Oocyte BiochemistryOocyte Biochemistry

Yves JR MENEZOYves JR MENEZO

Ph D, Dr Sci, TCPh D, Dr Sci, TC

UNILABS Scientific Adviser UNILABS Scientific Adviser (Geneve, Paris)(Geneve, Paris)

Nurilia LyonNurilia Lyon

25

8 162 4Fertilization

40 60 70

Stage

50 90 110 140 Hrs

Maternal mRNA Embryonic mRNA

Evolution of maternal and embryonic mRNA (human) during preimplantation development

mR

NA

(n

g/e

mb

ryo)

20

40

60

80

100

M Bl

YX

Timing and quality of MZT maternal to zygotic transition/transcription

RRegulation of mRNA Polyadenylation

M2 St 1(20h)

No of copies (105)

St 4(48h)

St 8(72h)

M(96h)

E Bl(120h)

X Bl(144h)

ICSIICSI NormalNormal IVF IVF

DelayedDelayed IVFIVF

EmbryonicEmbryonictranscriptstranscripts

6h6h 6h6hFERTILISATIONFERTILISATION

250

MaternalMaternaltranscriptstranscripts

mRNAs saved by ICSI process

Oocytemembrane

Nucleus

TGFBeta

R1, R3

SMADs

Oocyte cytoplasm

Activating factors in the oocyte and early preimplantation embryo…. In vivo

Endogenous pool

Aminoacids

Albumin

Lipids

ENVIRONMENT: O2 relative%

EMBRYO

Glucose, Lactate*, pyruvate*

Anabolism

Catabolism(NaHCO3) CO2

NH3 (Alanine)

Specific transport systems

Albumin Albumin (lipid transport)(lipid transport)

Anti-oxydants (hypotaurine) Anti-oxydants (hypotaurine)

(Fe++: Fenton reaction)(Fe++: Fenton reaction) YM/KE 2004

*In the oocytes and early preimplantation *In the oocytes and early preimplantation embryo, (embryo, (as in almost all the cellsas in almost all the cells)….)….

*Synthesis of a compound is usually more *Synthesis of a compound is usually more time and energy consuming than its uptake….time and energy consuming than its uptake…. Even Even if uptake requires energy….if uptake requires energy….

*Synthesis and uptake co-exist but Inhibition *Synthesis and uptake co-exist but Inhibition of synthesis will stop development even if the of synthesis will stop development even if the compound is supplied (cholesterol, nuclear compound is supplied (cholesterol, nuclear bases…)bases…)

Some basic statements…..

Activation: what for?Activation: what for?

glycolysis and glucose uptake

Up regulation of Up regulation of Pentose Pentose Phosphate Phosphate pathwayspathways

PPP increased PPP increased production of C5 production of C5 sugars (DNA sugars (DNA synthesis)synthesis)

Quality of S Quality of S Phase?Phase?

H+ + NADPH

H+ + NADPH

PPP up regulation : what for?PPP up regulation : what for?

PPP influences the onset of the first S-phase in both male and

female pronuclei, and continues to influence embryo development up to

the blastocyst stage.

PPP generates NADPH, involved in the majority of anabolic

pathways:

1 mole of Glucose 6 phosphate 2 moles of NADPH

NADPH allows methionine to be recycled from homocysteine,

( methylene tetrahydrofolate reductase) This pathway influences

imprinting process and is involved in thymidine synthesis (5 Methyl-

Uracyl,).

NADPH is also required to reduce oxidized glutathione (GSSG).

Glutathione is necessary for sperm head swelling,

Blastocyst formation,

cell number per blastocyst formed.

Glutathione is an universal metabolite in protection against

oxidative stress.

SugarsSugars

High incorporation of C3 monocarboxylic High incorporation of C3 monocarboxylic acidsacids (lactate , Pyruvate) (lactate , Pyruvate)

Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development.Hérubel F et al. Zygote. 2002Lactate allows regeneration of NADH (reduced form) and forms pyruvate(High concentration in tubal flud)

But: The oocyte and early embryos are not well equipped to resist to an acidic pHDale B, et al.1998 Intracellular pH regulation in the human oocyte. Hum Reprod.1998

Glucose is not toxic Glucose is not toxic per seper se, but it is, when , but it is, when added at a too high concentration, in a too added at a too high concentration, in a too simple medium Earle or other saline mediasimple medium Earle or other saline media

Leading to metabolic « cul de sac » and Leading to metabolic « cul de sac » and apoptosisapoptosis

Amino acidsAmino acids

Concept of « Essential amino acid toxicity » Concept of « Essential amino acid toxicity » oocyte and early stage: a terrible statement!!!oocyte and early stage: a terrible statement!!!

