal oocyte, and some homologous chromosomes seem to be ‘at risk’ because they are tethered by exchanges at suboptimal locations (near telomeres or centromeres). Indeed, when homologs are segregated years later in the human oocyte, exchanges that are either too proximal or too distal are more likely to be associated with nondisjunctional events [3]. Thus, the establishment of recombination sites in the fetal ovary is an important window of vulnerability with respect to the genetic quality of the oocyte. Can environmental factors influence the number and placement of exchange events? Although considerable information exists about patterns of meiotic recombination, little is known regarding the mechanisms that control the number and placement of these exchanges. Most mammals exhibit sex-specific differences in recombination, and these are especially pronounced in the human. The rate of recombination is 1.6 times higher in human females than males [36], and the placement of exchanges differs, with more exchanges in distal regions in males. Furthermore, direct studies of prophase in humans provide evidence of considerable variation in recombination levels both within and among individuals of the same sex [21,37]. In the mouse, the availability of inbred strains can be exploited to examine the genetic control of recombination. The available data indicate that there is significant variation among inbred stains, that recombination levels are likely controlled by a few genes in the male, and that different control mechanisms likely operate in the female [38,39]. Although the strain-specific differences suggest that genetic factors are largely responsible for variation in recombination patterns, recent studies in mice provide evidence that environmental factors also can influence recombination. Exposure of pregnant females to low, environmentally relevant levels of the estrogenic chemical bisphenol A (BPA) (Box 3) disrupts prophase events in the fetal ovary [40]. Specifically, oocytes from treated females exhibit a high frequency of synaptic defects and increased levels and altered placement of crossovers. Importantly, these abnormalities herald subsequent errors in segregation, as a dramatic increase in meiotic aneuploidy is observed in oocytes from adult females exposed to BPA in utero [40]. This not only provides the first direct evidence of an environmental effect on recombination in mammals but raises concerns about the potential effects of environmental contaminants on human fertility. The follicle: developing a long-term relationship Before an oocyte can resume and complete the first meiotic division, it must undergo an extensive, hormone-dependent growth phase [41]. Oocyte growth and maturation
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Box 3. Bisphenol A Bisphenol A (BPA) is one of the highest volume chemicals produced, with >6 billion pounds manufactured annually. It is the major component of polycarbonate plastic, an additive in other forms of plastic, and a component of both resins used to line food and beverage containers and of some composites used in dentistry. Its presence in a wide variety of common household products (e.g. baby bottles, food storage containers, plastic wrap and canned foods and beverages) means that BPA is a part of everyday life. BPA has been shown to leach from food and beverage containers, and it is widely believed that the bulk of human exposure is through the ingestion of contaminated food and beverages. Recent studies, however, have shown that BPA contaminates our air, water and soil [66]. Human populations in many different countries in the developed world have now been screened for BPA, and detectable levels of the chemical have been found in human blood, urine, amniotic fluid, cord blood, breast milk and follicular fluid [66]. Although the levels of BPA vary widely among individuals within a population and among studies, analyses using the most sensitive detection methods suggest that the average level of BPA circulating in the blood of adults is in the 2- to 4-ng/ml range [66]. Unfortunately, these values provide no means of accurately determining current human exposure levels. A troubling aspect of the human BPA data are results of studies of pregnant women and their fetuses, which have reported high levels in some placental tissues and amniotic fluid samples. Indeed, in some cases, substantially higher levels were found in fetal tissues than in maternal serum. Studies in rodents suggest that the fetus is exquisitely sensitive to BPA; low-dose exposure has been reported to produce a variety of adverse reproductive effects, including morphological alterations of the reproductive tract, accelerated onset of puberty, disruption of estrus cycles and reduced sperm counts. These and other in vivo effects of BPA on laboratory rodents have recently been summarized in a review from an NIH-sponsored meeting [67].
