| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2009: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2008: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2007: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
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| Research Abstract |
To elucidate molecular mechanisms underlying oogenesis and oocyte quality, we first employed in silico analysis of global gene expression profiling data and an EST frequency database, and selected genes that are specifically expressed in oocytes and preimplantation embryos. We further studied the differences of gene expression profiles (1) between oocytes from aged mice and those from young mice, and (2) between post-ovulatory aged oocytes and fresh oocytes to extract genes that are important for oocyte quality. Finally, we focused on two oocyte-specific gene families (Nlrp [NACHT, leucine-rich repeat and PYD containing] genes and KRAB [Kruppel-associated box] zinc finger genes).
As results of quantitative RT-PCR and in situ hybridization assays, we confirmed oocyte-specific expression of the Nlrp and the KRAB genes at the follicle stages later than the primary-follicle stage. To analyze gene function of the Nlrp genes, we used the RNA interference approach : injection with siRNAs into
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mouse fertilized eggs. The knockdown of a Nlrp gene lead to cleavage arrest during preimplantation development. On the other hand, we are now in the process of generating knockout mice for an oocyte-specific KRAB zinc finger gene.
In addition, we study a preimplantation-stage-specific gene Hmgpi which is a putative chromosomal protein with two high-mobility-group boxes. Hmgpi is zygotically transcribed during zygotic genome activation, but not transcribed after implantation. The Hmgpi-encoded protein (HMGPI), first detected at the 4-cell stage, remains highly expressed in pre-implantation embryos. Interestingly, HMGPI is expressed in both the inner cell mass (ICM) and the trophectoderm, and translocated from cytoplasm to nuclei at the blastocyst stage, indicating differential spatial requirements before and after the blastocyst stage. siRNA (siHmgpi)-induced reduction of Hmgpi transcript levels caused developmental loss of preimplantation embryos and implantation failures. Furthermore, reduction of Hmgpi prevented blastocyst outgrowth leading to generation of embryonic stem cells. The siHmgpi-injected embryos also lost ICM and trophectoderm integrity, demarcated by reduced expressions of Oct4, Nanog and Cdx2. The findings implicated an important role for Hmgpi at the earliest stages of mammalian embryonic development. Less
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