Inhibitory effects of astaxanthin on postovulatory porcine oocyte aging in vitro

Mammalian oocytes represent impaired quality after undergoing a process of postovulatory aging, which can be alleviated through various effective ways such as reagent treatment. Accumulating evidences have revealed the beneficial effects of astaxanthin (Ax) as a potential antioxidant on reproductive biology. Here, porcine matured oocytes were used as a model to explore whether Ax supplement can protect against oocyte aging in vitro and the underlying mechanism, and therefore they were cultured with or without 2.5 μM Ax for an additional 24 h. Aged oocytes treated with Ax showed improved yield and quality of blastocysts as well as recovered expression of maternal genes. Importantly, oxidative stress in aged oocytes was relieved through Ax treatment, based on reduced reactive oxygen species and enhanced glutathione and antioxidant gene expression. Moreover, inhibition in apoptosis and autophagy of aged oocyte by Ax was confirmed through decreased caspase-3, cathepsin B and autophagic activities. Ax could also maintain spindle organization and actin expression, and rescue functional status of organelles including mitochondria, endoplasmic reticulum, Golgi apparatus and lysosomes according to restored fluorescence intensity. In conclusion, Ax might provide an alternative for ameliorating the oocyte quality following aging in vitro, through the mechanisms mediated by its antioxidant properties.

www.nature.com/scientificreports/ of SCNT embryos 26 or the in vitro growth of oocytes derived from early antral follicles 27 . In humans, treatment with Ax can enhance sperm capacitation 28 and prevent papillomavirus L1 protein binding in sperm membrane 29 . However, it is unknown whether Ax can protect the oocytes against aging in vitro. Porcine oocytes provide a more ideal model for the investigation of reproductive biology, as they have developmental and physiological similarities to human oocytes 30 . Therefore, the present study was designed to determine the cytoprotective effect of Ax on porcine oocytes during aging in vitro, and explore the underlying cellular and molecular mechanisms.

Results
Ax improves developmental competence of aged porcine oocytes. At the end of in vitro maturation (IVM), cumulus cells were removed by repeated pipetting in Tyrode's lactate-HEPES-polyvinyl alcohol (TLH-PVA) medium 31 containing 0.1% (w/v) hyaluronidase, and oocytes with evenly granular cytoplasm and a first polar body were selected for the following experiments. For aging in vitro, oocytes were cultured in porcine zygote medium-3 (PZM-3) 32 supplemented with or without 2.5 μM Ax (Aged + Ax and Aged groups, respectively) for an additional 24 h at 39 °C in an atmosphere of 5% CO 2 with saturated humidity. The optimal concentration of Ax was based on our preliminary experiments. The fresh oocytes without any prolonged culture were used as control (Fresh group).
In this experiment, we determined whether Ax could improve the developmental competence of aged oocytes following parthenogenetic activation (PA). As shown in Fig. 1A-C, percentages of cleavage and blastocyst formation were significantly lower in the Aged group (68.2 ± 3.1% and 28.3 ± 3.3%, respectively) than in the Fresh group (88.7 ± 2.1% and 55.7 ± 2.3%, respectively), while the Aged + Ax group increased the cleavage and blastocyst formation rates to 80.4 ± 2.5% and 46.2 ± 2.9%, respectively. There was a significant decrease in blastocyst diameter in the Aged group (147.1 ± 4.2 μm) when compared to the Fresh (185.5 ± 5.1 μm) and Aged + Ax (166.2 ± 4.6 μm) groups (Fig. 1D). Moreover, the Aged group (35.3 ± 4.3) also showed a significant reduction in total cell number per blastocyst than the Fresh (52.6 ± 3.9) and Aged + Ax (44.1 ± 4.5) groups (Fig. 1E).
To further confirm the effects of Ax on the quality of aged oocytes and resultant blastocysts, quantitative realtime reverse transcription polymerase chain reaction (qRT-PCR) was submitted to detect the mRNA expression of oocyte maturation and/or embryo development related genes (C-MOS, CCNB1, BMP15, GDF9, ZAR1, PCNA, CDX2, POU5F1, DNMT3A and uPA). As shown in Fig. 1F, the mRNA levels of C-MOS, CCNB1, BMP15, ZAR1, CDX2 and POU5F1 in oocytes were significantly lower in the Aged group than in the Fresh group, but no differences in gene expression for GDF9, PCNA and DNMT3A. Compared with the Aged group, the Aged + Ax group had significantly up-regulated mRNA expression of these genes, except for ZAR1 and PCNA. For the resultant blastocysts, the mRNA levels of PCNA, CDX2, POU5F1, DNMT3A and uPA were similar in the Fresh and Aged + Ax groups, but CDX2 and POU5F1 showed significantly decreased expression in the Aged group (Fig. 1G).
