Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway

RESEARCH ARTICLE

Molecular Reproduction & Development 81:497–513 (2014)

Bovine Embryo Survival Under Oxidative-StressConditions is Associated With Activity of theNRF2-Mediated Oxidative-Stress-Response Pathway

AHMED AMIN,1 AHMED GAD,2 DESSIE SALILEW-WONDIM,1 SIGIT PRASTOWO,1 EVA HELD,1 MICHAEL HOELKER,1

FRANCA RINGS,1 ERNST THOLEN,1 CHRISTIANE NEUHOFF,1 CHRISTIAN LOOFT,1 KARL SCHELLANDER1,AND DAWIT TESFAYE1*

1 Animal Breeding and Husbandry Group, Institute of Animal Science, University of Bonn, Bonn, Germany2 Faculty of Agriculture, Department of Animal Production, Cairo University, Giza, Egypt

SUMMARY

In present study, we sought to examine the ability of preimplantation bovine embryosto activate the NF-E2-related factor 2 (NRF2)-mediated oxidative-stress responseunder anoxidative stress environment. Invitro 2-, 4-, 8-, 16-cell-, andblastocyst-stageembryoswereculturedunder low (5%)or high (20%)oxygen levels. TheexpressionofNRF2, KEAP1 (NRF2 inhibitor), antioxidants downstream of NRF2, and genesassociated with embryo metabolism were analyzed between the embryo groupsusing real-time quantitative PCR. NRF2 and KEAP1 protein abundance, mitochon-drial activity, and accumulation of reactive oxygen species (ROS) were also investi-gated in blastocysts of varying competence that were derived from high- or low-oxygen levels. The expression levels of NRF2 and its downstream antioxidant geneswere higher in 8-cell, 16-cell, and blastocyst stages under high oxygen tension,whereas KEAP1 expression was down-regulated under the same conditions. Higherexpression of NRF2 and lower ROS levels were detected in early (competent)blastocysts compared to their late (noncompetent) counterparts in both oxygen-tension groups. Similarly, higher levels of active nuclear NRF2 protein were detectedin competent blastocysts compared to their noncompetent counterparts. Thus, thesurvival and developmental competence of embryos cultured under oxidative stressare associatedwith activity of theNRF2-mediated oxidative stress response pathwayduring bovine pre-implantation embryo development.

Mol. Reprod. Dev. 81: 497�513, 2014. � 2014 Wiley Periodicals, Inc.

Received 16 December 2013; Accepted 18 February 2014

�Correspondence to:Department of Animal Breedingand Husbandry

Institute of Animal ScienceEndenicherallee 15Bonn 53115, Germany.E-mail: [email protected]

Grant sponsor: German AcademicExchange Programme (DAAD)

Published online 21 March 2014 in Wiley Online Library(wileyonlinelibrary.com).DOI 10.1002/mrd.22316

INTRODUCTION

Despite significant improvements over the last decade inin vitro fertilization and culture technologies for mammalianembryo production, in vitro-derived embryos still differfrom their in vivo counterparts in many aspects. This ismainly attributed to differences in various factors includingmedia composition, pH, and oxygen tension between invitro and in vivo conditions. These deviations from the in

Abbreviations: ROS, reactive oxygen species. Genes: ACACA1, acetyl-CoA carboxylase alpha; CAT, catalase; CPT2, carnitine palmitoyl transferase-2;HMOX1, heme oxygenase decycling-1; KEAP1, kelch-like ECH-associatedprotein 1; NRF2, nuclear factor (erythroid-derived 2)-like 2; PPARAa, peroxi-some proliferator-activated receptor alpha; PRDX1, peroxiredoxin-1; SOD1,superoxide dismutase-1; SREBP1, sterol regulatory element binding proteintranscription factor1; TXN1, thioredoxin-1.

� 2014 WILEY PERIODICALS, INC.

vivo environment��especially in terms of metabolites andoxygen tension��are known to create suboptimal in vitroembryo culture conditions by inducing oxidative stressthrough the accumulation of reactive oxygen species(ROS; Goto et al., 1993). An imbalance between the con-centration of intracellular ROS and the ROS scavengingcapability of cells, through antioxidants mechanisms, canlead to a state of oxidative stress (Agarwal et al.,2003, 2005b; Van Guilder et al., 2006).

Oxidative stress can be triggered by various types ofROS, such as superoxide (O2

�), hydrogen peroxide(H2O2), and hydroxyl radicals (OH), which originate bothfrom the environment aswell as fromwithin the oocytes andembryos as by-products of the energy metabolism path-ways, particularly mitochondrial phosphorylation and gly-colysis (Guerin et al., 2001; Balaban et al., 2005). ROS arehighly reactivemolecules that interactwith andcandamageother molecules, including DNA (Mello Filho et al., 1984),proteins and lipids (Wu and Cederbaum, 2003), and cellstructures such as cell membranes, which can impairmitochondrial integrity and activity via lipid peroxidation(Kadenbach et al., 2004; Powers and Jackson, 2008).High oxidative stress conditions can negatively affect invitro oocyte maturation and early embryo development,resulting in increased apoptosis, embryo fragmentation(Du and Wales, 1993; Johnson and Nasr-Esfahani, 1994;Khurana and Niemann, 2000; Liu and Keefe, 2000; Guerinet al., 2001; Bedaiwy et al., 2004); changes in gene ex-pression and embryonic metabolism (Du andWales, 1993;Rinaudo et al., 2006; Balasubramanian et al., 2007; Harveyet al., 2007); lipid accumulation; and reduced embryoquality (Abe et al., 2002; Barcelo-Fimbres and Seidel,2007; Sudano et al., 2011). Thus, activation of embryonicmechanisms that protect against oxidative stress isone approach to improving embryo quality, and therebyembryo developmental competence, under in vitro condi-tion (Takahashi, 2012).

