The role of micro-RNA, complement proteins, and immune cells in bovine luteal function and rescue
Open Access
- Author:
- Hellmers, Adelaide
- Graduate Program:
- Integrative and Biomedical Physiology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 13, 2022
- Committee Members:
- Joy Pate, Chair & Dissertation Advisor
Connie Rogers, Outside Unit & Field Member
Francisco Diaz, Major Field Member
Troy Ott, Major Field Member
Donna Korzick, Program Head/Chair - Keywords:
- bovine
corpus luteum
reproduction
ovary
complement
miRNA
mitochondria
pregnancy associated glyocoproteins
immune cells - Abstract:
- The corpus luteum (CL) is a transient endocrine tissue on the ovary that secretes the hormone progesterone that is essential for the maintenance of pregnancy in mammals. Functional maintenance of the CL during the estrous cycle is important for the continued secretion of progesterone to allow for the establishment of pregnancy. Luteal rescue in the cow takes place between days 17-20 of the estrous cycle. If luteal rescue does not occur during early pregnancy, the CL regresses and the expectant pregnancy will be lost, which can lead to major financial losses for farmers. The interplay between different molecular mechanisms, including the role of micro-RNA (miRNA) and the role of different immune components, by which the CL is maintained are not fully understood. Understanding these mechanisms and how they aid in maintaining the CL could help elucidate answers to prevent early pregnancy loss in dairy cows. The objectives of this study were to 1) Understand if let-7, a miRNA, can modulate mitochondrial respiration and progesterone production in the functional CL; 2) To identify a phenotype of resident immune cells in the CL exposed to uterine infused interferon-tau (IFNT) and/or pregnancy specific protein B (PSPB) using a transcriptomic profiling approach; and 3) To characterize the expression and the role of complement components in the CL. In the first study, the abundance of let-7a and let-7b were characterized in isolated mitochondria from midcycle luteal cells. Neither let-7a nor let-7b were expressed in isolated luteal mitochondria except in one animal. Additionally, neither let-7a nor let-7b abundance changed over 5 days of culture, or in response to luteinizing hormone (LH) or prostaglandin F2a (PGF2A). However, when luteal cells were transfected with a let-7b mimic, mitochondrial respiration markers decreased including basal respiration and proton leak. Production of production and spare capacity tended to decrease. There was no change in mitochondrial respiration in response to let-7a. The let-7b mimic also decreased progesterone production from luteal cells on day 5 and 7 of culture, while let-7a mimic increased progesterone production on day 3 of culture. The abundance of EGR1 and PGRMC1, both targets of let-7a and let-7b, were also assessed via flow cytometry after transfection with the let-7 mimics and inhibitors. Interestingly, the let-7a inhibitor increased the percentage of EGR1 positive cells and the mean fluorescence intensity (MFI) of EGR1. There was no effect of let-7b on EGR1. Both the let-7a mimic and inhibitor increased the MFI of PGRMC1 with no change on the percentage of PGRMC1 positive luteal cells. The let-7b mimic and inhibitor also increased the MFI of PGRMC1, and the inhibitor increased the percentage of PGRMC1 positive cells. In the second chapter, first experiment evaluated whether treatment of luteal cells with luteotropic hormones could alter T cell activation. This experiment measured the abundance of T cell activation molecules CD3e, IL2 inducible T cell kinase (ITK), LCK proto-oncogene (LCK), and protein tyrosine phosphatase non-receptor type 7 (PTPN7) were measured in T cells cocultured with luteal cells previously treated with interferon-tau (IFNT), prostaglandin E2 (PGE2), and a combination treatment. Both CD3e and LCK mRNA was less in T cells that had been cocultured with PGE2-treated luteal cells. PTPN7 mRNA tended to be less in T cells exposed to luteal cells treated with IFNT, PGE2, and a combination treatment, while ITK mRNA did not change. The second experiment was a transcriptomic profiling study comparing luteal tissue and resident immune cells isolated from CL exposed to uterine infusions of IFNT and/or PSPB for either 3-days or 6-days. The purpose was to evaluate if PSPB could alter the transcriptome of the CL and/or resident immune cells, and if a phenotype of resident immune cells could be elucidated