Ax alleviates oxidative stress in aged porcine oocytes. For evaluating the efficacy of Ax in reducing oxidative stress, we first measured the levels of intracellular ROS including hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 •-) with the fluorescent probes of CM-H 2 DCFDA and Dihydroethidium, respectively. As shown in Fig. 2A-C, the levels of H 2 O 2 and O 2 •were significantly higher in the Aged group than in the Fresh group. In the Aged + Ax group, the two values decreased significantly compared to these in the Aged group. Reduced glutathione (GSH) is an important intracellular antioxidant component for counteraction of oxidative stress injury, and so GSH level was subsequently detected by ThiolTracker Violet probe. Surprisingly, intracellular GSH level did not differ between the Fresh and Aged groups, and was significantly lower compared to that in the Aged + Ax group ( Fig. 2A,D). In addition, we also examined intracellular nitric oxide (NO) production by DAF-FM Diacetate, because of its responsibility for oxidative stress. Both the Aged and Aged + Ax groups showed similar level of NO, but significantly higher compared to the fresh group ( Fig. 2A,E).
Finally, we determined the mRNA expression of antioxidant and oxidative stress related genes (SOD1, SOD2, GPX4, CAT , SIRT1 and NRF2) by qRT-PCR analysis. Compared to the Fresh group, the Aged group showed significantly decreased mRNA levels of GPX4, CAT and SIRT1, but had a similar expression for SOD1, SOD2 and NRF2 genes (Fig. 2F). As expected, the mRNA levels of these genes except for GPX4 were significantly increased in the Aged + Ax group than in the Aged group (Fig. 2F).
Ax inhibits apoptosis and autophagy in aged porcine oocytes. It is generally known that a series of cellular apoptotic events occur during oocyte aging in vitro. Caspase-3 is a key mediator of apoptosis; therefore its activity was measured using a CaspGLOW Fluorescein Active Caspase-3 Staining Kit. As shown in Fig. 3A,B, the caspase-3 activity in the Aged group was significantly higher than that in the Fresh group, and the Aged + Ax group significantly decreased this level. In addition, cathepsin B as a lysosomal cysteine protease ascribes a proapoptotic function. We next quantified the cathepsin B activity by Magic Red Cathepsin B Assay Kit. Notably, the cathepsin B activity was significantly decreased in the Aged + Ax group compared with the Aged group, similar to the Fresh group (Fig. 3A,C). Furthermore, the mRNA expression of apoptosis related genes (CASP3, CSTB, BAD, BCL2L1 and SURVIVIN) were also analyzed. qRT-PCR results showed that the mRNA levels of these genes in the Aged group were significantly decreased than those in the Fresh group, and the Aged + Ax group had significantly increased mRNA levels for CSTB, BCL2L1 and SURVIVIN when compared with the Aged group (Fig. 3E).
On the other hand, autophagy is reported to be involved in the process of oocyte aging. As Ax can modulate autophagy by regulating signaling pathways, we examined whether it continued to inhibit the autophagy in aged oocytes. Firstly, the autophagic activity was measured by a CYTO-ID Autophagy Detection Kit. As shown in Fig. 3A,D, the Aged group presented a significantly higher autophagic activity compared to the Fresh group, and www.nature.com/scientificreports/ the level was remarkably reduced in the Aged + Ax group. Moreover, the autophagy related genes (ATG5, ATG7, LAMP2 and LC3) of the Aged group also showed significantly lower mRNA expression compared to those of the Fresh group. Expectedly, the mRNA levels of ATG5 and LAMP2 were significantly higher in the Aged + Ax group than in the Aged group (Fig. 3F).
