The Role Of Btg4 In Oogenesis And Early Mammalian Embryogenesis

Oocyte as the largest cell in mammals provides half of the nuclear genetic material and nearly all membrane and cytoplasmic determinants, which are needed in oogenesis and early embryogenesis. There is a hiatus between oocyte and embryonic gene transcription-from 12h before ovulation to the 2-cell stage. Transition from meiosis to mitosis, which is one of most dramatic and complicated cell transformations, occurs during this period. During this period, female and male gametes fuse to form a zygote and then switches to mitosis. The mRNAs and proteins, which are needed in these physiological events, are provided by oocytes. Thus, mRNAs and proteins in oocyte cytoplasm plays vital role in meiosis, fertilization and early embryogenesis. Maternal effect genes were not described in mammals until 2000 and they play vital role in early embryonic development. For example, deletion of Hsfl(a maternal effect gene) in mouse oocytes leads to embryos arrest in the zygote stage. And deletion of another maternal effect gene-Mater in mouse oocytes leads to embryos arrest in the 2-cell stage. Fourteen maternal effect genes were identified by traditional gene knockout animals. And then, other fifteen maternal effect genes were found by RNAi and conditional gene knockout methods. Although these maternal effect genes are very important in embryonic development, there is no comprehensive and specific molecular mechanisms and signaling pathways which can explain working mechanisms of these maternal effect genes. Thus, we aim to clarify functions of maternal effect genes and obtained Btg4-/-mouse through TALEN-a gene knockout method. And Btg4-/-mouse is female sterility. The result of histomorphology shows that oogenesis in ovary is normal. Though oocyte in vitro culture、MII oocyte immunofluorescence and oocyte chromosome spreading, we found that Btg4-/-oocytes would resume meiosis and arrest in the MII stage. And Btg4-/- oocytes could be ovulated normally. And then, we collected embryos of different stages. The result shows that deletion of Btg4 in mouse oocytes leads to embryos arrest in the zygote stage. The result of zygotic immunofluorescence shows that there are defects in pronucleus nuclear envelope breakdown and meiosis to mitosis transition. Realtime result shows that mRNAs of zygotes came from Btg4-/-oocytes are more stable than WT zygotes. And the result of RNA sequencing confirms the result of Realtime. Through IP assay, we confirm that BTG4 could interact with CNOT7, which is a important component of CCR4-NOT complex. And BTG4 could enhance deadenylation activity of CNOT7. Thus, we obetain result is that:1. Btg4 is a typical maternal effect gene, and Btg4-/-mouse is female sterility.2. Deletion of Btg4 in mouse oocytes would no effect oogenesis, meiosis and fertilization.3. Deletion of Btg4 in oocytes would leads to embryos arrest in the zygote stage and pronuleus envelope fail to break down.4. BTG4 facilitates maternal mRNAs decay in through CCR4-NOT complex. This paper has a great importance in biology. It finds a new maternal gene and discovers the molecular mechanism it involved. And it finds a new maternal mRNA decay pathway in mammals.