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RESEARCH |
Department of Physiology, National University of Ireland, Galway, University Road, Galway, Ireland
Correspondence should be addressed to A C Hynes; Email: ailish.hynes{at}nuigalway.ie
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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There are a number of lines of evidence which strongly suggest that in vitro follicle culture conditions are not yet optimal. Fully grown mouse follicles in vivo reach diameters of approximately 500 µm (reviewed by Murray & Spears 2000) whereas in vitro grown follicles generally reach approximately 400 µm (Spears et al. 1996, 1998, Murray et al. 1998). In addition, the developmental competence of oocytes from cultured follicles is far below what one would expect from healthy in vivo produced mouse follicles (Spears et al. 1994). There have also been very few live births from in vitro grown oocytes using follicle culture systems (reviewed by Smitz & Cortvrindt 2002). The greatest degree of success in producing live births from in vitro grown oocytes has being achieved using organ culture of whole ovaries of newborn mice followed by culture of isolated cumulus–oocyte complexes (Eppig & O’Brien 1996, O’Brien et al. 2003). However, since their method does not involve whole follicle culture, it is not a useful model for the study of follicle growth and maturation.
In most follicle culture systems the follicle is cultured, under a gas phase of about 20% oxygen, at the bottom of a culture well in which the medium is covered by an oil overlay. Since a fully grown mouse follicle contains about 50 000 cells (Pedersen, 1970), the question arises as to whether oxygen diffusion through the oil overlay down to the bottom of the culture well is capable of supplying enough oxygen for follicular growth. In the work reported here, we investigated the use of a novel inverted system without oil for follicle culture and compared it with upright systems with and without an oil overlay (Fig. 1
). In this inverted system, culture is carried out with the culture dish in the upside down or inverted position. Since the follicle in the inverted system lies at the medium/gas interface during culture, one effect of the inverted system should be to maximize access of oxygen to the cultured follicle.
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Materials and Methods |
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Follicle isolation
Follicles were isolated as previously described (Boland et al. 1993) with minor modifications. Briefly, prepubertal mice aged between 23 and 26 days of age were killed by cervical dislocation and their ovaries were removed to a 35 mm Petri dish (Nunc A/S, Roskilde, Denmark) with 4 ml Leibovitz L-15 medium (Sigma-Aldrich, Poole, Dorset, UK) cell culture grade supplemented with 3 mg/ml bovine serum albumin (fraction V; Sigma-Aldrich) at 37 °C. Ovaries were cleaned of any surrounding tissue and individual follicles (diameters from 180 to 240 µm) were dissected out with acupuncture needles (Carbo EZY-5, 0.22–0.30 gauge, 25 mm in length; Helio Medical Supplies, Santa Clara, CA, USA), attached to microbiological loop holders. Only follicles with an intact theca layer with some stromal cells attached and no visible signs of atresia were used for culture.
Follicle culture
Follicles were cultured in 96-well round-bottomed suspension cell tissue culture plates (Sarstedt, Drinagh, Co. Wexford, Ireland) in 100 µl droplets of 
-minimal essential medium (Sigma-Aldrich) supplemented with 5% female mouse serum, human follicle-stimulating hormone (FSH; 5 IU or 1 IU/ml; National Hormone Pituitary Program, Harbor-UCLA Medical Centre, Torrance, CA, USA) and 25 µg/ml ascorbic acid (Sigma-Aldrich). Serum was prepared as described by Boland et al.(1993) except that the mice were killed by carbon dioxide asphyxiation and blood was collected from the heart immediately postmortem.
All media were made up weekly from powdered stocks with Milli-Q water from a Milli-Q system (Millipore, Watford, Herts, UK). Follicles were cultured in three ways depending on the experiment: in a 100 µl drop overlaid with 70 µl mineral oil (upright with oil system), a 100 µl drop not overlaid (upright without oil system) or a 100 µl drop with the plate turned upside-down without oil (inverted system). 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 because of surface tension. Sterile Milli-Q water (100 µl) was put into the first and last row of wells and into the spaces between the wells to ensure a humid atmosphere over the culture medium. Follicle diameters were measured daily on a Nikon Axiovert inverted microscope (The Micron Optical Co. Ltd, County Wexford, Ireland) at 100 x and follicles were transferred every other day to a fresh row of wells. For any follicles that were not spherical, the average of the long and short axes was taken as the diameter. Follicles grown in inverted plates were turned right side up for transfer and measurement. All plates were cultured in a humidified incubator under a 5% CO2 atmosphere at 37 °C.
