Folate metabolism and epigenetic programming in preimplantation embryos

Lead Research Organisation: University of Nottingham
Department Name: Sch of Biosciences

Abstract

As above

Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M008770/1 01/10/2015 30/09/2023
1796056 Studentship BB/M008770/1 01/10/2016 30/09/2020 Constance Emily Clare
 
Description Dietary-induced disturbances to one-carbon (1C) metabolism adversely affect the development and lifelong health of humans and animals. Deficiencies in key 1C metabolites, such as folate (vitamin B9), vitamin B12 (cobalamin), vitamin B6 (pyridoxine) and vitamin B2 (riboflavin) can lead to long-term deleterious health consequences. We hypothesise that mutations in 1C genes will affect 1C metabolism and the sensitivity of animals to micronutrient deficiency. With this in mind, the first experimental chapter of my PhD thesis involved an animal study whereby Texel sheep (n = 90) were maintained on a methyl-deficient diet for several weeks and a comprehensive quantitative analysis of 1C metabolites in their liver was conducted to assess the impact of dietary methyl-deficiency on 1C metabolism. In collaboration with the School of Pharmacy, I developed and optimised a hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS/MS) method for the quantification of folates, B-vitamins and 1C-related amines in sheep liver. This method is sufficiently novel, robust and reliable to measure low abundance analytes in a complex biological matrix and may be adapted to measure such compounds in other physiological tissues. The results of this feeding trial clearly demonstrate that moderate dietary methyl-deficiency can affect system-wide 1C metabolism and physiology with potential consequences for long-term health.

Considering the above, ongoing research focuses on 1C metabolism in the bovine preimplantation embryo. Studies within the following three experimental chapters of my thesis assess the impact of different concentrations of essential amino acid, methionine, during in vitro embryo culture on early embryo development. As the precursor for the universal methyl donor in 1C metabolism, S-adenosyl methionine (SAM), we hypothesise that altering methionine concentrations during culture will have downstream epigenetic consequences in embryos via changes to DNA methylation and the regulation of gene expression.

To date, our findings indicate that physiological levels of methionine during culture alter early embryo development. Germinal-vesicle oocytes were matured, fertilised and zygotes cultured to Day 8 in custom-made media containing 0, 10, 50, and 500 µM methionine. Whilst there was no effect of methionine on the proportion of oocytes cleaved following insemination, the proportion Day 7 blastocysts of inseminated and cleaved increased (P<0.001) with methionine up to 50 µM. This indicates that of the concentrations tested, the high physiological methionine concentration (50 µM) appears to be optimal for development. Immunocytochemistry demonstrated that total cell number in Day 8 blastocysts also increased (P<0.001) with methionine up to 50 µM. The proportion of inner cell mass (ICM; ~0.30) was unaffected by methionine. In addition, the proportion of Grade 1 (transferable quality) blastocysts increased (P<0.05) with methionine up to 50 µM.

DNA methylation analysis within the inner cell mass (ICM) and trophectoderm (TE) cell lineages of the Day 8 bovine preimplantation embryo revealed a global decrease in cytosine methylation when methionine concentration is decreased within physiological limits, from 50 to 10 µM, during embryo culture. Ongoing bioinformatic analyses aims to investigate the genome-wide effects of low methionine during culture, with a particular focus on the methylation status of imprinted genes. By way of example, insulin-like growth factor 2 receptor (IGF2R) is an imprinted gene that has been identified as deferentially methylated as a result of methionine deficiency in our analyses. As IGF2R is important for embryonic/placental growth and development, its imprinted status has been implicated in the aetiology of large offspring syndrome (LOS) in various mammalian species. In order to better understand the effect of methionine during culture on the imprinted status of IGF2R, further characterisation of the differentially methylated region (DMR2) and antisense non-coding RNA transcript (AIRN) that regulates IGF2R expression remains the focus of foregoing experiments in our laboratory.
Exploitation Route Considering that physiological levels of methionine during culture alter early embryo development, it is important to assess how the epigenome of embryos is affected by subtle changes in methionine concentration. This is of particular importance in the clinical field of assisted reproduction (IVF) in humans and the commercial production of livestock since we aim to improve understanding of how the in vitro culture environment may programme early embryo development, predisposing humans and animals to detrimental adult health phenotypes, such as cardiovascular disease, type II diabetes and cancer. This research is also relevant to humans and animals exhibiting dietary methionine deficiency. In humans, this specifically refers to vegetarians and vegans who may be at increased risk.

