Maternal gene Ooep may participate in homologous recombination-mediated DNA double-strand break repair in mouse oocytes

2018·2.1 MB·

by  He, Da-Jian

ZOOLOGICAL RESEARCH
Maternal gene Ooep may participate in homologous
recombination-mediated DNA double-strand break
repair in mouse oocytes
Da-JianHe1,3,#,LinWang1,#,Zhi-BiZhang2,#,KunGuo1,Jing-ZhengLi1,Xie-ChaoHe1,Qing-HuaCui2,*,PingZheng1,*
1YunnanKeyLaboratoryofAnimalReproduction,KunmingInstituteofZoology,ChineseAcademyofSciences,KunmingYunnan650223,
China
2LaboratoryofBiochemistryandMolecularBiology,SchoolofLifeSciences,YunnanUniversity,KunmingYunnan650091,China
3KunmingCollegeofLifeScience,UniversityofChineseAcademyofSciences,KunmingYunnan650204,China
ABSTRACT and can substantially impair genomic integrity and cell
DNAdamageinoocytescancauseinfertilityandbirth viability. Two major pathways are involved in DNA DSB
repair, non-homologousendjoining(NHEJ)-andhomologous
defects. DNAdouble-strandbreaks(DSBs)arehighly
recombination (HR)-mediated repair. In oocytes, defection
deleterious and can substantially impair genome
of the error-free HR pathway can cause a decline in oocyte
integrity. Homologousrecombination(HR)-mediated quantity and quality in natural and pathological reproductive
DNADSBrepairplaysdominantrolesinsafeguarding aging in both mice and humans (Day et al., 2015; Perry et
oocyte quantity and quality. However, little is known al., 2013; Titus et al., 2013; Wang et al., 2014). Therefore,
HR-based DNA DSB repair in oocytes plays a key role in
regarding the key players of the HR repair pathway
regulating female reproductive performance. This repair has
in oocytes. Here, we identified oocyte-specific gene
been intensively studied in somatic cells, with many players
Ooep as a novel key component of the HR repair
identified(Adamsonetal.,2012);however,littleinformationis
pathway in mouse oocytes. OOEP was required for availableregardingthecomponentsoftheHRrepairpathway
efficient ataxia telangiectasia mutated (ATM) kinase inoocytes. Identifyingessentialparticipantswillshedlighton
activation and Rad51 recombinase (RAD51) focal understandingoocyteandovarianaging.
Oocyte-expressed protein (Ooep) is an oocyte-specific
accumulation at DNA DSBs. Ooep null oocytes were
maternal-effect gene. The OOEP protein localizes at the
defective in DNA DSB repair and prone to apoptosis
subcortical region of an oocyte and interacts with four other
uponexogenousDNAdamageinsults. Moreover,Ooep
maternalproteins,includingMATER,FILIA,PADI6,andTLE6,
null oocytes exhibited delayed meiotic maturation. toformasubcorticalmaternalcomplex(SCMC)inmouseand
Therefore, OOEP played roles in preserving oocyte humanoocytes(Lietal.,2008;Zhuetal.,2015). Depletionof
quantity and quality by maintaining genome stability. OOEP or its interaction proteins can lead to embryo arrest at
the2-cellstagewithuncharacterizedmolecularmechanismsin
Ooep expression decreased with the advance of
maternalage,suggestingitsinvolvementinmaternal
aging.
Received: 02March2018;Accepted: 09April2018;Online: 15June
Keywords: Ooep;Homologousrecombination;DNA
2018
double-strandbreakrepair;ATM;RAD51
Foundation items: This work was supported by the National Key
INTRODUCTION ResearchandDevelopmentProgramofChina(2017YFC1001102)and
NationalNaturalScienceFoundationofChina(81760507)
During the long period of meiotic arrest, oocytes are
#Authorscontributedequallytothiswork
exposed to endogenous and exogenous genotoxic insults
and tend to accumulate DNA damage. DNA damage can *Corresponding authors, E-mail: [email protected]; Cuiqing
occur as single-stranded breaks (SSBs) or double-strand [email protected]
breaks (DSBs), the latter of which are highly deleterious DOI:10.24272/j.issn.2095-8137.2018.067
SciencePress ZoologicalResearch39(6):387–395,2018 387
mice (Li et al., 2008; Tong et al., 2000; Yu et al., 2014; conditions. All experimental procedures and animal care
Yurttas et al., 2008; Zheng & Dean, 2009). We recently were performed according to the protocols approved by the
utilizedmouseembryonicstemcellsasamodeltoexplorethe EthicsCommitteeoftheKunmingInstituteofZoology,Chinese
functionofFILIAanduncovereditscriticalrolesinsafeguarding AcademyofSciences.
the genomic stability of pluripotent stem cells (Zhao et al.,
2015). This finding prompted our hypothesis that OOEP may DNAdamagetreatmentofoocytesandovaries
beinvolvedinregulatinggenomestabilityinoocytes. Thus,in Germinalvesicle(GV)-stageoocyteswereculturedaccording
thisstudy,wecarefullyexaminedthefunctionofOOEPinDNA to standard procedures (Marangos & Carroll, 2012). Ovaries
DSBrepairinoocytes. werecollectedfromnewbornmiceatpostnatalday5(P5)and
culturedaccordingtostandardprotocols(Gonflonietal.,2009).
MATERIALSANDMETHODS
Animals Antibodies
The mouse line with targeted mutation of Ooep (background Rabbit anti-OOEP serum was raised against the 1–19 amino
C57BL/B6×Sv129) was kindly provided by Dr. Jurrien Dean acids of the OOEP protein (Li et al., 2008), and antibody
from the National Institutes of Health, USA. These Ooeptm/tm specificity was verified by utilizing Ooep null oocytes. Other
female mice do not express the OOEP protein in oocytes (Li primaryandsecondaryantibodieswereobtainedcommercially,
et al., 2008). Mice were maintained in specific pathogen-free withrelevantinformationshowninTable1.
