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

Departments of Biology and ¹ Technology Facility, University of York, York YO10 5DJ, UK

Correspondence should be addressed to R G Sturmey; Email: r.g.sturmey{at}gmail.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
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, E=22.2 ± 3.18%). 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 ß-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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Oxidisable substrates for ATP generation can be broadly categorised into those which are available exogenously (i.e. present within the extracellular milieu) and those which are endogenous, typically as glycogen or lipid in the form of triglycerides. This distinction is particularly relevant in the oocyte, which is the largest cell in the female mammal. Early work demonstrated the importance of oxidisable exogenous energy sources to the oocyte, such as glucose, lactate and pyruvate (reviewed by Sutton et al. 2003), however, as a large mammalian cell and through its ancestry to amphibians and birds, which have yolk-laden eggs, the oocyte is likely to contain considerable endogenous energy stores (Leese 1991). This is particularly evident in the oocytes of the domestic pig, where very high levels of lipid have been reported (156 ng; (McEvoy et al. 2000); the majority in the form of triglyceride (TG) (Homa et al. 1986)). The potential contribution of endogenous energy substrates to ATP generation in mammalian oocytes and the early embryos to which they give rise has been largely ignored, reflecting the traditional experimental approach of examining the metabolism of exogenously supplied nutrients. Global energy metabolism may be determined by measuring oxygen consumption (Leese et al. 2001). Using such an approach, Sturmey & Leese (2003) reported a fall in intracellular TG content and oxygen consumption in porcine oocytes during in vitro maturation with sufficient O₂ consumed to account for the fall in TG.

TG is metabolised by ß-oxidation and the TCA cycle within the mitochondrial matrix. For this to occur, mitochondria and lipid droplets, the primary source of TG, should ideally reside in close proximity. This has been observed in oocytes of domestic species (Cran et al. 1980, Kruip et al. 1983, Hyttel et al. 1986, Sun et al. 2001), where mitochondria and lipid droplets are seen to associate, forming ‘metabolic units’ which tend to accumulate at the edge of the oocyte, thus ensuring a steady and readily accessible supply of O₂ for oxidative processes. However, while these studies suggest mitochondrial:lipid co-localisation, the data are based on subjective morphological assessment – true mitochondrial:lipid association, on a molecular scale, is yet to be demonstrated.

Fluorescence resonance energy transfer (FRET) with confocal microscopy allows the study of co-localised organelles in living cells at a molecular level (Jares-Erijman & Jovin 2003). FRET relies on labelling organelles with fluorescent probes whose spectral profiles overlap. If the organelles in question are in close proximity, typically 6–10 nm or closer, the amount of light emitted from the donor probe will be reduced since some of its energy is passed on to a second, acceptor probe, leading to its excitation and the emission of light. By removing the acceptor probe, emission from the donor probe is increased as energy transfer can no longer take place. Acceptor bleaching (i.e. destroying the acceptor probe) FRET can be used to demonstrate whether organelles are within the FRET-distance (i.e. 6–10 nm), thus showing true association on a molecular scale.

The aim of the present study was to examine the location of lipid droplets and mitochondria using porcine oocytes as a model system by laser scanning confocal microscopy, using fluorescent probes and FRET techniques. Oocytes have been individually stained for mitochondria with Mitotracker Green (MTG) and lipid droplets with Nile Red (NR) to identify any association between them. Difficulties in separating the emission of the two probes have been resolved using emission fingerprinting and spectral unmixing. In order to examine the role of TG metabolism during maturation, oocytes matured in the presence of inhibitors of lipid metabolism were fertilised and grown to the blastocyst stage. The inhibitors used were methyl palmoxirate, a stereospecific irreversible inhibitor of the carnitine palmitoyl transferase complex responsible for transporting fatty acyl CoA across the mitochondrial membrane, and mercaptoacetate, a competitive inhibitor of the 3-hydroxyl CoA dehydrogenase enzyme of ß-oxidation. We report that mitochondria associate with lipid droplets on a molecular level in immature and in vitro-matured oocytes, and that inhibition of lipid transport into mitochondria during maturation inhibits subsequent embryo development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Unless otherwise stated, all chemicals were supplied by Sigma-Aldrich.

