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BOR - Papers in Press, published online ahead of print January 22, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012096
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BIOLOGY OF REPRODUCTION 68, 2157–2163 (2003)
DOI: 10.1095/biolreprod.102.012096
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Ovarian Modulators of 11ß-Hydroxysteroid Dehydrogenase (11ßHSD) Activity in Follicular Fluid from Bovine and Porcine Large Antral Follicles and Spontaneous Ovarian Cysts1

Lisa M. Thurston2,3, Kim C. Jonas4, D. Robert E. Abayasekara3, and Anthony E. Michael4

Department of Veterinary Basic Sciences,3 Royal Veterinary College, Camden Town, London NW1 0TU, United Kingdom Department of Biochemistry & Molecular Biology,4 Royal Free & University College Medical School, University College London, London NW3 2PF, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the ovary, cortisol is oxidized to cortisone by 11ß-hydroxysteroid dehydrogenase (11ßHSD). The present study investigated whether follicular fluid (FF) from large antral follicles and spontaneous ovarian cysts, isolated from bovine and porcine ovaries, contained modulators of 11ßHSD activity. Whereas FF from antral follicles had no significant effect over 1 h on NADP+-dependent 11ßHSD activity in rat kidney homogenates, enzyme activity was inhibited by FF from bovine and porcine ovarian cysts (80.5% ± 2.3% and 72.8% ± 3.4% of control, respectively). Following C18 reverse-phase chromatography, the hydrophilic fractions of FF from bovine and porcine antral follicles stimulated NADP+-dependent 11ßHSD activities (111.5% ± 21.6% and 55.2% ± 5.7% respectively). Hydrophobic compounds inhibited NADP+-dependent cortisol oxidation by 58.2% ± 5.1% (bovine) and 45.7% ± 2.0% (porcine). In both species, FF from ovarian cysts appeared to contain less of the hydrophilic stimuli to 11ßHSD activity and more of the hydrophobic inhibitors. The FF from antral follicles and ovarian cysts, and the C18 fractions thereof, had no significant effect on NAD+-dependent cortisol oxidation. The ovarian modulators of NADP+-dependent 11ßHSD activities did not coelute with cortisol, cortisone, estradiol, testosterone, progesterone, pregnenolone, and cholesterol. However, the 11ßHSD stimuli in porcine FF from both antral follicles and cysts coeluted with prostaglandin (PG) E2 and PGF2{alpha}. We conclude that large antral follicles and spontaneous ovarian cysts, in both the cow and the pig, contain ovarian modulators of the NADP+-dependent 11ßHSD activity. Moreover, FF from spontaneous ovarian cysts, because of decreased content of the 11ßHSD stimulus accompanied by increased content of the 11ßHSD inhibitors, exerts a net inhibitory effect on 11ßHSD activity.

cortisol, follicle, ovulation, ovulatory cycle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The current decline in the fertility of domestic livestock, which has major financial implications for the farming industry [1], underscores the need to explore those factors that could be manipulated to increase productivity. One explanation for subfertility is the presence of anovulatory cystic follicles within the ovary. Between 10% and 15% of dairy cows develop follicular cysts [2], and approximately 10% of sows sent for slaughter because of infertility have cystic ovaries [3, 4]. Animals with cystic ovaries exhibit extended intervals between conceptions and require an increased number of inseminations for fertilization to occur [5].

During the bovine estrous cycle, normal follicular growth occurs in two or three waves [6, 7]. Each follicular wave involves the recruitment of from two to six subordinate follicles of approximately 4 mm in diameter, followed by the development of one dominant follicle. Unlike the cow, which produces a single preovulatory follicle, the pig develops 15–20 mature follicles, each of which will ovulate during a single cycle [8]. Luteinizing hormone is thought to regulate development of dominant follicles [6, 7], with an increase in LH pulse frequency being associated with maturation and subsequent ovulation of the dominant follicle [9]. However, in cystic ovary disease (COD), a reduced LH pulse frequency acts to maintain estradiol production by the mature follicles, which fail to ovulate and continue to grow into ovarian follicular cysts [10].

