Mitochondrial isolation, cryopreservation and preliminary biochemical characterisation from placental cytotrophoblast and syncytiotrophoblast

Abstract

Placental function and homeostasis is very much dependent upon trophoblast mitochondria. Mitochondria are responsible for a myriad of functions including energy production, endocrine functions, nutrient and oxygen transfer as well as protecting the placenta from oxidative insult and cell stress. The materno-fetal interface is comprised of 2 related cell lineages, cytotrophoblast cells which fuse to form the overlaying syncytium. Mitochondrial morphology and function in these 2 lineages is different and, in this study, we describe methods for mitochondrial isolation, cryopreservation and preliminary biochemical characterisation of these discrete forms of mitochondria. Villous tissue was collected from normal term placentae following vaginal delivery (n=7). Differential centrifugation was used to isolate 2 fractions, larger mitochondria predominantly from the cytotrophoblast cells and smaller mitochondria from the syncytiotrophoblast. Freshly isolated mitochondria were assessed for Complex I and Complex II respiration and maximal respiratory capacity and these were compared to equivalent isolates that had been frozen, stored at-80 o C and revived. Other important functional parameters of mitochondrial activity were also assessed post cryopreservation including membrane potential, ATP production, progesterone biosynthesis and anti-oxidant expression. The methods described resulted in 2 mitochondrial populations which match the types of mitochondria observed in situ with election microscopy. Both forms of mitochondria were metabolically active and recovered well from cryopreservation. There were important functional differences between cyto-mitochondria and syncytio-mitochondria including decreased respiration, decreased oxidative phosphorylation, decreased anti-oxidant expression but increased expression of the enzyme CYP11A1 and increased progesterone production. This study highlights the need to consider different types of trophoblast mitochondria in placental studies and provides methods to facilitate this.

Proteomic Analysis of Placental Mitochondria Following Trophoblast Differentiation

“…The ETC generates ATP, and lower levels of ETC proteins in the Syncytio-Mito may have implications for bioenergetic capacity and ability to supply cellular energy. Indeed, our recently published data on mitochondrial respirometry demonstrated decreased bioenergetic parameters in Syncytio-Mito including; maximum respiratory capacity and ATP production (Fisher et al, 2019). This supports existing literature, which showed altered dimerization of ATP synthase and F 1 to F 0 ratios associated with decreased ATPase activity in mitochondria from Syncytio-Mito and Cyto-Mito (De los Rios Castillo et al, 2011).…”

“…Isolation of enriched mitochondrial fractions was carried out using sequential differential centrifugation at 1,500 × g for 10 min, the supernatant collected and subsequently spun at 4,000 × g for 15 min to produce a cytotrophoblast (Cyto-Mito) pellet and 12,000 × g to produce syncytiotrophoblast (Syncytio-Mito) pellet as recently described (Fisher et al, 2019).…”

“…As previously published, differential centrifugation resulted in highly enriched populations of cytotrophoblast and syncytiotrophoblast mitochondria (Fisher et al, 2019). Their respective protein profiles were determined via proteomics and results were cross-referenced against the MitoCarta2.0 database to identify solely mitochondrial proteins and enable analysis of varied expression between mitochondrial populations.…”

“…However, mitochondria in the syncytium appear smaller, spherical and punctate with little to no definition in cristae structure. What drives this process at a molecular level remains unclear although it is known that alterations in morphology are accompanied by changes in mitochondrial bioenergetics, changes in membrane potential and ATP production (Kolahi et al, 2017;Fisher et al, 2019).…”

Maternal selenium deficiency during pregnancy in mice increases thyroid hormone concentrations, alters placental function and reduces fetal growth

“…; Fisher et al . ) with minor modifications. Briefly, placental tissue samples were placed immediately in ice‐cold BIOPS solution (2.77 m m CaK 2 EGTA, 7.23 m m K 2 EGTA, 5.77 m m Na 2 ATP, 6.56 m m MgCl 2 ⋅6H 2 O, 20 m m taurine, 15 m m Na 2 phosphocreatine, 20 m m imidazole, 0.5 m m dithiothreitol and 50 m m Mes, pH 7.1).…”