Preeclampsia is an increasingly prevalent disorder of placentation that has very limited options for treatment. The disease is characterized by aberrant invasion of placental trophoblasts into the decidualized maternal endometrium. In order to identify pathways of therapeutic interest during placentation, we are focusing on the pathway of the neuropeptide kisspeptin and its receptor KISS1R, both highly expressed in the human placenta. Early functional studies of the ligand-receptor system identified a role for kisspeptin in the inhibition of cancer metastasis. Parallels exist between cancer and placentation, suggesting the possibility of an inhibitory role for kisspeptin during pregnancy as well.
Existing functional data supports kisspeptin's inhibitory influence on cellular invasion, but the mechanism remains unknown. Evidence for the localization of the KISS1R receptor in the current literature was established via a nonspecific antibody and requires further investigation. Current literature suggests involvement of the ERK (extracellular signal-regulated kinase) pathway as well. Our work aims to solidify the localization of kisspeptin and KISS1R, avoiding the use of KISS1R antibodies. Using immunohistochemistry for protein localization of kisspeptin and placental fractionation followed by quantitative PCR analysis for gene expression, we provide evidence of kisspeptin's restriction to the syncytiotrophoblast layer of the placenta, and KISS1R gene expression limited to the villous cytotrophoblast layer. This distribution of ligand and receptor suggests a paracrine mechanism for kisspeptin action, with syncytiotrophoblasts secreting kisspeptin to act on its receptor on the villous cytotrophoblast layer, and thus restricting cytotrophoblast invasion.
We further attempt to support these data with the use of laser capture microdissection of placental tissue to isolate the different layers, followed by quantitative PCR. This technique introduced a particularly challenging aspect of working with the placenta: maintaining tissue morphology while also preserving RNA integrity. This thesis outlines our troubleshooting process for that technique and introduces alternatives for future work. We also employed Western blot analysis of ERK activation to establish the mechanism of kisspeptin's inhibitory effect on fractionated trophoblasts.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/16262 |
| Date | 08 April 2016 |
| Creators | Masheeb, Zahrah |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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