Many different compounds have been implicated infacilitating cellular defense against oxidative stress. Amongthese, nonenzymatic compounds such as vitamins A, C,and E (Pascoe et al., 1987; Schweigert and Zucker, 1988),pyruvate (Morales et al., 1999), cysteine (Ali et al., 2003),glutathione (GSH), and GSH ethyl ester (Takahashiet al., 1993; Luvoni et al., 1996; Curnow et al., 2010)have been used as culture media supplements to improveembryo development. In addition, antioxidant enzymes,including glutathione peroxidase (GPX), catalase (CAT),superoxide dismutase (SOD), and thioredoxin (TXN),have been shown to protect embryos from oxidative stressby scavenging ROS (Nonogaki et al., 1991, 1992; Liet al., 1993; Natsuyama et al., 1993; Ozawa et al., 2006;Abedelahi et al., 2010).

We recently showed stage-specific transcriptome ef-fects of culturing bovine embryos to the blastocyst stagein vitro or in vivo (Gad et al., 2012). Functional classificationand pathway analyses of genes affected by the alternateculture environment revealed the NRF2-mediated oxida-tive-stress-response pathway to be the dominant respon-sive cascade (Gad et al., 2012). Within this response

pathway, the nuclear factor (erythroid-derived 2)-like 2(NFE2L2 or NRF2) is a key transcription factor that regu-lates many antioxidant genes in mammalian cell (Wildet al., 1999; Nguyen et al., 2003, 2009; Tanaka et al.,2008). Kelch-like ECH-associated protein 1 (KEAP1), inturn, is known to regulate NRF2 activity (Zhang, 2006). Asthe effect of an alternative culture environment has beeninvestigated only for embryos that have survived andreached the blastocyst stage (Gad et al., 2012), it wasnot possible to assess NRF2 activity in those embryos thatdid not reach the experimental endpoint. Nevertheless, wehypothesized that embryos that were able to activate theiroxidative-stress defense mechanism via the NRF2-medi-ated response pathway are more likely to reach the blasto-cyst stage within an acceptable developmental period. Asthe oxygen tension between in vitro and in vivo environ-ments differ (20% in vitro and 5�7% in the mammalianoviduct), and as high oxygen levels can be one source ofoxidative stress, we evaluated how varying oxygen levels inour in vitro culture system affects oxidative stress duringembryo development, specifically assessing the activity ofthe NRF2-mediated oxidative-stress-response pathway inrelationship to embryo survival and metabolism.

RESULTS

In Vitro Development of Bovine Embryos Under5% and 20% Oxygen Conditions

Embryo developmental competence, evaluated in termsof cleavage and blastocyst rates, under 5% or 20% oxygenwere compared. The cleavage rate did not differ signifi-cantly between groups, namely 79.1% for 20% and 77.8%for 5% oxygen conditions (Table 1). On the other hand,culturing embryosunder 5%oxygen resulted in significantlyhigher (P< 0.05) blastocyst rates than those cultured at20% oxygen (37.8% vs. 25.4%, respectively).

Activity of NRF2-Mediated Oxidative-StressResponse in Bovine Embryos Under OxidativeStress Conditions

Oxidative stress activated the embryonic NRF2-mediated oxidative-stress response in a stage-specific manner A significantly higher level of ROSwas detected in embryos cultured at 20% oxygen com-pared to those cultured at 5% (Fig. 1). In order to investi-gate if the elevatedROS levels triggered aNRF2-mediatedoxidative-stress response in embryos, we compared theexpressionofNRF2and its inhibitor (KEAP1) in embryos ofdifferent ages that developed under 20% versus 5% oxy-gen conditions. There was no significant difference inNRF2 transcript abundance between the groups at 2-and 4-cell stages, but NRF2 mRNA levels became signifi-cantly higher in embryos cultured at 20% oxygen thanthose from 5% oxygen at the 8-cell, 16-cell, and blastocyststages. KEAP1 mRNA levels showed reciprocal enrich-ment in all stages of development compared to the expres-sion of NRF2 (Fig. 2).

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Immunofluorescence staining for NRF2 and KEAP1proteins at blastocyst-stage embryos from 20% versus5% oxygen conditions paralleled the transcript abundance.Higher florescence intensity of nuclear NRF2 protein wasdetected in blastocysts from the 20% oxygen culture com-pared to the 5% oxygen condition (Fig. 3). In contrast,blastocysts from 20% oxygen conditions expressed lessKEAP1 protein (Fig. 3).

Oxidative stress induced changes in the expressionof NRF2-regulated genes and genes related to lipidmetabolism We next investigated the gene expressionof antioxidant enzymes downstream of NRF2 activation,namelySOD1,CAT,PRDX1,HMOX1,NQO1, andTXN1, inpre-implantation-stage embryos derived from 20% or 5%oxygen environments. Interestingly, the expression ofSOD1, CAT, and PRDX1 genes was higher in young,cleavage-stage embryos cultured in 5% than in 20% oxy-gen until the 8-cell-stage, when the expression patternreversed. Higher expression levels of NQO1, TXN1, andHMOX1 were detected at the 8- and 16-cell stages forembryos cultured at 20% compared to 5% oxygen level(Fig. 2).

We also looked at the effect of oxidative stress on thelipogenic pathway, specifically evaluating the expression ofthe lipid metabolism genes SREBP1, ACACA1, CPT2, andPPARAa in the same groups of embryos. The expressionSREBP1, ACACA1, CPT2, and PPARAa genes were sig-nificantly higher in embryos derived from the 20% oxygencondition compared to those from 5% oxygen at variousstages of development (Fig. 2).

