Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/234070876

SubcellulardynamicsofthematernalcelldeathregulatorBCL2L10inhumanpreimplantationembryos

ARTICLEinHUMANREPRODUCTION·JANUARY2013

ImpactFactor:4.57·DOI:10.1093/humrep/des443·Source:PubMed

CITATION

1

READS

18

6AUTHORS,INCLUDING:

Jean-FrancoisGuerin

CentreHospitalierUniversitairedeLyon

129PUBLICATIONS2,477CITATIONS

SEEPROFILE

AurélieCornut-Thibaut

EcolenormalesupérieuredeLyon

4PUBLICATIONS15CITATIONS

SEEPROFILE

SandrinePouvreau

UniversitéVictorSegalenBordeaux2

27PUBLICATIONS429CITATIONS

SEEPROFILE

AbdelAouacheria

FrenchNationalCentreforScientificResearch

43PUBLICATIONS623CITATIONS

SEEPROFILE

Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate,

lettingyouaccessandreadthemimmediately.

Availablefrom:AbdelAouacheria

Retrievedon:03February2016

ORIGINAL ARTICLE Reproductive biology

Subcellular dynamics of the maternalcell death regulator BCL2L10 in humanpreimplantation embryosJean-Francois Guerin1, Aurelie Cornut-Thibaut2,3,Sandrine Giscard-Destaing1, Sandrine Pouvreau4,5, Yannis Guillemin3,6,and Abdel Aouacheria2,*1Service de Medecine de la Reproduction, Hopital Femme-Mere-Enfant, Bron F-69500, France 2Laboratoire de Biologie Moleculaire de laCellule of Ecole Normale Superieure de Lyon, LBMC UMR 5239 CNRS – UCBL – ENS Lyon – HCL, 46 Allee d’Italie, Lyon Cedex 07F-69364, France 3IBCP Institut de Biologie et Chimie des Proteines, UMR 5086, CNRS, Universite de Lyon, Lyon F-69397, France4Physiologie Integrative Cellulaire et Moleculaire, Universite Lyon 1, UMR CNRS 5123, Batiment R. Dubois, 43 boulevard du 11 novembre1918, Villeurbanne F-69622, France5Present address: Interdisciplinary Institute for Neuroscience, University of Bordeaux, CNRS UMR 5297, Bordeaux F-33000, France6Present address: Laboratoires Genevrier, 280, Rue de GOA Z.I. Les Trois Moulins, Antibes F-06600, France

*Correspondence address. LBMC UMR5239 CNRS/ENS Lyon/UCBL/HCL, Faculte de Medecine Lyon Sud, 165 Chemin du Grand Revoyet,69495 Pierre-Benite, France. Tel: +33-4-26-23-59-43; Fax: +33-4-26-23-59-00; E-mail: abdel.aouacheria@ens-lyon.fr

Submitted on September 5, 2012; resubmitted on November 26, 2012; accepted on November 30, 2012

study question: What is the expression status and subcellular localization of the maternally expressed Bcl-2 family member,BCL2L10, in early human embryos of diverse developmental stages and quality?

summary answer: The anti-apoptotic protein, BCL2L10, is expressed in human preimplantation embryos at least until the blastocyststage and appears to be differentially distributed at the subcellular level between viable embryos and fragmented or arrested embryos.

what is known already: BCL2L10 is an anti-apoptotic member of the BCL-2 family that shows abundant expression in humanoocytes and limited sequence conservation to its mouse homologue.

study design, size, duration: Embryos donated with informed consent by couples consulting for infertility in the Departmentof Reproductive Medicine (Hopital Femme Mere Enfant, Bron, France) were divided into two groups: high quality embryos (n ¼ 18) and poorquality embryos (n ¼ 30). Semen samples (n ¼ 4) were obtained after informed consent from men consulting for couple infertility. Experi-ments involving human preimplantation embryos were performed between January and December 2009.

participants/materials, setting, methods: We examined BCL2L10 expression and subcellular localization in earlyhuman embryos by using immunofluorescence and confocal microscopy. The subcellular distribution of BCL2L10 was also studied in ejacu-lated sperm cells and in isolated mouse skeletal muscle fibres.

main results and the role of chance: The BCL2L10 protein was detectable in healthy human preimplantation embryos atleast until the blastocyst stage. In high-quality embryos, BCL2L10 was predominantly cytoplasmic with mitochondrial localization. In contrast,BCL2L10 exhibited extra-mitochondrial localization in abnormal embryos, and was nuclear–cytoplasmic in approximately half (17/30) of thepoor-quality embryos. Morphologically fragmented embryos showed coexistence of blastomeres with BCL2L10-positive expression and blas-tomeres or fragments negative for BCL2L10.

limitations, reasons for caution: Future studies are needed to evaluate whether embryo quality is related to an exclusivemitochondrial localization of BCL2L10. Mechanisms mediating the nuclear translocation of BCL2L10 in abnormal embryos and functions ofthis nuclear pool of BCL2L10 are currently unknown.

wider implications of the findings: The nuclear localization of BCL2L10 in abnormal embryos suggests a potential role forthis protein in pathological conditions resulting in embryo arrest.

study funding/competing interest(s): No external funding was obtained for this study. There are no competing interests.