Oestradiol assay
Follicle-conditioned medium was collected on each day of transfer and stored at –70 °C for oestradiol analysis. Oestradiol production was measured for individual follicles using an enzyme immunoassay (Tamate et al. 1997) according to the manufacturer’s instructions (BioResearch Ireland, Dublin, Ireland). Serial dilutions of the sample were made in a matrix provided by the manufacturer and all dilutions were parallel to the standard curve. Absorbance was read on an Anthos 2010 microplate reader (AGB, Dublin, Ireland) at 492 nm and oestradiol values were read from a standard curve.
Quantification of the number of cells per follicle in vitro and in vivo
At the end of every 24 h in culture a sample of follicles was removed, washed once in phosphate-buffered saline and treated with 5 mg/ml collagenase type I (Sigma-Aldrich) at 37 °C for 5 min. At the end of the 5 min an equal volume of trypsin–EDTA solution (trypsin 0.5 g/l, EDTA 0.2 g/l; 1 x liquid; GibcoBRL, Life Technologies, Paisley, Strathclyde, UK) was added and incubated for a further 5 min. The clumped cells were then pipetted gently to form a single cell suspension. The suspension was centrifuged for 3 min, the supernatant discarded and the pellet re-suspended in an appropriate volume of phosphate-buffered saline for counting using a haemocytometer. Because all the cells from intact follicles were counted, cell counts include both granulosa and theca cells.
For purposes of comparison with in vitro grown follicles, follicles grown in vivo were dissected out of adult mice in a similar fashion to that described above except that the largest follicles (3–4 per ovary) were chosen and great care was taken to remove as much stroma as possible from the follicle. The dispersion and counting procedure was the same as for in vitro grown follicles.
Measurement of lactate
Lactate production was measured using a colorimetric lactate diagnostic kit (Sigma-Aldrich) based on the conversion of lactate to pyruvate and hydrogen peroxide by lactate oxidase. Sample volume was 10 µl, reagent volume was 1 ml and the absorbance was measured at 540 nm on a Pharmacia Ultraspec 4000 (Amershampharmacia Biotech, Cambridge, UK).
Statistical analysis
All experiments were analysed by analysis of variance in which the effects of culture treatment were examined separately for either each day of culture or every second day of culture. Where appropriate, means were further analysed by the Bonferroni–Dunn post hoc test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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The effects of the culture system on follicle growth and secretion of oestradiol and lactate into the culture medium are shown in Fig. 2. Examination of the effects of the culture system on follicle diameter (Fig. 2a) and volume (Fig. 2b) showed that the inverted system significantly increased follicle growth on days 2–6 (P < 0.001). The percentage increases in follicle diameter and volume on day 6 in the inverted system were 29.6% and 120% as compared with the upright with oil system. There were no significant differences between the two upright systems.
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), since oestradiol is soluble in oil, oestradiol content of the medium as a measure of follicular oestradiol secretion is only valid for the oil-free upright and inverted systems and statistical comparisons were only made for these two systems. On day 6, oestradiol secretion per follicle into the culture medium (Fig. 2c) was markedly increased (+177%) in the inverted system as compared with the upright without oil system (P < 0.01). There was no significant difference between inverted and upright without oil systems at days 2 or 4. When oestradiol secretion was expressed on a per nl follicle basis, there was no significant difference between inverted and upright without oil systems at any day of culture, indicating that the increased secretion in the inverted system on day 6 relative to the upright without oil system was due mainly to the increase in follicle volume. However, the capacity to secrete oestradiol per unit volume of follicle increased linearly with duration of culture in both inverted (P < 0.001) and upright (P < 0.05) without oil systems. Interestingly, the increase in day 6 secretion as compared with day 2 secretion was much greater for the inverted (5.6-fold increase) than for the upright without oil system (2.6-fold increase).
Lactate production and secretion is an indicator of anaerobic metabolism. Lactate secretion into the medium per follicle increased in both the upright systems on day 4 as compared with day 2 but either leveled off or decreased again on day 6 (Fig. 2e). However, in the inverted system, secretion per follicle increased on both day 4 and day 6. The relative effect of the culture systems on lactate secretion per follicle varied with the day of culture; on day 2, secretion in the inverted system was significantly lower than that in the upright with oil system (P < 0.05), not significantly different on day 4 and significantly greater than both upright systems (P < 0.01) on day 6.