Oocyte culture and staining
Porcine oocyte-cumulus complexes (OCCs) were harvested and matured according to the method of Sturmey & Leese (2003). Briefly, OCCs were aspirated from slaughter house-derived ovaries and those with evenly granulated cytoplasm and at least two complete layers of cumulus cells were selected for maturation in chemically defined in vitro-matured (IVM) medium (Abeydeera et al. 2000). Where appropriate, porcine maturation medium (pMM) was modified by the inclusion of MTG dye or the addition of TG metabolism inhibitors, methyl palmoxirate (MP) or mercaptoacetate (Merc). MP, a kind gift from RW Johnson Pharmaceutical Research, was dissolved in acetone and added to pMM at concentrations between 0.1 and 5 mM; Merc was added to pMM directlyat concentrations ranging between 0.1 and 10 mM (see Results for specific concentrations). When cultured in the presence of inhibitors, oocytes were fertilised and placed into embryo culture in NSCU-23 as described by Sturmey & Leese (2003); cleavage was assessed on day 2 and blastocyst rates were recorded 6 days post-insemination.

MTG and NR (Molecular Probes, Invitrogen) were used to label mitochondria and lipid droplets respectively. All stages were performed under minimal ambient lighting. For mitochondrial staining, oocytes were gently denuded by passing through hand-drawn glass pipettes of gradually decreasing diameter in the presence 0.25% (w/v) hyaluronidase in PBS. They were then incubated in chemically defined pMM (Abeydeera et al. 2000) supplemented with 2 µM MTG (an optimised concentration) for 30 min at 39 °C, 5% CO₂ in air. Following MTG staining, oocytes were washed in PBS, pre-warmed to 39 °C and attached to poly-L-lysine-coated No. 1 glass coverslips (VWR, Leicester, UK) glued to a steel washer, to form a viewing chamber. Lipid droplets were stained using 100 nM NR in pre-warmed PBS, 2 ml of which was gently washed over the surface of chamber and incubated at room temperature for 10 min. Excess stain was washed off with pre-warmed PBS and the chambers were sealed with a second glass coverslip. Single-stained control samples were prepared where either MTG or NR staining step was omitted.

Image acquisition
All images were acquired on a Zeiss LSM 510 Meta confocal system with a Zeiss Axiovert inverted microscope. Oocytes were viewed within 5 min of staining with a 63x (1.4NA) Oil DIC lens, using a 488 nm laser for excitation of MTG and a 543 nm laser for NR excitation. Dual-stained images were viewed and emissions were separated using ‘spectral unmixing’; this relies on obtaining reference spectra, or spectral sticks, from single-labelled control samples. A ‘lambda stack’ of a single-stained control oocyte (either NR or MTG) was obtained by exciting the sample with 488 nm laser-line, using a HFT 488/543 nm dichroic beam splitter. A ‘region of interest’ (ROI) was drawn around a positive-stained area of the image, and the spectral profile of the stain in situ was extracted and saved to a spectral database. The technique was repeated with a sample labelled with the other stain and the spectral stick recorded. Dual-stained samples were then imaged using the settings used for obtaining spectral sticks and the image was ‘unmixed’. Using algorithms within the software, the spectral sticks were applied to the dual-stained image and the image was unmixed on a pixel-by-pixel means. Representative images are presented.

