64 OOPLASMIC TRANSFER AFTER INTERSPECIES NUCLEAR TRANSFER: PRESENCE OF FOREIGN MITOCHONDRIA, PATTERN OF MIGRATION, AND EFFECT ON EMBRYO DEVELOPMENT
Bibliographic record
Abstract
Interspecies somatic cell nuclear transfer (iSCNT) using the bovine cytoplast as universal recipient has potential applications in the conservation of exotic species. However, an in vitro developmental block has been observed using this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could be a cause for the embryonic developmental arrest. A series of experiments were conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of cloned embryos. In Experiment 1, we examined the effect of combining the technique of ooplasmic transfer (OT) with somatic cell nuclear transfer (SCNT) in the bovine model. In addition, presence and pattern of migration of foreign mitochondria after OT were examined by MitoTracker® (Molecular Probes, Inc., Eugene, OR, USA) staining. In Experiment 2, we examined the effect of transferring caprine ooplasm into bovine enucleated oocytes (iOT) used as recipients for goat iSCNT. Ooplasm from donor oocytes was aspirated until the oolema was ruptured and filled about 200 µm of the micropipette. Aspirated ooplasm was injected into recipient oocyte; the oolema of the recipient oocyte was also ruptured by partial aspiration into the micropipette to ensure mixing. Mean cleavage rates and embryo development were compared by chi-square analysis. Percentages (except for parthenogenic controls) were calculated from number of fused couplets. In Experiment 1, there was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst (BLST), or hatched blastocyst (HCHD) embryos (Table 1). MitoTracker Green FM staining of donor oocytes used for OT revealed foreign mitochondria were introduced by the procedure. Their pattern of distribution remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. However, when goat ooplasm was injected into bovine enucleated oocytes used for iSCNT, there was a significant reduction in fusion (52 vs. 82%) and cleavage rates (55 vs. 78%) (P < 0.05). In addition, the procedure of iOT prior to iSCNT was not effective in overcoming the 8- to 16-cell in vitro developmental block and only parthenogenic cow and goat controls reached blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively (Table 1). This study demonstrates that foreign mitochondria are introduced at the time of OT and these mitochondria remain in a cluster without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at genomic activation. To our knowledge, this is the first scientific report of iOT used in combination with iSCNT in an attempt to overcome the in vitro developmental block. Further research is needed to determine characteristics of foreign mitochondrial dynamics as well as replication of foreign mitochondria introduced into NT embryos. Table 1. Intraspecies (cow) and interspecies (goat-cow) ooplasmic transfer and nuclear transfer
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| 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) | 0.001 | 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 source (direct Gemma or distilled Codex), 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".