Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles

G. Wycherley1, M.T. Kane1 and A.C. Hynes1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium–gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial µm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. METHODS: All inhibitors were tested in the inverted system using 100 µl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. RESULTS: The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 µmol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. CONCLUSIONS: Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.

Key words: culture/glycolysis/inhibitors/mouse follicles/oxidative phosphorylation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Culture of intact ovarian follicles in vitro provides a powerful model for the study of follicle growth and ovulation. Systems of follicle culture are numerous (Hartshorne, 1997Go), but most involve culture in upright drops of medium under mineral oil (Nayudu et al., 2001Go; Spears et al., 2004Go). We recently reported a novel inverted culture system, which involves culture of follicles at the medium–gas interface in inverted 100 µl drops of culture medium (Wycherley et al., 2004Go) (see Figure 1). This culture system was designed to increase the supply of oxygen to the growing follicle by minimizing the diffusion distance for oxygen to the cultured follicle. This inverted system markedly improved follicle growth as compared with growth in the upright systems (either with or without an oil overlay) as evidenced by increased follicle diameter, cell number and estradiol production. In contrast, these follicles produced significantly less lactate per unit follicular volume than follicles cultured in the upright systems, suggesting that follicles in the inverted system may be relying less on glycolysis and more on the tricarboxylic acid (TCA) cycle and oxidative phosphorylation for the provision of energy than follicles cultured in the upright systems. It therefore seemed reasonable to hypothesize that the improved follicle growth and estradiol production in the inverted system were due to an improved energy supply resulting from increased TCA cycle and oxidative phosphorylation activity due, in turn, to improved oxygen supply to the follicle in the inverted system.



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Figure 1. Diagrammatic representation of the three follicle culture systems including the proposed oxygenation state of follicles cultured in each system.

 
A serious objection to this hypothesis stems from the work of Gosden and collaborators who investigated the energy metabolism of the mouse follicle using an upright culture system with drops of medium overlaid with mineral oil (Boland et al., 1993Go, 1994aGo,bGo). The results of these studies, which are the most comprehensive studies to date on the relative importance of glycolysis versus oxidative phosphorylation for growth of the mouse follicle, suggested that follicles utilize a predominantly glycolytic mode of energy production. For instance, Boland et al. (1993)Go found that mouse follicles growing in vitro produce large quantities of lactate as a metabolic by-product, and went on to show that all of the glucose consumed by the ovarian follicle was converted to lactate (Boland et al., 1994aGo). They concluded that pre-antral follicles could undergo development to the pre-ovulatory stage using glycolysis alone, a feature which they suggested might allow them to conserve their limited supply of oxygen for other vital biosynthetic processes. There is a clear example of this kind of thing happening in other cells. Brison et al. (1994)Go found that inhibitors of oxidative phosphorylation did not prevent rat morulae developing to blastocysts; glycolytic activity was stimulated by the presence of inhibitors and compensated for the inhibition of oxidative phosphorylation.

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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Animals
Inbred MF1 mice were used for these experiments and were housed under a photoperiod of 14 h light:10 h dark and provided with food and water ad libitum.

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., 2004Go). 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., 2004Go) in 96-well round-bottomed suspension cell tissue culture plates (Sarstedt, Drinagh, County Wexford, Ireland) in 100 µl drops of {alpha}-minimal essential medium ({alpha}-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 medium–gas 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., 2004Go) 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)Go. 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, 1963Go; Ballantyne and Marrs, 1987Go). 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, 1977Go).

Rotenone. Rotenone is a respiratory chain complex I inhibitor which blocks the flow of electrons from NADH to coenzyme Q (Machinist and Singer, 1965Go; Degli Esposti et al., 1996Go). 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., 1963Go).

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, 1963Go). 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, 1963Go). 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, 1977Go) and ovarian follicles (Boland et al., 1994aGo).

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, 1949Go; Quastel, 1963Go). 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, 1977Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Experiment 1(a): the effect of the respiratory chain inhibitor sodium cyanide on the growth of mouse follicles in the inverted culture system
Follicles were cultured for 6 days in the inverted system in culture medium with or without sodium cyanide (1 mmol/l). The effect of cyanide on follicle growth in the inverted system is shown in Figure 2a. There was no significant difference in diameter between follicles cultured with or without cyanide on any day of culture in the inverted culture system. Since cyanide is volatile in aqueous solutions, it was possible that the cyanide evaporated from the culture dish before or soon after the follicles were placed into culture.



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Figure 2. Effect of cyanide (1 mmol/l) on the growth of follicles as measured by follicle diameters. Treatments are shown as follows: cyanide, filled circles; no cyanide, open circles, and values are means ± SEM. (a) Inverted culture system n = 13–24. There was no significant effect of cyanide at any day of culture. (b) Upright with oil system, n = 10–25; all follicles in cyanide were dead after 48 h.

