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RESEARCH |
The Department of Obstetrics and Gynaecology, University of Aberdeen, Aberdeen, UK and 1 The Department of Biomedical Sciences, University of Edinburgh, Edinburgh, UK
Correspondence should be addressed to Paul Fowler; Email: p.a.fowler{at}abdn.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Two publications have questioned a direct link between FSH and GnSAF. In the first, Tio et al.(1998) immunoneutralised FSH in female rats and concluded that there was no difference in circulating GnSAF. In the second, Schuiling et al.(1999) found that FSH treatment in female rats suppressed oestradiol-augmented LH responses to GnRH without affecting GnRH self-priming, a definite aspect of GnSAF from in vitro pituitary models (Fowler & Templeton 1996). In both of these studies the interpretation of the data is complex and debatable, due to multiple factors operating in vivo. What is required is an analysis of FSH actions on the production of GnSAF from a model system demonstrating normal function in vitro. The intact rodent follicle model (Spears et al. 1994, McGee et al. 2001, Murray et al. 2001, Spears et al. 2002) retains the cell–cell interactions and structural framework within which the granulosa cell normally functions.
The aim of this study was to investigate the relationship between follicle development, gonadotrophin exposure and GnSAF production in intact rodent follicle in vitro models. The mouse follicle model was initially investigated because follicular growth can be maintained to the late antral stage, allowing the production of GnSAF from both pre-antral and antral follicles to be investigated. However, this necessitates the presence of mouse serum in the culture medium which unfortunately renders it impossible to utilise a defined medium. Thus, the pre-antral rat follicle model was used to investigate gonadotrophin regulation of GnSAF production under highly controlled conditions. The fact that there are far fewer contaminating proteins in the culture medium makes the rat follicle culture model more suitable for the investigation of the regulation of GnSAF production.
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Materials and Methods |
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Follicle isolation and culture
Mice and rats were killed by cervical dislocation and their ovaries removed to watch-glasses containing Leibovitz L-15 medium (Gibco-BRL, Renfrew, UK) supplemented with 3 mg/ml BSA (Fraction V, Sigma, Poole, Dorset, UK). Individual pre-antral follicles (<200 µm in diameter) were manually dissected using fine needles, randomly allocated to microtitre plates and cultured at 37°C in a water-saturated atmosphere of 5% CO2/95% air mixture. Mouse follicles were placed in 'U'-shaped wells of microtitre plates (Iwaki, Osaka, Japan) containing 30 µl minimal essential medium (Gibco-BRL) overlaid with 75 µl silicone fluid (Gibco-BRL; Merck, Poole, Dorset, UK). Culture medium for the mouse follicles was supplemented with 5% mouse serum and 1 IU recombinant human FSH (hrFSH) per ml (Serono-Ares, Geneva, Switzerland). Follicles were moved to fresh medium daily. Rat follicles were placed in poly-carbonate membrane-lined flat-bottomed wells in 150 µl minimal essential medium overlaid with 75 µl silicone fluid. Rat follicle culture medium was supplemented with 3 mg/ml BSA and ITS+ (insulin, 10 mg/l; transferrin, 5.5 mg/l; linoleic acid, 4.7 mg/l; selenium, 5 mg/l; all from Sigma-Aldrich Company Ltd, Poole, Dorset, UK). Fifty percent of used medium was replaced with 50% fresh medium daily. hrFSH and/or recombinant human LH (hrLH) (Serono-Ares) were added as described below. A minimum of 12 follicles were cultured and assessed independently for every treatment in each follicle culture experiment.
Quality control follicular fluid
All protocols employing human subjects were given Joint Ethical Committee Approval at Aberdeen and patients all gave informed consent. Follicular fluid (hFF) was aspirated from follicles 
18 mm in diameter from 40 women undergoing IVF in Aberdeen and pooled and desalted as previously described (Fowler et al. 2001). Subsequently, 500 µl aliquots of these hFF pools were stored at –20 °C and used as a GnSAF bioactivity quality control (QC), producing a 40–60% reduction in GnRH-induced LH at 25 µl/well, as previously described (Fowler et al. 2001). This preparation is subsequently referred to as QC hFF.