When lactate secretion was expressed on a per nl follicle basis, the increase in lactate secretion per follicle with day of culture in the inverted system can be seen to have been totally due to the increase in follicle volume (Fig. 2f). Secretion on a per unit volume basis remained constant in the inverted system over days 2, 4 and 6 and was significantly (P < 0.001) less than secretion in both the upright systems. There was no significant difference between the two upright systems. There was a tendency in both upright systems for secretion of lactate per nl follicle to decrease with day of culture; in the upright with oil system, secretion at days 4 and 6 was decreased significantly (P < 0.01) as compared with secretion at day 2, and in the without oil system, secretion at day 6 was significantly decreased as compared with secretion at day 4.
The results of this experiment showed that the inverted culture system, as compared with either of the two upright systems, markedly improved follicle growth (as determined by diameter and volume) and oestradiol secretion, and decreased lactate production (as expressed on a per unit volume basis).
Experiment 2: effect of the culture system on follicle cell proliferation
Follicles from purebred MF1 mice were cultured for 6 days in the two culture systems (inverted and upright with oil). FSH concentration was 1 IU/ml. Follicle cell numbers were measured daily. The effect of the culture system on cell proliferation of follicles is shown in Fig. 3. Data are also shown for cell numbers of large in vivo grown follicles from adult mice. The mean follicle diameter of in vivo grown follicles was 525.9 ± 12.7 µm. Cell proliferation was markedly increased in the inverted system as compared with the upright with oil system; the increases in cell numbers were significant on day 3 (P < 0.01) and on all subsequent days (P < 0.001) with a 1.76-fold increase in cell numbers on day 5. Cell numbers of in vitro grown follicles in the inverted system at days 5 and 6 did not differ from the in vivo grown follicles from adult mice. Growth rate per day (calculated as the difference in cell numbers between successive days) in the inverted system increased rapidly up to day 3, was maintained from day 3 to day 5 and then decreased markedly from day 5 to day 6 (Fig. 3 inset). In contrast, growth rate in the upright with oil system increased up to day 3 but then declined markedly from day 3 to day 6.
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); the R2 value was 0.808, indicating that about 81% of the statistical variation in follicle cell numbers could be accounted for by follicle diameter.
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Experiment 3: effects of mouse strain and culture system on follicle growth
Follicles from purebred MF1 and crossbred C57BL/6 x CBA/ca F1 mice were cultured for 6 days in the two culture systems (inverted and upright with oil). FSH concentration was 1 IU/ml. Follicle diameters were measured daily. The effect of mouse strain and culture system on follicle growth is shown in Fig. 5. Follicular size was significantly greater in the inverted system as compared with the upright with oil system at all stages (P < 0.05 for day 1 and P < 0.001 for days 2–6). There was no significant interaction between culture system and strain of mice, indicating that the inverted system was equally beneficial for growth of both pure-bred MF1 and crossbred C57BL/6 x CBA/ca follicles.
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The result of this experiment showed that, in both inbred and crossbred mouse strains, the inverted system produced markedly increased follicle growth as compared with the upright with oil system.
Experiment 4: effect of FSH concentration on growth of follicles from purebred MF1 mice
Because the level of FSH used in experiment 1 was 5 IU/ml but 1 IU/ml in experiments 2 and 3, in this experiment we compared the effects of 1 and 5 IU/ml FSH on the growth of MF1 mouse follicles cultured for 6 days in the inverted system. There was no significant effect of concentration of FSH at any day of culture (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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) offers the advantage of overcoming both the problem of limited nutrient availability due to a small drop size and the problem of relatively long diffusion distances for oxygen at the bottom of an upright well. An alternative to the inverted system is the system used by Qvist et al.(1990) in which follicles were grown in culture dishes with a gas permeable Petriperm base and the medium just covered the follicle.
The results described here using a drop size of 100 µl have demonstrated that an inverted culture system without oil results in superior growth of mouse follicles as compared with upright systems with or without an oil overlay; this superiority was obvious whether the measure of follicular growth was volume (2.2-fold), cell number (1.7-fold) or oestradiol secretion (2.8-fold). The mean cell number per follicle by day 5 of culture in the inverted system (47 250) was greater than the mean cell number of the large follicles from adult mice (44 629; Fig. 3). It also compares favourably with the cell number of 50 000 reported by Pedersen (1970) for large type 7 mouse preovulatory follicles about 13 h before the expected time of ovulation.
