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 and their potential roles in bovine reproduction specifically, oocyte quality. With the past five months 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 identified significant differences between maturity stage in expression of two of the six lncRNAs, 18208.1 and 25823.1. Literature states follicle size can be an indicator of oocyte quality and that RNAs accumulated during maturation are crucial to achieve developmental competence. Thus, I believe lncRNAs, 18208.1 and 25823.1 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 five months of funding I have started to address 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. Based on previous RNA-sequencing data, our lab discovered 1535 lncRNAs in bovine oocytes. My aim was to to verify our RNA-seq data and if present, characterize the expression of six highly abundant lncRNAs (2160.1, 41165.1, 5379.1, 25823.1, 18208.1, and 34876.2) in single oocytes aspirated from varying size follicles. Single oocytes were aspirated from small (<4 mm) and large (6-18 mm) follicles and were denuded (cumulus cells removed) at the germinal vesicle (GV) or MII stage. Quantitative real-time PCR (RT-qPCR) analysis, using RPL-19 as a housekeeping gene, revealed detectable expression for each lncRNA in single oocytes from small (SF) and large (LF) follicles at both stages. Average expression of each lncRNA from SF was used as a calibrator in normalization. Effect of maturity stage on the lncRNA expression was examined using a two-sided t-test assuming unequal variances.
We found significant differences between maturity stages in expression of lncRNA 18208.1 (p=0.01), where the fold change of LF to SF was higher in MIIs (2.05) than in GVs (0.89). In addition, we found a similar significant difference between stages in expression of lncRNA 25823.1 (p=0.007). The GV from LF were about half (0.54) the expression of the GV from SF, while the MII from LF had a 1.3-fold increased expression with respect to the SF. In summary, both lncRNA 18208.1 and 25823.1 had higher expression in oocytes aspirated from the larger follicles during later developmental stage.
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 significant difference between maturity stage in two of the six highly abundant lncRNAs. Literature states follicle size can be an indicator of oocyte quality and that RNAs accumulated during maturation are crucial to achieve developmental competence. Therefore, lncRNAs 18208.1 and 25823.1 shows 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 has been submitted for as an abstract for the 19th International Congress on Animal Reproduction (ICAR 2020) in Bologna, Italy. If accepted I will be traveling this summer to present my work as a poster at an international conference. 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 going according to plan, I will be submitting that data as an abstract for the 53rd Annual Meeting organized by the Society for the Study of Reproduction (SSR). If accepted, I will also be traveling to Ottawa, Ontario, Canada 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.