Expression of MicroRNAs in the Bovine Corpus Luteum their Impact on Luteal Cell Function

Open Access
- Author:
- Smith, Courtney Lynne
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- July 13, 2010
- Committee Members:
- Joy Lee Pate, Thesis Advisor/Co-Advisor
Joy Lee Pate, Thesis Advisor/Co-Advisor - Keywords:
- bovine
CL
corpus luteum
miRNA
MicroRNAs
cow - Abstract:
- The corpus luteum is a temporary endocrine structure that produces progesterone for the maintenance of pregnancy and has many regulatory processes that are involved in its development, function, and regression. MicroRNAs are noncoding RNAs that are 17-25nt in length and function in translational repression by binding to the 3’ untranslated region of its target mRNA. Because of this repression, microRNAs are able to regulate the expression of many different genes. Drosha, an RNase III enzyme needed for processing the primary microRNA transcript into the precursor microRNA form, is considered one of the most important components in microRNA biogenesis. Little is known about microRNA expression and regulation pertaining in the corpus luteum. Even less is known about miRNA expression in livestock species. The studies presented here are the first studies focusing on microRNA expression in the bovine corpus luteum and miRNA regulatory abilities in bovine luteal cells. To investigate the expression of different miRNAs in luteal tissue, corpora lutea were removed from cyclic Holstein cows on days 4, 10, and 18 of the estrous cycle, and on day 18 of pregnancy. Total RNA enriched with small RNAs were isolated from each treatment and were hybridized to Affymetrix microRNA microarrays. Quantitative PCR was used to confirm microarray results. To assess the importance of microRNAs to luteal cell function, luteal cells were collected after the dissociation of midcycle (day 10-11) luteal tissues and luteal cells were transfected with siRNAs against Drosha to inhibit microRNA biogenesis. Transfected cells were treated with or without LH or TNF-á and IFN-ã to assess the effect of decreased microRNA expression on progesterone production and apoptosis, respectively. Bovine and nonbovine specific microRNAs were found to be differentially expressed among luteal tissue collected on day 4, 10, and 18 of the estrous cycle and on day 18 of pregnancy. Three microRNAs, miR-10a, miR-103, and miR-99a were confirmed to have increased expression in luteal tissue on day 18 of pregnancy. Overall, thirteen nonbovine miRNAs not previously reported as present in bovine tissues were detected only in luteal tissue on day 18 compared to three miRNAs expressed only in luteal tissue on day 18 of pregnancy. Nonbovine microRNAs were also identified in bovine luteal tissue on day 10 and day 4, with 42 miRNAs expressed only on day 10 and thirteen miRNAs expressed only on day 4. The knockdown of Drosha mRNA expression in luteal cells cultured with LH resulted in a significant decrease (P ˂ 0.05) in progesterone production compared to luteal cells transfected with Drosha siRNAs cultured with no LH. Also, the percentage of apoptosis for cells transfected with Drosha siRNAs and treated with or without TNF-á and IFN-ã significantly increased (P ˂ 0.05) compared to the controls. Overall, these data confirm the presence and differential expression of miRNAs in luteal tissue during the luteal phase and suggest that microRNAs have a role in luteal cell function and survival.