The most probable explanation for the improved growth in the inverted system is improved oxygenation of the follicles (Fig. 1). The distance between the bottom of the well in a 96-well round-bottomed culture plate and the medium/gas phase interface in our upright system without oil is 4.9 mm. Diffusion of oxygen in aqueous media is very slow with a diffusion coefficient of about 0.00003 cm2/s at 37 °C (Himmelbau 1964). In considering the supply of oxygen to follicles in an upright culture system, the important question is whether the oxygen consumption of a follicle at the bottom of a well is balanced by the flux of oxygen down from the top of the drop. The oxygen concentration in the medium surrounding the follicle must be maintained at a level adequate to allow an uptake of oxygen by the follicle at least equal to normal follicular oxygen consumption. There appears to be no information available in the literature on what is the optimal concentration of dissolved oxygen in the medium for follicular growth; however, for a number of cell types, optimal levels of dissolved oxygen in the vicinity of cultured cells are in the region of 25–50% air saturation, i.e. 25–50% of that in medium fully equilibrated with room air (Spier & Griffiths 1983). Since some follicle diameters are up to 500 µm, it is probable that the optimal dissolved oxygen concentration for follicular growth is at the upper end of that range or possibly even higher.
The flux of oxygen from the medium/gas interface to the medium in the vicinity of the follicle will depend on the difference in concentration of oxygen at the top and bottom of the drop; if oxygen consumption by the follicle markedly lowers oxygen partial pressure and concentration at the bottom of the drop, this will result in an increased diffusional flux of oxygen. However, this lower oxygen concentration in the vicinity of the follicle may be insufficient to adequately oxygenate follicular cells, particularly cells at the interior of the follicle.
If one assumes that the concentration of oxygen at the bottom of the drop is 50% of that at the medium/gas phase interface (where the medium should be in equilibrium with the oxygen in the gas phase), i.e. about 50% air saturation, it is possible to calculate the maximum possible flux for that situation in our upright without oil system as follows, based on the flux equation (Stein 1990); J = DA(SI – SII)/d, where J = flux of oxygen molecules (µmol/s), D = diffusion coefficient (0.00003 cm2/s), A = cross-sectional area of the well (0.322 cm2; well radius is 0.32 cm), SI = 178 µmol/l (based on solubility of oxygen in culture medium (Hütter et al. 2002) under conditions of equilibrium with a gas phase with a PO2 of approximately 142 mmHg; this PO2 is based on a gas phase with a dry gas composition of 5% CO2 and 95% air at 37 °C, a water vapour pressure of 47 mmHg and an atmospheric pressure of 760 mmHg), SII = 89 µmol/l, d = distance from medium/gas phase interface to the bottom of the well (0.49 cm) and J = 0.0000017545 µmol/s = 2.36 nl/min (volume at standard temperature (0°C) and pressure (760 mmHg)).
It must be emphasized that the flux of 2.36 nl/min is an upper limit; the reason for this is because the well radius is 0.32 cm, for a follicle with a diameter of about 200 µm or 0.02 cm, much of the oxygen at the bottom of the well will still be up to 0.3 cm from the follicle; thus the actual effective flux is much less than 2.36 nl/min. If the follicle oxygen consumption is greater than this flux, then we can expect the oxygen concentration in the vicinity of the follicle to drop below 50% of the concentration at the top of the well.
The question then arises – is this flux sufficient to meet oxygen consumption by the follicle? It is unfortunate that there seems to be no available data on oxygen consumption by mouse follicles. One approach to estimating the oxygen consumption of mouse follicles would be to take data from the literature on the oxygen consumption of mouse granulosa cells; unfortunately again such data appear to be lacking. However, there are data on the oxygen consumption of other types of mouse cells, e.g. mouse hepatocytes (51.4 nl O2/106 cells per min; Porter & Brand 1995) or mouse hybridoma cells (130.7 nl/106 cells per min; Miller et al. 1988). If one takes the case of the day-6 follicles at the end of culture in the inverted system (Fig. 3), which had a mean follicular cell number of 40 783, using data from both of these types of cells, one gets an oxygen consumption of either 2.10 or 5.33 nl/follicle per min. When it is considered that the calculated upper limit to the flux of O2 due to a 50% drop in O2 concentration from top to bottom of the culture drop is 2.36 nl/min and this limit, which is probably much greater than the actual flux to the follicle, is compared with these estimates of follicle oxygen consumption above (2.10 or 5.33 nl/follicle per min), it would seem that the oxygen tension in the vicinity of a large cultured follicle resting at the bottom of a well in either of the upright systems is likely to be much less than a PO2 of 71 mmHg which would correspond to 50% of the oxygen concentration at the gas phase/culture medium interface (a PO2 of about 142 mmHg). The resultant hypoxic conditions are made worse by the fact that, as the follicle grows, the oxygen in the vicinity of the follicle must also diffuse through the theca cell layer to supply the granulosa cells. This contrasts with the in vivo situation where diffusion of oxygen from thecal capillaries presents a rapidly renewable source of oxygen.
