Foundational questions Oocyte-derived functional mediators of early embryonic development (EST and candidate gene) JY-1 Nobox Importin 8 Oocyte and cu
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1 Models for study of oocyte competence: George W. Smith
2 Foundational questions Oocyte-derived functional mediators of early embryonic development (EST and candidate gene) JY-1 Nobox Importin 8 Oocyte and cumulus cell gene expression signatures associated with egg quality Functional significance Diagnostic and therapeutic potential Bovine, rhesus and human
3 Oocyte regulation of follicular development Follicle formation Proliferation Differentiation Steroid production Cumulus expansion Ovulation AA transport, Cholesterol synthesis Energy metabolism (Eppig et al., 2008)
4 Species specific oocyte regulation of the maternal-to to-embryonic transition Polyovulatory mouse 1 st PB. Sperm 2 nd PB Zar1 Mater Fertilization Zygote Embryonic genome activation Blastocyst Monoovulatory human (4-cell) Monoovulatory cattle (8-cell) (Tong et al., 2000; Wu et al., 2003)
5 Oocyte quality and success of ART 2007 Report: Assisted Reproductive Technology Success Rates
6 Oocyte quality (competence) Ability to resume meiosis Ability to cleave following fertilization Ability to develop to blastocyst stage Ability to induce pregnancy and bring to term Ability to develop to term in good health (Sirard et al., 2006)
7 Criteria for classification of oocyte competence (quality) Size Follicle Color Cumulus cells Cytoplasm Failure rate (ART) ~70%
8 Overall hypothesis: Characteristics ti of the RNA transcript t profiles of the oocyte and adjacent cumulus cells are reflective of competence of the oocyte
9 Approach RNA transcript profiling eggs (bovine) and cumulus (bovine and human) Data analysis (genes, gene families & pathways) Functional significance of putative markers Ablation replacement and pharmacological (bovine and rhesus) Predictive value of putative markers Cumulus cell assay (cow and human) Therapeutic potential (bovine and rhesus)
10 Advantages of bovine model system Similarities with human Oocyte size Embryo developmental kinetics Availability of large # s of oocytes Reliable protocols for in vitro maturation, fertilization and culture Multiple well-characterized experimental models of poor oocyte competence
11 Functional genomics studies of bovine oocyte competence Differences in oocyte transcriptome profile associated with egg quality (Patel et al., 2007) follistatin Differences in cumulus cell transcriptome associated with egg quality (Bettegowda et al., 2008) cathepsins
12 Experimental approaches Functional role and therapeutic potential
13 Effect of cathepsin inhibitor treatment during in vitro maturation on early embryonic development % Blasto ocyst rate Parthenogenetic embryos 40 b 30 a b e % Blast tocyst rat a IVF embryos b b 0 0 (1 M) (10 M) 0 0 (1 M) (10 M) cathepsin inhibitor cathepsin inhibitor (Bettegowda et al., 2008)
14 Potential mechanism of action of cathepsins in influencing oocyte competence Bright field Apoptotic Total Control 1 µm inhibitor 10 µm inhibitor Positive control Negative control (Bettegowda et al., 2008)
15 Zygote sirna injection: Ablation/replacement studies in early embryos c astocysts s (d 7) % bl a b a 0 Uninjected Follistatin sirna Without follistatin Uninjected With follistatin Follistatin sirna (Lee et al., 2009)
16 Zygote sirna injection: Ablation/replacement studies in early embryos # of ce ells a Uninjected Trophectoderm cells b Follistatin sirna c Uninjected a Follistatin sirna ells # of ce a Uninjected b Follistatin sirna Total cells c Uninjected a Follistatin sirna Without follistatin With follistatin Without follistatin With follistatin (Lee et al., 2009)
17 Oocyte sirna injection: ablation/replacement studies of egg maturation and cumulus expansion Microinjection of cumulus enclosed GV oocytes JY-1 sirna sham and/or negative control sirna and uninjected (control) JY-1 sirna rescue experiment (+/-) JY-1 protein COCs Maintenance of meiotic arrest for 48 h in maturation medium with 50 μm MS-roscovitine Confirm GV arrest In vitro maturation for 24 h in absence of S- roscovitine Levels of JY-1 mrna and protein Nuclear maturation Cumulus expansion In vitro fertilization and embryo culture (72 h) Same developmental endpoints Early cleavage, total cleavage, development to 8-16 cell and blastocyst stages (Wee et al., unpublished)
18 Translational studies in rhesus monkey Cathi VandeVoort California National Primate Research Center
19 Follistatin effects on rhesus monkey early embryonic development % blastocy ysts ( d 7) a Control b Follistatin (10 ng/ml) (VandeVoort et al., 2009) (P < 0.05)
20 Human clinical studies Jose Cibelli Michigan State university
21 Can factors produced by cumulus cells influence the quality of the oocyte and/or embryo in women? Gene expression signature associated with successful pregnancy
22 RNA Arrives to Laboratory Egg-RNA #1 Gene expression analysis Ranked results are Sent to IVF clinic Egg# Egg-RNA #2 Egg-RNA #3 Egg-RNA #11 Egg-RNA #12 Egg-RNA #13 Statistical Analysis Best Worst
23 Thank you!!!!!!!!
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