Download Diapositive 1

Oocyte Biochemistry
Yves JR MENEZO
Ph D, Dr Sci, TC
UNILABS Scientific Adviser
(Geneve, Paris)
Nurilia Lyon
mRNA (ng/embryo)
100
80
60
Timing and quality of
MZT maternal to zygotic
transition/transcription
Regulation of mRNA
R
Polyadenylation
Maternal mRNA
Embryonic mRNA
40
20
YX
25
Fertilization
40 50
2
4
60
70
90
8 16
110
M
Bl
140 Hrs
Stage
Evolution of maternal and embryonic mRNA (human) during preimplantation
development
mRNAs saved by ICSI process
No of copies (105)
250
ICSI
FERTILISATION
Normal
IVF
6h
Delayed
IVF
6h
Embryonic
transcripts
Maternal
transcripts
M2
St 1
(20h)
St 4
(48h)
St 8
(72h)
M
(96h)
E Bl
(120h)
X Bl
(144h)
Activating factors in the oocyte and early preimplantation
embryo…. In vivo
Oocyte
membrane
TGF
Beta
R1, R3
SMADs
Oocyte cytoplasm
Nucleus
Glucose, Lactate*, pyruvate*
ENVIRONMENT: O2 relative%
Specific
transport
systems
Aminoacids
Anabolism
Albumin
Endogenous
pool
Albumin
(lipid transport)
Lipids
Catabolism
EMBRYO
Anti-oxydants (hypotaurine)
(Fe++: Fenton reaction)
(NaHCO3) CO2
NH3 (Alanine)
Some basic statements…..
*In the oocytes and early preimplantation embryo,
(as in almost all the cells)….
*Synthesis of a compound is usually more time and
energy consuming than its uptake…. Even if uptake requires
energy….
*Synthesis and uptake co-exist but Inhibition of
synthesis will stop development even if the compound is
supplied (cholesterol, nuclear bases…)
Activation: what for?
H+ + NADPH



glycolysis and
glucose uptake
Up regulation of
Pentose Phosphate
pathways
PPP increased
production of C5
sugars (DNA
synthesis)
Quality of S Phase?
H+ + NADPH
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.
Sugars
High incorporation of C3 monocarboxylic acids
(lactate , Pyruvate)
Genetic expression of monocarboxylate transporters during
human and murine oocyte maturation and early embryonic
development.
Hérubel F et al. Zygote. 2002
Lactate 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 pH
Dale B, et al.1998 Intracellular pH regulation in the human oocyte. Hum
Reprod.1998
Glucose is not toxic per se, but it is, when added
at a too high concentration, in a too simple
medium Earle or other saline media
Leading to metabolic « cul de sac » and apoptosis
Amino acids

Concept of « Essential amino acid toxicity » oocyte and
early stage: a terrible statement!!!
Methionine

All the AAs at all stage. The Ratio between them is of
major importance
Glucose
glutamine
pyruvate
acCoA
oxaloacetate
malate
lactate
glycine
glycine
pyruvate
alanine
oxaloacetate
glutamate
(glutamine)
TGP
glyoxylate
TCA
cycle
AAT
oxoglutarate
(oxoglutaramate)
aspartate
alanine
TGP=transaminase glutamate-pyruvate
AAT=aspartate aminotransferase
Ammonium removal from oocyte and preimplantation embryo
250 µM
100 µM
Methionine
50 µM
carrier
time
Glycine
Glycine uptake
AA uptake
AMINOACIDS: Factors influencing aminoacids
endogenous pool in the embryo
with methionine
without methionine
Glycine Glycine
free In proteins
1-AA External
concentration
2-AA Competition
Aminoacid
pool
3-PROTEIN
PROTEIN
SYNTHESIS
SYNTHESIS
4-PROTEIN
CATABOLISM
Export (Ala: NH3)
ffect of methionine on incorporation and metabolism
of glycine
SAM, S Adenosyl methionine: Methyl donor for
imprinting
Methionine
CH3
SAH
Homocysteine
H
Met Uptake and conversion to SAM/SAH (fmoles/embryo/hr)
The conversion is similar from oocyte to 4-cell stage
Met Uptake
Conversion
250
350
650
2335
9 (3.6%)
12 (3.4%)
33 (5.1%)
41
770
26.2 (3.4)
Mouse
2-Cell
Early Morula
Compact. Morula
Blastocyst
(1.8%)
Human
4-cell
Menezo et al 1989
Genomic Imprinting/ DNA methylation/ DNA methyl
transferase “in the early human embryo”
Non-Imprinted genes
Imprinted genes
Bi-allelic expression
Mono-allelic expression
M
M
P
P
Maternally
expressed
5-methyl
CH3
cytosine
Viville Ménézo 2006
M
Paternally
expressed
DNA methylation
5 Methyl
Cytosine
P
Methionine and imprinting



