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Melatonin prevents post-ovulatory oocyte aging in the mouse and extends the window for optimal fertilization in vitro1

Short title: Melatonin prevents oocyte aging in vitro

Tessa Lord,3 Brett Nixon,3 Keith T. Jones,4 and R. John Aitken2,3

3Priority Research Centre for Reproductive Biology, 2School of Environmental and Life Sciences, University of Newcastle, Callaghan, New South Wales, Australia 4School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, New South Wales, Australia 1Supported by a grant from National Health and Medical Research Council of Australia (Grant #494802). 2Correspondence: E-mail: john.aitken@newcastle.edu.au ABSTRACT 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 an increased likelihood of embryonic resorption. As oxidative stress has been shown to accelerate the onset of apoptosis in oocytes and influence their capacity for fertilization, this study aimed to characterize the significance of such stress in the post-ovulatory aging of mouse oocytes in vitro. In the course of these studies, 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, and is followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence (P < 0.001). 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 h 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. Summary: Addition of the antioxidant melatonin to oocyte culture media delays the onset of post-ovulatory oocyte apoptosis; it not only extends the window for optimum fertilization but also improves the quality of resulting blastocysts. Keywords: Oocyte aging, apoptosis, antioxidants, melatonin, oxidative stress. INTRODUCTION The health and integrity of the oocyte can greatly influence the success of fertilization as well as the developmental competence of the embryo [1]. This dependence on oocyte quality is of little surprise when considering that this cell contains factors responsible for not only remodelling the

BOR Papers in Press. Published on January 30, 2013 as DOI:10.1095/biolreprod.112.106450

Copyright 2013 by The Society for the Study of Reproduction.

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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 [6]. 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 occur [7, 8]. Unfortunately, with increasing time following ovulation the MII stage 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 also becomes evident, particularly maturation promoting factor (MPF) and mitogen-activating protein kinase (MAPK) [19]. Additionally, these oocytes commonly exhibit spindle abnormalities and losses of chromosomal integrity [20] and are increasingly susceptible to polyspermy [21, 22]. As a result, fertilization of aged oocytes is associated with poor developmental potential of embryos [22], an elevated risk for early pregnancy loss [23] 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 [26], caspase activation [27], accumulation of the apoptotic signalling protein Bax, suppression of Bcl-xL [28, 29] and DNA fragmentation [30]. The intracellular signals controlling post-ovulatory oocyte aging have not been well defined, however, as oxidative stress is purportedly a prominent mediator of aging and disease in many cell and tissue types [31], 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 [32], while oocytes under oxidative stress exhibit a decreased fertilization rate [32] and are more likely to enter into apoptosis [33]. Despite these data suggesting an involvement of oxidative stress in the post-ovulatory oocyte aging process, little success has been achieved in delaying aging with antioxidant supplementation [34]. Although some non-antioxidant compounds such as caffeine have been shown to extend the window for fertilization by ICSI (intracytoplasmic sperm injection) [35] by preventing inactivation of MPF within the cell [19] and alleviating the deterioration of calcium release mechanisms [36], 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 [37] and has been demonstrated to reduce the ability of hamster oocytes to repair extensive DNA damage in human spermatozoa [38]. This is clearly a worrisome factor when considering the ART patient population, because many male patients exhibit significantly elevated levels of DNA damage in their spermatozoa when compared to donor populations [39]. The present study demonstrates that oxidative stress does indeed play an integral role in curtailing the structural and functional integrity of the post-ovulatory 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

