Oocyte RNA Research Articles

Discovery, identification and sequence analysis of RNAs selected for very short or long poly A tail in immature bovine oocytes

A major challenge in applying genomics to oocyte physiology is that many RNAs are present but will not be translated into proteins, making it difficult to draw conclusions from RNAseq and array data. Oocyte maturation and early embryo development rely on maternal storage of specific RNAs with a short poly(A) tail, which must be elongated for translation. To resolve the role of key genes during that period, we aimed to characterize both extremes of mRNA: deadenylated RNA and long polyA tails mRNA population in immature bovine oocytes. Using magnetic beads coupled to oligodT, we isolated deadenylated (A-, 20-50 adenosines) from polyadenylated (A+, up to 200 adenosines) RNAs. After transcriptomic analysis, we observed that A+ candidates are associated with short-term processes required for immediate cell survival (translation or protein transport) or meiotic resumption, while several A- candidates are involved in processes (chromatin modification, gene transcription and post-transcriptional modifications) that will be extremely important in the development of the early embryo. In addition to a list of candidates probably translated early or late, sequence analysis revealed that cytoplasmic polyadenylation element (CPE) and U(3)GU(3) were enriched in A- sequences. Moreover, a motif associated with polyadenylation signals (MAPS, U(5)CU(2)) appeared to be enriched in 3'untranslated regions (UTR) with CPE or U(3)GU(3) sequences in bovine but also in zebrafish and Xenopus tropicalis. To further validate our methodology, we measured specific tail length of known candidates (AURKA, PTTG1, H2A1) but also determined the poly(A) tail length of other candidate RNAs (H3F3A, H1FOO, DAZAP2, ATF1, ATF2, KAT5, DAZL, ELAVL2). In conclusion, we have reported a methodology to isolate deadenylated from polyadenylated RNAs in samples with small total RNA quantities such as mammals. Moreover, we identified deadenylated RNAs in bovine oocytes that may be stored for the long-term process of early embryo development and described a conserved motif enriched in the 3'UTR of deadenylated RNAs.

GSE Is a Maternal Factor Involved in Active DNA Demethylation in Zygotes

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5mC) and the accumulation of 5-hydroxymethylcytosine (5hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5mC and 5hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5mC to 5hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

256 HOXB9 KNOCK-DOWN DURING OOCYTE MATURATION IN CATTLE

HOXB9 is a transcription factor playing a major role in patterning the main body-axis of vertebrate embryos from the gastrulation stage. HOXB9 transcripts have been detected in cattle oocytes and early embryos (Paul et al. 2011 Mol. Reprod. Dev. 78, 436–449), but their role at those stages has remained unknown. The aim of the study was to inject small interfering RNA (siRNA) in immature oocytes to investigate HOXB9 functions during oocyte maturation and early development. Immature cow oocytes were injected with 7 pL of a HOXB9 siRNA mixture (Integrated DNA Technologies, Coralville, IA, USA) or with a negative control siRNA (SCR) or were not injected (control). To allow microinjection, the size of the cumulus was reduced by pipetting; however, the corona radiata was left intact. The efficiency of the knock-down was measured 24 h post-injection using RT-qPCR, and the results were normalized with an internal normalization factor corresponding to the mean expression of three housekeeping genes (Goossens et al. 2005 BMC Dev. Biol. 5, 27). The injection of HOXB9 siRNA induced a 60.7% reduction of HOXB9 relative expression in mature oocytes by comparison with the injection of SCR siRNA (Kruskall-Wallis; P < 0.05). The nuclear maturation rates, assessed 24 h post-injection after Hoechst staining, were not statistically different between the HOXB9 group (48.8%) and the SCR group (53.6%; chi-square; P > 0.05). After in vitro fertilization and culture (Paul et al. 2011 Mol. Reprod. Dev. 78, 436–449), the developmental rates of the HOXB9 and the SCR groups were not statistically different in terms of achieving the first cleavage (64.4% v. 57.0%, respectively) and 5- to 8-cell stages (25.1% v. 25.3%, respectively) at 48 h post-insemination and the blastocyst stage on Day 7 (IVF = Day 0; 22.0% v. 20.6%, respectively) and Day 8 (26.9% v. 23.1%, respectively; two-way ANOVA; P > 0.05). Differential staining of Day 8 blastocysts showed no difference between the HOXB9 and the SCR groups regarding the mean number of cells in the inner cell mass (53.9 v. 49.4, respectively), the trophectoderm (125.5 v. 104.3, respectively), or the total number of cells (two-way ANOVA; P > 0.05). However, the microinjection procedure impaired the developmental competence after fertilization of the injected oocytes (HOXB9 + SCR) compared with the non-injected ones, as shown by their lower ability to cleave (60.7% v. 72.1%, respectively), to reach the 5- to 8-cell stage (25.2% v. 35.1%, respectively) or the blastocyst stage on Day 7 (21.3% v. 29.8%, respectively) and Day 8 (25.0% v. 35.8%, respectively; two-way ANOVA; P < 0.05). The injection did not affect blastocyst cell numbers on Day 8 (two-way ANOVA; P > 0.05). In conclusion, microinjection of siRNA in immature oocytes is a valuable method to efficiently knock down the maternal messenger RNA during oocyte maturation. Indeed, HOXB9 knock-down in immature oocytes led to a significant and important reduction in the amount of transcript in mature oocytes. The absence of effect of the knock-down on nuclear maturation and embryo development could be explained by the persistence of the maternal proteins. This work was funded by the Fonds National de la Recherche Scientifique (Belgium).

