NuMA expression and function in mouse oocytes and early embryos

Nuclear mitotic apparatus protein (NuMA), originally described as a nuclear protein, is an essential component in the formation and maintenance of mitotic spindle poles. In this study, we analyze the expression pattern and function of NuMA in mouse oocytes and early embryos. In germinal vesicle-stage occytes, NuMA was detected both at the centrosome and in the nucleus. However, after nuclear maturation and extrusion of the first polar body, NuMA was concentrated at the broad meiotic spindle poles and at cytasters (centers of cytoplasmic microtubule asters) of mature metaphase II oocytes. Cold-induced depolymerization of microtubules appeared to disassociate NuMA foci from the cytoplasmic cytasters. During fertilization, NuMA was relocated into the reformed male and female pronuclei. Microinjection of anti-NuMA antibody into 1 of 2 cells of 2-cell-stage embryos inhibited normal cell division. These results suggest that NuMA might play an important role in cell division during early embryonic mitosis.     

The E3 ubiquitin ligase RNF114 and TAB1 degradation are required for maternal‐to‐zygotic transition

The functional role of the ubiquitin‐proteasome pathway during maternal‐to‐zygotic transition (MZT) remains to be elucidated. Here we show that the E3 ubiquitin ligase, Rnf114, is highly expressed in mouse oocytes and that knockdown of Rnf114 inhibits development beyond the two‐cell stage. To study the underlying mechanism, we identify its candidate substrates using a 9,000‐protein microarray and validate them using an in vitro ubiquitination system. We show that five substrates could be degraded by RNF114‐mediated ubiquitination, including TAB1. Furthermore, the degradation of TAB1 in mouse early embryos is required for MZT, most likely because it activates the NF‐κB pathway. Taken together, our study uncovers that RNF114‐mediated ubiquitination and degradation of TAB1 activate the NF‐κB pathway during MZT, and thus directly link maternal clearance to early embryo development.     

Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo

We describe a fluorimetric method for measuring the level of H2O2 in individual mouse oocytes and early embryos. Levels of H2O2 are low but detectable in unfertilized oocytes recovered freshly from the female reproductive tract. The levels in early cleaving embryos (1-cell to 8-cell stages) immediately after recovery from the female tract seem to be slightly higher the later the stage examined. However, when embryos are cultured in vitro from the 1-cell or early 2-cell stage, H2O2 levels rise when the embryos reach the mid-2-cell stage and remain elevated until they enter the early 4-cell stage. No equivalent elevation of H2O2 is seen during the transition from 1-cell to 2-cell or from 4-cell to 8-cell stages. Embryos that are able to develop successfully in vitro, as well as those that show a developmental block at the 2-cell stage on culture in vitro, both show this rise in H2O2 levels after in vitro culture. The relationship between the rise in H2O2 and the '2-cell block' to development is discussed.     

The role of miRNAs and endogenous siRNAs in maternal‐to‐zygotic reprogramming and the establishment of pluripotency

RNA silencing is a complex of mechanisms that regulate gene expression through small RNA molecules. The microRNA (miRNA) pathway is the most common of these in mammals. Genome‐encoded miRNAs suppress translation in a sequence‐specific manner and facilitate shifts in gene expression during developmental transitions. Here, we discuss the role of miRNAs in oocyte‐to‐zygote transition and in the control of pluripotency. Existing data suggest a common principle involving miRNAs in defining pluripotent and differentiated cells. RNA silencing pathways also rapidly evolve, resulting in many unique features of RNA silencing in different taxonomic groups. This is exemplified in the mouse model of oocyte‐to‐zygote transition, in which the endogenous RNA interference pathway has acquired a novel role in regulating protein‐coding genes, while the miRNA pathway has become transiently suppressed.     

Aurora-A is a critical regulator of microtubule assembly and nuclear activity in mouse oocytes, fertilized eggs, and early embryos

Aurora-A is a serine/threonine protein kinase that plays a role in cell-cycle regulation. The activity of this kinase has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this study, the changes in aurora-;A expression were revealed in mouse oocytes using Western blotting. The subcellular localization of aurora-A during oocyte meiotic maturation, fertilization, and early cleavages as well as after antibody microinjection or microtubule assembly perturbance was studied with confocal microscopy. The quantity of aurora-A protein was high in the germinal vesicle (GV) and metaphase II (MII) oocytes and remained stable during other meiotic maturation stages. Aurora-A concentrated in the GV before meiosis resumption, in the pronuclei of fertilized eggs, and in the nuclei of early embryo blastomeres. Aurora-A was localized to the spindle poles of the meiotic spindle from the metaphase I (MI) stage to metaphase II stage. During early embryo development, aurora-A was found in association with the mitotic spindle poles. Aurora-A was not found in the spindle region when colchicine or staurosporine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. Aurora-A antibody microinjection decreased the rate of germinal vesicle breakdown (GVBD) and distorted MI spindle organization. Our results indicate that aurora-A is a critical regulator of cell-cycle progression and microtubule organization during mouse oocyte meiotic maturation, fertilization, and early embryo cleavage.     

