RNAi in mouse oocytes and preimplantation embryos: effectiveness of hairpin dsRNA

Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO.     

Efficient delivery of dsRNA into zona-enclosed mouse oocytes and preimplantation embryos by electroporation

Conditions for the electroporation of mouse oocytes and preimplantation embryos have been optimised by following the incorporation of rhodamine labeled dextran. This procedure includes a step to weaken but not remove the zona pellucida that helps achieve good survival. This approach has been applied to introduce double-stranded RNA for c-mos into oocytes and green fluorescent protein (GFP) into transgenic GFP-expressing embryos at the 1- and 4-cell stages. In both cases we were able to observe sequence-specific interference with the expression of the target gene--a failure of oocytes to arrest at metaphase II and a loss in the green fluorescence of embryos by the morula or blastocyst stages. These effects could be observed in multiple oocytes or embryos allowed to develop together following electroporation.     

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.     

Transgenic RNAi in mouse oocytes: The first decade

RNA interference (RNAi), a sequence-specific mRNA degradation induced by double-stranded RNA (dsRNA), is a common approach employed to specifically silence genes. Experimental RNAi in plant and invertebrate models is frequently induced by long dsRNA. However, in mammals, short RNA molecules are used preferentially since long dsRNA can provoke sequence-independent type I interferon response. A notable exception are mammalian oocytes where the interferon response is suppressed and long dsRNA is a potent and specific trigger of RNAi. Transgenic RNAi is an adaptation of RNAi allowing for inducing sequence-specific silencing upon expression of dsRNA. A decade ago, we have developed a vector for oocyte-specific expression of dsRNA, which has been used to study gene function in mouse oocytes on numerous occasions. This review provides an overview and discusses benefits and drawbacks encountered by us and our colleagues while working with the oocytes-specific transgenic RNAi system.     

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.     

Expression of specific genes in early mouse embryos blocked by cytochalasin

Summary Mouse embryos at the two cell stage derived from C57BL/6 × C3H/Aa F₁-females heterozygous at the X-linked phosphoglycerate kinase locus (Pgk-1) were cultured continuously in the presence of cytochalasin B or D. Further cleavage of the two cell embryos was thus prevented and the embryos became polyploid during culture. The onset of expression of the maternally inherited Pgk-1 gene and of the paternally inherited glucosephosphate isomerase (Gpi-1) gene was determined in these polyploid embryos by cellulose acetate gel electrophoresis of single embryos. In contrast to euploid preimplantation embryos developing normally in utero or in culture without cytochalasins, expression of maternal Pgk-1 was never observed at days 4 and 5 of gestation in polyploid two cell embryos, showing that the Pgk-1 allele on the maternally inherited X chromosome is not activated independently of cytokinesis and morphogenesis. Expression of paternally derived Gpi-1, however, occurred in cleavage blocked embryos von day 5 of development. This may indicate that the activation of two genes which are both expressed during preimplantation development and which both code for glycolytic enzymes, is initiated by different signals.     

Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes

Mouse oocytes have proven useful in experiments aimed at studying gene function. They have been used to analyze the gain-of-function acquired after microinjection of RNA transcribed in vitro from specific gene constructs, and for establishing loss-of-function mutation obtained by injecting in vitro transcribed antisense RNA and/or synthetic oligonucleotides. This article presents protocols utilized in these studies. Specifically, the acquisition of mouse oocytes and/or embryos, the genesis of the necessary DNA and/or RNA to be used, and procedures for microinjection.     

Developmentally acquired PKA localisation in mouse oocytes and embryos

Localisation of Protein Kinase A (PKA) by A-Kinase Anchoring Proteins (AKAPs) is known to coordinate localised signalling complexes that target cAMP-mediated signalling to specific cellular sub-domains. The cAMP PKA signalling pathway is implicated in both meiotic arrest and meiotic resumption, thus spatio-temporal changes in PKA localisation during development may determine the oocytes response to changes in cAMP. In this study we aim to establish whether changes in PKA localisation occur during oocyte and early embryo development.Using fluorescently-labelled PKA constructs we show that in meiotically incompetent oocytes PKA is distributed throughout the cytoplasm and shows no punctuate localisation. As meiotic competence is acquired, PKA associates with mitochondria. Immature germinal vesicle (GV) stage oocytes show an aggregation of PKA around the GV and PKA remains co-localised with mitochondria throughout oocyte maturation. After fertilisation, the punctuate, mitochondrial distribution was lost, such that by the 2-cell stage there was no evidence of PKA localisation. RT-PCR and Western blotting revealed two candidate AKAPs that are known to be targeted to mitochondria, AKAP1 and D-AKAP2. In summary these data show a dynamic regulation of PKA localisation during oocyte and early embryo development.     

Triglyceride content of bovine oocytes and early embryos

A microfluorescence technique was used to measure the triglyceride content of a minimum of two bovine oocytes or preimplantation embryos up to the hatched blastocyst stage. Embryos were produced in vitro from abattoir-derived ovaries and grown in medium containing synthetic oviductal fluid, amino acids and BSA (SOFaaBSA medium); 10% fetal calf serum was added to some of the embryos at the four-cell stage. Before maturation, the triglyceride content of oocytes was 59 +/- 1.37 ng and it decreased (P < 0.05) after maturation to 46 +/- 0.85 ng. A decrease in triglyceride content (P < 0.05) was also observed after fertilization with the formation of the two-cell embryo (34 +/- 1.80 ng). In the absence of serum, the triglyceride content remained relatively constant from the two-cell to the hatched blastocyst stage. The triglyceride content of blastocysts produced in vivo was similar (33 +/- 0.70 ng) to that of blastocysts produced in vitro in the absence of serum. In contrast, the triglyceride content of embryos grown with 10% fetal calf serum increased steadily from the 9-16-cell stage to a value in hatched blastocysts (62 +/- 1.14 ng) almost double that in serum-free conditions. These results indicate that triglyceride may act as energy source during bovine oocyte maturation and fertilization and that the presence of serum causes excessive synthesis or accumulation of triglyceride in early embryos.     

Immunocytochemical detection of a murine leukemia virus-related nuclear antigen in mouse oocytes and early embryos.

The expression of murine leukemia virus (MuLV)-related antigens in oocytes and early embryos of Swiss mice was studied by the unlabeled antibody peroxidase-antiperoxidase method using an antiserum to the major core protein, p30, of AKR MuLV. This procedure resulted in an intense staining, at antibody dilutions up to 1:500, of the germinal vesicles of oocytes and the interphase nuclei of early embryos. Nuclear staining was restricted to a specific period of oocyte growth and embryo development. It developed gradually in oocytes having reached one third to one half the full-grown size and persisted until meiotic maturation. In early embryos, nuclear staining was present from the one-cell stage to the morula, but disappeared during transition from morula to blastocyst and was not seen in expanded blastocysts. Nuclear staining was abolished by absorption of the anti-p30 serum with detergent-disrupted virions of Gross(A), AKR, Kirsten and Moloney MuLV, and Kirsten MSV(MuLV), but not with the Friend and Rauscher strains of MuLV, the xenotropic BALB:virus 2, a mouse tropic and an amphotropic clone of wild mouse MuLV, or with FeLV and RSV. On the basis of these results, it is suggested that the nuclear antigen (termed germinal vesicle antigen) reacting with the anti-p30 serum is the product of a cellular gene having a normal function in early embryonic development, and that sequences related to this gene are incorporated into the genome of AKR-type MuLV.