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Original Studies |
Assisted Conception Unit (P.Y.K.Y., K.J.T.), Royal Infirmary of Edinburgh, Edinburgh EH3 9EF; Department of Medical Sciences (R.A., B.R.W.), University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU; and Department of Reproductive and Developmental Sciences (S.G.H.), University of Edinburgh, Centre for Reproductive Biology, Edinburgh EH3 9EW, Scotland, United Kingdom
Address all correspondence and requests for reprints to: Dr. Stephen G. Hillier, University of Edinburgh, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, Scotland, United Kingdom.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Cortisol (F) has well-established antiinflammatory properties (2, 3). It has been proposed that locally activated glucocorticoid
production may play a role in limiting tissue damage and mediating
repair/remodeling after human ovulation (4, 5). One
evidence for this proposal comes from the finding of elevated total F
concentration in follicular fluid relative to that in serum close to
follicular rupture (5). Additionally, the proinflammatory
cytokines tumor necrosis factor-
and interleukin-1ß were recently
reported to stimulate the expression of 11ß-hydroxysteroid
dehydrogenase type 1 (11ßHSD1) messenger RNA (mRNA) in glomerular
mesangial cells (6) and rat granulosa cells
(7) in vitro. Because 11ßHSD1 promotes
reversible formation of F from cortisone (E), we interpret this as
evidence that the proinflammatory cascade that leads to ovulation
induces a compensatory antiinflammatory response in the ovary, which
includes up-regulation of 11ßHSD1 and increased local availability of
F.
The two known isozymes of 11ßHSD are present in the human ovary, where their expression is development-dependent (8). Granulosa cells of immature follicles (IGC) express predominantly 11ßHSD type 2 (11ßHSD2) mRNA. In contrast, luteinized granulosa cells (LGC) in periovulatory follicles, after human (h)CG administration to induce ovulation, express mainly 11ßHSD1 mRNA but little or no 11ßHSD2 mRNA. Because 11ßHSD2 has unidirectional dehydrogenase activity, converting F to its inactive 11-keto metabolite E, whereas 11ßHSD1 is bidirectional, tending to preferentially convert E to F in vivo (9), this should result in enhanced production of F in periovulatory follicles, compared with immature follicles.
To test this hypothesis and thereby obtain further evidence for an antiinflammatory role for glucocorticoids in the ovary, it is necessary to measure E and F interconversion by ovarian cells directly. The aims of the present study were therefore: 1) to measure 11ßHSD enzymatic activity in IGC and LGC; 2) to measure follicular fluid levels of E and F in both immature and periovulatory follicles; 3) to determine whether 11ßHSD enzymatic activity is present in thecal cells (TC), which has hitherto not been reported; and 4) to determine whether granulosa cell 11ßHSD1 reductase activity (E-to-F conversion) is gonadotropically regulated in vitro.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Immature granulosa cells and TC were obtained from ovaries of
women undergoing oophorectomy as part of their surgery for various
benign gynecological conditions (Table 1
). These operations were performed
before 1130 h. LGC were obtained by transvaginal ultrasound-guided
aspiration of periovulatory follicles in patients undergoing in
vitro fertilization (IVF)-embryo transfer cycles (Table 1). IVF
treatment was performed according to the standard long protocol of
controlled ovarian stimulation (10). Human CG (5,000 or
10,000 IU) (Profasi, Serono, Welwyn-Garden City, Herts, UK) was
administered when there were at least three follicles 
17-mm diameter,
as determined by transvaginal ultrasonography. Oocyte recovery by
follicular aspiration was performed 35 h after hCG administration.
Blood samples for measurement of serum total E and F were obtained
between 0830 h and 1030 h, approximately 1 h before
surgery, in both groups of patients. All patients had given informed
consent, and the project was approved by the local ethics
committee.