Mitochondrial activity and cell number of bovineblastocysts under oxidative stress In order to evaluatethe effect of elevatedROSon themitochondria of embryos,mitochondrial activity was assessed in blastocysts derivedfrom 20% and 5% oxygen conditions. Blastocysts culturedin 5% oxygen exhibited higher mitochondrial activity com-pared to blastocysts derived from 20% oxygen culture,as evidenced by the high intensity of florescence dyedetected by confocal microscopy (Fig. 4). Blastocysts de-rived from 20% oxygen also had significantly fewercells (132� 29.5) compared to those from the 5% oxygencondition (155� 20).

Association of NRF2 Activity and EmbryoDevelopmental Competence UnderOxidative-Stress Conditions

NRF2 and KEAP1 expression in relation to embryocompetence To investigate if the oxidative-stress-activat-ed NRF2-mediated pathway in the pre-implantation bovineembryo is associated with embryo developmental compe-tence,weused blastocyst developmental rate as amodel todistinguish between competent and noncompetent embry-os. Embryos that reached the expanded-blastocyst stageat Day-7 post-insemination were considered early-devel-oping (competent) blastocysts, whereas those thatreached the same stage of development at Day-8 post-insemination were categorized as late-developing (non-competent). Accordingly, the expression levels of NRF2and KEAP1 were investigated in competent versus non-competent blastocysts, revealing that higher NRF2mRNAwas in early-developing compared to late-developing onesin both 5% and 20% oxygen conditions, with the conversetranscript abundance seen for its inhibitor, KEAP1 (Fig. 5).

Immunofluorescence staining showed that protein ex-pression of NRF2 and KEAP1 genes were in agreement

TABLE 1. Developmental Rates of Bovine Zygotes Generated Under 5% and 20% Oxygen Conditions

Oxygen level n

Blastocyst rate

Cleavage rate Day 7 Day 8

n (%) n (%) n (%)

5% O2 1,372 1,085 79.08 400 29.15a 118 8.6020% O2 1,381 1,074 77.77 224 18.13b 127 9.51

abValues with different superscripts within columns differ significantly.

Figure 1. ROS level of bovine blastocyst embryos derived from 5%and 20% oxygen culture environments. Scale bars, 70mm.

NRF2-MEDIATED OXIDATIVE STRESS RESPONSE ACTIVITY IN EMBRYOS

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with the mRNA expression pattern in competent and non-competent embryos under both oxygen conditions (Figs. 6and 7). Generally, high NRF2 protein levels could be de-tected in both competent and noncompetent blastocysts

derived from 20% compared to 5% oxygen conditions(Fig. 6). More NRF2 protein was found to be localized innuclei of early-developing blastocysts, whereas NRF2 pro-tein was predominantly cytoplasmic in late-developing

Figure 2. Expression levels of genes associated with the NRF2 pathway (NRF2, KEAP1, SOD1,CAT1, PRDX1,HMOX1,NQO1, and TXN1) andgenes related to lipid metabolism (SREBF1, ACACA1,CPT2, andPPARAa) at various developmental stages. Expression was compared betweenin vitro-produced bovine embryos cultured under 5% (white bars) and 20% (black bars) oxygen. Asterisks (�) represent statistically significantdifferences (P<0.05).

Figure 3. Protein expression of NRF2 and KEAP1 in bovine blastocyst-stage embryos cultured under 5% or 20% oxygen conditions. Greenfluorescence signals reveal NRF2 or KEAP1protein localization, while blue showed nuclear staining usingDAPI. Originalmagnification 40�. Scalebars, 50mm. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]


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blastocysts, independent of oxygen condition (Fig. 6). Incontrast, KEAP1 protein level was higher in blastocystsderived from 5% oxygen than those from 20% oxygenconditions (Fig. 7).

Expression of antioxidants and lipid metabolismgenes downstream of NRF2 in competent versusnoncompetent embryosConsistent with the expressionof NRF2, higher expression of antioxidants genes, namelyPRDX1, CAT, SOD1, and TXN1, was found in competentblastocysts compared to their noncompetent counterpartsat both oxygen levels (Fig. 5).

The expression of lipid metabolism genes SREBP1 andCPT2 revealed higher SREBP1 levels in competent blas-tocysts compared to noncompetent ones at 5% oxygen,whereas the expression ofCPT2mRNAwas higher in earlyblastocysts compared to the late ones in both 5% and 20%oxygen conditions (Fig. 5).

Intracellular ROS levels and mitochondrial activityof embryos under oxidative stress condition in re-lation to developmental competence IntracellularROS levels were higher for both early- and late-blastocystgroups cultured at 20%oxygen compared to the 5%oxygencondition. Under both conditions, competent blastocystsshowed relatively lower ROSsignal than the noncompetentones (Fig. 8). Yet, mitochondrial activity is higher in com-petent blastocysts compared to their noncompetent coun-terparts (Fig. 9).

Cryotolerance of embryos derived from culture un-der oxidative stressHere we used embryo-hatching rateas an indicator of an embryo’s ability to survive freeze-thaw.

Cryopreserved, competent blastocysts had a higher hatch-ing rate than the noncompetent ones after freeze-thaw,independent of oxygen culture conditions (Fig. 10).

