Mol. Hum. Reprod. Advance Access originally published online on January 19, 2008
Molecular Human Reproduction 2008 14(2):75-83; doi:10.1093/molehr/gam092
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Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids
1Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, UK 2Centre for Reproductive Biology, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4JT, UK
3 Correspondence address. Tel: +44-131-650-3267; Fax: +44-131-651-1706; E-mail: norah.spears{at}ed.ac.uk
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Abstract |
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Although there have been extensive studies on the effects of gonadotrophins and steroids on follicular development, less is known as to the effects these hormones have on the acquisition of oocyte developmental competence. This study investigates the effect of altering the gonadotrophin or steroidal environment on follicular development and on oocyte viability and DNA methylation. Oocytes were obtained from pre-ovulatory follicles after individual follicle culture from the pre-antral stage; gonadotrophin or steroid levels were manipulated during the culture period. Oocytes obtained from follicles grown in gonadotrophin free conditions were able to fertilize and develop to the blastocyst stage despite their impaired follicle development. There was no effect of luteinizing hormone or steroids on follicular growth. Altering the steroidal environment did, however, affect oocyte development. The oocytes of follicles exposed to high estrogen levels had lower fertilization rates, regardless of the presence or absence of high androgen levels. The combined presence of high levels of both steroids altered the level of global methylation. This study demonstrates that gonadotrophins and steroids influence the acquisition of developmental competence of the oocyte and suggests that optimal steroid exposure during follicle development is required for the oocyte to mature correctly.
Key words: DNA methylation/gonadotrophins/oocyte competence/steroids
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Introduction |
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The roles that follicle stimulating hormone (FSH) and luteinizing hormone (LH) play in ovarian development have been extensively studied for a number of years. Until the recent availability of recombinant forms of both gonadotrophins, it was difficult to define the precise and individual roles that FSH and LH played in follicular growth and development. From the growing number of reports that have now utilized these recombinant gonadotrophins it is clear that FSH on its own is capable of promoting follicular growth and development, while LH augments steroidogenesis and plays a critical role in the ovulatory process (Chappel and Howles, 1991; Hillier, 1994). Recombinant FSH alone has been used in both clinical and animal studies: results have indicated that inclusion of LH in ovarian stimulation protocols is required for adequate steroidogenesis and that inadequate estrogen synthesis is detrimental to embryo viability and endometrial development (Balasch et al., 1995, 2001; Spears et al., 1998; Shoham, 2002). The concentration of LH required to optimize stimulation protocols has been the subject of many debates. In humans, it has been suggested that follicular maturation is optimal when LH levels do not rise above a ‘ceiling’ which, when exceeded, leads to an arrest of follicular development (Loumaye et al., 2003). Elevation of LH levels above basal rates during ovarian stimulation protocols has also been proposed to impact directly on oocyte quality (Balasch et al., 2001; Shoham, 2002). Other studies, however, have failed to confirm any correlation between elevated LH levels and pregnancy rates (Westergaard et al., 1996). From these conflicting reports, it is still unclear what effect LH has on oocyte development within the follicle prior to ovulation and how this affects the quality of the resulting embryo after fertilization.
Gonadotrophin levels will directly affect ovarian steroidogenesis, LH stimulating the thecal cells of the follicle to produce androgens which are subsequently aromatized to estrogens within the FSH-responsive granulosa cells. Androgens and estrogens have been proposed as paracrine/autocrine regulators of follicular development, but, as with LH, there is a controversy over their roles in the ovary. Apart from serving as substrates for estrogen synthesis, androgens have been implicated in promoting follicular development (Murray et al., 1998; Vendola et al., 1998), and in up-regulating FSH receptor expression and enhancing FSH-stimulated follicular differentiation (Tetsuka and Hillier, 1997; Weil et al., 1999). However, excess androgen levels have also been associated with inducing follicular atresia, while high androgen:estrogen ratios in follicular fluid have been correlated with poor fertilization and development rates (Anderiesz and Trounson, 1995; Xia and Younglai, 2000). Estrogens have been shown to inhibit follicular atresia and to facilitate the proliferation of granulosa cells, the actions of gonadotrophins, gap junction formation and steroidogenesis (Merk et al., 1972; Roberts and Skinner, 1990; Billig et al., 1993; Dorrington et al., 1993; Bley et al., 1997). Other studies have indicated that estrogen is not obligatory for follicular development (Balasch et al., 1995; Spears et al. 1998). As both androgen and estrogen receptors have been reported on the oocyte, it is possible that steroids may also exert a direct effect (Szoltys et al., 2003; Gill et al., 2004).