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Resected ovaries were transported ice-cold to the laboratory and transferred to culture medium, which was also used for follicular dissection. The culture medium was Medium 199, containing Earle’s salts, 25 mmol/L HEPES buffer, supplemented with 2 mmol/L L-glutamine, 50 IU/mL penicillin, 50 ng/mL streptomycin (all from Life Technologies, Inc., Paisley, Renfrewshire, UK) and 0.1% BSA (ICN Biomedicals, Inc., High Wycombe, Buckinghamshire, UK). With the aid of zoom stereomicroscopic optics, follicles were dissected free from surrounding stroma, and their diameters were measured using calipers (11). IGC were gently scraped from the theca interna of hemisected follicles. All IGC from the same patient were pooled. Thecae from pooled follicles were rinsed in fresh medium and transferred to 2 mL of the same medium with added 0.1% collagenase type II from Cl. histolyticum and 0.01% deoxyribonuclease-I (both from Sigma, Poole, Dorset, UK). Complete dispersal into a cellular suspension was achieved by incubating the tissue in the enzyme solution for two successive 15-min periods, at 37 C, in a shaking water bath, with repeated pipetting after each incubation. Separated IGC and TC were sedimented by centrifugation at 800 x g for 5 min and resuspended in fresh culture medium.
Follicular aspirates from IVF patients were obtained between 1000 h and 1130 h. They were transported ice-cold to the laboratory, where LGC were recovered by unit-gravity sedimentation and washed several times with Dulbecco’s PBS until clear of red blood cells (12). After centrifugation at 800 x g for 5 min, granulosa cells were resuspended in fresh culture medium and counted.
Cell counting was performed in a hemocytometer using trypan blue exclusion to estimate viability, which ranged between 15–85%. Replicate portions of the cell suspensions, containing viable 1–5 x 104 granulosa cells or 5 x 104 TC, were then distributed into 24-well polystyrene culture dishes (Corning, Inc. Glass Works, Corning, NY) for measurement of 11ßHSD activity with or without culture, as described below.
Follicular fluid from individual antral follicles dissected from the ovarian specimens, and fluid from the first follicle aspirated at oocyte recovery were stored at -20 C for subsequent measurements of E, F, estradiol, and progesterone.
Measurement of 11ßHSD activity
Interconversion of [1,2,6,7-3H]E and [1,2,6,7-3H]F was studied in all granulosa and TC preparations as an index of 11ßHSD activity, as previously described (10). In brief, culture medium containing substrate E or F (50 pmol), including 0.1 µCi [3H]E or [3H]F, was added to each well to give a final vol of 0.5 mL. Control incubations containing no cells were also set up. All incubations were in triplicate. Incubation was for 4 h at 37 C in a humidified tissue culture incubator gassed with 95% air-5% CO2. Media from individual wells were then pipetted into glass tubes, to which was added diethyl ether (3 mL); and the samples were vortexed for 1 min. The extracts were transferred to tubes containing 10 µL 10-3 mol/L E and F. The dried etheric extracts were then transferred in ethyl acetate to silica gel-precoated plastic sheets (PE SIL G; Whatman Ltd., Maidstone, Kent, UK) for thin-layer chromatographic separation of precursor and product in the solvent system chloroform:ethanol (92:8 by vol) (BDH Laboratory Supplies, Poole, Dorset, UK). E and F bands were identified by visualization under UV light, and the corresponding areas were cut out and transferred to a scintillation counting vial for determination of radioactivity. Consistently greater than 90% of the total radioactivity added to the incubations was accounted for in the E and F bands. Enzymatic activity was expressed as percentage total radioactivity [(product cpm/substrate cpm + product cpm)x 100] after correction for values from control (no cell) incubations.
Effect of gonadotropins on 11ßHSD1 reductase activity in cell culture
In three cases (patients 6–8; Table 1
), IGC monolayers were
established in the presence and absence of gonadotropins before
determination of 11ßHSD activity. Serum precoated (13)
culture wells, inoculated with replicate portions of IGC suspension
(2–5 x 104 viable cells per well),
received additional medium containing gonadotropins, to give a total
vol of 0.5 mL. The gonadotropins used were recombinant hFSH (3860
IU/mg) and LH (6850 IU/mg) donated by Ares-Serono, Geneva, Switzerland.
Testosterone (10-6 mol/L)
was also added to the wells as an aromatase substrate. Incubation was
for 48 h, at 37 C, under humidified 5% CO2.