In COD, the reduction in the preovulatory LH surge has been ascribed to elevated concentrations of ACTH [11–13], which has, in turn, been attributed to a decrease in cortisol-mediated negative feedback because of increased peripheral metabolism. In a wide range of tissues, including the liver and kidneys, cortisol is oxidized to its inert metabolite, cortisone, by the enzyme 11ß-hydroxysteroid dehydrogenase (11ßHSD) (for review, see 14–16]). To date, two isoforms of 11ßHSD have been cloned. Type 1 11ßHSD is a bidirectional, NADP(H)-dependent enzyme with relatively low affinity for cortisol (Km = 27 µM) [17–19], whereas type 2 11ßHSD is an NAD+-dependent, high-affinity enzyme (Km for cortisol = 30–60 nM) that acts exclusively as an 11ß-dehydrogenase [20–22]. Changes in the activity of one or both isoforms of 11ßHSD may result in altered cortisol concentrations within the body, culminating in abnormal pituitary ACTH and LH secretion and formation of cystic follicles. In advancing this hypothesis, it is noteworthy that patients with polycystic ovarian syndrome (PCOS) have been reported to have decreased ratios of urinary cortisol to cortisone [23], reflecting either an increase in the 11ß-dehydrogenase activity of type 2 11ßHSD and/or a decrease in the 11-ketosteroid reductase activity of type 1 11ßHSD. However the mechanisms leading to altered 11ßHSD activities in women with cystic ovaries have yet to be elucidated.

We have recently reported the presence within follicular fluid (FF), aspirated from patients undergoing controlled ovarian hyperstimulation for in vitro fertilization-embryo transfer, of endogenous hydrophilic and hydrophobic compounds that can acutely stimulate and inhibit, respectively, the NADP(H)-dependent activities of type 1 11ßHSD [24]. Therefore, the primary aim of the present study was to determine whether FF aspirated from bovine and porcine large antral follicles contains endogenous ovarian modulators of type 1 11ßHSD activity and, if so, whether these compounds have biophysical properties similar to those of the modulators resolved from human FF. The secondary aim was to compare the levels of these endogenous ovarian 11ßHSD modulators in each species between FF aspirated from spontaneous ovarian cysts versus FF aspirated from large antral follicles.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ovarian Samples

Bovine and porcine ovaries were obtained from a local abattoir within 2 h of slaughter and were transported on ice to the Royal Veterinary College. Samples of acellular ovarian FF, aspirated from large antral follicles (diameter, >8mm) [25] and from ovarian cysts, were divided into 2-ml aliquots before being frozen at -20°C pending analysis. Spontaneous ovarian cysts were diagnosed as either single or multiple, anovulatory, fluid-filled structures with diameters of 25 mm or greater (range, 25–40 mm) in ovaries lacking corpora lutea [26–28]. In total, five FF samples from large antral follicles and five FF samples from individual ovarian cysts were collected from each species for study. In each case, only one follicle or cyst was aspirated from a given animal. Hence, for each of the two species, we collected five normal and cystic ovaries.

Effects of FF on Renal 11ßHSD Activities

The effects of bovine and porcine FF, aspirated from large antral follicles and ovarian cysts, on renal 11ßHSD activities were assayed using a modification of the radiometric conversion assay for glucocorticoid oxidation previously described in our laboratory [29–31]. Male Sprague-Dawley rats (weight, 200–250 g) were housed in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and were allowed ad libitum access to a standard rat chow diet and drinking water. Rats were killed by cervical dislocation, and kidney homogenates were prepared using established methods [32, 33]. Renal homogenates were utilized as a source of both NADP(H)-dependent, type 1 11ßHSD activities and NAD+-dependent, type 2 11ßHSD activity. For each assay, 1 g of rat kidney (containing approximately equal volumes of cortex and medulla) was homogenized in 18 ml of hypotonic Tris-EDTA lysis buffer [21, 32, 33]. After restoration of isotonicity by the addition of 2 ml of 1.5 M KCl (Merck, Dorset, U.K.), the homogenate was centrifuged at 250 x g (to precipitate intact tissue), and the supernatant was decanted into a fresh glass tube. From this supernatant, 100-µl volumes were transferred to glass screw-cap culture tubes, to each of which 600 µl of PBS (Life Technologies, Strathclyde, U.K.) were added. Triplicate tubes were also prepared as assay blanks containing 100 µl of BSA solution (1 mg/ml prepared in PBS) in place of renal homogenate. Each triplicate set of tubes then received 100 µl of FF or PBS (controls and blanks) before being preincubated for 30 min at 37°C in a gyratory waterbath. Previous studies in our laboratory have shown that the modulatory action of human FF on 11ßHSD activities increases with the concentration of fluid added to the assay [34]. Results from this study [34] suggested the optimal modulation of 11ßHSD activities in the rat kidney homogenate assay were achieved using a 10% (v/v) dilution of human FF samples. To produce consistent comparison of ovarian modulators across species, we have used this dilution in the bovine and porcine studies.