Ax rescues spindle defects and actin filaments in aged porcine oocytes. Cytoskeleton abnormality has been confirmed during oocyte aging in vitro. So, we detected spindle morphology and actin expression of the aged oocytes following Ax treatment. As shown in Fig. 4A, most fresh oocytes displayed a typical barrel-shaped spindle with a well-aligned chromosome. However, various forms of abnormal spindles such as condensation, elongation, dispersal, or disruption occurred in aged oocytes, and chromosome failed to align at the metaphase plate. The percentage of oocytes with abnormal spindles was significantly lower in the Aged + Ax group (20.7 ± 1.5%) than in the Aged group (32.8 ± 2.1%), and was still significantly higher compared to the Fresh group (8.2 ± 1.1%) (Fig. 4B). Actin filaments in oocytes were distributed mainly in the cortical region with accumulating strong signals (Fig. 4C). Moreover, the relative fluorescence intensity of actin filaments was www.nature.com/scientificreports/ significantly decreased in the Aged group compared with the Fresh group, and could be markedly increased in the Aged + Ax group (Fig. 4D).
Ax maintains organelle functional status in aged porcine oocytes. Aging often results in disturbances in morphology and function of intracellular organelles, thus impairing oocyte quality. Next, we intended to explore the beneficial effects of Ax on organelles in the aged oocytes, including mitochondria, endoplasmic www.nature.com/scientificreports/ reticulum (ER), Golgi apparatus and lysosomes. These organelles were labeled through respective fluorescent probes. The different values of fluorescence intensity can represent the distinction in organelle functional status. The relative fluorescence intensity showed that the values of mitochondria, ER and Golgi apparatus were significantly decreased in the Aged group compared with the Fresh group; meanwhile, these values were considerably improved in the Aged + Ax group (Fig. 5A-D). On the other hand, the relative fluorescence intensity of lysosomes in the Aged group were significantly higher than that in both the Fresh and Aged + Ax groups (Fig. 5A,E).

Discussion
Oxidative stress results from an imbalance of oxidant and antioxidant, which triggers a cascade of unfavorable factors to directly affect multiple aspects of biochemistry and functionality in the aged oocytes 33,34 . Moreover, the antioxidant Ax has been proved to alleviate oxidative stress in various cell types, including sperms, oocytes www.nature.com/scientificreports/ and embryos. In the present study, we revealed that Ax was able to delay oocyte aging in vitro, owing to possible mechanisms mediated by reducing oxidative stress. It is well known that aged oocytes exhibit a very poor ability to fertilization and embryo development, and the quality of resultant blastocysts is also compromised 35,36 . For identifying the beneficial effects of Ax on aged oocytes, the most straightforward way is to evaluate their developmental competence. In this study, the blastocyst yield, and diameter and total cell number per blastocyst were improved when the oocytes were treated with Ax during aging in vitro, suggesting that Ax could enhance the capacity of embryos to develop to the blastocyst stage and the embryo quality. As we know, the maternal gene in oocytes is required for preimplantation embryo development 37 . Differences in maternal gene expression lead to the lower developmental potential of aged oocytes. For instance, ZAR1 plays essential role in transition from oocyte to embryo 38 , and its expression in this study was found to be reduced in aged oocytes. Furthermore, Ax treatment was helpful to maintain the maternal gene expression for C-MOS, CCNB1, BMP15, CDX2 and POU5F1 in aged oocytes. Our results also showed that the mRNA levels of CDX2 and POU5F1 genes were also increased in resultant blastocysts from Ax-treated aged oocytes. These two genes are required for differentiation of trophectoderm during early embryo development 39 . Therefore, the above findings provided the most direct proof of the beneficial effects of Ax for aged oocytes.