occur in the ovary of the sexually mature female and require the coordinated actions of the oocyte and its somatic cell companions. Two distinct somatic cell compartments are required. Granulosa cells surround and are in direct contact with the oocyte and, together with the oocyte, comprise the follicle. The other key somatic cells in the ovary are the theca cells, which reside in the stromal tissue between follicles and, in conjunction with the granulosa cells, create an appropriate endocrine environment for oocyte growth and maturation. Forming follicles: a fetal event with direct relevance to reproductive lifespan Follicle formation occurs during the second trimester of human fetal development [42] and, in the mouse, immediately after birth [43]. The signal that triggers the formation of follicles is unknown. A sudden drop in estrogen levels (i.e. the loss of maternal estrogens at birth in the mouse or decreased availability of estrogens as a result of increased a fetal protein production in the human [44]) has been postulated to initiate the process. However, given the timing difference between humans and mice and the fact that the oocyte appears to regulate its own fate during follicle growth [45], it seems more likely that the signal comes from the oocyte and is linked to entry into dictyate arrest (Box 1). Despite our ignorance of the initiating event, the molecular control of follicle formation is beginning to be elucidated [46]. Although the specifics are beyond the scope of 89
Review this review, recent studies suggest that the packaging of oocytes into follicles can be perturbed by both genetic and environmental factors and that these perturbations might profoundly impact reproductive success. Before meiotic entry, primordial germ cells in the fetal ovary undergo a period of mitotic proliferation characterized by incomplete cytokinesis. This process creates groups of interconnected cells, called germ cell cysts, and these connections persist as oocytes enter meiosis [43]. Thus, the formation of a follicle requires two events: breakdown of intracellular bridges between oocytes and enclosure of individual oocytes by somatic cells (the precursors of granulosa cells). Intriguingly, recent data from studies of wildlife and experimental animals indicate that environmental contaminants can disrupt the process, resulting in the formation of follicles containing two or more oocytes (multioocyte or polyovular follicles) [47]. In alligators, the occurrence of multioocyte follicles in adult females exposed to environmental contaminants during fetal development is correlated with high levels of embryonic mortality [47]. In mice, neonatal exposure to both manmade and naturally occurring estrogenic substances increases the frequency of multioocyte follicles [44] and, as in alligators, a corresponding increase in embryo mortality has been reported [48]. Furthermore, there is evidence that multioocyte follicles might be selected against during oocyte growth [49]. Thus, the induction of multioocyte follicles might have reproductive repercussions by shortening the reproductive lifespan of the female and/or diminishing the quality of the oocytes she produces. Multioocyte follicles have been reported in a variety of species, including humans, [47,49], and available evidence suggests that environmental exposures are not the only precipitating event. In the mouse, defects in activin expression or signaling [50] and a variety of single gene mutations, including the growth factor genes bone morphogenetic protein 15 (Bmp15) [51] and growth differentiation factor-9 (Gdf9) [51] and the DM-domain encoding gene Dmrt4 [52], increase the frequency of multioocyte follicles. Recent studies of changes in mRNA and protein levels in response to neonatal estrogenic exposures showed suppression of the transforming growth factor b (TGF-b) superfamily member, activin [50]. Thus, although we are in the early stages of unraveling the molecular control of follicle development, understanding both activin and estrogen signaling and how these pathways are interrelated is key. In the interim, it is clear that follicle formation is a critical aspect of female fertility, and experimental studies raise concerns about the potential reproductive impact of environmental exposures during this critical developmental window. Oocyte atresia: programmed loss or a quality control mechanism? Around the time of follicle formation, a dramatic loss of oocytes, termed atresia, occurs in both humans and mice. In humans, it has been estimated that this loss reduces the pool of oocytes by >80% [42]. Because the pool of oocytes in the newborn ovary represents the reproductive resources of that female (Box 2), the amount of loss incurred influences the length of reproductive lifespan (i.e. the age at 90