Silencing of CDKN1C is associated with
hypomethylation at KvDMR1 in BeckwithWiedeman syndrom (Diaz-meyer et al 2003)
Normal maternal methylation imprints in
15q11-q13 (involved in PWS) are established
during or after fertilization in HUMAN
Methylation in PW1-C occurs during oogenesis
in the mouse (El-Maarri et al. 2001)
MOUSE model for HUMAN??
Ménézo 2007
Human Diseases Associated with Altered Methylation Profiles ‘
(> 80 imprinted genes (0.1-1% of all genes) Key role in embryonic growth and
placental function)
Cancer
Imprinting Diseases
- Angelman Syndrome
Inactivation of tumor
suppressor genes
Inactivation of
DNA repair
genes
- Prader-Willi Syndrome
- Beckwith-Wiedemann
Syndrome
ICF Syndrome
Immunodeficiency,
Centromeric region
instability, Facial anomalies
CpG island hypermethylation
Normal DNA methylation
Global hypomethylation
?
?
?
Mutation in DNMT3B 
hypomethylation of
centromeric chromatin
Chromosome
instability Retrotransposon
activation
Adapted from Strathdee et al.,
Expert Reviews in Molecular
Medicine (2002).
Oncogene
activation
Imprinting/apoptosis/ defense against oxydative
stress: importance of Zn and vitamins B
Met
Zn
Zn n
Hcy
B12
B6
B2
B6
Glutathion
Hypotaurine
Homocysteine recycling in the human oocyte and early preimplantation
embryo
No expression of
CBS pathway in
Human oocyte
And early embryo
Glutathione
(Benkhalifa, Monjean, Cohen-Bacrie and Ménézo 2010)
Vitamins B2, B6, B9, B12
co-effectors of methylation in the oocyte and early
embryo
B9
B6
B12
B2
4
Amonia detoxification
Methionine
Methionyl mRNA (starts protein synthesis)
1
Transsulfuration
Pathway
HCy
3
recycling
Cysteine
GLUCOSE