Currently, the adverse effects of oxidative stress occurred in aged oocytes have been well documented. The main reason for the oxidative stress is that aged oocytes often exhibit high levels of intracellular ROS. Ax acts as a scavenger of free radicals working on singlet oxygen, H 2 O 2 , O 2 •-, hydroxyl radical and reactive nitrogen species 40 . In the present study, Ax could effectively reduce the levels of H 2 O 2 and O 2 •in aged oocytes. In addition, we found that an accumulation of NO occurred in aged oocytes, but Ax treatment did not decrease its level, unlike in other cell types. GSH as an endogenous antioxidant not only maintain the intracellular redox balance, but also is an indicator of cytoplasmic maturation to reflect the oocyte fertilization and subsequent development 41 . There was no reduction of GSH content for aged oocytes in this study, similar to previous reports 42,43 ; however, several studies have showed the aged oocytes with lower level of GSH 44,45 . Moreover, the aged oocytes treated with Ax exhibited an increase in intracellular GSH content, which is similar to the result of antioxidant hesperetin treatment 42 . The genes related to antioxidation pathway are frequently detected to evaluate the oxidative stress in aged oocytes, but different results have been obtained due to different experimental conditions. It has been confirmed that Ax can play a major role in the modulation of redox signaling pathways such as SIRT1 and NRF2, suppressing oxidative damage from various stress conditions 46 . We found that the oocyte aging in vitro resulted www.nature.com/scientificreports/ in decreased mRNA levels of GPX4, CAT and SIRT1, but did not affect the SOD1, SOD2 and NRF2 expression. These genes except for GPX4 in aged oocytes showed increased mRNA expression after treatment with Ax. In a word, Ax exhibited a better radical scavenging capacity to attenuate oxidative stress during oocyte aging in vitro. Oxidative stress can lead to apoptosis through numerous mechanisms and then cell death. The degree of apoptosis has been confirmed to be increased in aged oocytes 9 . Caspase-3 acts as a key promoter of apoptosis, because all apoptotic pathways eventually converge on the caspase-3 activation 47 . Cathepsin B is closely associated with the process of apoptosis by activating caspases 48 . Similar to previous studies 44, 49 , we also observed a significant rise in the activities of caspase-3 and cathepsin B during oocyte aging in vitro. In addition, Ax is known to be able to regulate the mitochondria-mediated apoptosis probably caused by signaling pathways such as NF-κB and Wnt/β-catenin, involving a downregulation of pro-apoptotic proteins (eg. Bax and cleaved caspase-3) and an upregulation of anti-apoptotic proteins (eg. Bcl-2 and survivin) 50,51 . Treatment with Ax has also been reported to decrease the cathepsin B activity 27 . The present study found that the activities of these two proteases were efficiently inhibited in aged oocytes following Ax treatment. On the other hand, autophagy is induced to enable cellular survival under different stress conditions, but it also plays the functions in cell death 52 . It has been reported that the aging process of oocytes is accompanied by autophagy activation 53 . Several studies have demonstrated the potential of Ax in regulating autophagy 54 . Expectantly, Ax could meliorate the autophagic activity during oocyte aging in vitro. Our study also found that the aged oocytes treated with Ax exhibited a relatively stable expression for some genes related to apoptosis and autophagy, including CSTB, BCL2L1, SUR-VIVIN, ATG5 and LAMP2. Thus, the above results indicated the effective inhibition of Ax on apoptosis and autophagy in aged oocytes. www.nature.com/scientificreports/ The spindle as an important structure for accurate chromosomal distribution, is correlated with coordination interaction between the actin and microtubule cytoskeleton. The aged oocytes often present with spindle disruption and actin reduction 55 . In the present study, Ax treatment attenuated the spindle abnormality and restored the actin expression for aged oocytes, indicating its protection on cytoskeletal stability. On the other hand, cytoplasmic maturation of oocytes is dependent on the dynamic distribution and functional integrity of organelles 56 . The functional state changes in any kind of organelles including mitochondria, ER, Golgi apparatus and lysosomes can affect oocyte quality and early embryonic development. These oocyte organelles are especially vulnerable to oxidative stress. Still oocyte aging in vitro results in the abnormal distribution and function disorder of cytoplasmic organelles. For example, the most widely studied organelle is mitochondria, with confirmed dysfunction in aged oocytes such as low membrane potential, reduced ATP content and mutated mitochondrial DNA 57,58 . The aged porcine oocytes has been reported to exhibit disordered ER clusters and impaired ER calcium homeostasis 59 . In the present study, oocyte organelles were label with specific fluorescent probes, and the change in quantification of fluorescence intensity might reflect organelle status. The mitochondria, ER and Golgi apparatus in aged oocytes showed a decrease in the fluorescence intensity, indicating their functional decline. In addition, the fluorescence intensity of lysosomes was elevated in aged oocytes, which seemed to be associated with increased autophagic activity as described above. Because autophagy is a highly conserved lysosomedependent pathway 60 . We further found that Ax was able to effectively recover the fluorescence intensity of these organelles in aged oocytes, indicating powerful amelioration of dysfunctional organelles. The beneficial effects of Ax on various subcellular organelles may be attributed to its antioxidant properties, role as signaling molecule and gene regulator that are involved in the regulation of organellar functions such as mitochondrial redox state and ER stress 61,62 . In brief, these results suggested the positive effects of Ax treatment on cytoplasmic maturation for aged oocytes from the perspective of cytoskeleton and organelle status.