FRET determination
The present work utilised acceptor bleaching FRET. By using a fast scan speed and low resolution (512x 512), we were able to record images rapidly whilst minimising acquisitional bleach; however, this was at the expense of image quality. A bleach script was written and a ROI drawn around individual lipid droplets. The area within this ROI (i.e. the lipid droplet) was bleached using a 543 nm laser-line at 100% total power. The strength of bleach was set at 250 iterations. Using the 543 laser-line, it was possible to bleach most of the signal from NR within the ROI without any excitation of the MTG. A time series was also incorporated into the bleach script and images acquired every second for up to 3 min. Images were acquired using 4% of total laser output to minimise acquisitional bleach. Once acquired, all the images were linearly unmixed using pre-obtained spectral sticks. Bleaching of the NR and any associated increase in MTG signal were observed. FRET efficiency was calculated as a percentage increase in MTG signal intensity before and after photobleaching. In the present work, FRET efficiency was calculated according to Wouters et al.(1998) (see below). This determination of FRET coefficient assumes that all the acceptor fluorophores (i.e. NR in this case) are photodestroyed. FRET data were generated on four separate occasions from a total of 11 experiments on independent oocytes.

Therefore,

E%

FRET efficiency

MTG

difference in MTG intensity as measured pre – and post – bleach of NR (arbitrary units),

MTG_i

MTG intensity before NR bleach

MTG_f

MTG intensity post – bleach of NR (arbitrary units)

Statistical analysis
Cleavage and blastocyst rates of oocytes matured in the presence of MP or Merc are expressed as a percentage of the total number of zygotes selected for embryo culture post-fertilisation. All percentage data were subjected to angular transformation prior to analysis by one-way ANOVA with Fisher’s test post hoc. Statistical differences at the 5% level were assumed significant. All statistical analyses were performed on Minitab Version 12.1.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The pattern of mitochondrial distribution in porcine oocytes was studied at two time points: immediately after removal from the follicle (immature oocytes) and after 44 h of in vitro-maturation (mature oocytes). The selection of images, shown in Fig. 1, indicates differences between the distribution of mitochondria within the ooplasm at these stages of development. Immature oocytes (Fig. 1A) had a relatively homogenous distribution of mitochondrial foci throughout the ooplasm, with a higher number appearing at the cortical regions of the oocyte and relatively few mitochondrial foci around the germinal vesicle (GV) (Fig. 1A). Figure 1B is a representative image of mitochondrial distribution in in vitro-matured porcine oocytes. The GV is no longer visible and there are few mitochondrial foci within the very central region of the oocyte, however, peripheral and cortical distribution is not as obvious as in the immature oocyte. It appears that the mitochondria accumulate around spherical structures within the cytoplasm, a phenomenon more clearly visible in Fig. 1C (a highly magnified image of a peripheral region of mitochondrial staining).

View larger version (33K): [in this window] [in a new window]   Figure 1 Location and changes in distribution of mitochondria (stained green) and lipid droplets (red). Clear differences are apparent in mitochondrial (images A and B) and lipid droplet (images D and E) distribution between immature and in vitro-matured oocytes. Images G and H are dual-labelled oocytes. There is also subjective evidence of regions of ‘co-localisation’ between lipid droplets and mitochondria (images G and H); image C demonstrates mitochondrial association around unstained spherical objects; image I shows association in dual-stained oocyte.

Given the observed ‘spherical structures’ (e.g. Fig. 1C) and the hypothesised interaction between mitochondria and lipid droplets, oocytes were selected for dual staining. Figure 1G–I shows spectrally unmixed images of dual-stained oocytes. Close association between mitochondrial foci and lipid can be clearly seen in both immature (Fig. 1G) and in vitro-matured oocytes (Fig. 1H), with a more peripheral localisation apparent in the eggs post-maturation. Figure 1I shows this association in a magnified sample of the periphery of the oocyte (see figure legend for further details).

