Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

In vitro culture (IVC) of bovine embryos is one of the most important processes in the development of assisted reproductive techniques due to the fact that postfertilization culture conditions can dramatically alter the quality of the resulting blastocysts 1,2 . In vitro, gametes and embryos are exposed to spatial and temporal unnatural conditions, whose scope is not completely known 3 . Although many improvements have been made, in vitro culture systems are still not as efficient as in vivo embryo production 4 . In cattle, the proportion of embryos reaching the blastocyst stage is around 30-40% 5 and are often compromised in quality and competence manifested by a darker morphology 1 or altered gene expression patterns 6 when compared to their in vivo counterparts. The factors that most influence the quality of the embryos are the conditions after fertilization; which include physicochemical (temperature, osmolality, and pH), oxidative (antioxidant balance), and energetic (production, utilization, and storage) stresses 6 .
Under in vitro conditions, the dynamics of embryo development and the kinetics of cleavage are related to the subsequent developmental stages: the faster-cleaved embryos have a higher chance to develop to the blastocysts stage 7 . Therefore, the morphological and metabolic changes that occur during the first 4 days of preimplantation development of the bovine embryo are the most important; besides, during this same period the embryonic genome activation (EGA) occurs 2,8 . At the start of early embryogenesis, all mRNAs and proteins controlling development are of maternal origin, and as development progresses, these reserves gradually degrade while

Nobiletin during MN EGA or MJ EGA increases the quality of in vitro produced blastocysts.
Only the experimental groups that showed better blastocyst yield in the previous experiment (Nob5 and Nob10 during MN EGA or MJ EGA ) were used for embryo quality evaluation in comparison with both control groups (Control and C DMSO ).
The mitochondrial activity was higher (P < 0.001) in blastocysts from Nob5 and Nob10 groups, from either MN EGA or MJ EGA phase, compared with both control groups (Fig. 2).  54- www.nature.com/scientificreports/ When analyzing the lipid content, we observed that the total area of lipid droplets in blastocysts resulting from treatments during MN EGA or MJ EGA was significantly reduced (P < 0.001) in Nob5 and Nob10 groups compared with the control groups (Fig. 3).
Gene expression in ≥ 16-cell embryos and blastocysts produced with nobiletin during MJ EGA . The expression level of CDK2, H3-3B and NFE2L2 transcripts was significantly increased in 16-cell stage embryos from Nob10 group compared to Nob5 and both control groups. While the expression of GPX1 gene was higher in Nob5 and Nob10 compared to control groups (P < 0.05) (Fig. 5a). In blastocysts the expression of PPARα was significantly higher in Nob10 group compared to all other groups (P < 0.05), while CDK2 and GPX1 were upregulated in both nobiletin groups compared with controls (P < 0.05) (Fig. 5b). No significant differences were observed for PPARGC1A, PPARα, RPS6KB1 and H3-3A in 16-cell stage embryos, and for PPARGC1A, RPS6KB1, H3-3B, H3-3A and NFE2L2 in blastocysts.

Nobiletin during MN EGA or MJ EGA increases AKT phosphorylation in blastocysts produced in vitro.
Immunofluorescence analysis revealed immunoreactive proteins for p-AKT in bovine blastocysts. In Day 7 blastocysts, AKT increased its phosphorylation levels when nobiletin was present in the culture medium (Nob5 and Nob10 groups) during MN EGA or MJ EGA . While p-AKT levels were weaker in blastocysts produced from control groups during MJ EGA phase (Fig. 6).
Similarly, the western blot analysis showed that both p-AKT-Thr308 and p-AKT-Ser473 phosphorylation levels were significantly higher in blastocysts produced with nobiletin supplementation (Nob5 and Nob10) during MN EGA phase when compared with control groups (P < 0.05) (Fig. 7a-c). A similar pattern was observed in response to nobiletin treatment during MJ EGA , as p-AKT-Thr308 and p-AKT-Ser473 phosphorylation levels were significantly higher in blastocysts produced with Nob5 and Nob10 compared with control groups (P < 0.05) ( Fig. 7d-f).