Table1Informationonprimaryandsecondaryantibodiesforimmunofluorescencestaining(IF)andimmunoblotting(IB)
Antibody Company Host Catalognumber Dilution
Primary
1:200(IF)
γ-H2AX Cellsignaling,USA Rabbit 9718S
1:800(IB)
1:200(IF)
p-ATM(Ser1981) Novusbiologicals,USA Mouse NB100-306
1:800(IB)
1:200(IF)
RAD51 Abnova,China Mouse H00005888-B01P
1:500(IB)
DDX4 Abcam,USA Rabbit AB13840 1:200(IF)
Beta-ACTIN Zhongshan,China Mouse TA09 1:5000(IB)
GFP Abcam,USA Chicken AB13970 1:2000(IB)
Secondary
AlexaFluor488Donkeyanti-RabbitIgG Invitrogen,USA A-21206 1:500(IF)
AlexaFluor555DonkeyantiRabbitIgG Invitrogen,USA A-31572 1:500(IF)
AlexaFluor647GoatantiRabbitIgG Invitrogen,USA A-21244 1:500(IF)
AlexaFluor488GoatantiMouseIgG Invitrogen,USA A-11029 1:500(IF)
AlexaFluor555DonkeyantiMouseIgG Invitrogen,USA A-31570 1:500(IF)
AlexaFluor488GoatantiChickenIgG Invitrogen,USA A-11039 1:500(IF)
Immunofluorescence staining and foci intensity DetectionofapoptosisbyTUNELstaining
quantification Frozen ovarian sections were permeabilized with 0.1%
Immunofluorescence staining of oocytes and ovaries was Triton X-100 in phosphate-buffered saline (PBS), followed by
performed according to standard procedures (Guo et al., incubation with the TUNEL reaction solution from the In-Situ
2016; Xiong et al., 2008). DNA was labeled with 4(cid:48), Cell Death Detection Kit, Fluorescein (Roche Diagnostics,
6-diamidino-2-phenylindole(DAPI,Sigma, Germany). Images USA).Allstainingprocedureswereperformedaccordingtothe
were analyzed using FV10-ASW 2.1 viewer software. The manufacturer’sinstructions.DNAwaslabeledwithDAPI.
intensitiesofthefociwerequantifiedusingImageJsoftware.
Hematoxylin and eosin (HE) staining and primordial and
Cometassay primaryfolliclecounting
Alkaline comet assay was performed according to standard OvariansectionswereincubatedwiththeHEreactionsolution
procedures (Berthelot-Ricou et al., 2011; Tice et al., 2000). (BOSTER, USA) at room temperature (RT). All staining
Comets were analyzed using CASP comet assay analysis procedures were performed according to the manufacturer’s
software (Andor Technology, UK). Threeindependent repeats instructions.Oocytesresidinginprimordialandprimaryfollicles
wereperformed. were counted, as described previously (Myers et al., 2004;
388 www.zoores.ac.cn
Skaznik-Wikieletal.,2007). usedforstatisticalanalysis.
ConstructionofOOEP-GFPexpressionplasmid Statisticalanalyses
The protein-coding region of Ooep was amplified by Quantitative analyses were based on at least three
polymerase chain reaction (PCR) and inserted into independent repeats and results were represented as
pcDNA3.1/CT-GFP-TOPO (Invitrogen, USA). Integrity was means±SEM. Data were first tested by the homogeneity of
confirmedbyDNAsequencing. variance test. For non-normal distribution, the data were
subjected to nonparametric tests. Otherwise, the data were
In vitro transcription and mRNA microinjection of GV
analyzed by t-tests. We considered P<0.05 as statistically
oocytes
significant.
Ooep-GfpexpressingplasmidswerelinearizedwithScaI(New
England Biolabs, USA). The mRNA was synthesized using RESULTS
an in vitro transcription kit (mMessage mMachine T7 kit,
Ooep participates in DNA double-strand break repair in
Ambion, USA),andpurifiedwithaRNeasyMinEluteCleanup
mouseoocytes
Kit (Qiagen, Germany). The mRNA was then dissolved in
Upon DNA damage, histone H2AX is phosphorylated at
nuclease-free water and stored at −80 ◦C. We microinjected
Ser139 (γ-H AX) and recruited to the damaged sites to form
500ng/µLofmRNAininjectionbuffer(10mmol/LTris-HCl(pH 2
visible foci under confocal microscopy (Rogakou et al., 1998).
7.5)and0.1mmol/LEDTA)intothecytoplasmofOoep-/-GV
γ-H AX foci formation is generally considered as a marker of
oocytes. 2
DNA damage. In the fully-grown GV oocytes, depletion of
Single-oocytecDNAamplificationandquantitativeRT-PCR OOEP caused a significant increase in γ-H AX foci intensity
2
Briefly, cDNA was prepared from a single GV oocyte and compared to wild-type oocytes (Figure 1A), suggesting more
22
amplified by 20 cycles of PCR according to published endogenous DNA damage in Ooeptm/tm oocytes. To validate
protocols (Tang et al., 2010). Glyceraldehyde 3-phosphate thisobservation, weperformedcometassay,anunambiguous
dehydrogenase (Gapdh) was used as the housekeeping methodthatmeasurestheextentofDNAdamageonasingle
control.PrimersforamplificationofGapdhandOoepincluded: cell basis (Berthelot-Ricou et al., 2011; Tice et al., 2000).
Ooep forward: 5(cid:48)-GTCATAGGCACAGACCAAGCG-3(cid:48), Ooep TheGVoocytesfromOoeptm/tmfemalesdisplayedsignificantly
reverse: 5(cid:48)-GGCCGCCATGTTCAAGAGAAT-3(cid:48); Gapdh forward: longer comet tails than those from the wild-type counterparts
5(cid:48)-TTGAGGTCAATGAAGGGGTC-3(cid:48),Gapdhreverse: 5(cid:48)-TCG (Figure1B),confirmingthatOoeptm/tmoocytescontainedmore
TCCCGTAGACAFAAigATuGrGe-3s(cid:48) .GraphPadPrism5softwarewas endogenousDNAdamage.

Figure1OoepdepletioncausesDNAdamageinmouseoocytes
A:Immunostainingrevealedhigherlevelsofγ-H2AXfociinOoeptm/tmGVoocytesthaninwild-type(WT)counterparts.Quantificationoftheγ-H2AXfociintensity
isshownintheloweFrpiagneul.rBe:C 1om eOtaosseaypc odnfiermpedlethtaitoOone pctm/atmuGsVeoosc yDtesNshAow eddgarematearDgNeA dianm amge.oDuatasaere oproescenytetdeass m ean±SEM.Scale
bars:10µm,***:P<0.001.
