Density-mediated foraging behavioral responses of Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae).

Tribolium castaneum and Rhyzopertha dominica are cosmopolitan, destructive postharvest pests. Although research has investigated how high densities of T. castaneum affect attraction to the aggregation pheromone by conspecifics, research into the behavioral response of both species to food cues after high density exposure has been lacking despite its importance to foraging ecology. Our goal was to manipulate and observe the effects of crowding on the behavioral response of both species to common food and pheromonal stimuli and to determine how the headspace emission patterns from grain differed under increasing densities. Densities of colonies for both species was altered (10-500 adults) on a fixed quantity of food (10 g of flour or whole wheat), then the behavioral response to common food and pheromonal cues was evaluated in a wind tunnel and release-recapture experiment, while volatiles were examined through gas chromatography coupled with mass spectrometry. Importantly, at least for T. castaneum, crowded conditions attenuate attraction to food-based stimuli, but not pheromonal stimuli. Crowding seemed to have no effect on R. dominica attraction to food and pheromonal stimuli in the wind tunnel, but exposure to high density cues did elicit 2.1-3.8-fold higher captures in traps. The relative composition and abundance of headspace volatiles emitted varied significantly with different densities of beetles and was also species-specific. Overall, our results have implications for expanding our understanding of the foraging ecology of two economically important pests.

Flight capacity and behavior of Ephestia kuehniella (Lepidoptera: Pyralidae) in response to kairomonal and pheromonal stimuli.

Flight behavior is an important component to understand in the context of pest management. However, because of their small size, little is known about the flight capacity of most stored-product insects, and when a flight has been assessed, it usually consists of a propensity for initiating flight. Despite a priori expectations of the importance of flight for moths, there are no data about the flight capacity and little on the flight behavior of the Mediterranean flour moth, Ephestia kuehniella Zeller (Lepidoptera: Pyralidae). As a result, the objective of the current study was to (i) characterize the baseline flight capacity of E. kuehniella and (ii) determine how flight capacity is affected by the presence of kairomonal, pheromonal, or no stimuli. We found adult E. kuehniella flew a mean of 24-34 km in a 24-h period, and the distance flown per bout increased from 91 to 207 m in the presence of pheromones but decreased to 41 m when food was nearby compared to a negative control. The total number of flight bouts was 1.6-fold higher in the presence of pheromone compared to the negative control, but E. kuehniella flew significantly slower with pheromone and food cues present, suggesting they may be exhibiting an optimal foraging strategy. Our data on flight capacity results in qualitatively and quantitatively different conclusions about flight than those conclusions formed if only flight initiation is considered. Overall, this novel information is useful for understanding the spread within facilities and in the landscape (between facilities), as well as parameterizing ecological modeling.

How great thou ART: biomechanical properties of oocytes and embryos as indicators of quality in assisted reproductive technologies.

Assisted Reproductive Technologies (ART) have revolutionized infertility treatment and animal breeding, but their success largely depends on selecting high-quality oocytes for fertilization and embryos for transfer. During preimplantation development, embryos undergo complex morphogenetic processes, such as compaction and cavitation, driven by cellular forces dependent on cytoskeletal dynamics and cell-cell interactions. These processes are pivotal in dictating an embryo's capacity to implant and progress to full-term development. Hence, a comprehensive grasp of the biomechanical attributes characterizing healthy oocytes and embryos is essential for selecting those with higher developmental potential. Various noninvasive techniques have emerged as valuable tools for assessing biomechanical properties without disturbing the oocyte or embryo physiological state, including morphokinetics, analysis of cytoplasmic movement velocity, or quantification of cortical tension and elasticity using microaspiration. By shedding light on the cytoskeletal processes involved in chromosome segregation, cytokinesis, cellular trafficking, and cell adhesion, underlying oogenesis, and embryonic development, this review explores the significance of embryo biomechanics in ART and its potential implications for improving clinical IVF outcomes, offering valuable insights and research directions to enhance oocyte and embryo selection procedures.

The translational oscillation in oocyte and early embryo development.

Translation is critical for development as transcription in the oocyte and early embryo is silenced. To illustrate the translational changes during meiosis and consecutive two mitoses of the oocyte and early embryo, we performed a genome-wide translatome analysis. Acquired data showed significant and uniform activation of key translational initiation and elongation axes specific to M-phases. Although global protein synthesis decreases in M-phases, translation initiation and elongation activity increases in a uniformly fluctuating manner, leading to qualitative changes in translation regulation via the mTOR1/4F/eEF2 axis. Overall, we have uncovered a highly dynamic and oscillatory pattern of translational reprogramming that contributes to the translational regulation of specific mRNAs with different modes of polysomal occupancy/translation that are important for oocyte and embryo developmental competence. Our results provide new insights into the regulation of gene expression during oocyte meiosis as well as the first two embryonic mitoses and show how temporal translation can be optimized. This study is the first step towards a comprehensive analysis of the molecular mechanisms that not only control translation during early development, but also regulate translation-related networks employed in the oocyte-to-embryo transition and embryonic genome activation.

