The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.
The health and integrity of the oocyte can greatly influence the success of fertilization as well as the developmental competence of the embryo . This dependence on oocyte quality is of little surprise considering that this cell contains factors responsible not only for remodeling the maternal and paternal genomes [2, 3] but also for orchestrating the early stages of embryogenesis [4, 5]. Furthermore, the oocyte is the sole source of mitochondria in the developing embryo, with these organelles not only being responsible for ATP production but also conducting key cellular processes such as apoptosis in response to appropriate developmental cues .
Following ovulation, the prophase I oocyte resumes meiosis and undergoes a maturational process involving germinal vesicle breakdown, migration of the metaphase spindle, and extrusion of the first polar body. Following these events, meiosis is once again arrested—now at metaphase II (MII)—and remains in this state until fertilization occurs [7, 8]. Unfortunately, with increasing time following ovulation, the MII oocyte undergoes a process of deterioration in vivo and in vitro, referred to as oocyte aging. Oocyte aging is associated with many deleterious effects (for review, see [9, 10]); the aging oocyte experiences partial cortical granule exocytosis [11–14] and zona hardening [12, 14, 15], making it less receptive to fertilization [16–18]. A decrease in critical cell-cycle factors, particularly maturation-promoting factor (MPF) and mitogen-activating protein kinase, also becomes evident . Additionally, these oocytes commonly exhibit spindle abnormalities and losses of chromosomal integrity  and are increasingly susceptible to polyspermy [21, 22]. As a result, fertilization of aged oocytes is associated with poor developmental potential of embryos , elevated risk for early pregnancy loss , and abnormal/retarded development in offspring [24, 25]. The “end point” of this oocyte aging process is cell death via an apoptotic pathway characterized by phosphatidylserine externalization , caspase activation , accumulation of the apoptotic signaling protein Bax (Bcl-2-associated X) and suppression of Bcl-xL (B-cell lymphoma-extra large) [28, 29], and DNA fragmentation .
The intracellular signals controlling postovulatory oocyte aging have not been well defined; however, because oxidative stress is purportedly a prominent mediator of aging and disease in many cell and tissue types , reactive oxygen species (ROS) are certainly potential orchestrators of this process. Levels of ROS (particularly hydrogen peroxide) and lipid peroxidation have been shown to significantly increase with oocyte age , whilst oocytes under oxidative stress exhibit a decreased fertilization rate  and are more likely to enter into apoptosis . Despite these data suggesting involvement of oxidative stress in the postovulatory oocyte aging process, little success has been achieved in delaying aging with antioxidant supplementation . Although some nonantioxidant compounds such as caffeine have been shown to extend the window for fertilization using intracytoplasmic sperm injection (ICSI)  by preventing inactivation of MPF within the cell  and alleviating the deterioration of calcium release mechanisms , the clinical significance of such observations is questionable. For example, caffeine supplementation of in vitro culture medium has been shown to suppress DNA repair mechanisms in somatic cells  and has been demonstrated to reduce the ability of hamster oocytes to repair extensive DNA damage in human spermatozoa . This is clearly a worrisome factor when considering the assisted reproductive technology (ART) patient population, because many male patients exhibit significantly elevated levels of DNA damage in their spermatozoa compared to donor populations .
In the present study, we demonstrate that oxidative stress does indeed play an integral role in curtailing the structural and functional integrity of the postovulatory oocyte. We also show that the potent antioxidant melatonin can effectively relieve aging mouse oocytes of oxidative stress in vitro, delaying the onset of apoptosis and preventing fragmentation. Importantly, we also demonstrate that melatonin-supplemented oocytes experience an extended window for optimal fertilization and produce embryos of improved quality compared with their control aged oocyte counterparts. Finally, we showed that compounds targeted at maintenance of MPF levels (e.g., caffeine) are inadequate to delay all facets of oocyte aging; specifically, they do not prevent accumulation of oxidative stress in these cells. We propose that melatonin may be a compound that can be safely utilized to prevent oocyte aging in a clinical setting (e.g., by reducing the undesirable consequences associated with next-day rescue ISCI).