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which reproductive senescence occurs in the adult). Because the period of atresia coincides with follicle formation, it has been likened to the programmed cell death that occurs in Drosophila melanogaster females with the breakdown of germline cysts [43]. However, in flies, only one of the 16 cells in each cyst is destined to become an oocyte, with the others serving as nurse cells. Because a separate somatic cell compartment – the granulosa cells – serve the comparable nurse cell function in the mammalian ovary, it is difficult to know how far the analogy can be stretched. Furthermore, as detailed above, there is compelling evidence that at least a portion of the cells is lost because of the actions of prophase quality control mechanisms. Nevertheless, because the reduction in oocyte pool during this period directly influences the reproductive lifespan of the female, it is clear that we need to better understand oocyte atresia. Oocyte growth and meiotic maturation: the final frontier The complex events of prophase set the stage for chromosome segregation when meiosis resumes in the adult ovary. The completion of the first meiotic division and ovulation of the mature egg is preceded by oocyte growth, a process that rivals prophase in complexity. The trigger that recruits an arrested oocyte into the pool of growing follicles remains unknown. Two things, however, are clear: first, most follicles that initiate growth die before ovulation. Second, growth is protracted. There is a tendency to think of oocyte maturation as the culmination of a single ovarian cycle, but the entire process – from initiation of growth to ovulation – takes an estimated 85 days in humans and 2 weeks in mice [14]. The first group of oocytes initiates growth in the sexually immature female but, in the absence of appropriate endocrine support, these follicles cannot complete growth and die. On sexual maturation, pituitary gonadotropin levels become sufficient to support the late stages of follicle development and ovulation. The intraovarian control of oocyte growth involves both the interplay of signals between the steroidogenic cells (the granulosa and theca cells) and intricate bi-directional communication between the oocyte and the granulosa cells. The process is complex, but the pivotal roles of both estrogen and members of the TGF-b superfamily are beginning to be elucidated [53,54]. From a meiotic standpoint, the late stages of oocyte growth are crucial, because the ability to resume and complete the first meiotic division is acquired by the oocyte in a stepwise fashion [41]. Although this implies that the oocyte is passively nursed along by its granulosa cells, recently it has become evident that, through the production of growth factors, the oocyte influences both the proliferation and differentiation of the granulosa cells. Thus, although communication is bi-directional, the oocyte clearly participates in the regulation of its own development [45]. The endocrine environment, maternal age and human aneuploidy The decade preceding menopause is marked by hormonal changes that influence the length of the menstrual cycle.
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Accordingly, it has been suggested that the changing endocrine environment underlies the age-related increase in human aneuploidy. Perhaps it would be easier to understand age-related aneuploidy if we understood menopause. Although increasing FSH levels are the first indicator of reproductive senescence, it remains unclear whether FSH increases in response to declining follicle numbers in the ovary or whether rising FSH levels actively diminish the follicle pool. Indeed, there is some support for both ideas [55]. Recent studies of transgenic mice expressing pituitary-independent human FSH through the rat insulin II promoter, however, provided evidence that rising FSH levels alone might accelerate female reproductive failure [55]. Regardless of whether the brain or the ovary initiates menopause, oocyte quality suffers. Direct studies of human oocytes from both infertility patients [56] and normal donors [57] have provided evidence of age-related changes in spindle formation and chromosome alignment. Furthermore, in the assisted reproductive setting, increasing maternal age is the most important impediment to success [58], because it is associated with both a decline in the rate of fertilization and an increase in the frequency of aneuploidy.
and must be maintained until anaphase. In addition, sister centromeres must be constrained to act in tandem at metaphase I. During the past several years, meiosisspecific cohesins that mediate these events have been identified [59]. We remain ignorant, however, of a critical piece of the puzzle: cohesion is established during DNA replication and presumably modified during the repair of DNA double-stand breaks that produce crossovers [60]. Thus, the protein complex essential for maintaining connections between chromosomes until the onset of anaphase is established in utero. Does this mean that chromosome segregation in the oocyte of a 45-year-old woman is reliant on 45-year-old proteins? Attempts were made to address this question when an age effect was discovered in females homozygous for a targeted disruption of the meiosisspecific cohesin, Smc1b [61]. These studies revealed transcription of meiotic cohesins during oocyte growth; however, whether new proteins are synthesized and become incorporated into the cohesin complex remains unknown. The answer is pivotal. Human age-related aneuploidy could reflect the degeneration of components of the cohesin complex caused by the lack of protein turnover, or it could reflect inadequate synthesis or incorporation of essential replacement proteins during oocyte growth.
Can genetic quality be influenced by the growth environment? How compelling are the data that changes in oocyte growth influence the segregation of chromosomes in meiosis? At the beginning of oocyte growth, homologous chromosomes are joined together, but the cell is not yet capable of undergoing metaphase I. Thus, the above question is best considered in two parts: how might growth affect the connections between chromosomes and how might growth influence the metaphase machinery?