Telomeric repeat-containing RNA and telomerase in human fetal oocytes

What is the distribution of telomeric repeat-containing RNA (TERRA) and of telomerase in human fetal oocytes? TERRA forms discrete foci at telomeres of human fetal oocytes and it co-localizes with both the shelterin component telomeric repeat-binding factor 2 (TRF2) and the catalytic subunit of human telomerase at the telomeres of meiotic chromosomes. TERRA is a structural element of the telomeric chromatin that has been described in somatic cells of many different eukaryote species. The telomerase enzyme is inactive in adult somatic cells but is active in germ cells, stem cells and in the majority of tumors; however, its distribution in oocytes is still unknown. For this study, ovarian samples from four euploid fetuses of 22 gestational weeks were used. These samples were obtained with the consent of the parents and of the Ethics Committee of Hospital de la Vall d'Hebron. We analyzed the distribution of TERRA and telomerase in cells derived from human fetal ovaries. The co-localization of TERRA, telomerase and telomeres was performed by optimizing a combination of immunofluorescence (IF) and RNA-fluorescent in situ hybridization (RNA-FISH) techniques. The synaptonemal complex protein 3 (SYCP3), TRF2 and protein component of telomerase [telomerase reverse transcriptase (TERT)] were detected by IF, whereas TERRA was revealed by RNA-FISH using a (CCCTAA)(3) oligonucleotide. SYCP3 signals allowed us to identify oocytes that had entered meiosis and classify them into the different stages of prophase I, whereas TRF2 indicated the telomeric regions of chromosomes. We show for the first time the presence of TERRA and the intracellular distribution of telomerase in human fetal ovarian cells. TERRA is present, forming discrete foci, in 75% of the ovarian tissue cells and most of TERRA molecules (≈ 83%) are at telomeres (TRF2 co-localization). TERRA levels are higher in oocytes than in ovarian tissue cells (P = 0.00), and do not change along the progression of the prophase I stage (P = 0.37). TERRA is present on ≈ 23% of the telomeres in all cell types derived from human fetal ovaries. Moreover, ≈ 22% of TERRA foci co-localize with the protein component of telomerase (TERT). We present a descriptive/qualitative study of TERRA in human fetal ovarian tissue. Given the difficult access and manipulation of fetal samples, the number of fetal ovaries used in this study was limited. This is the first report on TERRA expression in oocytes from human fetal ovaries. The presence of TERRA at the telomeres of oocytes from the leptotene to pachytene stages and its co-localization with the telomerase protein component suggests that this RNA might participate in the maintenance of the telomere structure, at least through the processes that take place during the female meiotic prophase I. Since telomeres in oocytes have been mainly studied regarding the bouquet structure, our results introduce a new viewpoint of the telomeric structure during meiosis.