Recently, the phenomenon of genomic imprinting has received much attention, with maternal epigenetic imprints established during the oocyte growth period (Obata et al., 1998). Alterations to the mechanisms involved in imprinting can result in early embryonic lethality, but may become evident only during the later life of the organism. One of the most researched aspects of genomic imprinting is the role of DNA methylation in controlling gene transcription, with imprinted genes in the paternal and maternal genomes differentially methylated (Simon et al., 1999; Lucifero et al., 2002). Although these imprinted genes have been the focus of intensive research, they represent only a small percentage of the overall CpG methylation present in the genome, with the largest proportion of DNA methylation being found in the CpG rich repeat sequences of the mouse genome (Lees-Murdock et al., 2003; Rollins et al., 2006). The signals that mark certain genes for imprinting either via methylation or by other means have yet to be elucidated, however, paracrine signals arising from gonadotrophin or steroidal actions within the follicle are possible candidates. Recent data from a study by Sato et al. (2007) supports this possibility, with altered DNA methylation at imprinted gene loci being identified after superovulation of both mouse and human oocytes.
It has been proposed that the oocyte needs to be exposed to the correct sequence and pattern of steroids in order to acquire the molecular programming required for fertilization and further development (Moor et al., 1998). Alterations to the normal follicular sequence of FSH- and LH-stimulation, such as occurs during controlled ovarian stimulation, will inevitably perturb the pattern of steroid exposure which may in turn have an effect on oocyte competence. In mice, it has been suggested that the deleterious effects of exogenous administration of gonadotrophins on oocyte/embryo developmental competence is due to attendant increases in ovarian steroid levels (Ertzeid and Storeng, 2001) and that repeated ovarian stimulation disrupts synchronous oocyte and follicle maturation (Combelles and Albertini, 2003).
This study aims to investigate the effects of varying gonadotrophin and steroidal treatments on follicular development, oocyte viability and global DNA methylation levels, using an in vitro system that permits the development of individual follicles from the pre-antral to the Graafian stage. This system is highly physiological in that it closely mimics follicular development in vivo. Although mechanical isolation of follicles is labour-intensive and imposes some restrictions, it does ensure that individual follicles at a similar developmental stage can be placed in defined conditions thereby negating the effects of any extra follicular factors.
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Materials and Methods |
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Animals
F1 (C57BL/6J x CBA background) and hypogonadal (hpg) mutant mice were housed in an environmentally controlled room on a 12-h light:12-h dark photoperiod. Food and water were available ad libitum. Hpg mutant animals were generated by breeding heterozygous pairs (hpg+/–). Offspring were genotyped by extracting DNA from an ear clipping which was then analysed by polymerase chain reaction (Lang, 1991). All animals were maintained and treated according to UK Home Office requirements and the study received local ethics committee approval.
Culture supplements
In order to accommodate for batch-to-batch variation in recombinant human follicle stimulating hormone (rhFSH) potency, individual batches were titrated for use at the minimum concentration that allowed follicles to reach sizes of >400 µm over a 4–6 day period (doses are described for individual experiments).
The use of serum in the culture media potentially introduces extraneous gonadotrophins and/or steroids that might influence the results. In earlier experiments, serum was obtained from hpg mutant adult mice (which have no or very low levels of circulating gonadotrophins). Briefly, blood was withdrawn from anaesthetized adult animals via cardiac puncture, allowed to clot for a minimum of 10 min then centrifuged at 16 000 g for 10 min. Serum from each batch of animals was pooled before being aliquoted and stored at –70°C. As hpg serum proved erratic in its ability to support fertilization and cleavage to the 2-cell stage, charcoal-stripped fetal bovine serum (CSS) was used in later experiments. The use of activated charcoal to remove steroids has been previously described (Huot and Shain, 1988). Owing to the slower maturation of follicles in this serum, the culture period prior to oocyte collection for in vitro fertilization (IVF) was extended to 5 days.
Culture medium and chemicals
All chemicals were supplied by Sigma–Aldrich (St Louis, USA) with the exception of those noted here. Epidermal Growth Factor (EGF) was from Roche (Lewes, UK) and silicone fluid was from Merck (Lutterworth, UK). Arimidex, an aromatase inhibitor, was a gift from Astra Zeneca (Macclesfield, UK). Recombinant gonadotrophins (rhFSH and rhLH) were supplied by Serono (Geneva, Switzerland). Liebovitz L-15 and 
-minimal essential medium (-MEM) were supplied by Invitrogen (Renfrew, UK). Fetal bovine serum was supplied by Labtech (Uckfield, UK). 5-Methyl cytosine antibody was supplied by Eurogentec (Seraing, Belgium). FITC conjugated secondary antibody (Light chain specific) was supplied by Jackson ImmunoResearch (Pennsylvania, USA). Vectashield was supplied by Vector Laboratories (Peterborough, UK). Kits to detect Terminal Transferase-mediated dUTP Nick End labeling (TUNEL) were supplied by Boerhinger (Bracknell, UK).