Spent medium was removed and stored at -20 C for subsequent
measurement of steroid content. Prewarmed (37 C) culture medium (0.5
mL), containing substrate [3H]E or
[3H]F, was then added to the monolayers for the
11ßHSD assay described above.
Hormone assays
Total E and F were determined in serum and follicular fluid by
RIA after high-performance liquid chromatography (HPLC) separation.
After addition of epicortisol (11-hydroxy isomer of cortisol; 15 ng)
as an internal standard for validation of HPLC peaks, the samples (up
to 250 µL) were partitioned with 10 vol cyclohexane:dichloromethane
(95:5) to remove progesterone. The organic phase was discarded, and the
sample was reextracted with ethyl acetate (1:10). The extract was then
reduced to dryness under a stream of nitrogen at 60 C and reconstituted
in HPLC mobile phase (acetonitrile:methanol:water, 15:25:60). Fractions
containing E and F were collected, after separation by HPLC, with
conditions as follows: C18 Luna column (15 cm;
internal diameter, 4.6 mm; pore size, 5 µm; Phenomenex,
Maccleçfield, Chesire, UK), 0.7 mL/min, 37 C. The fractions were
reduced to half their volume, under nitrogen, as above and then
extracted with ethyl acetate (5 mL). Suitable proportions of the dried
extracts were then taken for RIA as previously described
(14). The inter- and intraassay precision
(coefficient of variation) was less than 10%.
Estradiol and progesterone in serum, follicular fluid, and culture medium were measured by standard RIA using previously described methods (11, 15). The inter- and intraassay coefficient of variation for each steroid was less than 12%.
Statistics
Statistical analysis was performed using Student’s t test. Differences assigned a P value less than 0.05 were regarded as statistically significant. Correlations between follicular fluid and serum concentration of steroids were examined using linear regression analysis.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Interconversion of E and F. The overall pattern of E-F
interconversion in patients 1–5 (IGC) and 9–15 (LGC) (Table 1) was
consistent with a markedly higher level of 11ßHSD1 reductase activity
in LGC, as compared with IGC. The converse was true for dehydrogenase
activity, which was higher in IGC, compared with LGC. As illustrated in
Fig. 1, the percent conversion (mean
± SEM) of F to E was not measurably influenced by the
stage of granulosa cell maturity (31.0 ± 2.9 in LGC
vs. 41.7 ± 7.4 in IGC; P = 0.15). In
contrast, percent conversion of E to F by LGC was over 60-fold greater
than that of IGC (36.3 ± 3.7 vs. 0.6 ± 0.4;
P < 0.001). In LGC, the percent conversion of E to F
consistently exceeded that of the reverse reaction, although the
difference was not statistically significant. This is in keeping with
the known predominant reductive role of 11ßHSD1 in vivo
(9). The high rate of F-to-E conversion and negligible
E-to-F conversion in IGC is consistent with a predominant 11ßHSD2
activity in these cells. TC from immature follicles (patients 1–5)
revealed minimal conversion of E to F (0.2 ± 0.1%), with a
significantly higher, but low-level (4.7 ± 0.9%), conversion of
F to E (P < 0.005) (Fig. 1).
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. The mean (±SEM)
concentration of F was significantly higher in periovulatory follicles
(n = 10) that yielded LGC, compared with immature follicles
(n = 15) that yielded IGC (254.8 ± 39.1 vs.
153.7 ± 20.0 nmol/L; P < 0.05). The converse was
true for E, the level being higher in immature follicles than
periovulatory follicles (76.2 ± 7.9 vs. 41.9 ±
5.2 nmol/L; P < 0.01). The F:E ratios were
significantly greater in follicular fluid from periovulatory follicles
(6.2 ± 0.8), compared with immature follicles (2.3 ± 0.3;
P < 0.001). The serum concentration of F was
significantly higher in the IVF patients, compared with patients
undergoing oophorectomy (721.2 ± 112.3 vs. 360.2
± 83.5 nmol/L; P < 0.05). The serum concentration of
E in the patients undergoing oophorectomy was variable (765.6 ±
388.2 nmol/L); and in comparison to the level in the IVF patients
(116.7 ± 20.1 nmol/L), the difference was not statistically
significant. Linear regression analysis revealed no correlation between
follicular fluid and serum concentrations of F in the IVF patients
(r = 0.08; P = 0.83). A similar lack of
correlation was obtained between E in follicular fluid of immature
follicles and serum (r = 0.09; P = 0.81).
