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RNA interference (RNAi) has been used for selective degradation of an mRNA transcript or inhibiting its translation to a functional protein in various species. Here, we applied the RNAi approach to suppress the expression of the maternal transcript C-mos and embryonic transcripts Oct-4 in bovine oocytes and embryos respectively, using microinjection of sequence-specific double-stranded RNA (dsRNA). For this, 435 bp C-mos and 341 bp Oct-4 dsRNA were synthesized and microinjected into the cytoplasm of immature oocytes and zygotes respectively. In experiment 1, immature oocytes were categorized into three groups: those injected with C-mos dsRNA, RNase-free water and uninjected controls. In experiment 2, in vitro produced zygotes were categorized into three groups: those injected with Oct-4 dsRNA, RNase-free water and uninjected controls. The developmental phenotypes, the level of mRNA and protein expression were investigated after treatment in both experiments. Microinjection of C-mos dsRNA has resulted in 70% reduction of C-mos transcript after maturation compared to the water-injected and uninjected controls (P<0.01). Microinjection of zygotes with Oct-4 dsRNA has resulted in 72% reduction in transcript abundance at the blastocyst stage compared to the uninjected control zygotes (P<0.01). Moreover, a significant reduction in the number of inner cell mass (ICM) cells was observed in Oct-4 dsRNA-injected embryos compared to the other groups. From oocytes injected with C-mos dsRNA, 60% showed the extrusion of the first polar body compared to 50% in water-injected and 44% in uninjected controls. Moreover, only oocytes injected with C-mos dsRNA showed spontaneous activation. In conclusion, our results demonstrated that sequence-specific dsRNA can be used to knockdown maternal or embryonic transcripts in bovine embryogenesis.
Department of Animal Science, Biotechnology and Reproduction of Farm Animals, Georg-August-University Goettingen, Burckhardtweg, Göttingen, Germany.
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Department of Animal Science, Biotechnology and Reproduction of Farm Animals, Georg-August-University Goettingen, Burckhardtweg, Göttingen, Germany.
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In brief
In the present study the sustainable effect of L-carnitine during the culture period on the post-transfer development was investigated. Taken together, we uncovered direct effects of L-carnitine on the bioenergetic profile of day 7 blastocysts along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.
Abstract
L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid-free conditions, one group with LC (LC embryos) and the control group without LC (control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates as regards early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analyses via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP-dependent activity, cellular stress, and respiration. Pathways like oxidative phosphorylation and thermogenesis, extracellular matrix receptor signaling, PI3K-Akt, and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in LC embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC embryos. In summary, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.
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Selecting developmentally competent oocytes and zygotes based on their morphology is more often influenced by personal judgments and lacks universal standards. Therefore, this experiment was conducted to investigate the rate of development and mRNA level of dielectrophoretically separated oocytes and zygotes to validate dielectrophoresis (DEP) as non-invasive option for selection of oocytes and zygotes. In the first experiment, metaphase II oocytes with (PB+) and without (PB−) first polar body and zygotes were subjected to DEP at 4 MHz and 450 μm electrode distance and classified into fast, very fast, slow, and very slow depending on the time elapsed to reach one of the electrodes in the electric field. Parthenogenetic activation was employed to monitor the embryonic development of dielectrophoretically classified oocytes. The result revealed that at 6 and 7 days of post-activation, the blastocyst rate of very slow dielectrophoretic PB+ and PB− oocytes was significantly (P < 0.05) lower than other groups. Similarly, in zygotes, the blastocyst rate at 7 days post-insemination was higher (P < 0.05) in the very fast dielectrophoretic categories when compared with the slow and very slow categories. In the second experiment, mRNA level was analyzed in the very fast and very slow dielectrophoretic PB+ oocytes and zygotes respectively using the bovine cDNA microarray. The result showed that 36 and 42 transcripts were differentially regulated between the very fast and very slow dielectrophoretic categories PB+ oocytes and zygotes respectively. In conclusion, dielectrophoretically separated oocytes and zygotes showed difference in the rate of blastocyst development accompanied by difference in transcriptional abundances.