A: Immunostaining revealed higher levels of γ-H AX foci in Ooeptm/tm GV oocytes than in
2
ZoologicalResearch39(6):387–395,2018 389
wild-type (WT) counterparts. Quantification of the γ-H AX foci intensity is shown in the
2
lower panel. B: Comet assay confirmed that Ooeptm/tm GV oocytes showed greater DNA
damage. Data are presented as mean±SEM. Scale bars: 10 µm, ***: P<0.001.
The accumulation of DNA damage in Ooeptm/tm oocytes than in the wild-type oocytes, indicating compromised DNA
suggested inefficiency in DNA damage repair. To test this damage repair (Figure 2A, B). To further validate the
hypothesis, we treated wild-type and mutant GV oocytes role of OOEP in DNA damage repair, we performed a
with 50 µg/mL of etoposide, a topoisomerase II inhibitor, rescue experiment by micro-injecting Gfp (green fluorescent
to induce DNA DSBs (Marangos & Carroll, 2012; Nagy & protein)-tagged Ooep mRNA into the Ooeptm/tm oocytes.
Soutoglou, 2009), and compared the dynamics of γ-H AX Consistently, the OOEP-complemented oocytes resolved
2
resolution. Immediately after treatment, wild-type and mutant γ-H AX more efficiently than the Ooeptm/tm oocytes after
2
oocytes had comparable γ-H AX levels, as measured by etoposide treatment and recovery (Figure 2C). These data
2
immunostaining (Figure 2A) and immunoblotting analyses together support the notion that maternal protein OOEP is
(Figure 2B). Following several hours of DNA repair recovery, necessaryforefficientDNADSBrepairinoocytes. Inlinewith
γ-H AX was significantly resolved in the wild-type oocytes, thisrole,theproteinexpressionofOOEPwasslightlyinduced
2
reflecting efficient DNA damage repair. In sharp contrast, byetoposidetreatmentinGVoocytes(Figure2D).
γ-H AX remained at a higher level in the Ooeptm/tm oocytes 23
2

Figure2OoepparticipatesinDNAdouble-strandbreakrepairinmouseoocytes
Figure 2 Ooep participates in DNA double-strand break repair in mouse oocytes
A:Ooeptm/tm andWToocytesweresubjectedtoetoposidetreatmentandrecovery. Immediatelyafteretoposidetreatment(norecovery),Ooeptm/tm andWT
oocytesshowedasimilarlevelofγ-H2AX.After6-hrecovery,Ooeptm/tmoocytesretainedmoreγ-H2AXfocithanWToocytes.B:Etoposidetreatmentinduced
A: Ooeptm/tm and WT oocytes were subjected to etoposide treatment and recovery.
asimilarincreaseinγ-H2AXinbothWTandOoeptm/tmoocytes.γ-H2AXwasbetterresolvedinWTbutnotOoeptm/tmoocytesafter4-hrecovery.Eachsample
contained80oocytes.C:OOEP-GFPwasre-expressedinOoeptm/tmoocytes(leftpanel).After3-hrecoveryfromthesameetoposidetreatment,OOEP-rescued
oocyteshadIamlomweerdleivaelteoflyγ -Ha2fAtXert heantoOpooepstimd/tme otorceytaetsm(reignhtt p(anneol) .rDe:cIomvmeurnyob)l,o ttOingoaenpatlyms/itsms haonwded WthaTtO oOoEPcyptroetse inslhevoewlweedre aup regulatedby
etoposidetreatment.Eachsamplecontained20oocytes.Dataarepresentedasmean±SEM.Scalebars:10µm.*:P<0.05.
similar level of γ-H AX. After 6-h recovery, Ooeptm/tm oocytes retained more γ-H AX foci than
2 2
OoepmaybeinvolvedinHR-mediatedDNADSBrepair 2015; Titus et al., 2013). ATM activation by DSBs initiates
In oocytesW, TD NoAocDySteBs. isB:p Eretdoopmoinsiadnetl ytrereaptamireendt viniaduthceed a sriombuilsatrc ihneccrkepaosinet isnig nγa-HlingAaXs wine llbaosthD SWBTr eapnadir processes
HR-mediatedpathway, inwhichataxiatelangiectasiamutated insomaticcells(Behrense2tal.,2014;Jackson&Bartek,2009)
(ATM) kinase plays a central orchestration role (Oktay et al., and germ cells (Di Giacomo et al., 2005; Oktay et al., 2015).
Ooeptm/tm oocytes. γ-H AX was better resolved in WT but not Ooeptm/tm oocytes after 4-h
2
390 www.zroeocroevs.earcy.c.n Each sample contained 80 oocytes. C: OOEP-GFP was re-expressed in Ooeptm/tm
oocytes (left panel). After 3-h recovery from the same etoposide treatment, OOEP-rescued
oocytes had a lower level of γ-H AX than Ooeptm/tm oocytes (right panel). D: Immunoblotting
2
analysis showed that OOEP protein level were upregulated by etoposide treatment. Each
sample contained 20 oocytes. Data are presented as mean±SEM. Scale bars: 10 µm. *: P<0.05.
InmouseGVoocytes,ATMcanbeactivatedbycertainlevels thewild-typeoocytes,despiteγ-H AXlevelbeingcomparable
2
of etoposide-induced DNA DSBs. We therefore treated the (Figure3A).Immunoblottinganalysisofp-ATMfurthervalidated
oocytes with 50 µg/mL of etoposide for 3 h, as per previous this observation. Under normal conditions, very little p-ATM
study(Marangos&Carroll,2012), andexaminedtheeffectof was detected in either wild-type or mutant oocytes. However,
OOEP on ATM activation by phosphorylation at serine 1981 higher levels of p-ATM were induced in wild-type oocytes
residue (p-ATM) (Lavin & Kozlov, 2007). Co-immunostaining compared with mutant oocytes following etoposide treatment
analysis showed that fewer p-ATM foci were formed and (Figure3B).TheseresultssuggestthatOOEPregulatesATM
co-localizedwithγ-H AXfociintheOoeptm/tm oocytesthanin activationuponDNADSBs.
