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

Abstract

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.

Age‐related differences in the translational landscape of mammalian oocytes

“…Four individual replicates were used for YF samples and three for AF (200 post-NEBD oocytes/sample). Using the SSP-profiling method (Masek et al, 2020) combined with RNA-Seq, we analysed mRNA content in polysomal and non-polysomal fractions from oocytes of the two different female groups.…”

“…All steps for this protocol were detailed in Masek et al (2020). Briefly, 200 oocytes per sample were collected in the presence of cycloheximide.…”

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

“…Exploiting alternative, oligo-dT versus random hexamer, cDNA synthesis priming strategies we uncovered evidence of cytoplasmic Wwc2 mRNA poly-adenylation, usually associated with increased translational efficiency of functionally important proteins during meiotic maturation (Salles et al, 1992) - Figure 3A. Consultation of our previously published assay of polysome associated transcripts during mid-meiotic maturation supported this interpretation, reporting ∼40% polysome association of detectable Wwc2 transcripts (Koncicka et al, 2018;Masek et al, 2020). We then confirmed by Q-RT-PCR that our siRNA construct could elicit robust Wwc2 transcript knockdown in microinjected GV oocytes blocked from re-entering meiosis I (using the cAMP/cGMP phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine/ IMBX) but then permitted to in vitro mature (IVM -via IBMX removal) to the MII equivalent stage (Figures 3B,C).…”

Expression of lamin C2 in mammalian oocytes

“…However, we detected Lmnc2 mRNA in the testicular and juvenile ovarian samples, but not in the fully grown GV oocytes ( Fig 4A). Next we validated our RT-PCR results by treating fully grown GV oocytes with the translational repressor cycloheximide PLOS ONE (CHX) [18][19][20][21][22]. LMN C2 protein levels were unaffected by inhibition of translation ( Fig 4B and 4C), indicating stability of the LMN C2 protein at this oocyte development stage and independence of LMN C2 protein level on new translation.…”

“…Oocytes were washed in PBS (Sigma-Aldrich) with polyvinyl alcohol (PVA, Sigma-Aldrich) and frozen to -80˚C. An exact number of oocytes (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and small piece of liver were lysed in 10 μL of 1x Reducing LDS Loading Buffer (lithium dodecyl sulfate sample buffer NP 0007 and reduction buffer NP 0004, Thermo Fisher Scientific, Waltham, MA, USA) and heated at 100˚C for 5 min. Proteins were separated by gradient precast 4-12% SDS-PAGE gel (NP 0323, Thermo Fisher Scientific) and transferred to Immobilon P membrane (IPVD 00010, Millipore, Merck group, Darmstadt, Germany) using a semidry blotting system (Biometra GmbH, Analytik Jena, Jena, Germany) for 25 min at 5 mA/cm -2 .…”