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demonstrate that melatonin supplemented oocytes experience an extended window for optimal fertilization and produce embryos of improved quality when compared with their control aged oocyte counterparts. Finally, this study showed that compounds targeted at maintenance of MPF levels, such as 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; for example, reducing the undesirable consequences associated with next-day rescue ISCI. MATERIALS AND METHODS Oocyte collection Three to five week old C57BL6/CBA F1 female mice were administered intraperitoneal injections of equine Chorionic Gonadotropin (eCG) (Intervet, Sydney, Australia), followed 48 h later by human Chorionic Gonadotropin (hCG) (Intervet, Sydney, Australia) to induce superovulation. Mice were culled 14-15 h post-hCG 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, MO) at 37°C. Oocytes were then washed 3-5 times in M2 media (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 In order to establish the effects of melatonin supplementation on oocyte aging, collected oocytes were immediately placed in a 20 µL droplet of either M2 media, M2 media containing 1 mM melatonin (Sigma Aldrich), or M2 media 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 which compared the ability of melatonin and caffeine to delay oocyte aging, the above protocol was followed however a portion of oocytes were also placed in M2 media containing 5 mM caffeine (Sigma Aldrich) as well as M2 media containing a combination of both 1 mM melatonin and 5 mM caffeine. The optimal concentration of 1 mM melatonin was pre-determined via a dose response study (Supplemental Figure S1, available online at www.biolreprod.org), whilst the optimal concentration of 5 mM caffeine was obtained from previously published studies on post-ovulatory oocyte aging [19, 35, 36, 40]. Carboxy-DFFDA In order to identify oxidative stress/ROS levels in aged oocytes, 5’-carboxy-2’, 7’-difluorodihydrofluorescein diacetate (carboxy-DFFDA, Molecular Probes, OR) 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 media for 15 min at 37˚C under gas. Oocytes were then washed 3 times in M2 media before mounting on a glass slide for microscopy. In order to compare ROS levels between different treatment groups, images were generated using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss MicroImaging Inc., Thornwood, NY) and a pixel intensity value was calculated for each oocyte using the public sector image processing program Image J (NIH, MD).

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Caspase assay A FAM FLICA Poly Caspases Assay Kit (ImmunoChemistry Technologies, Bloomington, MN) 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 3 times in M2 media before mounting for fluorescence microscopy. Pixel intensity values were again used to compare activated caspase levels between treatment groups. Annexin-V assay 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 propidium iodide (Sigma Aldrich) solution to allow for recognition of necrotic cells. Oocytes were washed 3 times in M2 medium before mounting and analysis via fluorescence microscopy. In Vitro Fertilization (IVF) Fertilization studies were conducted on oocytes aged for 8, 16 and 24 h in vitro with or without 1 mM melatonin supplemented into their culture medium. The IVF procedure followed was modified from that used by MRC Harwell which was based on a series of publications by Takeo et al. [41-43]. Spermatozoa were retrieved from the cauda epididymides from adult Swiss mice and capacitated in Biggers, Whitten and Whittingham medium (BWW) [44] supplemented with 1 mg/mL polyvinyl alcohol (PVA) and 1 mg/mL methyl-beta-cyclodextrin for 1 h at 37˚C and 6% CO2 under mineral oil. Immediately prior to completion of capacitation, aged oocytes were washed four times in human tubal fluid (HTF) medium [41-43] and were 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 4 times in HTF, and incubated overnight to the 2-cell stage. 2-cell embryos were transferred to G1 plus medium (Vitrolife, Göteborg, Sweden) on the morning of day 2, then transferred to G2 plus medium (Vitrolife) on the morning of day 4. The percentage of oocytes which fertilized and reached the blastocyst stage was calculated on the morning of day 5. TUNEL A terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Roche Diagnostics, IN) was conducted on day 5 blastocysts to identify DNA fragmentation related to apoptosis in nuclei. The TUNEL assay was conducted as described previously [45], and blastocysts were analysed using confocal microscopy (Carl Zeiss Laser Scanning Microscope 510, Thornwood, NY, USA). Statistical analyses All experiments were conducted at least 3 times on independent samples and results were analysed by ANOVA using JMP version 9.0.0. A post hoc comparison of group means was conducted using a Fisher’s Protected Least Significant Difference test. Analysis of paired

