Oocyte Biochemistry Yves JR MENEZO Ph D, Dr Sci, TC UNILABS Scientific Adviser (Geneve, Paris)...
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Transcript of Oocyte Biochemistry Yves JR MENEZO Ph D, Dr Sci, TC UNILABS Scientific Adviser (Geneve, Paris)...
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.
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
SAM, S Adenosyl methionine:SAM, S Adenosyl methionine: Methyl Methyl donor for imprintingdonor for imprinting
CH3
Methionine
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
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
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)
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