Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes

RNA interference (RNAi) is a conserved eukaryotic mechanism by which double-stranded RNA (dsRNA) triggers the sequence-specific degradation of homologous mRNAs. Recent concerns have arisen in mammalian systems about off-target effects of RNAi, as well as an interferon response. Most mammalian cells respond to long dsRNAs by inducing an antiviral response mediated by interferon that leads to general inhibition of protein synthesis and nonspecific degradation of mRNAs. Moreover, recent reports demonstrate that under certain conditions, short interfering RNAs (siRNAs, 21-25 bp) may activate the interferon system. Mouse oocytes and preimplantation embryos apparently lack this response, as potent and specific inhibition of gene expression triggered by long dsRNA is observed in these cells. In the present study, we analyzed the global pattern of gene expression by microarray analysis in transgenic mouse oocytes expressing long dsRNA and find no evidence of off-targeting. We also report that genes involved in the interferon response pathway are not expressed in mouse oocytes, even after exposure for an extended period of time to long dsRNA.     

Selective reduction of weak synaptic activity awakens dormant synapses

Slutsky et al. (this issue of Neuron) report that by selectively filtering out low-level uncorrelated synaptic activity at NMDA receptors in hippocampal cultures they can unlock a large reserve of quiescent synapses and make them available for potentiation with theta burst stimulation. These findings differ from previously reported activity-dependent mechanisms in that inactivity does not necessarily increase synaptic activity globally.     

RNA synthesis in preovulatory mouse oocytes

RNA synthesis, previously shown to take place during oocyte growth, has been demonstrated throughout the growth-quiescent period preceding ovulation of the mouse oocyte. In the final 7-day preovulatory period, the level of incorporation of (5,6-3H)uridine into ovulated oocytes decreased as the interval between exposure to precursor and ovulation decreased; significant incorporation was detectable within 2 days before ovulation. Analysis of the frequency and density of label in ovarian oocytes at successive stages of meiosis in relation to the interval between adminstration of labeled precursor and collection of oocytes revealed that RNA synthesis continues up to within 2 h before GVBD.     

Selective inhibition of hepatitis B virus replication by RNA interference

Small interfering RNA (siRNA) is a powerful tool to silence gene expression in mammalian cells including genes of viral origin. To evaluate the therapeutic efficacy of siRNA against the hepatitis B virus (HBV), we studied the effect of transfection of the HBV-inducible cell lines HepAD38 and HepAD79 with siRNA specific for the core gene of the HBV genome. HepAD38 cells produce wild-type HBV, whereas HepAD79 cells produce the lamivudine resistant YMDD variant. Transfection of HepAD38 cells with either 1.6 or 4 microg/ml siRNA resulted in a profound inhibition (72% and 98%, respectively) of viral replication (as assessed by real-time quantitative PCR). The inhibitory effect was corroborated by a marked reduction of HBV core protein synthesis in induced HepAD38 cells. In HepAD79 cells, transfected with 1.6 or 4 microg/ml HBV-specific siRNA, virus production was reduced by 75% and 89%, respectively.     

Amounts and modulation of actin mRNAs in mouse oocytes and embryos

In order to measure the content of beta- and gamma-actin mRNA in mouse oocytes and ovulated eggs, Northern and slot blots were hybridized to complementary RNA probes transcribed from mouse isotype-specific cDNA sequences. The blots included samples of isotype-specific sense strand RNA standards prepared from the same cDNA sequences. Total actin mRNA content was estimated to be 40 fg per preovulatory full-grown oocyte or egg, consisting of one-third beta-actin mRNA and two-thirds gamma-actin mRNA. Ninety per cent of the actin mRNA is on polysomes in full-grown oocytes. The per cent of actin mRNA in polysomal mRNA is similar to the per cent of actin in newly synthesized proteins. Measurements on other developmental stages showed that, in mid-growth-phase oocytes, each actin mRNA reaches a level twofold higher than in full-grown oocytes. Thereafter, all modulations of the two isotypic mRNAs occur in parallel; that is, they are maintained at constant levels during the late growth phase (oocytes from females 8-14 days old); gradually degraded in oocytes that have completed their rapid growth phase (oocytes from females 15-18 days old), in maturing oocytes, and in 1- and 2-cell embryos; and deadenylated after about 7 h of progression into meiotic maturation.     

Expression of mRNAs of the aquaporin family in mouse oocytes and embryos

The molecular basis of water and cryoprotectant permeability in mammalian oocytes and embryos is poorly understood. Therefore, we investigated the expression of mRNAs of water channel proteins (aquaporins) in mouse oocytes and embryos by RT-PCR. The total RNA of mouse oocytes at metaphase II and embryos at the 4-cell, morula, and blastocyst stages was isolated, reverse-transcribed, and subjected to nested PCR amplification. Aquaporins were expressed in both oocytes and embryos, but the types were different among the developmental stages: aquaporins 3 and 7 were expressed in oocytes and embryos at all stages examined, but aquaporins 8 and 9 were expressed only in blastocysts. On the other hand, aquaporins 1, 2, 4, 5, and 6 were not detected in any of the stages examined. The present study shows for the first time that aquaporins are expressed in mammalian oocytes and embryos. These aquaporins may play a role in water transport and conceivably also in cryoprotectant transport across the plasma membrane in these cells.     