2 24

Figure3OOEPisrequiredforATMactivationandRAD51recruitmenttoDNAdamagesites
A:Ooeptm/tm anFdigWuTroeo 3cy tOesOwEerPe siusb rjeecqteuditroetdhe fsoarm AeTetMopo asicdetitvreaattimoenn t.anImdm RunAoflDuo5re1s creenccreusitatimninegnsth toow eDdNthaAt adta3m-hapogset damage,Ooeptm/tm
oocytescontainedalowerlevelofactiveATM(p-ATM)comparedwithWTcounterparts. B:Immunoblottingdetectionofp-ATMlevelunderuntreatedand
etoposide-treatseidtecosn ditions. Inuntreatedconditions,rarep-ATMwasdetectedinWTandOoeptm/tmoocytes. At3hafteretoposidetreatment,higherlevelof
p-ATMweredetectedinWTbutnotOoeptm/tmoocytes.Eachsamplecontained80oocytes.C:Immunofluorescencestainingshowedthatat3-hpostetoposide
treatment,OoeAptm:/ tmOooocyetepstmh/atmd alaonwder leWvelTof RAoDo5c1yttheasn WwTecornet rolss.uDbajteacatreedp restoen tetdhaes mseaamn±eS EMe.toSpcaolesibdaers :1t0reµamtm.*e**n:tP.< 0.001,*:P<0.05.
RAD51 isImanmeusnsoefnlutioalrersecceonmcbei nsatasienidnogw snhsotrweaemd tohfatA TaMt 3-h poTshtu dsa,mthaegaeb, sOenoceeptomf/tmO OooEcPyitneso occoyntteasinimedp aair edRAD51focal
in the HR pathway (Oktay et al., 2015, Perez et al., 2007). accumulationatDNAdamagesites.Takentogether,thesedata
After DNA DSBs, the RAD51 protein is recruited and binds demonstratethatOOEPmayfunctionintheHR-mediatedDNA
lower level of active ATM (p-ATM) compared with WT counterparts. B: Immunoblotting
to resected single-strand DNA to form DNA-protein filaments DSBrepairpathwaybyregulatingATMactivationandRAD51
(RAD51foci),whichfacilitatethesearchingandpairingofDNA recruitmenttoDSBsitesinmouseoocytes.
homologuesddeutericntgiorne coofm bpi-nAaTtioMn (lJeavseinl &unRdoetrh sutenitnr,ea2t0e1d3 )a.nd etoposide-treated conditions. In untreated
Ooep protectsoocytesfromDNADSB-inducedapoptosis
Uponetoposidetreatment,RAD51fociwereformedatdamage
andmeiosisdelay
siteslabeledcwointhdiγti-Hon2As,X rianrew ipld--AtyTpeMo owcaytse sd.eHteocwteedve irn,f eWweTr and Ooeptm/tm oocytes. At 3 h after etoposide
RAD51 foci were detected in Ooeptm/tm oocytes (Figure 3C). Persistent DNA DSBs can evoke p63-mediated apoptosis of
oocytes in primordial and primary follicles (Gonfloni et al.,
treatment, higher level of p-ATM were detected in WT but not Ooeptm/tm oocytes. Each sample
contained 80 oocytes. C: Immunofluorescence staining showed Zthoaolto gaitc a3l-Rhe speoasrct he3to9(p6o):si3d8e7 –395,2018 391
treatment, Ooeptm/tm oocytes had a lower level of RAD51 than WT controls. Data are presented
2009;Suhetal.,2006). OOEPisnecessaryforHR-mediated advanced maternal age (Oktay et al., 2014, 2015; Titus et
DNA DSB repair in oocytes, suggesting that Ooeptm/tm al., 2013; Xu et al., 2015). We examined if Ooep expression
oocytes might be sensitive to exogenous or endogenous declinedwithmaternalaging. mRNAexpressionlevelsofthe
DNA damage insults and prone to apoptosis. To test Ooep gene measured in single GV oocytes of old females
this hypothesis, we compared the apoptosis susceptibility (40-week old) were lower than those from young females
of oocytes to exogenous DNA damage insults between (8-week old) (Figure 4E). Thus, Ooep expression exhibited
wild-type and Ooeptm/tm females at the early postnatal stage. age-dependent decline and may contribute to ovarian failure
Five-day-old (P5) mouse ovaries, which contained mostly and reproductive aging under physiological or pathological
primordial follicles, were collected and cultured for 12 h with conditions.
various doses of DNA cross-linking agent cisplatin (Gonfloni
DISCUSSION
et al., 2009). At the concentration of 3 mg/L, cisplatin was
able to induce mild apoptosis (∼13.5%) in wild-type ovaries
DNA damage in oocytes can cause infertility, abortion, and
and was therefore utilized in the following studies. After in
birth defects, leading to reproductive failure. Among all
vitro treatment,ovariansectionsfromOoeptm/tm andwild-type
types of DNA damage, DSBs are the most deleterious and
females were counterstained with DDX4 (a germ cell specific
can substantially impair genome integrity. Experimental and
marker) and TUNEL. The percentages of apoptotic oocytes
clinicalstudiesonmiceandhumanshaverevealedthatintact
(DDX4+TUNEL+)weresignificantlyhigherinOoeptm/tm infants
DNA DSB repair in oocytes is necessary to preserve oocyte
than in wild-type counterparts (Figure 4A), suggesting that
quantityandquality. DeclineinDNADSBrepairhasacausal
Ooeptm/tm oocytesweremoresensitivethanwild-typeoocytes
relationship with ovarian failure, menopause, and infertility
to exogenous DNA damage insults due to compromised HR
(Oktay et al., 2014, 2015; Titus et al., 2013; Xu et al., 2015).
repair. To understand the in vivo effect of OOEP depletion
Genome-wideassociationstudiesonmenopausetiminghave
under the physiological conditions, we collected ovaries from
also highlighted the strong association of early menopause
4-week-old and 16-week-old wild-type and Ooeptm/tm females
with DNA damage response pathways where many genes
and compared the number of oocytes within primordial and
are involved in DNA repair, particularly the HR pathway
primary follicles, which express P63 and are subject to DNA
(e.g., BRCA1 pathway) and checkpoint response (cell cycle
damage-induced atresia (Suh et al., 2006). Intriguingly, at
responseandapoptosis)(Dayetal.,2015;Perryetal.,2013).
