1 Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland
2 To whom correspondence should be addressed. E-mail: ailish.hynes{at}nuigalway.ie
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Abstract |
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Key words: culture/glycolysis/inhibitors/mouse follicles/oxidative phosphorylation
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Introduction |
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However, we considered that the results indicating that mouse follicles preferentially used glycolysis for energy production might be an artefact due to the follicles being cultured in hypoxic conditions at the bottom of upright drops of culture medium in the wells of 96-well plates. Thus, in the present study, we set out to determine the degree of follicular dependence on aerobic energy production by examining the effects of inhibitors of oxidative phosphorylation and the TCA cycle on follicular growth.
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Materials and methods |
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Reagents
Unless otherwise stated, all reagents and media were from Sigma-Aldrich (Poole, Dorset, UK).
Follicle isolation
Follicles were isolated in Leibovitz L-15 medium (cell culture grade) supplemented with 3 mg/ml bovine seum albumin (BSA; fraction V) at 37°C as previously described (Wycherley et al., 2004). Only follicles with an intact theca layer and no visible signs of atresia were used for culture. Any adhering pieces of stroma were dissected off; this was only necessary where large mature follicles were being dissected.
Follicle culture
Follicles were cultured individually as previously described (Wycherley et al., 2004) in 96-well round-bottomed suspension cell tissue culture plates (Sarstedt, Drinagh, County Wexford, Ireland) in 100 µl drops of
-minimal essential medium (
-MEM) supplemented with 5% female mouse serum, human FSH (1 IU/ml, National Hormone Pituitary Program, NIH) and 25 µg/ml ascorbic acid; this supplemented medium constituted the basic follicle culture medium.
Follicles were cultured in three ways depending on the experiment; they were cultured in either a 100 µl drop overlaid with 70 µl of mineral oil (upright with oil system), a 100 µl drop not overlaid with oil (upright without oil system) or a 100 µl drop with the plate turned upside-down without oil (inverted system). All plates were cultured in a humidified incubator at 37°C. The inverted system was used in all experiments; in some experiments, one or both of the upright systems was also used. When the plate was inverted, the follicles came to rest and were cultured sitting on the mediumgas interface thus maximizing gaseous exchange. The medium remained in the wells due to surface tension. Follicle diameters were measured daily on a Nikon Axiovert inverted microscope at 100x and follicles were transferred every other day to fresh medium in a new row of wells. Follicles grown in inverted plates were turned right side up for transfer and measurement.
Follicle diameter was used as the main index of follicle growth because we (Wycherley et al., 2004) found a correlation coefficient of R = 0.899 between follicle cell count and diameter (equivalent to an R2 value of 0.808), and similarly Spears et al. (1998) found a very high correlation (R = 0.85) between follicle DNA content and follicle diameter. Where a metabolic inhibitor caused death of all the follicles in a treatment, the dead follicles were not cultured for the entire 6 days but were discarded as soon as they were deemed to be dead. Follicles were deemed dead based on their morphological appearance under the dissecting and inverted microscopes and if there was no increase in diameter from the previous day. Dead follicles had a blackened appearance and had lost the clearly organized structure of the follicle (oocyte, granulosa and theca). Control follicles were cultured for the entire 6 days. In experiments where follicles continued to grow in the presence of inhibitor for all 6 days of culture and follicle diameters were measured on each day, effects of inhibitors were analysed by repeated measures analysis of the follicle diameters followed by analysis of variance (ANOVA) in which the effects of inhibitor were examined separately for each day of culture. Statistical analysis was not carried out for treatments in which growth in all follicles was abolished after 2 days culture in the presence of the metabolic inhibitor.
In separate experiments using small numbers of follicles as a check on the follicle diameter data, the effects of all inhibitors (except malonate) on follicle cell number were examined after 2, 4 and 6 days of culture, regardless of whether these follicles had ceased to grow in diameter or appeared morphologically dead after 2 days culture. Follicles were dissociated enzymatically and cell counting was carried out as described by Wycherley et al. (2004). In these experiments, because of the nature of the method of counting, measurements could not be repeated on the same follicle; the effects of inhibitors were analysed by standard ANOVA.