Discussion
Under in vivo conditions, cells have antioxidants levels in equilibrium and possess physiological mechanisms to hinder excessive free radical formation 20 . During in vitro culture this mechanism suffers disturbances, in which the redox balance is altered with an increase in the production of free radicals and, as a consequence, a decrease in embryo development 6 . Several studies, aiming to identify the most effective antioxidants to reduce the alteration of the redox balance and ROS levels during the in vitro production of embryos, have shown that the addition of quercetin, resveratrol, vitamin C or carnitine to the culture media have beneficial effects on early embryonic development 15,21 . To our knowledge, the present study is the first that investigates the antioxidant effects of nobiletin supplementation in the culture medium during the two main phases of EGA (MN EGA : minor activation from 2-to 8-cell stage and MJ EGA : major activation from 8-to 16-cell stage) 2,8 in bovine embryo developmental competence in vitro and quality of the produced blastocysts, as well as its possible interaction with the AKT signaling pathway.
Irrespective of concentration, addition of nobiletin to culture media during MN EGA phase (21-54 hpi) did not affect cleavage rates at 54 hpi as well as the percentage of embryos reach the 8-cell stage but increased blastocyst production, whereas nobiletin supplementation in culture media during MJ EGA phase (54-96 hpi) significantly increased the percentage of embryos that reach the16-cell stage and blastocyst production. Several studies have shown that during EGA the bovine embryo actively synthesize transcription factors and this process directly links to chromatin changes, protein allocation, nuclear reorganization and cell proliferation 8,22 . Since in our results developmental kinetics were stimulated with more 16-cell embryos by nobiletin during MJ EGA , we could hypothesize that nobiletin activates early embryonic genes important for the proper genomic function of the embryo during major EGA. Although with our experimental design we cannot link this effect specifically with either of the two activation phases of the embryonic genome.
The evidence that nobiletin supplementation improves blastocyst production is in line with other studies showing increased embryo development in vitro when culture medium was supplemented with biological antioxidants similar to nobiletin 16,23,24 . Another effect of nobiletin was to induce a significant increase in mitochondrial activity and a lower content of lipid droplets in blastocysts from both EGA phases analyzed. Mitochondria play a central role in the generation of adenosine triphosphate (ATP), so, they are considered as energy control units necessary for cell division, pluripotency and differentiation 25  www.nature.com/scientificreports/  www.nature.com/scientificreports/ and alter their efficiency to respond to oxidative phosphorylation 6 . Mitochondria also sense changes in redox potential and force embryos to adapt versus the decreased production of ATP by oxidative phosphorylation during the transition from morula to blastocyst 6,25 . Besides, some studies reported that changes in mitochondrial activity may affect the development of energetic metabolism in the embryo, in terms of availability of glucose, lipids, amino acids and DNA methylation 6,26 . Although the nobiletin action mechanism in mitochondria has not been fully elucidated, in a previous study we observed that increased oocyte mitochondrial activity was related to the cytoprotective effects of nobiletin and its intrinsic ROS-scavenging property 18 . Nevertheless, the effect in the blastocyst could be explained based on the fact that nobiletin is a hydrophobic compound, which easily penetrates through cell membranes directly affecting mitochondrial bioenergetics. Nobiletin can modify intramitochondrial proteins (e.g. acetylated proteins localized within the mitochondria in the brain of rats) 27 or alter the mitochondrial membrane potential by changing the activities of mitochondrial enzymes, like succinate dehydrogenase and cytochrome c oxidase as it has been demonstrated in human blood lymphocytes 28 . However, to verify if this mechanism occurs in bovine blastocysts, further investigation is necessary. Lipid content is a crucial factor for early embryo development in vitro in bovine since energy metabolism is abnormal under such conditions, resulting in an excessive accumulation of lipids associated with reduced embryonic quality 29 . Lipids are stored in intracellular droplets and are metabolized via β-oxidation in the mitochondrial matrix. A large amount of lipid droplets increases the production of ATP necessary for the formation of blastocysts but this can affect its quality; thus, a lower number of lipid droplets in blastocysts is considered as a criterion of good quality embryos 16 . Our results showed for both EGA phases, nobiletin supplementation in culture medium reduced the amount of lipids in blastocysts. Furthermore, we analyze the expression of peroxisome proliferator-activated receptor alpha transcript (PPARα), belonging to one of the 3 key nuclear receptors www.nature.com/scientificreports/ in the modulation of transcription for lipid metabolism-related genes 30 . PPARα was previously detected in cattle embryos and has been associated with embryo quality 31 . In our study, PPARα was significantly upregulated in blastocysts produced with both concentrations