Studies On Cloning, Localization And Function Of The Maternal Genes Of GV Oocytes In The Mouse

Objective:During oocyte growth, the oocyte synthesizes and accumulates organelles and macromolecules(e.g., mitochondria, mRNAs, and proteins) that will constitute the maternal contribution to early development. Following fertilization these maternal components direct early development prior to zygotic genome activation (ZGA). ZGA must be initiated and successfully executed in order for further development to continue, and in fact, if this transition does not occur, the embryo will arrest development usually within one cleavage division. Oocyte maturation initiates the destruction of maternal mRNA and this destruction proceeds through the two-cell stage. In mice, embryonic transcription is first detected in the late one-cell zygote stage and broad embryonic genome activation occurs at the late two-cell stage. Hence, the oocyte-to-embryo transition depends on maternal transcripts and proteins that accumulate during oogenesis. Despite their many important functions, few mammalian maternal-effect genes have been identified.To clone and screen maternal genes of mouse GV-ooc)tes, we performed suppression subtractive hybridization (SSH) to construct subtractive cDNA library of maternal genes from GV-oocytes withGV-intact oocytes as tester and 8-cell embryos as driver, which would provide a foundation for my further researches to study the function of the maternal genes in oogenesis and early embryonic development in the mouse. Methods:1. Suppression subtractive hybridization(SSH) was performed to construct subtractive cDNA library of maternal genes from GV-oocytes with GV-intact oocytes as tester and 8-cell embryos as driver. Dot blot was carried out to further screen the subtractive cDNA library of maternal genes. The positive colonies were recovered, sequenced and compared with known genes in Genebank. RT-PCR using total RNA isolated from equal numbers of oocytes and 8-cell embryos was employed to comfirm the differential expression of the selected three genes ramdonly from the subtractive cDNA library of maternal genes.2. RT-PCR and sequencing were performed to analyze a cDNA fragment encoding of Peroxiredoxin II from Germinal Vesicle intact oocytes (GV oocytes). Mouse ovaries were fixed by incubation in 4% paraformalddhyde in phosphated-buffered saline (PBS) and embedded in paraffin. Paraffm-embeded ovaries were cut into 4um section. In situ hybridization and Immunohistochemistry were used to show the distribution and localization of Peroxiredoxin II mRNA and protein in the follicles at different stages. Moreover, RT-PCR was conducted to display Peroxiredoxin II mRNA in GV oocytes, MII eggs and Preimplantation embryos. Immunocytochemistry was carried out to observe the localization of Peroxiredoxin II in GV oocytes, MII eggs and preimplantation embryos.3. One-cell embryos collected from the oviduct of thesuperovulated mice were cultured in microdrops of medium for 48h. The effects of Peroxiredoxin II antibody on the development of embryo in vitro were observed, and the percentage of embryos developing to 2- and 4-cell stage of embryos was used as evalution criteria. With redox-sensitive fluorescene probe 2', 7'-dichloroflurescin diacete (DCFH-DA), reactive oxygen species (ROS) induced by Peroxiredoxin II antibody was monitored in mouse embryos after 24h culture by laser confocal scanning microscopy. Results:1. 40 positive colonies were recovered and sequenced after SSH and dot blot. Sequencing results showed that eighteen genes and eight homologous ESTs were expressed specifically in GV-oocytes. We need further studies for the function of them in oogenesis and early embryonic development.2. RT-PCR and sequencing displayed that the cDNA from GV oocytes is 684 base pairs(bp) and possesses 99% identity with the mRNA encoding Peroxiredoxin II (Accession No: BC002034). The deduced amino acids sequence from the cDNA sequence is the same as Peroxiredoxin II protein(Accession NO: Q61171).3. In situ hybridization of ovary section revealed distinct signals for P...