4-weekold,thenumbersofoocytesinprimordialandprimary
Thus, investigating how oocytes respond to DNA DSBs and
follicles were significantly higher in Ooeptm/tm females than in
what molecules are involved in HR-mediated DSB repair will
wild-type females. This probably reflected the developmental
shed light on oocyte and ovarian aging. In this study, we
haltcausedbytheaccumulatedendogenousDNAdamagein
presentedthefollowinglinesofevidencetosupportthenotion
partoftheoocytes. Incontrast, at16-weekold, thenumbers
that OOEP may participate in HR-mediated DNA DSB repair
of oocytes in primordial and primary follicles were statistically
by regulating the ATM-RAD51 axis in oocytes. Firstly, after
lower in Ooeptm/tm females than in wild-type females (Figure
etoposide treatment, γ-H AX foci, which monitor DNA DSBs,
4B). Together, these results support that OOEP protects 2
were gradually resolved in wild-type oocytes but persisted
oocytes from endogenous or exogenous DNA DSB-induced
in Ooep null oocytes, although this defect was rescued by
apoptosis and preserves oocyte quantity under DNA damage
re-expression of OOEP. Secondly, wild-type oocytes evoked
insults.
theactivationofATM,acentralcoordinatorinDNADSBrepair
DNA damage in fully grown oocytes compromises the
processes,inresponsetoexogenousDNAdamage. However,
completionofmeiosisbyinducingadelayinmeiosisresumption
Ooep-/- oocytes failed to efficiently ignite ATM activation and
(germinalvesiclebreakdown,GVBD)(Marangos&Carroll,2012)
this defect could be rescued by the re-introduction of OOEP
and the activation of spindle assembly checkpoint (SAC) at
proteins into mutant oocytes. Finally, RAD51, a recombinase
metaphase I (Collins et al., 2015; Marangos et al., 2015).
essential for HR-mediated DNA DSB repair, was recruited to
We investigated the influence of OOEP loss on the kinetics
damagesitesandformedfociinwild-typeoocytesinresponse
of GVBD and the first polar body extrusion (PBE) during
toetoposidetreatment.Incontrast,fewerRAD51proteinswere
oocyte in vitro maturation. Under normal conditions, the
recruitedtoandformedfociatDNADSBsinOoep-/-oocytes,
Ooeptm/tmoocytesshoweda4-hdelayinreaching50%-GVBD
although this defect could also be rescued by exogenous
compared with wild-type oocytes. In addition, ∼20% less
OOEP.
oocytescompletedGVBDintheabsenceofOOEPcompared
Due to the compromised DNA damage repair, Ooep-/-
with the wild-type counterparts at the end of culture (Figure
oocytes displayed an accumulation of endogenous DNA
4C). Likewise, the Ooeptm/tm oocytes displayed a 2-h delay in
damage,asdeterminedbythecometassay,anunambiguous
reaching 50%-PBE. At the end of culture, 20% less oocytes
method that measures the extent of DNA damage on a
completed PBE compared with the wild-type counterparts
single cell basis. As DNA damage can induce atresia
(Figure 4D). Thus, OOEP was essential for efficient meiosis
of oocytes in primordial and primary follicles through the
resumption and completion by preserving oocyte genomic
p63-mediated pathway (Gonfloni et al., 2009; Suh et al.,
integrity.DeclineinDNADSBrepaircompetenceisconsidered
2006), we proposed that the accumulation of endogenous
a major factor contributing to oocyte and ovarian aging at
DNA damage in Ooep-/- oocytes may drive the atresia
392 www.zoores.ac.cn
of oocytes and decrease the oocyte number in primordial derivedfromOoep-/-oocyteswerearrestedatthe2-cellstage
and primary follicles under physiological conditions. Indeed, (Li et al., 2008). This developmental arrest could be due
in young adult mice (16-week old), a mild but statistically to the DNA damage-induced cell cycle checkpoint (Xu et
significant decrease in oocyte quantity within the primordial al., 2015). Thus, OOEP is essential for preserving oocyte
and primary follicles was detected in the Ooep-/- ovaries quality. Interestingly,themRNAexpressionofOoepinmouse
comparedtothewild-typecounterparts. Oocytesinprimordial oocytes was reduced with advanced maternal age. Several
and primary follicles from Ooep-/- females also displayed key HR pathway genes, including Brca1, Mre11, Atm, and
higher sensitivity to exogenous DNA insults and were prone Rad51, also display age-dependent expression decrease in
to apoptosis. These observations suggest that OOEP is both human and mouse oocytes (Day et al., 2015; Oktay
necessaryforthepreservationofoocytequantityinnormaland et al., 2015; Titus et al., 2013). Therefore, the coordinated
DNA-damaged conditions. OOEP depletion not only impairs decrease in the expression of Ooep and other canonical HR
the survival of oocytes within primordial and primary follicles, genes contributes to ovarian failure and reproductive aging
but also affects meiotic maturation and early embryonic under physiological and pathological conditions. OOEP is
development. Compared with wild-type oocytes, Ooep-/- GV conservativelyexpressedinhumanoocytes(Zhuetal.,2015),
oocytes exhibited delays in meiosis resumption as well as implying that it may play similar roles in repairing DNA DSBs
26
progression to the metaphase II stage. Moreover, embryos viatheHRpathwayinhumanoocytes.
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Figure4OoepprotectsoocytesfromDNADSB-inducedapoptosisandmeiosisdelay
A:PostnataldFayig5u(Pr5e) 4ov Oarioesepfro pmrWoTteacntdsO oooepctym/ttmesfe fmraolemsw DerNesAu bDjecSteBd-tiontdheuscaemde acipspolaptitnotrseiast maenndt. TmUeNiEoLseisxa dmeinlaatiyonshowedthatthenumbers
ofapoptoticgermcells(DDX4+TUNEL+,arrowheads)werehigherinOoeptm/tmovariesthaninWTovaries.B:HEstainingrevealedthatovariesfrom4-week-old
Ooeptm/tmfemAal:e sPcoosnttaninaetadlm doareyp 5rim (oPrd5ia)l oanvdaprriiemsa rfyrofomllic leWstTha annthdo sOeofroemptmW/Ttmf efmeamleasl(elesf twpaenreel) .sAutb1j6e-cwteeedk otold ,tOhoee pstamm/tmeovariescontainedless
primordialandprimaryfolliclesthanWTovaries(rightpanel).Tenovariansectionswereexaminedforeachanimalandfivefemaleswereusedateachstage.C:
Thegerminalcviesspiclleatbirne atkrdeoawtnm(GenVBt.D )ToUfONoEepLtm /temxoaomcyitensawtiaosnd eslahyoedwceodm ptahraedt ttohtehe nWuTmoboceyrtse soufn daepronoprtmoatlicco ngdeitriomns .cTehlelsre wasa4-hdifference
inthetimingof50%-GVBDbetweenthetwosamples. Morethan80oocyteswereusedineachsample. D:Kineticsofthefirstpolarbodyextrusion(PBE)in
WTandOoep(tDm/tmDGXV4o+oTcyUteNsuEnLde+r, naorrmroawlchonedaitdiosn)s .wOeoreept mh/tmigohoecry teisn dOispolaeypetdm2/t-mh doevlaayriinerse atchhainng i5n0% W-PTBE o.Mvoarreiethsa.n B50: oHocEyt eswereusedineach
sample.E:mRNAexpressionofOoepdeclinedwithmaternalaginginGVoocytes.FourteensingleGVoocytesfromtwofemaleswereexaminedateachstage.
