Progress 09/01/13 to 08/31/17
Outputs
Target Audience:The target audiences for this research were1) the community of animal reproductive biologists; 2) the broader group of all reproductive biologists in general, including clinical scientists and practitioners, with interests in fertility/infertility issues, and early embryonic mortality and nuclear reprogramming in somatic cell nuclear transfer embryos in particular; and 3) the animal agriculture production industries that would have interest in cloning animals commercially. Changes/Problems:The major changes we made to the original proposed research werean alteration to the embryo transfer timeline, and a genome-scale rather than a targeted DNA methylation analysis. Rather than transferring at the blastocyst stage, we are proposing FLICA analysis on day 3 to correlate to the time of maternal to zygotic transition where altered epigenetic marks could start wreaking havoc in the embryo. From a technical standpoint, we did not feel that we would be successful transferring at the blastocyst stage as we would not be able to transfer an acceptable number of blastocysts due to low development rates and only transferring a portion of the blastocysts. With this in mind, we felt that analyzing earlier would optimize the number of embryos transferred from each cloning session while continuing to test the hypothesis that altered epigenetic marks causes aberrant gene expression and increased apoptosis leading to eventual embryo failure. The final major challenge to the project is getting the embryo transfer system successful. With a limited number of surrogate females available for transfer, estrus synchronization is critical to success. We believe that we have developed the correct protocol for successful synchronization. What opportunities for training and professional development has the project provided?This project has provided one full-time graduate student and three part-time undergraduate students with excellent training opportunities. Also, a visiting scientist and a post-doc have contributed effort, both technical and scientific in nature. How have the results been disseminated to communities of interest?Thus far, our work has been presented 3 times at 2 scientific meetings. These presentations include a poster at the 2017 ASAS-SSR Joint Symposium and a poster and 3 minute flash talk at the 2017 Society for the Study of Reproduction Annual Conference. Our progress has been reported in local and regional meetings, with definite plans for peer-reviewed publication in the near future. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The goals of this project were to: Adapt a non-invasive apoptosis bioassay for use in porcine embryos generated using SCNT. After developing a slightly modified protocol to detect caspase activity in live oocytes as well as parthenogenote and SCNT embryos at various stages of development, we employed the SR-FLICA™ Poly Caspase Assay Kit from ImmunoChemistry Technologies (Bloomington, MN, USA) in various experiments. We demonstrated a wide range of FLICA fluorescence separation between individual oocytes, early embryos at the maternal-to-zygotic transition and blastocyst stage embryos. In an assessment of apoptosis in 30 individual PA blastocysts, raw FLICA "scores" for individual embryos ranged from 66 (arbitrary fluorescence units) to 255, with an average of 138, and a SD of 47.5. Apoptosis rates tracked nicely with day of development as demonstrated previously: early cleavage-stage embryos showed very little caspase activation, whereas by d4 and d5, more embryos would show up as highly apoptotic. Embryos cultured with the FLICA compound during cleavage-stage development were still able to develop to blastocyst at an acceptable rate, confirming the non-toxic and "non-invasive" aspect of the assay, which was critical to our experimental aims. The kit was then used to detect active caspase activity in day 6 SCNT porcine blastocysts. Blastocysts from 3 replicates (n=13) were categorized as high or low apoptosis if they were in the top or bottom 20% of detected caspase activity, respectively. The FLICA values ranged from 79.6 to 244.8. The low apoptosis blastocysts had an average FLICA value of 94.00 with a standard deviation of 13.40. The high apoptosis blastocysts had an average FLICA value of 221.32 with a standard deviation of 19.01. DNA and RNA was collected from these blastocysts for molecular analysis discussed below. Using porcine parthenogenetic blastocysts as a model (3 replicates, total n=23), no difference in cell number was detected between high and low apoptosis blastocysts (P=0.1839). Additionally, no correlation was detected between cell number and caspase detection (3 replicates, total n= 132, R square = 0.003). Determine whether key developmental genes are differentially expressed in individual SCNT embryos sorted according to the apoptotic index. RNA was isolated from individual blastocyst for gene expression analysis. Nanoscale qPCR was performed using the Fluidigm Biomark RT PCR system on low and high SCNT blastocysts (3 replicates, total n=13) and in vivo produced embryos (2 replicates, total n=11) to detect RNA expression of 96 genes including those involved in apoptosis, epigenetic regulation, imprinted genes, maternal effect, pluripotency, and trophoblast differentiation and