Postovulatory Oocyte Aging Research Articles

In vitro postovulatory oocyte aging affects H3K9 trimethylation in two-cell embryos after IVF

BackgroundThe physiological time axis of oocyte maturation comprises highly sensitive processes. A prolonged time span between ovulation and fertilization may impair oocyte developmental competence and subsequent embryo development, possibly due to epigenetic modifications. Since post-translational histone modifications can modify chromatin activity, and trimethylation of H3K9 (H3K9me3) has been shown to increase in the murine oocyte during maturation, here the effect of postovulatory oocyte aging on H3K9me3 was analyzed. MethodsThe competence of murine oocytes which were aged for 2, 4, 6 and 8 h in vitro after oocyte retrieval to develop to the two-cell and blastocyst stage was determined. Degree of H3K9me3 was analyzed in the postovulatory aged oocytes as well as in the resulting two-cell embryos after IVF. ResultsThe current study shows that postovulatory aging of oocytes for up to eight hours after oocyte retrieval exhibited no effect on two-cell embryo and blastocyst rate; however, changes in H3K9me3 in the resulting two-cell embryos were observed. ConclusionProlonged postovulatory oocyte aging leads to epigenetic modifications of H3K9. Such modifications may affect the developmental capacity of embryos at post-implantation developmental stages.

Coenzyme Q10 ameliorates the quality of postovulatory aged oocytes by suppressing DNA damage and apoptosis

Postovulatory aging is known to compromise the oocyte quality as well as subsequent embryo development in many different animal models, and becomes one of the most intractable issues that limit the outcome of human assisted reproductive technology (ART). However, the strategies to prevent the deterioration of aged oocytes and relevant mechanisms are still underexplored. Here, we find that supplementation of CoQ10, a natural antioxidant present in human follicular fluids, is able to restore the postovulatory aging-induced fragmentation of oocytes and decline of fertilization. Importantly, we show that CoQ10 supplementation recovers postovulatory aging-caused meiotic defects such as disruption of spindle assembly, misalignment of chromosome, disappearance of actin cap, and abnormal distribution patterns of mitochondria and cortical granules. In addition, CoQ10 protects aged oocytes from premature exocytosis of ovastacin, cleavage of sperm binding site ZP2, and loss of localization of Juno, to maintain the fertilization potential. Notably, CoQ10 suppresses the aging-induced oxidative stress by reducing the levels of superoxide and DNA damage, ultimately inhibiting the apoptosis. Taken together, our findings demonstrate that CoQ10 supplementation is a feasible and effective way to prevent postovulatory aging and preserve the oocyte quality, potentially contributing to improve the successful rate of IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection) during human ART.

Absence of mitochondrial DNA methylation in mouse oocyte maturation, aging and early embryo development

Mitochondrial DNA (mtDNA) is important for oxidative phosphorylation; dysfunctions can play a role in many mitochondrial diseases and can also affect the aging of cells and individuals. DNA methylation is an important epigenetic modification that plays a critical role in regulating gene expression. While recent studies have revealed the existence of mtDNA methylation there are still controversies about mtDNA methylation due to the special structure of mtDNA. Mitochondria and DNA methylation are both essential for regulating oocyte maturation and early embryo development, but whether mtDNA methylation changes during this process is unknown. By employing bisulfite sequencing, we found that in the process of mouse oocyte maturation, postovulatory oocyte aging, and early embryo development, all analyzed mitochondrial genes, including 16S-CpGI, DCR, ND6, 12S, and ATP8, lacked 5’mC. Thus, mtDNA methylation does not occur in the oocyte and early embryo.

Overgrowth of mice generated from postovulatory-aged oocyte spindles.