Key words: apoptosis / cell death / human preimplantation development / BCL-2 family / embryo quality

& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oup.com

Human Reproduction, Vol.28, No.3 pp. 729–739, 2013

Advanced Access publication on January 4, 2013 doi:10.1093/humrep/des443

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

IntroductionApoptosis, a form of cell death by self-destruction, has been reportedin human oocytes, and in mouse in vivo embryos and human in vitroembryos (Levy et al., 1998; Hardy, 1999; Morita and Tilly, 1999; Jur-isicova and Acton, 2004). Recent evidence suggests that the develop-mental potential of the embryo could be related to its apoptosis rate.Thus, the investigation of apoptosis-associated genes and mechanismsin early embryos is crucial. BCL-2 family proteins are major regulatorsof cell death and survival (Youle and Strasser, 2008). The balancebetween the relative expression and activity levels of anti- andpro-apoptotic BCL-2 family members is considered to be an import-ant determinant of cell death sensitivity in many cell types and may bea central determinant of oocyte survival as well (Boumela et al., 2011).Most BCL-2 family genes are developmentally regulated and displayconserved homologues in diverse vertebrate organisms. Among themembers of the BCL-2 family, the anti-apoptotic protein BCL2L10(Aouacheria et al., 2001; Ke et al., 2001; Lee et al., 2001; Zhanget al., 2001) is abundantly and selectively expressed in humanoocytes and non-human primate early embryos (Guillemin et al.,2009). Interestingly, this BCL-2-related gene evolved rapidly at themolecular level (Aouacheria et al., 2005; Guillemin et al., 2009,2011), like many other reproductive factors (Wyckoff et al., 2000;Swanson et al., 2001), but has a conserved cellular specificity of ex-pression at the oocyte-to-zygote transition throughout the vertebratelineage from zebrafish (Arnaud et al., 2006) to rodents (Burns et al.,2003; Hamatani et al., 2004; Yoon et al., 2009) and primates, includinghumans (Guillemin et al., 2009). Notably, knockdown of zebrafishBCL2L10 by antisense morpholinos caused early embryonic lethality(Arnaud et al., 2006) and neutralization of endogenous humanBCL2L10 by specific antibodies enhanced oocyte degeneration invitro (Guillemin et al., 2009), indicating that this protein probablyacts as a survival factor in female germ cells. However, RNA interfer-ence targeting mouse BCL2L10 resulted in a higher frequency ofspindle defects and abnormal chromosome configurations in meiosis(Yoon et al., 2009), rather than in massive oocyte death, which sug-gests that the cellular role of this BCL-2 family member can extendbeyond controlling cell death or that its role has changed in thelineage leading to mice. At the moment, at least two observationssupport this latter possibility. First, BCL2L10 knockout mice demon-strated no overt phenotype at the whole animal level (Russell et al.,2002). Secondly, BCL2L10 exhibits particularly high evolutionaryrates in mice and rats (Aouacheria et al., 2005; Guillemin et al.,2009, 2011). These results do not exclude the possibility thatBCL2L10 has as yet undefined regulatory functions in oocytes, butmake clear that rodent models may not be well suited to characterizebiochemical and cell biological aspects of the human BCL2L10 homo-logue. On the other hand, it is important to underline that theserodent models offer important advantages for functional studies,which are not easy to perform with human oocytes and embryos(because of difficulties in obtaining ethical approvals, leading to delayin the experiments, small sample size and restrictive study design in-cluding major limitations to performing genetic engineering in mostcountries).

In humans, the mode of action of the molecular actors drivingoocyte maturation, fertilization and preimplantation developmentremains largely unknown at the cellular and subcellular levels. In

particular, it is not precisely known to what extent potential embryon-ic phenotypes are associated with altered distribution of maternallycontributed proteins, nor at what stages of development these mater-nal supplies may be depleted. The aim of the present work was there-fore to examine BCL2L10 expression and subcellular localization inearly human embryos of diverse developmental stages and quality.We report that this prominent maternally expressed BCL-2 familyprotein could be detected in human preimplantation embryos atleast until the blastocyst stage, with a differential distribution at thesubcellular level between viable embryos and non-evoluting orarrested embryos.