of nobiletin supplementation during MN EGA phase or 10 µM of nobiletin supplementation during MJ EGA compared to controls. These results together are in line with other studies which demonstrated that antioxidant supplementation in IVC medium, like crocetin 23 and L-carnitine 21 , improved embryo quality by decreasing their lipid content. Regarding nobiletin, studies in mice showed its ability to reduce hepatic lipid accumulation, prevent lipoprotein overproduction and normalize insulin sensitivity when supplied in the diet 32 . Moreover, it has been demonstrated that nobiletin reduces lipid accumulation and regulates lipidic metabolism in hepatic cell lines 17,33 . There is evidence that nobiletin upregulates the expression of PPARα in white adipose tissue of mice 17 . An explanation for the reduction of lipids by nobiletin has been proposed indicating that full methoxylation of the A-ring of nobiletin seems to be the most optimal structure to express potent effects on modulating hepatic lipid metabolism via primarily suppressing lipoprotein secretion in HepG2 cells 33 . Therefore, it appears that the ability of nobiletin to reduce lipid content and improve mitochondrial activity in blastocysts may be related to the properties of its chemical structure that allows modulation of lipid metabolism and mitochondrial activity. Moreover, activation of PPARα by nobiletin could result in increased embryo lipid turnover through the β-oxidative pathway, preventing accumulation of lipoperoxides despite peroxisomal induction. Recent studies have shown that response of embryos to IVC involves a variety of metabolic factors that act as signals of extracellular and intracellular conditions to which the early embryos can adapt cell programming, signaling pathways, mitochondrial metabolism (mitochondrial production of Acetyl-Coenzyme A (Acetyl-Co A) and methyl groups, which are dependent on the availability of glucose, lipids and amino acids) or peroxisome proliferator-activated receptors (PPARs) in response to lipid content. These factors in the embryo are translated into effects on developmental speed or epigenetic modifications 6,34,35 . Consequently, these results  www.nature.com/scientificreports/ can reinforce the antioxidant-defense role of nobiletin during early embryo development in vitro in bovine and could indicate an improvement of the quality of the produced blastocysts. Embryo cell number is a parameter correlated with embryonic development and quality. Also, it has been reported for different cellular lines (MOLT-4, HUVEC, PC12D, K-N-SH cells) that nobiletin exert its activity by modulation of cell cycle progression 17 . We observed that regardless of EGA phase (MN EGA and MJ EGA ), nobiletin supplementation in culture media increased the total cell number of produced blastocysts. This increase rate was similar to that observed with other antioxidants such as vitamin C 15 or crocetin 16,23 , suggesting that nobiletin could directly stimulate the cell cycle during EGA and improve embryo quality.
To verify if the effects of nobiletin during MN EGA or MJ EGA were related to gene expression changes, we analyzed the expression of candidate genes for oxidative stress, embryo development and quality. Glutathione peroxidase (GPX1) and Nuclear Factor Erythroid 2-Like 2 (NFE2L2), are oxidative-stress-response-related genes. GPX1, considered the major antioxidant enzyme within the Glutathione peroxidase family, is ubiquitously expressed in the cytosol and also has been found in mitochondria 20 . Furthermore, GPX1 acts as a scavenger of hydrophilic peroxide species, can be transformed into an enzymatically inactive cellular structural component, and protects cells against oxidative damage 20 . During in vitro production ROS generation increases and one of the defenses to counter excess ROS in the embryo is GPX1; therefore, GPX1 overexpression has been positively linked with embryo quality 36,37 . In our study, gene expression analysis revealed the upregulation of GPX1 in 8-and 16-cell embryos as well as in blastocysts produced with nobiletin supplementation during MN EGA or MJ EGA phases. A similar response has been reported in sheep and bovine embryos treated with other types of antioxidants like L-carnitine 21 or crocetin 16 . NFE2L2 transcript (also known as Nrf2) is important for embryo tolerance to oxidative stress during EGA as well as for its competence for development 2 . PI3K/AKT pathway plays a role in regulating NFE2L2 activation and is involved in the regulation of protein kinases, which may induce nuclear translocation 38 . Harris and Hansen 39 , and Ghanem et al. 29 reported in mice that up-regulation of NFE2L2 transcript may protect embryos from oxidative stress through preservation of intracellular redox states to ensure normal embryonic development. In the same line, our results showed the relative abundance of NFE2L2 transcript increased in 16-cell stage embryos cultured with 10 µM nobiletin during MJ EGA , while remained unaltered in 8-cell stage embryos as well as in blastocysts from both treatments. Moreover, data obtained in cancer cells of mice showed that NFE2L2 mRNA levels were upregulated when nobiletin was supplemented in culture medium 40 . Taken together, these data suggest that nobiletin plays an antioxidant-defense role via distinct pathways during the different phases of early embryo development in vitro. However, it is necessary to confirm this antioxidant action by measuring ROS levels in the embryos.
