Dynamic changes of DNA epigenetic marks in mouse oocytes during natural and accelerated aging

Aging is a complex time-dependent biological process that takes place in every cell and organ, eventually leading to degenerative changes that affect normal biological functions. In the past decades, the number of older parents has increased significantly. While it is widely recognized that oocyte aging poses higher birth and reproductive risk, the exact molecular mechanisms remain largely elusive. DNA methylation of 5-cytosine (5mC) and histone modifications are among the key epigenetic mechanisms involved in critical developmental processes and have been linked to aging. However, the impact of oocyte aging on DNA demethylation pathways has not been examined. The recent discovery of Ten-Eleven-Translocation (TET) family proteins, thymine DNA glycosylase (TDG) and the demethylation intermediates 5hmC, 5fC and 5caC has provided novel clues to delineate the molecular mechanisms in DNA demethylation. In this study, we examined the cellular level of modified cytosines (5mC, 5hmC, 5fC and 5caC) and Tet/Tdg expression in oocytes obtained from natural and accelerated oocyte aging conditions. Here we show all the DNA demethylation marks are dynamically regulated in both aging conditions, which are associated with Tet3 over-expression and Tdg repression. Such an aberrant expression pattern was more profound in accelerated aging condition. The results suggest that DNA demethylation may be actively involved in oocyte aging and have implications for development of potential drug targets to rejuvenate aging oocytes.This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

Oocyte aging is controlled by mitogen‐activated protein kinase signaling

Oogenesis is one of the first processes to fail during aging. In women, most oocytes cannot successfully complete meiotic divisions already during the fourth decade of life. Studies of the nematode Caenorhabditis elegans have uncovered conserved genetic pathways that control lifespan, but our knowledge regarding reproductive aging in worms and humans is limited. Specifically, little is known about germline internal signals that dictate the oogonial biological clock. Here, we report a thorough characterization of the changes in the worm germline during aging. We found that shortly after ovulation halts, germline proliferation declines, while apoptosis continues, leading to a gradual reduction in germ cell numbers. In late aging stages, we observed that meiotic progression is disturbed and crossover designation and DNA double‐strand break repair decrease. In addition, we detected a decline in the quality of mature oocytes during aging, as reflected by decreasing size and elongation of interhomolog distance, a phenotype also observed in human oocytes. Many of these altered processes were previously attributed to MAPK signaling variations in young worms. In support of this, we observed changes in activation dynamics of MPK‐1 during aging. We therefore tested the hypothesis that MAPK controls oocyte quality in aged worms using both genetic and pharmacological tools. We found that in mutants with high levels of activated MPK‐1, oocyte quality deteriorates more rapidly than in wild‐type worms, whereas reduction of MPK‐1 levels enhances quality. Thus, our data suggest that MAPK signaling controls germline aging and could be used to attenuate the rate of oogenesis quality decline.     

Epigenetic clock and methylation study of oocytes from a bovine model of reproductive aging

Cattle are an attractive animal model of fertility in women due to their high degree of similarity relative to follicle selection, embryo cleavage, blastocyst formation, and gestation length. To facilitate future studies of the epigenetic underpinnings of aging effects in the female reproductive axis, several DNA methylation‐based biomarkers of aging (epigenetic clocks) for bovine oocytes are presented. One such clock was germane to only oocytes, while a dual‐tissue clock was highly predictive of age in both oocytes and blood. Dual species clocks that apply to both humans and cattle were also developed and evaluated. These epigenetic clocks can be used to accurately estimate the biological age of oocytes. Both epigenetic clock studies and epigenome‐wide association studies revealed that blood and oocytes differ substantially with respect to aging and the underlying epigenetic signatures that potentially influence the aging process. The rate of epigenetic aging was found to be slower in oocytes compared to blood; however, oocytes appeared to begin at an older epigenetic age. The epigenetic clocks for oocytes are expected to address questions in the field of reproductive aging, including the central question: how to slow aging of oocytes.     