Standard follicle culture
F1 female animals of 21 to 25 days of age were sacrificed by cervical dislocation, their ovaries removed and placed in watch glasses containing Leibovitz L-15 medium supplemented with 3 mg/ml bovine serum albumen (BSA) (dissection medium). Individual pre-antral follicles (175 ± 15 µm) were dissected and cultured as previously described (Murray et al., 1998). On average, 25–30 follicles were obtained from each mouse and these were randomly allocated across all treatment groups. Follicles were incubated for periods of between 4 and 6 days in a humidified 5% CO2, 95% air incubator at 37°C. Media were supplemented with 5% serum and gonadotrophins or steroids as detailed for each experiment.
Follicular growth, morphology and steroid production
Follicles were examined daily; extent of antral formation was noted and follicles were measured using an ocular micrometer. At the end of the culture period, spent culture media were frozen at –20°C until analysed for steroids. Samples of unused medium containing all supplements were also frozen and analysed for steroids. The various media from the follicle cultures were then analysed for the presence of estradiol (E2) and androstenedione. E2 was measured by ELISA (Murray et al., 1998) and androstenedione by radioimmunoassay as previously described (Hillier et al., 1991).
Oocyte maturation and in vitro fertilization
Oocyte–granulosa cell complexes (OGCs) were dissected from cultured follicles and transferred to -MEM medium supplemented with 20 ng/ml EGF, 5% serum and rhFSH. The serum type and rhFSH dose were the same as those used in the preceding follicle culture. The OGCS were then incubated for 18 h before being transferred into droplets of T6 medium to await fertilization (Quinn et al., 1982). Spermatozoa suspensions were prepared from F1 males as described previously and allowed to capacitate for 2 h (Spears et al., 1994). Spermatozoa were added to the droplets of T6 medium containing oocytes. After 4–5 h incubation, the oocytes were transferred into droplets of glutamine-free KSOM medium supplemented with 1 mg/ml fatty acid free BSA (Devreker and Hardy, 1997). As a control for the IVF system, oocytes were obtained from 6-week-old F1 females that had been superovulated, as described previously (Spears et al., 1994). Oocyte–cumulus complexes were recovered from the oviducts, transferred into droplets of T6 medium and treated in the same manner as experimental oocytes. All oocytes were examined the following day and the number of 2-cell embryos counted. These were transferred to fresh drops of KSOM medium at a ratio of 1 embryo to 2 µl of medium. A maximum of 10 x 2-cell embryos were transferred to a 20 µl drop of medium. Where appropriate, more than one 20 µl droplet of medium was set up and if necessary the droplet volume adjusted according to the number of 2-cell embryos found. All droplets were covered with silicone fluid and incubated in an environment of 5% CO2 in air, at 37°C. Incubation was continued to the blastocyst stage of development. The number of oocytes fertilizing and reaching the blastocyst stage of development was noted.
Methylation immunohistochemistry
At the end of the culture period, OGCs were isolated by rupturing the follicles with fine needles. Oocytes were then denuded by repeated pipetting. Oocytes were fixed overnight in paraformaldehyde (4% in phosphate buffered saline (PBS)) at 4°C before being permeabilized in Triton-X 100 (0.5% in PBS) for 30 min and then denatured with 4 N HCl at 37°C for 1 h. Non-specific binding was blocked using BSA (2% in PBS) for 1 h. Oocytes were then incubated with a primary 5-methyl cytosine antibody (1:50 in PBS/BSA) for 1 h at 37°C, washed and stained with a FITC-C conjugated secondary antibody (1:200 in PBS/BSA) for 1 h. After a final wash (Tween 20, 0.05% in PBS), the oocytes were mounted on positively charged slides using vectashield.
Confocal microscopy
At the end of experiments examining follicular growth, representative follicles from each of the groups were labeled to detect apoptotic cells using the TUNEL labeling technique and counterstained using propidium iodide (Baker et al., 2001). Follicles were examined using the Leica TCSNT Confocal microscope (Leica Microsystems, Milton Keynes, UK) fitted with a 63X water-corrected PL APO lens. Simultaneous scans at 488 and 568 nm were taken to produce an amalgamated, true color RGB image.