To initiate the 11ß-dehydrogenase assays, each tube received 100 µl of pyridine nucleotide (either NADP+ or NAD+, 4 mM in PBS; Sigma, Dorset, U.K.) and 100 µl of PBS containing 0.5 µCi [1,2,6,7-3H]cortisol (Amersham, Aylesbury, Bucks, U.K.) plus unlabelled cortisol (to a final steroid concentration of 100 nM; Sigma). Tubes were then returned to the waterbath for 60 min, after which reactions were terminated by the addition to each tube of 2 ml of ice-cold chloroform (Merck). To partition the organic and aqueous phases, these tubes were centrifuged at 1000 x g for 30 min at 4°C. After aspirating the aqueous supernatant, the organic extracts were evaporated to dryness under nitrogen at 60°C. The steroid residues were resuspended in 20 µl of ethyl acetate containing 1 mM cortisol and 1 mM cortisone (Sigma) and were resolved by thin-layer chromatography (TLC) using Silica 60 TLC plates (Merck) in an atmosphere of 92:8 (v/v) chloroform:95% (v/v) ethanol (Merck). After quantifying [3H]cortisol and [3H]cortisone using a Bioscan 200 TLC radiochromatogram scanner (LabLogic, Sheffield, U.K.), 11ßHSD activities were calculated as picomoles of cortisol oxidized to cortisone over 60 min and standardized per milligram of protein in the renal homogenate, in which protein concentrations were measured using the Bio-Rad (Hemel Hempstead, U.K.) protein assay [35, 36].

Fractionation of FF by C18 Column Chromatography

Each FF sample tested in the assays described above was subsequently fractionated using the method previously described by Thurston et al. [24]. Aliquots (1 ml) of independent FF samples were applied to separate C18 Sepak cartridges (Amersham) that had previously been conditioned with 20 ml of methanol and washed with 20 ml of double-distilled water (DDW). After collecting the loading eluent (i.e., that fraction of the sample not retained by the column), the column was sequentially eluted with 1-ml volumes of a stepwise gradient of methanol (Merck) in DDW. All 1-ml fractions were collected into borosilicate tubes, and those samples eluted at methanol concentrations greater than 20% (v/v) methanol were evaporated to dryness under nitrogen before being resuspended in 1-ml volumes of 20% (v/v) methanol in DDW. Parallel samples of DDW and PBS only were similarly fractionated as negative controls.

Effects of FF Fractions on Renal 11ßHSD Activities

Assays of renal NADP+-dependent and NAD+-dependent 11ß-dehydrogenase activities were performed as described above with the following modification: Samples were incubated in triplicate in the presence of 1) 100 µl of a specific FF fraction, 2) 100 µl of 20% (v/v) methanol in DDW (i.e., final methanol concentration in 1 ml = 2%), or 3) 100 µl of DDW alone. Enzyme activities in the presence of the loading eluent, 0% and 10% (v/v) methanol fractions, were compared to those measured in the controls incubated with DDW alone, whereas enzyme activities in the presence of fractions eluted at 20% (v/v) methanol or greater were compared to the 20% (v/v) methanol control.

Elution Profiles of Standard Compounds by C18 Column Chromatography

The C18 elution profiles for a number of candidate ovarian modulators of 11ßHSD activities, each known to be present in FF, were assessed as follows: Samples of bovine and porcine FF were equilibrated (for 18 h at 4°C) with 1 µCi of each candidate compound (each purchased from Amersham or from NEN-DuPont Ltd., Stevenage, Herts, U.K.). The candidate compounds were [5,6,8,11,12,14,15-3H]prostaglandin (PG) E2 (130 Ci/mmol), [5,6,8,9,11,12,14-3H]PGF2{alpha} (130 Ci/mmol), 6-keto[5,8,9,11,12,14,15-3H]PGF1{alpha} (130 Ci/mmol), [1,2,6,7-3H]cortisol (50 Ci/mmol), [1,2,6,7-3H]cortisone (50 Ci/mmol), [2,4,6,7-3H]estradiol (70 Ci/mmol), [1,2,6,7-3H]testosterone (70 Ci/mmol), [1,2,6,7-3H]progesterone (60 Ci/mmol), [7-3H]pregnenolone (60 Ci/mmol), or [7-3H]cholesterol (20 Ci/mmol). The elution profile for each compound was assessed three times for bovine FF and three times for porcine FF, using an FF sample for a different large antral follicle on each occasion. Fractions were eluted from each C18 Sepak cartridge as described above, and duplicate 100-µl aliquots of each column fraction were transferred to scintillation vials. After adding Ultima-gold scintillant (2 ml/vial; Packard BioScience Ltd., Pangbourne, Berkshire, U.K.), the radioactivity present in each eluted C18 fraction was quantified using a LS500E liquid scintillation counter (Beckman, High Wycombe, Bucks., U.K.).

