- Title
- Regulation of expression of key genes in uterine myocytes
- Creator
- Ilicic, Marina
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2017
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Background: As term pregnancy approaches, the human uterus is transformed from a relaxed quiescent phenotype to a contractile phenotype capable of the powerful coordinated contractions of labor. Regulation of this transformation is yet to be fully understood, however, key contraction-associated genes have been identified. Understanding the regulation of these genes is a high priority. Ethical consideration of human pregnancy primarily limits researchers to in vitro investigations using human cell lines and biopsied tissues. Whilst informative, concerns have been raised about the ability of cells and tissues in culture to remain representative of the tissue of origin. This concern was realised in relation to pregnant human myometrium upon observing that expression of progesterone receptor (PR) isoforms in non-laboring tissue transitioned to a laboring state in vitro. This confounded the ability to study PR regulation in vitro, and raised questions as to whether other key contraction-associated genes underwent culture-induced expression changes. Hypotheses and Aims: It was hypothesised that the key contraction-associated genes; estrogen receptor 1 (ESR1), prostaglandin-endoperoxide synthase 2 (PTGS2), oxytocin receptor (OXTR), PR, human ether-à-go-go-related gene (KCNH2) and potassium voltage-gated channel subfamily E regulatory subunit 2 (KCNE2) underwent significant expression changes upon culturing term nonlaboring human myometrium in vitro. Furthermore, it was hypothesised that these culture-induced changes were consistent with transition toward a laboring myometrial phenotype. The aim of this thesis was to determine whether expression of ESR1, PTGS2, OXTR, PR, KCNH2 and KCNE2 changed upon placing non-laboring human myometrium into culture. In addition, this research aimed to identify culture conditions that blocked or minimized phenotypic transition of these genes in vitro. As newly identified contraction-associated genes, this thesis also aimed to determine whether expression of KCNH2 and KCNE2 in term non-laboring human myometrium correlate with the expression of ESR1, PTGS2, OXTR and PR, which was yet to be examined in pregnant human myometrium. Methods: For gene expression studies, quantitative RT-PCR was used to determine ESR1, PTGS2, OXTR, PR, KCNH2 and KCNE2 mRNA abundance in fresh term non-laboring human myometrial tissue and tissue cultured for 48 h under conditions routinely used within the field. Effects of specific treatments were examined, including; serum, progesterone, estrogen, cAMP, PMA, stretch, NF-ĸB inhibitors, PGF₂α and trichostatin-A (TSA). For contraction studies, strips of term non-laboring human myometrium were suspended in organ baths. Effects of hERG channel activators and inhibitors on spontaneous contractions were examined. Results: Placing myometrial tissue in culture for 48 h resulted in significant culture-induced up-regulation of ESR1, PTGS2, PR-T, PR-A and KCNE2 mRNA expression, as well as significant down-regulation of OXTR mRNA expression, relative to fresh tissue. Expression of PR-B and KCNH2 mRNA remained unchanged. Progesterone and estrogen prevented culture-induced increase in ESR1 mRNA expression; PMA maintained KCNE2 and OXTR mRNA expression; while TSA prevented culture-induced increase in PR-A mRNA expression and maintained the original PR-A/PR-B expression ratio. In term non-laboring human myometrium, KCNH2 and KCNE2 expression were found to correlate with expression of ESR1, PTGS2, OXTR and PR. Additionally, the hERG channel activators had no effect on spontaneous contractions in vitro, whereas inhibitors significantly increased contraction duration. Conclusion: Standard conditions routinely employed to perform in vitro culture of human myometrium results in significant changes in key contractionassociated genes, including the newly identified contraction-associated genes, KCNH2 and KCNE2. Overall, the altered expression of these genes wasconsistent with transition of non-laboring tissue toward a laboring phenotype in vitro. No single supplement was able to prevent all culture-induced changes of the genes examined. Culture-induced changes are anticipated to extend well beyond the limited cohort of genes examined. These findings have serious implications for in vitro studies attempting to advance our understanding of human parturition, and highlight the need to develop and validate robust in vitro models for the future.
- Subject
- genes; uterine myocytes; in vitro; pregnancy
- Identifier
- http://hdl.handle.net/1959.13/1349876
- Identifier
- uon:30455
- Rights
- Copyright 2017 Marina Ilicic
- Language
- eng
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 12 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 119 KB | Adobe Acrobat PDF | View Details Download |