Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

Vitrification, not cryoprotectant exposure, alters the expression of developmentally important genes in in vitro produced porcine blastocysts.

The vitrification of embryos is common practice in advanced livestock breeding programs and in human fertility clinics. Recent studies have revealed that vitrification results in aberrant expression of a number of stress related genes. However, few studies have examined the effect that vitrification has on developmentally important genes, and none have been conducted in porcine embryos. The aim of this study was to determine the effects that different vitrification procedures and cryoprotectant combinations have on the expression of imprinted genes in in vitro produced (IVP) porcine blastocysts. The transcript levels of insulin-like growth factor 2 (IGF2) were lower in all groups of vitrified blastocysts compared to that in non-vitrified control blastocysts (P < 0.05). Expression levels of IGF2 and IGF2 receptor (IGF2R) in blastocysts that had been exposed to cryoprotectants without being vitrified were similar to that in non-vitrified control blastocysts (P > 0.05). Furthermore, blastocysts vitrified using ethylene glycol and propanediol combined, and those vitrified in a closed device, had IGF2R transcript levels similar to that in non-vitrified control blastocysts (P > 0.05). In conclusion, vitrification, but not exposure to cryoprotectants, caused aberrant expression of the imprinted genes IGF2 and IGF2R. Vitrification protocols that incorporated propanediol or a closed device were found to be least disruptive of gene expression in IVP porcine blastocysts.

No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5'-phosphorylated guide DNA (gDNA) from 2.5 ng/µl to 50 ng/µl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

Cryotolerance of porcine blastocysts is improved by treating in vitro matured oocytes with L-carnitine prior to fertilization.

Sufficient generation of adenosine triphosphate (ATP) by oocytes is critical for fertilization and embryo development. The objective of this study was to determine the effects of supplementing media with L-carnitine, a co-factor required for the metabolism of fatty acids, during the peri-fertilization period on embryo development and energy generation. Firstly, in vitro matured (IVM) porcine oocytes were co-incubated with sperm in IVF medium supplemented with 0‒24 mM L-carnitine. The blastocyst formation rate of the control group was greater than those of the L-carnitine groups (P < 0.05), except for the 3 mM L-carnitine group. Subsequently, oocytes and/or sperm were treated without or with 3 mM L-carnitine for either the 1 h pre-IVF oocyte incubation; the pre-IVF sperm preparation; the first 30 min of IVF; or the entire 5.5 h of IVF. Despite similar fertilization rates among the groups, the cleavage rate of the pre-IVF oocyte group was significantly greater than those of the other groups, except for the pre-IVF sperm group. Additionally, the oocyte ATP content and the cryotolerance of the resulting blastocysts were examined following the pre-IVF oocyte treatment. Oocyte ATP content was also similar among the groups (P > 0.05). Following vitrification, the post-warming survival rate of blastocysts derived from L-carnitine-treated oocytes was greater than that of blastocysts derived from untreated oocytes (42.4% vs. 24.9%; P < 0.05). In conclusion, a 1 h oocyte exposure to 3 mM L-carnitine immediately prior to insemination enhanced cleavage and improved the cryotolerance of resulting blastocysts. While the findings are suggestive of a lipolytic action, further studies are required to clarify the contributions of lipid metabolism and oxidative mechanisms to the observed effects of the L-carnitine treatment.