DISCUSSION

Compared to the natural in vivo environment, in vitroculture conditions provide a suboptimal environment forbovine preimplantation embryo, which is manifested by adramatic change in the transcriptome profile and metabo-lism of the resulting embryos (Thompson et al., 1998;Khurana and Niemann, 2000; Niemann and Wrenzycki,2000; Rizos et al., 2001; Lazzari et al., 2002; Rizoset al., 2002a,b, 2003; Lonergan et al., 2003; Gardneret al., 2012). In addition to metabolite differences, thereis a significant difference in oxygen levels between in vivoand in vitro environments. Higher oxygen levels experi-enced during in vitro culture are known to be one of thesources of embryonic oxidative stress, and stage-specificexposure of embryos to in vitro or in vivo environmentsresults in blastocysts whose transcriptome profiles differmainly in the activity of their NRF2-mediated oxidative-stress-response pathway (Gad et al., 2012). Thus, to elu-cidate an embryo’s reaction to the elevated oxygen tension,we investigated the effect of 20% or 5% oxygen on embryodevelopment and the association of NRF2-mediated oxi-dative-stress-response pathway activity on an embryo’sability to survive and develop under an oxidative-stressenvironment.

Oxidative stress induced by ROS was previously re-ported to promote many detrimental effects during the invitro-production procedure, resulting in lower embryo qual-ity and a delay in embryonic development (Paszkowski and

Figure 4. Mitochondrial activity of bovine blastocysts derived from 5% and 20% oxygen conditions.Mitochondriawere stainedwithMitoTracker red (A andB) and nuclei were stainedwithDAPI (A1 andB1).A2andB2showmergedpicturesof the protein andnuclei.Originalmagnification40�. Scale bars, 50mm.[Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]


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Clarke, 1996; Khurana and Niemann, 2000; Liu andKeefe, 2000; Guerin et al., 2001; Harvey et al., 2002;Agarwal et al., 2003, 2005a; Das et al., 2006). Consistentwith reports from others (Goto et al., 1993; Favettaet al., 2007), here we showed that culturing embryos under20% oxygen in vitro resulted in accumulation of ROS andreduced blastocyst rate (Table 1).

The NRF2/KEAP1 system is a cellular self-defensemechanism activated under oxidative stress. NRF2 is atranscription factor that works with KEAP1 protein to acti-vate a battery of antioxidant genes in response to oxidativestress, with the final outcome of reducing ROS levels(Kobayashi et al., 2006); such involvement of the NRF2/KEAP1 signaling pathway in cytoprotection against oxida-tive stress has been verified using Nrf2 knock-out mice(Enomoto et al., 2001; Ramos-Gomez et al., 2001; Chanaset al., 2002; Gong andCederbaum, 2006; Khor et al., 2008;Cheung et al., 2014).

We recently identified NRF2/KEAP1 signaling as one ofthe dominant pathways affectedwhen embryos are exposeto various environments (Gad et al., 2012; Heldet al., 2012). In the present study, we used an in vitroembryo-culture model comparing 20% versus 5% oxygentension to investigate the effect of oxidative stress on theactivity of the NRF2-mediated oxidative-stress-responsepathway in early bovine embryos and on their survival anddevelopmental competence. It is widely accepted that pro-tection against oxidative stress during earlier stages ofdevelopment is, in part, driven by the maternal pool ofantioxidant enzymes stored in the oocyte during oogenesis(Harvey et al., 1995; El Mouatassim et al., 1999). Consis-tent with this model, NRF2 activity and antioxidant abun-dance was not significantly triggered by oxidative stressbefore the8-cell-stage in this study.Rather, genesencodingproteins with an antioxidant function such as SOD1, CAT,and PRDX1 showed significantly lower levels in 2- and 4-cell-stage embryos under 20% oxygen conditions com-pared to those cultured under 5% oxygen. This may bedue to the use ofmorematernally stored antioxidants by theembryo under oxidative stress, particularly before embry-onic genome activation, to prevent the accumulation ofROS (Harvey et al., 1995; El Mouatassim et al., 1999),which is vital for their development and survival under high-oxygen conditions. NRF2 and its downstream antioxidant-target mRNA abundance was higher in response to theirexposure to higher oxygen tension starting at the 8-cell-stage. In contrast, under low oxidative stress conditions(5% oxygen), the inactive form of NRF2 is abundant and issequestered to the cytoplasmbyKEAP1protein. The abilityof NRF2 to activate its downstream antioxidant genesisalso regulated by KEAP1 activity, which acts as a redoxsensor (Itoh et al., 1999, 2003; Dhakshinamoorthy andJaiswal, 2001). Under oxidative stress conditions, an activeform of NRF2 is released from KEAP1 and is translocatedfrom the cytoplasm into the nucleuswhere it can activate itstarget antioxidant genes by binding to their antioxidantresponse elements (AREs; Nguyen et al., 2003; Lauet al., 2008; Osburn and Kensler, 2008). Knockdown ofKEAP1 by siRNA is reported to enhance NRF2 transcrip-tional activity in the 36M2 human epithelial ovarian cancercell line in vitro (Konstantinopoulos et al., 2011). In line withthis observation, a reciprocal expression of NRF2 andKEAP1 genes and corresponding protein were observedin blastocysts derived from 20% versus 5% oxygenconditions.

As shown previously (Gad et al., 2012), expression ofseveral lipid-metabolism genes was altered between invitro- or in vivo-cultured embryos in a stage-specific man-ner. Recent reports suggest the involvement of NRF2 inlipid metabolism as well as lipid accumulation in the mouseliver (Tanaka et al., 2008; Okada et al., 2009; Huang et al.,2010). Lipid accumulation is oneproblemassociatedwith invitro embryo production, and leads to a reduced cryotol-erance after freezing and thawing (Abe et al., 2002). Thecause of lipid accumulation in in vitro-produced embryoscan be either due to application of serum as a sourceof excess lipid (Ferguson and Leese, 1999; McEvoy

Figure 5. Relative abundance of NRF2, KEAP1, antioxidant genesdownstream of NRF2, and genes related to lipid metabolism in early orcompetent blastocysts (EB, white bars) and late or noncompetentblastocysts (LB, black bars) derived from 5% or 20% oxygen level.Asterisks (�) represent statistically significant differences (P<0.05).