 
Experiment 1(b): the effect of the respiratory chain inhibitor sodium cyanide on the growth of mouse follicles cultured under oil overlay
Since the volatility of cyanide in aqueous solutions was a possible explanation for its lack of effect on follicles cultured in the inverted system, we next tested the effect of cyanide on follicles grown under oil overlay which would be expected to act as a barrier to the movement of cyanide out of the culture medium; cyanide is known to be poorly soluble in organic solvents as compared with water (Merck Index, 1989Go). It is also known that the inhibitory effect of cyanide is potentiated by hypoxic conditions (Hewitt and Nicholas, 1963Go). The effect of cyanide on follicle growth under oil overlay is shown in Figure 2b.

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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Figure 3. Effect of rotenone (0, open circles; 0.1, closed circles; 0.5, open squares; and 1 µmol/l, filled squares) on the growth of follicles in the inverted culture system. Values are means ± SEM; n = 8–10; all follicles in rotenone were dead after 48 h.

 

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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Figure 4. Effect of DNP (0, open circles; 0.1, filled circles; 0.5, open squares; and 1 mmol/l, filled squares) on the growth of follicles in the inverted culture system. Values are means ± SEM; n = 7–12. The overall effect of DNP (0.1 mmol/l) was significantly different (P < 0.001, repeated measures analysis); this level of DNP significantly decreased follicular growth on day 2 (P < 0.05) and on each subsequent day (P < 0.001). The higher levels of DNP (0.5 and 1 mmol/l) completely abolished follicular growth.

 

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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Figure 5. Effect of malonate (0, open circles; 10 mmol/l, filled circles) on the growth of follicles in the inverted culture system. Values are means ± SEM; n = 13–16. There was no significant effect of malonate at any day of culture.

 

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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Figure 6. Effect of the culture system and MFA (0, open circles; 10 mmol/l, filled circles) on the growth of follicles: (a) inverted, (b) upright without oil and (c) upright with oil systems. Values are means ± SEM; n = 7–18. Effects of the culture system. In the absence of MFA, growth in the inverted system was significantly greater than in the upright without oil (P < 0.01) and the upright with oil system (P < 0.001, repeated measures analysis); also the upright without oil system was superior to the with oil system (P < 0.05). In the presence of MFA, growth in the inverted system was not significantly greater than in the upright without oil system (P > 0.05) but was greater than in the upright with oil system (P < 0.05); also the upright without oil system was superior to the upright with oil system (P < 0.05). Effects of MFA. (a) Inverted culture system: the overall effect of MFA in decreasing follicle growth was significant (P < 0.001, repeated measures analysis); MFA significantly decreased follicular growth on day 2 and on each subsequent day (P < 0.001). (b) Upright system without oil: the overall effect of MFA was significant (P < 0.01); MFA significantly decreased follicular growth on days 2 and 3 (P < 0.05), day 4 (P < 0.01), day 5 (P < 0.001) and day 6 (P < 0.05). (c) Upright system with oil: the overall effect of MFA was significant (P < 0.001); MFA significantly decreased follicular growth on day 2 (P < 0.001), days 3 and 4 (P < 0.01), and days 5 and 6 (P < 0.001).

 

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).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The major question asked in these experiments was whether aerobic metabolism involving both the TCA cycle and oxidative phosphorylation is essential for normal mouse follicle growth in vitro. From the results of these experiments, the answer is clear—TCA cycle and oxidative phosphorylation are essential for normal follicular growth. First, the results of the experiments with cyanide, rotenone and DNP, in which these inhibitors totally abolished follicular growth, provide conclusive evidence that normal oxidative phosphorylation is essential. Since the TCA cycle is necessary to feed energy-rich electrons into the respiratory chain, this in turn provides strong evidence for the essentiality of the TCA cycle. Secondly, the inhibitory effect of the TCA cycle inhibitor, MFA, on follicular growth provides further evidence that the TCA cycle is essential.

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, 1977Go), 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)Go 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., 1993Go, 1994aGo,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., 1994aGo); 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., 1979Go; Wice et al., 1981Go); in certain circumstances, HeLa cells derived >98% of their energy from oxidative metabolism of glutamine. The medium used by Boland et al. (1994a)Go was {alpha}-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)Go could have come from metabolism of glutamine and other amino acids.

Support for this view comes from the work of Kelly and West (2002)Go 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, 1982Go). 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., 1981Go).

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., 2004Go). This implied that in the upright systems, follicles became hypoxic due to the distance of the follicle from the medium–gas interface. Some recent work with LLC-PK1 porcine renal cells in culture supports this idea that distance of cultured cells from the medium–gas 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)Go varied the distance of the medium–gas 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 medium–gas 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 medium–gas 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, 1982Go). 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., 1993Go), but the situation varies with type of cell and cell density (Metzen et al., 1995Go). However, since the distance between the bottom of the well and the medium–gas 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 high—examples of figures in mmHg as follows: rat, 23.5 (Fennema et al., 1986Go); pig, 51; and human, 50–80 (Fischer et al., 1992Go) and 107–116 (Verbessem et al., 1988Go). 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., 1999Go) and follicular cells in particular (Boland et al., 1993Go) can adapt to some degree of hypoxia by relying on glycolysis for the production of energy. However, our previous work (Wycherley et al., 2004Go) 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This research was funded by a Health Research Board of Ireland grant to A.C.H.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on November 1, 2004; resubmitted on April 19, 2005; accepted on May 10, 2005.





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