Sequences in the H19 ICR that are transcribed as small RNA in oocytes are dispensable for methylation imprinting in YAC transgenic mice

Allele-specific methylation of the endogenous H19 imprinting control region (ICR) is established in sperm. We previously showed that the paternal H19 ICR in yeast artificial chromosome (YAC) transgenic mice (TgM) was preferentially methylated in somatic cells, but not in germ cells, suggesting that differential methylation could be established after fertilization. In this report, we discovered small RNA molecules in growing oocytes, the nucleotide sequences of which mapped to the H19 ICR. To test if these small RNA sequences play a role in the establishment of differential methylation, we deleted the sequences from the H19 ICR DNA and generated YAC TgM. In somatic cells of these mice, methylation imprinting of the transgene was normally established. In addition, the mutant fragment was not methylated in sperm and eggs. These data demonstrate that sequences in the H19 ICR that correspond to the small RNA sequences are dispensable for methylation imprinting in YAC TgM.

Factors Affecting Oocyte and Embryo Transcriptomes

The most important factor affecting the oocyte and early embryo transcriptome is the legacy from the follicular environment prior to meiotic resumption. Up to the 8-cell stage, the oocyte responds to maternal instructions stored before resumption of the meiotic division. Recent evidence suggests that properly prepared or programmed oocytes (in vivo) can achieve close to 100% blastocyst rates in standard in vitro conditions/media. Therefore, the optimal oocyte requires perfect follicular timing and differentiation, but the intra-oocyte mechanisms involved in such preparation are not completely understood. In addition, the influence of maternal mRNA storage and degradation, as well as the length of the poly A tail that influences the general pattern of the oocyte/early embryo transcriptome, is an important factor. Several hypotheses have been put forth to explain the depletion of the maternal store, including the potential role of microRNA (miRNA) in this process. The activation of the embryonic genome could be dependent on, or associated with, the process of maternal mRNA degradation, but obviously other functions are being activated at this critical time point. This review will focus on the period from full-size oocytes to the eight-cell stage and will summarize the impact of the important factors, that is, follicle, maternal RNA storage and embryonic genome activation, on the transcriptome.

Discovery of Centrosomal RNA and Centrosomal Hypothesis of Cellular Ageing and Differentiation

In 2006, a group of scientists studying centrosomes of Spisula solidissima mollusc oocytes under the leadership of Alliegro (Alliegro, M.C.; Alliegro, M.A.; Palazzo, R.E. Centrosome-associated RNA in surf clam oocytes. Proc. Natl. Acad. Sci. USA 2006, 103(24), 9034–9038) reliably demonstrated the existence of specific RNA in centrosome, called centrosomal RNA (cnRNA). In their first article, five different RNAs (cnRNAs 11, 102, 113, 170, and 184) were described. During the process of full sequencing of the first transcript (cnRNA 11), it was discovered that the transcript contained a conserved structure—a reverse transcriptase domain located together with the most important centrosomal protein, γ-tubulin. In an article published in 2005, we made assumptions about several possible mechanisms for determining the most important functions of centrosomal structures and referred to one of them as a “RNA-dependent mechanism.” This idea about participation of hypothetic centrosomal small interference RNA and/or microRNA in the process was made one year prior to the discovery of cnRNA by Alliegro's group. The discovery of specific RNA in a centrosome is indirect evidence of a centrosomal hypothesis of cellular ageing and differentiation. The presence of a reverse transcriptase domain in this type of RNA, together with its uniqueness and specificity, makes the centrosome a place of information storage and reproduction.

Seasonal Effect on Germinal Vesicle-Stage Bovine Oocytes Is Further Expressed by Alterations in Transcript Levels in the Developing Embryos Associated with Reduced Developmental Competence1

Physiological perturbations of bovine follicle-enclosed oocytes during the lengthy period of follicular development can lead to reduced oocyte developmental competence. It is suggested that heat stress-induced alterations in germinal vesicle (GV)-stage oocytes are further expressed in the transcriptional levels of genes involved in oocyte maturation and early embryonic development. Bovine oocytes were collected during cold (December-April) and hot (May-November) seasons, matured, fertilized, and cultured in vitro. The percentage of fertilized oocytes cleaving to the 2- to 4-cell stage was higher in the cold vs. hot season (89.0% ± 2.63% vs. 75% ± 2.63%, respectively; P < 0.05), as was the percentage of cleaved embryos further developing to blastocysts (26.6% ± 0.9% vs. 10.1% ± 1.8%, respectively; P < 0.05). Total RNA and poly(A) mRNA of oocytes and developing embryos were isolated and subjected to semiquantitative and real-time PCR for MOS, GDF9, and POU5F1 genes. In GV-stage oocytes, their mRNA levels did not differ between the seasons. However, following maturation, mRNA levels were higher in oocytes collected in the cold season (P < 0.05). In 4-cell-stage embryos, GDF9 and POU5F1 showed opposite mRNA patterns between seasons (higher and lower levels, respectively) in the hot season (P < 0.05). In both 8-cell-stage embryos and blastocysts, POU5F1 expression was lower during the hot season (P < 0.05). Exposing the ovarian pool of oocytes to environmental stress appears to impair maternal mRNA storage and/or the mechanism of transcription renewal, in turn affecting embryo gene expression before and after embryonic genome activation. Such impairment might partially explain the carry-over effect of summer heat stress on dairy cow conception rates.