from this experimental model of pregnancy. There were only 3 differentially abundant transcripts found in resident immune cells from the 6-day IFNT and 6-day IFNT+PSPB infusion treatments, while there were 5 differentially abundant transcripts identified in luteal tissue from the same treatment. The upregulated pathways associated with transcripts with Q < 0.15 in resident immune cells were eNOS signaling and the complement system, while IL17 signaling, coagulation, and iNOS signaling were the upregulated pathways in luteal tissue. In the 3-day IFNT and 6-day IFNT infusion comparison, 8 differentially abundant transcripts were identified in resident immune cells and 56 differentially abundant transcripts were identified in luteal tissue. The upregulated pathways associated with transcripts with Q < 0.15 in resident immune cells were ephrin A signaling, sphingosine-1-phosphate signaling, and IL15 production, while ILK signaling and IL8 signaling were upregulated pathways in luteal tissue. Lastly, there were 83 differentially abundant transcripts identified in resident immune cells from the 3-day BSA and 3-day IFNT infusion treatments and 117 differentially abundant transcripts identified in luteal tissue from the 3-day infusions. Interferon signaling and antiviral response pathways were upregulated pathways associated with the differentially abundant transcripts in both resident immune cells and luteal tissue. In the third study, the abundance of complement C3 and C4 were characterized across the estrous cycle and during early pregnancy and an attempt was made to determine a functional role of C3 in the CL. The assessment of C3 and C4 abundance during luteal regression found that C3 and C4 did not change at any time point after an in vivo injection of PGF2A, but there was a tendency for C3 to increase 24 hours after injection compared to the 0 hour control. There was also no change in abundance of C3 and C4 during acquisition of luteolytic capacity, however, C3 tended to be less abundant in the day 6 CL compared to the day 4 CL. During early pregnancy, on day 17, there was a 2-fold numerical decrease in C3 between the cyclic and pregnant CL, and no change in C3 between the CL of the cycle and pregnancy on day 18. While there was no change in C3 on day 17, there was an increase in C3 in the CL of pregnancy compared to the cycle on day 18. C4 abundance did not change between the CL of the cycle and pregnancy on day 17, but there was a 5-fold numerical increase in C4 abundance on day 18 of pregnancy compared to the cycle. There were no changes in C4 abundance on day 17 or day 18. In response to various luteotropic and luteolytic hormones, C3 abundance per cell (MFI) decreased with IFNT+PGE2 treatment and PGF2A+PGE2 treatment compared to individual treatments with these hormones. However, C3 decreased in response to PGF2A compared to control, and the effect of PGF2A remained when PGE2 was added to the treatment. C4 abundance increased in response to IFNT compared to control, and remained increased when PGF2A was added to the treatment. There was a tendency for C4 abundance to increase with IFNT+PGE2. Lastly, the effect of C3 on luteal cell induced T cell proliferation was tested by knocking down C3 in both luteal cells and T cells. There was no change in T cell proliferation after C3 knockdown. In summary, several mechanisms involving miRNA and immune components were investigated to understand how the CL can remain functional both during the estrous cycle and during the period of maternal recognition of pregnancy. One major finding was let-7b does reduce mitochondrial respiration and progesterone production in luteal cells. This finding is important and suggests critical roles for miRNA in facilitating luteal function. Additionally, the identification of differentially abundant transcripts in the CL in response to uterine infused PSPB is a novel finding that suggests PSPB does alter the transcriptome of the CL by altering several immune signaling pathways during early pregnancy. Along with these findings, the characterization of the expression of complement C3 and C4, suggest these proteins might play a role in the CL of pregnancy, but unfortunately the exact role was unable to be characterized in this study. These findings provide a framework for future directions of luteal research to further elucidate the role of the immune system in the CL of early pregnancy as well as further understanding how miRNA might facilitate the continued function of the CL.