Cyclin Dependent Kinase 2 (CDK2) is necessary for cell cycle progression, and is a major kinase that governs AKT phosphorylation, while it also participates on EGA 41 . In our study, CDK2 mRNA expression was upregulated in 8-cell (MN EGA ), and blastocysts (MJ EGA ) cultured with 5 µM or 10 µM of nobiletin as well as in 16-cell (MJ EGA ) cultured with 10 µM of nobiletin. This is in line with previous data obtained in bovine embryos www.nature.com/scientificreports/ that showed changes in the levels of transcription in genes associated with cell cycle and observed an increase in CDK2 expression during early embryo development (8 and 16-cell embryos, and blastocysts) 42 . Conversely, studies using nobiletin in cancer cells (U87, Hs683) showed a decrease in CDK2 expression 17,43 . Hence, nobiletin seems to respond differently depending on the cell type. Histone H3.3 is encoded by H3.3 histone A (H3-3A) and H3.3 histone B (H3-3B) genes and is related to DNA synthesis and integrated into embryonic nucleosomes to mark genes for subsequent expression in development 44 . We observed that supplementation of 5 or 10 µM nobiletin during MN EGA , increased the expression of H3-3B and H3-3A genes in 8-cell embryos while supplementation of 10 µM nobiletin during MJ EGA increased the expression of H3-3B gene in 16-cell embryos. This is in corroboration with results from a recent study were characterization of the expression of both genes that encode H3.3 (H3-3A and H3-3B) was performed in early bovine embryos, demonstrating that H3-3B mRNA is very abundant throughout early embryogenesis, being two to three times higher than H3-3A mRNA during the major wave of EGA 45 . Additionally, a higher abundance of H3-3B compared to H3-3A was found in mouse embryos 46 , suggesting that the protein encoded by H3-3B gene may be critical for initiating the transcription of embryonic genes during EGA. As mentioned above, EGA is crucial for further embryo development and regulated by several important factors 47 . One crucial factor is histone modification, including methylation and acetylation 48 .Likewhise, Acetyl-Co A is a central metabolite linking glucose oxidation and long-chain fatty acid or cholesterol synthesis, providing energy and materials for cell growth and proliferation. Furthermore, Acetyl-Co A, as a donor of an acetyl group, can be utilized by histone acetyltransferases for histone acetylation 49 . A recent study showed that Acetyl-CoA synthases are essential for maintaining histone acetylation under metabolic stress during EGA in pigs and they corroborated that β-oxidation is crucial for porcine embryo development by contributing to energy metabolism and histone acetylation 50 . This suggests one more time that nobiletin could prefer the β-oxidation pathway as an energy production mechanism.