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et al., 2000, 2001) or the inability of an embryo to metabo-lize the available lipid due to disturbedmitochondrial activitythat results from excessive ROS accumulation (Abeet al., 2002; Barcelo-Fimbres and Seidel, 2007). The lattermodel is consistent with our results, demonstrating thatexposure of embryos to 20% oxygen conditions resulted inthe up-regulation of SREBP1 and its downstream lipidbiosynthesis gene ACACA1 beyond 8-cell stages (Fig. 2)and that embryos from the same in vitro culture conditionpossess significant differences in ROS accumulation, mi-tochondrial activity, and lipid accumulation (data not shown)under 20% oxygen conditions.

The findings of the first part of thiswork revealed a stage-dependent variation in the embryonic NRF2/KEAP1 re-sponse to environmental oxidative stress. Since this analy-sis was performed using embryos that had survived andreached the required stage of development over an accept-

able timewindow, it was not possible to assess the status ofthe NRF2 defense pathway of those embryos that did notsurvive or reach the required stage of development underoxidative stress conditions. Therefore, we used rate ofblastocyst formation as a metric to distinguish betweenembryos with high or low competence for further develop-ment, which then allowed us to associate an embryo’sNRF2-mediated oxidative stress response to its develop-mental potential and survival under oxidative stress. Such amodel has been used in several species based on thecorrelation that early-developing blastocysts are compe-tent to induce higher rates of successful pregnancy aftertransfer as compared to their late-developing counterparts(Monson et al., 1992; Hasler et al., 1995; Bernardi andDelouis, 1996; Hasler, 2000; Shapiro et al., 2001; Barren-etxea et al., 2005; Muthukumar et al., 2013). These differ-ences in developmental competence are associated with

Figure 6. Localization of NRF2 protein in early (competent) (A,C) versus late (noncompetent; B,D)bovine blastocyst-stage embryos cultured under 5% or 20% oxygen conditions. Labels A�D showNRF2protein in green, while A1�D1 show nuclear staining in blue (DAPI). A2�D2 are merged images of theprotein and nuclei staining. Original magnification 40�. Scale bars, 50mm. [Color figure can be viewed inthe online issue which is available at wileyonlinelibrary.com]


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an alteration in expression of developmentally importantgenes associated with various embryonic physiologicalprocesses (Brevini et al., 2002; Fair et al., 2004; Dodeet al., 2006). In the present study, competent blastocystsshowed higher NRF2 activity both at the mRNA and active,nuclear protein levels. The enrichment of nuclear NRF2protein is accompanied bymore abundant antioxidant genetranscripts (CAT, PRDX1, SOD1, and TXN1) in competentblastocysts, and subsequently resulted in reduced ROSaccumulation. We also previously observed a clear associ-ation between antioxidant expression and ROS accumula-tion in invitro- produced, early- (competent) or late-cleaving(noncompetent) 2-cell embryos (Held et al., 2012).

Application of exogenous antioxidant agents is alsoknown to protect embryos against oxidative-stress-depen-dent damage by reducing intracellular ROS and apoptosislevels, which enhance embryo development. For instance,

supplementation of culture media with 0.5mg/ml of recom-binant human thioredoxin dramatically improved bovineembryo development to the blastocyst stage in vitro under5% oxygen conditions (Bing et al., 2003), with similarresults obtained in porcine embryos by adding 1.0mg/mlthioredoxin (Ozawa et al., 2006). Supplementation withsuperoxide dismutase in the culturemedia 7 hr after insem-ination resulted in increased mouse blastocyst rate (Non-ogaki et al., 1992; Orsi and Leese, 2001), while similarbeneficial effects on blastocyst and hatching rates werereported for murine zygotes cultured under 5% oxygen inthe presence of catalase (50�100U/ml) and superoxidedismutase (100�7,000U/ml) (Orsi and Leese, 2001).

Mitochondria are known to play a vital role in early-embryo metabolism, and their subsequent function is as-sociated with developmental competence (Barnett andBavister, 1998; Wilding et al., 2001; Tarazona et al.,

Figure 7. Localization of KEAP1 protein in early (competent; A,C) versus late (noncompetent; B,D)bovineblastocyst-stageembryosculturedunder5%or20%oxygenconditions. LabelsA�DshowKEAP1protein in green, while A1�D1 show nuclear staining in blue (DAPI). A2�D2 are merged images of theprotein and nuclei staining. Original magnification 40�. Scale bars, 50mm. [Color figure can be viewed inthe online issue which is available at wileyonlinelibrary.com]


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2006; Leese, 2012). Oxidative stress induced by ROSaccumulation has been implicated as a causative factorof mitochondrial deficiency (Kowaltowski and Vercesi,1999; Milei et al., 2001; Shokolenko et al., 2009; Cuiet al., 2012). In the present study, competent blastocysts,which have lower ROS accumulation, showed higher mi-tochondrial activity than their noncompetent counterpartswith elevated ROS levels. TheNRF2-mediated response ispostulated to help protect mitochondria against oxidative-stress damage and to regulate mitochondrial biogenesis inmouse and rat species (Piantadosi et al., 2008; Greco andFiskum,2010;Ungvari et al., 2011;MacGarveyet al., 2012),but adirect associationbetween thebovineNRF2-mediatedoxidative-stress-response pathway and embryo metabo-lism, in general and particularly in lipid accumulation, needsfurther investigation. Developmentally competent blasto-cysts derived from both oxygen conditions revealed a betterhatching rate after cryopreservation than their noncompe-tent counterparts. This may be attributed to the activatedNRF2 oxidative-stress response system in competent

Figure 8. ROS staining of early- and late-developing blastocystsderived form 5% and 20% oxygen conditions. [Color figure can beviewed in the online issue which is available at wileyonlinelibrary.com]

Figure 9. Mitochondrial activity of early- and late-developing blastocysts derived from5%or 20%oxygenconditions after staining byMitoTracker (red fluorescent). Nuclei are stainedwith DAPI (blue). Scale bars,50mm. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]


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embryos, which enables them to overcome the stressesinduced by the cryopreservation procedure itself (Laneet al., 2002; Rahimi et al., 2003; Zhao et al., 2012).