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samples was conducted using a paired Student’s t-test. A value of P < 0.05 was considered to be statistically significant. RESULTS Oxidative stress precedes the appearance of markers of aging and apoptosis in oocytes in vitro In order 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 48 h in vitro. MII stage 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 exteriorisation 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, achieving 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 it was expected that supplementation with a potent antioxidant compound, that would be stable under the culture conditions employed in this study, would 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 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 2C-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. 72% (±8.4) 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 in vitro culture with melatonin had a rate of 2-cell embryo formation that was 17% above that of the control (P < 0.05; Fig. 3B), while 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 those established from control oocytes. Thus 54% (±10.0) of oocytes aged for 8 h in the presence of melatonin reached the blastocyst stage while fertilization of control

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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 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 prior to insemination were of poor quality, displaying elevated levels of apoptosis (P < 0.05), with 18% (± 4.2) and 16% (± 4.6) of blastomeres being TUNEL positive respectively (Fig. 3D-H), compared to under 7% of blastomeres in embryos formed from fresh oocytes (Fig. 3D,F). Significantly, embryos originating from oocytes aged for the same periods of time in the presence of melatonin did not experience elevated levels of apoptosis, with results showing that the percentage of TUNEL-positive blastomeres was not significantly different in these melatonin-supplemented cultures from the fresh oocyte controls (Fig. 3D,E). A 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 [35], caffeine supplementation provided no relief from oxidative stress following 48 h 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). DISCUSSION With the demand for ART increasing exponentially in recent years, the drive to refine these technologies has become paramount; particularly when considering that under 20% of initiated IVF cycles result in the production of live offspring [46] and that these offspring have a significantly increased risk of possessing birth defects [47]. This study has focused specifically on the contribution of post-ovulatory oocyte aging in vitro to fertilization efficiency and embryonic developmental potential. We have identified oxidative stress as a major contributor to the post-ovulatory oocyte aging process and have found that supplementation of culture media 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 [48]. Our results have demonstrated that the onset of oxidative stress may be a relatively early event associated with in vitro culture, as a significant elevation in ROS levels was 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

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reported decline in GSH levels within the oocyte with time post-ovulation, as well as the absence of antioxidant-rich follicular fluid to provide protection [49]. 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 [52]. The free-radical theory of aging proposes that ROS which are produced by the mitochondria as a by-product 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 [53]. 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 (4HNE) and the latter then forming adducts with enzymes in the mitochondrial electron transport chain, stimulating yet more ROS generation in a self-perpetuating manner [54]. 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 [55]. 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 externalisation) appearing in a simultaneous manner with oxidative stress, while caspases, involved in the subsequent processing of cellular proteins [55], materialize later. In alignment with our research, previous studies have also identified oxidative stress as a factor which is likely to be involved in the aging of post-ovulatory 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 L-ascorbic acid and vitamin E to oocyte culture medium could not improve fertilization rates or embryo development in aged oocytes in vitro, whilst L-cysteine treatment caused a decreased developmental rate to the blastocyst stage [34]. In contrast to previous research, our study has found 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 our study may relate to several key features of this compounds chemistry. Firstly, melatonin is extremely stable in aqueous solution [57], meaning there is no rapid inactivation of its ROS scavenging capabilities 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 [58]. 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 [61]. Additionally, oocytes in oxidative stress have been demonstrated to experience a

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perturbation in calcium homeostasis, resulting in impaired calcium oscillations following fertilization, affecting their capacity for oocyte activation [32] and embryo development [62]. 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 are clearly more likely to default to the intrinsic apoptotic pathway than continue with embryonic development (i.e. will not pass through the G1 checkpoint of the cell cycle to reach S-Phase [63]). 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 this study appears to be directly linked with oxidative stress levels in the oocyte prior to insemination, as 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 [64] and a tendency for early embryos to undergo resorption [65, 66]. Therefore, oxidative stress in the oocyte prior to insemination may not only affect the pre-implantation embryo, but also events post-implantation, and hence the likelihood of producing healthy, live offspring. The final component of this 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 [35], our 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 [67]. It is worth noting that although compounds such as melatonin and caffeine are capable of delaying several aspects of post-ovulatory oocyte aging, a threshold window for development does still exist; 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 organisation. 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 for prolonged incubation periods in vitro prior to IVF/ICSI.