During in vitro development, embryos have a series of metabolic factors that are required in proliferation, differentiation, and survival of cells 13,51 . In this context, the quality of the embryos produced in vitro depends on many factors, among them the expression of different genes, as we have shown in this study. Gene expression depends of different signaling pathways that play important roles in the formation of the blastocyst, for example, PI3K/AKT 12,13,51 . AKT regulates cellular processes such as glucose metabolism, transcription, cellular growth and proliferation 51 . In blastocysts, AKT inhibition alters their development and AKT activation triggers the differentiation and migration of trophoblast cells 14,52 . Other studies showed that the AKT appear to have an important role in early embryonic development, in double-knockout mice deletion of any of the AKT isoforms leads to embryonic death or exhibiting more severe phenotype and earlier lethality 53 . In addition, PI3K/AKT regulates the development of preimplantation embryo by mediating the effects of autocrine factors 54 . Previous studies in cell lines have shown that nobiletin can act through various signaling pathways, including AKT 17 . However, as far as we know, nobiletin action on AKT pathway in bovine blastocysts produced in vitro is unknown. In this study, we established the presence of the AKT pathway in bovine blastocysts. Based on immunofluorescence images, p-AKT protein appears to be predominantly localized in the cytoplasm of embryos cultured with 5 and 10 µM nobiletin during MN EGA and MJ EGA , suggesting constant stimulation of this pathway during the preimplantation period. Expression of the AKT protein and its phosphorylation status were confirmed by western blot analysis of bovine blastocysts produced with or without nobiletin supplementation during MN EGA or MJ EGA phases. Similar results were found by Ashry et al. 14 , who investigated the relationship between AKT signaling and the embryotrophic actions of follistatin, and indicated that it plays an important role in the regulation of AKT signaling in early bovine embryos. Together, these results suggest that nobiletin is associated with increased AKT phosphorylation and, as it has been shown in cell lines studies, nobiletin has the ability to interact with this pathway, and regulate specific genes. In our study increased AKT phosphorylation might be related to the increase in the production and the expression of genes that favor the progression of the cell cycle (CDK2) and to the improvement in embryo quality by increasing mitochondrial activity and genes related to oxidative stress (NFE2L2), and reducing lipid content (PPARα). However, further studies are needed to fully elucidate its mechanism of action in early embryos.
In conclusion, nobiletin supplementation during MN EGA or MJ EGA has a positive effect on preimplantation bovine embryonic development in vitro by increasing blastocyst production and also corroborates on the increase in transcription level of genes related to cell division. Besides, this effect is reflected on the blastocysts quality improvement by (i) stimulating mitochondrial activity and expression of genes related to the protection of oxidative stress, and (ii) reducing the cytoplasmatic accumulation of lipids and promoting the expression of genes that regulate lipid metabolism. In addition, these positive responses of nobiletin on embryonic development and quality of the produced blastocysts in vitro could be modulated by the activation of AKT signaling pathway (Fig. 8). Therefore, nobiletin could constitute a suitable supplement to overcome oxidative stress in bovine IVP and improve ARTs in mammals.

Methods
Unless stated otherwise, all reagents were purchased from Sigma-Aldrich Corporation (St Louis, MO, USA).
Oocyte collection and maturation. Immature cumulus-oocyte complexes (COCs) were obtained by aspirating follicles (2-8 mm) from the ovaries of mature heifers and cows collected from a local abattoir. COCs (homogeneous cytoplasm and intact CCs) were selected and matured in four-well dishes (Nunc, Roskilde, Denmark) in 500 μL maturation medium (TCM-199), supplemented with 10% (v/v) fetal calf serum (FCS) and 10 ng/mL epidermal growth factor (EGF), in groups of 50 COCs per well for 24 h at 38.5 °C and an atmosphere of 5% CO 2 in the air with maximum humidity 55  www.nature.com/scientificreports/ Sperm preparation and in vitro fertilization (IVF). IVF was performed as described previously 56 .
Briefly, frozen semen straws (0.25 mL) from an Asturian Valley bull previously tested for IVF were thawed at 37 °C in a water bath for 1 min and centrifuged for 10 min at 280×g through a gradient of 1 mL of 40% and 1 mL of 80% Bovipure (Nidacon Laboratories AB, Göthenborg, Sweden) according to the manufacturer´s instructions. www.nature.com/scientificreports/ 'Experimental design' described below for more details). Culture took place at 38.5 °C in an atmosphere of 5% CO 2 , 5% O 2, and 90% N 2 .