Materials and Methods
Three- to five-week-old C57BL6/CBA F1 female mice were administered intraperitoneal injections of equine chorionic gonadotropin (Intervet) followed 48 h later by human chorionic gonadotropin (hCG; Intervet) to induce superovulation. Mice were culled 14–15 h after hCG injection using CO2 asphyxiation. Ovaries were removed immediately and placed in PBS at 37°C before cumulus mass retrieval from the ampullae. Oocytes were denuded from cumulus cells using a 5-min incubation in hyaluronidase (300 μg/ml; Sigma-Aldrich) at 37°C. Oocytes were then washed from three to five times in M2 medium (Sigma-Aldrich) to completely remove the cumulus cells. The use of animals in this project was approved by the University of Newcastle Animal Care and Ethics Committee, and all animals were obtained from breeding programs run in the University of Newcastle Central Animal House.
Aging of Oocytes In Vitro
To establish the effects of melatonin supplementation on oocyte aging, collected oocytes were immediately placed in a 20-μl droplet of either M2 medium, M2 medium containing 1 mM melatonin (Sigma-Aldrich), or M2 medium plus ethanol (vehicle control) under mineral oil (Sigma-Aldrich). Oocytes were “aged” in these droplets in groups of 8–10 at 37°C under gas (5% O2, 6% CO2 in N2) for 1, 8, 24, or 48 h from the time of oocyte retrieval. In experiments that compared the ability of melatonin and caffeine to delay oocyte aging, the above-described protocol was followed; however, a portion of the oocytes was also placed in M2 medium containing 5 mM caffeine (Sigma-Aldrich) as well as in M2 medium containing a combination of both 1 mM melatonin and 5 mM caffeine. The optimal concentration of 1 mM melatonin was predetermined via a dose-response study (www.biolreprod.org), whereas the optimal concentration of 5 mM caffeine was obtained from previously published studies on postovulatory oocyte aging [19, 35, 36, 40].
To identify oxidative stress/ROS levels in aged oocytes, 5′-carboxy-2′,7′-difluorodihydrofluorescein diacetate (carboxy-DFFDA; Molecular Probes), a fluorescent probe capable of detecting powerful oxidants such as H2O2 and peroxynitrite, was utilized. Oocytes were incubated in a 10 μM solution of carboxy-DFFDA in M2 medium for 15 min at 37°C under gas. Oocytes were then washed three times in M2 medium before mounting on a glass slide for microscopy. To compare ROS levels between different treatment groups, images were generated using an Axioplan 2 fluorescence microscope (Carl Zeiss MicroImaging GmbH.), and a pixel intensity value was calculated for each oocyte using ImageJ software (National Institutes of Health).
A FAM-FLICA Poly Caspase Assay Kit (ImmunoChemistry Technologies) was used to establish levels of caspase activation in aged oocytes. Oocytes were incubated in working solution (created as per the manufacturer's instructions) for 30 min at 37°C under gas. Following incubation, oocytes were washed three times in M2 medium before mounting for fluorescence microscopy. Pixel intensity values were again used to compare activated caspase levels between treatment groups.
An Annexin-V conjugate (Molecular Probes) was used to identify phosphatidylserine exteriorization in apoptotic cells. Oocytes were incubated in Annexin-V for 15 min before being transferred to a 0.25 mg/ml solution of propidium iodide (Sigma-Aldrich) to allow recognition of necrotic cells. Oocytes were washed three times in M2 medium before mounting and analysis via fluorescence microscopy.