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In conclusion, our research has demonstrated for the first time that by preventing oxidative stress using melatonin supplementation, aspects of the aging process that mouse oocytes undergo post-ovulation 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 when compared to their untreated, aged counterparts. This study also demonstrated that oxidative stress is not only 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 a more effective and safer alternative to caffeine for inhibiting the oocyte aging process in a clinical setting. ACKNOWLEDGEMENTS We are very grateful to the NHMRC for financial support (Grant #494802). REFERENCES 1. Wang Q, Sun QY. Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fertil Dev 2007; 19: 1-12. 2. Yoshida N, Brahmajisyula M, Shoji S, Amanai M, Perry AC. Epigenetic discrimination by mouse metaphase II oocytes mediated asymmetric chromatin remodelling independently of meiotic exit. Dev Biol 2007; 301: 464-477. 3. Torres-Padilla ME, Bannister AJ, Hurd PJ, Kouzarides T, Zernicka-Goetz M. Dynamic distribution of the replacement histone variant H3.3 in the mouse oocyte and preimplantation embryos. Int J Dev Biol 2006; 50: 455-461. 4. Minami N, Suzuki T, Tsukamoto S. Zygotic gene activation and maternal factors in mammals. J Reprod Dev 2007; 53: 707-715. 5. Schultz GA, Heyner S. Gene expression in pre-implantation mammalian embryos. Mutat Res 1992; 296: 17-31. 6. Cummins JM. Mitochondria in reproduction. Reprod Biomed Online 2004; 8: 14-15. 7. Stitzel ML, Seydoux G. Regulation of the oocyte-to-zygote transition. Science 2007; 316: 407-408. 8. Swann K, Yu Y. The dynamics of calcium oscillations that activate mammalian eggs. Int J Dev Biol 2008; 52: 585-594. 9. Miao Y, Kikuchi K, Sun Q, Schatten H. Oocyte aging: cellular and molecular changes, developmental potential and reversal possibility. Human reproduction update 2009; 15: 573-585. 10. Fissore RA, Kurokawa M, Knott J, Zhang M, Smytth J. Mechanims underlying oocyte activation and postovulatory ageing. Reproduction 2002; 124: 745-754. 11. Szolli D. Morphological changes in mouse eggs due to aging in the fallopian tube. Am J Anat 1971; 130: 209-226. 12. Dodson MG, Minhas BS, Curtis SK, Palmer TV, Robertson JL. Spontaneous zona reaction in the mouse as a limiting factor for the time in which an oocyte may be fertilized. J In Vitro Fert Embryo Transf 1989; 6: 101-106. 13. Ducibella T, Duffy P, Reindollar R, Su B. Changes in the distribution of mouse oocyte cortical granules and ability to undergo the cortical reaction during gonadotropin-stimulated meiotic maturation and aging in vivo. Biol Reprod 1990; 43: 870-876. 14. Xu Z, Abbott A, Kopf GS, Schultz RM, Ducibella T. Spontaneous activation of ovulated mouse eggs: time-dependant effects on M-phase exit, cortical granule exocytosis, maternal