Assessment of embryo development and quality
Embryo development. Developmental  Embryo quality: mitochondrial activity measurement, lipid content quantification and total cell number of blastocysts. Day 7 blastocysts (~ 30 per group) were simultaneously evaluated regarding mitochondrial activity, the number of lipid droplets, and total cell number. Blastocysts from each treatment were first suspended in 100 µL phosphate-buffered saline (PBS) without calcium or magnesium supplemented with 0.1% polyvinylpyrrolidone (PVP). Next, blastocysts were equilibrated for 15 minutes in culture media supplemented with 5% FCS and then incubated for 30 min at 38.5 °C in 400 nM MitoTracker DeepRed (Molecular Probes, Eugene, USA) for mitochondrial activity; blastocysts were then fixed in 4% paraformaldehyde (PF) for 30 min at room temperature. For lipid content analysis, fixed blastocysts were permeabilized with 0.1% saponin for 30 min and stained for 1 h with 20 μg/mL Bodipy 493/503. For analysis of total cell number, blastocysts were stained with Hoechst 33342 (10 μg/mL) for 30 min, washed in PBS + 0.1% PVP three times for 5 minutes each, and then mounted in 3.8 μL mounting medium between a coverslip and a glass slide which was sealed with nail polish. Slides were examined using a laser-scanning confocal microscope (Leica TCS SP2) equipped with an argon laser excited at 488 nm and with an emission spectrum of 500-537 nm for visualization of lipid droplets. For mitochondria, we used excitation and emission set at 644 nm and 625-665 nm, respectively. All images were captured using the same parameters, performing sequential acquisition. For the assessment of mitochondrial activity, the fluorescence signal intensity (pixels) was quantified. Serial sections of 5 µm were made for each blastocyst and a maximum projection was accomplished for each one. Images obtained were evaluated using the ImageJ program (NIH; ImageJ version 1.52k software (http:// rsbweb. nih. gov/ ij/)). After selection using the freehand selection tool, each blastocyst was measured to determine its area and its integrated density (IntDen), which corresponds to pixel intensity. Also, the background fluorescence of an area outside the blastocyst was measured. Fluorescence intensity in each blastocyst was determined using the following formula: Relative fluorescence = IntDen − (area of selected blastocyst x mean fluorescence of background readings). Fluorescence intensities are expressed in arbitrary units (a.u.).
The lipid quantification in blastocysts was obtained by analysis of the total area of lipids in each blastocyst. We captured three images of each blastocyst: one in the middle of the blastocyst (the image with the largest diameter) and the other two in the middle of the resulting halves. We used a 63× objective at a resolution of 1024 × 1024 and images were analyzed using the 'nucleus counter' tool, set to detect, distinguish, and quantify droplet areas with the ImageJ program. For blastocysts, lipid quantity was corrected by total embryo area, to account for varying blastocyst sizes. After verification of a significant correlation (r 2 = 0.84 and P < 0.0001 by Pearson's correlation test) between lipid quantity of three sections in 30 blastocysts (10 per group) we chose the section with the largest area per blastocyst to be analyzed 58 Table S4. The selection of genes to be evaluated was carried out considering the expression of key genes in preimplantation embryonic development. All primers were designed using Primer-BLAST software (http:// www. ncbi. nlm. nih. gov/ tools/ primer-blast/) to span exon-exon boundaries when possible. For quantification, RT-qPCR was performed as described previously 60 . The PCR conditions were tested to achieve efficiencies close to 1. Relative expression levels were quantified by the comparative cycle threshold (CT) method 61 . Values were normalized using two housekeeping genes (H2AFZ and ACTB) selected according to previous studies 56,62 , while their stabilities were evaluated using the geNorm software for microsoft 63,64 , ranking the genes based on the internal control gene stability parameter M. Fluorescence was acquired in each cycle to determine the threshold cycle or the cycle during the log-linear phase of the reaction at which fluorescence increased above background for each sample. Within this region of the amplification curve, a difference of one cycle is equivalent to a dou- www.nature.com/scientificreports/ bling of the amplified PCR product. According to the comparative CT method, the ΔCT value was determined by subtracting the mean CT value of the two housekeeping genes from the CT value of the gene of interest in the same sample. The calculation of ΔΔCT involved using the highest treatment ΔCT value (i.e. the treatment with the lowest target expression) as an arbitrary constant to subtract from all other ΔCT sample values. Fold-changes in the relative gene expression of the target were determined using the formula 2 −ΔΔCT.