The behavioral response to the putative necromones from dead Tribolium castaneum (Coleoptera: Tenebrionidae) in traps by conspecifics as a function of density and time since capture.

Long-term trapping programs of stored product pests provide information for timely and accurate pest management. Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a highly successful external-infesting grain pest and is frequently monitored using a commercial pitfall trap that combines pheromonal and kairomonal stimuli. However, an often overlooked component of lure-based traps is the potential for the volatile plume to change over time as individuals are captured. These now-dead insects may then release necromones altering the captures of conspecifics. In this study, we evaluated changes in (i) the behavior of T. castaneum and (ii) the relative change in volatiles over time since dead insects were added and among different densities of dead conspecifics in a commercially available kairomone oil. We used multiple behavior assays, including wind tunnel, release-recapture, and 2-way olfactometer, and performed chemical analyses via headspace collection and gas chromatography coupled with mass spectrometry. Tribolium castaneum response to the kairomone lure was not consistent among assays of density of conspecifics between 4 and 40 adults after 24 or 96 h, or time of seeding over 1-96 h or 8-11 days prior. Tested strains collected in 2012 and 2019 ruled out strain-specific differences. Oil batch effects were also ruled out as a factor contributing to the response of T. castaneum. The relative volatile composition was generally stable among the treatments despite using different seeding densities and seeding times. Given that attraction and relative volatile composition were generally unaffected by prior captures, long-term monitoring programs may be robust in their interpretability over time.

Spatial positioning of preimplantation mouse embryo cells is regulated by mTORC1 and m7G-cap-dependent translation at the 8- to 16-cell transition.

Preimplantation mouse embryo development involves temporal-spatial specification and segregation of three blastocyst cell lineages: trophectoderm, primitive endoderm and epiblast. Spatial separation of the outer-trophectoderm lineage from the two other inner-cell-mass (ICM) lineages starts with the 8- to 16-cell transition and concludes at the 32-cell stages. Accordingly, the ICM is derived from primary and secondary contributed cells; with debated relative EPI versus PrE potencies. We report generation of primary but not secondary ICM populations is highly dependent on temporal activation of mammalian target of Rapamycin (mTOR) during 8-cell stage M-phase entry, mediated via regulation of the 7-methylguanosine-cap (m7G-cap)-binding initiation complex (EIF4F) and linked to translation of mRNAs containing 5' UTR terminal oligopyrimidine (TOP-) sequence motifs, as knockdown of identified TOP-like motif transcripts impairs generation of primary ICM founders. However, mTOR inhibition-induced ICM cell number deficits in early blastocysts can be compensated by the late blastocyst stage, after inhibitor withdrawal; compensation likely initiated at the 32-cell stage when supernumerary outer cells exhibit molecular characteristics of inner cells. These data identify a novel mechanism specifically governing initial spatial segregation of mouse embryo blastomeres, that is distinct from those directing subsequent inner cell formation, contributing to germane segregation of late blastocyst lineages.

Immediate and delayed movement of resistant and susceptible adults of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) after short exposures to phosphine.

BACKGROUND: During the last decade, the evaluation of certain behavioral attributes has been utilized as an indicator of resistance to phosphine. In this context, an underappreciated challenge may be the development of behavioral traits that are related with resistance to phosphine such as the movement to refugia and recovery of stored product insects after short exposures. Thus, the aim of the current study was to track the movement of phosphine-resistant and -susceptible adults of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), which is a major pest of stored products, after brief exposures to phosphine. Exposures were followed for extended intervals to assess the recovery patterns and how those patterns are related to known resistance to phosphine. A video-tracking procedure coupled with Ethovision software was used to assess movement after exposure. RESULTS: Overall, we found baseline movement was less for phosphine-resistant T. castaneum, suggesting resistance comes at a considerable fitness cost. In the presence of phosphine (1000 or 3000 ppm), there was a much greater reduction in movement for phosphine-susceptible than phosphine-resistant T. castaneum adults immediately after brief 5-min exposures. Twenty-four hours later, these effects were more variable and less apparent, regardless of the susceptibility level. CONCLUSIONS: The initial knockdown associated with successful fumigation may just be a temporary state whereafter insects shortly resume movement and may be able to seek out refugia from phosphine, thereby promoting the development of resistance. Our results strengthen a growing consensus that it is the speed to knockdown that truly matters, with quick knockdown indicating slow recovery and a reduced likelihood for the occurrence of resistance. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Collagen-like Motifs of SasG: A Novel Fold for Protein Mechanical Strength.