MethionineMethionine

All the AAs at all stage. The Ratio between them All the AAs at all stage. The Ratio between them is of major importanceis of major importance

aspartateaspartate

(glutamine)(glutamine)

oxaloacetateoxaloacetate

pyruvatepyruvate

acCoAacCoA

lactatelactate

GlucoseGlucose

malatemalate

glutamateglutamate

oxoglutarateoxoglutarate

glycine glycine

glyoxylate

TGPTGP AATAAT

oxaloacetateoxaloacetate

(oxoglutaramate)(oxoglutaramate)

pyruvatepyruvate glutaminglutaminee

Ammonium removal from oocyte and preimplantation embryoAmmonium removal from oocyte and preimplantation embryo

AAT=aspartate aminotransferaseAAT=aspartate aminotransferase

TGP=transaminase glutamate-pyruvateTGP=transaminase glutamate-pyruvate

TCAcycle

alaninealanine

alaninealanine

PROTEINSYNTHESIS

Glycinefree

GlycineIn proteins

Gly

cin

e u

pta

ke

with methioninewithout methionine

Export (Ala: NH3)

2-AA Competition

50 µM

100 µM

250 µM

AA

up

take

Aminoacidpool

4-PROTEINCATABOLISM

carrier Glycine

1-AA External concentration

3-PROTEINSYNTHESIS

AMINOACIDS: Factors influencing aminoacids endogenous pool in the embryo

AMINOACIDS: Factors influencing aminoacids endogenous pool in the embryo

Methionine

time

Effect of methionine on incorporation and metabolism of glycine

SAM, S Adenosyl methionine:SAM, S Adenosyl methionine: Methyl Methyl donor for imprintingdonor for imprinting

CH3

Methionine

SAHSAH

H

Homocysteine

Met Uptake and conversion to SAM/SAH Met Uptake and conversion to SAM/SAH (fmoles/embryo/hr)(fmoles/embryo/hr)

The conversion is similar from oocyte to 4-cell stageThe conversion is similar from oocyte to 4-cell stage

Met Uptake Met Uptake ConversionConversionMouseMouse2-Cell2-Cell 250250 9 (3.6%)9 (3.6%)Early MorulaEarly Morula 350350 12 (3.4%)12 (3.4%)Compact. MorulaCompact. Morula 650650 33 (5.1%)33 (5.1%)BlastocystBlastocyst 23352335 41 (1.8%)41 (1.8%)HumanHuman4-cell4-cell 770770 26.2 (3.4)26.2 (3.4) Menezo et al 1989Menezo et al 1989

Ménézo et al.1989Ménézo et al.1989

Paternally expressed

M P

Imprinted genes

Mono-allelic expression

Maternally expressed

M P

Genomic Imprinting/ DNA methylation/ DNA methyl transferase “in the early human embryo”

DNA methylation5-methyl cytosine

Non-Imprinted genes

Bi-allelic expression

M P

Viville Ménézo 2006CH3

5 Methyl Cytosine

Methionine and imprintingMethionine and imprinting

Silencing of Silencing of CDKN1CCDKN1C is associated with is associated with hypomethylationhypomethylation at at KvDMR1KvDMR1 in in Beckwith-Beckwith-Wiedeman syndrom Wiedeman syndrom (Diaz-meyer et al 2003)(Diaz-meyer et al 2003)

Normal maternal methylation imprints in Normal maternal methylation imprints in 15q11-q1315q11-q13 (involved in PWS) are (involved in PWS) are established established during or after fertilizationduring or after fertilization in in HUMANHUMAN

Methylation in PW1-C occurs Methylation in PW1-C occurs during during oogenesisoogenesis in the mouse (El-Maarri et al. in the mouse (El-Maarri et al. 2001)2001)

MOUSE model for HUMAN??MOUSE model for HUMAN??Ménézo 2007

Imprinting DiseasesImprinting Diseases

- Angelman Syndrome

- Prader-Willi Syndrome

- Beckwith-Wiedemann Syndrome

ICF SyndromeICF Syndrome

Mutation in DNMT3B hypomethylation

of centromeric chromatin

IImmunodeficiency, CCentromeric region instability, FFacial

anomalies

Human Diseases Associated with Altered Methylation Profiles ‘Human Diseases Associated with Altered Methylation Profiles ‘((> 80 imprinted genes (0.1-1% of all genes) Key role in embryonic growth and > 80 imprinted genes (0.1-1% of all genes) Key role in embryonic growth and placental function)placental function) Human Diseases Associated with Altered Methylation Profiles ‘Human Diseases Associated with Altered Methylation Profiles ‘((> 80 imprinted genes (0.1-1% of all genes) Key role in embryonic growth and > 80 imprinted genes (0.1-1% of all genes) Key role in embryonic growth and placental function)placental function)

CancerCancerInactivation of tumor

suppressor genesInactivation of

DNA repair genes

CpG island hypermethylation

Normal DNA methylationNormal DNA methylation

Global hypomethylation

Chromosome instability Retrotransposon

activation

Oncogene activation

?? ?