function. Statistical analysis to compare gene expression values between high and low apoptosis SCNT blastocysts revealed three genes that were expressed differently between the two groups. These genes include ACTB (P=0.03), SIRT1 (P=0.04), and TAF11 (P=0.01). Two genes, HAT1 (P=0.086) and HDAC2 (P=0.056), trended towards significance. Interestingly, SIRT1, HAT1, and HDAC2 are all involved in epigenetic regulation of histone configuration through histone acetylation and deacetylation. An unsupervised bidirectional hierarchical clustering analysis of gene expression data revealed the in vivo produced embryos clustered together while the SCNT embryos showed no distinct clustering by apoptosis incidence. While a clear "finger-print" of gene expression was not detected as hypothesized, only a small subset of the total genes expressed in the porcine blastocysts was examined; a global analysis of RNA expression in high- vs low- apoptosis embryos will likely reveal many additional differences. Preliminary analysis of a follow-up experiment using RNA-seq to analyze global gene expression patternsin 26individual blastocysts(13 high apoptosis and 13 low apoptosis, as determined by FLICA staining)suggests that 114 genes were 'upregulated' and 15 genes were 'downregulated' in high vs low apoptotsis blastocysts. A Gene Set Enrichment Analysis of these differentially expressed genes suggests that receptor tyrosine kinase signaling pathways were somehow 'downregulated' in high apoptosis embryos, and that genes associated with the TAR RNA Binding Protein Containing complex are upregulated in high apoptosis embryos. Further investigation and validation of these findings is ongoing. Identify differences in patterns of gene promoter methylation for key development genes in embryos exhibiting high or low levels of apoptosis. Our initial attempts at generating PCR amplicons directly from bisulfite-treated gDNA from individual blastocysts were successful if we only did 2 or 3 amplicons, but when we tried to multiplex preamplify 20 or 30 or 40 amplicons together by PCR, we didn't get good results. Therefore, we shifted our approach. After developing a method for isolating enough high quality genomic DNA (gDNA) from individual SCNT embryos, a modified single-cell reduced-representation bisulfite sequencing (RRBS) protocol was adapted for methylation analysis of CpG-dense regions of the genome. This method utilized a restriction enzyme digest (MspI), two rounds of PCR amplification, and size selection to isolate abundant quantities of the CpG-dense regions. Individual blastocyst RRBS libraries were prepared from the high and low apoptosis blastocysts (3 replicates, total n=13) and in vivo produced embryos (2 replicates, total n=11.) The libraries were sequenced on the Illumina NextSeq using 150 paired-end reads. Approximately 40 million reads were obtained from each library. A rigorous bioinformatic analysis of the libraries is currently in progress. Preliminary data suggest a modest number of differences across all embryos of a given 'treatment'. Validate the utility of the non-invasive apoptosis bioassay as a tool for predicting the viability of porcine SCNT embryos. Establishing SCNT pregnancies proved to be a challenge. In order to optimize the number of embryos, two cloning sessions were preformed two days in a row and embryos are transferred on day 4 and 5 of development, following FLICA analysis on day 3. Our aim for earlier FLICA analysis was to analyze the embryo at the time of maternal-to-zygotic transition and minimize negative effects of in vitro culture. While the embryos still showed variation in FLICA values (and separated into statistically different high and low apoptosis groups), the variation was less intense compared to previous blastocysts analyzed. High and low apoptosis embryos sorted on day 3 showed no significant difference in blastocyst development on day 5, 6, or 7 (5 replicates). Additionally, no difference in cell number was found in the day 7 blastocysts from high and low apoptosis groups (4 replicates, total n=150). While struggling to implement a successful estrus synchronization protocol, three embryo transfer sessions occurred. The first transferred 307 embryos into one pig and failed to develop a pregnancy. Two transfers comparing the development of high and low apoptosis embryos have been performed at this time. The first of these transfers included 50 high apoptosis embryos into one pig and 50 low apoptosis embryos into a second pig. The second of these transfers included 87 high and low apoptosis embryos transferred into two separate pigs. All pigs returned to heat before 30 days. Additional embryo transfers will take place in the coming months. We have also begun to test the validity of this bioassay for predicting pregnancy success in the bovine model system.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gene expression and cell count characterization of porcine SCNT blastocysts with high and low incidence of apoptosis. Society for the Study of Reproduction (SSR) Annual Meeting. Washington DC. Poster AND Flash Oral Presentation
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Gene expression and cell count characterization of porcine SCNT blastocysts with high and low incidence of apoptosis. ASAS-SSR Joint Reproduction Symposium. Washington DC. Poster Presentation.