The Staphylococcus aureus surface protein G (SasG) is associated with host colonisation and biofilm formation. As colonisation occurs at the liquid-substrate interface bacteria are subject to a myriad of external forces and, presumably as a consequence, SasG displays extreme mechanical strength. This mechanical phenotype arises from the B-domain; a repetitive region composed of alternating E and G5 subdomains. These subdomains have an unusual structure comprising collagen-like regions capped by triple-stranded ß-sheets. To identify the determinants of SasG mechanical strength, we characterised the mechanical phenotype and thermodynamic stability of 18 single substitution variants of a pseudo-wildtype protein. Visualising the mechanically-induced transition state at a residue-level by ϕ-value analysis reveals that the main force-bearing regions are the N- and C-terminal 'Mechanical Clamps' and their side-chain interactions. This is tailored by contacts at the pseudo-hydrophobic core interface. We also describe a novel mechanical motif - the collagen-like region and show that glycine to alanine substitutions, analogous to those found in Osteogenesis Imperfecta (brittle bone disease), result in a significantly reduced mechanical strength.

Grain Inoculated with Different Growth Stages of the Fungus, Aspergillus flavus, Affect the Close-Range Foraging Behavior by a Primary Stored Product Pest, Sitophilus oryzae (Coleoptera: Curculionidae).

Although some research has investigated the interactions among stored product insects and microbes, little research has examined how specific fungal life stages affect volatile emissions in grain and linked it to the behavior of Sitophilus oryzae, the cosmopolitan rice weevil. Thus, our goals were to 1) isolate, culture, and identify two fungal life stages of Aspergillus flavus, 2) characterize the volatile emissions from grain inoculated by each fungal morphotype, and 3) understand how microbially-produced volatile organic compounds (MVOCs) from each fungal morphotype affect foraging, attraction, and preference by S. oryzae. We hypothesized that the headspace blends would be unique among our treatments and that this will lead to preferential mobility by S. oryzae among treatments. Using headspace collection coupled with GC-MS, we found the sexual life stage of A. flavus had the most unique emissions of MVOCs compared to the other semiochemical treatments. This translated to a higher interaction with kernels containing grain with the A. flavus sexual life stage, as well as a higher cumulative time spent in those zones by S. oryzae in a video-tracking assay in comparison to the asexual life stage. While fungal cues were important for foraging at close-range, the release-recapture assay indicated that grain volatiles were more important for attraction at longer distances. There was no significant preference between grain and MVOCs in a four-way olfactometer. Overall, this study enhances our understanding of how fungal cues affect the close and longer range foraging ecology of a primarily stored product insect.

Field Trapping and Flight Capacity of Eucosma giganteana (Riley) (Lepidoptera: Tortricidae) in Response to Behaviorally Active Congeneric Semiochemicals in Novel Silflower Agroecosystems.

Silphium integrifolium is a novel perennial crop being developed for oilseed and biofuel in the midwestern US. One of the primary pests in this system is Eucosma giganteana (Lepidoptera: Tortricidae). Little is known about the chemical ecology or flight behavior of E. giganteana, but many semiochemicals have been identified from other closely related Eucosma species. Some of these compounds include: (Z)- and (E)-8-dodecenyl acetate, (E)-9-dodecenyl acetate, (Z)-8-dodecenol, (E,E)-8,10-dodecadienyl acetate, and (Z,E)-9,12-tetradecadienyl acetate. The goals of this study were to evaluate whether any of these compounds could improve capture of E. giganteana on clear sticky cards in the field, and the most attractive volatiles might affect flight behavior on a computer-automated flight mill assay. We found that there was significant attraction to (E)-8-dodecenyl acetate in two years in the field, which may possibly be a component in the pheromone blend for E. giganteana. On flight mills, E. giganteana flew an average of 23 km in a 24 h period. The presence of attractive stimuli (e.g., (E)-8-dodecenyl acetate) had arresting properties and decreasing flight distance on the mill by 78 to 80%. The longest flight distances were registered in the morning (4:00-12:00) and were 1.8-fold greater than flight distances and durations at night (20:00-4:00). (E)-8-dodecenyl acetate may be useful in behaviorally based monitoring and management strategies for E. giganteana. Overall, our research expands the knowledge on the chemical ecology of adult E. giganteana.