Metabolic inhibitors
All inhibitors were added to the basic follicle culture medium at the start of follicle culture and every 48 h (immediately after follicle transfer to fresh wells).
Cyanide. Cyanide forms a reversible complex with the respiratory cytochrome oxidase enzyme system, an enzyme system essential for oxidative phosphorylation, and thus inhibits cell oxygen utilization (Hewitt and Nicholas, 1963; Ballantyne and Marrs, 1987
). Basic follicle culture medium was supplemented with 1 mmol/l sodium cyanide and culture medium without cyanide was used as a negative control. This concentration of cyanide was used previously to inhibit oxidative phosphorylation in rabbit preimplantation embryos (Kane and Buckley, 1977
).
Rotenone. Rotenone is a respiratory chain complex I inhibitor which blocks the flow of electrons from NADH to coenzyme Q (Machinist and Singer, 1965; Degli Esposti et al., 1996
). Rotenone was first dissolved in dimethylsulphoxide (DMSO) at a concentration of 1 mmol/l and was used at final concentrations of 0.1, 0.5 and 1 µmol/l in basic follicle culture medium. The highest level of DMSO that was used as a carrier for rotenone was 0.1% (v/v) and this level was used as a negative control. Rotenone was used previously at similar concentrations as a respiratory inhibitor (Ernster et al., 1963
).
2,4-Dinitrophenol (DNP). DNP is known as a mitochondrial uncoupler because it has the ability to block respiratory chain-linked ATP synthesis by dissipating the proton gradient which drives the phosphorylation of ADP by ATP synthase, while permitting electron transport to continue (Slater, 1963). DNP was used at a concentration of 0.1, 0.5 and 1 mmol/l in basic follicle culture medium, and culture medium without DNP was used as a negative control.
Malonate. Sodium malonate is a competitive inhibitor of the TCA cycle enzyme succinate dehydrogenase. The structure of malonate is similar to that of succinate but cannot be oxidized by succinate dehydrogenase, resulting in inhibition of the enzyme (Quastel, 1963). Sodium malonate was used at a final concentration of 10 mmol/l in basic follicle culture medium, and culture medium without malonate was used as a negative control. This concentration was used previously to inhibit the TCA cycle in rabbit preimplantation embryos (Kane and Buckley, 1977
) and ovarian follicles (Boland et al., 1994a
).
Monofluoroacetate (MFA). MFA is a TCA cycle inhibitor. In the mitochondria, MFA is converted to fluoroacetyl-CoA by acetyl-CoA synthetase. Fluoroacetyl-CoA is a substrate for citrate synthetase, which condenses it with oxaloacetate to form fluorocitrate. The fluorocitrate formed inhibits the TCA cycle enzyme aconitase, resulting in TCA cycle inhibition and an accumulation of citrate (Liébecq and Peters, 1949; Quastel, 1963
). The basic follicle culture medium was supplemented with 10 mmol/l MFA, and culture medium without MFA was used as a negative control. This concentration was used previously to inhibit the TCA cycle in rabbit preimplantation embryos (Kane and Buckley, 1977
).
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Results |
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Cyanide (1 mmol/l) completely abolished follicle growth in the under oil culture system. After 24 h in culture, the follicles treated with cyanide had a darkened appearance with many black speckles throughout the follicle. The theca layer of cells looked loosely packed and disorganized in the cyanide-treated follicles as compared with the negative controls. After 48 h in culture, these follicles looked black and atretic and showed no increase in size from the previous day. The oocyte was no longer visible and the organization of the follicle was disrupted with the theca layer no longer clearly distinguishable from the granulosa. These results were confirmed by examining the effects of cyanide (1 mmol/l) on follicle cell number. Cell numbers were as follows (means ± SEM, follicle numbers in parentheses), without cyanide values first, plus cyanide second: day 0, 1567 ± 833 (6), 1450 ± 180 (6); day 2, 4250 ± 250 (2), 2775 ± 1148 (4); day 4, 24 950 ± 2950 (2), 3000 ± 677 (4); day 6, 24 250 ± 3250 (2), 2850 ± 286 (6). Overall, cyanide significantly decreased follicle cell numbers (P < 0.001).
