MOS mutation causes female infertility with large polar body oocytes.

The Moloney sarcoma oncogene (MOS) encodes a protein serine/threonine kinase and MOS is expressed at high levels in oocytes undergoing meiotic maturation. The MOS/MAPK pathway is normally required for the maintenance of microtubules and chromatin in a metaphasic state during the meiotic divisions. To determine the pathogenic genes in a female infertile patient due to large polar body oocytes, whole-exome sequencing was performed on the patient and available family members. We identified a novel homozygous missense mutation c.591T > G in MOS. Bioinformatics analysis showed that the mutation is harmful. These findings suggest that MOS mutation results in oocytes with a large polar body and poor embryonic development in patients. The MOS variant may regulate oocyte asymmetric division by MAPK/WAVE2/Arp2/3/actin signaling pathway. This will help to understand the comprehensive role of MOS in early human reproductive process and provide genetic markers for future genetic counseling for more individualized treatments.

Single Molecule RNA Localization and Translation in the Mammalian Oocyte and Embryo.

During oocyte growth the cell accumulates RNAs to contribute to oocyte and embryo development which progresses with ceased transcription. To investigate the subcellular distribution of specific RNAs and their translation we developed a technique revealing several instances of localized translation with distinctive regulatory implications. We analyzed the localization and expression of candidate non-coding and mRNAs in the mouse oocyte and embryo. Furthermore, we established simultaneous visualization of mRNA and in situ translation events validated with polysomal occupancy. We discovered that translationally dormant and abundant mRNAs CyclinB1 and Mos are localized in the cytoplasm of the fully grown GV oocyte forming cloud-like structures with consequent abundant translation at the center of the MII oocyte. Coupling detection of the localization of specific single mRNA molecules with their translation at the subcellular context is a valuable tool to quantitatively study temporal and spatial translation of specific target mRNAs to understand molecular processes in the developing cell.

Cyclin B2 is required for progression through meiosis in mouse oocytes.

Cyclins associate with cyclin-dependent serine/threonine kinase 1 (CDK1) to generate the M phase-promoting factor (MPF) activity essential for progression through mitosis and meiosis. Although cyclin B1 (CCNB1) is required for embryo development, previous studies concluded that CCNB2 is dispensable for cell cycle progression. Given previous findings of high Ccnb2 mRNA translation rates in prophase-arrested oocytes, we re-evaluated the role of this cyclin during meiosis. Ccnb2-/- oocytes underwent delayed germinal vesicle breakdown and showed defects during the metaphase-to-anaphase transition. This defective maturation was associated with compromised Ccnb1 and Moloney sarcoma oncogene (Mos) mRNA translation, delayed spindle assembly and increased errors in chromosome segregation. Given these defects, a significant percentage of oocytes failed to complete meiosis I because the spindle assembly checkpoint remained active and anaphase-promoting complex/cyclosome function was inhibited. In vivo, CCNB2 depletion caused ovulation of immature oocytes, premature ovarian failure, and compromised female fecundity. These findings demonstrate that CCNB2 is required to assemble sufficient pre-MPF for timely meiosis re-entry and progression. Although endogenous cyclins cannot compensate, overexpression of CCNB1/2 rescues the meiotic phenotypes, indicating similar molecular properties but divergent modes of regulation of these cyclins.

Influence of interleukin 1 beta and tumour necrosis factor alpha on the in vitro growth, maturation and mitochondrial distribution of bovine oocytes from small antral follicles.

SummaryThis study aimed to investigate the effects of IL1ß and TNFα on growth and maturation of oocytes from small follicles (1-3 mm) during in vitro culture. To this end, cumulus-oocyte complexes (COCs) with diameters of ~110 µm were cultured in TCM-199 medium alone or supplemented with IL1ß (10 ng/ml), TNFα (10 ng/ml) or both for 48 h. The oocytes were measured at the beginning and at the end of the culture period. COCs were cultured for 20 h in pre-maturation medium and then half of the COCs of each group was destined for in vitro maturation and the remaining COCs were used to evaluate meiotic progression, mitochondrial distribution and the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. The results showed that COCs cultured with TNFα alone or together with IL1ß had higher diameters than those cultured in control medium alone or supplemented with IL1ß. Control oocytes isolated from large antral follicles (>5 mm) had heterogeneous distribution of mitochondria. Oocytes isolated from small antral follicles, that had been grown in vitro in TCM-199 alone or supplemented with TNFα had similar heterogeneous mitochondrial distribution before in vitro maturation (IVM). After IVM, mitochondria were heterogeneously distribution when cultured in TCM-199. However, when cultured with TNFα and/or IL1ß, mitochondria were homogeneously distributed. Presence of TNFα and/or IL1ß in TCM-199 culture medium did not influence the expression of mRNAs for GDF-9, c-Mos, Cyclin-B1 and H1foo. In conclusion, TNFα and a mixture of TNFα and IL1ß both stimulated the growth of bovine oocytes during their in vitro culture, but do not influence gene expression in grown oocytes.