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32. Takahashi T, Takahashi E, Igarashi H, Tezuka N, Kurachi H. Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization. Mol Reprod Dev 2003; 66: 143-152. 33. Chaube SK, Prasad PV, Thakur SC, Shirastav TG. Hydrogen peroxide modulates meiotic cell cycle and induces morphological features characteristic of apoptosis in rat oocytes cultured in vitro. Apoptosis 2007; 10: 863-874. 34. Tarin JJ, Ten J, Vendrell FJ, Cano A. Dithtiothreitol prevents age-associated decrease in oocyte/conceptus viability in vitro. Hum Reprod 1998; 13: 381-386. 35. Ono T, Mizutani E, Li C, Yamagata K, Wakayama T. Offspring from intracytoplasmic sperm injection of aged mouse oocytes treated with caffeine or MG132. Genesis 2011; 49: 460-471. 36. Zhang N, Wakai T, Fissore RA. Caffeine alleviates the deterioration of Ca(2+) release mechanisms and fragmentation of in vitro-aged mouse eggs. Mol Reprod Dev 2011; 78: 684-701. 37. Selby CP, Sancar A. Molecular mechanisms of DNA repair inhibition by caffeine. PNAS 1990; 87: 3522-3525. 38. Genesca A, Caballin MR, Miro R, Benet J, Germa JR, Egozcue J. Repair of human sperm chromosome aberrations in the hamster egg. Hum Genet 1992; 89: 181-186. 39. De Iuliis GN, Thomson LK, Mitchell LA, Finnie JM, Koppers AJ, Hedges A, Nixon B, Aitken RJ. DNA damage in human spermatozoa is highly correlated with the efficiency of chromatin remodeling and the formation of 8-hydroxy-2'-deoxyguanosine a marker of oxidative stress. Biol Reprod 2009; 81: 517-524. 40. Kikuchi K, Naito K, Noguchi J, Shimada A, Kaneko H, Yamashita M, Aoki F, Tojo H, Toyoda Y. Maturation/M-phase promoting factor: a regulator of aging in porcine oocytes. Biol Reprod 2000; 63: 715-722. 41. Takeo T, Hoshii T, Kondo Y, Toyodome H, Arima H, Yamamura K, Irie T, Nakagata N. Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells. Biol Reprod 2008; 78: 546-551. 42. Takeo T, Nakagata N. Combination medium of cryoprotective agents containing L-glutamine and methyl-beta-cyclodextrin in a preincubation medium yields a high fertilization rate for cryopreserved C57BL/6 mouse sperm. Lab Anim 2010; 44: 132-137. 43. Takeo T, Nakagata N. Reduced glutathione enhances fertility of frozen/thawed C57BL/6 mouse sperm after exposure to methyl-beta-cyclodextrin. Biol Reprod 2011; 85: 1066-1072. 44. Biggers JD, Whitten WK, Whittingham DG. The culture of mouse embryos in vitro. San Fransisco: W. H. Freeman; 1971. 45. Brison DR, Metcalfe AD, Bloor DJ, Hunter HR, Brady G, Kimber SJ. A laboratory guide to the mammalian embryo. New York: Oxford University Press; 2004. 46. Wang YA, Macaldowie A, Hayward I, Chambers GM, Sullivan EA. Assisted reproductive techonology in Australia and New Zealand 2009. In: AIHW (ed.), 15 ed. Canberra; 2011. 47. Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, Haan EA, Chan A. Reproductive technologies and the risk of birth defects. N Eng J Med 2012; 366: 1803-1813. 48. Koppers A, Mitchell LA, Wang P, Lin M, Aitken RJ. Phosphoinositide 3-kinase signalling pathway involvement in a trunctated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa. Biochem J 2011; 436: 687-698.

