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Stage Oocytes (stage + oocyte)
Selected AbstractsMolecular Reproduction & Development: Volume 76, Issue 8MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 8 2009Article first published online: 9 JUN 200 Early bovine development, from oocyte to blastocyst. Immature germinal vesicle stage oocyte mechanically stripped from the surrounding cumulus cells (top); cleavage-stage embryos, morulae as well as early, hatching and hatched blastocysts (the latter collapsed due to the stress of the procedure) were produced by in vitro culturing. See Gilbert et al. in this issue for details on how the RNA levels fluctuate between these developmental stages. [source] Advanced fluorescence in situ hybridization to localize and quantify gene expression in Japanese medaka (Oryzias latipes) exposed to endocrine-disrupting compoundsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2009June-Woo Park Abstract In an earlier study, we described the development of fluorescence in situ hybridization (FISH) using confocal microscopy to localize and quantify gene expression in fish. Here, we report the results of FISH application to investigate effects of model endocrine-disrupting chemicals (EDCs), 17,-ethinylestradiol (EE2) and 17,-trenbolone (TB), on expressions of EDC-responsive genes in Japanese medaka (Oryzias latipes) at the cellular/tissue level paired with histological observation. Gene expressions of vitellogenin-II (Vit-II), androgen receptor (AR), and cytochrome P450 gonadal aromatase (CYP19a) were determined after exposure to 5, 50, or 500 ng/L of EE2 or 50, 500, or 5,000 ng/L of TB for 7 d. Exposure to the greatest concentration of EE2 or TB significantly reduced fecundity and caused histological alterations in gonads. 17,-Ethinylestradiol induced Vit-II expression in both male gonads and liver relative to controls and resulted in greater intensity of hematoxylin staining in hepatocytes, which was significantly correlated with Vit-II induction in liver. When exposed to EE2 at less than 50 ng/L, CYP19a expression associated with early stage oocytes was greater than that in controls. However, at 500 ng/L, this trend was reversed. The greater Vit-II expression in testis from all EE2 groups, and the lesser expression of CYP19a in ovaries from the 500 ng/L group, likely is related to changes in the number of cells in which these genes are predominantly expressed rather than to an increase in expression per cell. 17,-Trenbolone significantly induced AR expression in ovaries but did not alter AR expression in female liver. It was concluded that FISH combined with histology enables advanced elucidation of molecular effects of chemicals by associating changes in gene expression with certain tissues and/or cell types and allows these changes to be related to histological effects. [source] Inactivation of MAPK affects centrosome assembly, but not actin filament assembly, in mouse oocytes maturing in vitroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 7 2007Seung-Eun Lee Abstract Mitogen-activated protein kinase (MAPK) plays a crucial role in meiotic maturation of mouse oocytes. In order to understand the mechanism by which MAPK regulates meiotic maturation, we examined the effects of the MAPK pathway inhibitor U0126 on microtubule organization, ,-tubulin and nuclear mitotic apparatus protein (NuMA) distribution, and actin filament assembly in mouse oocytes maturing in vitro. Western blotting with antibodies that detect active, phosphorylated MAPK revealed that MAPK was inactive in fully grown germinal vesicle (GV) oocytes. Phosphorylated MAPK was first detected 3 hr after the initiation of maturation cultures, was fully active at 6 hr, and remained active until metaphase II. Treatment of GV stage oocytes with 20 µM U0126 completely blocked MAPK phosphorylation, but did not affect GV breakdown (GVBD). However, the oocytes did not progress to the Metaphase I stage, which would normally occur after 9 hr in the maturation cultures. The inhibition of MAPK resulted in abnormal spindles and abnormal distributions of ,-tubulin and NuMA, but did not affect actin filament assembly. In oocytes treated with U0126 after GVBD, polar body extrusion was normal, but the organization of the metaphase plate and chromosome segregation were abnormal. In conclusion, the meiotic abnormalities caused by U0126, a specific inhibitor of MAPK signaling, indicate that MAPK plays an important regulatory role in microtubule and centrosome assembly, but not actin filament assembly. Mol. Reprod. Dev. © 2006 Wiley-Liss, Inc. Mol. Reprod. Dev. 74: 904,911, 2007. © 2007 Wiley-Liss, Inc. [source] Proteomic profiling of murine oocyte maturation,MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2007Alejandra M. Vitale Abstract In an effort to better understand oocyte function, we utilized two-dimensional (2D) electrophoresis and mass spectrometry to identify proteins that are differentially expressed during murine oocyte maturation. Proteins from 500 germinal vesicle (GV) and metaphase II-(MII) arrested oocytes were extracted, resolved on 2D electrophoretic gels, and stained with silver. Analysis of the gels indicated that 12 proteins appeared to be differentially expressed between the GV and MII stage. These proteins were then cored from the 2D gels and identified by mass spectrometry as: transforming acidic coiled-coil protein 3 (TACC3), heat shock protein 105 (HSP105), programmed cell death six-interacting protein (PDCD6IP), stress-inducible phosphoprotein (STI1), importin ,2, adenylsuccinate synthase (ADDS), nudix, spindlin, lipocalin, lysozyme, translationally controlled tumor protein (TCTP), and nucleoplasmin 2 (NPM2). Interestingly, PDCD6IP, importin ,2, spindlin, and NPM2 appear slightly larger in mass and more acidic on the MII oocyte gel compared to the GV oocyte gel, suggesting that they may be post-translationally modified during oocyte maturation. Given NPM2 is an oocyte-restricted protein, we chose to further investigate its properties during oocyte maturation and preimplantation development. Real-Time RT-PCR showed that NPM2 mRNA levels rapidly decline at fertilization. Indirect immunofluorescence analysis showed that, with the exception of cortical localization in MII-arrested oocytes, NPM2 is localized to the nucleus of both GV stage oocytes and all stages of preimplantation embryos. We then performed one-dimensional (1D) western blot analysis of mouse oocytes and preimplantation embryos and found that, as implicated by the 2D gel comparison, NPM2 undergoes a phosphatase-sensitive electrophoretic mobility shift during the GV to MII transition. The slower migrating NPM2 form is also present in pronuclear embryos but by the two-cell stage, the majority of NPM2 exists as the faster migrating form, which persists to the blastocyst stage. Mol. Reprod. Dev. 74: 608,616, 2007. © 2006 Wiley-Liss, Inc. [source] Specific maternal transcripts in bovine oocytes and cleavaged embryos: Identification with novel DDRT-PCR methodsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2005Kyu-Chan Hwang Abstract We used annealing control primer (ACP)-based differential display reverse transcription polymerase chain reaction (DDRT-PCR) to isolate differentially expressed amplicons in bovine germinal vesicle (GV) stage oocytes, 8-cell stage embryos produced in vitro, and blastocyst stage embryos produced in vitro. Four expressed sequence tags (ESTs) of genes that were specifically and predominantly expressed in GV oocytes were cloned and sequenced. We have used a fluorescence monitored real-time quantitative PCR (qPCR) to quantify and analyzed the temporal expression of the target differentially expressed transcripts throughout the preimplantation stages from oocytes to blastocysts. The cloned genes or ESTs all exhibited significant sequence similarity with known bovine genes (98%,100%; DNCL1 and ZP2) or ESTs (81%,97%; FANK1 and GTL3) of other species. As revealed by real-time qRT-PCR, DNCL1, FANK1, GTL3, and ZP2 transcripts were observed in the GV stage oocytes and expression gradually decreased up to the 8-cell stage embryo and the transcripts were not detected in later stages. Similarly, upregulation was observed in GV stage mouse oocytes and metaphase II, suggesting that these four differentially expressed orthologous genes play important roles in early preimplantation, as maternally-derived transcripts. Mol. Reprod. Dev. © 2005 Wiley-Liss, Inc. [source] Oocyte-selective expression of MT transposon-like element, clone MTi7 and its role in oocyte maturation and embryo developmentMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Chang-Eun Park Abstract Previously, we found MT transposon-like element, clone MTi7 (MTi7) is highly expressed in the mouse ovary. Here, we show that the MTi7 is expressed in the oocyte from the primordial to the preovulatory follicles. For RNA interference (RNAi), double stranded RNAs (dsRNAs) were prepared for MTi7 and c-mos, a control gene with known functions. Each dsRNA was microinjected into germinal vesicle (GV) stage oocytes or zygotes with pronuclei (PN), after which developmental changes, mRNA expression, and nuclear and microtubular organization were analyzed. We found a 43.4,53% GV arrest in the microinjected oocytes with a concomitant decrease in targeted mRNA expression. In MTi7 dsRNA-injected early and late PN zygotes, a 92.9% 1-cell arrest and 76.9% 2-cell arrest were observed, respectively. This is the first report of an oocyte-selective expression of MTi7 mRNA, and our results strongly suggest that MTi7 involved in the nuclear membrane breakdown during oocyte maturation and embryo development. Mol. Reprod. Dev. 69: 365,374, 2004. © 2004 Wiley-Liss, Inc. [source] | ||||||||||