Effects of tumour necrosis factor-alpha and interleukin-1 beta on in vitro development of bovine secondary follicles.

The objective of this study was to determine the effects of TNF-α and IL-1ß on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO2 in air, for 18 days, in TCM-199+ alone (cultured control) or supplemented with 10 ng/ml IL-1ß, 10 ng/ml TNF-α or both TNF-α and IL-1ß. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF-9, c-MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF-α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL-1ß and a mixture of IL-1ß and TNF-α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF-α had the best-preserved oocytes and granulosa cells. The presence of TNF-α, IL-1ß or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL-1ß, TNF-α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.

Cryptic diversity in Ptyodactylus (Reptilia: Gekkonidae) from the northern Hajar Mountains of Oman and the United Arab Emirates uncovered by an integrative taxonomic approach.

The Hajar Mountains of south-eastern Arabia form an isolated massif surrounded by the sea to the east and by a large desert to the west. As a result of their old geological origin, geographical isolation, complex topography and local climate, these mountains provide an important refuge for endemic and relict species of plants and animals. With 19 species restricted to the Hajar Mountains, reptiles are the vertebrate group with the highest level of endemicity, becoming an excellent model for understanding the patterns and processes that generate and shape diversity in this arid mountain range. The geckos of the Ptyodactylus hasselquistii species complex are the largest geckos in Arabia and are found widely distributed across the Arabian Mountains, constituting a very important component of the reptile mountain fauna. Preliminary analyses suggested that their diversity in the Hajar Mountains may be higher than expected and that their systematics should be revised. In order to tackle these questions, we inferred a nearly complete calibrated phylogeny of the genus Ptyodactylus to identify the origin of the Hajar Mountains lineages using information from two mitochondrial and four nuclear genes. Genetic variability within the Hajar Mountains was further investigated using 68 specimens of Ptyodactylus from 46 localities distributed across the entire mountain range and sequenced for the same genes as above. The molecular phylogenies and morphological analyses as well as niche comparisons indicate the presence of two very old sister cryptic species living in allopatry: one restricted to the extreme northern Hajar Mountains and described as a new species herein; the other distributed across the rest of the Hajar Mountains that can be confidently assigned to the species P. orlovi. Similar to recent findings in the geckos of the genus Asaccus, the results of the present study uncover more hidden diversity in the northern Hajar Mountains and stress once again the importance of this unique mountain range as a hot spot of biodiversity and a priority focal point for reptile conservation in Arabia.

Molecular systematics, species delimitation and diversification patterns of the Phyllodactylus lanei complex (Gekkota: Phyllodactylidae) in Mexico.

The description of cryptic gecko species worldwide has revealed both that many putative species are, in fact, conformed by a complex of morphologically conserved species that are genetically distinct and highly divergent, and that gecko species diversity could be underestimated. The taxonomy and species delimitation of geckos belonging to the genus Phyllodactylus is still controversial, 16 of which are distributed in Mexico and 13 are endemic. Although the large morphological variation shown by the Phyllodactylus species from Mexico has been amply documented, little is known about their genetic diversity and evolutionary relationships, and much less regarding cryptic speciation. Here, we included the most comprehensive sampling of populations and species of the Phyllodactylus lanei complex distributed in Mexico, and applied an analytical approach that included probabilistic phylogenetic analyses, jointly with species delimitation methods and Bayesian putative species validation analysis. Our results suggest the existence of 10 lineages within the complex, supporting the existence of cryptic species, and in great contrast with the current taxonomic proposal that includes only four subspecies. The most recent common ancestor (MRCA) for the P. lanei clade originated on the Early Eocene (∼54Mya), along the southern coasts of Mexico, followed by the highest diversification of the complex MRCA during the Eocene (34-56Mya). Lineages subsequently dispersed and diversified towards the northwest, and the diversification process ended with the most recent lineages inhabiting two islands on the coasts of Nayarit (Miocene; 5.5-23Mya). Our results highlight three vicariant events associated with the evolution of the lineages, two of them intimately related to the formation of the Sierra Madre del Sur and the Transmexican Volcanic Belt mountain ranges, main geographic barriers that isolated and facilitated the divergence and speciation in this group of geckos. Finally, we propose that there are 10 species in the P. lanei complex, from which four represent taxonomic changes and six are new species and require a formal description. We acknowledge that more analyses, including a detailed evaluation of morphological characters and use of more unlinked nuclear loci with enough variability, are needed to further support their taxonomic description.

