Epigenetics, Inheritance and Assisted Reproduction Technology Keith E. Latham

1. Epigenetics, Inheritance and Assisted Reproduction Technology Keith E. Latham Department of Animal Science MICHIGAN STATE UNIVERSITY

2. Oogenesis Fertilization Demethylation Genome Activation Metabolic Meioisis Meiosis Compl. Reprogramming Reprogramming Programming Mat. mRNA Transl. Prevent Apoptosis Zygotic Imprint modifications Epigenetic changes Genetic Factors Growth Differentiation Disease Reproduction Environment (gamete, prenatal, postnatal) Epigenetic Processes How might clinical procedures affect development?

3. Epigenetic modifications are changed by the ooplasm after fertilization Different strain oocytes modify pronuclei differently, leading to different phenotypes in uniparental embryos One study suggested that inter-strain nucleocytoplasmic hybrids display growth defects and malformations Clinical procedures (cytoplasm transfer and GV transfer) result in nucleocytoplasmic hybrids. What is epigenetic risk? Ooplasm Manipulation Studies

4. Negligible effect of Inter-strain oocyte cytoplasm transfer on growth in mice Female Transfer 10% volume of cytoplasm from one strain egg to another Male (Yong Cheng et al 2009)

5. Sign. Decrease in growth rate Female Effect of Germinal Vesicle Transfer(Yong Cheng et al 2009) GVT IVM, ICSI D2 sperm BBD DBD Male Females are affected; Males not affected; Transgenerational Effects?

6. Cytoplasm Manipulation Effects • Cytoplasm transfer was not associated with pronounced growth deficiency or changes in Mup or imprinted gene methylation, • Other studies indicate cytoplasm transfer can affect paternal genome function (not shown) • GVT yielded growth deficiency in females, but no difference in Mup or imprinted gene metehylation, or expression of Rasgfr1, Igf2r, or Mest; Growth deficiency seen at week 1, so unlikely to be related to GH deficiency

7. Mitochondria (MT) Aspects of Cloning and Embryo Studies • Donor MT can persist (Han et al., 2004; Do et al., 2002; Chen et al., 2002; Shitara et al., 2000; ) • Donor MT can increase in abundance, showing a replicative advantage (Takeda et al., 2003) • Donor MT may not be uniformly distributed • Heterotypic MT combinations in SCNT can be disadvantage (Yan et al., 2010) • Oocyte MT haplotype can affect bovine embryo development (Jiao et al., 2007)

8. Mitochondria (MT) Aspects of Cloning, Oocyte and Embryo Studies • MT mRNA expression correlates with nonhuman primate oocyte quality (Mtango et al., 2008) • MT impairments commonly seen in low quality oocytes (Eichenlaub-Ritter, 2012) • Inter-species MT transfer can be disadvantageous (Takeda et al., 2012) • Serum-starved MT can inhibit parthenote development (Takeda et al., 2010)

9. Outcomes from Mitochondria microinjection or other procedures • Granulosa cell MT can enhance development and blastocyst parameters of bovine embryos from poor quality oocytes (Hua et al., 2007) • MT injection can rescue EtBr treated embryos (Chiaratti et al., 2011) • MT injection can improve oocytes from aged mice (Kujjo et al., 2013) • Human pregnancy (46 y old patient) established after MT injection, but failed development to term (Kong et al., 2003) • Twins reported born to 37 y old patient after autologous MT transfer (Kong et al., 2003) • Human spindle transfer resulted in abnormal fertilization events, but some normal blastocysts and ES cells (Tachibana et al., 2013)

10. Adultbody weightisprogrammed by a redox-regulated and energy-dependentprocessduring the pronuclear stage in mouse. • Banrezes B, Sainte-Beuve T, Canon E, Schultz RM, Cancela J, Ozil JP. • PLoSOne. 2011;6(12):e29388. • Exogenous pyruvate induces NAD(P)H oxidation and stimulates mitochondrial activity with resulting offspring that are persistently and significantly smaller than controls. • Exogenous lactate stimulates NAD(+) reduction and impairs mitochondrial activity, and produces offspring that are smaller than controls at weaning but catch up after weaning. • Cytosolic alkalization increases NAD(P)(+) reduction and offspring of normal birth-weight become significantly and persistently larger than controls. • These results constitute the first report that post-natal growth rate is ultimately linked to modulation of NAD(P)H and FAD(2+) concentration as early as the PN stage. How might MT manipulation or other manipulations affect REDOX state?

11. Summary of points to bear in mind • Oocytes display genetic differences in composition • Maternal genome and ooplasm must be compatible • Paternal genome modification variable • Early stress to embryo leads to changes in growth and physiology • Alterations in REDOX state lead to long-term phenotypic (epigenetic?) effects (e.g., growth) • MT localization in zygote correlates with developmental potential and can influence subsequent partitioning to daughter cells

12. Questions to be addressed • How might somatic MT injection affect REDOX state, epigenetic state, phenotype? • Enhanced preimplantation development may not coincide with favorable adult phenotype • What is required match between MT and nuclear haplotypes? • What is impact of patient’s MT haplotype and how to manage it? • How “pure” is the MT preparation being used? • What other factors are co-transferred with MT? • What is risk of epigenetic modification or incompatibility? • How predictive are data from parthenote studies or studies limited to blastocyst characteristics as end points? • Efficiency and cost concerns? Animal models can provide answers to many of these questions. Multiple models may be required.

13. Animal modeling: • Can track long-term phenotypic consequences; particularly for organisms with shorter life cycle/span • Can track trans-generational effects more readily • Can deliberately vary MT-nuclear genetic combinations • Can undertake molecular analysis of oocytes and embryos before and after manipulation • Can use high quality embryos for diverse studies • Can collect fetal and post-natal tissues to assess epigenetic effects • Genetic tools (e.g., mouse) can eliminate certain experimental barriers • Can use multiple models to address differences in reproductive physiology