Oocytes from experiments to examine DNA methylation were serially scanned at 3 µm intervals through the stained region, using a 40X lens. The average threshold of the images was calculated using Imaris imaging software. Using this information, global DNA methylation levels were assessed with the Scion Image Software package. Specificity of staining was checked by examination of oocytes exposed only to secondary antibody. To allow comparisons between experimental runs, all raw data were normalized by the comparison to the mean of the control group (assigned unity) from that run.
Statistics
Hormone assays were analysed by ANOVA followed by Tukey–Kramer multiple comparison tests where appropriate. IVF results of oocytes were analysed by Chi-square. Methylation levels were analysed by non-parametric ANOVA, followed by Dunn's multiple comparison test.
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Results |
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Experiment 1: The effects of altered gonadotrophin regimes on follicular growth, development and subsequent oocyte developmental competence
Experiment 1A: Follicular growth and development
Follicles were allocated to control medium supplemented with 5% hpg serum either without gonadotrophin supplements (Gn free) or into one of the following treatment groups: FSH alone (control medium + 5 IU rhFSH/ml); FSH + low LH (control medium + 5 IU rhFSH/ml + 0.01 IU rhLH/ml); FSH + high LH (control medium + 5 IU rhFSH + 0.05 IU rhLH/ml). Follicles were moved into fresh wells daily and cultured for 6 days. The experiment was performed twice, with 30–40 follicles cultured per treatment. Damaged follicles were discarded within the first 2 days of the culture period.
FSH alone was capable of inducing follicular growth and development: the addition of low or high LH had no effect on the development of the follicles, with follicles reaching the same size and antral formation occurring at the same time and to the same extent in all gonadotrophin groups (Fig. 1). Gn free follicles initially began to grow, although growth was significantly restricted by Day 3 of culture when compared to all other group (P < 0.001). These follicles then began to regress from Day 4 onwards and no follicles with antral cavities were seen in this treatment (Fig. 1).
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16 for each group). Arrow indicates the start of antral formation in all gonadotrophin groupsFive representative follicles from each of the groups were labeled for apoptosis using TUNEL labeling and examined by confocal microscopy (Fig. 2). The only group of follicles that exhibited any high degree of apoptotic labeling was those in the Gn free group, with most granulosa cells near the basement membrane showing TUNEL labeling (Fig. 2A). In follicles from all other groups, any TUNEL labeled cells were almost always situated around the antral cavity; this is likely to indicate a healthy follicle: as the antral cavity enlarges, granulosa cells nearest to the cavity become apoptotic and release their contents into the antral fluid (Baker et al., 2001).
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Spent culture media from Days 4 and 5 of culture were analysed for the presence of E2 and androstenedione. Gonadotrophin treatment altered the production of androstenedione and E2 during follicular growth (Fig. 3). Neither steroid was detectable in unincubated culture medium or in that from the Gn free group (data not shown). Follicles from both the FSH + low LH and the FSH + high LH groups were producing androstenedione on Day 4 of culture, whereas none was detected in the medium until Day 5 of culture when follicles were treated with FSH alone (P < 0.05, Fig. 3A). This was reflected in the E2 results, as both the FSH + low LH and the FSH + high LH groups produced significantly higher concentrations of E2 compared to the FSH alone group at Day 4 (P < 0.05, Fig. 3B). Although androstenedione production increased significantly by Day 5 in all gonadotrophin groups, there was a marked increase in the production of this steroid by follicles cultured with FSH + low LH when compared to either the FSH alone or the FSH + high LH group (Fig. 3A). Similar levels of E2 were produced at this time point irrespective of treatment (Fig. 3B).