Given the observed co-localisation, we pursued the possibility that mitochondria stained with MTG would FRET with lipid droplets stained with NR. Figure 2 is a representative dual-stained image collected during the FRET experiments; images A, B and C were collected simultaneously prior to the bleach pulse and images D, E and F after the bleach. The circle indicates the ROI bleached with the 543 laser; the target lipid droplet can be seen in image A with its fluorescence being no longer visible in image D, following the bleach. Images B and E show the MTG signal before and after the bleach; the intensity is increased. This is more apparent in the merged, spectrally unmixed image showing the reduction in red, and the increase in green, signal, within the ROI. In this occasion, the FRET efficiency was determined to be 17.8%.

View larger version (21K): [in this window] [in a new window]   Figure 2 FRET occurrence in a dual-labelled immature oocyte. Images A and D show the NR signal pre- and post-bleach; a decrease in intensity can clearly be seen within the white circle. Images B and E show the MTG component pre- and post-bleach. A clear increase in intensity can be seen within the white circle. Images C and F represent the merged spectrally unmixed images. Graph G shows the mean changes in intensity for both probes during the image acquisition time; bleaching of the NR is apparent, along with an increase in MTG intensity. This is representative of all 11 repetitions of the technique, performed on multiple occasions. Graph H shows mean FRET efficiencies for MTG–NR interaction and corresponding control experiments. Control 1, bleach of ROI, represents a re-bleach of previously bleached region to investigate any recovery of NR post-bleach. The FRET efficiency was calculated at –0.4%, indicating no FRET. Control 2, bleach with 633 nm, represents the investigation of any heat effect (H). Control 3, bleach with 488 nm, represents bleaching the MTG signal with 100% 488 nm laser to show that MTG acted as a fluorophore donor. The negative FRET efficiency shows that MTG is bleached; there was no change in NR signal. Control 4, bleach away from lipid, shows that the increase in MTG signal was not an artefact of bleaching the cytoplasm indiscriminately.

FRET validation
It was possible that a true FRET signal was not observed and that MTG was entering the lipid droplets. However, we are confident that this was not the case as indicated by the spectral profiles of the two stains. Although we were unable to stain lipid droplets with NR and MTG in the absence of mitochondria, lipid droplets with fewer mitochondria present gave significantly lower FRET signals, strongly indicating that NR–MTG FRET was due to mitochondrial–lipid association. Another possibility was that the increase in MTG signal was due to local effects from the prolonged bleach. A region of MTG not associated with NR was ‘bleached’ with the 543 nm laser (i.e. the laser-line that elicits NR bleach and FRET reaction) but no significant increase in MTG signal was observed (E~2%; Fig. 2H). The increase in MTG signal may have been due to ‘false assignment’ of the MTG and NR when spectrally unmixed; once the NR was bleached, more green may have been assigned to MTG than previously observed. However, closer inspection of the individual wavelength images from the lambda stack (data not shown), where the intensity in the shorter wavelength images (510–520 nm) clearly increased after bleaching, confirmed that unmixing did not introduce artefactual results. A lipid droplet was bleached for a second time to test if there had been photorecovery of NR which could have caused FRET to recur. In this case, E was again ~0%, indicating that NR was being bleached and that no recovery was occurring. As a final control, the MTG signal was bleached with the 488 nm laser (i.e. at its excitation wavelength). The value of E given was ~–15% (i.e. the fluorescent signal was reduced) thus showing that the FRET changes were not due to spectral changes upon MTG bleaching.

Given these findings, we examined the effects of inhibiting TG metabolism during IVM on subsequent embryo development. Two inhibitors were selected: MP, an inhibitor of the carnitine shuttle responsible for transport of free fatty acids across the mitochondrial membrane, and Merc, competitive inhibitor of 3-hydroxyacyl CoA dehydrogenase, a key enzyme in ß-oxidation. Figure 3 shows cleavage and blastocyst rates of embryos derived from oocytes matured in the presence of MP. Mean cleavage in control groups (i.e. groups matured in pMM alone) was 43%; blastocyst rate was 23% (six replicates, n=637). When cultured in the presence of MP for the duration of maturation, there was neither cleavage (except in one replicate) nor production of blastocysts (six replicates, n~600 per group). Cleavage and blastocyst production rates of oocytes matured in the acetone control groups were similar to the standard control groups.