Statistics

The effects of particular FF samples or fractions thereof on 11ßHSD activities were assessed by comparison of the relevant enzyme activities in the presence of a treatment and in the matched, untreated control samples using one-way ANOVA followed by Dunnett or Bonferroni multiple comparison as appropriate. Although data are presented graphically as the percentage of control enzyme activities in the absence of treatments, all statistical evaluations were performed on absolute, nonreferenced data using GraphPad Prism2 software (San Diego, USA). In all cases, P < 0.05 was accepted to indicate statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of FF on Renal 11ßHSD Activities

Samples of FF (before fractionation), from bovine and porcine large antral follicles (from each of five different animals), had no net effect on the NADP+-dependent rate of cortisol oxidation when added to homogenates of rat kidney (P > 0.05) (Table 1). However, the addition of unfractionated FF from bovine and porcine cystic follicles to homogenates of rat kidney significantly inhibited NADP+-dependent 11ß-dehydrogenase activity (P < 0.01) (Table 1).


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  		TABLE 1. Effect of FF from bovine and porcine large antral follicles and spontaneous ovarian cysts on NADP+-dependent 11ß-HSD activities in rat kidney homogenates.a

With respect to NAD+-dependent inactivation of cortisol, samples of FF from bovine and porcine large antral follicles and cystic follicles (n = 5 in each case) had no significant effect on this type 2 11ßHSD activity within 1 h (P > 0.05). In these assays, the positive-control test compound, carbenoxolone, consistently inhibited the NADP+- and NAD+-dependent oxidation of cortisol by 96.6% ± 2.6% (P < 0.01).

Resolution of 11ßHSD Modulators by C18 Column Chromatography

When samples of FF from five bovine large antral follicles were applied to C18 columns, those fractions eluted at 0% and 10% (v/v) methanol each stimulated the NADP+-dependent oxidation of cortisol by 111.5% ± 21.6% and 82.7% ± 17.4%, respectively (P < 0.01) (Fig. 1a). Whereas the fractions eluted at 20%–75% (v/v) methanol had no significant effect on 11ßHSD activities, those eluted at methanol concentrations of 80% and 85% inhibited NADP+-dependent inactivation of cortisol by 47.6% ± 7.1% and 58.2% ± 5.1%, respectively (P < 0.01) (Fig. 1a).



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  		FIG. 1. Effect of C18 fractions of follicular fluids from (a) bovine and (b) porcine large antral follicles on NADP+-dependent 11ßHSD activities in rat kidney homogenates. Each data point is the mean ± SEM for five individual FF samples. The horizontal line indicates a control enzyme activity of 100%. *P < 0.05 and **P < 0.01 versus respective control enzyme activity.

When FF samples from five porcine large antral follicles were similarly fractionated, the fractions eluted at 20% and 30% (v/v) methanol stimulated NADP+-dependent 11ßHSD activity by 48.6% ± 7.1% and 55.2% ± 5.7%, respectively (P < 0.01) (Fig. 1b). Although fractions of porcine FF eluted at 0% and 10% (v/v) as well as 40%–75% (v/v) methanol had no significant effects on 11ßHSD activities, those eluted at methanol concentrations of 80%–90% (v/v) methanol inhibited NADP+-dependent inactivation of cortisol by between 41.9% ± 4.2% and 45.7% ± 2.0% (P < 0.01) (Fig. 1b).

When each of the five bovine FF samples (Fig. 2a) and five porcine FF samples (Fig. 2b) were applied to C18 columns, none of the fractions eluted at methanol concentrations of up to and including 100% (v/v) had any significant effect on NAD+-dependent, type 2 11ßHSD activity.