194 IN VITRO MATURATION AND RNA CONTENT AND DISTRIBUTION OF PORCINE OOCYTES DERIVED FROM SMALL AND MEDIUM FOLLICLES AND CLASSIFIED BY BRILLIANT CRESYL BLUE ASSAY

Oocytes collected from the surface of slaughterhouse ovaries clearly have heterogeneous quality. The objective was to characterize a change or difference in RNA distribution and the content of porcine oocytes that were collected from small (SF; 1–2 mm in diameter) and medium follicles (MF; 3–6 mm in diameter) and assessed for glucose-6-phosphate dehydrogenase (G6PD) activity by brilliant cresyl blue (BCB) assay. Following BCB staining, porcine cumulus–oocyte complexes (COC) with dark blue (DB; G6PD-inactive, suggesting good quality) and light blue (LB; G6PD-active, suggesting immaturity) ooplasm were then separately cultured in vitro to evaluate nuclear maturation and analyze the characteristics of the RNA aspect. RNA distributions in cumulus cell mass and ooplasm were labeled with fluorescence, SYTO RNA select green and then examined at different periods of culture for in vitro maturation (IVM; with gonadotropins and dbcAMP for 20 h and then without those for 24 h). Total RNA content of oocytes and cumulus cell mass were measured by using a manufacture's kit (Invitrogen Quant-iT RNA assay kit). Statistical analyses of results from 3 to 5 replicated trials were performed by ANOVA with a Bonferroni/Dunn post-hoc test (significance, P &lt; 0.05). When oocytes were classified by BCB assay, the percentage of oocytes with DB cytoplasm was much higher (P &lt; 0.05) in oocytes from MF (72.5% of 208) compared with those of SF (53.6% of 352). Regardless of the origin of oocytes (SF vs MF), the incidence of mature oocytes following culture was higher in DB than LB (64.0% of 147 and 72.1% of 133 vs 50.9% of 108 and 52.8% of 54, respectively). Twenty hours after the start of IVM, the DB oocytes had a higher proportion of RNA-free zone inside the germinal vesicle compared to the LB oocytes (66.1% of 193 and 26.8% of 159 in MF; 47.9% of 185 and 17.9% of 184 in SF; P &lt; 0.05). When the total content of RNA was examined during IVM, both oocytes from SF and MF contained higher levels of total RNA at the beginning of IVM as compared with 20 and 44 h after the start of IVM, whereas the content (40 denuded oocytes/sample) was higher in oocytes with DB cytoplasm from MF than those from SF (P &lt; 0.05). Regardless of the origin of oocytes from SF and MF, the total content of RNA in oocytes with LB cytoplasm was significantly lower than in oocytes with DB cytoplasm (P &lt; 0.05) before the start of IVM. The total RNA content of cumulus cells (40 COC/sample) before IVM culture was also higher in the cell mass surrounding the DB oocytes than the LB ones and in the cell mass surrounding the oocytes from MF rather than SF (P &lt; 0.05). These results demonstrate a possibility that differences in RNA distribution and content in oocytes, as well as the content in cumulus cells, reflect in the ability of porcine oocytes to mature in vitro.

Porcine CPEB1 is involved in Cyclin B translation and meiotic resumption in porcine oocytes

