1. UNILABS Scientific Adviser (Geneve, Paris)
Oocyte Biochemistry
Yves JR MENEZO
Ph D, Dr Sci, TC
UNILABS Scientific Adviser (Geneve, Paris)
Nurilia Lyon

2. Timing and quality of MZT maternal to zygotic transition/transcription
100
Timing and quality of MZT maternal to zygotic transition/transcription
Regulation of mRNA Polyadenylation R 80
mRNA (ng/embryo) 60
Maternal mRNA
Embryonic mRNA 40 20 YX 25 40 50 60 70 90
110
140
Hrs
Fertilization 2 4 8 16 M Bl
Stage
Evolution of maternal and embryonic mRNA (human) during preimplantation development

3. mRNAs saved by ICSI process
No of copies (105)
250
ICSI
Normal
IVF
Delayed
IVF
FERTILISATION 6h 6h
Embryonic
transcripts
Maternal
transcripts M2
St 1
(20h)
St 4
(48h)
St 8
(72h) M
(96h)
E Bl
(120h)
X Bl
(144h)

4. Activating factors in the oocyte and early preimplantation embryo…
Activating factors in the oocyte and early preimplantation embryo…. In vivo
Oocyte
membrane
TGF
Beta
R1, R3
SMADs
Nucleus
Oocyte cytoplasm

5. Albumin (lipid transport)
Glucose, Lactate*, pyruvate*
ENVIRONMENT: O2 relative%
Aminoacids
Specific
transport
systems
Anabolism
Endogenous
pool
Albumin
Albumin
(lipid transport)
Lipids
(NaHCO3) CO2
NH3 (Alanine)
Catabolism
EMBRYO
Anti-oxydants (hypotaurine)
(Fe++: Fenton reaction) YM/KE 2004

6. 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…)
Some basic statements…..

7. glycolysis and glucose uptake
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

8. 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.

9. Sugars

10. 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

11. 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

12. Amino acids Methionine
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

13. Ammonium removal from oocyte and preimplantation embryo
Glucose
glutamine
pyruvate
acCoA
TCA
cycle
oxaloacetate
malate
lactate
pyruvate
oxaloacetate
glycine
glycine
glutamate
(glutamine)
TGP
AAT
glyoxylate
aspartate
alanine
oxoglutarate
(oxoglutaramate)
alanine
TGP=transaminase glutamate-pyruvate
AAT=aspartate aminotransferase
Ammonium removal from oocyte and preimplantation embryo

14. AMINOACIDS: Factors influencing aminoacids endogenous pool in the embryo
with methionine
50 µM
100 µM
250 µM
without methionine
AA uptake
Methionine
Glycine uptake
carrier
Glycine
time
Glycine
free
Glycine
In proteins
1-AA External
concentration
2-AA Competition
Aminoacid
pool
3-PROTEIN
SYNTHESIS
PROTEIN
SYNTHESIS
4-PROTEIN
CATABOLISM
Export (Ala: NH3)

15. Effect of methionine on incorporation and metabolism of glycine

16. SAM, S Adenosyl methionine: Methyl donor for imprinting
CH3

17. SAH
Homocysteine H

18. Met Uptake Conversion Mouse 2-Cell 250 9 (3.6%)
Met Uptake and conversion to SAM/SAH (fmoles/embryo/hr) The conversion is similar from oocyte to 4-cell stage
Met Uptake Conversion
Mouse
2-Cell (3.6%)
Early Morula (3.4%)
Compact. Morula (5.1%)
Blastocyst (1.8%)
Human
4-cell (3.4)
Menezo et al 1989
Ménézo et al.1989 1

19. Bi-allelic expression Mono-allelic expression
Genomic Imprinting/ DNA methylation/ DNA methyl transferase “in the early human embryo”
Non-Imprinted genes
Bi-allelic expression M P
Imprinted genes
Mono-allelic expression
Maternally expressed M P
Paternally expressed M P
DNA methylation
5-methyl cytosine
CH3
Viville Ménézo 2006
5 Methyl Cytosine

20. Methionine and imprinting
Silencing of CDKN1C is associated with hypomethylation at KvDMR1 in Beckwith-Wiedeman 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