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Progress 09/01/15 to 08/31/16
Outputs
Target Audience:The target audiences for this research are 1) the community of animal reproductive biologists; 2) the broader group of all reproductive biologists in general, including clinical scientists and practitioners, with interests in fertility/infertility issues, and early embryonic mortality and nuclear reprogramming in somatic cell nuclear transfer embryos in particular; and 3) the animal agriculture production industries that would have interest in cloning animals commercially. Changes/Problems:One semi-major proposed change is an alteration to the embryo transfer timeline. Rather than transferring embryos at the blastocyst stage, we are proposing FLICA analysis on day 3 to correlate to the time of maternal to zygotic transition where altered epigenetic marks could start wreaking havoc in the embryo. From a technical standpoint, we did not feel that we would be successful transferring at the blastocyst stage as we would not be able to transfer an acceptable number of blastocysts due to low development rates and only transferring a portion of the blastocysts. With this in mind, we felt that analyzing earlier would optimize the number of embryos transferred from each cloning session while continuing to test the hypothesis that altered epigenetic marks causes aberrant gene expression and increased apoptosis leading to eventual embryo failure. The final major challenge to the project is getting the embryo transfer system successful. With a limited number of surrogate females available for transfer, estrus synchronization is critical to success. We believe that we have developed the correct protocol for successful synchronization. What opportunities for training and professional development has the project provided?This project has provided one full-time graduate student and three part-time undergraduate student with excellent training opportunities. Also, a visiting scientist and a post-doc have contributed effort, both technical and scientific in nature. How have the results been disseminated to communities of interest?Thus far, our work has been presented 3 times at 2 scientific meetings. These presentations include a poster at the 2017 ASAS-SSR Joint Symposium and a poster and 3 minute flash talk at the 2017 Society for the Study of Reproduction Annual Conference. Our progress has been reported in local and regional meetings, with definite plans for peer-reviewed publication in the near future. What do you plan to do during the next reporting period to accomplish the goals?With an RRBS approach to analyze the methylation of CpG-dense regions rather than an amplicon specific approach, we plan to further analyze gene expression at a genome-wide level to greater understand the connection of methylation and gene expression differences in high and low apoptosis SCNT embryos. We will be pursing RNA-seq on the same SCNT and in vivo samples, deepening not only our understanding of the differences between the high and low apoptosis groups, but also the differences between individual embryos within the groups. We will additionally continue to pursue embryo transfer success using SCNT embryos
Impacts
What was accomplished under these goals?