Formation of mos RNA granules in the zebrafish oocyte that differ from cyclin B1 RNA granules in distribution, density and regulation.

Many translationally repressed mRNAs are deposited in the oocyte cytoplasm for progression of the meiotic cell cycle and early development. mos and cyclin B1 mRNAs encode proteins promoting oocyte meiosis, and translational control of these mRNAs is important for normal progression of meiotic cell division. We previously demonstrated that cyclin B1 mRNA forms RNA granules in the zebrafish and mouse oocyte cytoplasm and that the formation of RNA granules is crucial for regulating the timing of translational activation of the mRNA. However, whether the granule formation is specific to cyclin B1 mRNA remains unknown. In this study, we found that zebrafish mos mRNA forms granules distinct from those of cyclin B1 mRNA. Fluorescent in situ hybridization analysis showed that cyclin B1 RNA granules were assembled in dense clusters, while mos RNA granules were distributed diffusely in the animal polar cytoplasm. Sucrose density gradient ultracentrifugation analysis showed that the density of mos RNA granules was partly lower than that of cyclin B1 mRNA. Similar to cyclin B1 RNA granules, mos RNA granules were disassembled after initiation of oocyte maturation at the timing at which the poly(A) tail was elongated. However, while almost all of the granules of cyclin B1 were disassembled simultaneously, a fraction of mos RNA granules firstly disappeared and then a large part of them was disassembled. In addition, while cyclin B1 RNA granules were disassembled in a manner dependent on actin filament depolymerization, certain fractions of mos RNA granules were disassembled independently of actin filaments. These results suggest that cytoplasmic regulation of translationally repressed mRNAs by formation of different RNA granules is a key mechanism for translational control of distinct mRNAs in the oocyte.

Association analysis for feet and legs disorders with whole-genome sequence variants in 3 dairy cattle breeds.

Identification of genetic variants associated with feet and legs disorders (FLD) will aid in the genetic improvement of these traits by providing knowledge on genes that influence trait variations. In Denmark, FLD in cattle has been recorded since the 1990s. In this report, we used deregressed breeding values as response variables for a genome-wide association study. Bulls (5,334 Danish Holstein, 4,237 Nordic Red Dairy Cattle, and 1,180 Danish Jersey) with deregressed estimated breeding values were genotyped with the Illumina Bovine 54k single nucleotide polymorphism (SNP) genotyping array. Genotypes were imputed to whole-genome sequence variants, and then 22,751,039 SNP on 29 autosomes were used for an association analysis. A modified linear mixed-model approach (efficient mixed-model association eXpedited, EMMAX) and a linear mixed model were used for association analysis. We identified 5 (3,854 SNP), 3 (13,642 SNP), and 0 quantitative trait locus (QTL) regions associated with the FLD index in Danish Holstein, Nordic Red Dairy Cattle, and Danish Jersey populations, respectively. We did not identify any QTL that were common among the 3 breeds. In a meta-analysis of the 3 breeds, 4 QTL regions were significant, but no additional QTL region was identified compared with within-breed analyses. Comparison between top SNP locations within these QTL regions and known genes suggested that RASGRP1, LCORL, MOS, and MITF may be candidate genes for FLD in dairy cattle.

Phylogeny of Neotropical Cercosaura (Squamata: Gymnophthalmidae) lizards.

Among Neotropical lizards, the geographically widespread gymnophthalmid Cercosaura as currently defined includes lowland and highland taxa from Panama to Argentina, with some species occurring in the northern Andes. In this study we analyze three mitochondrial (12S, 16S, ND4) and one nuclear (c-mos) gene using Bayesian methods to clarify the phylogenetic relationships among most species of Cercosaura based on a well-supported phylogenetic hypothesis that also includes a large sample of other taxa within Cercosaurini. The phylogenetic tree obtained in this paper shows that Cercosaura as currently defined is not monophyletic. Two species from the northern Andes (C. dicra and C. vertebralis) are nested within Pholidobolus, which has been formerly recognized as a major radiation along the Andes of Ecuador and Colombia. Therefore, Cercosaura has probably not diversified in the northern Andes, although the phylogenetic position of C. hypnoides from the Andes of Colombia remains unknown. Tree topology and genetic distances support both recognition of C. ocellata bassleri as a distinct species, C. bassleri, and recognition of C. argula and C. oshaughnessyi as two different species. In the interest of promoting clarity and precision regarding the names of clades of gymnophthalmid lizards, we propose a phylogenetic definition of Cercosaura.

