MicroRNA Expression in Bovine Cumulus Cells in Relation

15 Pages·2017·3.3 MB·English

non-coding
RNA
Article
MicroRNA Expression in Bovine Cumulus Cells in
Relation to Oocyte Quality
KarenUhde1,HelenaT.A.vanTol1,TomA.E.Stout1,2andBernardA.J.Roelen1,2,*
1 DepartmentofFarmAnimalHealth,FacultyofVeterinaryMedicine,UtrechtUniversity,Yalelaan104,
3584CMUtrecht,TheNetherlands;[email protected](K.U.);[email protected](H.T.A.T.);
[email protected](T.A.E.S.)
2 DepartmentofEquineSciences,FacultyofVeterinaryMedicine,UtrechtUniversity,Yalelaan112,
3584CMUtrecht,TheNetherlands
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:GeorgeA.Calin
Received:6January2017;Accepted:7March2017;Published:11March2017
Abstract: Cumulus cells play an essential role during oocyte maturation and the acquisition of
fertilizabilityanddevelopmentalcompetence. Micro(mi)RNAscanpost-transcriptionallyregulate
mRNAexpression,andwehypothesizedthatmiRNAprofilesincumuluscellscouldserveasan
indicator of oocyte quality. Cumulus cell biopsies from cumulus−oocyte−complexes that either
yielded a blastocyst or failed to cleave after exposure to sperm cells were analyzed for miRNA
expression. Onaverage,332miRNAspecieswithmorethan10readsand240miRNAspecieswith
more than 50 reads were identified in cumulus cells; this included nine previously undescribed
microRNAs. ThemosthighlyexpressedmiRNAsincumuluscellsweremiR-21, membersofthe
let-7familyandmiR-155. However,norepeatabledifferencesinmiRNAexpressionbetweenthe
cumuluscellsfromoocytesthat becameblastocystsversusthose fromnon-cleaved oocyteswere
identified. Furtherexaminationofindividualcumuluscellsamplesshowedawidevariabilityin
miRNAexpressionlevel. WethereforeconcludethatmiRNAexpressionincumuluscellscannotbe
usedasanoocytequalitymarker.
Keywords: microRNA;cumuluscells;oocytequality
1. Introduction
Mammalian oocytes acquire the capacity to become fertilized and acquire developmental
competence during their final maturation within the ovary. These oocytes originate from female
primordial germ cells that proliferated while migrating towards the gonadal ridges and continue
toproliferateuntilgonadalsexdifferentiation. Thecellsareclassifiedasoocytesfromthemoment
theystopdividingandentermeiosis[1]. Meiosisindevelopingoocytesarrestsatthefirstprophase,
and depending on the species, this meiotic arrest can be maintained for decades [2]. As early as
this time point, the oocyte acquires competence to develop into an embryo after fertilization [3].
The oocyte only resumes meiosis shortly before ovulation, in response to luteinizing hormone
stimulation. Aftertheresumptionofmeiosisandinadditiontochromosomesegregation,theoocyte
needs to redistribute organelles and proteins in a process known as cytoplasmic maturation [4].
Fortheseprocesses,theoocytereliesonmoleculespresentwithinthefollicularfluidandonintimate
contactwiththesomaticcellsthatsurroundtheoocyte,knownascumuluscells. Together,theyform
the cumulus−oocyte−complex (COC). Tightly-regulated communication between the oocyte and
surroundingcumuluscellsisimportantatvariousstages,includingoocytegrowth,maturationand
fertilization [5–7]. Exactly how the cumulus cells contribute to the fertilizability and developmental
competenceoftheoocyte,hereafterreferredtoasoocytequality,isnotknown.Frominvitromaturation
Non-codingRNA2017,3,12;doi:10.3390/ncrna3010012 www.mdpi.com/journal/ncrna
Non-codingRNA2017,3,12 2of15
andfertilizationexperiments,ithashoweverbecomeclearthatlargedifferencesexistbetweenthequality
ofoocytes,anditseemslikelythatcumuluscellsareatleastpartlyresponsiblefortheoocytequality.
Cumuluscellsareconnectedtoeachotherandtotheoocytebydirectcell-cellcontact.Exchangeof
small(<1kDa)molecules,likecyclicadenosinemonophosphate(cAMP),isthoughttooccurthrough
gap junctions [8–10], whereas the passage of larger molecules, including RNA, can occur via
zonula-adherens-like junctions [11,12]. Communication between the oocyte and cumulus cells is
bidirectional[13],withthecumuluscellsprovidingtheoocytewithfactorsandsignalingmolecules[6],
buttheoocytealsosendingsignalstothecumuluscells[9].Ifcommunicationbetweenthecumuluscells
andtheoocyteisdisturbed,oocytequalitycanbeaffected,whichcaninturncompromisesubsequent
embryo development [14]. Indeed, invitro maturation of oocytes in the absence of cumulus cells
markedlyreducesthedevelopmentalcompetenceofoocytes[15].
