Lipid metabolism during the in vitro production of porcine embryos

Currently, the in vitro production (IVP) of porcine embryos suffers from low efficiency and reduced blastocyst quality. Poor outcomes from in vitro matured oocytes and in vitro fertilised embryos have limited the use of assisted reproductive technologies (ARTs) within commercial porcine herds, reducing the potential for global genetic improvement programs. It is believed that this reduced developmental competency compared to in vivo embryos is attributable to altered metabolism resulting from in vitro culture. Improper or incomplete metabolic support from the culture media leads to production of inferior embryos. Much of the prior research centres on metabolism of carbohydrates by oocytes and embryos, with the formulation of media based on this knowledge. However, oocytes and embryos also contain endogenous lipid substrates, and there is a lack of understanding as to how and when these stores are utilised. Lipids are a dense form of energy storage, and there is evidence of their metabolism by oocytes and embryos for energy generation. Porcine oocytes and embryos have higher intracellular lipid content than other domestic livestock species, and this makes them an excellent model for studying aspects of lipid metabolism in vitro. The aim of this study was to examine the impact of lipid metabolism on the acquisition of developmental competence during porcine IVP, and how this is affected by the presence or absence of exogenous carbohydrates. Stimulation or inhibition of the β-oxidation pathway was used to discern the importance of fatty acid oxidation to oocyte maturation and embryo development during in vitro maturation (IVM), in vitro fertilisation (IVF) and in vitro embryo culture (IVC). During IVM, it was identified that porcine oocytes are capable of using different substrates to compensate for deficiencies in others. While pyruvate and glucose are preferentially utilised to support maturation, upregulation of β-oxidation can compensate for a low glucose concentration and an absence of pyruvate to support nuclear maturation. Although there was no discernible decrease in lipid content associated with this, lipids provide such a dense energy reserve that any usage may have been beyond the limit of detection. Inhibition of β-oxidation in the absence of carbohydrates had a greater effect on nuclear maturation compared to inhibition in complete media. This indicates that lipid metabolism plays a minor role during oocyte maturation in the presence of carbohydrates and is likely to be more important when other substrates are deficient. Energy generation prior to fertilisation is an important factor in the developmental outcomes of subsequent embryos. Upregulation of β-oxidation for the duration of IVF increased cleavage rates, but doses above 6mM L-carnitine led to decreased blastocyst development. This effect may be attributable to the antioxidant activity of L-carnitine, with low levels of reactive oxygen species (ROS) being required at fertilisation for normal sperm function and sperm-oocyte interactions. Oocyte incubation in media supplemented with 3mM L-carnitine for an hour prior to insemination increased cleavage and improved cryosurvival of Day 7 embryos after vitrification. While ATP content of oocytes did not increase over this period, it is unclear if lipid content was reduced. Previous studies have shown that L-carnitine treatment of oocytes and embryos decreased lipid content, thereby increasing cryotolerance. It would therefore appear that there is a limited role for β-oxidation during the IVF period itself, although upregulation immediately prior to fertilisation may have beneficial effects on metabolic processes and may provide antioxidant protection leading to improved development in early cleavage stage embryos. During embryo culture, there was a greater effect of upregulating lipid metabolism seen in the absence of carbohydrate substrates than in complete media. However, this could not support embryo development to the same extent as carbohydrate substrates. Changing nutrient requirements of embryos has led to the development of sequential media, leading to the production of better quality IVP embryos. Upregulation of β-oxidation for the first three days of culture in a single media system increased embryo quality to the same extent as a sequential carbohydrate media system, implying there is some level of plasticity to embryo metabolism allowing for adaptability to different substrates. Inclusion of L-carnitine for either a three day period or the duration of culture increased cryosurvival, suggesting decreased lipid content due to increased β-oxidation activity. Similarly for oocyte maturation, β-oxidation appears to be able to compensate for carbohydrate deficiencies during embryo culture to some extent, and oxidation of lipids has a greater role in promoting embryo quality over increasing production rates. The findings reported in this thesis represent a contribution to the understanding of lipid metabolism during the in vitro production of porcine embryos. These results provide evidence to support a level of adaptability of porcine oocytes and embryos to different substrates available during maturation and culture. There is a preference shown for carbohydrates substrates, with the ability to utilise lipids to compensate for certain deficiencies. This would justify the inclusion of co-factors of lipid metabolism such as L-carnitine in culture media, to ensure that any deficiencies in other substrates might be corrected for and to promote higher embryo quality. Upregulation of β-oxidation also increased the cryosurvival of porcine embryos following vitrification, with this being a major development in the global movement of superior genetics for herd improvement programs. These findings will also have implications for improving in vitro culture of oocytes and embryos of other species, most notably advancements in human ARTs where research is predominantly limited to work in animal models. The understanding of how lipids are metabolised alongside exogenous carbohydrates will contribute to improving media formulations for better metabolic support in vitro, further to improving embryo production and quality