Genetic modifiers of EGFR dependence in non-small cell lung cancer.

Lung adenocarcinomas harboring activating mutations in the epidermal growth factor receptor (EGFR) represent a common molecular subset of non-small cell lung cancer (NSCLC) cases. EGFR mutations predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs) and thus represent a dependency in NSCLCs harboring these alterations, but the genetic basis of EGFR dependence is not fully understood. Here, we applied an unbiased, ORF-based screen to identify genetic modifiers of EGFR dependence in EGFR-mutant NSCLC cells. This approach identified 18 kinase and kinase-related genes whose overexpression can substitute for EGFR in EGFR-dependent PC9 cells, and these genes include seven of nine Src family kinase genes, FGFR1, FGFR2, ITK, NTRK1, NTRK2, MOS, MST1R, and RAF1. A subset of these genes can complement loss of EGFR activity across multiple EGFR-dependent models. Unbiased gene-expression profiling of cells overexpressing EGFR bypass genes, together with targeted validation studies, reveals EGFR-independent activation of the MEK-ERK and phosphoinositide 3-kinase (PI3K)-AKT pathways. Combined inhibition of PI3K-mTOR and MEK restores EGFR dependence in cells expressing each of the 18 EGFR bypass genes. Together, these data uncover a broad spectrum of kinases capable of overcoming dependence on EGFR and underscore their convergence on the PI3K-AKT and MEK-ERK signaling axes in sustaining EGFR-independent survival.

Polyspermy block in jellyfish eggs: collaborative controls by Ca(2+) and MAPK.

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca(2+) and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm-egg fusion and initiation of an intracellular Ca(2+) rise from this site. The elevated Ca(2+) levels lasted for several minutes following the sperm-egg fusion. The Ca(2+) rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca(2+) rise with EGTA promoted polyspermy, and conversely, triggering a Ca(2+) rise by inositol 1,4,5-trisphosphate (IP3) or excess K(+) immediately abolished the egg's capacity for sperm-egg fusion. A Ca(2+) rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca(2+) rise at fertilization or by IP3 injection, and shut down the subsequent sperm-egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca(2+) rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca(2+) levels, and prevented sperm-egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm-egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm-egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca(2+) and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.

The Mos-MAPK pathway regulates Diaphanous-related formin activity to drive cleavage furrow closure during polar body extrusion in starfish oocytes.

Maintenance of spindle attachment to the cortex and formation of the cleavage furrow around the protruded spindle are essential for polar body extrusion (PBE) during meiotic maturation of oocytes. Although spindle movement to the cortex has been well-studied, how the spindle is maintained at the cortex during PBE is unknown. Here, we show that activation of Diaphanous-related formin mediated by mitogen-activated protein kinase (MAPK) is required for tight spindle attachment to the cortex and cleavage furrow closure during PBE in starfish (Asterina pectinifera) oocytes. A. pectinifera Diaphanous-related formin (ApDia) had a distinct localization in immature oocytes and was localized to the cleavage furrow during PBE. Inhibition of the Mos-MAPK pathway or the actin nucleating activity of formin homology 2 domain prevented cleavage furrow closure and resulted in PBE failure. In MEK/MAPK-inhibited oocytes, activation of ApDia by relief of its intramolecular inhibition restored PBE. In summary, this study elucidates a link between the Mos-MAPK pathway and Diaphanous-related formins, that is responsible for maintaining tight spindle attachment to the cortex and cleavage furrow closure during PBE.

Changes in aberrant DNA methylation after Helicobacter pylori eradication: a long-term follow-up study.

Changes of DNA methylation in gastric mucosae after eradication of Helicobacter pylori have not been clarified yet. From this background, we investigated time course of DNA methylation following H. pylori eradication in 221 successfully H. pylori eradicated subjects with endoscopic follow-up at least for 6 months, including 114 controls, 53 subjects with gastric dysplasia and 54 patients with early gastric cancer. All dysplasia and gastric cancer patients underwent endoscopic resection at the time of enrollment. The methylation levels in LOX, APC and MOS genes from noncancerous gastric mucosae using quantitative methylation-specific PCR, as well as the histologic findings of gastric mucosae, were compared before and after eradication. Average follow-up duration was 26.0 months (range: 6 to 76 months). H. pylori eradication decreased methylation levels in LOX (p-value for slope < 0.001) but not in APC. In MOS, decrease of its methylation level following H. pylori eradication was significant among controls without intestinal metaplasia (IM) (p-value for slope < 0.05); however, it was not observed among patients with IM or those with dysplasia or gastric cancer. After H. pylori eradication, methylation level in MOS persistently increased in patients with dysplasia or gastric cancer (p < 0.01). In conclusion, H. pylori eradication decreases aberrant DNA methylation with gene-specific manner. Methylation level in MOS is associated with IM and may be used as a surrogate marker for gastric cancer risk, regardless of H. pylori eradication history.