Gluthation
2
Oxydative
Stress ROS
DNA, Lipid
Peroxides
Me
SAM
APOPTOSE
Imprinting DNA Metransferase
OverMethylation (alkylation)
YM 2004
Apoptosis
Oxidative stress
DNA repair
Apoptosis, cell cycle arrest,
DNA repair, Tolerance
CDC25
Replication
Transcription
TP 53
Apoptosis
Tolerance
Apoptosis
Trans-lesion synthesis
Recombination
Menezo 2007
Mutagenesis, carcinogenesis)
Repair
Reversion
Excision
Recombination
OK +++
Apoptosis what for?
Removal of abnormal
cells
idem
CASPASES +/- AIF
CLEAVAGE
AGE
GAMETES : ADN - CHROMOSOMES
- AGE - NICOTINE
COMPACTION DIFFERENTIATION
CAVITATION
Suboptimal conditions : Medium, GLUCOSE, UV, T°, O2 STRESS
Relation Imprinting/Apoptosis
and defense against oxidative stress
Incorporation of
Uracyl into DNA
(/thymidine)
Hyperhomocysteinehemia
Defective
methylation
Folate deficiency
Genic
Expression
Alteration
Alteration of protein
function
Alteration of
membrane lipids
Cellular
growth
a
impairment
Anomalies in cellular function
Apoptosis
DNA
Fragmentation
Different types of DNA decays
DNA Oxidation products
+
O
ROS/ Free radicals
Source
e-
HN
H2N
N
N
H2O
O
H+
N
HN
H2N
O
HN
N
Deoxy
guanosine
N
OH°
N
H2N
O
HN
DNA oxidation through
Free radicals
H2N
8 oxo dG/8 OH dG
N
N
OH
H
Follicular fluid
mRNA storage
MnSOD CuZnSOD GPX
CuZnSOD
SOD, GPX
Oocyte
Vit. C
+
Hypotaurine
CSD
Tubal cells
MnSOD
SOD GPX
OH°
O2-°
MnSOD
CuZnSOD
GPX
Catalase
H2O2
GPX
O2-° OH°
Embryo
Tubal fluid
H202 Production by different culture media
AMPLEX RED + HORSERADISH PEROXIDASE
(From Alvarez-Miguel et al. 2005)
Different pathways involved in DNA repair
L
Ones step
Excision repair system, NER, BER , MMR
30 proteins
Alkyl guanine
DNA
Alkyltransferase
Age-related alteration of gene expression
patterns in mouse oocytes

5% of the 11,000 genes whose transcripts are detected in
oocytes shows statistically significant expression changes,
excluding a global decline in transcript abundance
Affects





Mitochondrial function,
oxidative stress
Chromatin structure,
DNA methylation,
genome stability
Hanatani et al. 2004 Human Mol. Genet
YM 2009
DNA repair pathways in the oocyte:
Nucleases
APEX: multifunctional nuclease

ERCC(6): Excision repair cross complementing
MDB4: methyl-CPG binding domain (ethenocytosine
glycosylase)
IMPRINTING!!!


OGG1: 8 oxoGuannieDNA Glycosylase

UNG: Uracyl-DNA glycosylase
Menezo 2007
DNA over-Methylation: repair by oocyte


Alkylation damages (Methyl to benzyl)
Overmethylation may induce alkylation of DNA. Then
DNA repair machinery is necessary MGMT 06-meGAlkyltransferase,

ABH2 and ABH3: 1 MeAdenine and 3 Me Cytosine

Or NER: 04-methylThymine
Nuclear bases
Nuclear bases can be incorporated by oocyte and preimplantation
embryos (Σ 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 DNA
Synthesis of pyrimidic bases by the embryo is active Block of the
pathway at carbamoyl transferase level stops development*
DNA repair pathways in the oocyte:



Highly quantitatively present
Repetitive
Finite capacity
Probably more than1.500 000 damage repairs in
the first 24 hrs at the PN stage
Need to understand effectors
acting on DNA repair during maturation
GH?
Antioxidants and fertility
The oocyte has a finite capacity to repair DNA damages. Its capacity
decreases with age
Decrease de charge of DNA repair by decreasing the ROS
linked DNA decays
Ménézo and Cohen 2011
Cumulus cells gene expression and oocyte quality
Several publications on relationship between CC Rna content and
oocyte quality (Hamel et al. 2008, Adrianssens et al. 2010)
In our hands: Measure of MTFs (metal responsive transcription factors)
The MTFs, especially MTF2 are highly expressed in oocytes, and are
completely absent in cumulus cells (CCs)
Cumulus cells have reached the end of their life’s journey and the tentative
correlation does not seem rational
Ménézo et al. RBMO 2011 in press
Conclusion




Improve storage of protein and mRNAs (GH?)
Timing in transcription and preparation for MZT
is of major importance
Avoid any delay *mishandling of oocyte (T°C)
or * culture media anomalies
Do not forget that there is a spontaneous
generation of ROS in the culture medium if not
well protected