A Biomass Pyrolysis Oil as a Novel Insect Growth Regulator Mimic for a Variety of Stored Product Beetles.

As fumigants face increasing regulatory restrictions, resistance, and consumer pushback, it is vital to expand the integrated pest management (IPM) chemical toolkit for stored products. The production of biomass derived insecticides (e.g., bio-oil fraction) from byproducts of biofuel production may be a promising alternative source of chemistries for controlling stored product insects. These potential insecticidal bio-oils were fractionated based on boiling points (ranging from 115 to 230°C in one series and 245-250°C in another). Fractions were analyzed using GC-MS, and were found to be unique in composition. The lethality of these fractions was tested on Tribolium castaneum, Tribolium confusum, and Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae). Fractions were tested at concentrations ranging from 5-260 mg/ml to screen for efficacy against adults for durations of 2-8 hr sprayed on concrete arenas. In addition, a separate assay evaluated adult emergence of larvae after 6 wk with supplemental food in arenas, while repellency was evaluated against four stored product insect species in a laminar wind tunnel. A greenhouse gas (GHG) emissions life cycle assessment was also performed, which found the use of the bio-oil fraction could reduce GHG emissions associated with the insecticide supply chain by 25-61% relative to a fossil-fuel based insecticide or pyrethroid. While adults were largely unaffected, we found that larval emergence was significantly suppressed compared to controls by roughly half or more. We also determined that there was minimal repellency to most fractions by most species. We conclude that the use of bio-oil fractions is a climate-friendly choice that may support IPM programs.

Measuring Digital Pathology Throughput and Tissue Dropouts.

BACKGROUND: Digital pathology operations that precede viewing by a pathologist have a substantial impact on costs and fidelity of the digital image. Scan time and file size determine throughput and storage costs, whereas tissue omission during digital capture ("dropouts") compromises downstream interpretation. We compared how these variables differ across scanners. METHODS: A 212 slide set randomly selected from a gynecologic-gestational pathology practice was used to benchmark scan time, file size, and image completeness. Workflows included the Hamamatsu S210 scanner (operated under default and optimized profiles) and the Leica GT450. Digital tissue dropouts were detected by the aligned overlay of macroscopic glass slide camera images (reference) with images created by the slide scanners whole slide images. RESULTS: File size and scan time were highly correlated within each platform. Differences in GT450, default S210, and optimized S210 performance were seen in average file size (1.4 vs. 2.5 vs. 3.4 GB) and scan time (93 vs. 376 vs. 721 s). Dropouts were seen in 29.5% (186/631) of successful scans overall: from a low of 13.7% (29/212) for the optimized S210 profile, followed by 34.6% (73/211) for the GT450 and 40.4% (84/208) for the default profile S210 profile. Small dislodged fragments, "shards," were dropped in 22.2% (140/631) of slides, followed by tissue marginalized at the glass slide edges, 6.2% (39/631). "Unique dropouts," those for which no equivalent appeared elsewhere in the scan, occurred in only three slides. Of these, 67% (2/3) were "floaters" or contaminants from other cases. CONCLUSIONS: Scanning speed and resultant file size vary greatly by scanner type, scanner operation settings, and clinical specimen mix (tissue type, tissue area). Digital image fidelity as measured by tissue dropout frequency and dropout type also varies according to the tissue type and scanner. Dropped tissues very rarely (1/631) represent actual specimen tissues that are not represented elsewhere in the scan, so in most cases cannot alter the diagnosis. Digital pathology platforms vary in their output efficiency and image fidelity to the glass original and should be matched to the intended application.

Microbial Volatile Organic Compounds from Tempered and Incubated Grain Mediate Attraction by a Primary but Not Secondary Stored Product Insect Pest in Wheat.

There has been a dearth of research elucidating the behavioral effect of microbially-produced volatile organic compounds on insects in postharvest agriculture. Demonstrating attraction to MVOC's by stored product insects would provide an additional source of unique behaviorally-relevant stimuli to protect postharvest commodities at food facilities. Here, we assessed the behavioral response of a primary (Rhyzopertha dominica) and secondary (Tribolium castaneum) grain pest to bouquets of volatiles produced by whole wheat that were untempered, or tempered to 12%, 15%, or 19% grain moisture and incubated for 9, 18, or 27 days. We hypothesized that MVOC's may be more important for the secondary feeder because they signal that otherwise unusable, intact grains have become susceptible by weakening of the bran. However, contrary to our expectations, we found that the primary feeder, R. dominica, but not T. castaneum was attracted to MVOC's in a wind tunnel experiment, and in a release-recapture assay using commercial traps baited with grain treatments. Increasing grain moisture resulted in elevated grain damage detected by near-infrared spectroscopy and resulted in small but significant differences in the blend of volatiles emitted by treatments detected by gas chromatography coupled with mass spectrometry (GC-MS). In sequencing the microbial community on the grain, we found a diversity of fungi, suggesting that an assemblage was responsible for emissions. We conclude that R. dominica is attracted to a broader suite of MVOC's than T. castaneum, and that our work highlights the importance of understanding insect-microbe interactions in the postharvest agricultural supply chain.

