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Endocrinology Vol. 142, No. 2 687-693
Copyright © 2001 by The Endocrine Society

ARTICLES

Endocrine-Regulated and Protein Kinase C-Dependent Generation of Superoxide by Rat Preovulatory Follicles1

Pinar H. Kodaman and Harold R. Behrman

Reproductive Biology Section, Departments of Obstetrics/Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Dr. Pinar H. Kodaman, Reproductive Biology Section, Department of Obstetrics and Gynecology, Yale University School of Medicine, P.O. Box 208063, New Haven, Connecticut 06520-8063. E-mail: kodamaph{at}biomed.med.yale.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The ovulatory LH surge results in follicular inflammation with an increase in cytokines and PGs. Reactive oxygen species (ROS) are also produced during inflammatory processes. To study ROS generation during the ovulatory cascade, preovulatory follicles were dissected from immature female rats primed with PMSG. Follicles were isolated, and ROS generation was assessed by luminol-amplified chemiluminescence. Immature rat granulosa cells were also subjected to luminometry after isolation from immature rats treated with diethylstilbestrol. Phorbol ester-stimulated ROS generation by follicular cells was completely suppressed by superoxide dismutase and the NADPH/NADH oxidase inhibitor diphenylene iodonium bisulfate, whereas catalase was without effect. Fractionation of granulosa cells with an antibody against leukocyte common antigen-1 showed that leukocyte-enriched cells produced more than 95% of the superoxide measured. In vivo treatment with LH produced a 5-fold increase in phorbol-stimulated superoxide production by isolated follicles. This response was maximal within 4 h and was blocked by indomethacin. In vivo administration of PGE2 and PGF2{alpha} did not reverse the blockade by indomethacin; however, isolated follicles incubated with PGE2 produced a time-dependent increase in phorbol-stimulated superoxide generation. Thus, a superoxide generator is present in the preovulatory follicle that is leukocytic in origin, hormone regulated, and activated by a protein kinase C-dependent pathway. The regulated generation of superoxide by preovulatory follicles may indicate a role for ROS in the periovulatory period.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVULATORY SURGE of LH results in follicular hyperemia, edema, vasodilatation, and extravasation of leukocytes, all of which are characteristic of an inflammatory response (1). Various inflammatory mediators play a role in ovulation, including cytokines (2, 3, 4), peptides (5), PGs (6), and other phospholipid-derived compounds, such as platelet-activating factor (7, 8, 9). The importance of PGs for ovulation was first demonstrated by blockade of ovulation with cyclooxygenase inhibitors (10, 11), and similar studies have subsequently shown that antihistamines (12), antagonists of platelet-activating factor (7, 8, 9) and lipoxygenase (13), and antibodies directed against interleukin-8 (IL-8) (4) are also able to block or at least suppress the ovulatory rate.

At the time of ovulation, follicular macrophages increase by 5-fold, whereas neutrophils increase by 8-fold (14), and this may be influenced by the local production of various chemotactic cytokines, including IL-1, tumor necrosis factor-{alpha}, IL-8, monocyte chemotactic protein-1, and granulocyte monocyte colony stimulating factor (2, 15, 16). The significance of ovarian leukocytes is underscored by the finding that infusion of leukocytes into the perfused rat ovary produced an increased ovulatory rate compared with infusion of LH alone (17), and subsequent studies by Brannstrom et al. (18) demonstrated that IL-1 and tumor necrosis factor-{alpha} also increased the ovulation rate in this model. Therefore, in addition to their traditional roles in scavenging cell debris, antigen processing, and promoting repair, leukocytes appear to enhance the physiological processes of follicle growth and ovulation (19).