Materials and Methods

Embryo recoveryEmbryos were donated with fully informed consent by couples who wereconsulting for infertility in our Center of Reproductive Medicine (HopitalFemme Mere Enfant, Bron, France) and who were proposed to undergoIVF or ICSI procedure. In most cases, the origin of infertility was due toboth male and female factors (abnormal sperm characteristics and alter-ation of ovarian function). Women received a daily gonadotrophin-releasing hormone agonist (0.1 mg ss Decapeptyl, Beaufour IpsenPharma, Paris, France) on cycle day 21. After verification of complete pi-tuitary desensitization 3 weeks later, recombinant FSH therapy wasinitiated. Administration of recombinant hCG (Ovitrelle, Merck Serono)was performed according to the classical criteria: several follicles exceeding17 mm in diameter and estradiol levels per mature follicle .200 pg/ml.Oocyte retrieval was performed 36 h after hCG injection by transvaginalultrasound-guided aspiration. Either conventional IVF or ICSI was per-formed according to sperm characteristics after the washing and selectionprocedure.

The research protocol has been authorized by the French BiomedicineAgency. A total of 48 embryos were used, including appropriate controls.Of these 48 embryos, 18 were considered to be of high quality since theywere cultured from supernumerary embryos which were selected andfrozen at the 4-cell stage or blastocyst stage in order to be transferredlater in the uterus of the female partner. The other 30 embryos were con-sidered as poor-quality embryos, and were to be discarded; they were nottransferred nor frozen, either because they were of Grade 3 or 4 (n ¼ 20)or because they were early-arrested embryos, exhibiting no evolution afterat least 24 h (n ¼ 10). The assessment of cleavage-stage embryo qualitywas done according to the classical classification, i.e. Grade 1: 0–10% frag-mentation; Grade 2: 11–25%; Grade 3: 26–50%; Grade 4: .50%. Theassessment of blastocyst stage embryo quality was done according tothe Gardner classification (1999).

Freezing protocolCleavage-stage embryos were frozen using a slow-freezing procedurederived from Testart et al. (1986): Embryo Freezing Pack (Origio,Denmark) containing 1,2-Propanediol as cryoprotectant, then1,2-propanediol plus sucrose, was used in a two-step process. Only good-quality embryos (Grades 1 and 2) with normal kinetics of development(four blastomeres for 2 days embryos) were cryopreserved. Embryo freez-ing was performed using a programmed cooler (Freezal, Air Liquide,France). Manual seeding of the straws was done at 278C with an ironruler that was plunged in liquid nitrogen. Then the embryos were slowlycooled (0.38C/min until 2358C, then 108C/min until 21508C, thenthe straws were plunged in liquid nitrogen). For the thawing procedure,the straws were removed from liquid nitrogen and thawed at room

730 Guerin et al.

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

Dynamic redistribution of BCL2L10 in human embryos 731

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

temperature. Removal of the cryoprotectant was done in a four-step pro-cedure using embryo thawing pack (Origio). The embryos were finallyplaced in Universal IVF medium (Origio). Embryo transfer occurredwithin 2 h after thawing. During the period corresponding to the study,the rates of clinical pregnancies following the transfer of fresh and cryopre-served embryos were 31.4 and 21.5%, respectively.

Origin of sperm samples and spermpreparationFour sperm samples were provided from men consulting for couple infer-tility, who gave their informed consent. The sperm concentrations rangedfrom 5 to 13 million/ml. After liquefaction, ejaculates were centrifugated

at 300g for 20 min on a discontinuous three-layer density gradient [Pure-Sperm (Nicadon): 90, 70 and 50%] in order to eliminate debris and selecta high-quality sperm fraction. The higher density fraction (90%) waswashed with Ferticult flushing medium (FertiPro N.V.), then centrifugatedat 600g for 10 min; finally the sperm pellet was resuspended in UniversalIVF medium (Origio).

Immunofluorescent staining and confocalimagingEmbryos were fixed with 4% paraformaldehyde, permeabilized, blockedand processed for double immunolabelling with selected primary anti-bodies and Alexa-conjugated secondary antibodies as previously described

Figure 1 BCL2L10 protein expression in healthy human preimplantation embryos at various stages of development. Embryos were stained withanti-mitoHSP70 and anti-BCL2L10 antibodies. The embryos were then incubated with Alexa Fluor 488 anti-mouse (green fluorescence) andAlexa Fluor 568 anti-rabbit (red fluorescence) secondary antibodies. Co-localization of green and red fluorescence is indicated by yellow colouration(OVERLAY). This experiment was repeated with at least three embryos per group. Representative embryos are shown. Scale bars: 25 mm. Highermagnification images of the boxed regions are shown.

Figure 2 Confocal 3D projection images showing the mitochondrial localization of BCL2L10 in healthy human preimplantation embryos. Embryoswere stained for DNA (magenta), mitoHSP70 (green fluorescence) and BCL2L10 (red fluorescence). Direct green/red overlay appears yellow. Imagesshow representative 3D projections reconstructed from confocal z-stacks (25 optical sections) superimposed on corresponding phase-contrastimages. Scale bars: 25 mm. High-resolution views through the stack of confocal sections are available as supplementary material (movies S1–S6).