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49. Yoshida M, Ishigati K, Nagai T, Chikyu M, Pursel VG. Glutathione concentration during maturation and after fertilization in pig ooctes: relevance to the ability of oocytes to form male pronucleus. Biol Reprod 1993; 49: 89-94. 50. Mass DHA, Storey BT, Mastroianni L. Oxygen tension in the oviduct of the rhesus monkey (Macaca mulatta). Fertil Steril 1976; 27: 1312-1317. 51. Mastroianni L, Jones R. Oxygen tension within the rabbit fallopian tube. J Reprod Fertil 1965; 9: 99-102. 52. Goto K, Noda Y, Mori T, Nakano M. Increased generation of reactive oxygen species in embryos cultured in vitro. Free Radic Biol Med 1993; 15: 69-75. 53. Barja G. Free radicals and aging. Trends Nuerosci 2004; 27: 595-600. 54. Aitken RJ, Whiting S, De Iuliis GN, McClymont S, Mitchell LA, Baker MA. Electrophilic aldehydes generated by sperm metabolism activate mitochondrial reactive oxygen species generation and apoptosis by targeting succinate dehydrogenase. J Biol Chem 2012; 287: 33048-33060. 55. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radic Biol Med 2000; 29: 323-333. 56. Tatone C, Emidio GD, Barbaro R, Vento M, Ciriminna R, Artini PG. Effects of reproductive aging and postovulatory agins on the maintenance of biological competence after oocyte vitrification: insights from the mouse model. Theriogenology 2011; 76: 864-873. 57. Cavallo A, Hassan M. Stability of melatonin in aqueous solution. J Pineal Res 1995; 18: 90-92. 58. Tan DX, Reiter RJ, Manchester LC, Yan MT, El-Sawi M, Sainz RM, Mayo JC, Kohen R, Allegra M, Hardeland R. Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger. Curr Top Med Chem 2002; 2: 181-197. 59. Reiter RJ, Calvo J, Karbownik M, Qi W, Tan DX. Melatonin and its relation to the immune system and inflammation. Ann N Y Acad Sci 2000; 917: 376-386. 60. Reiter RJ, Tan DX, Acuna-Castroviejo D, Burkhardt S, Karbownik M. Melatonin: Mechanisms and actions as an antioxidant. Curr Top Biophys 2000; 24: 171-183. 61. Tarin JJ. Potential effects of age-associated oxidative stress on mammalian oocytes/embryos. Mol Hum Reprod 1996; 2: 717-727. 62. Takahashi E, Igarashi H, Kawagoe J, Amita M, Hara S, Kurachi H. Poor embryo development in mouse oocytes aged in vitro is associated with impaired calcium homeostasis. Biol Reprod 2009; 80: 493-502. 63. Menezo Y, Dale B, Cohen M. DNA damage and repair in human oocytes and embryos: a review. Zygote 2010; 18: 357-365. 64. Hardy K, Handyside AH, Winston RML. The human blastocyst: cell number, death and allocation during late preimplantion development in vitro. Development 1989; 107: 597-604. 65. Chi M, Pingsterhaus J, Carayannopoulos M, Moley KH. Decreased glucose transporter expression triggers BAX-dependant apoptosis in the murine blastocyst. J Biol Chem 2000; 275: 40252-40257. 66. Wuu YD, Pampfer S, Becquet P, Vanderheyden I, Lee KH, De Hertogh R. Tumor necrosis factor alpha decreases the viability of mouse blastocysts in vitro and in vivo. Biol Reprod 1999; 60: 479-483.

