Progress report for GNE19-196
Oocyte quality remains an undefined obscure topic in reproductive research. My goal is to take a new approach to tackling this decade old problem. As technology has increased more and more aspects of everyday life has started to utilize technology and take a turn more towards science. My goal is to incorporate molecular physiology with whole animal applications. I hope to investigate long noncoding RNAs (lncRNAs) and their potential roles in bovine reproduction specifically, oocyte quality. With the past year and a half of funding I have successfully verified the presence and differential expression of six highly abundant lncRNAs in single bovine oocytes aspirated from small and large follicles at varying developmental stages. More specifically, I documented the significance of maturity stage in all six lncRNAs and graphically showed their accumulation and degradation during maturation. When examining each for the interaction of follicle size and maturity stage, there was a significant interaction in expression of OOSNCR5 (P < 0.05) and a tendency for significant interaction in expression of OOSNCR2 . The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieve developmental competence. Therefore, lncRNAs OOSNCR2 and OOSNCR5 show promise to be linked with oocyte quality. Studies of this nature are the first steps in determining potential bio-markers that can one day be utilized to predetermine which oocytes will produce viable embryos saving the farmer money by removing the oocytes that would not survive the culture process.
1. To characterize the temporal and spatial expression of oocyte-specific lncRNAs during early embryogenesis and determine if they are involved in fertilization.
2. To investigate oocyte quality by comparing lncRNA expression profiles in “good and bad” oocytes based on previously published methodologies; follicle size and cytoplasmic morphology.
3. To elucidate the regulatory role of specific lncRNAs in meiotic maturation, fertilization, cleavage, and developmental events characteristic to the maternal-to-embryonic transition.
The purpose of my project is to make novel discoveries investigating the potential roles of long noncoding RNAs in bovine reproduction. Up to this point in time, oocyte quality remains difficult to define. My goal is to approach it from a different perspective. I plan to use molecular genetics to screen oocytes of different qualities to try to find similarities between gene expression and oocyte quality. Although this research may seem too far away from sustainable agriculture, I believe that thinking in itself is the problem. I believe that the future of sustainable agriculture lies in the utilization of technology. There comes a point when farmers and scientist must work together to tackle the issues seen among their herds. Scientist have the scientific knowledge and the means to make great new discoveries while the farmers have the agricultural knowledge and the animals needed to mesh the two fields. I believe my research is important for the future of sustainable agricultural because it takes the new generation of thinking to combine science with hundreds of years of agricultural knowledge. I do not have the means or knowledge to run a farm, however I know how to perform in vitro embryo production which when used properly, has the means to produce genetically superior animals that can yield hundreds of thousands of dollar worth of profit for a farmer. By combining a scientists knowledge with an open minded farmer who's willing to try something new. I believe that is the future of sustainable agriculture.
With the past year and a half of funding I have completed objective 2.) To investigate oocyte quality by comparing lncRNA expression profiles in "good and bad" oocytes based on previously published methodologies, follicle size and cytoplasmic morphology. Although I started this objective when I received funding and it was reported in last year's project report, due to the pandemic this year my research was placed on hold for a majority of the year. The break in my lab work allowed me to go back over my data and correct some errors. The originally submitted data was added to and re-analyzed. The methodology remained the same and is as followed. Our laboratory identified 1535 lncRNAs in bovine oocytes using RNA-sequencing (Table 1 below). The objective of this study was to verify our RNA-seq data and if present, characterize the expression of six highly abundant lncRNAs (OOSNCR1, OOSNCR2, OOSNCR3, OOSNCR4, OOSNCR5, and OOSNCR6) in single oocytes aspirated from varying size follicles at different developmental stages. Single oocytes were aspirated from small (<4 mm) and presumably oestrogen active (6–18 mm) follicles and were denuded at the germinal vesicle (GV) or MII stage. Single oocytes were aspirated from small (<4 mm) (SF) and presumably estrogen active (6–18 mm) (EA) follicles and were denuded at the germinal vesicle (GV) or MII stage. MII stage was determined by cumulus expansion and the extrusion of the first polar body. Real-time quantitative PCR analysis was performed, using RPL-19 as an endogenous control for data normalization analyzed using the standard curve method. Average expression of each lncRNA from SF was used as a calibrator in determination of fold change. Effect of follicle size (SF, EA) and maturity stage (GV and MII) and their interaction on the lncRNA expression were examined using a two-way factorial ANOVA followed by Tukey’s HSD post hoc test.