732 Guerin et al.

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

Dynamic redistribution of BCL2L10 in human embryos 733

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

(Guillemin et al., 2009). The primary antibodies were as follows: rabbitpolyclonal anti-BCL-B (Biovision), mouse polyclonal anti-BCL2L10(Abnova), rabbit polyclonal anti-BAX(N20) (Santa Cruz), monoclonalanti-mitochondrial-HSP70 (Abcam) and rabbit polyclonal anti IP3R-I(Santa Cruz). For staining of DNA, the preparation was treated withTO-PRO-3 iodide or DAPI nuclear stain. Note that paraformaldehyde-fixed, zona-intact embryos could be difficult to penetrate with thesenuclear stains, leading to weak staining or loss of microscopic details. Torelease calcium from internal stores and induce cell death, embryoswere incubated with thapsigargin (1 mM, 1 h) prior to fixation and im-munofluorescence analysis. Sperm cells were incubated with 100 nM Mito-Tracker Red CMXRos for 20 min prior to fixation and indirectimmunofluorescence staining using rabbit polyclonal anti-BCL-B (Biovi-sion). Confocal analysis was performed on Zeiss (LSM510) or Leica(TCS-SP2) confocal microscopes. Three-dimensional (3D) reconstructionswere created from sequential confocal sections. Images were importedinto ImageJ for analysis of intensities within nuclear and mitochondrialcompartments. Images acquired with constant exposure settings andshowing embryos with clearly delimited single blastomeres were chosenfor analysis. One individual blastomere per embryo (of a total of 10embryos per condition, healthy or pathological) was outlined usingImageJ and the fluorescence intensity (red channel) was measured. The in-tensity measured in an adjacent area in the background was subtracted,and the background-subtracted intensity was considered as the total fluor-escence signal. Nuclear fluorescence signal (corresponding to the intensitymeasured in a selected area corresponding to the nucleus) was deter-mined after background subtraction and expressed as the percentage oftotal fluorescence. Fluorescence intensity of the red channel was quanti-fied in merged images and the extra-mitochondrial signal was obtainedafter background subtraction. The mitochondrial signal was calculated asthe difference, normalized to the total fluorescence, between the totalfluorescence and the extra-mitochondrial signal area. A Student’s t-testwas used to calculate the P values.

Results

Expression and subcellular distributionof BCL2L10 in high-quality human embryosof various developmental stagesBCL2L10 transcripts specifically accumulate in mouse and humanoocytes cultured in vitro and decline gradually after fertilization inrhesus monkey (Macaca mulatta) (Guillemin et al. 2009; Yoon et al.2009). These data prompted us to check whether the BCL2L10protein could be detected in human early embryos. Using confocalimmunofluorescent microscopy with previously well-characterizedantibodies (Guillemin et al., 2009), the BCL2L10 protein was detectedfrom the 4-cell through to the blastocyst stage in the cytoplasm ofhealthy preimplantation embryos (Fig. 1). In contrast, no significantsignal was detected in negative-control experiments that lackedprimary antibody [(Guillemin et al., 2009) and data not shown]. Bydouble staining with anti-mitoHSP70, the punctate fluorescence

pattern of BCL2L10 was found to coincide with the mitochondria(Fig. 1). For most developmental stages (4-cell, 8-cell, 16-cell andblastocyst), a number of 3D projection images from stacks of z-axisoptical scans were reconstructed (see Fig. 2A and Supplementarydata, Movies S1–S6). In all the embryos examined, the vast majorityof BCL2L10-specific fluorescence was associated with the mitochon-dria. Moreover, the cellular staining patterns for anti-BCL2L10and anti-mitoHSP70 were relatively homogeneous among blasto-meres. Altogether, these observations indicate that BCL2L10 predom-inantly adopts a mitochondrial localization in viable embryos.

Interestingly, treatment of viable 4-cell embryos with thapsigargin,an inhibitor of endoplasmic reticulum (ER) Ca2+-ATPase, causedboth morphological defects and a marked shift in the subcellulardistribution of BCL2L10, with a more diffuse staining than thatfound in the control condition (Fig. 2B and Supplementary data,Movies S5 and S6). The fact that thapsigargin induced exclusion ofBCL2L10 from the mitochondrial localization indicates that apoptoticstimuli (such as ER stress) can lead to internal defects in BCL2L10localization.