Elucidating the effect of low methionine during periconceptional development on the methylation status of imprinted genes, such as IGF2R, offers some insight into the long-lasting adult health outcomes caused by subtle nutrient deprivation, as described by the developmental origins of adult health and disease (DOHaD) hypothesis.
Sectors Agriculture, Food and Drink,Chemicals,Environment,Pharmaceuticals and Medical Biotechnology

URL https://pubmed.ncbi.nlm.nih.gov/33673278/
 
Description 'Open Science' opens doors: How #Scidata18 helped me unlock career opportunities 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact As #SciData19 approaches, this article was published to Springer Nature's 'The Source' blog, a page providing insight and discussion on publishing in the academic world. I was invited to share my personal story of how attending last year's conference (#SciData18) has unlocked some exciting career opportunities in the field of 'Open Science'.
Year(s) Of Engagement Activity 2019
URL https://www.springernature.com/gp/researchers/the-source/blog/blogposts-open-research/open-science-o...
 
Description 'Open Science' opens doors: How #Scidata18 helped me unlock career opportunities 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A lightning talk about my personal story, 'Open Science opens doors: How #Scidata18 helped me unlock career opportunities', is to be presented at 'Better Science through Better Data 2019' on 6th November 2019. The annual one-day event, hosted by Springer Nature and the Wellcome Trust, brings together international researchers to discuss innovative approaches to data sharing, open science and reproducible research, together with demonstrations of exemplary projects and tools.
Year(s) Of Engagement Activity 2019
URL https://doi.org/10.5281/zenodo.3527201
 
Description A talk at an international meeting on one carbon metabolism and clinical outcomes 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 12th International conference on One carbon metabolism, B vitamins and homocysteine held in Reus, Catalonia in June 2019. Attended by around 300 delegates including fellow scientists, practitioners, health-care professionals and policy makers from across the world. Delivered a 30 minute talk and discussion on genetics, one carbon metabolism and epigenetic programming. Considerable debate around long-term health implications and some discussion around European policies towards fortification of flour-based foodstuffs with folic acid.
Year(s) Of Engagement Activity 2019
URL http://wwwa.fundacio.urv.cat/congressos/homocysteine-2019/
 
Description Benefits and risks to careers of practising open research 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This post was my winning entry to the 'Better Science Through Better Data' writing competition in 2018. This entry led to my invitation to attend the #Scidata18 conference and further participation as a contributing writer to Springer Nature's Research Data blog page.
Year(s) Of Engagement Activity 2019
URL https://researchdata.springernature.com/users/206394-connie-clare/posts/43156-benefits-and-risks-to-...
 
Description Book Sprint Success: A Team Writing Exercise For The Win 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact On 10th July our multidisciplinary team of volunteers checked-in at a hotel in the Hague, Netherlands, to participate in a three-day book sprint to draft the first version of 'Engaging Researchers with Data Management: The Cookbook.' This blog article was written and published to the Research Data Alliance website to report on the book sprint, and to explain the positive aspects and challenges associated with collaborative writing exercises.
Year(s) Of Engagement Activity 2019
URL https://www.rd-alliance.org/blogs/book-sprint-success-team-writing-exercise-win.html
 
Description Community building to champion Open Research 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact An article written for publication on the University of Nottingham Postgraduate Placement blog to inform about my Biotechnology and Biological Sciences Doctoral Training Programme (BBSRC DTP) Professional Internship Placement (PIP).
Year(s) Of Engagement Activity 2019
URL http://blogs.nottingham.ac.uk/postgraduateplacements/2019/12/11/community-building-to-champion-open-...
 
