201 SUPPRESSION OF Suz12 IN BOVINE PREIMPLANTATION EMBRYOS VIA CYTOPLASMIC SMALL INTERFERING RNA INJECTION
Bibliographic record
Abstract
The proper removal of gametic epigenetic marks and coordinated reestablishment of the epigenome are critical to mammalian embryonic development. Suz12 is a member of Polycomb repressive complex 2, known to catalyse trimethylation of lysine 27 on histone 3 (H3K27me3; Pasini et al. 2004); Suz12 has also been shown to interact with Suv39h1 for proper trimethylation of H3K9 (de la Cruz et al. 2007). Our objective in this study was to suppress expression of Suz12 via cytoplasmic injection of small interfering RNA (siRNA) targeted at this gene, and to evaluate the effect on embryo development rates. Bovine zygotes were produced in vitro via standard laboratory procedures. Nineteen hours post-fertilization, presumptive zygotes (n = 3979) were divided into 3 treatment groups: noninjected control (CTL), or injected with a fluorescent dextran marker combined with either a nontargeting siRNA (NULL) or an Suz12-targeting siRNA (SUZ). Embryos were cultured in Bovine Evolve (Zenith Biotech, Canada) with 4 mg mL–1 of BSA (Probumin, Millipore, Billerica, MA) and collected at the 4-cell, 8-cell, morula, and blastocyst stages. Ribonucleic acid was isolated with an RNeasy® Mini Kit (Qiagen, Valencia, CA) from 3 replicates of pooled embryos at each stage (4-cell, n = 15; 8-cell, n = 20; morula, n = 10), except for the blastocyst stage [2 samples (n = 10) collected from the CTL and NULL groups, and 1 sample (n = 3) from the SUZ group]. Each RNA sample was reverse-transcribed into cDNA and diluted for use by quantitative real-time PCR. Relative gene expression levels from each sample were calculated in triplicate using the SYBR Green comparative Ct method (Applied Biosystems, Foster City, CA), adjusted according to individual PCR efficiencies for each primer pair (R2 > 0.95) and normalized to the geometric mean Ct of 3 endogenous controls (GAPDH, YWHAZ, and SDHA), to account for differences in both cell number and amount of total mRNA present in each sample (Goossens et al. 2005). Our data indicate that Suz12 expression was suppressed to undetectable levels in SUZ-treated zygotes at all embryo stages analysed. The blastocyst rate of the SUZ group was extremely low (0.88 ± 0.16% SEM) compared with the CTL (19.87 ± 0.36% SEM) and NULL (5.09 ± 0.36% SEM) groups. Morphologically, SUZ morulae appeared fragmented with fewer larger cells than expected, whereas the NULL and CTL morulae seemed to develop normally. We presume the loss of Suz12 expression during this important developmental time is detrimental to embryo morphology and results in a decreased rate of blastocyst formation. Because of this decrease, we were able to collect only 3 SUZ blastocysts from a total of 227 injected. The microinjection procedure also contributed to significant (P < 0.05) decreases in blastocyst rates of the injected groups as compared with the CTL. Future experiments will explore potential alterations in histone methylation, as well as other epigenetic modifiers in bovine preimplantation embryos, to further elucidate the role of the epigenome in early embryonic development.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".