After 48 h in culture, follicles cultured without cyanide had increased in size and had the characteristics of healthy follicles with spherical oocytes, few black speckles and a highly organized structure.
Experiment 2: the effect of the respiratory chain inhibitor rotenone on the growth of mouse follicle
Follicles were cultured in the inverted drop system in medium supplemented with varying levels of rotenone (Figure 3). Rotenone completely abolished follicle growth in the inverted culture system at all three concentrations used (0.1, 0.5 and 1 µmol/l). The rotenone-treated follicles had a darkened appearance after 24 h in culture, and after 48 h the follicles were dead as evidenced by the total lack of growth and fully blackened appearance similar to the cyanide-treated follicles.
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Again these results were confirmed by examining the effects of rotenone (0.1 µmol/l) on follicle cell number. Cell numbers were as follows (means ± SEM, follicle numbers in parentheses), without rotenone values first, plus rotenone second: day 0, 2600 ± 726 (4), 3460 ± 460 (5); day 2, 17 500 ± 1000 (2), 5800 ± 982 (4); day 4, 40 000 ± 6429 (3), 5125 ± 898 (4); day 6, 39 000 ± 4041 (3), 6875 ± 718 (4). Overall, rotenone significantly decreased follicle cell numbers (P < 0.001).
Experiment 3: the effect of the mitochondrial uncoupler DNP on the growth of mouse follicles
Follicles were cultured for 6 days in inverted drops in culture medium supplemented with varying levels of DNP (Figure 4). The lowest level of DNP (0.1 mmol/l) decreased follicular growth as compared with the 0 control on day 2 (P < 0.05) and on each subsequent day of culture (P < 0.001). The follicles treated with 0.1 mmol/l DNP looked darker than those in the control, but not completely degenerated. There was also no visible antral formation in these follicles. The two highest levels of DNP (0.5 and 1 mmol/l) completely abolished follicular growth from day 1 of culture. The appearance of these follicles was similar to the blackened appearance after cyanide and rotenone treatment.
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Again these results were confirmed by examining the effects of DNP (0.1 mmol/l) on follicle cell number. Cell numbers were as follows (means ± SEM, follicle numbers in parentheses), without DNP values first, plus DNP second: day 0, 2250 ± 629 (4), 2820 ± 610 (4); day 2, 18 000 ± 4000 (2), 8750 ± 1250 (4); day 4, 31 333 ± 5812 (3), 14950 ± 1924 (4); day 6, 46 000 ± 5292 (3), 14 650 ± 1498 (4). Overall, DNP significantly decreased follicle cell numbers (P < 0.001).
Experiment 4: the effect of the TCA cycle inhibitor sodium malonate on follicle growth in vitro
Follicles were cultured for 6 days in the inverted system in culture medium with and without sodium malonate (10 mmol/l). Malonate had no significant effect on follicle diameter as compared with follicles cultured without malonate on any day of culture (Figure 5).
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Experiment 5: the effect of the TCA cycle inhibitor MFA on follicle growth
Follicles were cultured for 6 days in the three culture systems in culture medium with and without MFA (10 mmol/l). The effects of MFA on follicle growth are shown in Figure 6a, b and c. MFA markedly decreased follicular growth in all three culture systems. In the inverted culture system (Figure 6a), this effect was significant on day 2 of culture and on each subsequent day (P < 0.001). In the upright culture system without oil overlay (Figure 6b), the decrease first became significant on day 2 and remained significant on each subsequent day of culture (day 3, P < 0.05; day 4, P < 0.01; day 5, P < 0.001; day 6, P < 0.05). In the upright system with oil overlay (Figure 6c), the decrease first became significant on day 2 (P < 0.001) and remained significant on each subsequent day of culture (days 3 and 4, P < 0.01; days 5 and 6, P < 0.001). MFA reduced growth in the two oil-free systems, upright and inverted, to about the same low level, i.e. the beneficial effect of the inverted system on follicle growth was not seen in the presence of MFA.