Description Community-based models for engaging researchers with research data 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Upon completion of my internship, on 23rd September 2019 I was invited to the conference, 'Researchers Engagement: Use cases for engaging researchers with research data', at the University of Vienna, Austria, to give a keynote talk about the use of 'Community-based models for engaging researchers with research data.'
Year(s) Of Engagement Activity 2019
URL https://doi.org/10.5281/zenodo.3457080
 
Description Data (Stewardship) Makes The Difference: Towards A Community-Endorsed Data Stewardship Profession 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact The 14th RDA Plenary, held in Helsinki, invited research, industry and policy experts from all corners of the globe to explore diverse ways by which 'Data Makes the Difference'. As this year's theme addressed the potential of research data to facilitate decision making, tackle grand societal challenges and engage citizens in knowledge creation for the betterment of society, this article was published to highlight the importance of 'Professionalising data stewardship in the RDA community' in helping to achieve these aims.
Year(s) Of Engagement Activity 2019
URL https://www.rd-alliance.org/blogs/data-stewardship-makes-difference-towards-community-endorsed-data-...
 
Description Data in everyday life: A Reflection on Data Justice 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A blog article published to Springer Nature's Research Data blog page to celebrate 'Love Data Week 2019'. The piece provides a reflection on 'data justice', i.e. fairness in the way people are made visible and represented as a result of their production of and interaction with digital data.
Year(s) Of Engagement Activity 2019
URL https://researchdata.springernature.com/users/206394-connie-clare/posts/44025-data-in-everyday-life-...
 
Description Genetics of one-carbon metabolism: Explaining inter-individual and ethnic variation in epigenetic responses to peri-conceptional diet 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract submitted in collaboration with PhD Student, Amey Brassington, for 'Fertility 2018' conference - Society for Reproduction and Fertility, Liverpool, UK

Genetics of one-carbon metabolism: Explaining inter-individual and ethnic variation in epigenetic responses to peri-conceptional diet

Introduction
Dietary disturbances to one-carbon metabolism during the peri-conceptional period can lead to early miscarriage and compromise offspring health, in part due to epigenetic dysregulation of chromatin methylation in developmentally important genes. We hypothesise that inter-individual and ethnic variability in epigenetic gene regulation arises because of single-nucleotide polymorphisms (SNPs) within metabolic genes, epigenetic regulators and differentially-methylated target DNA sequences. We report on ongoing studies that seek to test this hypothesis in sheep.

Methods
Twenty unrelated Texel ewes were sequenced to a depth of 30X in two pools. Reads were mapped to the reference sheep genome assembly (Oar_v3.1), identified variants were used to construct an Illumina Infinium? iSelect? Custom Array (6,000 probes), capturing 140 one-carbon metabolism genes and 116 related epigenetic regulators. This array was used to SNP profile 270 Texel-sheep liver samples for which we also quantified 116 one-carbon and related metabolites by LC-MS/MS. SNP variants were also determined in 190 unrelated pedigree sheep from three traditional UK breeds (Suffolk, Bluefaced-Leicester and Swaledale).

Results and Discussion
Genetic quantitative trait association analyses (by GenABEL in R) on metabolite concentrations were adjusted for substrates, co-factors and allosteric regulators. Following Bonferroni correction, and re-estimation in ASReml, a prioritised list of 27 SNPs in 15 metabolic genes and 54 SNPs in 25 epigenetic-regulator genes was generated, consistent across covariate categories. Of these, four SNPs (upstream variants) in PCMTD1 (protein methylation; allele frequencies (q) of 0.43) and three SNPs (two missense variants; q of 0.26 for each) in RRP8 (a histone methyltransferase) were linked to tetrahydrofolate. K-means clustering and phylogenetic analyses indicated a high degree of breed divergence for our 256 genes; with the three UK breeds clustering separately from Texels. Future studies will confirm/extend prioritised SNPs in one-carbon deficient animals and undertake a prospective proof-of-concept pregnancy study with each breed.

