The impact of microRNAs and LPS on bovine luteal function
Publication Date
July 17, 2023
Creator
Abstract
The corpus luteum (CL) is a transient endocrine gland with critical roles in the establishment and maintenance of pregnancy, therefore, an increased understanding of the mechanisms regulating luteal function is key to improving reproductive outcomes in the future. This study investigated both physiological and pathological factors which regulate luteal function, namely microRNAs (miRNAs) and lipopolysaccharide (LPS).
MiRNAs are small non-coding molecules that bind to the 3’ untranslated regions (3’UTRs) of target messenger RNAs (mRNAs) to regulate their post-transcriptional translation. MiRNAs mediate a wide range of ovarian function, hence the possibility of ovarian miRNAs being used as biomarkers for reproductive efficiency. LPS acts through its receptor Toll like receptor 4 (TLR4) to negatively affect luteal function. This study determined the effect of LPS on luteal gene expression and cytokine production and assessed the role of TLR4 signalling in regulating the expression of critical luteal genes in vitro using TAK242 (a specific TLR4 inhibitor). For this project, a model that mimics early bovine luteal angiogenesis and steroidogenesis was used. Bovine luteal cells (early corpus luteum) were cultured for 7 days. Bovine luteal cells were either transfected (with miRNAs) or treated with LPS on days 1, 3 and 5 and cells collected for RNA sequencing, quantitative polymerase chain reaction (qPCR), immunocytochemistry, luciferase, and apoptosis assays on days 3, 5 and 7 of culture. Conditioned media was also collected on days 3, 5 and 7 for progesterone enzyme linked immunosorbent assay (ELISA), and cytokine arrays. Firstly, a systematic review on published work on ovarian miRNA studies was performed. MiRNAs identified were subjected to bioinformatic prediction (via FunRich) to determine the biological pathways regulated by these miRNAs at different stages/phases of the reproductive cycle. This review revealed that 146 miRNAs ovarian miRNAs from 9 animal species regulate various critical biological pathways most of which have established roles in ovarian function. The expression of nine critical luteal miRNAs was determined. MiR-378b, miR-221, miR- 96, miR-202, let-7a, let-7b, miR-7, miR-378a and miR-210 were expressed throughout the period of culture. The expression of miR-378b increased on day 7 following vascular endothelial growth factor receptor 2 (VEGFR2) inhibition on day 5. There was a decreased expression of miR-221 and miR-378a in conditioned media compared to control media. There was an increased expression of let-7b, let-7a and miR-202 in conditioned media compared to control media. The effects of miR-378b on bovine luteal angiogenesis and progesterone production was further investigated. Luciferase assays were performed to measure the functional binding of miR-378b to predicted target genes. Luciferase assay results showed that miR-378b binds to the 3’UTR of STAR and IGF2 to repress their expression and function in bovine luteal cells. MiR-378b mimic also caused a significant increase in the caspase 3/7 activity. In addition, we developed a fluorescence- activated cell sorting (FACS) protocol to sort bovine endothelial cells and steroidogenic cells to determine which cell type expressed miR-378b and the other eight critical luteal miRNAs. There was a significantly increased expression of miR-378b, miR-96 and miR-210 in enriched endothelial cells. In response to LPS treatment, differential gene expression analysis using a log2 fold change of <-1 or >1 revealed 33 downregulated genes and 18 upregulated genes on day 3 of culture. On day 5, 4 genes were down regulated while 33 genes were upregulated in LPS treated cells. RNA sequencing and qPCR analyses revealed decreased expression of roundabout guidance receptor 4 (ROBO4), epidermal growth factor-like domains protein 7 (EGFL7), secretogranin II (SCG2), 2’-5’- oligoadenylate synthetase (OAS1Y), delta like canonical notch ligand (DLL4), platelet and endothelial Cell adhesion molecule 1 (PECAM1) and C-X-C motif chemokine ligand 5 (CXCL5) in LPS treated cells on day 3. There was decreased expression of cadherin 5 (CDH5), luteinizing hormone/choriogonadotropin receptor (LHCGR), PECAM1, CXCL5 and SCG2 in LPS treated cells on day 5. TLR4 inhibition using TAK242 reversed the effect of LPS on the expression of DLL4 and PECAM1 on days 3 and 5. The effect of LPS on the expression of ROBO4 and EGFL7 on day 3, LHCGR, CDH5, SCG2 and CXCL5 on day 5 was partially reversed following TLR4 inhibition. Cytokine arrays revealed an increased concentration of VEGF in conditioned media from LPS treated cells on days 3 and 5. In conclusion, this thesis highlights the complexity of the CL and demonstrates that specific miRNAs and LPS are key regulators of bovine luteal function.