GnSAF bioassay
For each cell culture, 15 rats, selected at random during the oestrous cycle, were killed by stunning and cervical dislocation. Dispersion and culture of the pituitary cells was carried out as described in Fowler et al.(1994, 2002) and only preparations with >75% viability of dispersed cells were used for bioassay. Primary pituitary cells were cultured at 30 000 viable cells/200 µl culture medium per well in 96-well tissue culture plates. The cells were cultured under sterile conditions for 24 h at 37 °C in a water-saturated atmosphere of 5% CO2/95% air mixture with serum-free defined culture medium (SFDM) as defined in Fowler et al.(1994). Media and non-attached cells were removed by aspiration and the wells were washed with 500 µl SFDM. All experiments were then carried out on quadruplicate wells as follows: 200 µl fresh SFDM were added, together with the treatments made up to 25 µl with SFDM. All the culture plates contained at least four control wells receiving SFDM only. After 24-h incubation with the test substances, the wells were washed with fresh medium and then treated with 0.1 µM GnRH (Fertagyl; Intervet UK Ltd, Cambridge, Cambs, UK). Cultures were terminated after 4-h incubation by collecting the media which were stored at –20 °C for determination of GnRH-induced LH and FSH secretion.
Dose–responses for assessment of bioactivities
Doses of 0, 1, 5, 25 µl/well of rodent follicle-conditioned medium and QC hFF, with and without incubation with GnSAF antiserum, were added randomly to 5 independent rat pituitary cell cultures in quadruplicate wells. Each culture dish contained 4 control wells receiving SFDM only and 4 wells receiving 25 µl SFDM instead of the 25 µl GnRH challenge. This enabled the calculation of LH release in the absence of GnRH stimulation in order to ensure comparable GnRH responses from one culture to another. The QC hFF ensured that comparable detection of GnSAF bioactivity occurred across the different cultures. In all experiments, specific controls for each dose of gonadotrophin were samples of medium + gonadotrophin at the relevant doses, but without conditioning by rodent follicles. This design controlled for the exposure of the pituitary cells to FSH and LH where appropriate. Because the wells were washed prior to the GnRH challenge, the presence of LH or FSH during the treatment period could not artefactually elevate LH or FSH concentrations in the GnRH-treated samples.
Two-dimensional gel electrophoresis
Proteins in rat follicle-conditioned medium (±FSH) were freeze-dried and resuspended at a ratio of 250 µl unprocessed conditioned medium per 100 µl lysis buffer (0.01 M Tris–HCl, pH 7.4, 1 mM EDTA, 8 M urea, 0.05 M dithiothreitol (DTT), 10% (v/v) glycerol, 5% (v/v) NP40, 6% (w/v) pH 3–10 Resolyte (VWR International Ltd, Poole, Dorset, UK)). Following centrifugation at 11 000 g, the supernatant was stored at –70 °C. Soluble proteins were analysed by two-dimensional gel electrophoresis (2-D gels) gels using a small format gel system (Cash et al. 1997) with immobilised pH gradient (IPG) gels for the first dimension separation. Briefly, 40 µl rat follicle-conditioned medium (±FSH) containing 100 µg total protein were adjusted to a final volume of 125 µl in IPG re-swelling buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 0.3% (w/v) DTT, 2% (w/v) pH 4–8 Resolyte). The proteins were separated in the first dimension using 7 cm, pH 4–7 IPG gel strips (Amersham Biosciences UK Ltd, Chalfont St Giles, Bucks, UK). The dehydrated IPG strips were re-hydrated overnight in the IPG re-swelling buffer containing the conditioned media (Rabilloud et al. 1994). Following their re-hydration, the IPG gel strips were electrophoresed on a Multiphor II apparatus (Amersham Biosciences UK Ltd), the IPG gels were then equilibrated in buffer containing 50 mM Tris–HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 1% (w/v) DTT for 30 min followed by a second equilibration for 30 min in the same buffer + 2.5% (w/v) iodoacetamide. The equilibrated strips were overlayed onto 7 x 8 cm 10–15% gradient polyacrylamide slab gels and processed as described previously (Cash et al. 1999). Proteins were located by silver staining and Western blot. For the latter, gels were transferred onto 0.45 µm nitrocellulose at 200 V for 75 min in transfer buffer (0.25 M Tris–HCl, 19.2 mM glycine, 0.01% SDS, 2% methanol), washed in blocking buffer containing 2% non-fat powdered milk in Tris-buffered saline (TBS) for 45 min then washed for 3 x 10 min in TBS containing 3 g Tris, 8 g NaCl, 0.2 g KCl/l, pH 8 prior to incubation with GnSAF polyclonal antibody (pAb) at room temperature for 90 min. The membranes were washed (3 x 10 min, TBS + Tween) and incubated with alkaline phosphatase-conjugated IgG diluted in TBS for 90 min. Recognised proteins were directly visualised with 5-bromo-4-chloro-3-indolylphosphate with nitroblue dimethyl formamide (Sigma-Aldrich Company Ltd).