Expression and subcellular distribution ofBCL2L10 in poor-quality human embryosApproximately two-thirds of the embryos used for the study were ab-normal embryos with .25% fragmentation (n ¼ 20) or arrestedembryos (n ¼ 10). All these embryos were discarded and used as‘fresh’ samples, since they were considered as unsuitable for eithertransfer or cryopreservation. Staining for the BCL2L10 protein waspresent in all of these pathological embryos, albeit at different levels.We performed image analysis on images of individual blastomeres(one per embryo), in which the immunostaining intensities allowedclear differentiation between background, nuclei, mitochondrial andextra-mitochondrial staining. Quantification of the mitochondrial andnuclear staining of BCL2L10 was possible in all of the representativelyselected cases. In the group of abnormal embryos, we found that astatistically significant proportion of BCL2L10 was extra-mitochondrial(Fig. 3 and 4), whereas blastomeres from viable embryos were almostdevoid of diffuse cytoplasmic fluorescence (Fig. 5). More strikingly,bright fluorescence was detected in the nuclei of approximately half(17/30) of the poor-quality embryos (which were all at the 8-cellstage or beyond) whereas nuclear labelling was absent in intactviable embryos (Figs 3 and 5). Degenerating embryos showingstrong nuclear staining for BCL2L10 exhibited a fainter mitochondrialsignal (see for example Fig. 3, 8-cell). In highly fragmented embryos,we observed the coexistence of blastomeres showingBCL2L10-positive expression and some embryonic cellular bodiesexhibiting high fluorescence intensity, with blastomeres or fragmentsnegative for BCL2L10 (Fig. 3, 10/12-cell). Hence, while theBCL2L10 protein clearly co-localizes with the mitochondria in

Figure 3 BCL2L10 protein expression in pathological human preimplantation embryos at various stages of development. Embryos were stained withanti-mitoHSP70 and anti-BCL2L10 antibodies. The embryos were then incubated with Alexa Fluor 488 anti-mouse (green fluorescence) and AlexaFluor 568 anti-rabbit (red fluorescence) secondary antibodies. Co-localization of green and red fluorescence is indicated by yellow coloration(OVERLAY). This experiment was repeated with at least three embryos per group. Representative embryos are shown. Scale bars: 25 mm.Higher magnification images of the boxed regions are shown.

734 Guerin et al.

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

Dynamic redistribution of BCL2L10 in human embryos 735

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

healthy embryos, its subcellular distribution appears to be profoundlyaltered in abnormal embryos.

Next, poor-quality embryos were stained with a polyclonal antibodythat recognizes total BAX (i.e. both the native and conformationallyactive forms of the protein) as well as anti-BCL2L10 antibodies. Asshown in Fig. 4A, the subcellular localization of BAX was cytosolic,while there was a clear nuclear distribution of BCL2L10 in the samefragmented or arrested embryos. These data indicate that the proapop-totic BCL-2 family member BAX, a specific dimerization partner ofBCL2L10, did not undergo nuclear translocation in abnormalembryos displaying a nuclear pool of BCL2L10. Interestingly, althoughBCL2L10 localized to the nucleus in almost all blastomeres within agiven abnormal embryo, this was not an absolute rule, as evidencedby the double immunostaining analysis shown in Fig. 4B.

Subcellular distribution of BCL2L10 inejaculated sperm cells and isolated mouseskeletal muscle fibresWe sought to determine the subcellular distribution of BCL2L10,either endogenously or ectopically expressed, in cellular contexts dif-ferent from that of embryos, and different from cultured cells wherethe BCL2L10 localization has previously been extensively studied.To this end, ejaculated sperm aliquots from four patients were ana-lysed by confocal immunofluorescence microscopy usinganti-BCL2L10 antibody. As shown in Fig. 6, endogenous BCL2L10was expressed in all four sperm cell preparations and was mainlydetected at the level of the mitochondria-containing midpiece of thesperm tails. Interestingly, for one patient, strong immunofluorescencestaining of the nucleus was also observable in some sperm cells (Fig. 6,bottom panel, asterisks), suggesting that BCL2L10 could be a nucleus-resident protein under certain conditions in this cellular type as well.

Last, a recombinant construct encoding the green fluorescentprotein (GFP) open reading frame fused to the N-terminus ofBCL2L10 was transfected into mouse skeletal muscle fibres and thesubcellular localization of the chimeric protein was examined by con-focal microscopy. Similar to an ER-targeted GFP (Supplementary data,Fig. S7, B), GFP-BCL2L10 localized to the longitudinal sarcoplasmic re-ticulum of the striated muscle cells (Supplementary data, Fig. S7, A), asevidenced by the periodicity of fluorescence. Confocal images of iso-lated muscle fibres loaded with TMRM, a mitochondrial membranepotential-sensitive probe, showed that the regions of highGFP-BCL2L10 expression were associated with low TMRM fluores-cence (Supplementary data, Fig. S7, C). These findings indicate thatthe smooth ER represents another extra-mitochondrial subcellular lo-cation for BCL2L10 and that excess BCL2L10 protein can lead to dis-sipation of mitochondrial DCm, a phenomenon often associated withcell death.