Oocyte spindle transfer (OST) is a potent reproductive technology used for mammals that enables the spindle in a deteriorated oocyte at the metaphase of the second meiotic division (MII) to serve as the genetic material for producing descendants. However, whether postnatal growth is achieved via OST using developmentally deteriorated MII oocytes remains unclear. At 16 h after human chorionic gonadotropin administration, denuded MII oocytes immediately after retrieval from oviducts (0 h‐oocytes) were used for in vitro fertilization (IVF) as controls. For IVF using postovulatory‐aged oocytes, the 0 h‐oocytes were further incubated for 12 h and 24 h (12 h‐ and 24 h‐oocytes). These mouse oocytes served as a model for assessing the postnatal growth of individuals produced via OST from developmentally deteriorated oocytes. The embryos from 12 h‐ and 24 h‐oocyte spindles exhibited high rates of development up to the neonatal stage as good as the non‐manipulated controls. However, the mice derived from the 24 h‐oocyte spindles displayed heavier body weights and greater feed consumption than both controls and mice derived from 12 h‐oocyte spindles. Our results demonstrate the feasibility of OST as a potent reproductive technology and its limitation in the use of excessively aged postovulatory oocytes in mammalian reproduction.

N-acetyl-L-cysteine (NAC) delays post-ovulatory oocyte aging in mouse.

The quality of post-ovulatory oocytes decreases with aging. In this study, we aimed to investigate the effects of N-acetyl-L-cysteine (NAC), a broadly used antioxidant, on oocyte quality in mouse post-ovulatory oocyte aging in vitro. NAC at 0.6mM concentration was added to culture medium (M2), and the quality of oocytes was analyzed at 6h, 12h, 18h and 24h of culture. We found that the frequency of spindle defects decreased in NAC-treated oocytes compared to those without NAC treatment. NAC treatment significantly decreased abnormal distribution of cortical granules (CGs) in oocytes during aging for 18h and 24h. Decreased intracellular reactive oxygen species (ROS) was also observed. Increased intracellular ATP levels and decreased abnormal distribution of mitochondria could be observed with NAC supplementation during post-ovulatory oocyte aging in vitro. These results indicate that NAC will maintain the quality of oocytes, and delay post-ovulatory oocyte aging as studied in the mouse.

Evaluation of gamete quality indicators for Arctic charr Salvelinus alpinus

The reproductive performance of farmed Arctic charr exhibits substantial individual variation. The causes behind this variation have not yet been properly explained, despite the species relatively long history in aquaculture. The aim of the present study was to provide better understanding of the issue by evaluating parent and gamete traits in relation to fertility and offspring viability under routine hatchery conditions. Are losses mainly due to failed fertilisation or mortality? Are maternal or paternal factor more predominant? The study covered sex hormone status of the broodstock, egg traits (egg size and wateriness of egg batches), milt traits (volume, density, and sperm motility characteristics), and symmetry of early cell division.We found that the majority of offspring loss occurred as embryo mortality, although a significant additional loss could be assigned fertilisation failure. Fertilisation and mortality rates were not inter-correlated. Neither of the evaluated egg traits, egg size and wateriness of newly stripped egg batches, proved to be useful indicators of egg quality. With regard to sperm traits, milt density as well as several computer assisted sperm analysis (CASA) parameters, i.e. swimming speed (VAP, VSL, and VCL) and beat cross frequency (BCF), were correlated with reproductive performance and could potentially be used as quality indicators for Arctic charr. Together, male 17,20β-P levels and BCF explained 69.5% of the between male variation in proportion of successfully eyed eggs.The results support previous indications that post-ovulatory aging of oocytes is contributing to the impairment of reproductive performance in farmed Arctic charr. In addition, a significant paternal factor to this impairment has been exposed. However, the cause and mechanism involved are still far from clear.

Putrescine delays postovulatory aging of mouse oocytes by upregulating PDK4 expression and improving mitochondrial activity.

If fertilization does not occur for a prolonged period in vivo or in vitro, the postovulatory oocytes will deteriorate, which called the postovulatory aging. This process disrupts the developmental competence. In the present study, we showed that the reactive oxygen species (ROS) was accumulated in oocytes during the postovulatory aging. ROS inhibited Sirt1 expression, and then increased oxidative stress by downregulating the intracellular Sirt1-FOXO3a-SOD2 axis. Moreover, the inhibited Sirt1 expression was related to the decreased mitochondrial function and the lowered level of autophagy. The mitochondrial-related apoptosis was increased by inhibiting the AKT and ERK1/2 pathways, due to the accumulation of ROS in the postovulatory oocytes. The mitochondrial pyruvate dehydrogenase kinase-4 (PDK4) can reduce ROS by inhibiting the tricarboxylic acid (TAC) cycle. We found that PDK4 was significantly decreased in the postovulatory aging oocytes. Putrescine, one of the abundant biogenic amines, ameliorated the effects of ROS and therefore improved the quality of the postovulatory aging oocytes by increasing the expression of PDK4. When PDK4 was downregulated using siRNAs, the effects of putrescine were significantly receded. We concluded that putrescine delayed the aging process of postovulatory oocytes by upregulating PDK4 expression and improving mitochondrial activity.