Hormone assays
Concentrations of LH in cell-conditioned media from rat anterior pituitary cell cultures were determined using a homologous rat time-resolved fluoro-immunoassay (DEL-FIA) with sensitivity and intra- and interassay coefficients of variation values of 0.2 ng LH/ml (NIDDK-rLH-RP3) using NIDDK-anti-rLH-S11 and 5.4% and 7.9% respectively.
Experiments
Experiment 1 was carried out on mouse follicles, using follicles dissected from 21-day-old mice. Five percent mouse serum was added to the medium. Subsequent experiments (experiments 2–5) were then carried out using a rat follicle culture model which supports follicle growth in the absence of serum (although BSA is still required in the culture medium). This allowed possible confounding effects of serum proteins on the culture system to be removed from the model. This culture system used follicles dissected from 12-day-old rats.
Experiment 1: effect of duration of mouse follicle culture on GnSAF production
Pre-antral mouse follicles were cultured for 6 days in culture medium. The medium was collected every 24 h and stored at –20 °C for subsequent bioassay. The aim of this experiment was to investigate how the profile of GnSAF production changed as follicles grew, from less than 200 µm to around 400 µm in diameter (Spears et al. 1994, 1998, 2002, Murray et al. 2001).
Experiment 2: effect of duration of rat follicle culture on GnSAF production
Pre-antral rat follicles were cultured in the presence or absence of 5 IU FSH/ml for 3 days. The medium was collected daily and stored at –20°C for subsequent bioassay. The aim of this experiment was to establish the pattern of GnSAF production over time in the presence and absence of FSH.
Experiment 3: effect of gonadotrophins on GnSAF production by intact rat follicles
(a) Pre-antral rat follicles were cultured in the presence of 0, 0.5, 1, or 5 IU FSH/ml for 3 days. The medium from the last 24 h was collected and stored at –20 °C for subsequent bioassay. The aim of this experiment was to investigate the FSH dose–response behaviour of rat follicles in terms of GnSAF production. (b) Pre-antral rat follicles were cultured in the presence of (i) no gonadotrophin, (ii) 5 IU FSH/ml, (iii) 0.5 IU LH/ml, (iv) 5 IU LH/ml, (v) 0.5 IU LH/ml + 5 IU FSH/ml or (vi) 5 IU LH/ml + 5 IU FSH/ml, for 3 days. The medium from day 2 was collected and stored at –20 °C for subsequent bioassay. The aim of this experiment was to investigate whether LH treatment affected GnSAF production both in the presence and absence of FSH.
Experiment 4: effect of anti-human GnSAF polyclonal antibody
In order to investigate whether the GnSAF bioactivity produced by the rodent follicles was homologous to that produced by human granulosa cells and detected in human serum and hFF, an antibody bioactivity-blocking experiment was carried out as follows. Both the rat polycloncal antibody (pAb) which previously blocked in vitro GnSAF bioactivity in human material (Fowler et al. 2002) and a rat pAb raised against whole serum from post-menopausal women (PMWS) and employed as a control antiserum in Fowler et al.(2002), were used. These antibodies were mixed gently at 1:1 in 100 µl aliquots with 2-day and 3-day rat follicle-conditioned medium (obtained from follicles cultured in the presence of 5 IU FSH/ml), for 1 h at 37 °C in 24-well dishes on an orbital shaker. The incubation experiment was repeated twice, combined and the resulting samples were bioassayed in two separate monolayer cultures. If the bioactivity produced by the rodent follicles had good homology with human GnSAF then we predicted that it would reduce apparent GnSAF bioactivity in rodent follicle-conditioned medium while the PMWS antiserum would have no effect since post-menopausal women do not produce GnSAF.
Experiment 5: effect of FSH on the protein secretion profile of rat follicles
Six rat follicles were cultured for 3 days both with and without 5 IU FSH/ml. For each treatment the media were collected and concentrated from 20 ml to 1000 µl, desalted and dehydrated in a vacuum centrifuge. The two residues were resuspended in 250 µl of 2-D lysis buffer and electrophoresed using 2-D gels as outlined above.