In Vitro Fertilization
Fertilization studies were conducted on oocytes aged for 8, 16, and 24 h in vitro with or without 1 mM melatonin supplemented into the culture medium. The in vitro fertilization (IVF) procedure followed in the present study was modified from that used by the MRC Harwell (Oxfordshire, U.K.), which is based on a series of publications by Takeo and colleagues [41–43]. Spermatozoa were retrieved from the caudae epididymides from adult Swiss mice and capacitated in Biggers, Whitten, and Whittingham medium  supplemented with 1 mg/ml of polyvinyl alcohol and 1 mg/ml of methyl-beta-cyclodextrin for 1 h at 37°C and 6% CO2 under mineral oil. Immediately before completion of capacitation, aged oocytes were washed four times in human tubal fluid (HTF) [41–43] and then placed in fertilization medium (HTF containing 1 mM reduced glutathione [GSH]). Denuded, freshly retrieved oocytes were also fertilized and used as a control. Aliquots (3–5 μl) of capacitated sperm suspension were added to the fertilization dishes before a 4-h period of incubation at 37°C under gas. Following fertilization, oocytes were washed four times in HTF and incubated overnight to the 2-cell stage. The 2-cell embryos were transferred to G-1 PLUS medium (Vitrolife) on the morning of Day 2, then transferred to G-2 PLUS medium (Vitrolife) on the morning of Day 4. The percentage of oocytes that fertilized and reached the blastocyst stage was calculated on the morning of Day 5.
A TUNEL assay (Roche Diagnostics) was conducted on Day 5 blastocysts to identify DNA fragmentation related to apoptosis in nuclei. The TUNEL assay was conducted as described previously , and blastocysts were analyzed using confocal microscopy (Zeiss LSM 510; Carl Zeiss MicroImaging GmbH).
All experiments were conducted at least three times on independent samples, and results were analyzed by ANOVA using JMP version 9.0.0 (SAS Institute Inc.). A post hoc comparison of group means was conducted using a Fisher protected least significant difference test. Analysis of paired samples was conducted using a paired Student t-test. A value of P < 0.05 was considered to be statistically significant.
Oxidative Stress Precedes the Appearance of Markers of Aging and Apoptosis in Oocytes In Vitro
To establish a relationship between the onset of oxidative stress and the onset of apoptosis in C57BL6/CBA F1 aging mouse oocytes, levels of ROS and of apoptotic markers were assessed at a series of time points over the course of 48 h in vitro. MII oocytes showed a significant time-dependent increase in ROS levels (P < 0.001) as detected by the carboxy-DFFDA fluorescent probe (Fig. 1A). ROS levels were significantly elevated after only 8 h in culture and continued to rise exponentially up to 48 h. Oocytes therefore appear to experience oxidative stress from as early as 8 h following retrieval. Our results further suggested that oxidative stress is a factor associated with the onset of apoptosis in oocytes as they age in vitro. Thus, a significant increase in the percentage of Annexin-V-positive cells was observed simultaneous with the aforementioned early increase in ROS levels, with the Annexin-V conjugate identifying a significant increase in the exteriorization of phosphatidylserine after 8 h (P < 0.05), 24 h (P < 0.05), and 48 h (P < 0.001) in culture (Fig. 1B). Furthermore, a significant rise in levels of caspase activation (a late apoptotic marker) was observed; this rise achieved statistical significance after 24 h (P < 0.05) and 48 h (P < 0.001) in culture (Fig. 1C). The onset of oxidative stress in these aging oocytes was also demonstrated to precede morphological features of cellular stress (fragmentation related to apoptosis or spontaneous activation), which were significantly elevated after 24 h (P < 0.001) and 48 h (P < 0.001) of culture, respectively (Fig. 1D).
Melatonin Relieves Oxidative Stress in Aging Oocytes and Delays the Onset of Apoptosis
If oxidative stress is indeed a prominent mediator of oocyte aging, then supplementation with a potent antioxidant compound that would be stable under the culture conditions employed in the present study should stop or delay the aging process. By supplementing the culture medium with 1 mM melatonin, we did indeed detect a 41% decrease (P < 0.05) in ROS levels following 48 h of in vitro culture (Fig. 2A). This prominent reduction in ROS levels was accompanied by a delay in the onset of apoptosis, with a 52% decline in levels of caspase activation (P < 0.05) (Fig. 2B) as well as a 32% decrease in morphological abnormalities (P < 0.001) (Fig. 2, C–E). It was concluded from these results that oxidative stress certainly influences at least some facets of oocyte aging and that a degree of attenuation of this process could be enforced using the antioxidant melatonin.