Adapted from Strathdee et al., Expert Reviews in Molecular Medicine (2002).

Imprinting/apoptosis/ defense against Imprinting/apoptosis/ defense against oxydative stress: oxydative stress: importance of Zn and importance of Zn and vitamins Bvitamins B

Met

Hcy

GlutathionHypotaurine

nB12

Zn

B6

B6

Zn

Menezo and Cohen 2010

B2

Homocysteine recycling in the human oocyte and early preimplantation embryo

No expression ofCBS pathway inHuman oocyteAnd early embryo

(Benkhalifa, Monjean, Cohen-Bacrie and Ménézo 2010)Glutathione

Vitamins B2, B6, B9, B12

co-effectors of methylation in the oocyte and early embryo

B12

B6B2

B9

Methionine

Cysteine Gluthation

Oxydative Stress ROS

DNA,

APOPTOSE

3GLUCOSE

TranssulfurationTranssulfurationPathwayPathway

2

Imprinting DNA MetransferaseImprinting DNA Metransferase

OverMethylation (alkylation)OverMethylation (alkylation)

SAMSAM

1 Methionyl mRNA (Methionyl mRNA (starts protein synthesisstarts protein synthesis))

Me

Lipid

Peroxides

YM 2004

4Amonia detoxification

HCyrecycling

ApoptosisApoptosisOxidative stressOxidative stressDNA repairDNA repair

Apoptosis, cell cycle arrest,Apoptosis, cell cycle arrest, DNA repairDNA repair,, ToleranceTolerance

TP 53

CDC25

Menezo 2007 OK ++OK ++++

Mutagenesis, carcinogenesis)

Replication Transcription

Tolerance RepairApoptosisApoptosisTrans-lesion synthesisRecombination

ReversionExcisionRecombination

Apoptosis what for?Apoptosis what for?

idemRemoval of abnormal cells

CLEAVAGE COMPACTION - DIFFERENTIATION

CAVITATION

GAMETES : ADN - CHROMOSOMES - AGE - NICOTINE

Suboptimal conditions : Medium, GLUCOSE, UV, T°, O2 - STRESS

CASPASES +/- AIF

AGE

Relation Imprinting/ApoptosisRelation Imprinting/Apoptosisand and defensedefense against oxidative stress against oxidative stress

Hyperhomocysteine-hemia

Folate deficiency

Genic

Expression

Alteration

Alteration of protein function

Alteration of membrane lipids a

Cellular growth

impairment

DNAFragmentation

Anomalies in cellular function

Apoptosis

Defectivemethylation

Incorporation of Uracyl into DNA (/thymidine)

Different types of DNA decaysDifferent types of DNA decays

DNA Oxidation productsDNA Oxidation products

Deoxy guanosine

NHN

NNH2

ROS/ Free radicalsSource e-

NHN

NNH2

O

O+

NHN

NNH2

O

H

OH

H2O

H+

OH°

NHN

NNH2

O

O

8 oxo dG/8 OH dG

DNA oxidation throughFree radicals

MnSODMnSODCuZnSODCuZnSOD

GPXGPX

SOD GPXSOD GPX

MnSODMnSODCuZnSODCuZnSODGPXGPX

SOD, GPXSOD, GPX

Follicular fluidFollicular fluid

Tubal fluidTubal fluid

Vit. CVit. C

CSDCSD

HypotaurineHypotaurine++

OH°OH°

Tubal cellsTubal cells

OocyteOocyte

EmbryoEmbryoMnSODMnSODCuZnSODCuZnSODGPXGPXCatalaseCatalase

OO22-°-°

HH22OO22

OH°OH°OO22-°-°

mRNA storagemRNA storage

H202 Production by different culture mediaH202 Production by different culture media

AMPLEX RED + HORSERADISH PEROXIDASEAMPLEX RED + HORSERADISH PEROXIDASE

((From Alvarez-Miguel et al. 2005From Alvarez-Miguel et al. 2005))

LDifferent pathways involved in DNA

repairExcision repair system, NER, BER , MMR Ones step

30 proteins

Alkyl guanineDNAAlkyltransferase

Age-related alteration of gene expression Age-related alteration of gene expression patterns in mouse oocytespatterns in mouse oocytes

5% of the 11,000 genes whose transcripts are detected 5% of the 11,000 genes whose transcripts are detected in oocytes shows statistically significant expression in oocytes shows statistically significant expression changes, excluding a global decline in transcript changes, excluding a global decline in transcript abundanceabundance