To our knowledge, this is the first study demonstrating apotential role for the NRF2-mediated oxidative-stress-re-sponse pathway during bovine preimplantation embryodevelopment and survival under oxidative stress condi-tions. Our findings are summarized schematically toshow the relationship between the activity of the NRF2-mediated oxidative-stress defense mechanisms and em-bryo developmental competence under suboptimal culturecondition (Fig. 11). Results from this study support thehypothesis that the fate of early bovine embryos undersuboptimal environmental conditions is associated with itsability to activate the oxidative-stress defense mechanism,which is mainly driven by the NRF2-mediated stress-re-sponse pathway.

MATERIALS AND METHODS

Experimental Design

Expression analysis of NRF2-mediated oxidative-stress-response- and lipid-metabolism-pathwaygenes in bovine embryos cultured at 5% and 20%oxygen To elucidate if the bovine embryos at the earlydevelopmental stages are able to activate NRF2 and itsdownstream antioxidant-related genes as a response tohighoxidative stress,wecultured embryos invitro under 5%(comparable to the oxygen level in the oviduct) or 20%oxygen (as commonly used in in vitro culture system).Embryos from 2-, 4-, 8-, 16-cell-, and blastocyst stageswere collected from both oxygen conditions. The expres-

sion of NRF2-pathway genes, including antioxidants (CAT,HMOX1, NQO1, PRDX1, SOD1, and TXN1), the NRF2cytoplasmic inhibitor KEAP1, and genes involved in lipidmetabolism (SREBP1, ACACA1, ARAPPa, and CPT2)were quantified in all stages of embryos. In addition,NRF2 and KEAP1protein of was determined at the blasto-cyst stage. The ROS level, mitochondrial activity, andlipid content were also evaluated in blastocyst-stageembryos.

Figure 10. Hatching rate of early- and late-developing blastocystsderived from 5% or 20% oxygen conditions after vitrification freeze-thaw. Asterisks (�) represent statistically significant differences(P<0.05) between early- and late-developing blastocysts at eachoxygen condition.

Figure 11. Model of the NRF2-mediated oxidative stress responsepathway in competent (embryos that reach the expanded-blastocystsstage at 6�7 days post-insemination) and noncompetent (embryosthat reached the same stage at 8�9 days post-insemination) under asuboptimal culture environment. Competent blastocysts have anactive NRF2 protein that localizes to the nucleus, where it binds tothe antioxidants reactive element (ARE) in the promoters of antioxi-dant genes. This activates antioxidant gene expression so the re-spective proteins can scavenge the ROS and subsequently maintainthe activity of mitochondria for better metabolism of lipids (L). In thecase of noncompetent embryos, however, inactive NRF2 leads tolower levels of antioxidants and subsequent accumulation of ROS,which impair the activity of mitochondria and embryo metabolism. Asa result of this disturbed metabolism, lipid (L) accumulates in thoseembryos. [Color figure can be viewed in the online issue which isavailable at wileyonlinelibrary.com]


506 Mol. Reprod. Dev. 81:497–513 (2014)

Association of NRF2-mediated oxidative-stress-re-sponse pathway activity with the developmentalcompetence of embryos To determine if the ability ofembryos to tolerate high oxidative stress under in vitroculture conditions is associated with NRF2 activity, weanalyzed the expression of NRF2-mediated oxidative-stress-response-pathway-related genes in developmental-ly competent versus noncompetent blastocyst-stage em-bryos derived from20%or 5%oxygen conditions. The earlyblastocysts, which reached the expanded-blastocyst stageat Day 7 post-insemination, were considered competentwhereas those that reached the same stage at Day 8 post-insemination were categorized as noncompetent, based oncharacterization by Hasler et al. (1995) and Bernardi andDelouis (1996). Blastocyst-stage embryos from each oxy-gen condition (5% and 20%) and competence category(competent and noncompetent) were used to investigatethe expression of NRF2, KEAP1, antioxidant genes down-stream of NRF2 (CAT, PRDX1, SOD1, and TXN1), andgenes related to lipid metabolism (SREBP1 and CPT2).The amount and distribution of NRF2 and KEAP1 proteinswere evaluated in blastocysts from each category by immu-nohistochemistry. The level of ROS, mitochondrial activity,lipid content, and cryotolerance were determined for eachblastocyst category as well.

Oocyte Collection and In Vitro EmbryoProduction

Bovine ovaries were collected from a nearby slaughter-house and transported to the laboratory in 378C salinesolution within 3 hr of slaugher. The ovaries were washedtwice in fresh phosphate buffer saline (PBS), and cumulus-oocyte complexes (COCs) were then aspirated from fol-licles 2�8mm in size. Only COCs with a homogenouscytoplasm and surrounded by at least three layers ofcompacted cumulus cells were used for in vitro maturationin groups of 50 in modified TCM199 culture media(Sigma-Aldrich, Munich, Germany) supplemented with4.4mM HEPES, 33.9mM NaCHO3, 2mM pyruvate,2.9mM calcium lactate, 55mg/ml gentamicin, and 12%(v/v) heat-inactivated oestrus cow serum (OCS). Followingin vitro maturation, fertilization was performed in Fert-TALP medium (Parrish et al., 1986, 1988) supplementedwith 20mM penicillinamine, 10mM hypotaurine, 2mMnoradrenaline, 6mg/ml bovine serum albumin (BSA),50mg/ml gentamicin, and 1mg/ml heparin. Frozen-thawed bull sperm at a concentration of 2� 106 sperm/ml were used for in vitro fertilization, which was initiated byco-culture of sperm and COCs for 20 hr. Both maturationand fertilization procedures were performed under 20%oxygen. Presumptive zygotes were subsequently trans-ferred to CR-1aa culture medium (Rosenkrans andFirst, 1994) supplemented with 10% OCS, 10ml/mlBME (essential amino acids), and 10ml/ml MEM (nones-sential amino acids). Zygoteswere cultured either at 5% or20% oxygen levels until Day 8 (blastocyst stage) at 398C.Embryosat the 2-, 4-, 8-, 16-cell, andblastocyst stagewereharvested at 32, 48, 72, 92, and 168�192 hr post-insemi-