Melatonin Extends the Optimal Window for Fertilization and Improves Embryo Quality
In preventing the accumulation of ROS during the aging process, melatonin effectively extended the optimal window for fertilization of the oocyte. Specifically, 72% ± 8.4% (values are ± SEM throughout) of oocytes aged for 8 h in the presence of melatonin reached the 2-cell stage following insemination, compared to only 49% ± 10.8% of oocytes aged in control media (P < 0.05) (Fig. 3A). Similarly, oocytes inseminated after 16 h of in vitro culture with melatonin had a rate of 2-cell embryo formation 17% greater that of the control (P < 0.05) (Fig. 3B), whereas after 24 h of culture, the rate of 2-cell embryo formation was increased by 26% in the presence of melatonin (P < 0.05) (Fig. 3C). The developmental progression of embryos generated from oocytes aged in the presence of melatonin was also superior to the progression of those established from control oocytes. Thus, 54% ± 10.0% of oocytes aged for 8 h in the presence of melatonin reached the blastocyst stage, whereas fertilization of control aged oocytes resulted in a 29% ± 13.8% blastocyst formation rate (P < 0.01) (Fig. 3A). Melatonin also increased the rate of blastocyst formation in oocytes inseminated following 16 h of aging in vitro by 16.8% (P < 0.01) (Fig. 3B). Despite an increased 2-cell embryo formation rate in oocytes exposed to melatonin for 24 h relative to aged controls, very few of these embryos progressed to produce viable blastocysts, indicating a dramatic loss of developmental potential when oocytes are aged for such prolonged periods of time (Fig. 3C).
Blastocysts formed from control oocytes aged for 8 and 16 h before insemination were of poor quality, displaying elevated levels of apoptosis (P < 0.05), with 18% ± 4.2% and 16% ± 4.6% of blastomeres, respectively, being TUNEL positive (Fig. 3, D–H) compared to under 7% of blastomeres in embryos formed from fresh oocytes (Fig. 3D). Significantly, embryos originating from oocytes aged for the same periods of time in the presence of melatonin did not experience elevated levels of apoptosis; the percentage of TUNEL-positive blastomeres was not significantly different in these melatonin-supplemented cultures compared with the fresh oocyte controls (Fig. 3, D and E).
Comparison Between the Effects of Melatonin and Caffeine on Oocyte Aging
In the interest of comparing mechanisms by which aging is delayed in oocytes supplemented with melatonin versus oocytes supplemented with caffeine, a comparison of the oxidative and apoptotic status of oocytes exposed to these reagents was conducted. Interestingly, despite its ability to extend the window in which murine oocytes can be fertilized , caffeine supplementation provided no relief from oxidative stress following 48 h of in vitro culture; with ROS levels decreasing below that of control aged oocytes only in the presence of melatonin (P < 0.05) (Fig. 4A). Additionally, caffeine-treated aged oocytes did not experience any relief from the onset of apoptosis. Again, levels of caspase activation were only significantly reduced with melatonin supplementation or when caffeine and melatonin were combined in culture medium (P < 0.05) (Fig. 4B).
With the demand for ART increasing exponentially in recent years, the drive to refine these technologies has become paramount, particularly considering that less than 20% of initiated IVF cycles result in the production of live offspring  and that these offspring have a significantly increased risk of possessing birth defects . The present study has focused specifically on the contribution of postovulatory oocyte aging in vitro to fertilization efficiency and embryonic developmental potential. We have identified oxidative stress as a major contributor to the postovulatory oocyte aging process and have found that supplementation of culture medium with the potent antioxidant melatonin can delay multiple facets of oocyte aging and apoptosis in vitro, thereby extending the optimal window for fertilization and preventing an age-associated decline in embryo quality.