View larger version (16K): [in this window] [in a new window]   Figure 3 Cleavage and blastocyst formation rates for oocytes matured in the presence of MP and subsequently fertilised. No differences were observed between Control groups and ‘Acetone Controls’. Maturation in the presence of pMM led to complete failure of subsequent embryo development as assessed in terms of cleavage or blastocyst formation. The data were collected from six replicates, with approximately 600 oocytes per treatment, and are expressed as mean ± S.E.M.

View larger version (11K): [in this window] [in a new window]   Figure 4 Blastocyst formation rates from oocytes matured in the presence of the competitive inhibitor of TG metabolism, Merc and subsequently fertilised. Blastocyst rates did not differ between control groups and oocytes cultured in 0.1 or 1.0 mM Merc. Oocytes matured in the presence of 10 mM Merc failed to generate blastocysts. The data were collected from five replicates with approximately 530 oocytes per treatment group and are expressed as mean ± S.E.M.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

Mitochondrial distribution
Immature oocytes showed a relatively homogenous distribution of mitochondria with some localisation at the periphery, generally consistent with other findings for porcine (Sun et al. 2001), bovine (de Paz et al. 2001) and ovine (Cran et al. 1980) immature oocytes. In contrast, in vitro-matured oocytes tended to display a slightly more peripheral localisation of mitochondrial foci with few mitochondria in the central region of the ooplasm (Fig. 1B, E and H); a pattern of mitochondrial distribution reported previously in porcine (Sun et al. 2001, Brevini et al. 2006) and murine (Van Blerkom et al. 2002) oocytes. Bavister & Squirrell (2000) found that mitochondria actively relocate during oocyte maturation in several species and our data suggest that this occurs in the porcine oocyte. Sun et al.(2001) considered that morphological and biochemical changes in the cytoplasm during maturation were coordinated with the successful resumption of meiosis and Abe et al.(2002) reported that the presence of serum in oocyte culture medium could have adverse effects on the distribution and the structure of mitochondria.

Observations of a peripheral distribution of mitochondria may relate to the fate of the mitochondria within the pre-implantation embryo, all of which are derived from the oocyte with de novo mitochondrial synthesis, not occurring until the blastocyst stage (Ebert et al. 1988). Blastomeres deficient in mitochondria fail to divide and lyse during culture (Van Blerkom et al. 2000). Whilst there is some mitochondrial clustering around the pronuclei during fertilisation, we postulate that a pronounced peripheral arrangement ensures that mitochondria are distributed equally between the blastomeres during embryo cleavage; if all the mitochondria were to cluster in the centre of the oocyte, unequal cell division might leave some blastomeres deficient in mitochondria.

Van Blerkom & Runner (1984) suggested that mito-chondrial relocalisation to areas that require elevated levels of ATP is a necessary feature of oocyte maturation. The peripheral distribution of mitochondria may therefore also be linked to the need to metabolise compounds supplied to the periphery of the oocyte (de Paz et al. 2001). For example, Donahue & Stern (1968), and Leese & Barton (1985) reported that follicular-derived cells produced pyruvate in quantities sufficient to support murine oocyte maturation. During maturation, the association between accessory cells is maintained by gap junctions from cumulus cell processes which terminate on the surface of the oolemma (Fleming & Saacke 1972, Eppig 1982). Moreover, Moor & Dai (2001) described the oocyte membrane as poorly equipped to transport metabolites, thus reinforcing the importance offollicular cells. While it is unclear how long the gap-junctional contact is maintained during oocyte maturation, mitochondria may localise to the periphery of the oocyte to enable rapid metabolism of exogenous compounds taken up. In addition, mitochondrial relocalisation may be linked to intracellular oxygen gradients. Mitochondrial clustering results in steep oxygen gradients within the cell (Aw 2000) and altered physiological states can lead to mitochondrial redistribution (Jones et al. 1990). These factors may account for the peripheral mitochondrial distribution we observed since oxygen availability will be highest in this region of the oocyte.