Statistical analysis
The in vitro pituitary cell responses are expressed as percentages of the relevant control gonadotrophin concentrations secreted from wells on the same culture dishes. These controls were either wells exposed to SFDM alone (basal secretion) or wells exposed to SFDM + 0.1 µM GnRH. The differences between treatment groups and dose–responses were assessed using two-way analysis of variance (ANOVA). Differences between treatments and controls were tested by Dunnet’s post hoc test and between treatments by the Bonferroni–Dunn post hoc test. The analyses were performed using the Statview 5 programme (Abacus Concepts Inc., Berkley, CA, USA). All results are presented as means±S.E.M. Since all bioassays were carried out in quadruplicate wells using two separate cell cultures, n is stated as 2 x 4. For the rodent follicle growth curves n 
12 individual follicles.
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), resulting in a marked drop in LH secretion at the 25 µl conditioned medium/well dose. GnSAF production was highest on day 1 with even the 1 µl conditioned medium/well dose significantly reducing GnRH-induced LH secretion (P < 0.001). Between days 4 and 6 of culture the stimulatory effect of the follicle-conditioned medium increased, with GnRH-induced LH secretion increased to up to 180 ± 16% of control, P < 0.01 at 5 µl/well. Thus during days 4–6 of culture, decreases in LH secretion were only induced by the highest dose of conditioned medium/well.
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). In the absence of FSH there was only detectable GnSAF production by the follicles on day 1 of culture (P < 0.05). In contrast, FSH treatment stimulated GnSAF production on both days 1 and 2 of culture (P < 0.01 and P < 0.05 respectively). Although there was a statistically significant decline (P < 0.05) in GnSAF production as the follicles grew from around 170 to 180 µm in diameter, this difference was reflected in an 8% difference in the suppression in GnRH-induced LH secretion at the highest dose of conditioned medium. These findings show that GnSAF production by rat follicles occurs in the presence of FSH and suggest that this occurs primarily in smaller follicles, as in the mouse.
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). All FSH doses stimulated follicle growth over a 3-day period (P < 0.001). In the absence of FSH, follicle diameter fell slightly over the course of the culture (P < 0.05). GnSAF production on day 2 was stimulated most by 1 IU FSH/ml (P < 0.001), a significantly greater production than that of other doses (P < 0.05), although there was no difference between the growth rates of any of the follicles grown in the presence of FSH. In contrast, experiment 3b showed that LH alone (at both 0.5 and 5 IU/ml) had no significant effect on GnSAF secretion (Fig. 4a) and that these follicles did not grow over the culture period (Fig. 4b). The presence of FSH stimulated growth (P < 0.01), as in experiment 3a, although growth was unaffected by the additional presence of LH. Co-treatment of LH with 5 IU FSH/ml did, however, further increase GnSAF bioactivity significantly at the 5 µl conditioned medium/well dose (P < 0.05).
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). This suggests that there is relatively close homology between rodent and human GnSAF.
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). Since the culture medium had to be supplemented with BSA to support follicle health, heavily stained areas coinciding with the established position of serum albumin were obvious. Despite this, the FSH could clearly be seen to both stimulate and inhibit a range of protein spots. When probed with the anti-human GnSAF pAb in a Western blot (Fig. 6c), several proteins in a band between 50 and 65 kDa and between 4.5 and 5.5 pH were stained – an area similar to that observed when this antiserum was used to Western blot human granulosa cell-conditioned medium (Fowler et al. 2002). These Western blot-recognised protein spots matched several less abundant spots that increased with FSH treatment but are not highlighted in Fig. 6b in order to retain clarity. Unfortunately, the protein yield was not sufficient for any of the up-regulated protein spots to be excised and subjected to peptide mass mapping.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Follicles produced GnSAF only if cultured in the presence of FSH, the presence of LH alone being insufficient to induce its production. Since FSH, but not LH, is required for antral follicle survival in mice (Spears et al. 1998), the lack of GnSAF production in the absence of FSH is likely to be due to the induction of granulosa cell apoptosis in these follicles (Baker et al. 2001). Similarly, McGee et al.(1997) demonstrated increased apoptosis in cultured pre-antral rat follicles (using the same culture method as in these experiments), while Wang et al.(2003) showed that FSH is essential to protect early antral rat follicles from apoptosis. While the inability of LH to induce GnSAF production in the absence of FSH could be an indirect effect due to atresia in these follicles, we also showed that LH was unable to stimulate increased GnSAF production in the presence of FSH. The follicles in these experiments remained at the pre-antral stage throughout the culture period, and it is possible that these follicles do not yet express active LH receptors. Our findings, therefore, emphasise that FSH (but not LH) is required for GnSAF production by pre-antral follicles.
