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

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

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)

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

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

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…..

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

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 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

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

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)

Effect of methionine on incorporation and metabolism of glycine

SAM, S Adenosyl methionine: Methyl donor for imprinting CH3

SAH Homocysteine H

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 250 9 (3.6%) Early Morula 350 12 (3.4%) Compact. Morula 650 33 (5.1%) Blastocyst 2335 41 (1.8%) Human 4-cell 770 26.2 (3.4) Menezo et al 1989 Ménézo et al.1989 1

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

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

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

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

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

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

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

Apoptosis Oxidative stress DNA repair

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)

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

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

Different types of DNA decays

DNA Oxidation products

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

+ 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

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

Different pathways involved in DNA repair 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-meG-Alkyltransferase, ABH2 and ABH3: 1 MeAdenine and 3 Me Cytosine Or NER: 04-methylThymine Ménézo 2011

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 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

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