AffectsAffects Mitochondrial function, Mitochondrial function, oxidative stressoxidative stress Chromatin structure, Chromatin structure, DNA methylation, DNA methylation, genome stabilitygenome stability

Hanatani et al. 2004 Human Mol. GenetHanatani et al. 2004 Human Mol. Genet

YM YM 20092009

DNA repair pathways in the oocyte: DNA repair pathways in the oocyte: NucleasesNucleases

APEX: multifunctional nucleaseAPEX: multifunctional nuclease

ERCC(6): Excision repair cross complementingERCC(6): Excision repair cross complementing

MDB4: methyl-CPG binding domain MDB4: methyl-CPG binding domain (ethenocytosine glycosylase)(ethenocytosine glycosylase)

IMPRINTING!!!IMPRINTING!!!

OGG1: 8 oxoGuannieDNA GlycosylaseOGG1: 8 oxoGuannieDNA Glycosylase

UNG: Uracyl-DNA glycosylaseUNG: Uracyl-DNA glycosylase

Menezo 2007

DNA over-Methylation: repair by oocyteDNA over-Methylation: repair by oocyte

Alkylation damages (Methyl to benzyl)Alkylation damages (Methyl to benzyl)

Overmethylation may induce alkylation of DNA. Overmethylation may induce alkylation of DNA. Then DNA repair machinery is necessary MGMT Then DNA repair machinery is necessary MGMT 0066-meG-Alkyltransferase,-meG-Alkyltransferase,

ABH2 and ABH3: 1 MeAdenine and 3 Me CytosineABH2 and ABH3: 1 MeAdenine and 3 Me Cytosine

Or NER: 0Or NER: 044-methylThymine-methylThymine

Ménézo 2011

Nuclear bases can be incorporated by oocyte and Nuclear bases can be incorporated by oocyte and preimplantation embryos (preimplantation embryos (ΣΣ Nucleosides) Nucleosides)

The nucleotide pool sanitization enzymes are the first defences against mutagenesis, and the human oocyte is well equipped with NUDT (nucleoside diphosphate linked moiety X), the major enzyme involved (Removal of 8-oxo guanosine) If not the oxidized base is re-incorporated in the DNAIf not the oxidized base is re-incorporated in the DNA

Synthesis of pyrimidic bases by the embryo is active Block of Synthesis of pyrimidic bases by the embryo is active Block of the pathway at carbamoyl transferase level stops the pathway at carbamoyl transferase level stops developmentdevelopment**

Nuclear bases

DNA repair pathways in the oocyte:DNA repair pathways in the oocyte:

Highly quantitatively presentHighly quantitatively present RepetitiveRepetitive Finite capacityFinite capacity

Probably more than1.500 000 damage Probably more than1.500 000 damage repairs in the first 24 hrs at the PN stagerepairs in the first 24 hrs at the PN stage

Need to understand effectors Need to understand effectors acting on DNA repair during acting on DNA repair during maturationmaturation

GH?GH?

Antioxidants and fertilityAntioxidants and fertility

The oocyte has a finite capacity to repair DNA damages. Its The oocyte has a finite capacity to repair DNA damages. Its

capacity decreases with agecapacity decreases with age

Ménézo and Cohen 2011

Decrease de charge of DNA repair by decreasing the ROS linked DNA decays

Cumulus cells gene expression and oocyte qualityCumulus cells gene expression and oocyte quality

Several publications on relationship between CC Rna Several publications on relationship between CC Rna content and oocyte quality (Hamel et al. 2008, Adrianssens content and oocyte quality (Hamel et al. 2008, Adrianssens et al. 2010)et al. 2010)

In our hands: Measure of MTFs (In our hands: Measure of MTFs (metal responsive transcription metal responsive transcription factors)factors)

The MTFs, especially MTF2 are highly expressed in oocytes, The MTFs, especially MTF2 are highly expressed in oocytes, and are completely absent in cumulus cells (CCs)and are completely absent in cumulus cells (CCs)

Cumulus cells have reached the end of their life’s journey Cumulus cells have reached the end of their life’s journey and the tentative correlation does not seem rationaland the tentative correlation does not seem rational

Ménézo et al. RBMO 2011 in pressMénézo et al. RBMO 2011 in press

ConclusionConclusion

Improve storage of protein and mRNAs Improve storage of protein and mRNAs (GH?)(GH?)

Timing in transcription and preparation Timing in transcription and preparation for MZT is of major importancefor MZT is of major importance

Avoid any delay *mishandling of oocyte Avoid any delay *mishandling of oocyte (T°C) or * culture media anomalies(T°C) or * culture media anomalies

Do not forget that there is a spontaneous Do not forget that there is a spontaneous generation of ROS in the culture medium generation of ROS in the culture medium if not well protectedif not well protected