Progress 04/15/18 to 04/14/20
Outputs
Target Audience:Scientific community Changes/Problems:n/a for final report What opportunities for training and professional development has the project provided?There have been several opportunities for training due to this project, including two presentations at the annual meeting for the Society for the Study of Reproduction (2018 and 2019). Other professional development that has occurred has been several workshops, seminars, and a conference (T4L) attended on improving teaching skills aimed at a career in academia. The project has also encouraged one on one work with professors and other students in the programming skills necessary to process large data sets like the RNA sequencing data we are working with. Unfortunately, because of restrictions surrounding the national and state response to covid-19, Jocelyn was unable to present her work publicly to a lay audience, as we had proposed in her fellowship project. However, she is currently working in a teaching/research post-doctoral position at Utah State, and we hope that she can meet this training goal at a later date as circumstances allow. How have the results been disseminated to communities of interest?This work has been published as part of Jocelyn Cuthbert's dissertation. Two manuscripts are in preparation for submission May 2020 to the journal Molecular Reproduction and Development. What do you plan to do during the next reporting period to accomplish the goals?n/a for final report
Impacts
What was accomplished under these goals?
The student supported by this fellowship has completed her dissertation research and successfully defended her dissertation on April 10, 2020. Here is a summary of accomplishments and major findings: The efficiency of embryo production by somatic cell nuclear transfer (scNT) is far below that of in vitro fertilized embryos (IVF), likely due to the accumulation of errors in genome reprogramming, which may encompass the coding genes for small non-coding RNAs (sncRNAs). While sncRNAs have been reported to be important in mammalian embryo development, their potential functions have not been assessed through the critical maternal-to-embryonic transition (MET) in cattle embryos. The objectives of this study were to examine changes in expression of sncRNA during the MET in bovine IVF embryos, to assess the dysregulation of sncRNA and transcriptome profiles in scNT bovine embryos compared to IVF embryos, and to correlate transcript expression profiles with micro-RNA expression profiles. First, RNA sequencing was utilized to profile sncRNAs in bovine IVF oocytes and 8-cell- and blastocyst-staged bovine embryos. The analyses revealed a strong dynamic shift in relative abundance of differentially expressed sncRNAs, suggesting important roles for sncRNAs during the MET. Next, small and large RNA fractions were isolated from the same samples of cattle embryos produced via IVF or scNT at the 2-cell, 8-cell, morula and blastocyst developmental stages, and from MII oocytes, donor cell fibroblasts and either IVF or scNT blastocyst-derived cells (BDCs). RNA sequencing showed that few sncRNAs were differentially expressed between scNT and IVF embryos, with all significant differences occurring at the morula stage. Differential expression of sncRNAs was apparent between stages of development within type (scNT or IVF), with changes in sncRNA populations appearing to be based on the relative differentiation status of developmental stage. Last, transcriptome data analysis revealed large-scale differences between scNT and IVF embryos at each developmental stage examined. Interestingly, altered transcripts at the 8-cell stage were associated with biological functions critical for the MET. The abundance of two miRNAs differentially expressed in scNT morulae, miR-34a and miR-345, was negatively correlated with the expression of some predicted mRNA targets. However, broad changes in mRNA expression were not consistently correlated with aberrations in miRNA expression, suggesting that other mechanisms leading to altered expression of mRNA in scNT embryos may be at play.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Cuthbert, J. (Presenter & Author), Russell, S. (Author Only), Polejaeva, I. (Author Only), White, K. L. (Author Only), Benninghoff, A. (Author Only), Research Week: Student Research Symposium, "Somatic cell nuclear transfer in early bovine embryo development is associated with changes in small non-coding RNA species," Utah State University, Logan, UT. (April 8, 2019 - April 12, 2019)