Accumulation and localisation of maternal RNA in oocytes of Japanese quail

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25-2.0 mm in diameter (correlated with the presence of lampbrush chromosomes), followed by a plateau in 2.0-3.0 mm oocytes (correlated with the condensation of lampbrush chromosomes). Resumption of the RNA accumulation in the 3.0-5.0 mm oocytes is interpreted as a result of external RNA delivery by the granulosa cells. Most of the RNA (approximately 3/4, 3-4 micrograms) contained in the ovulated oocyte was found under the vitelline membrane surrounding the yolk. This RNA has been designated as 'extraembryonic RNA', as it is located outside the germinal disc region where the embryo is formed. The extraembryonic RNA is rapidly degraded within 24 h, from ovulation until oviposition, during egg passage through the oviduct, while the RNA present in a germinal disc (approximately 1.1 micrograms) is stable. In bird oocytes the presence of two, territorially separated pools of RNA has been postulated: one contained in a germinal disc and needed for early embryo development, and the second present in the cytoplasmic layer around the yolk supporting oocyte growth and development during oogenesis.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Specific interference with gene function by double-stranded RNA in early mouse development

The use of double-stranded (ds) RNA is a powerful way of interfering with gene expression in a range of organisms, but doubts have been raised about whether it could be successful in mammals. Here, we show that dsRNA is effective as a specific inhibitor of the function of three genes in the mouse, namely maternally expressed c-mos in the oocyte and zygotically expressed E-cadherin or a GFP transgene in the preimplantation embryo. The phenotypes observed are the same as those reported for null mutants of the endogenous genes. These findings offer the opportunity to study development and gene regulation in normal and diseased cells.     

RNA interference as a tool to study gene function in bovine oocytes

RNA interference (RNAi) has become a well-established technique to study gene function in several species. Our objective was to develop a RNAi approach to study gene function in bovine oocytes. In the first experiment, three different treatments including a 20 min exposure to cytochalasin B, a 6 hr maturation in cycloheximide, and a combination of these two treatments were tested to improve oocyte survival following microinjection. The cycloheximide/cytochalasin B treatment greatly increased (P<0.02) the survival rate of the microinjected oocytes. In the second experiment, we assessed the effect of both cyclin B1 and GFP dsRNA on cyclin B1 mRNA and protein expression. The injection of cyclin B1 dsRNA resulted in a decrease in cyclin B1 mRNA and protein, while the cyclin B2 mRNA remained unaffected. Furthermore, the injection of GFP dsRNA did not interfere with cyclin B1 mRNA or protein nor with the ability of the oocyte to mature properly. In addition, the lack of cyclin B1 in the oocyte led to activation in 10% of the oocytes as evidenced by the presence of a pronucleus. However, the use of an additional 10 hr of maturation in the presence of 6-dimethylaminopurine (6-DMAP) prevented germinal vesicle breakdown and allowed a longer exposure to dsRNA. This procedure increased the percentage of activated oocytes to 33% and is likely to result from an increased length of time for dsRNA processing and for degradation of the cyclin B1 mRNA to occur. In conclusion, RNAi represents a useful technique to study gene function in the bovine oocyte.     

The influence of maternal age on radiation-induced chromosome aberrations in mouse oocytes

The relative sensitivities of dictyate oocytes from young and old female mice to radiation-induced chromosome damage were examined in 2 separate experiments. Firstly, females were given either 2 or 4 Gy of X-rays and metaphase I stage oocytes collected 16.5 days later. Analysis of these cells showed dose-related increases in chromosome aberrations in both age groups. The response was significantly greater in oocytes of older females. In the second experiment, females were given 4 Gy of X-rays and metaphase I stage oocytes collected 3.5 days later. Again, a significantly larger frequency of aberrations was present in cells from older animals. Overall, these 2 experiments provide unambiguous evidence that the radiosensitivity of mouse dictyate oocytes increases with advancing maternal age.     

RNA interference in mammalian cells by chemically-modified RNA

RNA interference (RNAi) is proving to be a robust and versatile technique for controlling gene expression in mammalian cells. To fully realize its potential in vivo, however, it may be necessary to introduce chemical modifications to optimize potency, stability, and pharmacokinetic properties. Here, we test the effects of chemical modifications on RNA stability and inhibition of gene expression. We find that RNA duplexes containing either phosphodiester or varying numbers of phosphorothioate linkages are remarkably stable during prolonged incubations in serum. Treatment of cells with RNA duplexes containing phosphorothioate linkages leads to selective inhibition of gene expression. RNAi also tolerates the introduction of 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) nucleotides. Introduction of LNA nucleotides also substantially increases the thermal stability of modified RNA duplexes without compromising the efficiency of RNAi. These results suggest that inhibition of gene expression by RNAi is compatible with a broad spectrum of chemical modifications to the duplex, affording a wide range of useful options for probing the mechanism of RNAi and for improving RNA interference in vivo.     

Lack of effect of maternal age on UV-induced DNA repair in mouse oocytes

Oocytes at the dictyate stage young (8–14 weeks) and old (12–15 months) BALB/c mice were manually isolated and UV-irrdiated. They were cultured for 1 h in medium containing tritiated thymidine and chased for a furthur hour in cold thymidine medium before being incubated for 18–20 h in medium with no added thymidine. Oocytes which had developed to metaphase II were analysed following autoradiography. Pooled results from 14 replicate experiments revealed no significant age-related difference between the mean corrected grain count per cell [159.2 ± 8.5 (86 cells) for young mice and 164.6 ± 9.8 (70 cells) for the old animals]. Thus in the female mouse the oocyte's capacity to repair UV-induced damage is apparently maintained at a high level throughout reprodcutive life.