Dataarepressetnateindiansgm reeavn±eaSlEeMd. *t:hPa<t 0o.0v5a,r**i:ePs <f0r.o01m,* *4*:-Pw<e0e.0k0-1o.ld Ooeptm/tm females contained more primordial
OOEPparontde inpsriamreardyis tfroiblulitceldesin tchyatno ptlhaosmse afnrdomloc WalizTe dfeamt ales D(lSefBt rpeapnaeirl)a. rAetl ik1e6ly-winedeikresc to.ldO, OOEoPepistmp/tmredicted to contain
the subcortex of mouse and human oocytes (Li et al., 2008; an atypical hnRNP K homology (KH) domain implicated in
Zhu et al., 2015). OOEP was also excluded from the DNA RNAbinding(Wangetal.,2014). RNAbindingproteinshave
ovaries contained less primordial and primary follicles than WT ovaries (right panel). Ten
DSBsitesinoocytestreatedwithetoposide(datanotshown). been recognized as crucial players in the HR repair pathway
Thus, the functions of OOEP in regulating HR-mediated DNA (Adamsonetal.,2012).TheinvolvementofRBPsinregulating
ovarian sections were examined for each animal and five females were used at each stage. C:
The germinal vesicle breakdown (GVBD) of Ooeptm/tm oocytes wasZ dooelloagyiceadl Rcoesmeparacrhed3 9t(o6 )t:h3e8 7–395,2018 393
WT oocytes under normal conditions. There was a 4-h difference in the timing of 50%-GVBD
between the two samples. More than 80 oocytes were used in each sample. D: Kinetics of the
first polar body extrusion (PBE) in WT and Ooeptm/tm GV oocytes under normal conditions.
Ooeptm/tm oocytes displayed 2-h delay in reaching 50%-PBE. More than 50 oocytes were used
ovarian aging has also been reported (Day et al., 2015). For AlbrechtE,AminN,CorreT,HottengaJJ,ManginoM,SmithAV,Tanaka
instance, RNA binding protein FMRP encoded by fragile X T,AbecasisGR,AndrulisIL,Anton-CulverH,AntoniouAC,ArndtV,Arnold
mental retardation (Fmr1) gene plays an essential role in
AM, Barbieri C, Beckmann MW, Beeghly-Fadiel A, Benitez J, Bernstein
preservingovarianfunctioninmiceandhumans(Ascanoetal.,
L, Bielinski SJ, Blomqvist C, Boerwinkle E, Bogdanova NV, Bojesen SE,
2012;Luetal.,2012). Inthefuture,elucidatingthemolecular
BollaMK,Borresen-DaleAL,BoutinTS,BrauchH,BrennerH,BrüningT,
mechanism of OOEP is warranted to better understand the
Burwinkel B, CampbellA, Campbell H, ChanockSJ, Chapman JR, Chen
function of RBPs in preserving the genome integrity of germ
Y,Chenevix-TrenchG,CouchFJ,CovielloAD,CoxA,CzeneK,DarabiH,
cellsandearlyembryos.
De Vivo, Demerath EW, Dennis J, Devilee P, Dörk T, Dos-Santos-Silva I,
CONCLUSIONS Dunning AM, Eicher JD, Fasching PA, Faul JD, Figueroa J, Flesch-Janys
D,GandinI,GarciaME,García-ClosasM,GilesGG,GirottoGG,Goldberg
To the best of our knowledge, this work is the first to
provide evidence that maternal OOEP may participate in MS,González-NeiraA,GoodarziMO,GroveML,GudbjartssonDF,GuénelP,
HR-mediated DNA DSB repair by regulating the ATM-RAD51 GuoXQ,HaimanCA,HallP,HamannU,HendersonBE,HockingLJ,Hofman
axisinoocytes. Thus,OOEPmayparticipateintheregulation A,Homuth G,HooningMJ, HopperJL,HuFB, HuangJY,Humphreys K,
ofgenomestabilityinoocytesandcontributetoovarianfailure Hunter DJ, Jakubowska A, Jones SE, Kabisch M, Karasik D, Knight JA,
and reproductive aging under physiological or pathological Kolcic I, Kooperberg C, Kosma VM, Kriebel J, Kristensen V, Lambrechts
conditions. D,LangenbergC,LiJM,LiX,LindströmS,LiuYM,LuanJA,LubinskiJ,
MägiR,MannermaaA,ManzJ,MargolinS,MartenJ,MartinNG,Masciullo
COMPETINGINTERESTS
C,MeindlA,MichailidouK,MihailovE,MilaniL,MilneRL,Müller-Nurasyid
Theauthorsdeclarethattheyhavenocompetinginterests. M,NallsM,NealeBM,NevanlinnaH,NevenP,NewmanAB,Nordestgaard
BG, Olson JE, Padmanabhan S, Peterlongo P, Peters U, Petersmann A,
AUTHORS’CONTRIBUTIONS
PetoJ,PharoahPDP,PirastuNN,PirieA,PistisG,PolasekO,Porteous
P.Z.designedthestudy.P.Z.andQ.H.C.supervisedthestudy.D.J.H.,L.W., D,PsatyBM,PylkäsK,RadiceP,RaffelLJ,RivadeneiraF,RudanI,Rudolph
Z.B.Z.,K.G.,J.Z.L.,andX.C.H.performedtheexperiments. P.Z.wrotethe A, Ruggiero D, Sala CF, Sanna S, Sawyer EJ, Schlessinger D, Schmidt
manuscript. MK,SchmidtF,SchmutzlerRK,SchoemakerMJ,ScottRA,SeynaeveCM,
SimardJ,SoriceR,SoutheyMC,StöcklD,StrauchK,SwerdlowA,Taylor
ACKNOWLEDGEMENTS
KD,ThorsteinsdottirU,TolandAE,TomlinsonI,TruongT,TryggvadottirL,
WethankDr. JurrienDeanfromtheNationalInstitutesofHealth,USA,for TurnerST,VozziD,WangQ,WellonsM,WillemsenG,WilsonJF,Winqvist