The goals of this project are to: 1) Adapt a non-invasive apoptosis bioassay for use in porcine embryos generated using SCNT. After previously employing the SR-FLICA™ Poly Caspase Assay Kit from ImmunoChemistry Technologies (Bloomington, MN, USA) with a slightly modified protocol to detect caspase activity in live oocytes and parthenogenote and SCNT embryos at various stages of development, the kit was utilized to detect active caspase activity in day 6 SCNT porcine blastocysts. Blastocysts from 3 replicates (n=13) were categorized as high or low apoptosis if they were in the top or bottom 20% of detected caspase activity, respectively. The FLICA values ranged from 79.6 to 244.8. The low apoptosis blastocysts had an average FLICA value of 94.00 with a standard deviation of 13.40. The high apoptosis blastocysts had an average FLICA value of 221.32 with a standard deviation of 19.01. DNA and RNA was collected from these blastocysts for molecular analysis discussed below. Using porcine parthenogenetic blastocysts as a model (3 replicates, total n=23), no difference in cell number was detected between high and low apoptosis blastocysts (P=0.1839). Additionally, no correlation was detected between cell number and caspase detection (3 replicates, total n= 132, R square = 0.003). 2) Determine whether key developmental genes are differentially expressed in individual SCNT embryos sorted according to the apoptotic index. RNA was isolated from individual blastocyst for gene expression analysis. Nanoscale qPCR was performed using the Fluidigm Biomark RT PCR system on low and high SCNT blastocysts (3 replicates, total n=13) and in vivo produced embryos (2 replicates, total n=11) to detect RNA expression of 96 genes including those involved in apoptosis, epigenetic regulation, imprinted genes, maternal effect, pluripotency, and trophoblast differentiation and function. Statistical analysis to compare gene expression values between high and low apoptosis SCNT blastocysts revealed three genes that were expressed differently between the two groups. These genes include ACTB (P=0.03), SIRT1 (P=0.04), and TAF11 (P=0.01). Two genes, HAT1 (P=0.086) and HDAC2 (P=0.056), trended towards significance. Interestingly, SIRT1, HAT1, and HDAC2 are all involved in epigenetic regulation of histone configuration through histone acetylation and deacetylation. An unsupervised bidirectional hierarchical clustering analysis of gene expression data revealed the in vivo produced embryos clustered together while the SCNT embryos showed no distinct clustering by apoptosis incidence. While a clear "finger-print" of gene expression was not detected as hypothesized, only a small subset of the total genes expressed in the porcine blastocysts was examined; a global analysis of RNA expression in high- vs low- apoptosis embryos will likely reveal many additional differences. 3) Identify differences in patterns of gene promoter methylation for key development genes in embryos exhibiting high or low levels of apoptosis. After developing a method for isolating enough high quality genomic DNA (gDNA) from individual SCNT embryos, a modified single-cell reduced-representation bisulfite sequencing (RRBS) protocol was adapted for methylation analysis of CpG-dense regions of the genome. This method utilized a restriction enzyme digest (MspI), two rounds of PCR amplification, and size selection to isolate abundant quantities of the CpG-dense regions. Individual blastocyst RRBS libraries were prepared from the high and low apoptosis blastocysts (3 replicates, total n=13) and in vivo produced embryos (2 replicates, total n=11.) The libraries were sequenced on the Illumina NextSeq using 150 paired-end reads. Approximately 40 million reads were obtained from each library. Analysis of the libraries is currently in progress. 4) Validate the utility of the non-invasive apoptosis bioassay as a tool for predicting the viability of porcine SCNT embryos. Establishing SCNT pregnancies has proven to be a challenge. In order to optimize the number of embryos, two cloning sessions are preformed two days in a row and embryos are transferred on day 4 and 5 of development, following FLICA analysis on day 3. Our aim for earlier FLICA analysis is to analyze the embryo at the time of maternal-to-zygotic transition and minimize negative effects of in vitro culture. While the embryos still show variation in FLICA values (and separate into statistically different high and low apoptosis groups), the variation is less intense compared to blastocysts analyzed. High and low apoptosis embryos sorted on day 3 showed no significant difference in blastocyst development on day 5, 6, or 7 (5 replicates). Additionally, no difference in cell number was found in the day 7 blastocysts from high and low apoptosis groups (4 replicates, total n=150). Embryo transfers are challenging as we have struggled to implement an estrus synchronization protocol for successful embryo transfer as the pigs continually come into heat 24 to 48 hours later than anticipated. We believe that recent changes to the protocol will be more successful in the future as they have worked correctly in the last attempt. Three embryo transfer sessions have occurred. The first transferred 307 embryos into one pig. The second transfer included 50 high apoptosis embryos into one pig and 50 low apoptosis embryos into a second pig. The third transfer included 87 high and low apoptosis embryos transferred into two separate pigs. All pigs returned to heat before 30 days.
