Extra follicular oocytes spontaneously resume meiosis in vitro, but the intact germinal vesicle (GV) is retained if the oocytes are cultured in medium containing phosphodiesterase (PDE) inhibitors or cAMP analogues. On the basis of our finding that Obox4 is prominently expressed in oocytes, the present study was conducted to determine the functional role of the homeodomain-containing factor Obox4 during in vitro oocyte maturation. After microinjection of Obox4 dsRNA into the cytoplasm of GV oocytes cultured in M16 medium, oocytes were arrested at metaphase I (MI, 77.7%) and metaphase II (MII, 22.3%). Surprisingly, however, 89% of Obox4 RNAi-treated oocytes resumed meiosis and developed to MI and MII when cultured in medium containing 0.2 mM 3-isobutyl-1-methyl-xanthine (IBMX), in which untreated oocytes maintain intact GVs. Spindles were aberrant, and chromosomes were severely aggregated with decreased MPF and MAP kinase activities in arrested MI oocytes after exposure to Obox4 RNAi. Oocytes overexpressing Obox4 retained intact GVs when cultured in M16 medium. Taken together, for the first time to our knowledge, these findings indicate that Obox4 plays a key role in the cAMP-dependent signaling cascades that maintain GV arrest. Oocytes not expressing Obox4 failed to maintain intact GVs in IBMX-supplemented medium, while GVs remained intact when oocytes were kept in plain medium and overexpressing Obox4, suggesting that Obox4 plays a critical role in cAMP-dependent cascade for maintaining intact GVs.
"To determine the role played by target genes in oocyte maturation and preimplantation embryo development, RNAi was used to knockdown target gene expression in the mouse. While we carried out RNAi for several genes, we could categorize transcripts in oocytes into three groups, with loss-of-function stopping meiosis at GV , MI [91,92,93,94,95], and MII [2,3]. Therefore, transcripts in oocytes could be categorized as genes required for embryonic development and for oogenesis, as suggested by Dean . "
[Show abstract][Hide abstract] ABSTRACT: Stored maternal factors in oocytes regulate oocyte differentiation into embryos during early embryonic development. Before zygotic gene activation (ZGA), these early embryos are mainly dependent on maternal factors for survival, such as macromolecules and subcellular organelles in oocytes. The genes encoding these essential maternal products are referred to as maternal effect genes (MEGs). MEGs accumulate maternal factors during oogenesis and enable ZGA, progression of early embryo development, and the initial establishment of embryonic cell lineages. Disruption of MEGs results in defective embryogenesis. Despite their important functions, only a few mammalian MEGs have been identified. In this review we summarize the roles of known MEGs in mouse fertility, with a particular emphasis on oocytes and early embryonic development. An increased knowledge of the working mechanism of MEGs could ultimately provide a means to regulate oocyte maturation and subsequent early embryonic development.
Clinical and Experimental Reproductive Medicine 06/2014; 41(2):47-61. DOI:10.5653/cerm.2014.41.2.47
"We used a GFP fusion expression system to investigate the subcellular localization of Obox4. As we previously reported, Obox4 is specifically expressed in mouse oocytes and plays important roles in oocyte maturation . Therefore, further studies are required to define the minimal and fully functional nuclear localization signal of Obox4 and other cellular localization signals such as nuclear export signals to understand the multiple roles of Obox4 in cells and eventually in oocytes in particular. "
[Show abstract][Hide abstract] ABSTRACT: Oocyte-specific homeobox 4 (Obox4) is preferentially expressed in oocytes and plays an important role in the completion of meiosis of oocytes. However, the Obox4 expression pattern has not been reported yet. In this study, we investigated the subcellular localization of Obox4 using a green fluorescent protein (GFP) fusion expression system.
Three regions of Obox4 were divided and fused to the GFP expression vector. The partly deleted homeodomain (HD) regions of Obox4 were also fused to the GFP expression vector. The recombinant vectors were transfected into HEK-293T cells plated onto coated glass coverslips. The transfected cells were stained with 4',6-diamidino-2-phenylindol and photographed using a fluorescence microscope.
Mutants containing the HD region as well as full-length Obox4 were clearly localized to the nucleus. In contrast, the other mutants of either the N-terminal or C-terminal region without HD had impaired nuclear localization. We also found that the N-terminal and C-terminal of the Obox HD contributed to nuclear localization and the entire HD was necessary for nuclear localization of Obox4.
Based on the results of the present study, we demonstrated that the intact HD region of Obox4 is responsible for the nuclear localization of Obox4 protein in cells.
Clinical and Experimental Reproductive Medicine 03/2013; 40(1):1-6. DOI:10.5653/cerm.2013.40.1.1
"The Obox family consists of 6 members and is exclusively expressed in the ovary and testes [19-21]. Kim et al.  reported that the Obox family is expressed in mESCs and Obox4 regulates histone family gene expression. "
[Show abstract][Hide abstract] ABSTRACT: Lin28 has been known to control the proliferation and pluripotency of embryonic stem cells. The purpose of this study was to determine the downstream effectors of Lin28 in mouse embryonic stem cells (mESCs) by RNA interference and microarray analysis.
The control siRNA and Lin28 siRNA (Dharmacon) were transfected into mESCs. Total RNA was prepared from each type of transfected mESC and subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis to confirm the downregulation of Lin28. The RNAs were labeled and hybridized with an Affymetrix Gene-Chip Mouse Genome 430 2.0 array. The data analysis was accomplished by GenPlex 3.0 software. The expression levels of selected genes were confirmed by quantitative real-time RT-PCR.
According to the statistical analysis of the cDNA microarray, a total of 500 genes were altered in Lin28-downregulated mESCs (up-regulated, 384; down-regulated, 116). After differentially expressed gene filtering, 31 genes were selected as candidate genes regulated by Lin28 downregulation. Among them, neuropeptide Y5 receptor and oocyte-specific homeobox 5 genes were significantly upregulated in Lin28-downregulated mESCs. We also showed that the families of neuropeptide Y receptor (Npyr) and oocyte-specific homeobox (Obox) genes were upregulated by downregulation of Lin28.
Based on the results of this study, we suggest that Lin28 controls the characteristics of mESCs through the regulation of effectors such as the Npyr and Obox families.
Clinical and Experimental Reproductive Medicine 06/2012; 39(2):87-93. DOI:10.5653/cerm.2012.39.2.87
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