Research in my laboratory aims at: 1) understanding the cellular and molecular mechanisms underlying mammalian oocyte meiosis, activation, and aging; 2) discovering novel genes, factors, and signaling pathways that are functionally required during mammalian preimplantation embryo development; 3) creating animal models of human genetic diseases using knock-out, knock-in, and transgenesis strategies.

Mammalian oocytes enter meiosis during fetal development, and arrest at diplotene stage of meiotic prophase I - also called germinal vesicle (GV) stage, until puberty. During this prolonged arrest, oocytes grow, differentiate and stockpile maternal components. Following puberty, oocytes resume meiosis under the action of gonadotropin hormones. For most mammals, after going through germinal vesicle breakdown (GVBD), metaphase I (MI), as well as anaphase/telophase I, oocyte extrudes the first polar body and reaches metaphase II (MII). The matured MII oocyte is then ovulated and is ready for fertilization. In some cases, if fertilization or parthenogenetic activation cannot take place right away, oocyte will arrest at MII stage but postovulatory aging then occurs. However, in some species (rat, hamster, some mouse strains, and some patients), spontaneous exit from MII arrest - also named spontaneous activation, can occur quickly in vitro and/or in vivo. A tremendous investment and complicated network (cell-cell interactions, hormones, second messenger molecules, cell cycle regulators, organelle and cytoskeletal systems, transcription and translation factors, ion channels, etc.) has been generated by the body to ensure a high quality oocyte, however, this machinery is also being challenged by environmental exposures, diet, lifestyle, and age. Thus, only understanding the full developmental network, specifically under current challenging situations, can guide our management of human reproduction, including assisted reproductive technology (ART).  

Preimplantation development refers to the period from fertilization to implantation, during which the fertilized oocyte progresses through a number of cleavage divisions and major transcriptional and morphogenetic events that lead to the first cell-fate decision (inner cell mass [ICM] and trophectoderm [TE]) and development into a blastocyst capable of implantation. During this brief but dynamic time window, several events occur, which include: 1) maternal-to-zygotic transition (MZT), namely, degradation of maternal mRNAs and replacement with zygotic transcripts; 2) embryo compaction and polarization, which initiate during the 8-cell stage in mouse embryos; 3) blastomere allocation and ICM/TE separation, which is the very first cell fate determination of our life. Previous microarray and current RNA-seq both confirmed more than 10,000 genes expressed during this period; however, for most of these genes, we do not know their function. More importantly, this time window is right clinically relevant: after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), human preimplantation embryos need to be cultured in a dish and incubator for several days, then transferred into mother’s uterus. Thus, understanding the role of each expressed gene and what happens during the culture is an essential next step for elucidating developmental networks at play and improving human assisted reproduction.

There are multiple strategies to create genetically modified animal models. The earliest one is microinjection of foreign DNA into pronucleus to make transgenic animals. This method is straightforward (direct injection into 1-cell embryo); however, integration locus and copy numbers are random. When gene targeting technology is available, we can accurately modify specific locus of interest; but, due to the very low rate of homologous recombination, we have to rely on an excess of somatic cells or stem cells cultured in vitro, then, positive cells are selected to perform somatic cell nuclear transfer (SCNT) to get cloned animals, or through stem cell blastocoel injection to make chimera. In other words, these methods are effective but indirect and not straightforward, thus time consuming. When engineered nucleases (ZFN, TALEN, CRISPR/Cas9) are utilized, things become easier. These nucleases can accurately find precise locus of interest with high efficiency, so we can do direct microinjection into 1-cell embryos for directed gene editing. Compared with ZFN and TALEN, CRISPR/Cas9 system is easier to design, more flexible and cost effective. Currently, we are taking advantage of CRISPR/Cas9 system to perform gene editing. In addition to knock-out, knock-in and transgenic models, my laboratory also works on generation of patient-derived xenograft (PDX) cancer models.