In contrast to other species, localized maternal mRNAs are not believed to be prominent features of mammalian oocytes. We find by cDNA microarray analysis enrichment for maternal mRNAs encoding spindle and other proteins on the mouse oocyte metaphase II (MII) spindle. We also find that the key translational regulator, EIF4EBP1, undergoes a dynamic and complex spatially regulated pattern of phosphorylation at sites that regulate its association with EIF4E and its ability to repress translation. These phosphorylation variants appear at different positions along the spindle at different stages of meiosis. These results indicate that dynamic spatially restricted patterns of EIF4EBP1 phosphorylation may promote localized mRNA translation to support spindle formation, maintenance, function, and other nearby processes. Regulated EIF4EBP1 phosphorylation at the spindle may help coordinate spindle formation with progression through the cell cycle. The discovery that EIF4EBP1 may be part of an overall mechanism that integrates and couples cell cycle progression to mRNA translation and subsequent spindle formation and function may be relevant to understanding mechanisms leading to diminished oocyte quality, and potential means of avoiding such defects. The localization of maternal mRNAs at the spindle is evolutionarily conserved between mammals and other vertebrates and is also seen in mitotic cells, indicating that EIF4EBP1 control of localized mRNA translation is likely key to correct segregation of genetic material across cell types.T HE oocytes of many species, both invertebrate and vertebrate, contain a large collection of localized determinants in the form of proteins and translationally inactive maternal mRNAs. Similar localized determinants in mammalian oocytes have been proposed (Ciemerych et al. 2000), but this aspect of mammalian reproduction remains controversial (Hiiragi et al. 2006). Indeed, early mammalian embryogenesis is considered to be quite plastic and regulative in nature, so that localized determinants would not be expected to play essential functions. Embryo splitting can be used for twinning, and blastomere extirpation does not prevent elaboration of normal body plans and term development. Additionally, much of the volume of the mammalian oocyte eventually becomes allocated to cells that do not contribute to embryonic development, being destined instead to generate the placenta. Accordingly, prepatterning of the mammalian oocyte through localization of maternal mRNAs or proteins, if it occurs, appears to be dispensable for mammalian embryogenesis.One potential exception to this would relate to localization within the oocyte of maternal mRNAs that support a vital process that is evolutionarily conserved between mammals and other species, namely the formation and maintenance of the meiotic spindle. Recent studies in Xenopus revealed enriched localization to spindle microtubules of mRNAs encoding spindle proteins (Blower et al. 2007). The spindle is a complex structure; proteomic studies of is...

Abstract. Aberrant gene expression in somatic cell nuclear-transferred (NT) embryos due to abnormal epigenetic modifications of the donor nucleus likely accounts for much of the observed diminished viability and developmental abnormalities. We compared the expression of 13 developmentally important genes in individual 8-cell and blastocyst stage NT embryos produced from adults female cumulus cells and adult male skin fibroblast cells with low and high incidences of neonatal abnormalities [1,2]. In vitro-fertilized (IVF) embryos were used as control embryos. Among the genes tested, the relative abundance of Glut-1, IGF-1R, E-cad, and Cx43 transcripts varied significantly between the two types of NT embryos at the 8-cell stage. The relative abundance of manganese super oxide dismutase (MnSOD) and Stat3 transcripts was significantly higher in IVF embryos compared with both types of NT embryos. At the blastocyst stage, there was a significant difference in the relative expression of only one gene, Bcl-2, between the two types of NT embryos. Although the level of Glut-1 expression did not vary between the two types of NT blastocysts, its expression in both types of NT blastocysts was significantly lower than that in IVF blastocysts. The MnSOD expression level tended to be higher in NT blastocysts. The gene expression profile for any single gene, however, was highly variable among individual embryos and was independent of embryo morphology. The present study demonstrated that the expression profiles of the 13 genes examined in Day 9 NT blastocysts produced from two different types of donor cells with different incidences of neonatal abnormalities are largely indistinguishable.

The high incidence of abnormalities in cloned calves is a most serious problem for bovine somatic cell nuclear transfer (NT) technology. Because there is little information on the differences in mRNA expression in cloned blastocysts with donor cells of different sex and origin, we compared development-related gene expression in two types of cloned bovine blastocysts with different potentials to develop into normal calves, a female adult cumulus cell line (high potential to develop into live calves) and a male fibroblast cell line (low potential to develop into live calves) to examine the correlation between the normality of cloned calves and blastocyst mRNA expression patterns. We analyzed 12 genes involved in apoptosis, growth factor signaling, metabolism, and DNA methylation in blastocysts originating from two types of donor cells and in vitro-fertilized blastocysts using quantitative real-time polymerase chain reaction. Expression of the pro-apoptotic Bax gene and anti-apoptotic Bcl-2 and Glut-1 genes in fibroblast-derived blastocysts was significantly higher than in cumulus cell-derived and in vitro-fertilized blastocysts. The high Bcl-2 and Glut-1 gene expression suggests that some embryonic cells with damaged DNA in fibroblast-derived blastocysts are not removed, and their descendants later manifest abnormal placenta or fetus formation. Transfer of pre-selected cloned blastocysts into recipients is required, however, to determine whether the expression pattern of these apoptosis-related genes reflects differences in the potential to develop into normal calves.

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The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus-oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P<0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

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