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  		FIG. 2. Effect of C18 fractions of follicular fluids from (a) bovine and (b) porcine large antral follicles on NAD+-dependent 11ßHSD activities in rat kidney homogenates. Each data point is the mean ± SEM for five individual FF samples. The horizontal line indicates a control enzyme activity of 100%.

When five FF samples from bovine cysts were applied to C18 columns, the 0% (v/v) methanol fraction of each was found to stimulate the NADP+-dependent oxidation of cortisol (by 22.3% ± 1.8%, P < 0.01) (Fig. 3a). The degree of stimulation of 11ßHSD activity exerted by the hydrophilic fraction was decreased for FF from cysts as compared to large antral follicles. Fractions of bovine follicular cyst fluid eluted at methanol concentrations of 75%–95% (v/v) methanol inhibited NADP+-dependent inactivation of cortisol by between 8.1% ± 1.9% and 70.6% ± 2.4% (P < 0.01), with peak inhibitory activity eluted at 85% (v/v) methanol (Fig. 3a). Hence, the degree of inhibition of 11ßHSD activity exerted by the hydrophobic fraction was greater for bovine FF from cysts as compared to that from large antral follicles. Additional inhibitory compounds present in bovine ovarian cysts were eluted from C18 columns at methanol concentrations of 45%–55% (v/v) methanol (Fig. 3a). Fractions of bovine cyst fluid eluted at all other concentrations of methanol had no significant effects on NADP+-dependent 11ß-dehydrogenase activities.



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  		FIG. 3. Effect of C18 fractions of follicular fluids from (a) bovine and (b) porcine ovarian cysts on NADP+-dependent 11ßHSD activities in rat kidney homogenates. Each data point is the mean ± SEM for five individual FF samples. The horizontal line indicates a control enzyme activity of 100%. *P < 0.05 and **P < 0.01 versus respective control enzyme activity

When five FF from porcine cysts were applied to C18 columns, the 20%–30% (v/v) methanol fractions of each stimulated NADP+-dependent 11ßHSD activity (by 44.6% ± 6.2% and 26.4% ± 4.1%, respectively; P < 0.01) (Fig. 3b). However, this stimulation was decreased when compared to porcine FF samples from large antral follicles. All other fractions of fluid from porcine cysts eluted at concentrations of 0% and 10% as well as 40%–100% (v/v) methanol inhibited NADP+-dependent inactivation of cortisol by between 11.2% ± 1.7% and 51.8% ± 9.4% (P < 0.01), with peak inhibitory activity eluted at 75% (v/v) methanol (Fig. 3b). Both the total number of inhibitory fractions and the degree of inhibition by those fractions eluted at 75%–85% (v/v) methanol was significantly greater for the FF aspirated from porcine cysts as compared to porcine large antral follicles.

After five different bovine (Fig. 4a) and five porcine (Fig. 4b) fluids from cystic follicles were applied to C18 columns, none of the eluted fractions had any significant effect on NAD+-dependent, type 2 11ßHSD activity.



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  		FIG. 4. Effect of C18 fractions of follicular fluids from (a) bovine and (b) porcine ovarian cysts on NAD+-dependent 11ßHSD activities in rat kidney homogenates. Each data point is the mean ± SEM for five individual FF samples. The horizontal line indicates a control enzyme activity of 100%

When C18 Sepak cartridges were loaded with either DDW or PBS in place of FF, none of the eluted fractions had any significant effect on the NADP+-dependent oxidation of cortisol (data not shown).

Elution Profiles of Standard Compounds by C18 Column Chromatography

In general, the peak elution of [3H]PGs and of [3H]steroids was at methanol concentrations of 30% (v/v) and 55%–70% (v/v), respectively (Fig. 5). The peak elution of [3H]pregnenolone and of [3H]cholesterol occurred at 75% (v/v) and 100% (v/v) methanol, respectively (Fig. 5b). The elution profiles for all candidate compounds did not differ when used to spike either porcine or bovine FF. All elution profiles were in agreement with those previously published for human FF by Thurston et al. [24].