The difference in the biological effect of secreted proteins from small and large mouse follicles is interesting beyond the observation of decreasing GnSAF production as the follicles grow. The suppression of GnRH-induced LH secretion by proteins secreted from small follicles rather than large ones is further indirect evidence that GnSAF activity is not due to inhibin, since inhibin production increases as mouse follicle cultures progress (Smitz & Cortvrindt 1998).
The stimulatory effects of proteins from large murine follicles on GnRH-induced LH secretion may possibly be due to the combined effects of increasing activin and decreasing GnSAF bioactivities. The reasoning behind this is that, because activin secretion is known to increase as the follicles grow (Newton et al. 2002) and since activin stimulates GnRH-induced LH secretion from rat pituitary cells (Weiss et al. 1993), conditioned medium from larger follicles would be more likely to stimulate GnRH-induced LH secretion. In the present study, therefore, the lower doses of conditioned medium might favour the action of activin bioactivity, causing an increase in GnRH-induced LH secretion. In contrast, at the higher doses of conditioned medium, even though GnSAF bioactivity is reduced, there might be sufficient GnSAF bioactivity present to counteract the stimulatory effects of activin, which could have already exerted its maximal effect at the lower dose of medium. Data in the human suggest that GnSAF production declines rapidly as the follicle grows past 10 mm in diameter in spontaneously cyclic women (Fowler et al. 2001) or 13 mm in diameter in women undergoing IVF treatment (Fowler et al. 1994). This represents a decline in GnSAF production post-dominance or following major waves of follicular development (Baerwald et al. 2003) in the human. In the rodent, while there is no evidence for follicular waves, the wave of atresia due to follicle dominance appears to occur around the mid antral stage of development (Baker et al. 2001), with the fall in GnSAF shown here occurring immediately after that. GnSAF production is, therefore, falling as follicles mature towards the pre-ovulatory stage, with a concomitant increase in the production of oestrogens (Spears et al. 1998). Taken together these data are suggestive of the likely role of GnSAF in the rodent. The fact that GnSAF secretion will be highest prior to follicular maturity indicates that the proposal by Fowler et al.(2003) that GnSAF acts to keep LH pulsatility in check is feasible for the rodent. Declining GnSAF production during the last phase of follicular maturation would represent a fall in the overall negative feedback at the level of the pituitary, concomitant with increasing oestradiol secretion and the resulting positive feedback effects on both hypothalamus and pituitary.
The suggestion by Schuiling et al.(1999) that FSH stimulates rat ovarian production of an oestradiol-antagonising factor rather than GnSAF is only one of the two most likely interpretations of their data. The present study clearly demonstrates that FSH treatment of rodent follicles stimulates production of GnSAF bioactivity, and that this bioactivity is completely inhibited by co-incubation with an antibody that blocks human GnSAF (Fowler et al. 2002). This finding also casts doubt on the interpretation of the effects of FSH immunoneutralisation that Tio et al.(1998) reported did not affect the indirectly determined GnSAF bioactivity in rats.
It was unfortunate that we were unable to recover sufficient protein to identify some of the proteins secreted in response to FSH treatment, especially those recognised by the anti-GnSAF antiserum. These proteins fell within the expected pI (5.5–5.8 pH) and molecular mass (60–70 kDa) ranges proposed for GnSAF (Fowler et al. 2003) and it would be reasonable to predict that one of them may have been GnSAF. However, this study has clearly demonstrated that the intact rodent follicle culture model is suitable for the investigation of both GnSAF and the regulation of GnSAF production. To investigate the GnSAF molecule itself, a scaling-up of the rat follicle cultures would be required in order to produce sufficient protein for identification using serum protein depletion steps and proteomic techniques. In contrast, with respect to the study of the regulation of GnSAF production, the ability to control steroidal, gonadotrophin and inhibin signalling in the intact follicles and then subsequently determine changes in GnSAF bioactivity would greatly enhance our understanding of the factors regulating GnSAF during the ovarian cycle.
To summarise, the intact rodent follicle culture model demonstrates that GnSAF production is positively regulated by FSH and preferentially produced by smaller follicles, matching existing GnSAF data for the human in particular.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
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Baerwald AR, Adams PG & Pierson RA 2003 Characterisation of ovarian follicular wave dynamics in women. Biology of Reproduction 69 1023–1031.
Baker SJ, Srsen V, Lapping R & Spears N 2001 Combined effect of follicle–follicle interactions and declining follicle-stimulating hormone on murine follicle health in vitro. Biology of Reproduction 65 1304–1310.
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