B. SIGNIFICANCE Oocyte maturation and the transition from differentiated germ cells to a totipotent one-cell fertilized zygote mark the beginning of the development of a metazoan. Understanding the earliest stages of development, particularly the oocyte-to-embryo transition, is of fundamental importance to reproductive medicine. Defects in human germ cells and preimplantation embryos lead to clinical infertility, miscarriage, and potentially birth defects as well. Our findings, along with methodologies that are developed, will provide robust, functionally-rich, and annotated datasets that will not only be valuable resources for the reproductive medicine/biology communities, but will also serve to bring reproductive biology to the forefront of the broad scientific audience. A. BACKGROUND AND SPECIFIC AIMS Most, if not all, of the mRNAs and proteins required for the initial development of the mammalian embryo come from the oocyte, the female germ cell. The fertilized, one-cell zygote is thought to be transcriptionally silent. Maternal factors support and direct the earliest stages of development, including the first mitotic cell cycle(s) and nuclear reprogramming, at least until the embryonic genome is activated. The precise timing of embryonic genome activation (EGA) varies among mammalian species. In humans, EGA initiates at the 4- to 8-cell stages, while in the mouse, EGA occurs by the end of the 2-cell stage[1]. Since there is no or minimal embryonic transcription prior to EGA, the embryo relies on various posttranscriptional mechanisms for initial development. In particular, previous work has indicated that control of mRNA translation may be crucial for the oocyte-to-embryo transition. In the growing oocyte, some mRNAs are deadenylated and stored in the ooplasm for later translation. The deadenylated mRNAs of immature oocytes are bound and kept silent by protein complexes containing inactive CPEB, which is a highly conserved RNA-binding protein that promotes elongation of the polyadenine tail of mRNAs, the 5'cap-binding factor elF4E, and maskin, which is a protein that interacts with and prevents elF4E from recruiting other 5'cap elements necessary for translation initiation. Upon oocyte maturation, a series of molecular events results in the addition of several hundred adenine residues to the 3'tail and dissociation of maskin from elF4E to allow for translation. At least two c/s-elements are necessary for this process, namely the nuclear polyadenylation signal and the cytoplasmic polyadenylation element (CPE). An unusually large fraction of transcripts in the two-cell embryo contains CPE sequences in their 3'UTRs[2]. Understanding post-transcriptional and translational control during the oocyte-to-embryo transition is an important and new direction in our laboratory's research. We recently discovered that the master transcription factor Oct4 may function to switch the early embryonic developmental program from one that is dependent on post-transcriptional control to one that is predominantly regulated by a transcriptional network[3]. This potential new role of Oct4 is distinct from its well established and critical function to regulate pluripotency in embryonic stem cells and to reprogram somatic cells into a more pluripotent state. When we knocked down Oct4 by injecting morpholino oligonucleotides (MOs), which could simultaneously silence both maternal and embryonic transcripts rapidly, into the one-cell zygote, we found that most of the embryos arrested by the multicell stage. Significantly, we found that Oct4-regulated genes in the two-cell embryo were enriched for translation (e.g. eukaryotic translation initiation factors (elFs) and elF4E2 or 4EHP, which inhibits translation by binding to the 5' cap but not to the elFs) and RNA processing functions. The Drosophila homolog of elF4E2, or d4EHP, may potentially interact with Rbp4, which is required for translational repression of cyclin B (see Project 3 from the Fuller laboratory). These observations suggest that post-transcriptional control is a highly conserved and critical mechanism for the oocyte-to-embryo transition. However, it remains poorly understood in mammalian models because most of the previous studies on mouse oocyte-to-embryo transition were based on a candidate gene approach and progressed considerably slowly. With the advent of genomics technologies, we can now begin to interrogate important biological questions in a high throughput manner. Hypothesis: We hypothesize that post-transcriptional and translational control of at least some genes is critical during the oocyte-to-embryo transition, and is controlled by Oct4, the master regulator of reprogramming and pluripotency. In the pilot project, we propose to adopt next generation sequencing (NGS) to understand the extent of post-transcriptional and translational control during the oocyte-to-embryo transition and to determine how much of this control is mediated by Oct4.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54HD068158-02
Application #
8377046
Study Section
Special Emphasis Panel (ZHD1-DSR-L)
Project Start
Project End
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
2
Fiscal Year
2012
Total Cost
$122,374
Indirect Cost
$50,427
Name
Stanford University
Department
Type
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Durruthy-Durruthy, Jens; Sebastiano, Vittorio; Wossidlo, Mark et al. (2016) The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming. Nat Genet 48:44-52
Ramathal, Cyril; Angulo, Benjamin; Sukhwani, Meena et al. (2015) DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs. Sci Rep 5:15041
Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T (2015) Cell type-specific translational repression of Cyclin B during meiosis in males. Development 142:3394-402
Briggs, Sharon F; Dominguez, Antonia A; Chavez, Shawn L et al. (2015) Single-Cell XIST Expression in Human Preimplantation Embryos and Newly Reprogrammed Female Induced Pluripotent Stem Cells. Stem Cells 33:1771-81
Cheng, Yuan; Feng, Yi; Jansson, Lina et al. (2015) Actin polymerization-enhancing drugs promote ovarian follicle growth mediated by the Hippo signaling effector YAP. FASEB J 29:2423-30
Cheng, Yuan; Kim, Jaehong; Li, Xiao Xiao et al. (2015) Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators. PLoS One 10:e0117769
Hsueh, Aaron J W; Kawamura, Kazuhiro; Cheng, Yuan et al. (2015) Intraovarian control of early folliculogenesis. Endocr Rev 36:1-24
Briggs, Sharon F; Reijo Pera, Renee A (2014) X chromosome inactivation: recent advances and a look forward. Curr Opin Genet Dev 28:78-82
Durruthy-Durruthy, Jens; Briggs, Sharon F; Awe, Jason et al. (2014) Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions. PLoS One 9:e94231
Durruthy Durruthy, Jens; Ramathal, Cyril; Sukhwani, Meena et al. (2014) Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules. Hum Mol Genet 23:3071-84

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