Evaluation of Follicular Synchronization Caused by Estrogen Administration and Its Reproductive Outcome

To evaluate multiple follicular development synchronization after estrogen stimulation in prepubertal mice, follicular responsiveness to gonadotropin superovulation, the prospective reproductive potential and ovarian polycystic ovary syndrome (PCOS)-like symptoms at adulthood, prepubertal mice were intraperitoneally injected with estrogen to establish an animal model with solvent as control. When synchronized tertiary follicles in ovaries, in vitro oocyte maturation and fertilization rates, blastocyst formation rate, developmental potential into offspring by embryo transfer, adult fertility and PCOS-like symptoms, and involved molecular mechanisms were focused, it was found that estrogen stimulation (10μg/gBW) leads to follicular development synchronization at the early tertiary stage in prepubertal mice; reproduction from oocytes to offspring could be realized by means of the artificial reproductive technology though the model mice lost their natural fertility when they were reared to adulthood; and typical symptoms of PCOS, except changes in inflammatory pathways, were not remained up to adulthood. So in conclusion, estrogen can lead to synchronization in follicular development in prepubertal mice, but does not affect reproductive outcome of oocytes, and no typical symptoms of PCOS remained at adulthood despite changes related to inflammation.     

Mitochondrial dysfunction in reproduction

The mitochondrial genome passes from one generation to the next by way of the egg's cytoplasm, so ordinarily an individual's mitochondrial DNA (mtDNA) is entirely derived from his or her mother. A potential mother has a finite number of eggs, or oocytes, all of which were formed when she herself was still a fetus, many years before she can conceive. The eggs are progressively depleted through childhood and her reproductive years at a much faster rate than is accounted for by ovulation. Up to a decade before the ultimate depletion of ovarian follicles (and hence oocytes) at or soon after menopause, cytoplasmic senility of the remaining eggs leads to physiological sterility; a phenomenon that is suspected of being mitochondrially based and has been termed the oopause. When ovulation and conception occur, oxidative phosphorylation and other mitochondrial functions of the fertilized oocyte are thought to be essential to the early embryo well before it implants in the uterus. The competition between follicles to deliver the oocyte that will be fertilized and which will found a new generation could also be mitochondrially based, but the mechanism remains to be elucidated. Increasing experience with the culture of human embryos in vitro is highlighting the importance of mitochondrial metabolism generally, and the avoidance of excessive generation of reactive oxygen species in particular. Paradoxes abound in the experimental data, however. Although natural selection operates on mitochondria only in females (and in extreme cases through the survival of their offspring), reproductive disturbance from mitochondrial mutations is most obvious in males, who typically have reduced sperm motility. mtDNA point mutations such as T8993G, which is serious enough to cause the death of infants from Leigh disease in the first few years of life, can carry through the female germ line apparently unhindered; yet mtDNA deletions that cause a less severe phenotype, and which typically manifest at a later age, are effectively blocked from transmission to offspring--a phenomenon in accord with early experimental observations that deleted mtDNA species are less common in cleaving embryos than in unselected preovulatory oocytes. A mitochondrial basis for ooplasmic aging has not been convincingly established, but the novel IVF-based practice of micro-aspiration and transfer of ooplasm from younger eggs to older eggs, which includes the transfer of mitochondria, appears in preliminary studies to have some clinical efficacy in rejuvenating fertility in older women.     

Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model

Cryopreservation of female reproductive cells allows preservation of fertility and provides materials for research. Although freezing protocols have been optimized, and there is a high survival rate after thawing, the in vitro fertilization (IVF) pregnancy rate is still lower in cycles with cryopreserved oocytes, thus highlighting the importance of identifying intrinsic limiting factors characterizing the cells at time of freezing. The aim of the present study is to investigate in the mouse model the impact of reproductive aging and postovulatory aging on oocyte biological competence after vitrification. Metaphase II oocytes were vitrified soon after retrieval from young and reproductively old mice. Part of the oocytes from young animals was vitrified after 6 h incubation (in vitro aged oocytes). All classes of oocytes showed similar survival rate after vitrification. Moreover, vitrification did not alter chromosomal organization in young cells, whereas in vitro aged and old oocytes presented an increase of slightly aberrant metaphase configurations. Compared to fresh young oocytes, in vitro aged and old oocytes showed increased ROS levels which remained unchanged after vitrification. By contrast, cryopreservation significantly increased ROS production in young oocytes. Both the aging processes negatively impacted oocyte ability to undergo pronucleus formation and first cleavage after vitrification by stimulating cellular fragmentation. These results could be helpful for establishing the correct time table for cryopreservation in the laboratory routine and improving its application in reproductively old females. Moreover, our observations highlight the importance of oxidative stress protection during vitrification procedures.     

The mare model for follicular maturation and reproductive aging in the woman

Reproductive aging and assisted reproduction are becoming progressively more relevant in human medicine. Research with human subjects is limited in many aspects, and consequently animal models may have considerable utility. Such models have provided insight into follicular function, oocyte maturation, and reproductive aging. However, models are often selected based on factors other than physiological or functional similarities. Although the mare has received limited attention as a model for reproduction in women, comparisons between these species indicate that the mare has many attributes of a good model. As the mare ages, cyclic and hormonal changes parallel those of older women. The initial sign of reproductive aging in both species is a shortening of the reproductive cycle with elevated concentrations of FSH. Subsequently, cycles become longer with intermittent ovulations and elevated concentrations of FSH and LH. Reproduction ceases with failure of follicular growth and elevated gonadotropins, apparently because of ovarian failure. In the older woman and mare, oocytes have been maintained in meiotic arrest for decades -- approximately four to five for the woman and two to three for the mare; in both species, reduced oocyte quality is the end factor identified in age-associated infertility. After induction of oocyte maturation in vivo, the timeline to ovulation is the same for the mare and woman, suggesting a comparable sequence of events. The mare's anatomy, long follicular phase and single dominant follicle provide a foundation for studies in oocyte and follicular development. The aim of this review is to evaluate the mare as an animal model to study age-associated changes in reproduction and to improve our understanding of oocyte and follicular maturation in vivo.     

Advancing maternal age predisposes to mitochondrial damage and loss during maturation of equine oocytes in vitro

In many mammalian species, reproductive success decreases with maternal age. One proposed contributor to this age-related decrease in fertility is a reduction in the quantity or functionality of mitochondria in oocytes. This study examined whether maternal age or (in vitro maturation). IVM affect the quantity of mitochondria in equine oocytes. Oocytes were collected from the ovaries of slaughtered mares categorized as young (<12 years) or aged (≥12 years) and either denuded and prepared for analysis immediately (not-IVM) or matured in vitro for 30 hours before preparation (IVM). The mean oocyte mitochondrial DNA copy number was estimated by quantitative polymerase chain reaction and found to be significantly lower in oocytes from aged mares and that had been subjected to IVM than in any other group. Transmission electron microscopy demonstrated that mitochondria in aged mare oocytes subjected to IVM experienced significantly more swelling and loss of cristae than in other groups. We conclude that maternal aging is associated with a heightened susceptibility to mitochondrial damage and loss in equine oocytes, which manifests during IVM. This predisposition to mitochondrial degeneration probably contributes to reduced fertility in aged mares.     

Effects of maternal age on oocyte developmental competence

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.     