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  		FIG. 5. Elution profiles for candidate hydrophobic modulators of 11ßHSD in bovine FF eluted from a C18 Sepak cartridge with increasing concentrations of methanol. Candidate modulators used were (a) PGE2, PGF2{alpha}, and 6 keto-PGF1{alpha} (PGF1{alpha}) and (b) cortisol (F), cortisone (E), estradiol (E2), testosterone (T), progesterone (P4), pregnenolone (P5), and cholesterol (C). Each data point is the mean ± SEM for three independent profiles


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The principal finding of the present study was that FF samples aspirated from both bovine and porcine large antral follicles contain hydrophilic compounds that can acutely (i.e., within 1 h) increase NADP+-dependent cortisol oxidation by in excess of 2-fold and hydrophobic compounds that can acutely inhibit NADP+-dependent 11ß-dehydrogenase activity by up to 58% in homogenates of rat kidney. The fact that these same fractions of FF had no significant effect on the NAD+-dependent oxidation of cortisol to cortisone suggests these ovarian compounds selectively stimulate or inhibit the activities of the type 1 isoform of 11ßHSD without affecting the activity of type 2 11ßHSD. Whereas these findings are consistent with our recent studies of enzyme modulators in human FF [24], it is noteworthy that the studies reported herein were conducted on FF samples aspirated from abattoir material of animals that had undergone no endocrine therapy before slaughter. Hence, on the basis of our current findings, it seems unlikely that the presence of endogenous ovarian modulators of type 1 11ßHSD activity in human FF was an artifact of the controlled ovarian hyperstimulation protocol required for assisted conception.

Although the profile of ovarian enzyme modulators as resolved from bovine large antral follicles was remarkably similar to that for the endogenous ovarian 11ßHSD modulators resolved from human FF [24], the profile of enzyme modulators as resolved from porcine large antral follicles was quite different. In large antral follicles from both cows and pigs, the predominant ovarian inhibitor(s) of NADP+-dependent 11ßHSD activity eluted maximally from C18 cartridges at between 80% and 90% (v/v) methanol, as was the case for human FF samples. However, whereas the hydrophilic ovarian stimuli to type 1 11ßHSD activity from bovine large antral follicles eluted in the 0% and 10% (v/v) methanol fractions (consistent with the 11ßHSD stimuli in human FF), the fractions of porcine antral FF that contained the ovarian stimulators of NADP+-dependent cortisol oxidation eluted at 20% and 30% (v/v) methanol.

A number of hydrophobic molecules that are present at high concentrations in FF, such as steroids, sterols, and bile pigments, have previously been identified as potent inhibitors of glucocorticoid metabolism by the cloned isoforms of 11ßHSD [37–42]. To establish which (if any) of these ovarian compounds might be a candidate for the hydrophobic inhibitor(s) of 11ßHSD, we compared the C18 elution profiles for known enzyme modulators to those of the endogenous enzyme modulators as resolved from bovine and porcine FF. In large antral FF from both species, the ovarian inhibitors of 11ßHSD eluted maximally at methanol concentrations greater than those required to elute cortisol, cortisone, estradiol, and testosterone but lower than that required to elute cholesterol. Given that the bovine and porcine FF fractions eluted at 70% and 75% (v/v) methanol contained progesterone and pregnenolone, we were not able to exclude these C21 steroids as contributing to the hydrophobic inhibitory fraction of FF from large antral follicles solely on the basis of their elution profiles. However, we have noted that progesterone inhibits the activities of both cloned isoforms of 11ßHSD [24]. Therefore, the fact that the endogenous ovarian inhibitors of 11ßHSD significantly inhibit NADP+-dependent cortisol oxidation without affecting NAD+-dependent 11ßHSD activity suggests that progesterone and pregnenolone are not likely to be the predominant endogenous ovarian inhibitor of 11ßHSD in bovine, porcine, or human FF.

In the case of FF from bovine large antral follicles, those fractions that would be expected to contain PGs, eluted at 30% and 40% (v/v) methanol, had no significant effect on renal NADP+-dependent 11ßHSD activities. This suggests that in bovine FF samples, these fractions did not contain sufficient concentrations of PG to affect 11ßHSD activities in the in vitro assay of enzyme modulators. In contrast, in FF from porcine large antral follicles, those fractions that exerted the greatest acute stimulation of NADP+-dependent cortisol oxidation coeluted with PGE2 and the stable prostacyclin metabolite, 6-keto-PGF1{alpha}. It is relevant to note that in recent studies of placental glucocorticoid metabolism, PGs have been shown to stimulate the activity of type 1 11ßHSD [43]. Hence, PGs may contribute to the endogenous ovarian stimulus to type 1 11ßHSD activity in porcine follicles.