One important consequence of leukocyte infiltration is the generation of reactive oxygen species (ROS) that are associated with acute inflammation. There is indirect evidence that ROS play a role in ovulation; for example, the expression of superoxide dismutase isozymes (Mn-SOD and Cu/Zn-SOD) varies during the periovulatory period (20, 21), and Mn-SOD activity decreases during this time (21). Furthermore, inactivation of superoxide (SO) by long-acting SOD administration blocks ovulation (21, 22). Yet, there is no information regarding the generation of SO during the periovulatory period and its source. Therefore, the objectives of the present studies were to examine the nature and regulation of ROS production by the preovulatory follicle in response to LH and other mediators of ovulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Highly purified human FSH (AFP 4822-B; 3100 U/mg) was obtained from the NIAMDD, Pituitary Hormone Distribution Program (Bethesda, MD). Penicillin and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY). Luminol was obtained from Aldrich (Milwaukee, WI). Diphenylene iodonium bisulfate was a gift from A. R. Cross (The Scripps Research Institute, La Jolla, CA). PGE2, PGF2{alpha}, and all other chemical reagents (analytical grade or better) were purchased from Sigma (St. Louis, MO).

Animals
Immature female rats (21–23 days old; Sprague Dawley strain, Taconic Farms, Inc., Germantown, NY) were housed and cared for in the fully accredited facilities operated by the Animal Resource Center (Yale University School of Medicine, New Haven, CT). All treatments and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by the Yale University animal care committee.

Before death, animals were anesthetized with a solution of ketamine hydrochloride (Quad Pharmaceuticals, Indianapolis, IN), xylazine hydrochloride (Rompun, Miles, Inc., West Haven, CT), and 0.9% NaCl (0.5:0.11:1.39, vol/vol/vol; 0.25 ml/animal), which was injected ip. Animals were perfused with saline at the time of death to remove circulating blood cells. In brief, thoracotomy was performed, and a blunt 18-gauge needle was inserted into the right atrium, allowing saline to flow by gravity into the cardiovascular system. Subsequently, the ventricle was incised to permit exsanguination, and perfusion was continued until clear saline flowed from the ventriculotomy site. The adequacy of perfusion was confirmed by the observation of ovarian and liver blanching.

Isolation and incubation of preovulatory follicles
Follicular development was induced in immature female rats on days 25–28 of life by sc injection of 10 IU PMSG (Gestyl; Organon Pharmaceuticals, West Orange, NJ). Animals were killed 48 h after injection of PMSG, and ovaries were removed and placed in Earle’s MEM (MEM 2360, Life Technologies, Inc.) supplemented with BSA (1 mg/ml), glutamine (0.29 mg/ml), FSH (50 ng/ml), insulin-like growth factor I (30 ng/ml), penicillin (100 U/ml), and streptomycin (100 µg/ml). After removing the bursa from the ovaries, the largest follicles were dissected from each ovary under a stereomicroscope.

Isolated follicles were placed in sterile 25-ml Erlenmeyer flasks (six follicles per flask) containing 2.5 ml medium and the various treatments described in the text. Follicles were incubated in a shaking water bath at 37 C under an atmosphere of 95% oxygen-5% CO2 for the indicated time periods. Follicles were subsequently digested with collagenase (0.53 mg/ml) and deoxyribonuclease (0.05 mg/ml) for 1 h to permit luminol penetration during subsequent luminol-amplified chemiluminescence.

In some experiments animals were treated in vivo with agents described in the text before death and follicle isolation. These rats were simultaneously treated with phenobarbital (4.5 mg/animal, sc) to prevent the endogenous LH surge. All agents administered in vivo were dissolved in saline, except for indomethacin and PGs, which were dissolved in 5% sodium bicarbonate and sodium carbonate (0.04 mg/ml)/10% ethanol, respectively.

Isolation and incubation of granulosa cells
Granulosa cell proliferation was induced by pretreating immature rats for 4 days with diethylstilbestrol (DES) delivered by 1-cm SILASTIC brand capsules (Dow Corning Corp., Midland, MI) implanted under the skin of the upper back (23) after anesthesia with methoxyflurane (Malinckrodt Veterinary, Mundelein, IL). After the rats were killed, ovaries were removed and placed in ice-cold DMEM-Ham’s F-12 culture medium (Life Technologies, Inc.) supplemented with 0.1% BSA, 100 U/ml penicillin, and 100 µg/ml streptomycin. Ovaries were trimmed of their bursae and surrounding fat tissue, and follicles were punctured with a 27-gauge needle to release granulosa cells, which were harvested as previously described (24, 25). Cells were cultured in 12 x 75-mm polystyrene tubes (3 x 106 cells/ml/tube) in a shaking water bath under 95% air-5% CO2 in the presence and absence of various treatments for the indicated time periods before luminometry.