Lipid droplet distribution
Spherical structures in the oocyte cytoplasm, measuring 1–4 µm in diameter, appeared to be surrounded by mitochondria (Fig. 1C). The position of these putative lipid droplets pre- and post-maturation was therefore studied using the probe NR, a lipid-specific stain. Lipid droplets tended to show peripheral distribution in immature oocytes (Fig. 1D), with very few droplets detectable in the inner cortex. A broadly similar pattern was observed in oocytes post-maturation, although the peripheral localisation was more pronounced (Fig. 1E). The presence of lipid droplets has previously been shown in porcine (Cran 1985) and bovine oocytes (Kruip et al. 1983, Abe et al. 2002). However, we believe that this is the first study of lipid distribution in porcine oocytes during maturation. Cran (1985) reported changes in the number and the size of lipid droplets within the cytoplasm of immature and matured porcine oocytes, but did not refer to the positioning of the droplets. Campagna et al.(2006), who examined the effects of organochlorines during oocyte maturation, reported that although lipid droplets did not change in size, alteration in the electron density ratio was suggestive of lipolysis at the periphery of the lipid droplets; these lipid droplets were associated with smooth endoplasmic reticulum and mitochondria. The peripheral distribution of lipid droplets seen in the porcine oocyte might again relate to oxygen gradients within the cell or as a consequence of mitochondrial positioning per se. By locating to the periphery, lipid–mitochondrial units ensure that oxidative metabolism is not limited by oxygen availability, since there will be a higher cytoplasmic oxygen concentration at the edge of the oocyte (Van Blerkom et al. 2002).

Mitochondrial:lipid interaction – FRET analysis
Single-stained images showed that mitochondria and lipid droplets reside in similar areas within the oocyte during maturation (Fig. 1A–E). However, using dual-staining methods, a clear association between lipid droplets and mitochondria within the porcine oocyte was observed (Fig. 1G–I). This association has previously been reported in adipocytes (Cinti 2001), the cytoplasm of bovine oocytes (Kruip et al. 1983), ovine and bovine embryos (Crosier et al. 2001, Rizos et al. 2002), and more recently in porcine oocytes (Campagna et al. 2006). However, these studies have relied either on electron microscopy, where the use of processed fixed cells may inadvertently form artificially close associations, or on traditional confocal imaging. Confocal microscopy has an x, y resolution of 200 nm at best, and the z resolution is only around 700 nm. Moreover, chromatic aberration using two colours further decreases the resolution of x, y and z. The possibility of true spatial interaction and tight association was therefore pursued using FRET methods.

The FRET data show that those mitochondria that are associated with lipid droplets lie within 10 nm and most probably closer, to each other, indicating true ‘co-localisation’ on a molecular scale. This finding does not suggest that all mitochondria in a given oocyte are associated with lipid droplets, but that of those that are the association between the two organelles represents true molecular-level co-localisation. The data were collected without the need for fixatives and prolonged sample-processing stages which may introduce artefacts. The efficiency of FRET relies on the Förster equation (Forster 1965) which states that ‘the efficiency of FRET is dependent on the inverse sixth power of intermolecular separation’ (Sekar & Periasamy 2003). In real terms, the Förster radius is the distance at which half the excitation energy is passed to the donor, typically 3–6 nm (Sekar & Periasamy 2003), although this distance can be as high as 10 nm. Whilst co-localisation between lipid droplets and mitochondria is generally inferred from visualisation of electron micrographs (i.e. sublight resolution), it is believed that the present work provides the first evidence for such tight association. The distance at which FRET occurs (<60 Å, Selvin 2000) corresponds to the thickness of a single phospholipid monolayer (10–60 Å) (Stryer 1995). Since lipid droplets are most probably surrounded by a phospholipid monolayer (Murphy & Vance 1999, Ostermeyer et al. 2001), the data suggest that mitochondria have direct contact with the hydrophilic tail of the monolayer, allowing efficient direct transport of free fatty acids from the lipid droplet, an arrangement that would be necessary for efficient diffusion with minimal loss to the surrounding cytoplasm.

