Mitochondrial adaptions in the placenta during pregnancy

Abstract

The placenta is a unique organ critical for the growth and development of the fetus. The placenta provides the link between maternal and fetal circulations, supplying nutrients and oxygen, removing waste and regulating metabolic and hormonal responses in both mother and fetus. The placenta develops from a single cell mass with a common cell progenitor diverging down multiple pathways responsible for the development of many cell lineages. Among these cell lineages is the villous trophoblast which form the junction for the aforementioned maternal and fetal interface. This junction consists of underlying cytotrophoblast cells which fuse and differentiate into a multinucleated syncytium. During this transformation from cytotrophoblast to syncytiotrophoblast, the organelles which accompany these cell types also undergo morphological and functional changes. This PhD will investigate these changes in organelle morphology and function. Cell studies on transformed placental trophoblast cells, Swan-71’s, in Chapter 2 examined the effect of endoplasmic reticular (ER) stress on mitochondrial function and dynamics under acute and chronic exposure. The aim of this work was to address the interactions between mitochondria and endoplasmic reticular stress which converges on pathways often associated with mitochondria dysfunction, that have been proposed to play a role in gestational disorders. It has been established that alterations in ER stress effects mitochondrial functionality in different ways dependent on exposure time and the pathway of ER stress initiated. Mitochondrial alterations in bioenergetics, reactive oxygen species production, mitochondrial dynamics and key metabolic proteins associated with differentiation were assessed under normal conditions and after induction of ER stress. While finding that ER stress does initiate mitochondrial dysfunction, the translatability of such a finding was difficult to ascertain due to examining a trophoblast precursor cell (most similar to the cytotrophoblast) containing a mostly uniform mitochondrial population in contrast to the vastly different populations observed in the placenta. Investigations in Chapter 3 aimed to optimise isolation methodology enabling the characterisation of mitochondria from the cytotrophoblast and syncytiotrophoblast cell lineages. These studies provided an experimental model to assess the bioenergetic and metabolic alterations between these different mitochondria, laying a foundation for future studies to be translated and the continued examination of mitochondrial interactions in pathologies. Chapter 4 utilised the optimized protocol to generate a proteomic profile of the mitochondrial populations from the placenta post differentiation, identifying key proteins involved in the mechanisms which may drive this previously speculative process. Through this characterisation we established a more comprehensive understanding of mitochondrial transformations associated with trophoblast differentiation. We further investigated altered protein expression patterns in carbohydrate, fatty acid and amino acid metabolism, in combination with key structural proteins within the electron transport chain that were significantly altered. These findings expanded our knowledge and proposed mechanisms by which the previously observed alterations in mitochondrial functionality may occur. This was followed by an investigation into gestational disorders in Chapter 5 through examination of gestational diabetes mellitus (GDM) in placental samples. The aim of this investigation was to examine bioenergetic and metabolic pathways to determine if mitochondrial dysfunction occurred as a result of alterations in the previously identified mechanisms and proteins; with the specific intent to assess mitochondria from both cell lineages individually. The purpose of this was to ascertain if contradictory findings on mitochondrial function in GDM were as a result of whole tissue analysis, therefore not accounting for the differing mitochondrial populations present in the placenta. Overall, the findings presented in this thesis establishes that mitochondria and the ER are intrinsically linked through functionality, the implication of which require further investigation in the placenta. However, with the comprehensive characterisation of the two mitochondrial populations present in the cytotrophoblast and syncytiotrophoblast, this interaction can now be more thoroughly studied. This body of work identifies key mitochondrial pathways which are altered between the two populations, identifying mechanisms which may drive mitochondrial transformation within the placenta. Observations which have previously been speculative based on morphological appearance alone. This thesis provides a well characterised physiological basis for future work into placental mitochondria. Further, this body of work forms a critical foundation when assessing the importance of mitochondria in pathologies. While often linked to pathologies through mitochondrial dysfunction, this thesis highlights the importance of a sessing isolated mitochondria from pathological placentae in order to minimise the often contradicting results in the literature obtained due to examination of whole tissue, thereby, not accounting for the different physiology of the mitochondrion in the placenta. This body of work identified that mitochondrial appear dysfunctional in GDM and proposed mechanisms which may be involved in the pathogenesis of the disorder although mechanisms underpinning this require further examination.