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.This work was supported from the National Collaborative Research Infrastructure (NCRIS) through the Australian Phenomics Network in Australi

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: A multi-center evaluation

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.This work was supported by the National Collaborative Research Infrastructure (NCRIS) via the Australian Phenomics Network (APN) (to Gaetan Burgio and Paul Thomas), by an Institutional Development Award (PI: Shelley Smith) P20GM103471 (to CBG, RMQ, DWH, JDE, and RR), by NIGMS 1P30GM110768-01 and P30CA036727 (as part of support to University of Nebraska Mouse Genome Engineering and DNA Sequencing Cores), the British Heart Foundation FS12-57, FS12/57/29717, and CH/13/2/30154 and the program grant RG/15/12/31616 (to Kathryn Hentges and Bernard Keavney), the Wellcome Trust grants 107849/Z/ 15/Z, 097820/Z11/B, and 105610/Z/14/Z, the Medical Research Council MR/ N029992/1 (to DB and CBL), the National BioResource Project of Ministry of Education, Culture, Sports, Science and Technology/Japan Agency for Medical Research and Development (MEXT/AMED), Japan, the Canadian Institutes of Health Research MOP#142452 (MCB and LJM). LJM is a member of the Research Centre of the McGill University Health Centre which is supported in part by FQRS. Dr. William Thompson was supported by the Indiana Clinical and Translational Sciences Institute, funded in part by grant #UL1 TR001108 from the National Institute of Health (NIH), National Center for Advancing Translational Sciences, Clinical and Translational Sciences Award. KC Kent Lloyd is supported by the NIH (UM1OD023221), and work contributed by staff from the UC Davis Mouse Biology Program (MBP) is supported by a grant from the American College of Laboratory Animal Medicine. The work contributed from Xiande Liu, Chad Smith, Eric Jonasch, Xuesong Zhang, and Jan ParkerThornburg is supported by the NIH under the award number P30CA16672 (XL, CS, EJ, XZ, JPT) and R50CA211121 (JPT). Joseph Miano is supported by the NIH under the award number HL138987. R Sedlacek was supported by LM2015040 (Czech Centre for Phenogenomics), CZ.1.05/1.1.00/02.0109 (BIOCEV), and CZ.1.05/2.1.00/19.0395 by the Ministry of Education, Youth and Sports (MEYS) and by Academy of Sciences of the Czech Republic (RVO 68378050). David Ray was supported by a Wellcome Trust Investigator (107849/Z/15/Z) and the Medical Research Council (MR/P011853/1 and MR/P023576/) grants. Andrew Loudon was supported by a Wellcome Trust Investigator (107849/Z/15/Z), Biotechnology and Biological Sciences Research Council (BB/N015584/1), Medical Research Council (MR/P023576/1). The work contributed from Gloria Lopez-Castejon is supported by the Wellcome Trust (104192/Z/14/Z) and the Royal Society. Pilar Alcaide was supported by the NIH (HL 123658). The work contributed from Surinder K. Batra is supported by the NIH under the award number P01 CA217798