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Experiment 1B: In vitro fertilization
Follicles were allocated to treatment groups as described above in Experiment 1A (i.e. Gn free, FSH alone, FSH + low LH and FSH + high LH). These were cultured for 4 days, after which the OGCs were placed in maturation medium prior to IVF. Maturation medium was supplemented with 1 IU rhFSH/ml. Four experimental runs were performed with a total of 160 follicles cultured in each treatment. The number of oocytes available for IVF at the end of the culture period was as follows: Gn free, 114; FSH alone, 123; FSH + low LH, 128 and FSH + high LH, 98. There was little difference in fertilization and 2-cell cleavage rate amongst the culture groups regardless of treatment (Fig. 4A). In contrast, later embryo development was more variable across treatments. The percentage of 2-cell embryos developing to the blastocyst stage was similar in the FSH alone and FSH + low LH group, but there was a substantially lower rate of 2-cell embryos derived from the FSH + high LH group that developed into blastocysts (Fig. 4B). This low percentage was similar to the percentage of blastocysts derived from the Gn free group. The differences between all groups did not, however, reach statistical significance (P = 0.08). For reference, a further group of 30 follicles between 200 and 300 µm in diameter (i.e. the same size as that attained by the Gn free group at the end of the culture period) were freshly dissected but not cultured. The oocytes from these follicles were removed immediately and placed into maturation medium before undergoing IVF. In comparison to the 20% fertilization and 2-cell cleavage rate and 23.5% blastocyst rate obtained from the Gn free group oocytes, 29 out of 30 of the oocytes from the uncultured late pre-antral follicles did not fertilize, whereas the remaining oocyte did not proceed past the 2-cell stage, even though both the Gn free oocytes and the uncultured oocytes were obtained from similar sized follicles. It appears, therefore, that the oocytes from the Gn free group had undergone a degree of maturation during the culture period despite the fact that they were from atretic looking follicles. In addition, as a control for the IVF system, 184 oocytes were obtained from superovulated F1 mice and underwent IVF alongside the cultured oocytes; 76% of these cleaved to 2-cells, with 87% of these reaching the blastocyst stage. The results obtained from the superovulated F1 mice were not included in the statistical tests.
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Experiment 2: Effect of alteration in steroid regime during follicular growth in vitro on subsequent oocyte developmental competence
Experiment 2A: Follicular growth and development
The gonadotrophin treatment received by the follicles resulted in an alteration to the concentration and production of steroids during the culture period. In order to investigate the effects of this alteration more directly an aromatase inhibitor, Arimidex (Zeneca ZD 1033), was used to elevate androgen levels and lower estrogen levels. This compound selectively blocks the aromatase reaction without inhibiting other steroidal enzymes and without having any direct steroidal actions (Lonning et al., 1998). To elevate estrogen levels, diethylstilboestrol (DES) was added to culture medium. This compound is structurally related to 17-β-E2 and has been used extensively to study reproductive function. DES was used rather than E2 as it was not possible to obtain a solution of E2 at a sufficiently high concentration to add to the culture medium in a form that the follicles would tolerate in this culture system. To elevate levels of both steroids, culture medium was supplemented with both DES and Arimidex. As both DES and Arimidex were solubilized in ethanol before addition to culture medium, control medium was supplemented with the same concentration of ethanol. Control follicles were allocated to control medium supplemented with 5% hpg serum and 2 IU/ml FSH. Treatment groups were High A (high androgen: control medium + 0.1 µm Arimidex); High E (high estrogen: control medium + 0.004 nmol DES); High A + E (control medium + 0.1 µm Arimidex + 0.004 nmol DES). Follicles were moved daily into fresh wells of medium and cultured for 6 days. At the end of the culture period, five representative follicles from each group were labeled for apoptosis using TUNEL. Spent media from all groups were analysed for steroids.
Elevating androgen, estrogen or both steroids had no effect on follicular growth or rate of antral development, with all follicles growing well and developing large antral cavities during the culture period, irrespective of treatment (Fig. 5). Similarly, altering steroid treatments had no effect on the degree of apoptotic cell labeling within the follicles (data not shown as these follicles were very similar to those shown in Fig. 2B–D).
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In order to check that the treatments had successfully altered the level of steroids to which the follicles had been exposed, media samples from Day 6 of culture were analysed for androstenedione and E2. Comparison of the levels of androstenedione in the media showed that the concentration of Arimidex used successfully inhibited aromatase activity. In the High A and High A + E groups, androstenedione levels were significantly raised when compared to the control group or High E group (P < 0.01, Fig. 6). As expected, inclusion of Arimidex (High A group) also significantly reduced the level of 17-β-E2 production by the follicles compared with the mean and SEM of the control group (73.0 ± 19.0 pmol/ml, compared to 229.1 ± 31.7 for the control group; P < 0.001). The estrogen assay used is highly specific for 17-β-E2 and does not cross react significantly with any other estrogen. We were, therefore, unable to measure the total increase in estrogen levels in response to the addition of DES (in both the High E and the High A + E groups). There were no detectable steroids in the unincubated culture medium.
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Experiment 2B: In vitro fertilization
In order to minimize any influence of steroids found in serum, CSS was used for these cultures. As the majority of follicles could not maintain their basement membranes on the final 2 days of culture in CSS, the culture system was modified; on Days 1 and 2 of culture, follicles were transferred into wells of fresh medium and damaged or atretic follicles discarded. On Day 3 of culture, they were then transferred into 60 µl of medium overlaid with 75 µl of silicone fluid.