For human invitro fertilization (IVF) in particular, it would be beneficial if fertilizable and
developmentally-competentoocytescouldbeidentifiedbeforefertilization,withoutdamagingthe
oocyte. Oocytemorphologyhasbeendemonstratedtobeapoorpredictorofqualityandisrather
subjective[16,17]. Amolecularmarkertopredictoocytequalitybeforefertilizationwouldimprove
embryodevelopmentandpregnancyratesandcouldbeusedtoreducethenumberofsurplusembryos
producedandtheresultingethicalconflictregardingtheultimatefateoftheseembryos[18].Oneuseful
model animal for human oocyte development is the cow since, like man, it is a mono-ovulatory
species;inaddition,ithasacomparabletimecourseofoogenesis[19,20]. Furthermore,theblastocyst
developmentrateafterinvitrofertilizationincattleis~35%,comparablewiththatobtainedinhuman
IVFprograms[21,22]. Notsurprisingly,effortshavebeenundertakentoidentifynon-invasivequality
markersforoocytes,includinganalysisofgeneexpressionincumuluscells[23–27]andinvestigation
ofthecompositionoffollicularfluid[25,28].
MicroRNAs(miRNAs)arehighlyconserved, small(21–25nucleotidesinlength), non-coding
RNAsthatcanregulategeneexpression[29,30]. Viasequencecomplementarity,theseedsequencesof
miRNAscanbindtothe3’UTRoftargetmRNAsand,incombinationwithanArgonautefamilyprotein,
the miRNA can regulate the expression of the target gene by degrading that mRNA or inhibiting
itstranslation. IthasbeensuggestedthatasinglemiRNAspeciescanregulate~100differenttarget
genes[31],enablingabroadlevelofregulation.
miRNAsareprocessedfromlongertranscriptsbytheRNaseIII-likeenzymesDicerandDrosha.
GeneticdeletionofDicerresultsinembryoniclethalityinmice[32],andwhenDicerwasspecifically
deleted in growing oocytes, they failed to cleave after fertilization, indicating the importance of
miRNAstooocytecompetence[32].Apartfromexpressioninoocytes,miRNAshavealsobeendetected
incumuluscells[14,33],andithasbeendemonstratedthatthemiRNAexpressionprofilesofboth
oocyteandcumuluscellschangeduringmaturation[33–37]. Identificationofdifferentially-expressed
miRNAsincoronaradiatacellsandtheoutercumuluscellsfromhumanoocytesindicatedarolefor
thesemiRNAsinnutritionalandregulatorysignalingbetweenoocytesandcumuluscells[38].
Inthisstudy,weexaminedthemiRNAexpressionprofilesofbovinecumuluscellsandcompared
thosefromCOCsthatyieldedblastocystafterfertilizationversusthosethatremainednon-cleaved
afterexposuretosperm.
2. Results
2.1. ComparisonofIndividualversusGroupCulture
In order to identify miRNA species expressed in cumulus cells that could potentially predict
qualityoftheenclosedoocyte,cumulusbiopsieswereobtainedfromCOCsafterinvitromaturation.
SubsequentindividualfertilizationofCOCsandindividualembryoculturewereperformedtofollow
the progression of eachoocyte individually and therebyallow correlation of the cumulus miRNA
expression profile to oocyte quality, retrospectively. First, to analyze whether individual embryo
culture affected the efficiency of blastocyst formation, embryos were cultured either individually,
Non-codingRNA2017,3,12 3of15
butwithsharedculturemedium,oringroupsof30–50. Thepercentagesofoocytesthatcleavedor
developedintoblastocystsweresimilarforthetwocultureconditions(Table1),demonstratingthe
validityofthesingleembryoculturesystem.
Table 1. Comparison of group and individual embryo culture. Comparison of cleavage and
further development of bovine cumulus−oocyte−complexes (COCs)/zygotes cultured in groups
orindividually.
Day5 Day8
Culturetype Total Not–cleaved 2to8 >8 Blastocyst
Group n 481 121 207 153 129
% 25.2 43 31.8 26.8
Individual n 188 35 86 67 53
% 18.6 45.7 35.6 28.2
2.2. SequencingofmiRNAsinCumulusCells
InordertodeterminewhetherthemiRNAexpressionprofilesofcumulusbiopsiesfromCOCs
thatcontainedfertilizableanddevelopmentally-competentoocytes(i.e.,yieldedablastocyst)differed
fromthosethatcontainedoocytesofpoorquality,cellswereharvestedforsmallRNAsequencing.
After COCs had been matured, a biopsy of the cumulus cells was obtained, and the COCs were
culturedindividuallyforfertilizationandembryodevelopment.
Cumuluscellssurroundinganoocytethat(1)developedintoablastocystor(2)failedtocleave
after exposure to sperm were pooled to ensure sufficient amounts of RNA (each pooled sample
contained cumulus cells from 10 to 16 individual COCs). Three pooled samples of cumulus cells
harvestedfromoocytesthatdidnotcleave(referredtoasA,BandC)andthreepooledsamplesof
cumuluscellsthathadenclosedoocytesthatdevelopedintoblastocysts(D,EandF)weresequenced
forsmallRNAs;theyyieldedanaverageof14millionreads(Figure1).
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 4 of 18

Figure 1. Total number of micro RNA (miRNA) reads in each sample. The total number of miRNA
Figure1. TotalnumberofmicroRNA(miRNA)readsineachsample. ThetotalnumberofmiRNA
sized reads for each sample of cumulus cells pooled from 10 to 16 bovine cumulus-oocyte complexes;
sizedreadsfoorn eavaecrhagsea, 1m4 mplilelioonf rceaudms wuelrue sobctealinlsedp poeor lseamdpflreo, ams in1d0ictaoted1 6byb tohev binlaeckc luinme. Tuhleu rse–do doocttyedte complexes;
onaverage,14linme imllairokns frievae dmsilwlioenr reeaodbst, athine eadmopuenrt scaonmsipdelere,da ssuifnfidcieicnat tfoerd mbeyantihnegfbull aacnkalylisnise. .AT, Bh,e Cr e= ddottedline
cumulus pieces surrounding oocytes that failed to cleave after exposure to sperm; D, E, F = cumulus
marksfivemillionreads,theamountconsideredsufficientformeaningfulanalysis.A,B,C=cumulus
pieces surrounding oocytes that developed into a blastocyst after fertilization.
piecessurroundingoocytesthatfailedtocleaveafterexposuretosperm;D,E,F=cumuluspieces
surroundingmooiRcNytAess wthearet dideevnetilfoiepde adccionrtdoinag btola esntotrcieyss tina mfteiRrBfaesret iRliezlaeatisoe n2.0. A mean of 332 miRNA
species had more than 10 reads (Figure 2, blue bars), while 240 miRNA species had more than 50
reads per sample (Figure 2, red bars). The majority of the miRNAs were present in all samples; of the
miRNA species identified, 276 with more than 10 counts and 205 with more than 50 counts were
present in all samples. No repeatable differences in the numbers of miRNAs identified were present
between the two types of sample (Figure 2).
Non-codingRNA2017,3,12 4of15
miRNAs were identified according to entries in miRBase Release 20. A mean of 332 miRNA
specieshadmorethan10reads(Figure2,bluebars),while240miRNAspecieshadmorethan50reads
persample(Figure2,redbars). ThemajorityofthemiRNAswerepresentinallsamples;ofthemiRNA
speciesidentified,276withmorethan10countsand205withmorethan50countswerepresentinall
samples. NorepeatabledifferencesinthenumbersofmiRNAsidentifiedwerepresentbetweenthe
twotypesofsample(Figure2).
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 5 of 18
400
s
A 300
N
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200

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o

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u
N 100
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A B C D E F
Not cleaved Blastoccyst

FigurFeig2u.re 2N. Numumbbeerr ooff mmiRiRNNAA reardesa.d Ns.umbNeur mof bkenrowonf mkniRoNwAns wmiitRh NmAorse twhaitnh a mgivoerne ntuhmanbera ogf iven
mapped reads: >0, but <50 reads per sample for blue bars and >50 per sample for red bars.
numberofmappedreads: >0, but<50readspersampleforbluebarsand>50persampleforred
A, B, C = cumulus pieces surrounding oocytes that failed to cleave after exposure to sperm;
bars.A,B,C=cumulus pieces surrounding oocytes that failed to cleave after exposure to sperm;
D, E, F = cumulus pieces surrounding oocytes that developed into blastocysts after fertilization.
D,E,F=cumuluspiecessurroundingoocytesthatdevelopedintoblastocystsafterfertilization.
In short, cumulus cells harvested from the COCs of oocytes that failed to cleave after exposure
Itno sspheorrmt, acnudm tuhlouses ctheallts yhiealrdveeds ate bdlafsrtoomcystth sehCowOeCds ao sfimooilcayr teexsptrhesastiofani loefd mtioRNclAeasv. eOfa fatlel roef xthpeo sure
tospeidremntaifniedd tmhioRsNeAths,a mtyiRie-2ld1-e5dp awabsl athsteo mcyosstt asbhuonwdeadnt a(>s2i,m00i0l,a0r00e xrepardess,s Tioabnleo 2f)m, biuRtN thAe sn.uOmfbearl loof fthe
identriefiaedds mwiaRs NsiAmsil,amr biRet-w21ee-5np thwe atswtoh egrmouopsst. aDbiuffnedreannt tm(>em2,0be0r0s, 0o0f0 threea ldets-,7T faabmleily2,) ,mbiuRt-1t5h5e annudm ber
miR-99a-5p also gave more than 100,000 reads in each group (Table 2).
of reads was similar between the two groups. Different members of the let-7 family, miR-155 and
miR-99a-5palsogavemorethan100,000readsineachgroup(Table2).

WhencomparingthemiRNAsexpressedincumuluscellsfromaroundanoocytethatdeveloped
into a blastocyst after fertilization with those surrounding an oocyte that had not cleaved, a high
variabilitybetweenthedifferentpooledsamplesandnorepeatabledifferencesbetweenthegroups
wereobserved(Figure3). SamplesAandB(cumuluscellsenclosingoocytesthatdidnotcleaveafter
exposuretosperm)andSamplesFandD(cumuluscellssurroundingoocytesthatdevelopedinto
blastocysts) grouped together after hierarchical clustering. By contrast, Samples C (cumulus cells
enclosing oocytes that did not cleave) and E (cumulus cells surrounding oocytes that developed
intoblastocysts)exhibitedamarkedlydifferentmiRNAexpressionprofile; thiswasalsoapparent
afterprincipalcomponentanalysis(Figure4). Ingeneral,withinthetestedgroups,theexpressionof
miRNAswasvariable,whichpresumablycontributedtotheabsenceofdifferencesintheexpression
profilesbetweenthegroups.
Non-codingRNA2017,3,12 5of15
Table2.Mostabundantly-expressedmicroRNAs(miRNA).
miRNA CountsNC CountsBlast
bta-miR-21-5p 2,324,236 2,915,237
bta-let-7f 431,569 335,113
bta-let-7i 302,000 256,981
bta-let-7g 228,393 177,529
bta-miR-155 225,179 158,161
bta-let-7a-5p 217,033 158,238
bta-miR-99a-5p 129,482 112,348
bta-miR-30d 85,620 75,966
bta-miR-26a 73,820 56,991
bta-miR-320a 59,660 53,015
bta-miR-92a 55,108 47,255
bta-miR-10b 54,473 66,439
bta-miR-202 38,887 32,030
bta-miR-148a 38,574 40,607
bta-let-7b 36,653 29,117
bta-miR-532 34,632 32,815
bta-miR-99b 29,386 31,287
bta-let-7e 25,088 20,497
bta-miR-125a 22,159 20,436
bta-let-7c 23,668 17,542
Thelistofthe20mostabundantly-expressedmiRNAsinbovinecumuluscells.NC=cumuluspiecesenclosing
oocytesthatfailedtocleaveafterexposuretosperm;Blast=cumuluspiecesenclosingoocytesthatdevelopedintoa
blastocystafterfertilization.
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 7 of 18

FigureFi3g.urHe e3a. tHmeaatp moafpt hofe tthees tteedstecdu mcuumluulsusc eclellls asammpplleess.. HHeeaatt mmaapp anand dunusnuspueprveirsvedis ehdierhairecrhaicraclh ical
clustecrliunsgterbinyg sbaym spamleplaen adndm miRiRNNAA.. TThhee clculsutesrtienrgin wgasw paesrfopremrfeodr mone adll osanmapllless aanmdp olne sthaen tdop o5n0 the
top 5m0iRmNiRANs wAisthw thieth higthheesth %ig CheVs tba%sedC oVn TbPaMse (dtraonnscrTipPtsM per(t mrainllsiocnri)p ntosrmpaelrizemd irlleiaodns). An,o Br,m Ca =li zed
cumulus pieces surrounding oocytes that failed to cleave after exposure to sperm; D, E, F = cumulus
reads. A,B,C=cumuluspiecessurroundingoocytesthatfailedtocleaveafterexposuretosperm;
pieces surrounding oocytes that developed into blastocysts after fertilization.
D,E,F=cumuluspiecessurroundingoocytesthatdevelopedintoblastocystsafterfertilization.
Non-codingRNA2017,3,12 6of15
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 8 of 18

Figure 4. Principal component analysis (PCA) plot. The PCA was performed using the 50 miRNAs
Figure4. Principalcomponentanalysis(PCA)plot. ThePCAwasperformedusingthe50miRNAs
that had the largest variation across all samples. The features have been shifted to be zero centered
thathadthelargestvariationacrossallsamples. Thefeatureshavebeenshiftedtobezerocentered
(i.e., the mean value across samples was shifted to zero) and scaled to have unit variance (i.e., variance
(i.e.,themeanvalueacrosssampleswasshiftedtozero)andscaledtohaveunitvariance(i.e.,variance
across samples was scaled to one) before the analysis. The groups do not cluster, not least because
acrosssampleswasscaledtoone)beforetheanalysis. Thegroupsdonotcluster,notleastbecause
Samples B and E differ greatly from the rest for the primary component and from each other for the
SamplesBandEdiffergreatlyfromtherestfortheprimarycomponentandfromeachotherforthe
sseeccoonnddaarryy ccoommppoonneennt.t .RReedd cciricrcleles s== ccuummuululus sppieiececes ssusurrrorouunnddiningg oooocycytetse sththaat tfafailieledd toto ccleleaavvee aaffteterr
eexxppoossuurree ttoo ssppeerrmm; ;bbluluee ccirircclleess == ccuummuululuss ppieiecceess susurrrorouunnddiningg ooooccyytetess ththaat tddeevveeloloppeedd inintoto bblalasstotoccyysststs
aaffteterr ffeerrttiliilzizaattioionn. .
2.3. Quantitative real-time reverse transcription-PCR (qRT-PCR) from Pooled Cumulus Complex Samples
2.3. QuantitativeReal-TimeReverseTranscription-PCR(qRT-PCR)fromPooledCumulusComplexSamples
Although miRNA expression did not differ significantly between the two groups, expression of
AlthoughmiRNAexpressiondidnotdiffersignificantlybetweenthetwogroups, expression
miRNAs for which the abundance appeared to differ according to the heat map (Figure 3) was further
of miRNAs for which the abundance appeared to differ according to the heat map (Figure 3) was
examined by qRT-PCR. The selected miRNAs had low or moderate total reads at sequencing, and to
furtherexaminedbyqRT-PCR.TheselectedmiRNAshadlowormoderatetotalreadsatsequencing,
compensate for the anticipated low expression levels, pooled samples of cumulus cells from
andtocompensatefortheanticipatedlowexpressionlevels,pooledsamplesofcumuluscellsfrom
individually-cultured COCs were analyzed. The expression of miR-214, miR-424 and 2284ab was
individually-cultured COCs were analyzed. The expression of miR-214, miR-424 and 2284ab was
below the level of detection by qRT-PCR, and indeed, the read numbers for these miRNAs were
belowthelevelofdetectionbyqRT-PCR,andindeed,thereadnumbersforthesemiRNAswerearound
around or below 100 (Table 3). For miR-342, miR-2478 and miR424-3p, expression levels (Table 3)
orbelow100(Table3). FormiR-342,miR-2478andmiR424-3p,expressionlevels(Table3)weresimilar
were similar in the cumulus cells surrounding an oocyte that gave rise to a blastocyst to those in
inthecumuluscellssurroundinganoocytethatgaverisetoablastocysttothoseincumuluscells
cumulus cells around oocytes that did not cleave (Figure 5).
aroundoocytesthatdidnotcleave(Figure5).
Non-codingRNA2017,3,12 7of15
Non-Coding RNA 2017, 3, 12 7 of 15
Table3.SequencingcountsofchosenmiRNAs.
Table 3. Sequencing counts of chosen miRNAs.
Not-cleaved Blastocyst
miRNA
Not-cleaved Blastocyst
miRNA A B C D E F
A B C D E F
btab–tma–imR–i2R1–42 14 35 35 33 33 30 30 111199 2222 131 3
btab–tma–imR–i2R1–82 18 4,5446,5 46 1,4511, 451 2,1928, 198 33,0,00044 8866 1,14,1411 1
btab–tma–imR–i2R2–22 22 3,2238,2 28 1,1715, 175 2,0920, 090 11,7,72244 11,2,9219 1 1,11,5115 1
btab–tma–imR–i3R4–23 42 1,2617,2 67 802 802 963 963 22,8,85566 2200 51551 5
btab–tma–imR–i4R2–44–234p– 3p 3543 54 62 62 166 166 115599 22008 8 18118 1
btab–tma–imR–i4R2–44–254p– 5p 27 27 26 26 34 34 111133 2200 161 6
btab–tma–imR–i2R2–8242a8b4 ab 32 32 20 20 41 41 7700 1122 131 3
btab–tma–imR–i2R4–7284 78 3573 57 92 92 66 66 110055 11554 4 15105 0
btab–tlae–t–le7ti– 7i 4284,52485, 545 199,169490, 640 277,287175 ,815 339966,8,8818 1 22225,54,2402 0 14184,86,4634 3
btab–tlae–t–le7tg– 7g 3323,13620, 160 154,185940, 890 198,119381 ,131 223377,1,1414 1 11663,39,4904 0 13113,15,0560 6
SequeSnecqeuceonucnet scofournmts ifRoNr AmsiReNxaAmsi neexdamfuinrtehde rf.uNrtohtecrl. eNavoet dcl=eacvuemdu =l ucsupmiuecluess spuirercoeus nsduirnrgouonodcyintegs otohcayttfeasi ledto
cleaveth(Sata mfapilleeds Ato, Bc,lCea);vBe la(sStaomcypslte=s cAu,m Bu, luCs);p iBelcaesstoscuyrsrot u=n dcuinmguoloucsy tpeisectheas tsduervreoluonpdeidngin tooobclyatsetso ctyhsatts after
fertilizdaetvioenlo(pSeadm ipnlteos bDla,sEt,oFc)y.sts after fertilization (Samples D, E, F).
A miR-342 B miR-424-3p
2.0 3.5
3.0
n
expression 11..05 e expressio 22..05
Relative 0.5 Relativ 11..05
0.5
0.0 0.0
Not-cleaved Blastocyst Not-cleaved Blastocyst
C
miR-2478
2.5
2.0
n
o
si
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x
e
e
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ati
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0.5
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Not-cleaved Blastocyst
Figure 5. miRNA expression of pooled cumulus cell samples. Mean miRNA expression (qRT-PCR)
Figure5.miRNAexpressionofpooledcumuluscellsamples.MeanmiRNAexpression(qRT-PCR)for
for (A) miR-342, (B) miR-424-3p and (C) miR-2478 in pooled cumulus cell samples enclosing oocytes
(A)miR-342,(B)miR-424-3pand(C)miR-2478inpooledcumuluscellsamplesenclosingoocytesthat
that failed to cleave (black bars) or that developed into blastocysts (white bars) after exposure to
failedtocleave(blackbars)orthatdevelopedintoblastocysts(whitebars)afterexposuretosperm.
sperm. All samples were normalized for let-7a, miR-26a and miR-191 expression.
Allsampleswerenormalizedforlet-7a,miR-26aandmiR-191expression.
2.4. qRT-PCR from Individual Cumulus Complex Samples
2.4. qRT-PCRfromIndividualCumulusComplexSamples
For both the sequencing and the initial qRT-PCR analysis, pooled samples of cumulus cells from
individually-cultured COCs were used. However, since oocyte quality is likely to be dependent on
ForboththesequencingandtheinitialqRT-PCRanalysis,pooledsamplesofcumuluscellsfrom
many different factors acting at different times and on different pathways, pooling of the samples
individually-culturedCOCswereused. However,sinceoocytequalityislikelytobedependenton
might obscure miRNAs that are involved in regulating oocyte competence. Therefore, expression
manydifferentfactorsactingatdifferenttimesandondifferentpathways, poolingofthesamples
levels of candidate miRNAs were determined using qRT-PCR in cumulus cells from individual
might obscure miRNAs that are involved in regulating oocyte competence. Therefore, expression
COCs. The selection of miRNAs examined was based on the sequencing results; four of the selected
levelsofcandidatemiRNAsweredeterminedusingqRT-PCRincumuluscellsfromindividualCOCs.

TheselectionofmiRNAsexaminedwasbasedonthesequencingresults;fouroftheselectedmiRNAs
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 10 of 18
C
miR-2478
2.5
2.0
n
o
si
s
re 1.5
p
x
e
e
v 1.0
ati
el
R
0.5
0.0
Not-cleaved Blastocyst

Figure 5. miRNA expression of pooled cumulus cell samples. Mean miRNA expression (qRT-PCR)
for (A) miR-342, (B) miR-424-3p and (C) miR-2478 in pooled cumulus cell samples enclosing oocytes
that failed to cleave (black bars) or that developed into blastocysts (white bars) after exposure to
sperm. All samples were normalized for let-7a, miR-26a and miR-191 expression.
2.4. qRT-PCR from Individual Cumulus Complex Samples
For both the sequencing and the initial qRT-PCR analysis, pooled samples of cumulus cells from
individually-cultured COCs were used. However, since oocyte quality is likely to be dependent on
Non-codinmgRaNnyA d20if1f7e,re3n,1t 2factors acting at different times and on different pathways, pooling of the samples 8of15
might obscure miRNAs that are involved in regulating oocyte competence. Therefore, expression
levels of candidate miRNAs were determined using qRT-PCR in cumulus cells from individual
hadbeeCnOhCigs.h Tlhye esxelpecrteisosne odf maniRdNsAhso ewxaemdinaemd wodase braasteedd oinff ethree nsecqeuiennceinxgp rreessuslitos;n fobuert owf etheen se‘nleoctne-dc leaved’
miRNAs had been highly expressed and showed a moderate difference in expression between
COCsandCOCsthatgaverisetoablastocyst,namelylet-7g,let-7i,miR-222andmiR-218. Forallfour
‘non-cleaved’ COCs and COCs that gave rise to a blastocyst, namely let-7g, let-7i, miR-222 and
miRNAs,ahighvariabilityinmiRNAexpressionbetweenthetwelveindividualsampleswithina
miR-218. For all four miRNAs, a high variability in miRNA expression between the twelve individual
groupwasobserved(Figure6).
samples within a group was observed (Figure 6).
let-7g
A
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7 2 3126 1 8 4 9 11 510 4 5 9 3 8 1 6 2 1071211
Non-Coding RNA 2017, 3, x FOR PEER REVINEWo t -cleaved Blastocyst 11 of 18
B let-7i
2.0
1.8
1.6
n
sio 1.4
s
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miR-222
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Figure6.Cont.
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Figure 6. miRNA expression of individual miRNA samples. miRNA expression for cumulus cells
from individual COCs. Expression levels of (A) let-7g, (B) let-7i, (C) miR-218 and (D) miR-222 as
Non-Coding RNA 2017, 3, x FOR PEER REVIEW 11 of 18
B let-7i
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Figure 6. miRNA expression of individual miRNA samples. miRNA expression for cumulus cells
Figure6.miRNAexpressionofindividualmiRNAsamples.miRNAexpressionforcumuluscellsfrom
from individual COCs. Expression levels of (A) let-7g, (B) let-7i, (C) miR-218 and (D) miR-222 as
individualCOCs.Expressionlevelsof(A)let-7g,(B)let-7i,(C)miR-218and(D)miR-222asdetermined

byqRT-PCR.X-axesdepictindividualcumuluscomplexsamplesfromCOCsthathadenclosedan
oocytethateitherfailedtocleave(blackbars)ordevelopedintoablastocyst(whitebars)afterexposure
tosperm.Allsampleswerenormalizedforexpressionoflet-7a,miR-26aandmiR-191.
Finally,analysisofthesequencingdataindicated178novelputativemiRNAs. Notallofthese
miRNAsweredetectedinthecumuluscellsamples;however,ninenovelmiRNAspeciesweredetected
withameanof>10reads,indicatingthatthemiRNAsareindeedgeneratedandexpressed(Table4).
However,theexpressionlevelsofthesenovelmiRNAsdidnotdifferbetweenthenon-cleavedand
blastocystgroups.
Table4.NovelexpressedmiRNAs,predictedandexpressedmiRNAsidentifiedfromreaddatabased
onreadcountandsecondarystructureaccordingtothemiRPara(miRNApredictionsoftwaretool)
classificationscore.ThedescriptionofchromosomallocationisshownwithChr(chromosome),start
andstop.
Name Chr START STOP SEQUENCE
bta-put-45 chr7 12981779 12981801 CCGUGCCUACUGAGCUGAAACAC
bta-put-129 chr21 69641635 69641655 UGCAAGCAACACUCUGUGGCA
bta-put-53 chr7 53516556 53516579 UAUACUCUGAUUGGUUCAUUAUGA
bta-put-79 chr29 1067001 1067020 AUGGUCAUUACCAAGGCUUU
bta-put-168 chr7 5206807 5206828 UCAAAGUGAAUUUGGAGGUUCU
bta-put-44 chr7 5206802 5206823 UCAAAGUGAAUUUGGAGGUUCU
bta-put-82 chr29 41693966 41693989 GAUCCGCGUAAUGUACGGAGGUAG
bta-put-156 chr5 103546363 103546386 GGACCUCAGUUCCAAACCUCUGCC
bta-put-25 chr5 104238656 104238676 AUGUGGACCCAGGGAGCUGGG
3. Discussion
Inthisstudy,themiRNAexpressionprofilesofcumuluscellsfromdevelopmentally-competent
COCswerecomparedwiththosefromaroundoocytesdidnotcleaveafterexposuretosperm. Asfar
asweknow,thisisthefirststudytoexaminewhethercumuluscellmiRNAexpressioncanbeused
to predict the quality of oocyte before fertilization. In our study, samples of cumulus cells from
individually-fertilizedandculturedCOCswereusedandretrospectivelyassignedtopost-fertilization
developmentalcompetencegroups.
ForidentificationofmiRNAsexpressedincumuluscells,nextgenerationsequencingwasused.
Frombothgroups,threepooledsamplesofcumuluscells,eachconsistingofcellsfromaround10COCs,
Non-codingRNA2017,3,12 10of15
were analyzed. Cumulus cells surrounding an oocyte that did not cleave after sperm exposure or
developed into a blastocyst were chosen, to have the two most different stages to see a change in
miRNAexpression. However,nospecificmiRNAspecieswereidentifiedthatcouldbecorrelatedto
oocytequality,suggestingthatmiRNAexpressionincumuluscellsisnotpredictiveofoocytequality.