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:Scientific community Changes/Problems:Aim1 of the project addresses the question of whether any small non-coding RNA (sncRNA) targets are differentially expressed in cloned embryos compared to IVF generated embryos. While data processing and analysis for that project were still ongoing when the Aims for this fellowship were designed, we have since completed the analysis of the data. Our recently published data showed dynamic changes in miRNA, piRNA, and snoRNA during the MZT in IVF bovine embryos (Cuthbert JM, Russell JR, White KL, Benninghoff AD, 2019, Biology of Reproduction, 100(2):p331-350, https://doi.org/10.1093/biolre/ioy190). The significant changes in specific miRNA families population at the MZT supports the hypothesis that miRNA may be regulating transcripts detrimental to embryonic development during this period of development. While ScNT miRNA data processing was still ongoing, the well-known large-scale errors in the reprogramming of the donor nuclear genome made it a reasonable assumption that a subpopulation of regulatory miRNA would be aberrantly expressed in these embryos. However, our results revealed no targets of interest by comparison of cloned versus IVF embryos. Due to this unexpected finding, we propose major changes for Aim 3-1. Aim 3-1 was to utilize Reduced Representation Bisulfite Sequencing to identify aberrant DNA methylation sequencing in ScNT embryos that may be causing aberrant small non-coding RNA expression. In our data, we saw variable changes in snc-RNA expression early on in development, which suggested randomness in the reprogramming errors occurring during early development, when in IVF embryos the maternal genome is still driving development before the MZT takes place. We saw fewer aberrant changes in stages of development post MZT, which could suggest a selection for embryos that correctly underwent the MZT. ScNT embryos that had aberrant errors before the MZT likely would not continue to develop to the morula and blastocyst stage, which may select for embryos that were healthy and correctly reprogrammed. Another possibility is that the errors in epigenetic reprogramming are random in nature and therefore with our experimental design we would not have captured statistically significant patterns of miRNA differences. Due to this finding, DNA methylation information on these embryos would not give us more information on the dynamics of epigenetic reprogramming in relation to the sncRNA expression changes. However, we have the large RNA (mRNA) samples from each sample that were isolated in conjunction with the sncRNA. The sequencing of these large RNA samples could give us valuable information about the way random changes in sncRNA may impact the mRNA transcriptome during the MZT and early development. This would be valuable information to better understand how sncRNA populations could be impacting the expression of mRNA and therefore changing the developmental potential of these embryos. It could also give us valuable information about whether random and variable changes in sncRNA have the ability to functionally change the mRNA landscape, which could impact developmental potential of ScNT embryos. What opportunities for training and professional development has the project provided?There have been several opportunities for training due to this project, including presentations at the annual meeting for the Society for the Study of Reproduction. A poster on the population of small non-coding RNA was presented at the 2018 meeting, and an abstract has been accepted for a second poster presentation for the 2019 meeting happening this July. Other professional development that has occurred has been several workshops, seminars, and a conference (T4L) attended on improving teaching skills aimed at a career in academia. The project has also encouraged one on one work with professors and other students in the programming skills necessary to process large data sets like the RNA sequencing data we are working with. How have the results been disseminated to communities of interest?The RNA sequencing data has not been analyzed, so dissemination has been limited to conference presentations of processed preliminary data and plans of further study. This preliminary data was disseminated at the Society for the Study of Reproduction's 2018 meeting. Once the data has been appropriately analyzed, further dissemination to other communities of interest will begin. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan on completing the processing of the RNA sequencing data and to begin writing up a publication on those results. As RNA sequencing data is being processed and written up this summer, we will begin designing the synthetic micro-RNA to be injected into embryos, and optimize the microinjection process. We will also design and optimize the luciferase-binding assay for those individual micro-RNA, as well as design and optimize PCR primers and antibodies for protein analysis. Aim 2 should be then carried out this fall, with publication written in the spring as the student prepares to graduate at the end of the project period.
Impacts
What was accomplished under these goals?
The major activities on this grant included the completion of sample preparation for sequencing. (Note section on changes below.) The RNA sequencing library preparation was optimized and all samples were processed through the protocol, then run on the Illumina mi-seq sequencer. The completion of these lab activities meets our objective to optimize an RNA sequencing protocol for use with 20 pooled embryos, which provide a very limited input amount of RNA. As the RNA sequencing results are currently being processed, we do not yet have major findings or conclusions yet, but expect to have these results within the next 2 months.
Publications
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