Follicles were isolated, placed in the control medium and treatment groups as described in Experiment 2A (i.e. Control, High A, High E and High A + E) and cultured for 5 days after which OGCs were removed from the follicular masses and placed in maturation medium prior to IVF. In all cases, medium was supplemented with 1 IU/ml rhFSH. Three experimental runs were performed with a total of 120 follicles placed in culture for each treatment. The number of available oocytes taken for IVF at the end of the culture period was as follows: control, 160; High A, 148; High E, 117 and high A + E, 117. As in Experiment 1B, oocytes (181) from superovulated F1 mice were included as a control for the IVF system; 83% cleaved to 2-cells, with 78% of these proceeding to the blastocyst stage. The results for the F1 oocytes were not included in the statistical analysis. Steroid treatment significantly altered the rate of fertilization and cleavage to the 2-cell stage (P < 0.01). The presence of DES, whether in the High E or the High A + E group, yielded fewer oocytes capable of development to the 2-cell stage when compared to the control group (where steroid production could be expected to follow a more physiological pattern) (Fig. 7). It was not possible to determine if the oocytes that did not proceed to the 2-cell stage had in fact fertilized or not. The percentage of 2-cell embryos able to complete development to the blastocyst stage was similar irrespective of steroid treatment.
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Experiment 3: Effect of alteration in steroid regime during follicular growth in vitro on the global DNA methylation levels of the oocyte
Experiment 3A: DNA methylation during culture
Follicles were cultured in medium supplemented with CSS and 1 IU/ml rhFSH for 0, 2, 4 or 6 days before they were ruptured and the oocytes collected and fixed. After immunohistochemistry, the DNA methylation pattern and level could be seen to intensify with time in culture (Fig. 8): the level of DNA methylation showed a large increase between the start and end of culture, with the staining around the edge of the nucleolus becoming more defined.
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Experiment 3B: Global DNA methylation levels after exposure to altered steroidal regime in culture
Follicles were isolated, placed in the control and steroid treatment groups as described in Experiment 2A (i.e. Control, High A, High E and High A + E) and cultured for 5 days. At the end of the culture period, OGCs were removed, oocytes mechanically denuded and used for immunohistochemistry. The experiment was performed a minimum of three times. Differences in global DNA methylation level were evident after steroid treatment (Fig. 9). High A resulted in a significantly lowered methylation level when compared to the controls (P < 0.001), whereas when the oocytes were exposed to High A + E, there was an increase in global methylation over that of the controls (P < 0.0001). The High E treatment did not significantly alter the global DNA methylation level.
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Discussion |
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The results presented here confirm that FSH alone can drive follicular growth and development, with the addition of LH having no effect. Other in vitro studies have reported that the inclusion of LH in culture medium accelerates the formation of the antral cavity (Cortvrindt et al., 1998). We found no evidence to support this, as antral formation was similar in all treatment groups, perhaps due to the difference in culture technique (where follicles remain intact, rather than being allowed to rupture). As could be expected, the inclusion of LH in the culture medium augmented the production of androstenedione and E2 at an earlier time point in follicular development. Interestingly, FSH alone supported the production of androstenedione, and hence E2, by the follicles, albeit at a later time-point than when LH was also present. We have previously found that under the conditions used in this culture system, follicles exposed to FSH alone produce inhibin (unpublished data) which has been shown to augment androgen synthesis during pre-ovulatory follicle growth (Smyth et al., 1994); this pathway could explain the production of androstenedione (and hence E2) in follicles exposed to FSH alone. Some reports have suggested that high levels of androgens and estrogens can induce atresia in developing follicles (Billig et al., 1993; Dierschte et al., 1994) while others have shown that these steroids promote follicular development and health (Murray et al., 1998; Vendola et al., 1998). By employing an estrogen agonist and/or an aromatase inhibitor to alter levels of estrogens, androgens or both steroids throughout the pre-antral to antral growth phase of follicles, we found that there was no effect on follicular growth, development or health.
There has been some controversy over whether the actions of LH are necessary during follicular development in order to optimize oocyte maturation (Balen et al., 1993; Shoham et al., 1993; Homburg, 1998). Transgenic mice over-expressing LH are infertile due primarily to anovulation, however, their oocytes appear to be fertile and can give rise to offspring (Mann et al., 1999). Conversely, other studies have indicated that high intra-follicular LH permits the premature resumption of meiosis resulting in ‘aged’ oocytes (Homburg, 1998) and promotes spontaneous germinal vesicle breakdown (Cortvrindt et al., 1998). The addition of high levels of LH to the culture medium in these experiments did not confer any immediate advantage to the oocytes, as similar rates of fertilization and cleavage to the 2-cell stage were seen in all the treatment groups and fertilized oocytes from all treatment groups were able to develop to the blastocyst stage.