Destabilization of spindle assembly checkpoint causes aneuploidy during meiosis II in murine post-ovulatory aged oocytes

Mammalian oocyte quality degrades over time after ovulation in vitro, which can cause fatal defects such as chromosomal aneuploidy. As various oocyte manipulations employed in assisted reproductive technology are time consuming, post-ovulatory aging is a serious problem to overcome in reproductive medicine or ova research. In this study, we investigated the effects of postovulatory aging on the incidence of chromosome aneuploidy during meiosis II, with a focus on the expression of functional proteins from the spindle assembly checkpoint (SAC). Chromosome analysis was used to assess the rate of aneuploidy in in vitro aged oocytes, or in early embryos derived from aged oocytes. Immunofluorescent staining was used to detect the localization of MAD2, which is a SAC signal that monitors the correct segregation of sister chromatids. Immunoblotting was used to quantify cohesin subunits, which are adhesion factors connecting sister chromatids at the metaphase II (MII) centromere. It was shown that post-ovulatory oocyte aging inhibits MAD2 localization to the sister kinetochore. Furthermore, oocyte aging prevented cohesin subunits from being maintained or degraded at the appropriate time. These data suggest that the destabilization of SAC signaling causes sister chromatid segregation errors in MII oocytes, and consequently increases the incidence of aneuploidy in early embryos. Our findings have provided distinct evidence that the post-ovulatory aging of oocytes might also be a risk factor for aneuploidy, irrespective of maternal age.

Putrescine delays postovulatory aging of mouse oocytes by regulating PDK4 expression and mitochondrial activity

Putrescine delays postovulatory aging of mouse oocytes by regulating PDK4 expression and mitochondrial activity

Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes.

Although previous studies indicated that cumulus cells (CCs) accelerate oocyte aging by releasing soluble factors, the factors have yet to be characterized. While demonstrating that CCs promoted oocyte aging by releasing soluble Fas ligand (sFasL), our recent study suggested that CCs might secrete other factors to mediate oocyte aging as well. This study tested whether CCs accelerate oocyte aging by secreting tumor necrosis factor (TNF)-α. The results showed that mouse CCs undergoing apoptosis released soluble TNF-α (sTNF-α) during in vitro aging. While ethanol activation rates were higher, the maturation-promoting factor (MPF) activity was lower significantly after culture of cumulus-denuded oocytes (DOs) in medium conditioned with CCs for 36 h than in medium conditioned for 24 h. Aging mouse oocytes expressed TNF-receptor 1. The CCs released equal amounts of sTNF-α and sFasL during aging in vitro, and the TNF-α-knockdown CCs secreted less sFasL than the control CCs did. Treatment of DOs in vitro with sTNF-α significantly accelerated their aging. The aging-promoting effect of sTNF-α was significantly reduced in TNF-α-knocked-down CCs and in CCs from the TNF-α-knockout mice. It is concluded that mouse CCs accelerate oocyte aging by secreting sTNF-α as well as sFasL.

Resveratrol increases resistance of mouse oocytes to postovulatory aging in vivo.

After ovulation, metaphase II oocytes undergo a time-dependent deterioration in vivo or in vitro, which is referred to as postovulatory oocyte aging, a process during which a series of deleterious molecular and cellular changes occur. In this study, we found that short-term injection of resveratrol (3,5,4'-trihydroxystilbene) effectively ameliorated oxidative stress-induced damage in postovulatory oocyte aging of middle-aged mice in vivo. Resveratrol induced changes that delayed the aging-induced oocyte deterioration including the elevated expression of the anti-aging molecule Sirtuin 1 (SIRT1); it reduced intracellular reactive oxygen species (ROS) level, and improved mitochondria function. In addition, these beneficial changes may also help to prevent apoptosis. Taken together, our data suggest that resveratrol can effectively protect against postovulatory oocyte aging in vivo primarily by preventing ROS production.