DiscussionThis report presents several findings that are novel. First, we haveshown that the BCL2L10 protein is detected in human preimplanta-tion embryos at least until the blastocyst stage. Secondly, our resultsindicate that the BCL2L10 protein appears to be differentially distrib-uted at the subcellular level between viable embryos, where it has amitochondrial distribution, and non-viable or arrested embryos,where it adopts extra-mitochondrial locations, such as in thenucleus. This nuclear distribution of BCL2L10 is not a mere conse-quence of the cryopreservation procedure since poor-qualityembryos, in which such subcellular localization was observed, werenot submitted to cryopreservation. Rather, by considering the evolu-tion of embryos of different grades after thawing, the nuclear localiza-tion of BCL2L10 was shown to be in relation mainly to embryofragmentation and developmental stage (from the 8-cell stage to the

Figure 4 Confocal images of human preimplantation embryos oocytes showing pathological accumulation of BCL2L10 in the nuclei of blastomeres.Abnormal human preimplantation embryos were double stained with antibodies against BCL2L10 and BAX (A) or BCL2L10 and IP3R1 (B). Theembryos were then incubated with Alexa Fluor 488 anti-rabbit (green fluorescence) and Alexa Fluor 568 anti-mouse (red fluorescence) secondaryantibodies. Co-localization of green and red fluorescence is indicated by yellow coloration (OVERLAY). Scale bars: 25 mm. Higher magnificationimages of the boxed regions are shown.

Figure 5 Quantification of the mitochondrial and nuclear localiza-tion of BCL2L10 in healthy and pathological embryos. Graph showsmitochondrial (black bars) and nuclear (white bars) BCL2L10 as apercentage of total BCL2L10 fluorescence intensity within blasto-meres (one blastomere per embryo) of healthy (n ¼ 10) and patho-logical embryos (n ¼ 10). ****P , 0.0001 versus healthy (Student’st-test).

736 Guerin et al.

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

blastocyst stage). Moreover, no relationship could be establishedbetween nuclear BCL2L10 localization and the clinical context: therewas no difference, regarding the origin of infertility, between thegroup with the nuclear localization of BCL2L10 and the group withnormal mitochondrial staining. This does not mean that embryoorigin might not exert an influence on BCL2L10 localization; thisissue remains to be formally explored. Interestingly, our dataprovide evidence for a nuclear location of the BCL2L10 protein incertain sperm samples from patients, suggesting that BCL2L10 subcel-lular location change may extend to other tissues and conditions. Inthat respect, we report that BCL2L10 can be ectopically expressedin membranous organelles other than the mitochondria, such as inthe sarcoplasmic reticulum of mouse skeletal muscle fibres. In thissystem, high expression levels of GFP-BCL2L10 appear to trigger ab-errant dissipation of the mitochondrial membrane potential, raising theintriguing possibility that BCL2L10 may contribute to embryo demisein some cases, for instance in the embryonic cellular corpses that were

present within highly fragmented embryos and that were shown tocontain abundant endogenous BCL2L10.

As in our study, growing evidence indicates that, besides their local-ization at intracellular membranes (including the mitochondria, ER andthe nuclear envelop) or in the cytoplasm (Akao et al., 1994), severalBCL-2 structural homologues can be detected as nuclear proteins.For instance, in CHO cells BCL2L12 was retrieved in the nuclei andinduced cell growth inhibition and G2/M phase arrest (Hong et al.,2010). Moreover, both anti-apoptotic MCL-1 (Pawlikowska et al.,2010) and pro-apoptotic BID (Kamer et al., 2005; Zinkel et al.,2005) were found to translocate to the nucleus as part of the DNAdamage response pathway in mammalian cell lines. Nuclear localiza-tion of the BAX protein has also been reported in human cancercells (Nishita et al., 1998; Huang et al., 2006) as well as in early cleavingembryos (Metcalfe et al., 2004), though the biological significance ofthis finding remains unknown. Because the mitochondrial distributionof BCL2L10 has been found to be associated with healthy and

Figure 6 The subcellular localization of endogenous BCL2L10 in sperm cells. Brightfield and fluorescence images of human ejaculated spermatozoastained with anti-BCL2L10 antibody, MitoTracker Red CMXRos and TOPRO-3 for DNA detection. Immunostaining with anti-BCL2L10 antibodyshowed intense staining (green) in the midpiece where the mitochondria (red) are abundant. Two sperm cells from patient 4 show BCL2L10 stainingover the entire head (asterisks).