This research provides confirmation that oxidative stress is indeed a key mediator of oocyte aging in vitro and appears to act as a trigger for induction of the intrinsic apoptotic pathway, just as has been observed for spermatozoa . Our results demonstrate that the onset of oxidative stress may be a relatively early event associated with in vitro culture, because a significant elevation in ROS levels is evident from the 8-h time point. The onset of oxidative stress early in the oocyte aging process is not particularly surprising when taking into consideration the reported decline in GSH levels within the oocyte with time postovulation as well as the absence of antioxidant-rich follicular fluid to provide protection . Additionally, the in vitro environment itself is a potential promoter of oxidative stress in oocytes due to increases in oxygen tension [50, 51] and exposure to light . The free-radical theory of aging proposes that ROS produced by the mitochondria as by-products of oxidative phosphorylation may result in the progressive accumulation of toxic oxidative metabolites in the cell with age, inflicting damage on these organelles and initiating further ROS production in a damaging redox cycle . Such a chain of cause-and-effect has recently been demonstrated in human spermatozoa, with mitochondrial ROS generation triggering an increase in cellular 4-hydroxynonenal and the latter then forming adducts with enzymes in the mitochondrial electron-transport chain, stimulating yet more ROS generation in a self-perpetuating manner . As a further consequence of this process, the intrinsic apoptotic cascade ensues, characterized by mitochondrial pore formation, cytochrome c release, and subsequent cellular degradation by caspases and endonucleases . The onset of oxidative stress observed following the culture of oocytes for 8 h in vitro is consistent with the notion that ROS represent the instigator of apoptosis in these cells, with early markers for apoptosis (phosphatidylserine externalization) appearing simultaneously with oxidative stress whereas caspases, involved in the subsequent processing of cellular proteins , materialize later.
In alignment with our research, previous studies have also identified oxidative stress as a factor that likely is involved in the aging of postovulatory oocytes [32, 56]. However, until now, little success had been achieved in delaying the onset of this phenomenon using antioxidant supplementation. Tarin et al.  showed that supplementation of oocyte culture medium with l-ascorbic acid and vitamin E could not improve fertilization rates or embryo development in aged oocytes in vitro, whereas treatment with l-cysteine caused a decreased developmental rate to the blastocyst stage . In contrast to previous research, we found in the present study that the antioxidant melatonin was able to successfully delay aging and apoptosis in oocytes retrieved from the hybrid mouse strain C57BL6/CBA F1, thereby increasing both embryo formation rate and embryo quality after in vitro incubation periods of up to 16 h (the comparable effect of melatonin on oocytes retrieved from other mouse strains has not yet been assessed).
The superior ability of melatonin to influence oocyte quality in the present study may relate to several key features of this compound's chemistry. First, melatonin is extremely stable in aqueous solution , meaning that no rapid inactivation of its ROS scavenging capabilities occurs in oocyte culture medium. Importantly, unlike many other antioxidant agents, melatonin does not produce pro-oxidant by-products from its interaction with ROS. In fact, all known intermediates generated through the reaction between melatonin and ROS are free-radical scavengers themselves, meaning that even at low concentrations, melatonin induces a powerful “free radical scavenging cascade” within the cell . Finally, melatonin's amphiphilic nature means it can penetrate to all components of the cell, where it can then scavenge a wide range of ROS [59, 60].