Melatonin attenuates postovulatory oocyte dysfunction by regulating SIRT1 expression.

The quality of postovulatory metaphase II oocytes undergoes a time-dependent deterioration as a result of the aging process. Melatonin is considered to be an anti-aging agent. However, the underlying mechanisms of how melatonin improves the quality of postovulatory aged oocytes remain largely unclear. In this study, by using mouse model, we found that there were elevated reactive oxygen species levels and impaired mitochondrial function demonstrated by reduced mitochondrial membrane potential and increased mitochondrial aggregation in oocytes aged 24 h, accompanied by an increased number of meiotic errors, unregulated autophagy-related proteins and early apoptosis, which led to decreased oocyte quality and disrupted developmental competence. However, all of these events can be largely prevented by supplementing the oocyte culture medium with 10-3 M melatonin. Additionally, we found that the expression of sirtuin family members (SIRT1, 2 and 3) was dramatically reduced in aged oocytes. In addition, in vitro supplementation with melatonin significantly upregulated the expression of SIRT1 and antioxidant enzyme MnSOD, but this action was not observed for SIRT2 and SIRT3. Furthermore, the protective effect of melatonin on the delay of oocyte aging vanished when the SIRT1 inhibitor EX527 was used to simultaneously treat the oocytes with melatonin. Consistent with this finding, we found that the postovulatory oocyte aging process was markedly attenuated when the oocytes were treated with the SIRT1 activator SRT1720. In conclusion, our data strongly indicate that melatonin delays postovulatory mouse oocyte aging via a SIRT1-MnSOD-dependent pathway, which may provide a molecular mechanism support for the further application of melatonin in the assisted reproductive technology field.

Survival rates and the occurrence of larval malformations, including Siamese twins, following fertilization of post-ovulatory aged oocytes in ide Leuciscus idus.

Embryonic development of lower vertebrates can be influenced by many factors, especially when outside of the mother's body and under the influence of variable abiotic and biotic factors. In this study, the effects of fertilization of post-ovulatory aged (overmature) oocytes of ide Leuciscus idus on the risk of developing anomalies in ide larvae were assessed under controlled conditions. There was a negative effect of delayed fertilization of post-ovulatory aged (overmature) oocytes in the ovary on the quality of offspring, which caused reduced survival 3 days post-fertilization and at the hatching stage (68.3 vs. 81.2% and 47.2 vs. 79.7% for the overmature and mature groups, respectively). It also increased the occurrence of numerous developmental defects (19.3 vs. 0.8% for the overmature and mature groups, respectively), including the development of Siamese twins. This is the first reported case of the occurrence of Siamese twins in ide. The ide Siamese twins look like hen eggs with 2 heads and are different from 'typical' Siamese twins in fish.

Role of calcium-sensing receptor in regulating spontaneous activation of postovulatory aging rat oocytes.

Mechanisms for postovulatory aging (POA) of oocytes and for spontaneous activation (SA) of rat oocytes are largely unknown. Expression of calcium-sensing receptor (CaSR) in rat oocytes and its role in POA remain unexplored. In this study, expression of CaSR in rat oocytes aging for different times was detected by immunofluorescence microscopy, and western blotting and the role of CaSR in POA was determined by observing the effects of regulating its activity on SA susceptibility and cytoplasmic calcium levels. The results showed that CaSR was expressed in rat oocytes. Oocytes recovered 19 h post human chorionic gonadotropin (hCG) injection were more susceptible to SA and expressed more functional CaSR than oocytes recovered 13 h after hCG injection, although both expressed the same level of total CaSR protein. Treatment with CaSR antagonist significantly suppressed cytoplasmic calcium elevation and SA of oocytes. Activation of Na-Ca2+ exchanger with NaCl inhibited SA to a greater extent than suppression of CaSR with NPS-2143, suggesting that calcium sources other than CaSR-controlled channels contributed to the elevation of cytoplasmic calcium. Treatment with T- or L-type calcium channel blockers significantly reduced SA. Suppression of all calcium channels tested reduced SA to minimum. It is concluded that the level of CaSR functional dimer protein, but not that of the total CaSR protein, was positively correlated with the SA susceptibility during POA of rat oocytes confirming that CaSR is involved in POA regulation. Blocking multiple calcium channels might be a better choice for efficient control of SA in rat oocytes.