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Gonadotropic regulation of 11ßHSD1 reductase activity in vitro
In three of three cases, treatment of IGC cultures with hFSH
significantly increased the percent conversion of E to F, relative to
untreated controls (Fig. 3). In two of
these cases (patients 6 and 7), hFSH also stimulated aromatase activity
(estradiol production) and progesterone production (Fig. 3, A and B),
but neither of them showed a significant response to hLH. The third
case (patient 8) responded to hLH in the same way as hFSH, with
significantly increased percent conversion of E to F and progesterone
production but not aromatase activity (Fig. 3C).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Our results therefore suggest the existence of an E-F shuttle (16) in the ovary, similar to other glucocorticoid target tissues in which 11ßHSD activity gates the access of F to glucocorticoid receptors. However, in contrast to the constitutive expression of one or other 11ßHSD isozyme in other tissues, the ovary expresses both isozymes. While two studies to date have reported increased follicular fluid levels of F after the midcycle LH surge (17) or hCG injection (5), only one study has measured E in addition to F (18). This study reported concentrations of follicular fluid F similar to ours, and the F:E ratio was also greater in IVF periovulatory follicles (post-hCG), compared with follicles just before hCG administration. However, the latter group of patients all had polycystic ovarian syndrome and had received gonadotropin stimulation before follicle aspiration. They were therefore dissimilar to our patients in whom the immature follicles were studied. This is an important difference because serum levels of F and urinary excretion of F metabolites have been reported to be increased in polycystic ovarian syndrome (19, 20). Another major difference is that Andersen et al. (18) did not measure granulosa cell 11ßHSD activity.
It may be argued that the higher concentration of F in periovulatory follicles was attributable to higher stress levels in the IVF patients, because their serum F levels were also higher than those of patients undergoing elective gynecological surgery. However, if this was true, follicular fluid E should also have increased in parallel with F. On the contrary, we found the level of follicular fluid E to be significantly higher in immature follicles than periovulatory follicles. This is compatible with the predominance of 11ßHSD2 mRNA (8) and enzymatic activity in IGC. Additionally, there was no correlation between the levels of F in serum and follicular fluid in the IVF patients. It is not known whether the diurnal variation of glucocorticoid levels in blood also occurs in follicular fluid. However, all the blood samples and follicular fluid aspirates were collected in the morning. This would minimize any confounding influence caused by a diurnal variation in follicular fluid glucocorticoid levels.
Adrenal glucocorticoids can affect ovarian function through central inhibition of pituitary gonadotropin production (21), which is thought to explain the association of anovulation with stress and clinical states of glucocorticoid excess. At the ovarian level, the role of glucocorticoid metabolism presumably reflects the classic glucocorticoid actions in response to altered states of nutrition, injury, or stress seen elsewhere in the body. The present demonstration of increased intrafollicular F, in relation to ovulation, most obviously suggests involvement with final oocyte maturation and/or follicular rupture, but this remains to be determined. Fateh et al. (22) reported a positive correlation between oocyte maturity and follicular fluid levels of F. Cortisol metabolism by cultured LGC in vitro has been correlated with IVF outcome (23), but such a relationship was not observed when freshly isolated LGC were studied (10). Mice that survive to adulthood with inactivating11ßHSD1 (24) or 11ßHSD2 (25) mutations can reproduce. However, the ability to reproduce does not indicate unimpaired fecundity, because ovarian phenotypes and reproductive performance in these knockout animals have not been addressed in detail. On the other hand, the relative abundance of E in the follicular fluid of immature follicles and the almost exclusive expression of 11ßHSD2 mRNA and activity in IGC may imply a protective mechanism against the excessive accumulation of F in developing antral follicles, as in the case of the kidney (26, 27) and possibly placenta (28, 29).