Funded by BBSRC (BB/K017810/1)
Year(s) Of Engagement Activity 2018
 
Description How To Build A Community Of Data Champions: Six Steps To Success 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact During my BBSRC-funded professional internship placement undertaken at Delft University of Technology, I worked on the 'Data Champions Programme'. This initiative represents a community-based model that has been implemented across all faculties and departments at TU Delft in an aim to improve research data management (RDM) practice. I developed a 'toolkit to help other academic institutions to build a network of volunteers and this has been published on the Research Data Alliance website to reach a wider audience.
Year(s) Of Engagement Activity 2016,2019
URL https://www.rd-alliance.org/blogs/how-build-community-data-champions-six-steps-success.html
 
Description Mentoring open science: We can all become Guardians of the (Data) Galaxy 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact A news article reporting on the events of 'Better Science through Better Data', the annual one-day conference hosted by Springer Nature and the Wellcome Trust that brings almost 400 international delegates to London's Natural History Museum to champion open science.
Year(s) Of Engagement Activity 2019
URL https://researchdata.springernature.com/users/206394-connie-clare/posts/43157-mentoring-open-science...
 
Description Methionine, one-carbon metabolism and bovine preimplantation embryo development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Abstract submitted for '12th International Conference on one-carbon metabolism, B vitamins and Homocysteine' in Southern Catalonia, Spain (8th - 12th June)

Methionine, one-carbon metabolism and bovine preimplantation embryo development
Constance E Clare1, Desmond Tutt1, Wing Yee Kwong1 and Kevin D Sinclair1
1University of Nottingham, Sutton Bonington, Leicestershire, LE12 5RD

Introduction
Dietary deficiency of one-carbon metabolites during the periconceptional period can lead to epigenetic alterations in chromatin methylation affecting genes critical for early development. Mammalian embryo culture media are empirically derived with methionine concentrations ranging from 0 to >200 µM between formulations. We hypothesised that altering concentrations of methionine, the precursor of universal methyl donor, S-adenosylmethionine, will affect embryo development and the epigenetic regulation of gene expression during bovine in vitro culture with long-term developmental and metabolic consequences for offspring health. Initial findings from ongoing studies are presented.

Methods
Abattoir derived germinal-vesical oocytes were matured and fertilised, and zygotes cultured to Day 8, in a custom-made media containing 0 (non-physiological), 10 (low), 50 (high) and 500 µM (supraphysiological) methionine in 9 replicated experiments. Morphological stage and grade of Day 7/8 blastocysts was assessed visually according to IETS criteria. Cell lineage (i.e. inner-cell mass (ICM) and trophectoderm) allocation was determined by whole-mount immunofluorescence for SOX2 and DAPI, and sex ratio was determined by RT-PCR using primers for SRY and BSP. Proportions were analysed using logistic regression assuming binomial errors and count data assumed Poisson errors. Data are presented as adjusted means ± standard error of means.

Results and Discussion
Whilst there was no effect of methionine concentration on proportion oocytes cleaved following insemination, the proportion Day 7 blastocysts of both inseminated and cleaved increased (P<0.001) with increasing methionine concentration up to 50 µM, but decreased at 500 µM (e.g. Day 7 of cleaved: 0.03±0.011, 0.10±0.017, 0.16±0.021, 0.08±0.015 for 0, 10, 50 and 500 µM respectively). Similarly, total cell number in Day 8 blastocysts increased (P<0.001) with methionine up to 50 µM but decreased at 500 µM (70±2.5, 84±1.6, 89±1.6 and 71±4.0 cells). However, the proportion of these cells within the ICM (~0.30) was unaffected by methionine concentration. Ordinal regression analyses revealed that the proportion morphological Grade 1 (transferrable) blastocysts also increased (P<0.05) with methionine up to 50 µM but decreased at 500 µM (0.18±0.100, 0.49±0.085, 0.61±0.080, 0.45±0.113). Based on just three biological replicates, the proportion male embryos was statistically similar for each of the four methionine concentrations (0.23±0.095, 0.56±0.078, 0.60±0.078 and 0.50±0.066).

These findings demonstrate that physiological levels of methionine during culture alter early embryo development. The effect of methionine concentration on DNA and histone methylation remains the focus of ongoing studies. These experiments involve immunosurgery of Day 8 blastocysts to isolate the epiblast, hypoblast and trophectoderm for measurement of chromatin methylation in each of these lineages.