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These results also were confirmed by examining the effects of MFA (10 mmol/l) on follicle cell numbers using the inverted system. Cell numbers were as follows (means ± SEM, follicle numbers in parentheses), without MFA values first, plus MFA second: day 0, 2750 ± 372 (6), 2717 ± 415 (6); day 2, 99 67 ± 1017 (3), 5933 ± 636 (3); day 4, 36 333 ± 2848 (3), 18 667 ± 1202 (3); day 6, 40 750 ± 1931 (4), 28 000 ± 1151 (5). Overall, MFA significantly decreased follicle cell numbers (P < 0.001).
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Discussion |
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The failure of malonate, another TCA cycle inhibitor, to affect follicle growth is probably due to an inability to enter the follicular cells in sufficient amounts to cause inhibition. Questions over the adequacy of malonate entry into cells to cause inhibition are not new. In a study to determine the role of the TCA cycle in rabbit preimplantation embryo development, in which several inhibitors (cyanide, DNP, oligomycin, MFA and malonate) of both oxidative phosphorylation and the TCA cycle were used (Kane and Buckley, 1977), malonate was the only inhibitor that did not arrest growth at the 1-cell stage, possibly due to inadequate uptake. In some cases, researchers have used methylmalonate as an inhibitor instead of malonate in order to improve uptake by cells; the methylmalonate molecule is more cell permeable and intracellular esterases cleave the methyl group and release the parent malonate molecule inside the cell. McLaughlin et al. (1998)
found that in some neuronal cells, methylmalonate was more than twice as effective as malonate in causing cell death.
Earlier observations on follicular energy metabolism in vitro, involving an upright culture system, seemed to suggest a follicular preference for energy production via glycolysis (Boland et al., 1993, 1994a
,b). This hypothesis was based on two pieces of evidence. One piece of evidence was that malonate had no effect on follicular growth (Boland et al., 1994a
); however, this lack of effect can possibly be explained, as already discussed, by the problems of malonate entry into follicular cells. The second piece of evidence was that under their culture conditions, all of the glucose used by follicles was converted to lactate, suggesting that oxidative metabolism was of little importance for the provision of energy to follicles. However, two factors need to be considered here. One is that a molecule of glucose metabolized fully by oxidative metabolism to carbon dioxide and water provides 19 times as many ATP molecules as one molecule of glucose converted anaerobically to lactate. Thus, small amounts of glucose being metabolized by oxidative phosphorylation could provide significant amounts of ATP. A second factor is potentially much more important. There is widespread evidence that much of the energy needs of cells in culture are met, not by glucose but by amino acids and in particular by glutamine (Reitzer et al., 1979
; Wice et al., 1981
); in certain circumstances, HeLa cells derived >98% of their energy from oxidative metabolism of glutamine. The medium used by Boland et al. (1994a)
was
-MEM which, like the medium used in our work, contained 2 mmol/l glutamine (H.J.Leese and R.G.Gosden, personal communication). Thus, much of the energy needs of the follicles in the study of Boland et al. (1994a)
could have come from metabolism of glutamine and other amino acids.
Support for this view comes from the work of Kelly and West (2002) who found that follicular cells deficient in a key glycolytic enzyme, glucose isomerase, could survive if they were in contact with cells containing the enzyme. They concluded that follicular cells do not need an intact endogenous glycolytic pathway if they can obtain appropriate metabolites from an exogenous source, which they suggested could be glutamine or pyruvate.
It has been suggested by a number of workers that in such circumstances, the high rate of glucose uptake and aerobic glycolysis observed in some proliferating cells is a consequence of an overall metabolic strategy of the proliferating cell to maintain high cytostolic levels of glucose-6-phosphate, fructose-6-phosphate and triose-phosphates; these compounds are essential for nucleic acid, oligosaccharide and lipid synthesis (McKeehan, 1982). In the presence of adequate glutamine, glucose can be completely replaced in culture medium for HeLa cells and some other cell types by a pyrimidine nucleotide such as uridine or cytidine at
1 mmol/l (Wice et al., 1981
).