Turning to the spontaneous ovarian cysts, many factors, including season, nutrition, milk production, and puerperal stress, have all been associated with the occurrence of ovarian cysts in domestic animals, yet the mechanisms behind this reproductive disorder remain in question [44]. Cystic follicles are often associated with increased production of adrenal androgens [45], which are thought to result from excessive ACTH secretion prompted by a decrease in cortisol-dependent negative feedback because of increased peripheral metabolism of cortisol to cortisone by the type 2 11ßHSD enzyme [23]. In addition, studies in mice with targeted deletion of the gene encoding type 1 11ßHSD suggest that this reductive isoform of the enzyme has to act within the pituitary to regenerate cortisol from cortisone to exert appropriate negative feedback on ACTH secretion [46]. Therefore, the elevated pituitary secretion of ACTH (and LH) that is characteristic of COD states may be indicative of a decrease in type 1 11ßHSD activity rather than of an increase in type 2 11ßHSD activity. Recent studies have supported the view that cystic ovaries in hirsute women may be associated with decreased capacity of type 1 11ßHSD to reduce orally administered cortisone to cortisol [23], despite evidence that the gene encoding type 1 11ßHSD is unaltered in such patients [47].

In the present study, we report that the ovarian modulators of NADP+-dependent, type 1 11ßHSD activity resolved from FF aspirated from bovine and porcine large antral follicles are also present in bovine and porcine ovarian cysts. In both species, the levels of the ovarian stimuli to type 1 11ßHSD were significantly decreased in FF from spontaneous ovarian cysts as compared to large antral follicles. At the same time, the levels of the endogenous hydrophobic inhibitors of type 1 11ßHSD activity were increased relative to the FF fractions of large antral follicles, both in terms of absolute degree of inhibition of NADP+-dependent cortisol oxidation over 1 h and in terms of the range of methanol concentrations over which hydrophobic inhibitors of type 1 11ßHSD activity could be eluted. We propose that the decreased content of the endogenous ovarian 11ßHSD stimuli and the increased content of ovarian 11ßHSD inhibitors in ovarian cysts relative to large antral follicles explains why, before fractionation, FF from bovine and porcine ovarian cysts significantly inhibited NADP+-dependent cortisol oxidation, whereas FF from large antral follicles exerted no net effect (despite containing both hydrophilic enzyme stimuli and hydrophobic enzyme inhibitors).

We are not yet in a position to comment on whether these compounds originate from within or outside the ovary or in which tissues they might act. However, we would speculate that the modulators of type 1 11ßHSD activity might act within the follicle to modulate the interconversion of active cortisol and inert cortisone. In support of this proposal, a direct correlation between intrafollicular cortisol:cortisone ratios and levels of hydrophobic ovarian inhibitors of type 1 11ßHSD activity have recently been identified in the human [48]. Furthermore, ovarian inhibitors of NADP+-dependent cortisol oxidation resolved from FF might be secreted from the ovary to inhibit type 1 11ßHSD in the liver and, possibly, even in the pituitary gland, leading to increased net clearance of cortisol, decreased regeneration of cortisol from cortisone, and increased ACTH drive to the adrenal gland in CODs. Such a hypothesis could reconcile the strong evidence for decreased reduction of cortisone to cortisol in patients with PCOS, despite no obvious mutations in the gene encoding the hepatic, type 1 isoform of 11ßHSD. However, this hypothesis remains to be tested.


   ACKNOWLEDGMENTS
 
We wish to thank Mr. Andy Hartley at the Institute of Zoology (Zoological Society of London, London, U.K.) for obtaining the bovine and porcine ovaries used in the present study.


   FOOTNOTES
 
1 Supported by a grant from Freemedic plc (London, U.K.), BBSRC project grant 48/S15850, and MRC Studentship G69/1756. Back

2 Correspondence: Lisa Thurston, Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, Camden Town, London NW1 0TU, United Kingdom. FAX: 44 0 20 7388 1027; lthurston{at}rvc.ac.uk Back

Received: 9 October 2002.

First decision: 8 November 2002.

Accepted: 9 January 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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N. Sunak, D. F Green, L. R Abeydeera, L. M Thurston, and A. E Michael
Implication of cortisol and 11{beta}-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts
Reproduction, June 1, 2007; 133(6): 1149 - 1158.
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