Dynamic redistribution of BCL2L10 in human embryos 737

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

developing embryos, it is tempting to speculate that BCL2L10 may nolonger be active as a prosurvival factor when expressed in the extra-mitochondrial cytoplasm or in the nucleus. It is also possible that theseextra-mitochondrial pools of BCL2L10 could be implicated in particu-lar cellular processes within pathological embryos. In future studies,associations between BCL2L10 subcellular localization and apoptoticmarkers (e.g. Annexin V or TUNEL) will need to be determined inorder to gain a better understanding of BCL2L10 function in relationto embryo quality, and in particular whether and how embryoquality is related to an exclusive mitochondrial localization ofBCL2L10. The mechanisms mediating the nuclear translocation ofBCL2L10 also warrant closer scrutiny. Interaction with binding part-ners or post-translational modifications, such as phosphorylation,might initiate a cascade of events inducing BCL2L10 redistributioninto the nucleus. It is also possible that BCL2L10 subcellular traffickingmight be mediated by cleavage and loss of membrane-targeting seg-ments since many Bcl-2 family proteins were shown to be cleavedby proteases, a process that frequently triggers changes in their func-tion (Valero et al., 2012). Last, the BCL2L10 protein has so far beenmainly investigated in mammalian cell lines and in the context ofcancer. The present study examined the expression and subcellular lo-calization of BCL2L10 in early human embryos: proteins with multi-site distributions or dynamic features of this kind usually representinteresting research objects in both basic science and clinicalinvestigations.

Supplementary dataSupplementary data are available at http://humrep.oxfordjournals.org/.

AcknowledgementsWe wish to thank Thomas Van Den Heuvel and Arnaud Deguerra atAgence de la Biomedecine. Sandrine Magnetto and Djemilia Cavret atCNRS Delegation Rhone-Auvergne are thanked for their help. We grate-fully acknowledge Anne Beghin and Denis Ressnikoff at Centre Communde Quantimetrie, and Vincent Jacquemond for providing laboratory fa-cilities. We are grateful to Claire Lionnet and Christophe Chamot(PLATIM, ENS Lyon, UMS3444/US8). We are grateful to all thecouples who consented to donate embryos and gametes to this re-search and to the staff of Service de Medecine de la Reproductionat Femme Mere Enfant Hospital, Bron.

Authors’ rolesA.A. and J.F.G. conceived and designed the project. A.C.T., Y.G., S.P.and S.G.D. acquired the data. A.A., J.F.G., S.P. and A.C.T. interpretedthe data. Drafting of the article was done by A.A. J.F.G. provided crit-ical revision. All authors were involved in final approval of the versionto be published.

FundingNo external funding was either sought or obtained for this study.

Conflict of interestNone declared.

ReferencesAkao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular

localization of bcl-2: detection in nuclear outer membrane,endoplasmic reticulum membrane, and mitochondrial membranes.Cancer Res 1994;54:2468–2471.

Aouacheria A, Arnaud E, Venet S, Lalle P, Gouy M, Rigal D, Gillet G. Nrh,a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitorof apoptosis. Oncogene 2001;20:5846–5855.

Aouacheria A, Brunet F, Gouy M. Phylogenomics of life-or-death switchesin multicellular animals: Bcl-2, BH3-Only, and BNip families of apoptoticregulators. Mol Biol Evol 2005;22:2395–2416.

Arnaud E, Ferri KF, Thibaut J, Haftek-Terreau Z, Aouacheria A, LeGuellec D, Lorca T, Gillet G. The zebrafish bcl-2 homologue Nrzcontrols development during somitogenesis and gastrulation viaapoptosis-dependent and -independent mechanisms. Cell Death Differ2006;13:1128–1137.

Boumela I, Assou S, Aouacheria A, Haouzi D, Dechaud H, De Vos J,Handyside A, Hamamah S. Involvement of Bcl-2 family members inthe regulation of human oocyte and early embryo survival and death:gene expression and beyond. Reproduction 2011;141:549–561.

Burns KH, Owens GE, Ogbonna SC, Nilson JH, Matzuk MM. Expressionprofiling analyses of gonadotropin responses and tumor developmentin the absence of inhibins. Endocrinology 2003;144:4492–4507.

Guillemin Y, Lalle P, Gillet G, Guerin JF, Hamamah S, Aouacheria A.Oocytes and early embryos selectively express the survival factorBCL2L10. J Mol Med 2009;87:923–940.

Guillemin Y, Cornut-Thibaut A, Gillet G, Penin F, Aouacheria A.Characterization of unique signature sequences in the divergentmaternal protein BCL2L10. Mol Biol Evol 2011;28:3271–3283.

Hamatani T, Falco G, Carter MG, Akutsu H, Stagg CA, Sharov AA,Dudekula DB, VanBuren V, Ko MS. Age-associated alteration of geneexpression patterns in mouse oocytes. Hum Mol Genet 2004;13:2263–2278.

Hardy K. Apoptosis in the human embryo. Rev Reprod 1999;4:125–134.Hong Y, Yang J, Chi Y, Wang W, Wu W, Yun X, Kong X, Gu J. BCL2L12A

localizes to the cell nucleus and induces growth inhibition through G2/Marrest in CHO cells. Mol Cell Biochem 2010;333:323–330.