Biological aging and the etiology of aneuploidy

A prevalent hypothesis concerning the cause of the rise in aneuploid conceptions with maternal age is that the changes that accompany normal ovarian aging increase the rate of meiotic errors in the oocyte. Biological aging of the ovary is accompanied by a decline in both the total oocyte pool and the number of antral follicles maturing per cycle, as well as changes in the levels of circulating reproductive hormones. The biological aging hypothesis predicts that aneuploidy rates should be higher in women with a prematurely reduced oocyte pool, and that women with trisomic conceptions should show signs of earlier ovarian aging than women of the same chronological age without trisomic conceptions. Comprehensive studies of aneuploidy in groups of women with known causes of premature ovarian failure remain to be done, though anecdotal evidence does suggest increased rates of pregnancy loss and aneuploidy. Smoking, which is a well-documented cause of earlier ovarian aging, is not associated with an increase in aneuploid conceptions. Evidence from women with unilateral ovariectomies is inconsistent. Support for the biological aging hypothesis was provided by one study showing that menopause occurred about a year earlier in women with a trisomic spontaneous abortion compared to women with chromosomally normal conceptions. Associations between high FSH and pregnancies with Down syndrome and chromosomally abnormal spontaneous abortions have also been reported. However, the most direct test of the hypothesis, which compared antral follicle counts and hormonal levels in women with trisomic pregnancies and those with chromosomally normal pregnancies, failed to find a difference in the expected direction. A prospective study of FSH levels in women with subfertility also failed to find an association with the rate of pregnancy loss. The bulk of evidence thus suggests that, if the processes of biological aging are indeed related to aneuploidy, they probably involve factors other than those measured by oocyte or antral follicle pool size and reproductive hormone levels.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

Haploinsufficiency of the follicle-stimulating hormone receptor accelerates oocyte loss inducing early reproductive senescence and biological aging in mice

Female mice that are null for the FSH-receptor (FSH-R) gene are estrogen deficient, acyclic, and sterile. However, the heterozygous (+/-) mice initially have reduced fertility and stop breeding by 7-9 mo. The purpose of this study was to understand the basis of reduced fertility in mice with haploinsufficiency of the FSH-R. Heterozygous females were compared to +/+ females at 3, 7, and 12 mo of age. By 7 mo most of the +/- females were acyclic and <50% delivered pups. The wild-type females were normal in these respects. None of the 1-yr-old +/- females gave viable offspring (73% in +/+). Many degenerative changes, including atresia and apoptosis, and profound loss of oocytes, were apparent in +/- mice by 7 mo. The 1-yr-old +/- ovary had very few follicles and consisted mostly of fibroid tissue and cysts. Our data support the hypothesis that reproductive deficits in +/- FSH-R mice occur because of accelerated oocyte loss due to increased cell death in the ovary. These events contribute to early reproductive senescence and biological aging in mice. Thus FSH-R status is an important determinant of ovarian aging and all phenomena that arise from subsequent estrogen deficiency and other aberrations.     

Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns

It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.     

Maternal age effect on early human embryonic development and blastocyst formation

In humans, age-related decline in female fertility can be explained by a reduction in quality either of the older uterus or of the embryos arising from aging oocytes. The aim of this study was to examine the latter hypothesis, using in vitro fertilization (I.V.F.) and coculture of embryos until the blastocyst stage. We determined the blastocyst formation rate ([expanded blastocysts/blastocysts]*100) according to the patient's age the day of I.V.F. With increase in age, the number of retrieved oocytes decreased, without alteration of the cleavage rate. In patients above age 30 years, preimplantation development to blastocysts declined due to an increase in embryo arrest at the morula stage. If blastocyst stage was reached, a negative linear relationship between blastocyst expansion rate and patient age was observed. Drops in gamete production and embryo development with increasing age led to a drastic decrease in patients having at least one expanded blastocyst (<30 years, 82%; ≥40 years, 36%). A high delivery rate per oocyte retrieval (25.8%) was observed in patients above age 40 years after embryo transfer at the blastocyst stage. These results give a clear indication of decline in the quality of human embryos arising from aging oocytes. The origin of this alteration is discussed in terms of chromosome abnormalities, role of maternally-inherited products from the oocyte, timing of genomic activation, and temporal pattern of gene expression during initial development of the human embryo. © 1996 Wiley-Liss, Inc.     