Oocyte growth and the meiotic cell cycle During the final stages of growth, changes occur in both the organization of the chromatin and the microtubule network in the egg, and these changes coincide with the acquisition of meiotic competence [41]. In addition, both spindle assembly and the formation of functional kinetochores occur at the time of nuclear envelope breakdown. Is there compelling evidence that disturbances in growth can affect these processes and lead to malsegregation at either meiotic division? Studies designed to test the effects of altered growth on meiotic chromosome segregation in the mouse provided evidence of disturbances in spindle formation and chromosome alignment. Importantly, effects were evident in several different mouse mutants, and defects observed at MI were correlated with increases in aneuploidy in metaphase II arrested eggs [62].
Oocyte growth and chromosomal connections In contrast to mitosis, meiosis requires highly specialized chromosomal connections (Figure 2). The connections between homologs (chiasmata) forged during prophase in the fetal ovary are essential for homolog segregation during MI
Figure 2. Meiosis necessitates specialized chromosome connections. The unique features of meiotic chromosomes are shown in these images of mouse (a) mitotic, (b) meiosis I (MI) and (c) MII chromosomes. Chromosomes have been immunostained with an antibody to CENP-E to highlight the kinetochores (red) and counterstained with DAPI (blue) [68]. During mitotic division (a), kinetochores form at the centromeres of both sister chromatids (arrows), and cohesion at the centromere and along the chromosome arms keeps sister chromatids physically associated until anaphase. Degradation of the cohesin complex at anaphase allows the sisters to move to opposite poles. During MI metaphase (b), meiosis-specific cohesins perform two distinct functions: (i) they constrain the sister chromatids of an individual chromosome (arrows) to act in unison at the first division, so that their kinetochores are captured by microtubules from the same spindle pole, and (ii) they maintain a physical connection between homologs distal to the site of a crossover (arrowhead). At anaphase I, loss of cohesion at the sites of exchanges allows homologs to segregate. At metaphase II (c), the tight association between the kinetochores of sister chromatids (arrows) and cohesion along the chromosome arms have been lost. The retention of some cohesion at the centromere, however, is essential to maintain a connection between sister chromatids until anaphase II. Figure adapted, with permission, from Ref. [68]. Left and center images reprinted with permission from Chromosoma.
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Review Perhaps the most worrying evidence that changes during the final stages of oocyte growth can impact the genetic quality of the oocyte comes from studies of mice accidentally exposed to the estrogenic chemical, BPA. In the course of meiotic studies, a sudden change in meiotic chromosome alignment and segregation was observed in control animals. These effects proved to be the result of BPA exposure owing to inadvertent damage to the polycarbonate caging materials, and subsequent studies showed that low-dose exposures during the final stages of oocyte growth were sufficient to elicit the meiotic defects [63]. Although the data linking BPA to reproductive defects in humans remain equivocal [64,65], it is clear that current human exposure falls within levels that elicit effects in mice [66,67]. Concluding remarks Aneuploidy is the leading cause of reproductive failure and congenital birth defects in humans. The strong association between maternal age and trisomy, coupled with changing reproductive trends that have steadily increased the maternal age at first birth, suggests that the aneuploidy problem is not going to diminish. In this review, we tried to dispel the notion that there is a single cause of human nondisjunction or a single stage of oocyte development when chromosomes become destined to nondisjoin. Instead, we argue that events during at least three distinct stages of mammalian oogenesis have the potential to influence the genetic quality of the egg. We suggest that the interplay of these factors accounts for the complex etiology of human aneuploidy and explains why no single hypothesis has been able to adequately explain the effect of maternal age. We included a discussion of the potential impact of environmental factors on each of the three stages. The endocrine disrupting chemical bisphenol A has been used as an example because studies in mice suggest that it can adversely affect events at each stage. Our intent in summarizing these bisphenol A studies is not to focus attention on this chemical, but rather to use bisphenol A as a ‘poster child’ to make the point that environmental factors that contribute to human aneuploidy might well exist. Given the complexity of oogenesis, providing definitive evidence that environmental effects contribute to human chromosome abnormalities will be difficult indeed. Nevertheless, the clinical importance of any such association makes this an essential consideration in future studies. Acknowledgements We thank Smith Lutu and Crystal Lawson for help with the figures. This work was supported by National Institutes of Health Grants HD37502 and ES013527 (P.A.H.) and HD21341 (T.J.H.).
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