Early Embryogenesis Metabolism and Genes Imprinting

1. Early Embryogenesis Metabolism and Genes Imprinting Dr BenkhalifaMoncef (1, 2), PrMenezo Yves (1, 2) ATL R&D. Reproductive Biology & Genetics Laboratory. Paris. Eylau Laboratory/ UNILABS. Paris-Geneva

2. Imprinting and ART technology.??? Importance of pre-implantation embryo metabolism

3. Imprinting is a part ofbasic epigenetic mechanisms:DNA methylationhistone modificationchromatin remodelingRNA interference

4. Epigenesis/Epigenome Epigenesis: Changes in the genetic information not involving DNA sequence Modifications in gene expression - DNA methylation - Histone modification - Chromatin remodeling - RNA interference Nature 429:457, 2004

5. Genomic imprinting : Definition In mammals - Epigenetic ıs form of gene regulation. - Mono-allelic expression - Expression depends on the parental origin of the gene. - Genomes are complementary rather than equivalent. - Need to have one paternal genome and one maternal genome. - Parthenogenesis or androgenesis are impossible.

6. DNA methylation, gene silencing and links to diseases • 70à to 80% of the CpG sites are methylated in human • The 20% remaining are located in clusters: CpG islands • CpG islands represents 1% to 2% of the genome and contains 50% of the unmethylated cites • The estimated 29000 CpG islands in the genome are frequently found in the propoter regions of human genes either tissue specific or « house keeping »

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

8. Epigenetic Mark/ Imprint Genomic imprinting implies a differential marking of the two parental genomes : 1) Apposed during gametogenesis and early embryogenesis. 2) Specific. 3) Stable and transmissible. 4) Reversible, imprint resetting.

9. Genomic Imprinting and ART: Animal studies • Influence of the culture medium • - Large offspring syndrome LOS (sheep and cows) • Disturbance of gene profile expression of imprinted genes • H19, IGF2 Grb10, Grb7. • - Depend on the culture medium composition. • (Doherty et al 2000; Khosla et al 2001) • But…. • Care has to be taken of the animal models…

10. Genomic Imprinting • > 80 imprinted genes (0.1-1% of all genes) • Key role in embryonic growth and placental function, cognition and maternal behaviour • Defective imprinting involved in carcinogenesis and in human genetic diseases (Angelman, Prader-Willi and Beckwith-Wiedemann syndrome) • Maternal and paternal alleles carry different epigenetic modifications (“imprints”)

11. The importance of preimplantation embryo metabolism in relation with ART

12. Pre-embryo metabolism & Cuture medium,impact on Imprinting ???

13. Composition of culture medium impacts on: genome activation & regulation 100 Timing and quality of maternal to zygotic transition/transcription Regulation of mRNA Polyadenylation R 80 mRNA (ng/embryo) 60 Maternal mRNA Embryonic mRNA 40 20 YX Hrs 25 40 50 60 70 90 110 140 Fertilization M 8 16 Bl Stage 2 4 Evolution of maternal and embryonic mRNA (human)

14. + - - + + + + Growth factors and preimplantation development EGF TNFa TGFß ** GH increases DNA repair capacity Compacted morula 8-cell Blastocyst Hatching EGF, LIF (?) TGFß, PAF TGFß*, bFGF Insuline, EGF PDGF (c-myc), GH, PAF TGFß*, LIF (?) Insuline, PDGF (c-myc) TGFß*, bFGF Insuline, GH * Hyaluronic acid may act in synergy ; Role of Interleukines?

15. Imprint resetting Female Embryo P P M M P M P M Male DNA methylation patterns established during gametogenesis are largely erased during early embryogenesis and reestablished after implantation But….specific DNA/histone methylation occurs at fertilization SPERM OOCYTE

16. GenomicImprinting/ DNA methylation/ DNA methyl transferase Non-Imprinted genes Bi-allelic expression Imprinted genes Mono-allelic expression M P M P M P Maternally expressed Paternally expressed DNA methylation 5-methyl cytosine CH3 5 Methyl Cytosine

17. Embryo culture and gene expression (Mouse) • Microarray analysis • Culture from 1 cell to blastocyst, control with in vivo blastocysts Expression of 114 genes is affected when culture is performed in Whitten’s medium (No AA) 29 genes are mis expressed after culture in KSOM +AA MOUSE model for HUMAN ???? Rinaudo and Schultz 2004

18. Methionine and imprinting: A careful interpretation of the results observed on animal models is mandatory!!!! • In Human: Normal maternal methylation imprints in 15q11-q13 (involved in PWS) are established during or after fertilization. • Methylation in PW1-C occurs during oogenesis in the mouse (El-Maarri et al. 2001) MOUSE model for HUMAN??