Funder: BBSRC DTP
Year(s) Of Engagement Activity 2019
 
Description Methionine, one-carbon metabolism and bovine preimplantation embryo development 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Abstract submitted for 'Fertility 2020' Conference in January. Invitation to deliver a short paper presentation in the SRF Student Prize session:

Introduction
Deficiency of one-carbon metabolites during the periconceptional period can lead to epigenetic alterations in chromatin methylation affecting genes
critical for early development [1]. Methionine concentrations in mammalian embryo culture media range from 0 to >200µM between formulations.
We hypothesise that altering concentrations of methionine, the precursor of the universal methyl donor, S-adenosylmethionine (SAM), will affect
development and the epigenetic regulation of gene expression during bovine embryo culture with long-term health consequences. Here we report
the effects of altered methionine concentrations on preimplantation embryo development.

Methods
Germinal-vesical oocytes were matured, fertilised and zygotes cultured to D8 in custom-made media containing 0, 10, 50, and 500µM methionine
across 8 replicates. Morphological stage and grade of D7/8 blastocysts was assessed according to IETS [2]. Cell lineage allocation was
determined by immunofluorescence for SOX2, NANOG and SOX17. Sex ratio was determined by RT-PCR. Proportions were analysed using
logistic regression assuming binomial errors and count data assumed Poisson errors.

Results
There was no effect of methionine on proportion oocytes cleaved following insemination. The proportion D7 blastocysts of inseminated and
cleaved increased (P<0.001) with increasing methionine up to 50µM, but decreased at 500µM. Total cell number in D8 blastocysts increased
(P<0.001) with methionine up to 50µM, but decreased at 500µM (70±2.5, 84±1.6, 89±1.6 and 71±4.0 for 0, 10, 50 and 500µM, respectively).
Proportion of inner cell mass (ICM; ~0.30) was unaffected by methionine. Proportion of Grade 1 blastocysts increased (P<0.05) with methionine up
to 50µM, but decreased at 500µM (0.18±0.100, 0.49±0.085, 0.61±0.080 and 0.45±0.113). The proportion male embryos was statistically similar for
each concentration (0.23±0.095, 0.56±0.078, 0.60±0.078 and 0.50±0.066).

Conclusions
Altered methionine concentrations (within physiological limits) affects preimplantation embryo development. Consequences for epigenetic gene
regulation, based on DNA extracted from the immunodissected ICM and trophectoderm cell lineages, are under investigation. Funder: BBSRC
(BB/K017810/1) and DTP.
Year(s) Of Engagement Activity 2020
URL https://fertilityconference.org/
 
Description STEM Ambassador: Gold Crest Award Mentor 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact As a STEM Ambassador, I am involved in mentoring A-level students undertaking a Gold Crest Award. This means that I volunteer my time, enthusiasm and experience to help demonstrate the value of science, technology, engineering, and mathematics subjects in schools and provide support for students undertaking independent STEM research projects by offering one-to-one guidance and advice. I have recently been involved with two student projects which involved writing reports on 'the nutritional outcomes of drinking protein shakes' and 'an overview of diabetes: types, causes, symptoms and treatments'.
Year(s) Of Engagement Activity 2018
 
Description Talks at Fertility 2020: an international meeting involving basic scientists, clinicians, nurses and health care professionals - first of two 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Over 1000 delegates attended this event in Edinburgh. Talk initiated questions and discussion - increased awareness of importance of parental diet and nutrition during the periconceptional period. Of interest to IVF clinics and health-care professionals more generally.
Year(s) Of Engagement Activity 2020
URL https://fertilityconference.org/
 
Description Webinar: Engaging Researchers with Data Management: The Cookbook 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact In this Webinar, James Savage, Connie Clare and Raman Ganguly presented an overview of 'Engaging Researchers with Data Management: The Cookbook', a Research Data Alliance (RDA) output from the Libraries for Research Data Interest Group that contains 24 case studies on how institutions can effectively engage with researchers about research data. These case studies were selected from over 90 responses to a global survey of institutional support strategies conducted in early 2019, and are based on interviews with the staff members directly involved, each highlighting the essential ingredients that led to their strategy's success, and the challenges encountered along the way. The Webinar will also explore how the project developed from its inception at RDA P12 in Gaborone to completion by P14 in Helsinki, and discuss ongoing issues around engaging researchers with RDM.
Year(s) Of Engagement Activity 2020
URL https://www.rd-alliance.org/webinar-engaging-researchers-data-management-cookbook