Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the fourcell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer.oocyte extract and proteomics | reprogramming in mammalian oocytes E mbryonic cells differentiate into specific types of cells as development progresses. Once differentiated, the reversion of a differentiated cell state to an original undifferentiated state is strictly inhibited in normal development. However, it has been experimentally shown that differentiated nuclei can be returned to an undifferentiated embryonic state after nuclear transfer (NT) to enucleated eggs or oocytes (1, 2). Such experiments provide an opportunity to reprogram somatic cells as a means to prepare undifferentiated cells, which may be differentiated into any kinds of cells for cell-replacement therapy. Recently, nuclear reprogramming technology has been expanded by the production of induced pluripotent stem (iPS) cells (3). iPS cells can be obtained by overexpressing specific sets of transcription factors such as Oct4, Sox2, Klf4, and c-myc in cultured cells. The processes leading to establishment of iPS cell lines are being carefully examined and we are begining to understand how somatic cells acquire pluripotency by this method (4-6). The mechanisms leading to pluripotency may be different between iPS cells and NT embryos because somatic nuclei transferred into unfertilized metaphase II (MII) oocytes must undergo early embryonic development before the inner cell mass (ICM) can give rise to pluripotent embryonic stem (ES) cells. In addition, the molecules and mechanisms that induce somatic cell reprogramming are expected to be different between iPS cells and NT embryos (7,8). A recent study has shown that nuclear t...

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.

Abstract. Functional and structural changes in the mammalian ovary are coordinately regulated by the pituitary glycoprotein hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), leading to follicular development, ovulation and transformation of follicles into corpus lutea. To investigate protein profiles during these processes of the mouse ovarian cycle, we applied combined methods (two-dimensional gel electrophoresis for separation and visualization of proteins plus matrix laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF/MS] analysis for protein identification) for comparative proteomic analysis using immature mice at 3 weeks of age. Protein profiles were obtained from proteins extracted from intact ovaries that had been collected from pregnant mare serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed immature mice at 0 (no PMSG), 24 and 48 h post PMSG, as well as at 10 and 20 h post hCG. The results showed that 1028 common protein spots were found in representative gels that had been separated in the 3 to 11 pH range and the 15-200 kDa range, 253 protein spots (24.6%) of which were differentially expressed (p<0.05) during the mouse ovarian cycle. Of these 253 protein spots, 99 were identified by MALDI-TOF/MS. This comparative proteomic approach to identifying proteins that were potentially involved in the complex process of the ovarian cycle could contribute to our understanding of the molecular basis of functional and structural changes in the ovary in response to gonadotropins. Furthermore, the interesting ovarian proteins identified in this study may eventually serve as diagnostic biomarker candidates of ovarian function. Key words: Follicle stimulating hormone, Luteinizing hormone, Ovary, Proteome, Two-dimensional gel electrophoresis (J. Reprod. Dev. 55: [316][317][318][319][320][321][322][323][324][325][326] 2009) omplex biological processes in the mammalian ovary, such as follicular development, oocyte maturation, oocyte meiosis, ovulation and corpus luteum formation and demise, are coordinately regulated by autocrine, paracrine and endocrine factors of the hypothalamic-pituitary-ovarian axis [1][2][3]. Specifically, follicle-stimulating hormone (FSH) is a major promoter for orchestrating follicular development and differentiation in the granulosa cells of preovulatory follicles [4][5][6]. Luteinizing hormone (LH) plays a key role in initiation of the ovulatory process of preovulatory follicles by activating multiple cellular signaling pathways [5,7]. Detailed biological changes in gene expression and follicular structure occur due to the overlapping control of FSH and LH, and the theca, granulosa, cumulus and oocyte compartments of the ovarian follicle are interdependently regulated by them. Moreover, the universal and local actions of these gonadotropins are coordinated with signals from the oocyte to promote ovulation under multipartite control, facilitating synchronization of oocyte maturation with follicle rupture and resulting in select...

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Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.