Surprisingly, it was found that around 20% of oocytes obtained from follicles grown in Gn free conditions could fertilize and complete pre-implantation development even though the oocytes were obtained from follicles that had ceased growing by Day 3 and exhibited extensive apoptosis in their granulosa cell layers by the end of the culture period. In marked contrast to the oocytes from the Gn free group, oocytes from uncultured follicles of the same starting size were unable to fertilize, demonstrating that even oocytes from the Gn free group acquired further developmental competence during the culture period. Furthermore, these oocytes must have been relatively unaffected by the level of apoptosis in the somatic cells of the follicles. It was noticeable that the granulosa cells immediately surrounding the oocytes in the Gn free group tended to be non-apoptotic, perhaps benefiting from the close proximity of the healthy oocyte or perhaps helping to maintain the healthy status of the oocyte.
Under normal physiological conditions, levels of androgen are higher earlier in follicular development while higher estrogen levels become more prevalent as follicles progress towards ovulation (Moor et al., 1998). Disruption of this pattern has been correlated with poor fertilization and development rates (Anderiesz and Trounson, 1995; Xia and Younglai, 2000). Although there was some alteration in the steroid levels produced throughout the follicular growth stage when gonadotrophin regimes were altered, we could not detect an effect on oocyte developmental competence. However, we have shown here that by altering the concentration of steroids in vitro directly and more substantially, using DES and an aromatase inhibitor, the percentage of oocytes undergoing fertilization and cleavage to the 2-cell stage was altered, with fewer oocytes developing to the 2-cell stage under conditions of high levels of estrogen. In a previous study, we have demonstrated that under conditions that are marginal for follicular development, androgens exert a direct, stimulatory, effect on follicular growth (Murray et al., 1998). In this study, where we employed a culture system already optimal for follicular growth, no further effect was seen on the growth of the follicles in either experiment where steroids were altered. This would indicate that follicular development can proceed independently of oocyte developmental competence.
DES is structurally related to 17-β-E2 and equally (if not more) potent, with a longer half life. In addition, it has been shown to bind to both the alpha and beta estrogen receptor isoforms with a higher potency than E2 (Gutendorf and Westendorf, 2001). Although both low and high levels of LH stimulated steroid production earlier in the culture period, the effect was relatively minor and unlikely to be comparable to adding DES throughout the culture period (where high-level exposure of the oocyte to estrogen would be sustained throughout culture). This may well explain why LH treatment did not significantly affect fertilization or embryo development while DES treatment did. There is little information on the effects of elevated estrogen concentrations during follicular development on subsequent oocyte developmental competence. Studies from clinical situations, where supraphysiological estrogen levels often result as a consequence of ovarian stimulation, are difficult to interpret as any effects seen may be a result of poor oocyte quality or the effects of elevated estrogen on other areas of the reproductive tract. Although it is difficult to correlate in vivo and in vitro levels of estrogen, by using an individual follicle culture system, this current study has demonstrated for the first time that elevation of estrogen during the pre-antral to antral growth phase of the follicle is detrimental to the ability of the oocyte to undergo fertilization and cleavage to the 2-cell stage.
Both positive and negative effects of estrogen have been reported in the literature. Estrogen has been implicated in increasing free calcium stores within oocytes (Tesarik and Mendoza, 1995) and ensuring genomic integrity of oocytes before ovulation (Murdoch and Van Kirk, 2001). In primates, including humans, it has been suggested that lack of estrogen results in oocytes less able to undergo fertilization (Balasch et al., 1995) and it has been demonstrated that the addition of steroids during in vitro maturation can aid oocyte developmental competence (Zheng et al., 2003). However, a recent study on the maturation of bovine oocytes has shown that the inclusion of E2 in maturation medium decreases the percentage of oocytes progressing to metaphase II (Beker et al., 2002), while studies from transgenic mouse models that lack either of the estrogen receptors or that lack the aromatase gene have indicated that estrogen is not obligatory for follicular development or oocyte maturation (Couse et al., 1999; Huynh et al., 2004). The results presented here back the idea that lowering estrogen levels is not detrimental, since oocytes obtained from Arimidex-treated follicles (resulting not only in high levels of androgen, but also in lowered levels of estrogen) had equivalent fertilization and cleavage rates to controls. Similarly, a recent study utilizing Arimidex to inhibit aromatase activity in vivo concluded that rising estrogen levels were not a requirement for either follicular development or oocyte maturation (Guo et al., 2004).