Taken all together, our results demonstrated that Ax treatment could ameliorate the quality and developmental competence of aged porcine oocytes, which was probably at least partly attributable to the alleviation of oxidative stress, apoptosis and autophagy, and the stabilization of cytoskeleton and organelle status. This study provides new insights for the protective role of Ax against oocyte aging in vitro, and so will be quite helpful for ART.

Materials and methods
Reagents. All chemicals used in this study were purchased from Sigma-Aldrich Chemical Company Oocyte collection and IVM. The procedures and medium used for oocyte collection and IVM were essentially as described by us previously 63 . The porcine ovaries collected from pre-pubertal gilts at a local slaughterhouse (Shennong abattoir; Kunming, China) and transported to our laboratory at 35-37 °C in 0.9% NaCl (w/v) containing 75 mg/L penicillin G potassium and 50 mg/L streptomycin sulfate. Follicular fluid from the follicles (3-8 mm in diameter) was aspirated using a disposable syringe with an 18-gauge needle and placed in a conical tube to allow cumulus-oocyte complexes (COCs) to settle at the bottom. The precipitates containing COCs were washed twice in TLH-PVA medium and transferred to a petri dish. The COCs with compact cumulus cells and uniform cytoplasm were picked under a stereomicroscope (Olympus, Tokyo, Japan) and washed three times with IVM medium. Then, a group of 50-70 COCs was placed in each well of a 24-well plate (Costar, Corning, NY, USA) containing 500 μL IVM medium for 42-44 h at 39 °C in an atmosphere of 5% CO 2 with saturated humidity. The IVM medium was TCM-199 supplemented with 10% (v/v) porcine follicular fluid, 3.05 mM D-glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 10 ng/mL epidermal growth factor, 0.5 μg/mL each follicle-stimulating hormone and luteinizing hormone.
PA and embryo culture. Oocyte activation and embryo culture were conducted as previously reported 64 .
Those chosen oocytes were placed between two wires of a microslide 0.5 mm fusion chamber (Model 450; BTX, SanDiego, CA, USA) covered with activation medium (0.28 M mannitol, 0.1 mM MgSO 4 , 0.05 mM CaCl 2 and 0.5 mM HEPES) and then stimulated with a direct current pulse of 1.3 kV/cm for 80 μs using a BLS CF-150/B cell fusion machine (BLS, Budapest, Hungary). The oocytes were washed three times in PZM-3 supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide and incubated in the same medium for 4 h. Finally, they were cultured in PZM-3 at 39 °C in a humidified atmosphere with 5% CO 2 . Percentages of cleavage and blastocyst formation were recorded on Days 2 and 6, respectively (day of PA was designated Day 0). Blastocyst diameter was measured using an inverted microscope (IX71; Olympus, Tokyo, Japan) equipped with a CCD camera. Total cell number per blastocyst was examined by 10 μg/mL Hoechst 33,342 diluted in DPBS containing 0.3% (w/v) polyvinyl alcohol (DPBS-PVA). www.nature.com/scientificreports/ Statistical analysis. Images of oocytes stained in the same dye were captured with the same scan settings.
The average value of fluorescence intensity in each oocyte was quantified after deduction of the background fluorescence through ImageJ software (National Institutes of Health, Bethesda, MD, USA). Fluorescence values for both Aged and Aged + Ax groups were determined relative to that of the Fresh group, and Fresh group value was randomly set to 1. Data were evaluated by ANOVA (SPSS 20.0 software, SPSS Inc., Chicago, IL, USA) followed by Student-Newman-Keuls test for multiple comparisons, and percentage data such as cleavage and blastocyte formation rates were arcsine transformed prior to analysis. All results were presented as least squares mean ± SEM values, and the P-value < 0.05 was defined as significant.

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.