Lycodon and Dinodon: one genus or two? Evidence from molecular phylogenetics and morphological comparisons.

Based on a molecular phylogeny and a comparison of maxillary dentition and morphology, the relationship between the genera Lycodon and Dinodon was investigated. Bayesian Inference and Maximum Likelihood analysis of two mitochondrial genes (cyt b and ND4) and two nuclear genes (c-mos and Rag1) suggested that the two genera shared a most recent common ancestor. However, Dinodon was paraphyletic and Lycodon was polyphyletic, each with respect to the other. The results from counts of maxillary teeth indicated that the diagnostic characters used by previous authors to separate Dinodon and Lycodon were not reliable. Taking the molecular and morphological evidence together, we synonymized Dinodon with Lycodon. In addition, the validity of the species L. futsingensis was confirmed to be distinctly different from the other species of Dinodon and Lycodon.

Ringo/cyclin-dependent kinase and mitogen-activated protein kinase signaling pathways regulate the activity of the cell fate determinant Musashi to promote cell cycle re-entry in Xenopus oocytes.

Cell cycle re-entry during vertebrate oocyte maturation is mediated through translational activation of select target mRNAs, culminating in the activation of mitogen-activated protein kinase and cyclin B/cyclin-dependent kinase (CDK) signaling. The temporal order of targeted mRNA translation is crucial for cell cycle progression and is determined by the timing of activation of distinct mRNA-binding proteins. We have previously shown in oocytes from Xenopus laevis that the mRNA-binding protein Musashi targets translational activation of early class mRNAs including the mRNA encoding the Mos proto-oncogene. However, the molecular mechanism by which Musashi function is activated is unknown. We report here that activation of Musashi1 is mediated by Ringo/CDK signaling, revealing a novel role for early Ringo/CDK function. Interestingly, Musashi1 activation is subsequently sustained through mitogen-activated protein kinase signaling, the downstream effector of Mos mRNA translation, thus establishing a positive feedback loop to amplify Musashi function. The identified regulatory sites are present in mammalian Musashi proteins, and our data suggest that phosphorylation may represent an evolutionarily conserved mechanism to control Musashi-dependent target mRNA translation.

Regulation of maternal gene expression by MEK/MAPK and MPF signaling in porcine oocytes during in vitro meiotic maturation.

Mitogen-activated protein kinase (MAPK) and maturation/M phase promoting factor (MPF) play crucial roles in oocyte meiotic maturation in mammals. However, the underlying molecular mechanisms have not been addressed. In this study, the effects of the MEK/MAPK pathway inhibitor U0126 and the MPF inhibitor roscovitine on meiotic maturation and maternal gene expression in pig cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were investigated. Both inhibitors can reversibly block the resumption of meiosis in pig oocytes. COCs or DOs initially cultured in drug-free medium for 24 h and then transferred to medium containing U0126 showed normal progress to the Metaphase II stage (MII); (90.38 vs. 92.16% control group). In contrast, roscovitine treatment from 24 to 44 h significantly inhibited maturation of COCs and DOs. To explore the underlying molecular mechanisms, expression patterns and polyadenylation states of five representative maternal transcripts, cyclin B1, Cdc2, C-mos, GDF9 and BMP15, were examined by real-time PCR and poly(A)-test PCR (PAT assay). U0126 treatment resulted in aberrant expression of Cdc2 and GDF9, while roscovitine significantly maintained all five maternal transcripts at very high levels in treated COCs compared with the control group. The polyadenylation of these mRNAs increased as well. Furthermore, the experiments were repeated in DOs, and the results also indicated that both Cdc2 and GDF9 showed significantly higher expression in both mRNA and polyadenylation levels in the drug treatment groups. Together, these results provide the first demonstration in a mammalian system that MAPK and MPF play important roles in regulation of maternal gene expression during oocyte maturation.

Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes.

BACKGROUND: RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type. RESULTS: Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals. CONCLUSIONS: We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.