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67. Jahnke G, Marr M, Myers C, Wilson R, Travlos C, Price C. Maternal and developmental toxicity evaluation of melatonin administration orally to pregnant Sprague-Dawley rats. Toxicol Sci 1999; 50: 271-279. FIGURE LEGENDS Figure 1: Changes associated with post-ovulatory oocyte aging. A) From 8 h in vitro culture time onwards oocytes experienced a significant increase in ROS levels as detected using the carboxy-DFFDA fluorescent probe (P<0.001); ordinate axis represents percentage of the control value at 1 h. By 48 h culture time, levels of oxidative stress were increased by over 200% above the 1 h fresh oocyte control (n=18). Images below histogram depict a carboxy-DFFDA negative oocyte (1 h culture time) and an oxidatively stressed oocyte showing high levels of carboxy-DFFDA fluorescence at 48 h culture time. B) The percentage of Annexin-V positive, PI-negative oocytes increased in a time-dependent manner from oocyte retrieval. Less than 5% of oocytes were Annexin-V positive 1 h after retrieval, however this was significantly increased to 19.5% by 8 h and to over 50% by 48 h (P < 0.001) (n=5). Images below histogram depict staining patterns seen in Annexin-V negative (1 h culture time) and positive oocytes (48 h aging). C) A significant increase in levels of caspase activation was also detected in aging oocytes from 24 h (P < 0.05) and 48h (P < 0.001) ex vivo; ordinate axis represents percentage of the control value at 1 h (n=13). Images below histogram demonstrate low levels of caspase activity at 1 h culture time, and a 48 h aged oocyte showing high levels of fluorescence related to caspase activation. D) The percentage of oocytes which exhibited abnormal morphology (examples demonstrated in accompanying images) related to aging was elevated significantly from 1 h to 24 and 48 h culture time (P < 0.001) (n=12). Mean ±SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 40 oocytes per replicate. Bars = 50 µm. Figure 2: A reduction in oocyte aging is achieved with supplementation of 1 mM melatonin to culture medium. A) Melatonin supplemented oocytes which had been aged for 48 h in vitro showed a 40% reduction in ROS levels when compared to control aged oocytes (P < 0.05); ordinate axis represents percentage of the control value at 1 h (n=8). B) Melatonin significantly decreased levels of caspase activation in 48 h aged oocytes suggesting that the onset of apoptosis had been delayed (P < 0.05) (n=10). C) The percentage of oocytes which acquired abnormal morphology over time was significantly reduced below the control in the presence of melatonin after both 24 h and 48 h culture (P < 0.001) (n=15). D) Heavy cytoplasmic fragmentation could be visualized in control oocytes following extended periods of aging (> 48 h), while oocytes supplemented with melatonin during this time retained relatively normal morphology (E). Bar = 50 µm. Mean ±SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 50 oocytes per replicate. Figure 3: Embryo quality and oocyte ageing. A) Oocytes aged for 8 h in the presence of melatonin produced a significantly higher percentage of 2-cell embryos (P < 0.05), 4-cell embryos (P < 0.01) and blastocysts (P < 0.01) when compared to oocytes aged for the same period of time in control medium (n=8). B) A similar trend was detected in oocytes aged for 16 h in vitro, with melatonin supplemented oocytes producing an elevated percentage of 2-cell embryos (P < 0.05) and blastocysts (P < 0.01) (n=5). C) Following 24 h aging, all oocytes had lost the developmental competence to reach the blastocyst stage following fertilization, however a significantly increased percentage of melatonin treated oocytes were able to reach the 2-cell

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stage when compared to the aged oocyte control (P < 0.05) (n=4). D) The percentage of apoptotic nuclei (as detected by TUNEL staining) within blastomeres was found to be elevated in control-aged oocytes compared with melatonin-supplemented oocytes at both 8 h and 16 h (P < 0.05). E) A TUNEL negative blastocyst originating from an oocyte aged for 8 h in the presence of melatonin. F) A blastocyst originating from an 8 h aged control oocyte showing TUNEL positive blastomeres (white arrows). G) Negative control for TUNEL staining. H) Positive control for TUNEL staining (1 mg/ml DNase). Bar = 50 µm. Mean ±SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 100 oocytes per replicate. Figure 4: A comparison between the abilities of melatonin and caffeine to affect oocyte aging. A) Melatonin supplemented oocytes showed a significant reduction in ROS levels below the control (P < 0.05) while caffeine-supplemented oocytes displayed ROS levels similar to that of the control which could only be reduced with the combined supplementation of melatonin (P<0.05) (n=3). B) Caffeine supplementation also failed to decrease levels of caspase activation below the control in 48 h aged oocytes, whilst melatonin did significantly delay apoptosis in terms of caspase activity (P < 0.05) (n=7). Mean ±SEM values are plotted in histograms. Independent replicates were conducted with a minimum of 50 oocytes per replicate.