Fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte

Abstract

The role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes has been studied. Fluorescence resonance energy transfer (FRET) acceptor bleaching methods have been used to examine mitochondrial:lipid droplet co-localisation in live oocytes. FRET experiments demonstrate whether organelles are within the FRET-distance (i.e. 6-10 nm), thus showing true association on a molecular scale. Immature and in vitro-matured porcine oocytes were stained with Mitotracker Green (MTG; mitochondria) and Nile Red (NR; lipid droplets). The data indicated sufficient overlap between MTG emission and NR excitation to support a FRET reaction and that mitochondria and lipid droplets were sufficiently co-localised for a FRET reaction to occur. When NR-stained lipid droplets were specifically bleached, a significant increase in the MTG signal in stained mitochondria was observed (FRET efficiency, EZ22.2G3.18%). These results strongly suggest a metabolic role for lipid metabolism during oocyte maturation. This conclusion was reinforced by the use of inhibitors of fatty acid b-oxidation, methyl palmoxirate or mercaptoacetate, exposure to which during oocyte maturation led to developmental failure post-fertilisation. These data provide strong evidence that MTG and NR can act as a FRET pair and that in porcine oocytes, mitochondria and lipid droplets lie within 6-10 nm of each other, indicating association on a molecular scale. The findings also suggest that endogenous triglycerides play an important role in energy metabolism during porcine in vitro maturation. Reproduction (2006) 132 829-837

Not Just Fat: The Structure and Function of the Lipid Droplet

“…LDs are also in close proximity to mitochondria and peroxisomes, in which fatty acids liberated by lipolysis are used for b-oxidation. LDs and the associated organelles may form close membrane contacts as shown by Förster Resonance Energy Transfer (FRET) (Sturmey et al 2006). In S. cerevisiae T. Fujimoto and R.G.…”

The role of l-carnitine during oocyte in vitro maturation: essential co-factor?

“…These studies demonstrate a requirement for beta-oxidation in nuclear maturation (Downs et al, 2009;Valsangkar and Downs, 2013;Paczkowski et al, 2014;Sanchez-Lazo et al, 2014), as described above, and in the acquisition of oocyte developmental competence in mouse, bovine and porcine oocytes (Ferguson and Leese, 2006;Sturmey et al, 2006;Dunning et al, 2010;Paczkowski et al, 2014).…”

“…There is a growing body of literature describing the importance of fatty acid beta-oxidation as a source of energy during oocyte IVM (Ferguson and Leese, 2006;Sturmey et al, 2006;Downs et al, 2009;Dunning et al, 2010;Dunning and Robker, 2012;Valsangkar and Downs, 2013;Dunning et al, 2014b;Paczkowski et al, 2014). This is not surprising when one considers the energy dense nature of fatty acids with the potential to produce 106 ATP molecules from a single fatty acid, greater than 3.5-fold the capacity of a single glucose molecule.…”

Energy Metabolism Regulating Mammalian Oocyte Maturation

“…In fully grown-germinal vesicle oocyte, the mitochondria homogenously distribute throughout the cell with some localization at the periphery (Sun et al, 2001). As the oocyte progresses through meiotic maturation, mitochondria relocate into the perinuclear region and aggregate into cluster (Yu et al, 2010;Van Blerkom et al, 2002;Sturmey et al, 2006); this event coincides with the rise in ATP levels (Yu et al, 2010). Therefore, the change in localization of mitochondria during oocyte growth is probably in response to energy demand during a given stage of development (Ramalho-Santos et al, 2009).…”

“…Intracellular lipids play essential roles in oocyte development (Sturmey et al, 2009(Sturmey et al, , 2006Sturmey & Leese, 2003;Ferguson & Leese, 2006). Lipids serve as reservoir of endogenous energy source, substrates for water production during blastocoel development and precursors of second messengers modulating cell functions and purine synthesis (McEvoy et al, 2000).…”