nation, respectively, and frozen at�808C for gene expres-sion analysis.

RNA Isolation and cDNA SynthesisThree biological replicates each containing fifty 2-cell,

thirty 4-cell, twenty 8-cell, fifteen 16-cell-, and 10 blasto-cyst-stage embryos from each oxygen condition were usedfor RNA isolation. Total RNAwas isolated using a PicoPureRNA isolation kit (Arcturs, Munich, Germany) according tothe manufacturer’s instruction. On-column DNA digestionwas performed using RNase-free DNase enzyme (QiagenGmbH, Hilden, Germany). After two washes with washingbuffer, theRNAwaseluted in 11ml elution buffer, and storedat �808C until further use.

Equal quantities of RNA input (200 ng) were used forcDNA synthesis from each embryo stages derived from 5%or 20% culture conditions. The cDNA synthesis was per-formed using oligo (dT)-25 and random primers (Promega,Madison, WI) and Superscript reverse transcriptase II (In-vitrogen, Karlsruhe, Germany). The cDNA synthesis reac-tion, containing 4ml 5� first-strand buffer (375mM KCl,15mMMgCl2, 250mMTris�HCl, pH 8.3), 2ml dithiothreitol(Promega), 1ml dNTP, 0.7ml Superscript II reverse tran-scriptase, and 0.3ml RNase inhibitor (Promega), was incu-bated at 428C for 90min and then denatured at 708C for15min.

Real-Time Quantitative PCRA total of 12 genes��NRF2, KEAP1, NRF2-target anti-

oxidant genes [superoxide dismutase-1 (SOD1), hemeox-ygenase decycling-1 (HMOX1), peroxiredoxin-1 (PRDX1),NAD(P)H dehydrogenase quinine-1 (NOQ1), thioredoxin-1(TXN1), catalase (CAT)], and genes involve in lipid metab-olism (sterol regulatory element binding protein transcrip-tion factor1 (SREBP1), acetyl-CoA carboxylase alpha(ACACA1), peroxisome proliferator-activated receptor al-pha (PPARAa), and carnitine palmitoyl transferase-2(CPT2)��were investigated for their expression using se-quence-specific primers (Table 2) designed using Primer3online Software v.0.4.0 (http://frodo.wi.mit.edu/primer3/).AnABI PRISM1 7000 sequence detection system (AppliedBiosystems, Foster City, CA) instrument was used to quan-tify the transcript abundanceusingSYBRgreen fluorescentdetection dye.Standard curvesweregenerated using serialdilution (consisting of 101�109 molecules) of each geneproduct, including the endogenous control gene, glyceral-dehyde 3-phosphate dehydrogenase (GAPDH), to eluci-date the amount of transcript in unknown samples. Thesuitability of GAPDH as an endogenous control was vali-dated by testing its expression stability among the experi-mental samples (data not shown).

Quantitative PCRs were performed in 20ml reaction vol-umes containing 10ml SYBR green master mix (Eppendorf,Hamburg,Germany), 2ml cDNA,andanoptimizedamountofforward and reverse primers diluted in water to a volume of8ml. Following initial denaturation stepat 958C for 3min, then40 cycles of 15 sec at 958C and 45sec at 608C was usedas thermal cycling program. The relative-standard-curve


Mol. Reprod. Dev. 81:497–513 (2014) 507

http://frodo.wi.mit.edu/primer3/

method was used to determine the amount of transcriptsin the experimental samples, and the relative quantitywas normalized using GAPDH (Goossens et al., 2005)to compared transcript abundance differences between em-bryos cultured at 5% and 20% oxygen for each developmen-tal stage. The significance of mean differences in mRNAabundance between experimental samples was determinedby t-test.

Immunofluorescence Detection of NRF2 andKEAP1 Proteins

Immunohistochemistry was performed to detect andlocalize NRF2 and KEAP1 proteins in blastocyst-stageembryos as described previously (Ghanem et al., 2007;Goossens et al., 2011). Briefly, 15 embryos from eachblastocyst category were washed three times in phos-phate-buffered saline (PBS), then fixed overnight at 48Cin 4% (w/v) paraformaldehyde in PBS. Fixed blastocystswere washed twice with glycine in PBS (GPBS), thenpermeabilized with 0.5% (v/v) Triton-X100 (Sigma-Aldrich)in PBS for 4 hr at room temperature. The samples wereincubated in 3% normal donkey serum (Sigma-Aldrich) inPBS for 1 hr at room temperature, followed by incubationwith specific primary antibodies against NRF2 (1:100 dilu-tion, H300; Santa Cruz Biotechnology, Dallas, TX) orKEAP1 (1:100 dilution, H190; Santa Cruz Biotechnology)overnight at 48C. Then, embryos were further incubated atroom temperature for 1 hr in the dark with FITC-conjugatedrabbit anti-goat secondary antibody (1:100 dilution, Life-span Biosciences, Seattle, WA). A droplet of Vectashieldmounting medium (Dabco; Acros, Geel, Belgium) contain-ing 40,6-diamidino-2-phenylindole (DAPI) was used to stain

the nuclei. Finally the slides were mounted, and sampleswere visualized under a CLSM LSM-780 confocal laser-scanning microscope (Zeiss, Germany).