The observation that the beneficial effect of the inverted system on follicle growth was not seen in the presence of MFA because MFA reduced growth in the two oil-free systems to about the same low level is interesting and suggests that the major advantage of the inverted system is its ability to promote oxidative metabolism by an improved supply of oxygen to the follicle. This advantage is abolished by inhibition of the TCA cycle.
Our work in this study showing an essential role for oxidative metabolism in follicular growth is supported by our previous work showing that the inverted culture system which was designed to maximize follicular gaseous exchange resulted in a marked improvement in follicular development as compared with the commonly used upright culture systems (whether with or without oil overlay). The effect of the inverted system on lactate production per unit volume of follicle was of particular interest in that lactate production in the inverted system was in some cases less than half that in the upright systems (Wycherley et al., 2004). This implied that in the upright systems, follicles became hypoxic due to the distance of the follicle from the mediumgas interface. Some recent work with LLC-PK1 porcine renal cells in culture supports this idea that distance of cultured cells from the mediumgas interface can affect the oxygenation state of the cells and the rate of glycolysis as evidenced by their production of lactate. Gstraunthaler et al. (1999)
varied the distance of the mediumgas interface from cells cultured in monolayer in tissue culture dishes by varying the medium volume, and they also varied the glucose concentration in a factorial arrangement with medium volume. They found that lactate production and glycolytic enzyme activity increased markedly with distance of the mediumgas interface from the cells; they also found that glutamine consumption decreased markedly with increasing distance. Thus cells in tissue culture can become hypoxic (as evidenced by increased latate production) due to long diffusion distances from the mediumgas interface. There is evidence that proliferating epithelial cells in culture require a PO2 of >40 mmHg in their immediate environment; for some cells this may be up to 70 mmHg or more (Taylor and Camalier, 1982
). To achieve such oxygen tensions in the vicinity of the cells incubated in an air/5% CO2 atmosphere, fluid overlays not greater than 0.34 mm have been recommended (Wolff et al., 1993
), but the situation varies with type of cell and cell density (Metzen et al., 1995
). However, since the distance between the bottom of the well and the mediumgas phase interface in our upright system without oil is 4.9 mm, it can be readily seen how large follicles with large numbers of follicular cells might become hypoxic in such a system. In preliminary studies on the oxygen consumption of follicles in culture, we have found that a single large follicle in an upright culture system can significantly deplete the oxygen in its vicinity (G.Wycherley, unpublished data).
It is highly relevant to try to consider here the oxygenation state of the follicle in vivo. While data on oxygen tension in follicular fluid in vivo is very limited, the available evidence from a number of species suggests oxygen tensions are relatively highexamples of figures in mmHg as follows: rat, 23.5 (Fennema et al., 1986); pig, 51; and human, 5080 (Fischer et al., 1992
) and 107116 (Verbessem et al., 1988
). Since these are values found in follicular fluid which is situated at the interior of the follicle, this implies that oxygen tensions surrounding the follicular cells may be even higher.
There is clear evidence that cells in general (Gstraunthaler et al., 1999) and follicular cells in particular (Boland et al., 1993
) can adapt to some degree of hypoxia by relying on glycolysis for the production of energy. However, our previous work (Wycherley et al., 2004
) in straight forward direct comparisons showed that conditions resulting in improved oxygenation of cultured follicles resulted in increased follicular growth as evidenced by measurement of follicular diameter, cell count and estradiol production; this was associated with reduced lactate production
In conclusion, the mouse ovarian follicle appears to have an absolute dependence on aerobic respiration for normal growth and development, and the activities of oxidative phosphorylation and the TCA cycle are facilitated by an improved oxygen supply in the inverted culture system.
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Acknowledgements |
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References |
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Submitted on November 1, 2004; resubmitted on April 19, 2005; accepted on May 10, 2005.
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