providingtheOoeptm/tmmice. R,WolffenbuttelBBHR,WrightAF,YannoukakosD,ZemunikT,ZhengW,
ZygmuntM,BergmannS,BoomsmaDI,BuringJE,FerrucciL,Montgomery
REFERENCES
GW,GudnasonV,SpectorTD,VanDuijnCM,AlizadehBZ,CiulloM,Crisponi
AdamsonB,SmogorzewskaA,SigoillotFD,KingRW,ElledgeSJ.2012.A L, Easton DF, Gasparini PP, Gieger C, Harris TB, Hayward C, Kardia
genome-widehomologousrecombinationscreenidentifiestheRNA-binding SLR, Kraft P, McKnight B, Metspalu A, Morrison AC, Reiner AP, Ridker
proteinRBMXasacomponentoftheDNA-damageresponse.NatureCell PM,RotterJI,TonioloD,UitterlindenAG,UliviS,VölzkeH,WarehamNJ,
Biology,14(3):318–328. Weir DR, Yerges-Armstrong LM, The PRACTICAL Consortium, kConFab
AscanoMJr,MukherjeeN,BandaruP,MillerJB,NusbaumJD,CorcoranDL, Investigators, AOCS Investigators, Generation Scotland, EPIC-InterAct
LangloisC,MunschauerM,DewellS,HafnerM,WilliamsZ,OhlerU,Tuschl Consortium,LifeLinesCohortStudy,PriceAL,StefanssonK,VisserJA,Ong
T.2012.FMRPtargetsdistinctmRNAsequenceelementstoregulateprotein KK,Chang-ClaudeJ,MurabitoJM,PerryJRB,MurrayA.2015.Large-scale
expression.Nature,492(7429):382–386. genomicanalyseslinkreproductiveagingtohypothalamicsignaling,breast
BehrensA,VanDeursenJM,RudolphKL,SchumacherB.2014.Impactof cancer susceptibility and BRCA1-mediated DNA repair. Nature Genetics,
genomicdamageandagingonstemcellfunction.NatureCellBiology,16(3): 47(11):1294–1303.
201–207. DiGiacomoM,BarchiM,BaudatF,EdelmannW,KeeneyS,JasinM.2005.
Berthelot-RicouA,PerrinJ,DiGiorgioC,DeMeoM,BottaA,CourbiereB. DistinctDNA-damage-dependentand-independentresponsesdrivetheloss
2011.Cometassayonmouseoocytes: animprovedtechniquetoevaluate ofoocytesinrecombination-defectivemousemutants.Proceedingsofthe
genotoxicriskonfemalegermcells.FertilityandSterility,95(4):1452–1457. National Academy of Sciences of the United States of America, 102(3):
CollinsJK,LaneSIR,MerrimanJA,JonesKT.2015.DNAdamageinduces 737–742.
a meiotic arrest in mouse oocytes mediated by the spindle assembly Gonfloni S, Di Tella L, Caldarola S, Cannata SM, Klinger FG, Di
checkpoint.NatureCommunications,6:8553. Bartolomeo C, Mattei M, Candi E, De Felici M, Melino G, Cesareni G.
Day FR, Ruth KS, Thompson DJ, Lunetta KL, Pervjakova N, Chasman 2009.Inhibitionofthec-Abl-TAp63pathwayprotectsmouseoocytesfrom
DI, Stolk L, Finucane HK, Sulem P, Bulik-Sullivan B, Esko T, Johnson chemotherapy-induceddeath.NatureMedicine,15(10):1179–1185.
AD, Elks CE, Franceschini N, He CY, Altmaier E, Brody JA, Franke LL, GuoK,LiCH,WangXY,HeDJ,ZhengP.2016.Germstemcellsareactive
HuffmanJE,KellerMF,McArdlePF,NutileT,PorcuE,RobinoA,RoseLM, inpostnatalmouseovaryunderphysiologicalconditions.MolecularHuman
Schick UM, Smith JA, Teumer A, Traglia M, Vuckovic D, Yao J, Zhao W, Reproduction,22(5):316–328.
394 www.zoores.ac.cn
JacksonSP,BartekJ.2009.TheDNA-damageresponseinhumanbiology double-strandedbreaksinducehistoneH2AXphosphorylationonserine139.
anddisease.Nature,461(7267):1071–1078. TheJournalofBiologicalChemistry,273(10):5858–5868.
Jasin M, Rothstein R. 2013. Repair of strand breaks by homologous Skaznik-WikielM,TillyJC,LeeHJ,NiikuraY,Kaneko-TaruiT,JohnsonJ,Tilly
recombination.ColdSpringHarborPerspectivesinBiology,5(11):a012740. JL.2007.Seriousdoubtsover"Eggsforever?".Differentiation,75(2):93–99.
LavinMF,KozlovS.2007.ATMactivationandDNAdamageresponse.Cell SuhEK,YangAN,KettenbachA,BambergerC,MichaelisAH,ZhuZ,Elvin
Cycle,6(8):931–942. JA,BronsonRT,CrumCP,McKeonF.2006.p63protectsthefemalegerm
LiL,BaibakovB,DeanJ.2008.Asubcorticalmaternalcomplexessential lineduringmeioticarrest.Nature,444(7119):624–628.
for preimplantation mouse embryogenesis. Developmental Cell, 15(3): TangFC,BarbacioruC,NordmanE,LiB,XuNL,BashkirovVI,LaoKQ,
416–425. SuraniMA.2010.RNA-Seqanalysistocapturethetranscriptomelandscape
LuC,LinL,TanH,WuH,ShermanSL,GaoF,JinP,ChenD.2012.FragileX ofasinglecell.NatureProtocols,5(3):516–535.
premutationRNAissufficienttocauseprimaryovarianinsufficiencyinmice. TiceRR,AgurellE,AndersonD,BurlinsonB,HartmannA,KobayashiH,
HumanMolecularGenetics,21(23):5039–5047. MiyamaeY,RojasE,RyuJC,SasakiYF.2000.Singlecellgel/cometassay:
Marangos P, Carroll J. 2012. Oocytes progress beyond prophase in the guidelinesforinvitroandinvivogenetictoxicologytesting.Environmental
presenceofDNAdamage.CurrentBiology,22(11):989–994. andMolecularMutagenesis,35(3):206–221.