The use of mouse monoclonal antibody to 5-methyl cytosine allows reproducible assessments of the patterns and amounts of DNA methylation in cell lines as well as oocytes and embryos. Chromosomal regions with a high density of methylated cytosines will bind more of the anti-5-methyl cytosine antibody, thus the strength of the signal can be used to assess the distribution and level of DNA methylation at CpG dinucleotides (Dahl and Guldberg, 2003). We have shown that the levels of DNA methylation in oocytes increase during the culture period. It has recently been shown that changes in global methylation take place during oocyte growth in vivo (Kageyama et al., 2007). Similar increases in global DNA methylation levels over the equivalent stages of follicle growth in vivo have been observed (unpublished observations, Swales & Spears) and increases in the methylation level of specific sequences have been shown to occur over oocyte growth in vivo (Lucifero et al., 2004; Hiura et al., 2006). The steroidal environment affected oocyte global DNA methylation levels: the oocytes of follicles cultured in high levels of androgens and estrogens had raised DNA methylation levels, while those from follicles cultured in high levels of androgen alone had reduced DNA methylation levels. The differences in methylation pattern did not exactly mimic oocyte developmental competence, and cannot, therefore, be the sole cause of these effects. It is possible, though, that inappropriate DNA methylation is one of the causes of poor developmental competence given that the High A + E treatment significantly affected both fertilization and methylation levels of the oocytes. There is evidence that global DNA methylation of human sperm has an impact on IVF pregnancy rates (Benchaib et al., 2005), while in both human and mouse there are changes in DNA methylation at imprinted gene loci in oocytes retrieved after superovulaton (Sato et al., 2007), a condition where high levels of steroids are produced. This finding is of particular interest, as embryos derived from superovulated oocytes have been reported to have reduced viability (Van der Auwera and D'Hooghe, 2001), supporting the possibility that DNA methylation dynamics in the gamete can influence subsequent embryo development. DNA methylation is part of the mechanism involved in controlling normal expression patterns of imprinted genes: the correct establishment of genomic imprinting during oocyte growth and maturation is vital if normal fertilization and embryo development are to occur (Obata et al., 1998). It cannot be determined from the experiment described here whether the changes in DNA methylation are due to alterations in levels of imprinted genes, other regions of the genome or a combination of both. As the observed alterations in DNA methylation were quite large, though, it is probably unlikely that these changes would be due solely to changes in imprinted genes (as the DNA methylation associated with these imprinted genes is relatively small when compared to that of the non-imprinted genomic regions). The vast majority of studies which investigate DNA methylation dynamics of germ cells have concentrated on imprinted genes rather than global DNA methylation levels. Thus, less is known about DNA methylation that is found in non-imprinted regions of the genome or what effects are seen when global methylation levels at these repeat loci are altered in different developmental contexts. Theoretically, changes to global DNA methylation could have a range of potential effects, depending on the specific methylated sequences that were affected. For example, although satellite sequences are not transcribed they are thought to play a role in both centromere function and kinetochore assembly (Lees-Murdock et al., 2003). Other sequences that contain DNA methylation include SINEs (short interspersed elements), LINEs (long interspersed elements) and IAPs (intracisternal A-particles) which are all derived from transposable elements (Marchal et al., 2004). Alterations to global DNA methylation levels are associated with failure of cloned embryos to develop normally (Dean et al., 2001), whereas errors in the DNA methylation of specific imprinted genes can result in disorders or disease in subsequent offspring (Mizuno et al., 2001; Young et al., 2001).
Manipulating the ovary, such as through the administration of exogenous gonadotrophins, can alter the steroidal environment within those follicles and subsequently have an effect on the developmental competence of the oocyte. Although the idea that steroids influence oocyte competence is not new, these present studies have shown for the first time that inappropriate exposure of the oocyte to steroids during follicle maturation may be detrimental to oocyte developmental competence and impact upon DNA methylation of the genome.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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Grant Support. Supported by MRC grant (G9901514). AKES supported by BBSRC.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionFundingAcknowledgementsReferences |
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The authors wish to thank Vivian Allison for expert assistance with the follicle cultures and Martha Urquhart for performing the androstenedione assays. Dr Vlastimil Sr
e
kindly carried out the TUNEL staining of the follicles and Linda Wilson performed the confocal microscopy. Dr Nathalie Beaujean kindly advised on the methylation immunohistochemistry protocols. |
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Joint First Authors. 
4 Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.
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Submitted on August 6, 2007; resubmitted on September 21, 2007; accepted on September 27, 2007.
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