The Preliminary Study Of The Storage And Release Of Cyclin B1 MRNA In Mouse Oocyte

Processing bodies(P-bodies) are distinct foci within the cytoplasm of the eukaryotic cell consisting of many enzymes involved in mRNA turnover. P-bodies have been observed in somatic cells originating from vertebrates and invertebrates, plants and yeast. Fully grown mouse oocytes in Graafian follicles are transcriptionally silent during the period before the resumption of meiosis until after fertilization when most of the transcriptional reactivation occurs at the 2-cell stage. However, the maturation of oocytes needs a lot of proteins which are mainly generated from the translation of maternally stored mRNA. During the maturation of oocytes, different gene transcriptions expressed at specific time and precise regulation are vital. On the contrary, transcriptional regulation is not exist during the maturation of oocytes. Post-transcription regulation is the only way to regulate one gene. Metastatic Lymph Node 51(MLN51) is a core component of the exon junction complex(EJC), which is loaded on spliced mRNAs and plays an essential role in their fate. When overexpressed, MLN51 triggers P-body disassembly and mRNAs are released to the cytoplasm.Here, we review structure and function of P-bodies and stress granules, discuss the function of P-bodies and their similar granules in mouse oocyte maturation, come up with suppostion about how mRNAs are stored in P-bodies like granules and how mRNAs released from these granules, put forward that oocytes are novel model for the research of post-transcriptional regulation.Cyclin B1 is a regulatory protein involved in mitosis and meiosis. CyclinB1 plays an important role in oocyte maturation, it expresses highly in meiosis metaphase and expresses lowly in meiosis anaphase. It means it expresses highly twice in the whole maturation of oocyte. So it is of great value to do some research in CyclinB1 mRNA. So, we make several variants of CyclinB1 to detect their function in mRNA regulation and to figure out whether they can rebuilt P-body in oocyte.In this research, we use fluorescence in situ hybridization to detect CyclinB1 mRNA location in mouse P19 cells. We then investigate the function of MLN51, CyclinB1 and CyclinB1 variants to the maturation of oocyte. First of all, we build a full-sized mouse MLN51 and CyclinB1 eukaryotic expression vector :pVenus-MLN51 and pVenus-CyclinB1. We then use pVenus-CyclinB1 to build CyclinB1 variant eukaryotic expression vector: pVenus-D90-CyclinB1、pVenus-Y167a-CyclinB1 and pVenus-D90-Y167a-CyclinB1. All the eukaryotic expression vectors can be expressed in Hela after transfection. Using fluorescence in situ hybridization kit from Wuhan Boster company, we detect red fluorescence in the Cytoplasm of oocyte in mouse P19 cells. Using in vitro transcription, we get the cRNA of MLN51, CyclinB1 and CyclinB1 variants. We then microinject those cRNA into oocyte which have cultured for one hour. We observe the location of extraneous proteins and we calculate germinal vesicle breakdown rate and first polar exclusion rate of microinjected oocytes.