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification.

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.

p38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice.

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

Characterizing and predicting sublethal shifts in mobility by multiple stored product insects over time to an old and novel contact insecticide in three key stored commodities.

BACKGROUND: There has been a push to diversify integrated pest management (IPM) programs away from exclusive fumigant use in food facilities. Residual insecticides increasingly have been included among plans. In stored products, sublethal toxicity has been neglected in favor of evaluating direct mortality. Here, we evaluated the movement of Tribolium castaneum, Rhyzopertha dominica, Sitophilus oryzae and Sitophilus zeamais in response to aged residues of an existing (Diacon IGR+® with 11.4% methoprene + 4.75% deltamethrin) and novel (Gravista® with 2.85% methoprene + 1.2% deltamethrin + 33.3% piperonyl butoxide synergist) residual insecticide. RESULTS: Using the maximum labeled rate and two exposure times for each species, we assessed distance moved and velocity on wheat, rice and corn. Assessments were made from commodity residues aged between 0 and 12 months (at 3-month intervals). We found that after exposure, movement was reduced by 50-88% and equally by adults exposed to each insecticide formulation compared to untreated controls. After initial application, predicted distance moved increased from 4 to 14 m then 28 m in a 24 h period at 3 and 12 months post-application, respectively. Effectiveness of each insecticide at suppressing movement generally declined by 9-12 month post-application. CONCLUSIONS: Given the quick and dramatic increases in sublethal movement after initial application, our results suggest that sanitation programs in post-harvest environments are extremely important and it may be beneficial to pair chemical control with monitoring to prevent dispersal of affected insects to new areas of a facility. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Wwc2 Is a Novel Cell Division Regulator During Preimplantation Mouse Embryo Lineage Formation and Oogenesis.

Formation of the hatching mouse blastocyst marks the end of preimplantation development, whereby previous cell cleavages culminate in the formation of three distinct cell lineages (trophectoderm, primitive endoderm and epiblast). We report that dysregulated expression of Wwc2, a genetic paralog of Kibra/Wwc1 (a known activator of Hippo-signaling, a key pathway during preimplantation development), is specifically associated with cell autonomous deficits in embryo cell number and cell division abnormalities. Division phenotypes are also observed during mouse oocyte meiotic maturation, as Wwc2 dysregulation blocks progression to the stage of meiosis II metaphase (MII) arrest and is associated with spindle defects and failed Aurora-A kinase (AURKA) activation. Oocyte and embryo cell division defects, each occurring in the absence of centrosomes, are fully reversible by expression of recombinant HA-epitope tagged WWC2, restoring activated oocyte AURKA levels. Additionally, clonal embryonic dysregulation implicates Wwc2 in maintaining the pluripotent epiblast lineage. Thus, Wwc2 is a novel regulator of meiotic and early mitotic cell divisions, and mouse blastocyst cell fate.

Age-related differences in the translational landscape of mammalian oocytes.

Increasing maternal age in mammals is associated with poorer oocyte quality, involving higher aneuploidy rates and decreased developmental competence. Prior to resumption of meiosis, fully developed mammalian oocytes become transcriptionally silent until the onset of zygotic genome activation. Therefore, meiotic progression and early embryogenesis are driven largely by translational utilization of previously synthesized mRNAs. We report that genome-wide translatome profiling reveals considerable numbers of transcripts that are differentially translated in oocytes obtained from aged compared to young females. Additionally, we show that a number of aberrantly translated mRNAs in oocytes from aged females are associated with cell cycle. Indeed, we demonstrate that four specific maternal age-related transcripts (Sgk1, Castor1, Aire and Eg5) with differential translation rates encode factors that are associated with the newly forming meiotic spindle. Moreover, we report substantial defects in chromosome alignment and cytokinesis in the oocytes of young females, in which candidate CASTOR1 and SGK1 protein levels or activity are experimentally altered. Our findings indicate that improper translation of specific proteins at the onset of meiosis contributes to increased chromosome segregation problems associated with female ageing.

Identifying the Translatome of Mouse NEBD-Stage Oocytes via SSP-Profiling; A Novel Polysome Fractionation Method.

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)-a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.