MiRNAs are small non-coding molecules that bind to the 3’ untranslated regions (3’UTRs) of target messenger RNAs (mRNAs) to regulate their post-transcriptional translation. MiRNAs mediate a wide range of ovarian function, hence the possibility of ovarian miRNAs being used as biomarkers for reproductive efficiency. LPS acts through its receptor Toll like receptor 4 (TLR4) to negatively affect luteal function. This study determined the effect of LPS on luteal gene expression and cytokine production and assessed the role of TLR4 signalling in regulating the expression of critical luteal genes in vitro using TAK242 (a specific TLR4 inhibitor). For this project, a model that mimics early bovine luteal angiogenesis and steroidogenesis was used. Bovine luteal cells (early corpus luteum) were cultured for 7 days. Bovine luteal cells were either transfected (with miRNAs) or treated with LPS on days 1, 3 and 5 and cells collected for RNA sequencing, quantitative polymerase chain reaction (qPCR), immunocytochemistry, luciferase, and apoptosis assays on days 3, 5 and 7 of culture. Conditioned media was also collected on days 3, 5 and 7 for progesterone enzyme linked immunosorbent assay (ELISA), and cytokine arrays. Firstly, a systematic review on published work on ovarian miRNA studies was performed. MiRNAs identified were subjected to bioinformatic prediction (via FunRich) to determine the biological pathways regulated by these miRNAs at different stages/phases of the reproductive cycle. This review revealed that 146 miRNAs ovarian miRNAs from 9 animal species regulate various critical biological pathways most of which have established roles in ovarian function. The expression of nine critical luteal miRNAs was determined. MiR-378b, miR-221, miR- 96, miR-202, let-7a, let-7b, miR-7, miR-378a and miR-210 were expressed throughout the period of culture. The expression of miR-378b increased on day 7 following vascular endothelial growth factor receptor 2 (VEGFR2) inhibition on day 5. There was a decreased expression of miR-221 and miR-378a in conditioned media compared to control media. There was an increased expression of let-7b, let-7a and miR-202 in conditioned media compared to control media. The effects of miR-378b on bovine luteal angiogenesis and progesterone production was further investigated. Luciferase assays were performed to measure the functional binding of miR-378b to predicted target genes. Luciferase assay results showed that miR-378b binds to the 3’UTR of STAR and IGF2 to repress their expression and function in bovine luteal cells. MiR-378b mimic also caused a significant increase in the caspase 3/7 activity. In addition, we developed a fluorescence- activated cell sorting (FACS) protocol to sort bovine endothelial cells and steroidogenic cells to determine which cell type expressed miR-378b and the other eight critical luteal miRNAs. There was a significantly increased expression of miR-378b, miR-96 and miR-210 in enriched endothelial cells. In response to LPS treatment, differential gene expression analysis using a log2 fold change of <-1 or >1 revealed 33 downregulated genes and 18 upregulated genes on day 3 of culture. On day 5, 4 genes were down regulated while 33 genes were upregulated in LPS treated cells. RNA sequencing and qPCR analyses revealed decreased expression of roundabout guidance receptor 4 (ROBO4), epidermal growth factor-like domains protein 7 (EGFL7), secretogranin II (SCG2), 2’-5’- oligoadenylate synthetase (OAS1Y), delta like canonical notch ligand (DLL4), platelet and endothelial Cell adhesion molecule 1 (PECAM1) and C-X-C motif chemokine ligand 5 (CXCL5) in LPS treated cells on day 3. There was decreased expression of cadherin 5 (CDH5), luteinizing hormone/choriogonadotropin receptor (LHCGR), PECAM1, CXCL5 and SCG2 in LPS treated cells on day 5. TLR4 inhibition using TAK242 reversed the effect of LPS on the expression of DLL4 and PECAM1 on days 3 and 5. The effect of LPS on the expression of ROBO4 and EGFL7 on day 3, LHCGR, CDH5, SCG2 and CXCL5 on day 5 was partially reversed following TLR4 inhibition. Cytokine arrays revealed an increased concentration of VEGF in conditioned media from LPS treated cells on days 3 and 5. In conclusion, this thesis highlights the complexity of the CL and demonstrates that specific miRNAs and LPS are key regulators of bovine luteal function.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Veterinary Medicine and Science
eprints ID
74241
UoN Repository URI
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