Why co-localise?
Kruip et al.(1983) proposed that mitochondria and lipid droplets form ‘metabolic units’ during bovine oocyte maturation. TG is stored as lipid droplets (Londos et al. 1999, Murphy & Vance 1999), and free fatty acids cleaved from the TG molecules are transported across the mitochondrial membrane by the carnitine shuttle. Once internalised in the mitochondrial matrix, fatty acyl CoA molecules are oxidised by ß-oxidation and the TCA cycle. The data in Fig. 3 strongly suggest a metabolic role for TG during oocyte maturation; when carnitine palmitoyl transferase, the enzyme responsible for the transport of free fatty acids into the mitochondrial matrix, was inhibited by MP, oocyte maturation as indicated by subsequent embryo development was impaired, suggesting that endogenous fatty acid metabolism is important during porcine oocyte development in vitro. A similar effect in the bovine oocyte was reported by Ferguson & Leese (2006), when lipid metabolism was inhibited during bovine oocyte maturation, the blasto-cyst rate was reduced from 22 to 6%.

As with all inhibitors, there is a question of specificity. Kiorpes et al.(1984) reported that MP is a ‘site-directed inactivator of carnitine palmitoyl transferase I’, and that its inhibition is irreversible and stereospecific. However, to provide more evidence of the effects of inhibiting lipid metabolism during oocyte maturation, a second inhibitor, Merc, was used. Merc is a competitive inhibitor of ß-oxidation, inhibiting 3-hydroxyacyl CoA dehydrogenase by forming 2-mercaptoacyl CoA (Bauche et al. 1982). When cultured in Merc at low concentrations (0.1–1.0 mM), oocyte maturation was unaffected when measured in terms of the ability to form a blastocyst post-fertilisation. However, when oocytes were cultured in the presence of 10 mM Merc, no blastocysts were formed post-fertilisation. These data may be explained in terms of the mechanism of the two inhibitors. When MP was present, TG transport into the mitochondria was prevented since the compound binds irreversibly to the active site of transport (carnitine palmitoyl transferase I) (Kiorpes et al. 1984). This resulted in a failure of pre-implantation development. By contrast, Merc is a competitive inhibitor which competes for enzymatic activity by mimicking the structure of native substrates; if the native substrate is in excess of the competitor, the inhibition will be overcome and effects are only observed at high concentrations of inhibitor, as seen in the present work.

In conclusion, we have shown that mitochondria and lipid droplets within the porcine oocyte co-localise on a molecular scale and tend to localise at the periphery of the cell, most probably in response to oxygen availability. The data also suggest that lipid metabolism is important for porcine maturation since: (a) TG levels in the oocyte fall during maturation in vitro (Sturmey et al. 2003); (b) non-reversible inhibition of fatty acid metabolism prevents subsequent embryo development; and (c) low levels of competitive inhibition of TG metabolism may be overcome. Lipid:mitochondrial co-localisation, as demonstrated by FRET studies, facilitates rapid transport of free fatty acids to the site of metabolism (i.e. the mitochondria) and implies a metabolic role for TG stored in lipid droplets within the porcine oocyte during development in vitro.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The authors wish to thank Dr A Leech and Dr J Breton for helpful discussions in the preparation of this manuscript. The work was funded by the UK Biotechnology and Biological Sciences Research Council and Sygen International. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	Footnotes

 
Received 20 June 2006
First decision 25 July 2006
Accepted 18 August 2006

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