Experiments with rat granulosa cells have shown that treatment with LH
induces a switch from 11ßHSD2 to 11ßHSD1 expression in
vivo and in vitro (7, 30). However, this
only occurred when the cells were first exposed to sufficient amounts
of FSH to induce LH receptors and LH responsiveness. In the present
study of human granulosa cells, we found consistent up-regulation of
11ßHSD1 activity by hFSH, but not hLH, in vitro. However,
the exception proved the rule, because IGC from patient 8 (Fig. 3C),
which responded to hLH as well as hFSH, included cells from a
relatively mature (15-mm diameter) follicle with follicular fluid
estradiol (1500 nmol/L) and progesterone (18.0 µmol/L) in the late
preovulatory range. Although the contribution of granulosa cells from
this follicle to the overall hLH effect is not known, the
responsiveness to hLH, in terms of 11ßHSD1 activity as well as
progesterone synthesis, is consistent with advanced preovulatory
status. The inability of the same cells to increase aromatase activity
in the presence of hFSH or hLH can be explained by the high basal
aromatase activity that they displayed, leading to exhaustion of
substrate testosterone in the culture medium, even in the absence of
exogenous gonadotropins.
Low-level 11ßHSD2 activity was also present in TC from immature follicles. Similar to IGC, TC showed negligible 11ßHSD1 activity. This corroborates the work of Ricketts et al. (31), which showed positive immunocytochemical staining with the type 2, but not type 1, 11ßHSD antibody in follicular phase thecal and granulosa cells. Whether the midcycle LH surge or hCG administration modulates 11ßHSD activity in TC as ovulation approaches is not known, because TC from periovulatory follicles were unavailable for study. Moreover, the ability of gonadotropins to up-regulate granulosa cell 11ßHSD activity in vitro does not rule out the possibility that paracrine mechanisms emanating from TC contribute to this effect in vivo. Indeed, the potential for paracrine modulation of 11ßHSD activity in hLGC has previously been highlighted (32).
In conclusion, our findings provide direct evidence for a development-related switch in the expression of 11ßHSD1 and 2 enzymatic activity in human granulosa cells, favoring formation of F as ovulation approaches. We also show, for the first time, that 11ßHSD activity is gonadotropically regulated in human granulosa cells and present in TC. Overall, these results strengthen the case for a physiological role of 11ßHSD and its regulation of intrafollicular glucocorticoid metabolism in the human ovary. The role of glucocorticoids in ovulation remains unknown. However, in view of the inflammatory nature of ovulation and the potent antiinflammatory effects of cortisol, our findings are consistent with the hypothesis that glucocorticoids have a local antiinflammatory role, mediating repair and healing of the ovary before the next ovulatory process.
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Acknowledgments |
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Footnotes |
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Received May 8, 2000.
Revised August 2, 2000.
Accepted August 18, 2000.
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References |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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; n =
15 from 6 patients) and periovulatory (IVF, 
) follicles (n = 10
from 10 patients), with group means depicted by horizontal
bars. In periovulatory follicles, the levels of F and the F:E
ratios were significantly higher (P < 0.05 and
P < 0.001, respectively), and the levels of E were
significantly lower (P < 0.01), than the
corresponding values in immature follicles.
0.001) and b (P < 0.05)
denote statistically significant differences between treatment groups
and the corresponding control experiments. A and B, Results from
patients 6 and 7, respectively; C, results from patient 8. In contrast
to the findings in A and B, this shows a significant stimulatory effect
of hLH, as well as hFSH, on conversion of E to F and progesterone
production by IGC. Granulosa cells from a large follicle (15 mm) with
estradiol (1500 nmol/L) and progesterone (18.0 µmol/L) levels in the
late preovulatory range were among the pool of cells that were studied
in this experiment, possibly explaining the response to hLH. The high
basal aromatase activity, resulting in complete metabolism of substrate
testosterone to estradiol in control incubations, explains the
inability of these IGC monolayers to increase estradiol synthesis in
response to either hFSH or hLH.