Our results demonstrate that oxidative stress in the aged oocyte clearly affects its potential for embryo formation and development. The reduced rate of 2-cell embryo formation observed in oxidatively stressed aged oocytes may be attributed to a decreased fertilization rate resulting from peroxidation of lipids in the oocyte plasma membrane, with lipid peroxidation potentially causing a reduction in membrane fluidity and, thereby, a decreased capacity for sperm-oolemma fusion . Additionally, oocytes in oxidative stress have been demonstrated to experience a perturbation in calcium homeostasis, resulting in impaired calcium oscillations following fertilization, affecting their capacity for oocyte activation  and embryo development . In addition to preventing oxidative damage to lipids within the plasma membrane, melatonin may also act as a protector from oxidative attack for many other intracellular components such as proteins as well as nuclear and mitochondrial DNA. Oocytes with excessive DNA damage clearly are more likely to default to the intrinsic apoptotic pathway than to continue with embryonic development (i.e., will not pass through the G1 checkpoint of the cell cycle to reach the S phase ).
Further to its effect on embryo formation rate and developmental potential, we have found that the onset of oxidative stress in aging oocytes is linked with the production of poor-quality embryos. A high degree of apoptotic nuclei in blastomeres produced from aged oocytes in the present study appears to be directly linked with oxidative stress levels in the oocyte before insemination, because relieving this stress using melatonin decreased the incidence of TUNEL-positive blastomeres. Not surprisingly, high percentages of apoptotic blastomeres within the embryo have been associated with poor embryo quality  and with a tendency for early embryos to undergo resorption [65, 66]. Therefore, oxidative stress in the oocyte before insemination may affect not only the preimplantation embryo but also postimplantation events and, hence, the likelihood of producing healthy, live offspring.
The final component of the present study highlighted potential downfalls associated with utilizing compounds such as caffeine, which influence MPF levels within the oocyte, to inhibit aging. Although the temporal window for fertilization is purportedly extended with the prevention of MPF phosphorylation , the present study has demonstrated that other facets of oocyte aging such as the accumulation of oxidative stress and the onset of apoptosis are not controlled by this reagent. As previously discussed, the consequences of fertilizing an oxidatively stressed oocyte may include a higher rate of apoptotic nuclei in blastocysts and a subsequent elevated risk for embryo resorption. We propose that melatonin is likely to be a more effective and safer compound for potential utilization in ART, as, not only can this antioxidant positively affect embryo formation rate and blastocyst quality, but is also reported to have a lack of demonstrable toxicity .
It is worth noting that although compounds such as melatonin and caffeine are capable of delaying several aspects of postovulatory oocyte aging, a threshold window for development still exists beyond which a successful outcome is unlikely. This was demonstrated by our fertilization studies, which showed that although 24-h aged oocytes experienced increased levels of 2-cell embryo formation following melatonin supplementation, these embryos had mostly lost the capability to reach the 4-cell and blastocyst stages of development. The transient capacity for maintenance of high-quality oocytes with media supplementation is not surprising, particularly because any single compound is unlikely to hold the potential for prevention of all consequences associated with oocyte aging. For instance, although the relief from oxidative stress provided by melatonin delays the acquisition of morphological abnormalities and apoptosis, it may not exert influence on other facets of aging such as loss of cytoskeletal integrity and spindle organization. With future gains in understanding of the complex mechanisms controlling oocyte aging, it may be possible to create oocyte culture media containing a combination of active compounds, including an antioxidant agent such as melatonin, to target an extensive array of age-related changes and allow prolonged incubation periods in vitro before IVF/ICSI.
In conclusion, our research has demonstrated, to our knowledge for the first time, that by preventing oxidative stress using melatonin supplementation, aspects of the aging process that mouse oocytes undergo postovulation in vitro can be delayed. Oocytes aged in the presence of melatonin experienced a delay in the onset of apoptosis, an increased optimal window for fertilization, and improved embryo quality compared to their untreated aged counterparts. The present study also demonstrated not only that oxidative stress is a prominent mediator of the oocyte aging process but also that oxidative stress in aged oocytes is directly responsible for a decline in embryo formation rate and embryo quality. We have also provided evidence to suggest that melatonin may be more effective and safer than caffeine for inhibiting the oocyte aging process in a clinical setting.