Publications
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Progress 09/01/14 to 08/31/15
Outputs
Target Audience:Target Audience: The target audiences for this research are 1) the community of animal reproductive biologists; 2) the broader group of all reproductive biologists in general, including clinical scientists and practitioners, with interests in fertility/infertility issues, and early embryonic mortality and nuclear reprogramming in somatic cell nuclear transfer embryos in particular; and 3) the animal agriculture production industries that would have interest in cloning animals commercially. Changes/Problems:Changes/Problems: Two problems/challenges have slowed our progress on this project, but we have not yet proposed to make major changes to the objectives of the proposal. The first of these challenges has involved getting sufficient high quality genomic DNA isolated from individual porcine IVC blastocysts to do targeted DNA methylation (bisulfite PCR) analysis. It has taken some time, but we are now confident that we have a protocol in place that will give us sufficient DNA for targeted or whole genome DNA methylation analysis experiments. The second of our challenges has been one of logistics in getting enough cloned embryos for apoptosis monitoring. The recent addition of a visiting scientist to our team has helped to augment our cloned embryo production efficiencies, and so we have effectively addressed this concern as well. What opportunities for training and professional development has the project provided?This project has provided one full-time graduate student and one part-time undergraduate student with excellent training opportunities. Also, a visiting scientist and a post-doc have contributed effort, both technical and scientific in nature. How have the results been disseminated to communities of interest?Thus far, our work has been largely technical in nature, and hasn't been presented at national scientific meetings. But our progress has been reported in local and regional meetings, with definite plans for peer-reviewed publication and presentation at scientific venues in the near future. What do you plan to do during the next reporting period to accomplish the goals?What do you plan to do during the next reporting period to accomplish the goals? As we retain the services of the visiting scientist, we should have more access to cloned embryos than previously. We already are using parthenogenote embryos to fine-tune our techniques before using them on SCNT embryos, and we will continue to do this as appropriate. We will continue to improve upon our techniques for deriving DNA methylation data from individual SCNT embryos, as this has been the major technical bottleneck for this project thus far. We will continue our current work investigating the possibility of performing reduced representation bisulfite sequencing (RRBS) on bisulfite-converted DNA from individual porcine embryos.
Impacts
What was accomplished under these goals?