Maturation/M-phase promoting factor regulates aging of porcine oocytes matured in vitro

Control of oocyte aging during manipulation of matured oocytes should have advantages for recently developed reproductive technologies, such as cloning after nuclear transfer. We have shown that the enhanced activation ability and fragmentation of porcine in vitro matured and aged oocytes bore a close relationship to the gradual decrease in maturation/M-phase promoting factor (MPF) activity and that porcine aged oocytes contained plenty of MPF, but it was in an inactive form, pre-MPF, as a result of phosphorylation of its catalytic subunit p34(cdc2) and, therefore, had low MPF activity. We incubated porcine oocytes with vanadate and caffeine, which affected the phosphorylation status and MPF activity, and evaluated their activation abilities and fragmentation frequencies. Incubation of nonaged oocytes with vanadate increased p34(cdc2) phosphorylation and reduced MPF activity to levels similar to those of aged oocytes and increased their parthenogenetic activation and fragmentation rates compared with those of the control oocytes. Conversely, treating aged oocytes with caffeine reduced p34(cdc2) phosphorylation and increased MPF activity. These oocytes showed significantly lower parthenogenetic activation and fragmentation rates than aged mature oocytes. These results suggest that MPF activity is a key mechanism of oocyte aging and controlling MPF activity by altering p34(cdc2) phosphorylation with these chemicals may enable oocyte aging to be manipulated in vitro. We expect those ideas will be applied practically to pig cloning.     

A delay in age at first mating results in the loss of future reproductive potential via apoptosis

Summary Women who delay childbearing risk subfertility. However, this loss of fertility is not a simple function of aging. Women who have had children early in life tend to maintain fertility longer, measured as a later age at menopause. But why should otherwise healthy women lose reproductive capacity? Loss of fertility independent of senescence, menopause, has been approached from two perspectives: evolution and development. Evolutionary biologists focus on how natural selection favors survival after reproductive ability has ceased, whereas reproductive biologists examine mechanisms by which women lose fertility with age and factors that influence the rate of reproductive aging. Combining mechanistic studies with evolutionary theory should allow us to define principles of the evolution of postembryonic development of ovaries, including the role of reproductive timing relative to sexual maturation. Achieving this will require identifying appropriate, and more experimentally tractable, taxa in which to study how early reproductive events influence lifetime fertility. We work with an invertebrate species, the cockroach Nauphoeta cinerea, with a complex reproductive biology in which females experience reproductive cycles, give live birth, and show age‐related decline in fertility. Thus, N. cinerea provides an opportunity to use an experimental approach to examine mechanisms by which females lose reproductive potential as they delay reproduction. Our results demonstrate that the loss of both oocytes ready for fertilization and future oocytes in females that delay mating is because of apoptosis. We suggest that loss of fertility because of delayed mating may originate in a nonadaptive response in control of apoptosis through mistiming of reproduction.     

Mitochondrial DNA mutation exacerbates female reproductive aging via impairment of the NADH/NAD+ redox

Mammals' aging is correlated with the accumulation of somatic heteroplasmic mitochondrial DNA (mtDNA) mutations. Whether and how aging accumulated mtDNA mutations modulate fertility remains unknown. Here, we analyzed oocyte quality of young (≤30 years old) and elder (≥38 years old) female patients and show the elder group had lower blastocyst formation rate and more mtDNA point mutations in oocytes. To test the causal role of mtDNA point mutations on infertility, we used polymerase gamma (POLG) mutator mice. We show that mtDNA mutation levels inversely correlate with fertility, interestingly mainly affecting not male but female fertility. mtDNA mutations decrease female mice's fertility by reducing ovarian primordial and mature follicles. Mechanistically, accumulation of mtDNA mutations decreases fertility by impairing oocyte's NADH/NAD⁺ redox state, which could be rescued by nicotinamide mononucleotide treatment. For the first time, we answer the fundamental question of the causal effect of age‐accumulated mtDNA mutations on fertility and its sex dependence, and show its distinct metabolic controlling mechanism.