Maturation‐associated Dbf4 expression is essential for mouse zygotic DNA replication

Cdc7 is an S‐phase‐promoting kinase (SPK) that is required for the activation of replication initiation complex assembly because it phosphorylates the MCM protein complex serving as the replicative helicase in eukaryotic organisms. Cdc7 activity is undetectable in immature mouse GV oocytes, although Cdc7 protein is already expressed at the same level as in mature oocytes or early one‐cell embryos at zygotic S‐phase, in which Cdc7 kinase activity is clearly detectable. Dbf4 is a regulatory subunit of Cdc7 and is required for Cdc7 kinase activity. Dbf4 is not readily detectable in immature GV oocytes but accumulates to a level similar to that in one‐cell embryos during oocyte maturation, suggesting that Cdc7 is already activated in unfertilized eggs (metaphase II). RNAi‐mediated knockdown of maternal Dbf4 expression prevents the maturation‐associated increase in Dbf4 protein, abolishes the activation of Cdc7, and leads to the failure of DNA replication in one‐cell embryos, demonstrating that Dbf4 expression is the key regulator of Cdc7 activity in mouse oocytes. Dormant Dbf4 mRNA in immature GV oocytes is recruited by cytoplasmic polyadenylation during oocyte maturation and is dependent on MPF activity via its cytoplasmic polyadenylation element (CPE) upstream of the hexanucleotide (HEX) in the 3′ untranslated region (3′UTR). Our results suggest that Cdc7 is inactivated in immature oocytes, preventing it from the unwanted phosphorylation of MCM proteins, and the oocyte is qualified by proper maturation to proceed following embryogenesis after fertilization through zygotic DNA replication.     

Dynamic transition of Dnmt3b expression in mouse pre- and early post-implantation embryos

The de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns in mouse development. Dnmt3b is more highly expressed in early developmental stages than Dnmt3a, and is thought to have an important role in the epigenetic gene regulation during early embryogenesis. Previous reports suggest that Dnmt3b is expressed preferentially in the embryonic lineage, but less in the extra-embryonic lineage, in early post-implantation embryos. However, it is unclear when this lineage-specific differential expression is established. Here we demonstrate that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development. Contrary to the expectation, Dnmt3b is preferentially expressed in the trophectoderm rather than the inner cell mass at the mid blastocyst stage. Subsequently, the spatial Dnmt3b expression gradually changes during pre- and early post-implantation development, and finally Dnmt3b expression is settled in the embryonic lineage at the epiblast stage. The findings are consistent with the role for Dnmt3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.     

Differential regulation of G protein subunit expression in mouse oocytes, eggs, and early embryos

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.     

Maternal Nanos and Pumilio regulate zygotic vasa expression autonomously in the germ-line progenitors of Drosophila melanogaster embryos

vasa (vas) is transcribed earliest among reported genes expressed in the germ-line progenitors, or pole cells, in Drosophila melanogaster embryos. Its expression is detected in the germ-line cells throughout their development, making vas expression a useful marker for the establishment of germ-line fate. In the present report, it is shown that maternal Nos and Pum are required for normal expression of vas in pole cells. First, expression of enhancer-trap marker BC69, which reflects vas expression, is promoted by maternal Nos and Pum. Second, expression of vas mRNA in pole cells is promoted by maternal Nos and Pum. Third, pole cell transplantation experiments reveal that maternal Nos and Pum are required autonomously in pole cells for proper expression of vas. Finally, Nos and Pum are dispensable for vas expression in oogenesis, although they are expressed zygotically in adult ovaries. These observations show that germ-line-specific vas expression is promoted by autonomous function of maternal Nos and Pum in the germ-line progenitors during embryogenesis, and is regulated differentially in embryogenesis and oogenesis.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

Expression of cancer-testis antigens of the Mage family in mouse oocytes and early embryos

Cancer-testis antigens of the Mage family (Melanoma antigens) are expressed predominantly in the spermatogenic and cancer cells, but some genes of this family are expressed ubiquitously. Expression patterns and functional role of Mage family antigens in the regulation of cellular processes in normal embryonic and definitive cells are virtually unknown. Comparative immunofluorescent analysis of Mage expression in mouse oocytes and early embryos identified the expression of Mage antigens at all stages studied. The greatest intensity of the fluorescent staining was detected in the epiblasts and the extraembryonic structures of the egg cylinder at E6.5 stage. At all studied developmental stages of the mouse oocyte and the early embryo, the localization of Mage antigens was found predominantly in the cytoplasm. Quantitative real-time PCR showed that expression levels of most Mage genes in cells of the epiblast and ectoplacental cone were similar, while the gene expression levels of Mage-a10, Mage-b16, and Mage-b18 were higher in cells of the ectoplacental cone than in epiblast cells. Thus, for the first time, our analysis has shown that the Mage family antigens are expressed at the early stages of mouse development and may be involved in the regulation of earliest events of embryogenesis.