Melatonin prevents postovulatory oocyte aging and promotes subsequent embryonic development in the pig

Oxidative stress is known as a major contributing factor involved in oocyte aging, which negatively affects oocyte quality and development after fertilization. Melatonin is an effective free radical scavenger and its metabolites AFMK and AMK are powerful detoxifiers that eliminate free radicals. In this study, we used porcine oocytes to test the hypothesis that melatonin could scavenge free radicals produced during oocyte aging, thereby maintaining oocyte quality. We compared reactive oxygen species levels, apoptosis levels, mitochondrial membrane potential ratios, total glutathione contents and expression levels in fresh, aged and melatonin-treated aged porcine oocytes and observed the percentage of blastocyst formation following parthenogenetic activation. We found that melatonin could effectively maintain the morphology of oocytes observed in control oocytes, alleviate oxidative stress, markedly decrease early apoptosis levels, retard the decline of mitochondrial membrane potential and significantly promote subsequent embryonic development in oocytes aged for 24 hr in vitro. These results strongly suggest that melatonin can prevent postovulatory oocyte aging and promote subsequent embryonic development in the pig, which might find practical applications to control oocyte aging in other mammalian species including humans to maintain the quality of human oocytes when performing clinical assisted reproductive technology.

TCTP regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality

If no fertilization occurs for a prolonged time following ovulation, oocytes experience a time-dependent deterioration in quality both in vivo and in vitro due to processes called postovulatory aging. Because the postovulatory aging of oocytes has marked detrimental effects on embryo development and offspring, many efforts have been made to unveil the underlying mechanisms. Here we showed that translationally controlled tumor protein (TCTP) regulates spindle assembly during postovulatory aging and prevents deterioration in mouse oocyte quality. Spindle dynamics decreased with reduced TCTP level during aging of mouse oocytes. Knockdown of TCTP accelerated the reduction of spindle dynamics, accompanying with aging-related deterioration of oocyte quality. Conversely, overexpression of TCTP prevented aging-associated decline of spindle dynamics. Moreover, the aging-related abnormalities in oocytes were rescued after TCTP overexpression, thereby improving fertilization competency and subsequent embryo development. Therefore, our results demonstrate that TCTP-mediated spindle dynamics play a key role in maintaining oocyte quality during postovulatory aging and overexpression of TCTP is sufficient to prevent aging-associated abnormalities in mouse oocytes.

Expression profiles and function analysis of microRNAs in postovulatory aging mouse oocytes.

In this study, microRNA (miRNA) profiles in postovulatory aging mouse oocytes were analyzed by microarray screening and RT-qPCR. Hierarchical cluster analysis on the microarray data and KEGG pathway enrichment analysis on the mRNAs targeted by differentially expressed (DE) miRNAs between two adjacent egg-ages suggest that while only a mild alteration in miRNA expression occurred from 13 to 18 h, a great change took place from 18 to 24 h post hCG injection. Theoretical exploration on functions of the predicted target genes suggest that KEGG pathways enriched by 13-18 h DE miRNAs are correlated with early events of oocyte aging while pathways most enriched by 18-24 h or 24-30 h DE miRNAs are correlated with the late symptoms of aged oocytes. Experimental verification on functions of the key proteins predicted by the KEGG analysis and injection of miR-98 mimics or inhibitors further confirmed that miRNAs played stimulatory/inhibitory roles in postovulatory oocyte aging. In conclusion, marked changes in miRNA expression are associated with significant alterations in function and morphology of postovulatory aging oocytes.

Quercetin delays postovulatory aging of mouse oocytes by regulating SIRT expression and MPF activity.