Ovarian immature oocytes accumulate many dormant maternal mRNAs, which have short poly(A) tails. Cytoplasmic-polyadenylation-element binding protein (CPEB) has been reported to play key roles for the elongation of the tails and the translation of these mRNAs in Xenopus oocytes. However, the functions of CPEB in meiotic resumption have not yet been established in mammalian oocytes. The present study examined the roles of porcine CPEB in Cyclin B syntheses and meiotic resumption of porcine oocytes. Porcine CPEB1 (pCPEB1) cDNA was cloned from total RNA of immature oocytes by RT-PCR. The overexpression of pCPEB1 by mRNA injection into immature oocytes increased Cyclin B expression and the rate of meiotic resumption. Conversely, the inhibition of endogenous CPEB by expression of a dominant-negative mutant pCPEB1 (AA-CPEB), which replaced the expected phosphorylation sites with alanines, had the effect of inhibiting Cyclin B synthesis, ribosomal S6 kinase phosphorylation (an indicator of Mos activity), and meiotic resumption. The inhibition of porcine Aurora A by an injection of antisense RNA enhanced the inhibitory effects of AA-CPEB. These results suggest the involvement of mammalian CPEB1 in Cyclin B syntheses and meiotic resumption in mammalian oocytes. In addition, the phosphorylation sites of pCPEB1 were identified and are suggested to be phosphorylated by porcine Aurora A.

Molecular profiling of human oocytes after vitrification strongly suggests that they are biologically comparable with freshly isolated gametes

To assess the effects of vitrification on the biomolecular profile of oocytes, we analyzed through real-time reverse transcriptase-polymerase chain reaction eight genes encoding critically important proteins for embryo development and compared this partial transcriptome with that of freshly collected gametes isolated from the same women. The comparison of the molecular profiles demonstrated that our vitrification protocol does not alter the biomolecular quality of oocytes: in fact, between the two groups we found the absence of statistically significant variations. Accordingly, this cryopreservation technique might be helpful in preserving women's fertility.

Identification of germ plasm‐associated transcripts by microarray analysis of Xenopus vegetal cortex RNA

RNA localization is a common mechanism for regulating cell structure and function. Localized RNAs in Xenopus oocytes are critical for early development, including germline specification by the germ plasm. Despite the importance of these localized RNAs, only approximately 25 have been identified and fewer are functionally characterized. Using microarrays, we identified a large set of localized RNAs from the vegetal cortex. Overall, our results indicate a minimum of 275 localized RNAs in oocytes, or 2-3% of maternal transcripts, which are in general agreement with previous findings. We further validated vegetal localization for 24 candidates and further characterized three genes expressed in the germ plasm. We identified novel germ plasm expression for reticulon 3.1, exd2 (a novel exonuclease-domain encoding gene), and a putative noncoding RNA. Further analysis of these and other localized RNAs will likely identify new functions of germ plasm and facilitate the identification of cis-acting RNA localization elements.

An RNA Aptamer with High Affinity and High Specificity for the 5S RNA Binding Zinc Finger Proteins TFIIIA and p43

The Xenopus zinc finger proteins TFIIIA and p43 bind to 5S RNA in immature oocytes to form 7S and 42S ribonucleoprotein storage particles. To probe the similarities and differences in the RNA binding domains of these two proteins, a library of random RNA molecules was enriched using TFIIIA as the bait protein. One of the abundant aptamers isolated, RNA22, bound to both TFIIIA and p43 derived zinc finger peptides with high affinity and specificity even though the predicted secondary structure of the RNA was unrelated to that of 5S RNA. The interactions of TFIIIA and p43 peptides with RNA22 were compared to their interactions with 5S RNA by characterizing the effects of assay conditions, mutations in RNA22, and mutations in the zinc finger proteins. The similarities and differences in the mechanisms by which these two zinc finger proteins interact with 5S RNA compared to RNA22 suggest they share a common platform for RNA binding with enough flexibility to form specific interactions with both RNAs.

General Features of Certain RNA Populations from Gametes and Cumulus Cells

Results described herein provide insight regarding certain features of gamete RNA and how they compare to cumulus cell RNA. In particular, 28S/18S rRNA ratio and size distribution of RNA molecules differed in total RNA from oocytes versus surrounding cumulus cells. Specifically, oocyte total RNA had a lower rRNA ratio and an increased abundance of smaller RNA sizes compared to RNA from surrounding cumulus. Extensive efforts demonstrated that observed differences were repeatable whether oocyte maturation occurred in vitro or in vivo, and were similar between the nuclear stages examined. Features of oocyte RNA were conserved across six mammalian species, yet differed from surrounding cumulus. Profiles of sperm RNA were also examined but had no discernible ribosomal RNA peaks and were conserved across four mammalian species. Because the oocyte and spermatozoon are highly specialized cells representing unique molecular entities required for proper embryo development, dissimilarities described herein likely represent real gamete versus cumulus RNA differences.