The goals of this project are to: 1) Adapt a non-invasive apoptosis bioassay for use in porcine embryos generated using SCNT. We have employed the SR-FLICA™ Poly Caspase Assay Kit from ImmunoChemistry Technologies (Bloomington, MN, USA) with a slightly modified protocol to detect caspase activity in live oocytes and parthenogenote and SCNT embryos at various stages of development. We have demonstrated that there is, indeed, a wide range of separation between individual oocytes and blastocyst-stage embryos regarding FLICA fluorescence, which is indicative of apoptosis-associated caspase activation. In an assessment of apoptosis in 30 individual PA blastocysts, raw FLICA "scores" for individual embryos ranged from 66 (arbitrary fluorescence units) to 255, with an average of 138, and a SD of 47.5. Background-corrected FLICA values ranged from 56 to 236, with an average of 126 and SD of 46.4. Background corrected FLICA values corrected for DAPI intensity (putative measure of cell number) ranged from 1 to 12, with an average of 4 and a SD of 2.4. Apoptosis rates tracked nicely with day of development as demonstrated previously: early cleavage-stage embryos showed very little caspase activation, whereas by d4 and d5, more embryos would show up as highly apoptotic. Embryos cultured with the FLICA compound during cleavage-stage development are still able to develop to blastocyst at an acceptable rate, confirming the non-toxic and "non-invasive" aspect of the assay, which is critical to our experimental aims. 2) Determine whether key developmental genes are differentially expressed in individual SCNT embryos sorted according to the apoptotic index. We have validated 45 primer sets for qPCR analysis of important developmental genes in individual blastocysts using the BioMark HD platform from Fluidigm (South San Francisco, CA, USA). We are still in the process of collecting SCNT embryos separated by incidence of apoptosis for final gene expression analysis. But the BioMark system will give us exceptional power to investigate gene expression patterns in individual SCNT embryos. 3) Identify differences in patterns of gene promoter methylation for key development genes in embryos exhibiting high or low levels of apoptosis. We have worked hard to develop a method for isolating enough high quality genomic DNA (gDNA) from individual SCNT embryos so that we can generate locus-specific DNA methylation data at individual base pair resolution (bisulfite sequencing). Our initial attempts at generating PCR amplicons directly from bisulfite-treated gDNA from individual blastocysts were successful if we only did 2 or 3 amplicons, but when we tried to multiplex preamplify 20 or 30 or 40 amplicons together by PCR, we didn't get good results. We shifted our attention to doing a linear, non-PCR preamplification of bisulfite-treated gDNA from individual blastocysts, our results have been much better. In fact, were able to get significant methylation data from a small Illumina (MiSeq) sequencing run with this preamplified gDNA. We are currently fine-tuning the details of how we will best employ this preamplification technique to be most efficient in generating useful methylation data for this project (40-50 amplicons for traditional bisulfite sequencing vs. reduced representation bisulfite sequencing vs full genome shotgun bisulfite sequencing, e.g.). 4) Validate the utility of the non-invasive apoptosis bioassay as a tool for predicting the viability of porcine SCNT embryos. This being the final aim of our proposal, and largely dependent on the others, we have no progress to report yet for this particular goal.
Publications
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Progress 09/01/13 to 08/31/14
Outputs
Target Audience:1) the community of animal reproductive biologists, 2) the broader group of all reproductive biologists in general (clinical included) Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate and one graduate student have assisted on this work so far. They have been involved in learning new techniques such as in vitro embryo production, apoptosis assays, RNA extraction, cDNA synthesis, and DNA methylation analysis. One member of our research team has engaged in training to extend his expertise in micromanipulation of ruminant embryos to pig oocytes and embryos. This has been an adjustment and a valuable training opportunity for him. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1) we will continue to validate the utility of the FLICA kit for use with embryos and other sensitive and valuable tissue types. 3) we will dedicate significant time to establishing and perfecting the proper techniques necessary to generate bisulfite PCR amplicons for DNA methylation analysis from individual porcine embryos cultured in vitro.
Impacts
What was accomplished under these goals?
1) We tested the use of the SR FLICA(R) Poly Caspase Assay Kit (ImmunoChemistry Technologies LLC; Bloomington, MN, USA) for use in detecting caspase activation (indication of apoptosis) in porcine and bovine oocytes and embryos. 2) We continued to improve and extend our efforts to generate quantitative gene expression data from individual oocytes and preimplantation-stage embryos using the revolutionary microfluidics-based BioMark for single cell qPCR gene expression analysis from Fluidigm Inc (South San Francisco, CA, USA). We can generate robust and reliable data for up to 96 genes at a time for individual oocytes and embryos. This platform will allow us to query multiple members of many functional gene categoriesimportant to early development to get a more comprehensive evaluation of the magnitude and scope of gene expression changes in our different embryo groups. 3) We have only just started to test and validate the most appropriate methods for generating targeted bisulfite PCR amplicons for DNA methylation analysis from individual embryos. This is likely to be our biggest challenge for this project. 4) No progress on this goal
Publications
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