If no fertilization occurs at an appropriate time after ovulation, oocyte quality deteriorates rapidly as a process called postovulatory aging. Because the postovulatory aging of oocytes has detrimental effects on embryo development and offspring, many efforts have been made to prevent oocyte aging. Here we showed that quercetin prevented the decline in oocyte quality during postovulatory aging of oocytes. Quercetin treatment reduced aging-induced morphological changes and reactive oxygen species accumulation. Moreover, quercetin attenuated the aging-associated abnormalities in spindle organization and mitochondrial distribution, preventing decrease of SIRT expression and histone methylation. Quercetin also ameliorated the decrease in maturation-promoting factor activity and the onset of apoptosis during postovulatory aging. Furthermore, quercetin treatment during postovulatory aging improves early embryo development. Our results demonstrate that quercetin relieves deterioration in oocyte quality and improves subsequent embryo development.

Melatonin improves the fertilization ability of post-ovulatory aged mouse oocytes by stabilizing ovastacin and Juno to promote sperm binding and fusion.

What are the underlying mechanisms of the decline in the fertilization ability of post-ovulatory aged oocytes? Melatonin improves the fertilization ability of post-ovulatory aged oocytes by reducing aging-induced reactive oxygen species (ROS) levels and inhibiting apoptosis and by maintaining the levels and localization of the fertilization proteins, ovastacin and Juno. Following ovulation, the quality of mammalian metaphase II oocytes irreversibly deteriorates over time with a concomitant loss of fertilization ability. Melatonin has been found to prevent post-ovulatory oocyte aging and extend the window for optimal fertilization in mice. Mouse oocytes were randomly assigned to three groups and aged in vitro for 0, 6, 12 and 24 h, respectively. Increasing concentrations of melatonin (10-9 M, 10-7 M, 10-5 M and 10-3 M) were added to the 24 h aging group. Sperm binding assays, in-vitro fertilization, immunofluorescent staining and western blotting were performed to investigate key regulators and events during fertilization of post-ovulatory aged mouse oocytes. We found that the actin cap which promotes a cortical granule(CG) free domain is disrupted with a re-distribution of CGs in the subcortex of aged oocytes. Ovastacin, a CG metalloendoprotease, is mis-located and prematurely exocytosed in aged oocytes with subsequent cleavage of the zona pellucida protein ZP2. This disrupts the sperm recognition domain and dramatically reduces the number of sperm binding to the zona pellucida. The abundance of Juno, the sperm receptor on the oocyte membrane, also is reduced in aged oocytes. Exposure of aged oocytes to melatonin significantly elevates in-vitro fertilization rates potentially by rescuing the above age-associated defects of fertilization, and reducing ROS and inhibiting apoptosis. N/A. We explored the mechanisms of the decline in fertilization ability decline in aged mouse oocytes, in vitro but not in vivo. Our findings may contribute to the development a more efficient method, involving melatonin, for improving IVF success rates. This study was supported by the National Natural Science Foundation (31571545) and the Natural Science Foundation of Jiangsu Province (BK20150677). The authors have no conflict of interest to disclose.

Biochemical alterations in the oocyte in support of early embryonic development.

Notwithstanding the enormous reproductive potential encapsulated within a mature mammalian oocyte, these cells present only a limited window for fertilization before defaulting to an apoptotic cascade known as post-ovulatory oocyte aging. The only cell with the capacity to rescue this potential is the fertilizing spermatozoon. Indeed, the union of these cells sets in train a remarkable series of events that endows the oocyte with the capacity to divide and differentiate into the trillions of cells that comprise a new individual. Traditional paradigms hold that, beyond the initial stimulation of fluctuating calcium (Ca2+) required for oocyte activation, the fertilizing spermatozoon plays limited additional roles in the early embryo. While this model has now been drawn into question in view of the recent discovery that spermatozoa deliver developmentally important classes of small noncoding RNAs and other epigenetic modulators to oocytes during fertilization, it is nevertheless apparent that the primary responsibility for oocyte activation rests with a modest store of maternally derived proteins and mRNA accumulated during oogenesis. It is, therefore, not surprising that widespread post-translational modifications, in particular phosphorylation, hold a central role in endowing these proteins with sufficient functional diversity to initiate embryonic development. Indeed, proteins targeted for such modifications have been linked to oocyte activation, recruitment of maternal mRNAs, DNA repair and resumption of the cell cycle. This review, therefore, seeks to explore the intimate relationship between Ca2+ release and the suite of molecular modifications that sweep through the oocyte to ensure the successful union of the parental germlines and ensure embryogenic fidelity.