ROS DetectionBlastocyst-stage embryos from the two oxygen condi-

tions and competence categories were used to determineROS level using 6-carboxy- 20,70-dichlorodihydrofluorescindiacetate (H2DCFDA). Fifteen blastocysts from each groupwere incubated with 400ml of 5mMH2DCFDA for 20min indark at 378C. Embryo samples were then washed twicein PBS, and images were captured immediately under aninverted microscope (Leica DM IRB, Leica, Wetzlar,Germany) using a green-fluorescence filter.

Mitochondrial Activity DetectionMitochondrial activity of blastocyst-stage embryos was

determined using MitoTracker1 Red CMXRos (M7512;Invitrogen). Ten blastocysts from each group were incubat-ed with 200 nM MitoTracker red dye for 45min, followed bytwowashingwith PBS, andwere then fixed overnight at 48Cwith 4% formaldehyde. Fixed specimens were mounted onthe slide with Vectashield (H-1200) containing DAPI. Im-ages were acquired with the suitable wavelength for thedyes at 40�magnification under a CLSM LSM-780 confo-cal laser-scanning microscope (Zeiss).

Assessment of Blastocyst Cell NumberTotal blastocyst cell number was quantified using nucle-

ar fluorescence staining with the glycerol-based Hoechst33342 according to the manufacturer’s recommendation.

TABLE 2. Primers Designed for Quantitative Real-Time PCR Analysis

Gene Accession number Primer sequencesAnnealing

temperature (8C)Productsize (bp)

NFE2L2 NM_001011678 F 50-CCCAGTCTTCACTGCTCCTC 55 165R 50-TCAGCCAGCTTGTCATTTTG

KEAP1 NM_001101142.1 F 50-TCACCAGGGAAGGATCTACG 55 199R 50-AGCGGCTCAACAGGTACAGT

CAT NM_001035386.1 F 50-TGGGACCCAACTATCTCCAG 51 178R 50-AAGTGGGTCCTGTGTTCCAG

TXN1 NM_173968.3 F 50-AGCTGCCAAGATGGTGAAAC 55 215R 50-ACTCTGCAGCAACATCCTGA

HMOX1 NM_001014912 F 50-CAAGGAGAACCCCGTCTACA 55 225R 50-CCAGACAGGTCTCCCAGGTA

PRDX1 NM_174431.1 F 50-TGGATCAACACACCCAAGAA 55 217R 50-GTCTCAGCGTCTCATCCACA

SOD1 NM_174615 F 50-AGAGGCATGTTGGAGACCTG 53 189R 50-CAGCGTTGCCAGTCTTTGTA

NQO1 NM_001034535.1 F 50-AACCAACAGACCAGCCAATC 54 154R 50-CACAGTGACCTCCCATCCTT

SREBP1 NM_001113302.1 F 50-ACCGCTCTTCCATCAATGAC 56 190R 50-TTCAGCGATTTGCTTTTGTG

PPARAa NM_001034036.1 F 50-CCTACGGGAATGGCTTCATA 54 219R 50-GCACAATACCCTCCTGCATT

CPT2 NM_001045889.1 F 50-CACAACATCCTGTCCACCAG 54 209R 50-CCTTCCAAGGCATCAAACAT

ACACA1 NM_174224.2 F 50-CTCTTCCGACAGGTTCAAGC 55 248R 50-ACCATCCTGGCAAGTTTCAC


508 Mol. Reprod. Dev. 81:497–513 (2014)

Twenty blastocysts derived from 5% and 20% oxygenconditions were fixed for 5min in a solution containing2% formalin and 0.25% gluteraldehyde. The fixed blasto-cysts were mounted and stained for 10min with glycerol-based Hoechst 33342 (12.5mg/ml) solution on clean glassslides. Stained nuclei appeared blue when visualized usingan epifluorescent microscope (Olympus, Tokyo, Japan)fitted with a blue filter (excitation: 330�385 nm; emission:420 nm; dichromatic: 400 nm). The cell number was scoredfor individual blastocysts from each culture group.

Cryotolerance TestThe cryotolerance of bovine embryos was assessed as

described previously (Li et al., 2006). Briefly, blastocysts atDay 7 and 8 from 5% and 20% oxygen conditions wereequilibrated in a solution of 10% ethylene glycol and 10%DMSO for 2min. Sampleswere then exposed to vitrificationsolution containing 20% ethylene glycol and 20% DMSO.Blastocysts were loaded into an open-pulled straw, thenimmediately plunged into liquid nitrogen. After freezing, theembryoswere thawed by immersing the embryo-end of theopen pulled straw in 0.3mol/L sucrose for 5min, then in0.2mol/L sucrose for 5min, and then in holding medium for5min. Finally, the embryos were cultured to determinehatching rate after thawing.

Statistical AnalysisData were statistically analyzed using Statistical Analy-

sis System (SAS) version 9.1 (SAS Institute Inc., Cary, NC).Mean developmental and hatching rates of different treat-ment groups of embryos were analyzed by one-way AN-OVA followed by a multiple pairwise comparison (Tukeytest). SAS General Linear Model (GLM) was used to ana-lyze the relative mRNA expression data. Differences inmean valueswere tested among treatments using ANOVA,followed by a multiple pairwise comparison using t tests.Differences were considered significant at P< 0.05.

ACKNOWLEDGMENT

We are grateful to the German Academic ExchangeProgramme (DAAD) for financial support of Amin Ahmed.

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Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway