Models for study of oocyte competence:Models for study of oocyte competence:

George W. SmithGeorge W. Smith(Smithge7@msu.edu)(Smithge7@msu.edu)

Foundational questionsFoundational questionsFoundational questionsFoundational questions•• OocyteOocyte--derived functional mediators of early derived functional mediators of early yy yy

embryonic development (EST and candidate gene)embryonic development (EST and candidate gene)–– JYJY--11–– NoboxNobox–– ImportinImportin 88

Oocyte and cumulus cell gene expression signaturesOocyte and cumulus cell gene expression signatures•• Oocyte and cumulus cell gene expression signatures Oocyte and cumulus cell gene expression signatures associated with egg qualityassociated with egg quality–– Functional significanceFunctional significancegg–– Diagnostic and therapeutic potentialDiagnostic and therapeutic potential–– Bovine, rhesus and humanBovine, rhesus and human

OocyteOocyte regulation of regulation of follicular developmentfollicular developmentfollicular developmentfollicular development

Follicle formationFollicle formation

ProliferationProliferation DifferentiationDifferentiation

Cumulus expansionCumulus expansionOvulationOvulation

Steroid productionSteroid productionOvulationOvulation

AA transport Cholesterol synthesisAA transport Cholesterol synthesisAA transport, Cholesterol synthesisAA transport, Cholesterol synthesisEnergy metabolismEnergy metabolism

((EppigEppig et al., 2008)et al., 2008)

Species specific oocyte regulation of Species specific oocyte regulation of the maternalthe maternal toto embryonic transitionembryonic transitionthe maternalthe maternal--toto--embryonic transitionembryonic transition

Polyovulatory mousePolyovulatory mousePolyovulatory mousePolyovulatory mouse...

11stst PBPB

SpermSperm22ndnd PBPB

Zar1Zar1 MaterMater

FertilizationFertilization ZygoteZygote EmbryonicEmbryonic genomegenomeactivationactivation

BlastocystBlastocyst

Monoovulatory human (4Monoovulatory human (4--cell)cell)

Monoovulatory cattle (8Monoovulatory cattle (8--cell)cell)(Tong et al., 2000; (Tong et al., 2000; Wu et al., 2003)Wu et al., 2003)

OocyteOocyte quality and success of ARTquality and success of ART

2007 Report: Assisted Reproductive Technology Success Rates2007 Report: Assisted Reproductive Technology Success Rateswww.cdc.gov/ARTwww.cdc.gov/ART

Oocyte quality (competence)Oocyte quality (competence)

•• Ability to resume meiosisAbility to resume meiosis

Oocyte quality (competence)Oocyte quality (competence)

•• Ability to resume meiosisAbility to resume meiosis

•• Ability to cleave following fertilizationAbility to cleave following fertilizationy gy g

•• Ability to develop to Ability to develop to blastocystblastocyst stagestage

•• Ability to Ability to induce induce pregnancy and bring to termpregnancy and bring to term

•• Ability to develop to term in good health Ability to develop to term in good health

(Sirard et al., 2006)(Sirard et al., 2006)

Criteria for classification of Criteria for classification of oocyteoocyte competence (quality)competence (quality)

FollicleFollicle

oocyteoocyte competence (quality)competence (quality)

ColorColorSizeSize

Cumulus cellsCumulus cells CytoplasmCytoplasmCumulus cellsCumulus cells

Failure rate (ART)Failure rate (ART)~70%~70%

Overall hypothesis:Overall hypothesis:ypyp

Ch t i ti f th RNA t i t fil fCh t i ti f th RNA t i t fil fCharacteristics of the RNA transcript profiles of Characteristics of the RNA transcript profiles of the the oocyteoocyte and adjacent cumulus cells are and adjacent cumulus cells are

reflective of competence of thereflective of competence of the oocyteoocytereflective of competence of the reflective of competence of the oocyteoocyte

ApproachApproach•• RNA transcript profiling RNA transcript profiling

–– eggs (bovine) and cumulus (bovine andeggs (bovine) and cumulus (bovine andeggs (bovine) and cumulus (bovine and eggs (bovine) and cumulus (bovine and human) human)

•• Data analysis (genes gene families &Data analysis (genes gene families &Data analysis (genes, gene families & Data analysis (genes, gene families & pathways)pathways)

•• FunctionalFunctional significance of putativesignificance of putative markersmarkersFunctional Functional significance of putative significance of putative markersmarkers–– Ablation replacement and pharmacological Ablation replacement and pharmacological

(bovine and rhesus)(bovine and rhesus)(bovine and rhesus) (bovine and rhesus) •• Predictive value of putative Predictive value of putative markersmarkers

C l ll ( d h )C l ll ( d h )–– Cumulus cell assay (cow and human)Cumulus cell assay (cow and human)•• Therapeutic potential (bovine and rhesus) Therapeutic potential (bovine and rhesus)

Advantages of bovine model systemAdvantages of bovine model systemg yg y

•• Similarities with humanSimilarities with humanSimilarities with human Similarities with human –– Oocyte sizeOocyte size

Embryo developmental kineticsEmbryo developmental kinetics–– Embryo developmental kinetics Embryo developmental kinetics –– Availability of large #’s of oocytesAvailability of large #’s of oocytes–– Reliable protocols for in vitro maturation, Reliable protocols for in vitro maturation,

fertilization and culturefertilization and culture•• Multiple wellMultiple well--characterized experimental characterized experimental

models of poor oocyte competencemodels of poor oocyte competence

Functional genomics studies of Functional genomics studies of b i t tb i t tbovine oocyte competencebovine oocyte competence

•• Differences in oocyte transcriptome profileDifferences in oocyte transcriptome profileDifferences in oocyte transcriptome profile Differences in oocyte transcriptome profile associated with egg quality (Patel et al., associated with egg quality (Patel et al., 2007)2007)–– follistatinfollistatin

•• Differences in cumulus cell transcriptome Differences in cumulus cell transcriptome associated with egg quality (Bettegowda et associated with egg quality (Bettegowda et al., 2008)al., 2008)–– cathepsinscathepsins

Experimental approachesExperimental approaches

Functional role and therapeutic Functional role and therapeutic potentialpotential

Effect of Effect of cathepsincathepsin inhibitor treatment during inhibitor treatment during in vitro maturation on early embryonic in vitro maturation on early embryonic

developmentdevelopmentParthenogeneticParthenogenetic IVFIVF

embryosembryos

3030

4040 bbbb

%% 3030e %

e % bb bb

embryosembryos embryosembryos

2020

3030 aa

ocys

toc

ystr

ate

rate

2020

tocy

stto

cyst

rat

rat

aa

00

1010

00

Bla

sto

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Bla

stB

last

00(1 (1 MM)) (10 (10 MM))00

cathepsincathepsin inhibitorinhibitor(1 (1 MM)) (10 (10 MM))00

cathepsin inhibitorcathepsin inhibitor

((BettegowdaBettegowda et al., 2008)et al., 2008)

Potential mechanism of action of cathepsins in Potential mechanism of action of cathepsins in influencing oocyte competence influencing oocyte competence

ControlControl

ApoptoticApoptoticBright fieldBright field TotalTotal

1 µM1 µM

ControlControl

inhibitorinhibitor

10 µM10 µM

inhibitorinhibitor

PositivePositivePositivePositive

controlcontrol

NegativeNegativeNegativeNegative

controlcontrol ((BettegowdaBettegowda et al., 2008)et al., 2008)

Zygote siRNA injection: Ablation/replacement Zygote siRNA injection: Ablation/replacement studies in early embryosstudies in early embryosstudies in early embryosstudies in early embryos

3535

4040 c

2525

3030

3535ss

(d 7

)(d

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aaa

1515

2020

asto

cyst

sas

tocy

sts

b

00

55

1010

%

% b

lbl

b

UninjectedUninjected FollistatinFollistatinsiRNAsiRNA

UninjectedUninjected FollistatinsiRNA

Without Without follistatinfollistatin With With follistatinfollistatin

(Lee et al., 2009)(Lee et al., 2009)

Zygote siRNA injection: Ablation/replacement Zygote siRNA injection: Ablation/replacement studies in early embryosstudies in early embryos

TrophectodermTrophectoderm cellscells Total cellsTotal cells

studies in early embryosstudies in early embryos

pp

6060707080809090

ells

ells a

c

a100100120120140140160160

ells

ells

ab

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# of

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2020404060608080

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# of

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Uninjected FollistatinsiRNA

Uninjected FollistatinsiRNA

Without follistatinWithout follistatin With follistatinWith follistatin

00Uninjected Follistatin

siRNAUninjected Follistatin

siRNA

WithoutWithout follistatinfollistatin With follistatinWith follistatint o statt o stat Without Without follistatinfollistatin With follistatinWith follistatin

(Lee et al., 2009)(Lee et al., 2009)

Oocyte siRNA injection: ablation/replacement Oocyte siRNA injection: ablation/replacement studies of egg maturation and cumulus expansionstudies of egg maturation and cumulus expansion

Microinjection of cumulus enclosed Microinjection of cumulus enclosed GV GV oocytesoocytes

JYJY--1 1 siRNAsiRNA rescue experimentrescue experiment(+/(+/--) JY) JY--1 protein1 protein

JYJY--1 1 siRNAsiRNAsham and/or negative control sham and/or negative control siRNAsiRNA

and and uninjecteduninjected (control)(control)

Maintenance of meiotic arrest for 48 Maintenance of meiotic arrest for 48 h in maturation medium with h in maturation medium with

5050 μμM SM S--roscovitineroscovitine(72 h)(72 h)

COCs

50 50 μμM SM S roscovitineroscovitine

In vitro maturation for 24 In vitro maturation for 24 h in absence of Sh in absence of S--

roscovitineroscovitine

Confirm GV arrestConfirm GV arrest

Levels of JY-1 mRNA and protein Same developmental Same developmental

In vitro fertilization and In vitro fertilization and embryo cultureembryo culture

Nuclear maturationCumulus expansion

ppendpointsendpoints

Early cleavage, total cleavage, development to 8-

16 cell and blastocyst stages (Wee et al., unpublished)(Wee et al., unpublished)

Translational studies in rhesus Translational studies in rhesus monkeymonkey

CathiCathi VandeVoortVandeVoortCalifornia National Primate California National Primate

Research CenterResearch Center

FollistatinFollistatin effects on rhesus monkey effects on rhesus monkey early embryonic developmentearly embryonic development

6060

y y py y p

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bb

3030

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ControlControl FollistatinFollistatin

%%

(10(10 ngng/ml)/ml)(10 (10 ngng/ml)/ml)

(P < 0.05)(P < 0.05)((VandeVoortVandeVoort et al., 2009)et al., 2009)

Human clinical studiesHuman clinical studiesHuman clinical studiesHuman clinical studies

Jose Jose CibelliCibelliMichigan State universityMichigan State universityMichigan State universityMichigan State university

Can factors produced by cumulus cells influence thequality of the oocyte and/or embryo in women?

Gene expression signatureGene expression signatureassociated with successful pregnancyassociated with successful pregnancy

RNA Arrives toLaboratory

Gene expression analysis

Ranked results are Sent to IVF clinic

Egg-RNA #1Egg#

Best31

Egg-RNA #2

Egg-RNA #3

Best

10.

Statistical Analysis.

Egg-RNA #11

.

4Egg-RNA #12

2Egg-RNA #13 Worst

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