21. Cancer Imprinting Diseases ICF Syndrome
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
Inactivation of tumor suppressor genes
Inactivation of DNA repair genes
CpG island hypermethylation
Normal DNA methylation
Global hypomethylation
Chromosome instability
Retrotransposon activation
Oncogene activation ?
Adapted from Strathdee et al., Expert Reviews in Molecular Medicine (2002).
Imprinting Diseases
- Angelman Syndrome
- Prader-Willi Syndrome
- Beckwith-Wiedemann Syndrome
ICF Syndrome
Mutation in DNMT3B  hypomethylation of centromeric chromatin
Immunodeficiency, Centromeric region instability, Facial anomalies

22. Imprinting/apoptosis/ defense against oxydative stress: importance of Zn and vitamins B
Met Zn Zn n
Hcy
B12 B6 B2 B6
Glutathion
Hypotaurine
Menezo and Cohen 2010

23. Homocysteine recycling in the human oocyte and early preimplantation embryo
No expression of
CBS pathway in
Human oocyte
And early embryo
(Benkhalifa, Monjean, Cohen-Bacrie and Ménézo 2010)
Glutathione

24. co-effectors of methylation in the oocyte and early embryo
Vitamins B2, B6, B9, B12
co-effectors of methylation in the oocyte and early embryo B9
B12 B2 B6

25. Methionine Lipid DNA, 4 1 GLUCOSE 3 2 Cysteine  Gluthation APOPTOSE
Amonia detoxification
Methionine 1
Methionyl mRNA (starts protein synthesis)
Transsulfuration
Pathway 2 3
GLUCOSE
HCy
recycling
Cysteine  Gluthation
Oxydative Stress ROS
DNA,
Lipid
Peroxides Me
APOPTOSE
SAM
Imprinting DNA Metransferase
OverMethylation (alkylation)
YM 2004

26. Apoptosis Oxidative stress DNA repair

27. Apoptosis, cell cycle arrest, DNA repair, Tolerance
CDC25
Replication
Transcription
TP 53
Tolerance
Repair
Apoptosis
Apoptosis
Trans-lesion synthesis
Recombination
Reversion
Excision
Recombination
Menezo 2007
OK +++
Mutagenesis, carcinogenesis)

28. COMPACTION - DIFFERENTIATION
Apoptosis what for?
Removal of abnormal cells
idem
CASPASES +/- AIF
CLEAVAGE
COMPACTION - DIFFERENTIATION
CAVITATION
AGE
GAMETES : ADN - CHROMOSOMES - AGE - NICOTINE
Suboptimal conditions : Medium, GLUCOSE, UV, T°, O2 - STRESS

29. Relation Imprinting/Apoptosis and defense against oxidative stress
Incorporation of Uracyl into DNA (/thymidine)
Hyperhomocysteine-hemia
Folate deficiency
Defective
methylation
Genic
Expression
Alteration
Alteration of membrane lipids
Cellular growth
impairment
DNA
Fragmentation
Alteration of protein
function a
Anomalies in cellular function
Apoptosis

30. Different types of DNA decays

31. DNA Oxidation products

32. e- + DNA oxidation through Free radicals ROS/ Free radicals Source
H2O H2 N N O H+ N H N O N OH N H H2 N N H H2 N N
Deoxy
guanosine
OH° O N N H O
DNA oxidation through
Free radicals H2 N N
8 oxo dG/8 OH dG 2

33. + Follicular fluid Oocyte OH° O2-° H2O2 Embryo Tubal fluid Tubal cells
mRNA storage
MnSOD
CuZnSOD
GPX
SOD, GPX
CuZnSOD
MnSOD
GPX
SOD GPX
Oocyte
OH°
O2-°
OH°
Vit. C
O2-°
MnSOD
H2O2 +
Embryo
Hypotaurine
CuZnSOD
GPX
Catalase
Tubal fluid
CSD
Tubal cells

34. H202 Production by different culture media AMPLEX RED + HORSERADISH PEROXIDASE (From Alvarez-Miguel et al. 2005)

35. Different pathways involved in DNA repair
Ones step
Excision repair system, NER, BER , MMR
30 proteins
Alkyl guanine
DNA
Alkyltransferase

36. 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 Human Mol. Genet
YM 2009

37. 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

38. 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-meG-Alkyltransferase,
ABH2 and ABH3: 1 MeAdenine and 3 Me Cytosine
Or NER: 04-methylThymine
Ménézo 2011

39. 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*

40. DNA repair pathways in the oocyte:
Highly quantitatively present
Repetitive
Finite capacity
Probably more than damage repairs in the first 24 hrs at the PN stage
Need to understand effectors acting on DNA repair during maturation
GH?

41. Antioxidants and fertility
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 41

42. 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

43. 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