The hypothesis that the follicles cultured in the upright systems suffered from hypoxia is strongly supported by our finding that lactate production per unit volume of follicle in these systems was markedly higher (in some cases up to 2-fold higher) than in the inverted system at all stages of culture. A number of workers have found that hypoxic or anoxic conditions increase lactate production by follicles in vitro (rat follicles; Selstam & Gafvels 1987, mouse follicles; Boland et al. 1994); similar results were found by Surwilo & Doeg (1973) in sliced rat ovaries. The hypothesis is also supported by the fact that the growth rate (as measured by cell proliferation) of follicles cultured in the upright with oil system decreased steadily from day 3 to day 6 whereas growth rate in the inverted system was maintained from day 1 to day 5.
However, even in the inverted system, growth rate declined drastically and became negative from day 5 to day 6 in spite of the fact that follicular diameter and volume were still increasing. A possible explanation for this is that due to poor oxygen diffusion or nutrient transport to the centre of a large day-5 follicle, cells at the centre of the follicle begin to die even though cells in the outer layers of the follicle are still proliferating.
It is surprising that there is so little information in the literature on the effect of oxygen concentration on follicle growth and particularly on the use of gas phases with a higher oxygen concentration than 20%. In long-term culture of mouse follicles, Smitz et al.(1996) and Smitz & Cortvrindt (1998) found that a 20% oxygen gas phase was superior to 5% oxygen; a 5% concentration reduced survival of follicles and secretion of oestradiol and inhibin. Qvist et al.(1990) using Petriperm gas permeable dishes found that oxygen concentrations above 40% caused increased granulosa cell proliferation but also caused eventual necrosis of the cells and rapid disintegration of the ovum; they did not, however, specify the levels involved and did not compare 40% with 20%.
In studies on mouse oocyte granulosa cell complexes isolated from preantral follicles of 12-day mice and cultured for 10 days, followed by maturation and fertilization, Eppig & Wigglesworth (1995) concluded that concentrations of oxygen in the gas phase above 5% had a deleterious effect on oocyte development during the early stages of culture but increasing the concentration during later stages was critical in promoting normal oocyte development in vitro.
For the culture of sheep follicles, Cecconi et al.(1999) found that 5% oxygen was superior to 20%; 5% oxygen in association with a high concentration of FSH stimulated development of antral follicles and increased oestradiol secretion. The apparent differences between mouse and sheep follicles are perhaps not surprising since, because of the difference in body masses of the two species, one would expect the oxygen consumption of mouse follicles on a per unit mass basis to be about 4- to 5-fold that of sheep (Porter & Brand 1995).
One future avenue of research that must be investigated is whether the inverted system provides improved oocyte development and fertilizability as compared with the upright systems.
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Boland NI, Humpherson PG, Leese HJ & Gosden RG 1993 Pattern of lactate production and steroidogenesis during growth and maturation of mouse ovarian follicles in vitro. Biology of Reproduction 48 798–806.[Abstract]
Boland NI, Humpherson PG, Leese HJ & Gosden RG 1994 Characterization of follicular energy metabolism. Human Reproduction 9 604–609.
Cecconi S, Barboni B, Coccia M & Mattioli M 1999 In vitro development of sheep preantral follicles. Biology of Reproduction 60 594–601.
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and solid lines), upright without oil (
and solid lines) and upright with oil (
and broken lines); n = 25–26 follicles for each system. The inverted system was significantly different from the upright systems on all days of culture for both diameter and volume (day 1, P < 0.05; days 2–6, P < 0.001). (c and d) Effect of the culture system on oestradiol secretion into the medium with secretion expressed both (c) per follicle and (d) per nl follicle over 48 h. Culture systems are shown as follows: inverted system (solid bars), upright without oil (hatched bars) and upright with oil (open bars); n = 9–11 follicles for each system. Significant differences between inverted and upright system without oil, **P < 0.01, because oestradiol is soluble in oil, data for the upright with oil treatment were not included in the statistical analysis. (e and f) Effect of the culture system on lactate secretion into the medium with secretion expressed both (e) per follicle and (f) per nl follicle. Culture systems are shown as in (c and d); n = 16–18 follicles for each system. Significant difference from the upright culture systems, *P < 0.05, **P < 0.01, ***P < 0.001.
), upright with oil system in MF1 mice (