Sinceouraimwastoidentifycompetentoocytesbeforefertilization,wedecidedtocomparemiRNA
expressionpatternsincumuluscellsthatdidnotcleaveafterexposuretosperm,versusthosethat
developedintoablastocystafterexposuretosperm. Thisdidnotallowustodistinguishbetween
thoseoocytesthatwerefertilizableandthosethatwerenot. Fortheidentificationofcumulusmarkers
thatwouldpredictembryodevelopmentalcompetence,comparisonbetweencellsoriginatingfrom
embryosthatremainedatthetwo-cellstageversusthosethatbecomeablastocystwouldbeuseful,
butthiswouldalwaysrequirefertilizationandthegenerationofembryos. Oocytefertilizabilityand
developmental competence are dependent on various factors, and pooling of the cumulus pieces
mighthaveobscuredanyinfluenceofindividualmiRNAs. Inthisrespect,wefurthershowedthat
theexpressionlevelsofseveralselectedmiRNAsvariedgreatlybetweencumuluscomplexesfrom
individualCOCs;atleastforthesemiRNAs,thevariationinexpressionwassimilarforcompetentand
incompetentCOCs. Oocytesfromslaughterhouseovarieswereusedinthisstudy,whichrepresenta
heterogeneouspopulation. Possibly,amorehomogeneousoocytepopulation,forexampleoriginating
fromonebreed,wouldrevealspecificmiRNAs. Ourdataindicatehoweverthatthesecouldnotbe
usedasmarkersinaheterogeneouspopulation,suchasthehumanpopulation.
Astudyofhumanoocytesreportedachangeinexpressionlevelbetweenthegerminalvesicleand
themetaphaseIIstagefor15of722miRNAsexpressedduringmaturation[37],suggestingspecific
rolesduringmaturationandearlyembryodevelopment. Itwouldbeinterestingtodeterminewhich
miRNAexpressionlevelschangethemostinbovinecumuluscellcomplexesduringmaturation. Ona
similarnote,thedifferenceinmiRNAexpressionpatternsbetweenoutercumulusandinnercumulus,
i.e.,thecellsfurthestfromorclosesttotheoocyte,mightshedmorelightonthefunctionofmiRNAin
cumuluscells.
OursequencingresultsshowedthatmiR-21-5pisthemostabundantmiRNAinbovinecumulus
cells, followed by various members of the let-7 family and miR-155. In human cumulus cells,
membersofthelet-7familyandmiR-21arealsothemostabundantmiRNAs;however,theirfunctions
remain unclear [14]. An anti-apoptotic effect was proposed for miR-21 in mouse granulosa cells;
however, it was not clear how this effect was achieved, because no change in apoptotic protein
expression was observed after miR-21 knockdown [39]. A relatively low expression of miR-21
wasdetectedinbovineoocytes,buttheexpressionincreaseddramaticallyduringearlyembryonic
development[36];similarly,a25-foldincreaseinmiR-21expressionwasreportedinpigcumuluscells
duringmaturation[40]. ApotentialtargetofmiR-21isprogrammedcelldeathprotein4(PDCD4),
andinteractionbetweenmiR-21andPDCD4hasbeenreportedinpigoocytes[40]. WhethermiR-21
hasasimilarfunctioninbovinecumuluscellsremainstobedetermined.
AnalyzingmiRNAexpressioninoocytesandcumuluscellsusingaPCRarrayshowedanegative
fold change of miR-155 expression between bovine oocytes and cumulus cells, indicating higher
expressionincumuluscellsthaninoocytes.Interestingly,thisfoldchangeincreasedduringmaturation,
indicatingthateithertheexpressionlevelsinthecumuluscellsdecreasedorthelevelintheoocyte
increased,orboth[33]. miR-155wasalsofoundtobeabundantlyexpressedinexosomesrecovered
frombovinefollicularfluid,andtheexpressionlevelwashigherinfluidfromfolliclesthatcontained
agrowingoocytecomparedtothosewithafully-grownoocyte[41]. Basedonthehighexpression
levelsofmiR-155incumuluscells,itseemslikelythatthismiRNAisproducedbythecumuluscells
themselves,althoughitcannotbeexcludedthatcumuluscellstakeupmiR-155fromthefollicularfluid.
High expression levels of various let-7 family members have been reported in cumulus cells;
amongthemostabundantlet-7familymemberswerelet-7f,let-7iandlet-7g. Let-7wasalsoabundant
inhumanandmousecumuluscells[42]. Interestingly,let-7miRNAsarenegativelyassociatedwith
pluripotency[43],althoughtheyarehighlyexpressedinoocytes[35,44]. Thisindicatesahighdegree