19. Methylation and gene expression In mouse embryos, silent paternal alleles of H19, Igf2, Grb10 and Grb7 are aberrantly expressed and hypomethylated in simplex media but not media with AA (Koshla et al. 2001)

20. Association between Beckwith Wideman syndrom and ART (case series of 19 patients )Chang et al. Fertil Steril 2005) Absence of impact of the culture medium*?? Incorrect interpretation In common: No methionine + Maternal Age MetEndogenous pool regulation and maternal age Correlation between oocyte quality and environmental factors (culture medium) * 4 cases in P1 +/- SSR, 1 case in HTF + SSR, 3 cases in G1

21. Methionine and imprinting • The methionine content is the critical difference wich can affect DNA methylation and imprinting (Wolff et al. 1998, Waterland and Jirtle 2003, Niemitz and Feinberg 2004) • Silencing of CDKN1C is associated with hypomethylation at KvDMR1 in Beckwith-Wiedeman syndrom (Diaz-meyer et al 2003)

22. Beckwith-Wiedemann Syndrome BWS: 1/15 000 • 11p15 locus, cluster of genes : • IGF2; LIT1 (paternal expression) • H19; p57KIP2; KvLQT1 (maternal expression) • - Overgrowth disorder : Macrosomia, Macroglossia, • Organomegaly • midline abdominal defects (Omphalcele/exomphalos) • - Predisposition to nephroblastoma (Wilms tumor).

23. Histone methylation and imprinting Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting . Xin Z et al 2003 J Biol Chem. Histone H3-K9 Methyltransferase ASET is essential for early development Dodge JE et al 2004 Mol Cell Biol, “Methylation of histone H3 at lysine 9 (H3-K9) mediates heterochromatin formation by forming a binding site for HP1 and also participates in silencing gene expression at euchromatic sites 

24. Prader-Willi Syndrome PWS/ 1/30 000 • Paternal deletion or maternal disomy of the • 15q11- q13 locus; cluster of genes paternally expressed: • SNRPN; IPW; PAR1; PAR5; PAR7… • - Hypotonia infantile, • - Mental retardation (moderate), • - Short stature, • Behavioural troubles • (hyperphagia), • - hypogonadism.

25. Essential aminoacids Isoleucine, leucine, lysine, methionine, phenylalanine, threonine,tryptophan and valine Out of the 8 « essential aminoacids » Methionine Care has to be taken for culture media G1/G2 , Cook media, Blastassist

26. « Essential aminoacids »(Waymouth 1965)The terms « essential » and « non-essential » aminoacids, rather commonly used, are not, therefore, strict designations and should be accepted with scepticism unless carefully defined, in a particular context…. Menezo 2006

27. 4 Amonia detoxification Methionine 1 Methionyl mRNA (starts protein synthesis) Transsulfuration Pathway 2 3 GLUCOSE CysteineGluthation Oxydative Stress ROS DNA, Lipid (freezing) Peroxides Me APOPTOSE SAM Imprinting DNA Metransferase OverMethylation (alkylation)

28. Angelman Syndrome 1/10 000 1/30 000 • Maternal deletion or paternal disomy of the • 15q11- q13 locus; one gene UBE3A • maternally expressed • Severe mental retardation, • Characteristic facial feature • - motor defects, • - Behavioural troubles (Happiness),

29. Endogenous pool and aminoacids in the culture medium Pivotal Role of Methionine « Essential aminoacids ?» : aberrant approach in some culture media The absence of Met and other sulfur aminoacid is dangerous. It may increase imprinting problems: Niemitz and Feinberg (Am. J Human Genetics 2004), Summers and Biggers (Human Reprod Update 2003) Sulfur aminoacids involved in Imprinting and prevention of apoptosis are of major importance Homocysteine in the absence of folic acid induces apoptosis in trophoblast (De Simone et al. 2004)

30. 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 • Mitochondrial function, oxidative stress • Chromatin structure, DNA methylation, genome stability Hanatani et al. 2004 Human Mol. Genet

31. Conclusion: In ART procedure…. The « misfit » between embryo needs and culture medium will lead to developmental problems and apoptosis, especially in aged patients where the endogenous metabolic pool is border line, in different procedures of controlled ovarian stimulation protocols and in specific in vitro culture condition with defined media ( importance of methyl donor )

32. Dr I Giakoumakis & The laboratories team Mediterranean IVF & Genetics Center. Creta. Greece Dr Demirol A, Pr Gurgan T & the embryology Team Women’s Health Clinic. IVF & Genetics Centre. Ankara. Turkey