Huang YT, Huang DM, Chueh SC, Teng CM, Guh JH. Alisol B acetate, atriterpene from Alismatis rhizoma, induces Bax nuclear translocationand apoptosis in human hormone-resistant prostate cancer PC-3cells. Cancer Lett 2006;231:270–278.

Jurisicova A, Acton BM. Deadly decisions: the role of genes regulatingprogrammed cell death in human preimplantation embryodevelopment. Reproduction 2004;128:281–291.

Kamer I, Sarig R, Zaltsman Y, Niv H, Oberkovitz G, Regev L, Haimovich G,Lerenthal Y, Marcellus RC, Gross A. Proapoptotic BID is an ATMeffector in the DNA-damage response. Cell 2005;122:593–603.

Ke N, Godzik A, Reed JC. Bcl-B, a novel Bcl-2 family member thatdifferentially binds and regulates Bax and Bak. J Biol Chem 2001;276:12481–12484.

Lee R, Chen J, Matthews CP, McDougall JK, Neiman PE. Characterizationof NR13-related human cell death regulator, Boo/Diva, in normal andcancer tissues. Biochim Biophys Acta 2001;1520:187–194.

Levy R, Benchaib M, Cordonnier H, Souchier C, Guerin JF. Annexin Vlabelling and terminal transferase-mediated DNA end labelling (TUNEL)assay in human arrested embryos. Mol Hum Reprod 1998;4:775–783.

738 Guerin et al.

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from

Metcalfe AD, Hunter HR, Bloor DJ, Lieberman BA, Picton HM, Leese HJ,Kimber SJ, Brison DR. Expression of 11 members of the BCL-2 familyof apoptosis regulatory molecules during human preimplantationembryo development and fragmentation. Mol Reprod Dev 2004;68:35–50.

Morita Y, Tilly JL. Oocyte apoptosis: like sand through an hourglass. DevBiol 1999;213:1–17.

Nishita M, Inoue S, Tsuda M, Tateda C, Miyashita T. Nuclear translocationand increased expression of Bax and disturbance in cell cycleprogression without prominent apoptosis induced by hyperthermia.Exp Cell Res 1998;244:357–366.

Pawlikowska P, Leray I, de Laval B, Guihard S, Kumar R, Rosselli F,Porteu F. ATM-dependent expression of IEX-1 controls nuclearaccumulation of Mcl-1 and the DNA damage response. Cell DeathDiffer 2010;17:1739–1750.

Russell HR, Lee Y, Miller HL, Zhao J, McKinnon PJ. Murine ovariandevelopment is not affected by inactivation of the bcl-2 familymember diva. Mol Cell Biol 2002;22:6866–6870.

Swanson WJ, Yang Z, Wolfner MF, Aquadro CF. Positive Darwinianselection drives the evolution of several female reproductive proteinsin mammals. Proc Natl Acad Sci USA 2001;98:2509–2514.

Testart J, Lasalle B, Belaisch-Allart L, Hazout A, Foreman R, Rainhorn JD,Frydman R. High pregnancy rate after early embryo freezing. Fertil Steril1986;46:268–272.

Valero JG, Cornut-Thibaut A, Juge R, Debaud AL, Gimenez D, Gillet G,Bonnefoy-Berard N, Salgado J, Salles G, Aouacheria A et al. m-Calpainconversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factorreveals two distinct alpha-helices inducing mitochondria-mediatedapoptosis. PLoS One 2012;7:e38620.

Wyckoff GJ, Wang W, Wu CI. Rapid evolution of male reproductive genesin the descent of man. Nature 2000;403:304–309.

Yoon SJ, Kim EY, Kim YS, Lee HS, Kim KH, Bae J, Lee KA. Role of Bcl2-like10 (Bcl2l10) in regulating mouse oocyte maturation. Biol Reprod 2009;81:497–506.

Youle RJ, Strasser A. The BCL-2 protein family: opposing activities thatmediate cell death. Nat Rev Mol Cell Biol 2008;9:47–59.

Zhang H, Holzgreve W, De Geyter C. Bcl2-L-10, a novel anti-apoptoticmember of the Bcl-2 family, blocks apoptosis in the mitochondriadeath pathway but not in the death receptor pathway. Hum MolGenet 2001;10:2329–2339.

Zinkel SS, Hurov KE, Ong C, Abtahi FM, Gross A, Korsmeyer SJ. A role forproapoptotic BID in the DNA-damage response. Cell 2005;122:579–591.

Dynamic redistribution of BCL2L10 in human embryos 739

at INIST

-CN

RS on February 21, 2013

http://humrep.oxfordjournals.org/

Dow

nloaded from