Negative control was prepared to omit the primary antibody before adding the secondary antibody.
Western blot of AKT in blastocysts. The western blot analysis was performed as described previously The monoclonal anti-β-actin−peroxidase antibody produced in mouse was used as the loading control. Membranes were probed sequentially with primary p-AKT (Thr308), p-AKT (Ser473) and (t)AKT antibodies. For this purpose, after detection of an antibody membranes were stripped by washing extensively in TBS-T, three times for 10 minutes each, and repeating the blocking step, and then the membranes are re-probed with the next antibody. After detection of (t)AKT, membranes were stripped and re-probed with anti-β-actin−peroxidase antibody. In all cases, intensities of protein bands (optical density (OD)) were quantified by ImageJ software and the relative abundance of each protein was normalized to the total-actin expression in the corresponding lane and phosphorylation level was expressed as phosphorylated (p) AKT/(t) AKT. The ratio of the OD of the protein concerned (AKT/p-AKT) in relation to actin is presented in the form of bar charts. C DMSO : n = 331), embryos that reached the 8-or 16-cell stage, respectively, were transferred to SOF + 5% FCS and cultured until Day 8, maintaining the different experimental groups separately (Fig. 9). Embryos were cultured in groups of 50 under an atmosphere of 5% CO 2 , 5% O 2 and 90% N 2 at 38.5 °C.
Considering that during the experiment it was necessary to preselect the embryos at different stages of development (≥ 8 cells and ≥ 16 cells), the developmental parameters were calculated as follows:  www.nature.com/scientificreports/ Twelve and ten replicates for MN EGA and MJ EGA phases, respectively, were performed under the same assay conditions.
Only the experimental groups that showed higher blastocyst yield in this experiment (Nob5 and Nob10) in comparison with both control groups (Control and C DMSO ) were used for experiments 2 and 3.

Experiment 2: effect of nobiletin on the quality of in vitro produced blastocysts.
To evaluate blastocyst quality of embryos produced in vitro with or without nobiletin supplementation during MN EGA or MJ EGA , a representative number of Day 7 blastocysts (n ≈ 30 per group/Experiment 1) were stained with MitoTracker DeepRed, Bodipy and Hoescht to evaluate mitochondrial activity (intensity recorded in arbitrary units (a.u)), lipid content (lipid droplet area in μm 2 ) and total cell numbers, respectively. Blastocysts were examined using a laser-scanning confocal microscope or an epifluorescence microscope and images obtained were evaluated using the ImageJ program.
To evaluate if nobiletin induces changes in the expression levels of genes related to embryo development and quality, three independent pools of 10 embryos per stage (8-cell, 16-cell, and blastocyst) obtained from each experimental group cultured with or without nobiletin during MN EGA or MJ EGA (Experiment 1), were used for gene expression analysis by qRT-PCR according to the procedures described above.

Experiment 3: nobiletin effect on the AKT pathway in blastocysts produced in vitro.
To assess if nobiletin can interact with AKT pathway during in vitro embryo development, Day 7 blastocysts (n = 10 -Experiment 1) from each group were stained with p-AKT (Thr308/Ser473) for immunolocalization. To evaluate the phosphorylation level of AKT (Thr308 and Ser473), Day 7 blastocyst (n = 60 -Experiment 1) from each group were frozen in LN 2 for western blot analysis.
Statistical analysis. All statistical tests were performed using the software package SigmaStat (Systat Software Inc., San Jose, CA, USA). Cleavage rate, blastocyst yield, mitochondrial activity, lipid content, number of cells per blastocyst, relative mRNA abundance levels, and AKT phosphorylation level, were normally distributed with homogeneous variance, so one-way analysis of variance (ANOVA) with arcsine data transformation, followed by Tukey´s test, was performed to evaluate the significance of differences between groups. The correlation analysis for lipid quantification in blastocysts was determined by Pearson's correlation coefficient test. Values were considered significantly different at P < 0.05. Unless otherwise indicated, data are presented as the mean ± s.e.m.