Two randomized trials of effect of live attenuated influenza vaccine on pneumococcal colonization

The human nasopharynx is frequently colonized by Streptococcus pneumoniae (the pneumococcus), serving as the reservoir for transmission, a state which necessarily precedes invasive pneumococcal infection. Influenza infection increases pneumococcal colonization density and dysregulates host immune responses, increasing the risk of secondary bacterial pneumonia and death

Detectable Clonal Mosaicism from Birth to Old Age and its Relationship to Cancer

Clonal mosaicism for large chromosomal anomalies (duplications, deletions and uniparental disomy) was detected using SNP microarray data from over 50,000 subjects recruited for genome-wide association studies. This detection method requires a relatively high frequency of cells (>5–10%) with the same abnormal karyotype (presumably of clonal origin) in the presence of normal cells. The frequency of detectable clonal mosaicism in peripheral blood is low (<0.5%) from birth until 50 years of age, after which it rises rapidly to 2–3% in the elderly. Many of the mosaic anomalies are characteristic of those found in hematological cancers and identify common deleted regions that pinpoint the locations of genes previously associated with hematological cancers. Although only 3% of subjects with detectable clonal mosaicism had any record of hematological cancer prior to DNA sampling, those without a prior diagnosis have an estimated 10-fold higher risk of a subsequent hematological cancer (95% confidence interval = 6–18)

Phenotype harmonization and cross-study collaboration in GWAS consortia: the GENEVA experience

Genome-wide association study (GWAS) consortia and collaborations formed to detect genetic loci for common phenotypes or investigate gene-environment (G*E) interactions are increasingly common. While these consortia effectively increase sample size, phenotype heterogeneity across studies represents a major obstacle that limits successful identification of these associations. Investigators are faced with the challenge of how to harmonize previously collected phenotype data obtained using different data collection instruments which cover topics in varying degrees of detail and over diverse time frames. This process has not been described in detail. We describe here some of the strategies and pitfalls associated with combining phenotype data from varying studies. Using the Gene Environment Association Studies (GENEVA) multi-site GWAS consortium as an example, this paper provides an illustration to guide GWAS consortia through the process of phenotype harmonization and describes key issues that arise when sharing data across disparate studies. GENEVA is unusual in the diversity of disease endpoints and so the issues it faces as its participating studies share data will be informative for many collaborations. Phenotype harmonization requires identifying common phenotypes, determining the feasibility of cross-study analysis for each, preparing common definitions, and applying appropriate algorithms. Other issues to be considered include genotyping timeframes, coordination of parallel efforts by other collaborative groups, analytic approaches, and imputation of genotype data. GENEVA's harmonization efforts and policy of promoting data sharing and collaboration, not only within GENEVA but also with outside collaborations, can provide important guidance to ongoing and new consortia

Impact of maternal antibodies and microbiota development on the immunogenicity of oral rotavirus vaccine in African, Indian, and European infants: a prospective cohort study

Identifying risk factors for impaired oral rotavirus vaccine (ORV) efficacy in low-income countries may lead to improvements in vaccine design and delivery. We measured maternal rotavirus antibodies, environmental enteric dysfunction (EED), and bacterial gut microbiota development among infants receiving two doses of Rotarix in India (n = 307), Malawi (n = 119), and the UK (n = 60), using standardised methods across cohorts. ORV shedding and seroconversion rates were significantly lower in Malawi and India than the UK. Maternal rotavirus-specific antibodies in serum and breastmilk were negatively correlated with ORV response in India and Malawi, and this was mediated partly by a reduction in ORV replication. In the UK, ORV replication was not inhibited despite comparable maternal antibody levels. In both India and Malawi, pre-vaccination microbiota diversity was negatively correlated with ORV immunogenicity, suggesting that high early-life microbial exposure may contribute to impaired vaccine efficacy

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Investigation of SARS-CoV-2 faecal shedding in the community: a prospective household cohort study (COVID-LIV) in the UK

Background SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown. Methods In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR. Results A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding. Conclusions Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2