Systematic and single cell analysis of Xenopus Piwi-interacting RNAs and Xiwi

Piwi proteins and Piwi-interacting RNAs (piRNAs) are essential for germ cell development, but analysis of the molecular mechanisms of these ribonucleoproteins remains challenging in most animal germ cells. To address this challenge, we systematically characterized Xiwi, a Xenopus Piwi homologue, and piRNAs from Xenopus eggs and oocytes. We used the large size of Xenopus eggs to analyze small RNAs at the single cell level, and find abundant piRNAs and large piRNA clusters in the Xenopus tropicalis genome, some of which resemble the Drosophila piRNA-generating flamenco locus. Although most piRNA clusters are expressed simultaneously in an egg, individual frogs show distinct profiles of cluster expression. Xiwi is associated with microtubules and the meiotic spindle, and is localized to the germ plasm--a cytoplasmic determinant of germ cell formation. Xiwi associates with translational regulators in an RNA-dependent manner, but Xenopus tudor interacts with Xiwi independently of RNA. Our study adds insight to piRNA transcription regulation by showing that individual animals can have differential piRNA expression profiles. We suggest that in addition to regulating transposable elements, Xiwi may function in specifying RNA localization in vertebrate oocytes.

RNAi Experiments in Mouse Oocytes and Early Embryos

The discovery of RNA interference (RNAi) in 1998 ushered in a new era in biology. RNAi currently serves as a favorite approach for inhibition of gene function in many areas of research. This article provides a brief review of RNAi and discussion of the benefits and drawbacks of using long double-stranded RNA (dsRNA) in mammalian oocytes and early embryos. We also provide an introduction to protocols for RNAi experiments in mouse, including preparation and microinjection of dsRNA into mouse oocytes and early embryos, and preparation and testing of constructs for transgenic RNAi based on long hairpin RNA expression.

Global Transcriptional Repression in C. elegans Germline Precursors by Regulated Sequestration of TAF-4

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Contribution of Individual Amino Acids to the 5S RNA Binding Activity of the Xenopus Zinc Finger Protein p43

Xenopus zinc finger protein p43 binds to 5S RNA in immature oocytes to form a 42S ribonucleoprotein storage particle. To determine the role of individual zinc fingers of the protein in this RNA binding activity, a series of deletion and substitution mutants of p43 were constructed. The effects of the various mutations on the RNA binding activity of p43 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 1 and 4 of p43 are essential for the binding of the protein to 5S RNA. In the case of finger 1, four amino acids key to RNA binding are found on the same face of the alpha-helix, while in the case of finger 4, two key residues are clustered at the start of the alpha-helix. The similarities and differences in the mechanisms by which fingers 1 and 4 of p43 interact with 5S RNA are compared to the interaction of the zinc fingers of Xenopus transcription factor IIIA with 5S RNA.

Porcine Aurora A accelerates Cyclin B and Mos synthesis and promotes meiotic resumption of porcine oocytes

Full-grown oocytes arrested at germinal vesicle stage contain many dormant maternal mRNAs, and Aurora A has been reported to play a key role for the translation of these maternal mRNAs in Xenopus oocytes. Although the presence of Aurora A has been reported in mammals, the functions of Aurora A on the protein synthesis and the meiotic resumption have never been elucidated in mammalian oocytes. In the present study, the effects of porcine Aurora A on meiotic resumption of porcine oocytes were examined. At first, we cloned porcine Aurora A from total RNA of immature porcine oocytes by RT-PCR and obtained full-length cDNA that was 77%, 86% and 54% homologous with mouse, human and Xenopus Aurora A, respectively. The Aurora A mRNA and large amounts of protein were present throughout maturation period in porcine oocytes. The overexpression of porcine Aurora A by the mRNA injection into immature porcine oocytes had no effects on Cyclin B synthesis and meiotic resumption. Therefore we constructed a mutated Aurora A (AA-Aurora A), which was replaced the expecting inhibitory phosphorylation sites, serines 283 and 284, to non-phosphorylatable alanines. The oocytes expressed AA-Aurora A were accelerated their Cyclin B synthesis and Rsk phosphorylation, an indicator of Mos synthesis, then their meiotic resumption was promoted significantly. These results suggest for the first time in mammalian oocytes that mammalian Aurora A stimulates the protein synthesis and promotes the meiotic resumption. In addition, we identified the inhibitory phosphorylation sites of porcine Aurora A, and indicate the presence of phosphorylation-dependent regulation mechanisms in mammalian Aurora A.

Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes

Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.