Maturity stage was significant (P < 0.05) in all 6 lncRNAs. OOSCNR2, OOSNCR3, OOSCNR5 and OOSNCR6 were all higher in the GV oocytes and were degraded with maturation. OOSNCR1 and OOSNCR4 displayed opposite expression patterns in that they were accumulated during maturation. When examining each for the interaction of follicle size and maturity stage, there was a significant interaction in expression of OOSNCR5 (P < 0.05). Within it, SF oocytes at the GV stage had the highest relative expression. Significant difference was detected between SF MII and SF GV oocytes, indicating a higher expression in the earlier developmental stage (P < 0.05). SF oocytes at MII had a 0.63-fold decrease in its expression relative to the SF oocytes at GV. In addition, there was a tendency for significant interaction between stage and size in expression of OOSNCR2 (P < 0.1). OOSNCR2 exhibited a similar expression patterns as OOSNCR5. Although not significant, SF oocytes at the GV stage had the highest relative expression, indicating a higher expression in the earlier developmental stage. Here the SF oocytes at MII had a 0.43 -fold decrease in its expression relative to the SF oocytes at GV.
Due to the nature of this study, the data is very preliminary. However, based on our results it is very exciting to be able to pick up differential gene expression in single oocytes. Not only is it exciting that we were successful in single-cell PCR detection of lncRNAs, it is very exciting that we found a significant difference and a tendency for significant difference in the interaction of maturity stage and follicle size in two of the six highly abundant lncRNAs. The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieve developmental competence. Therefore, lncRNAs OOSNCR2 and OOSNCR5 show promise to be linked with oocyte quality. By characterizing lncRNAs associated with oocyte quality, one day we might be able to discover bio-markers of quality that allows farmers to test for "good" oocytes prior to spending the money necessary to perform in vitro embryo production. Farmers not only would spend less money by only submitting the "good" oocytes to undergo the expensive IVP procedure, but also they would be more willing to utilize the technology knowing it has a good outcome and removing the potential risk of failure.
Education & Outreach Activities and Participation Summary
The data provided here was originally submitted as an abstract for the 19th International Congress on Animal Reproduction (ICAR2020) in Bologna, Italy. However, due to the pandemic the conference was cancelled and pushed back to 2022. With the cancelling of the conference all abstracts were discarded and I was able to reanalyze my data and submit to a different conference. This data will now be presented as a poster at the virtual 47th Annual Conference of the IETS (International Embryo Transfer Society) on January 19th. This next semester I plan on continuing my research into oocyte quality looking at lncRNA expression as well as tackling portions of objective 1, characterizing the temporal and spatial expression of key lncRNAs. The lncRNAs found in objective 2 will be used for further examination during objective 1. Assuming all goes according to plan, I will be submitting that data as an abstract for the 54th Annual Meeting organized by the Society for the Study of Reproduction (SSR). If accepted, I will be traveling to St. Louis, MO to present my findings.
So far, we are in our preliminary steps and it is too soon for our research to have an affect on agricultural sustainability.
Due to the nature of this project the data is still very preliminary, however, it is very exciting to see differential gene expression in single oocytes at such an early phase of the project. We hope to continue on our path of looking into these long noncoding RNAs to discover potential whole animal applications. I hope to continue my work in the lab so that by next year I can start to translate my data into something farmers can actually utilize and understand. At the end of the day I hope that my research contributes to bovine reproduction and ultimately, helps to get healthy calves on the ground.