Marangos P, Stevense M, Niaka K, Lagoudaki M, Nabti I, Jessberger R, TitusS,LiF,StobezkiR,AkulaK,UnsalE,JeongK,DicklerM,Robson
CarrollJ.2015.DNAdamage-inducedmetaphaseIarrestismediatedbythe M,MoyF,GoswamiS,OktayK.2013.ImpairmentofBRCA1-relatedDNA
spindleassemblycheckpointandmaternalage.NatureCommunications,6: double-strand break repair leads to ovarian aging in mice and humans.
8706. ScienceTranslationalMedicine,5(172):172ra21.
Myers M, Britt KL, Wreford NG, Ebling FJ, Kerr JB. 2004. Methods for TongZB,GoldL,PfeiferKE,DorwardH,LeeE,BondyCA,DeanJ,Nelson
quantifyingfollicularnumberswithinthemouseovary.Reproduction,127(5): LM. 2000. Mater, a maternal effect gene required for early embryonic
569–580. developmentinmice.NatureGenetics,26(3):267–268.
Nagy Z, Soutoglou E. 2009. DNA repair: easy to visualize, difficult to WangET,PisarskaMD,BreseeC,ChenYDI,LesterJ,AfsharY,Alexander
elucidate.TrendsinCellBiology,19(11):617–629. C, Karlan BY. 2014. BRCA1 germline mutations may be associated with
Oktay K, Moy F, Titus S, Stobezki R, Turan V, Dickler M, Goswami reducedovarianreserve.FertilityandSterility,102(6):1723–1728.
S. 2014. Age-related decline in DNA repair function explains diminished Xiong B, Li S, Ai JS, Yin S, Ouyang YC, Sun SC, Chen DY, Sun QY.
ovarian reserve, earlier menopause, and possible oocyte vulnerability to 2008.BRCA1isrequiredformeioticspindleassemblyandspindleassembly
chemotherapyinwomenwithBRCAmutations.JournalofClinicalOncology, checkpoint activation in mouse oocytes. Biology of Reproduction, 79(4):
32(10):1093–1094. 718–726.
OktayK,TuranV,TitusS,StobezkiR,LiuL.2015.BRCAmutations,DNA XuQH,WangFC,XiangYL,ZhangXX,ZhaoZA,GaoZ,LiuWB,LuXK,
repairdeficiency,andovarianaging.BiologyofReproduction,93(2):67. LiuYS,YuXJ,WangHB,HuangJ,YiZH,GaoSR,LiL.2015.Maternal
PerezGI,ActonBM,JurisicovaA,PerkinsGA,WhiteA,BrownJ,Trbovich BCAS2protectsgenomicintegrityinmouseearlyembryonicdevelopment.
AM, Kim MR, Fissore R, Xu J, Ahmady A, D’Estaing SG, Li H, Kagawa Development,142(22):3943–3953.
W,KurumizakaH,YokoyamaS,OkadaH,MakTW,EllismanMH,Casper Yu XJ, Yi ZH, Gao Z, Qin DD, Zhai YH, Chen X, Ou-Yang YC, Wang
RF, Tilly JL. 2007. Genetic variance modifies apoptosis susceptibility in ZB, Zheng P, Zhu MS, Wang HB, Sun QY, Dean J, Li L. 2014. The
mature oocytes via alterations in DNA repair capacity and mitochondrial subcorticalmaternalcomplexcontrolssymmetricdivisionofmousezygotes
ultrastructure.CellDeathandDifferentiation,14(3):524–533. byregulatingF-actindynamics.NatureCommunications,5:4887.
PerryJRB,CorreT,EskoT,ChasmanDI,FischerK,FranceschiniN,He Yurttas P, Vitale AM, Fitzhenry RJ, Cohen-Gould L, Wu WZ, Gossen JA,
CY, Kutalik Z, Mangino M, Rose LM, Vernon Smith A, Stolk L, Sulem P, CoonrodSA.2008.RoleforPADI6andthecytoplasmiclatticesinribosomal
WeedonMN,ZhuangWV,ArnoldA,AshworthA,BergmannS,BuringJE, storage in oocytes and translational control in the early mouse embryo.
BurriA,ChenC,CornelisMC,CouperDJ,GoodarziMO,GudnasonV,Harris Development,135(15):2627–2636.
T, Hofman A, Jones M, Kraft P, Launer L, Laven JSE, Li G, McKnight B, ZhaoB,ZhangWD,DuanYL,LuYQ,CunYX,LiCH,GuoK,NieWH,LiL,
MasciulloC,MilaniL,OrrN,PsatyBM,ReproGenConsortium,RidkerPM, ZhangRG,ZhengP.2015.FiliaisanESC-specificregulatorofDNAdamage
RivadeneiraF,SalaC,SalumetsA,SchoemakerM,TragliaM,WaeberG, responseandsafeguardsgenomicstability.CellStemCell,16(6):684–698.
ChanockSJ,DemerathEW,GarciaM,HankinsonSE,HuFB,HunterDJ, ZhengP,DeanJ.2009.RoleofFilia,amaternaleffectgene,inmaintaining
LunettaKL,MetspaluA,MontgomeryGW,MurabitoJM,NewmanAB,Ong euploidyduringcleavage-stagemouseembryogenesis.Proceedingsofthe
KK,SpectorTD,StefanssonK,SwerdlowAJ,ThorsteinsdottirU,VanDam National Academy of Sciences of the United States of America, 106(18):
RM,UitterlindenAG,VisserJA,VollenweiderP,TonioloD,MurrayA.2013. 7473–7478.
Agenome-wideassociationstudyofearlymenopauseandthecombined ZhuK,YanLY,ZhangXX,LuXK,WangTR,YanJ,LiuXQ,QiaoJ,LiL.2015.
impactofidentifiedvariants.HumanMolecularGenetics,22(7):1465–1472. Identificationofahumansubcorticalmaternalcomplex.MolecularHuman
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. 1998. DNA Reproduction,21(4):320–329.
ZoologicalResearch39(6):387–395,2018 395