In the present studies, granulosa cells were used in addition to preovulatory follicles, because they were easier to obtain in great numbers, could be subjected to luminometry immediately without digestion, and were relatively pure, especially after cell sorting to remove leukocytes as described below. In addition to the practical reasons mentioned above, granulosa cells were examined to determine whether they, as follicular components, were responsible for any of the ROS production measured.

Fractionation of granulosa cell preparations
Granulosa cells were depleted of leukocytes using the MiniMACS magnetic cell sorting system (Miltenyi Biotec, Auburn, CA) and a polyclonal antibody directed against leukocyte common antigen-1 [LCA-1 (CD45); Chemicon, Temecula, CA]. In brief, cells (107) were washed at 4 C with MAC buffer [Dulbecco’s PBS (DPBS) without Ca2+ and Mg2+ supplemented with 0.5% BSA and 0.1 mM EDTA] and then resuspended in 80 µl of this buffer. LCA-1 antibody (20 µl; 1:300 final dilution) was added, and the cells were incubated for 30 min on ice. Subsequently, the cells were washed, resuspended in 80 µl buffer, and secondary antibody beads (20 µl) were added and incubated for 15 min at 4 C. The resulting suspension was loaded onto a MiniMACS column, and the leukocyte-depleted and leukocyte-enriched fractions were collected by eluting the column in the presence and then the absence of a magnetic field, respectively (26). The resulting cell fractions as well as identically treated but unfractionated controls were subjected to luminol-amplified chemiluminescence as described below.

Luminol-amplified chemiluminescence assay
ROS generation was measured by luminol-amplified chemiluminescence (27, 28). Cells were collected by centrifugation and washed once with phenol red-free DPBS containing sodium pyruvate (36 mg/liter), D-glucose (1 g/liter), Ca2+, and Mg2+ (Life Technologies, Inc.), which was further supplemented with BSA (1 mg/ml). The washed cells were suspended in 0.5 ml DPBS, and luminol was added to give a final concentration of 4.5 µM (27).

Luminescence was measured using a luminometer with a detection chamber maintained at 37 C (Turner Designs, Sunnyvale, CA). Readings were recorded at 1-min intervals with 5-sec delays. After 10 min of baseline recordings, the phorbol ester phorbol 12-myristate 13-acetate (TPA; 1 µM final concentration) was added to stimulate protein kinase C (PKC)-dependent luminescence, which was recorded for an additional 10 min. ROS production was previously standardized by measuring SO production with xanthine oxidase and xanthine (29), such that 1 relative light unit represented 106 pmol SO. SO generation was calculated by subtracting the summation of the baseline values from that of the TPA-stimulated values for each sample. With the exception of Table 1Go and Fig. 1Go, which show both basal and TPA-stimulated data, all data presented represent phorbol ester-stimulated SO production.


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  		Table 1. Superoxide production by preovulatory follicles and immature granulosa cells

 


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  		Figure 1. Phorbol ester stimulation of ROS generation by granulosa cells and inhibition by SOD. Freshly isolated cells (3 x 106) were incubated for 1 h and washed with DPBS buffer before addition of luminol (4.5 µM) in the presence and absence of catalase (0.127 mg/ml) and SOD (0.5 mg/ml) for measurement of luminol-amplified chemiluminescence. After baseline readings of basal ROS generation for 10 min, phorbol ester (TPA; 1 µM) was added, and ROS generation was recorded for an additional 10 min. Data are expressed as relative light units (RLU) per 3 million cells generated over a 20-min interval, with open symbols representing basal values, and closed symbols representing TPA-stimulated values. The graph illustrates a representative, replicated experiment.

 
Statistical analysis
Granulosa cells from several rats were pooled, and equal aliquots were exposed to each treatment in at least duplicate. Each experiment was repeated at least three times, and data are presented as the mean ± SEM. Experiments using follicles were also performed in duplicate and repeated at least three times. Statistical significance between treatments within an experiment was determined by t test or one-way ANOVA. The Bonferroni test was used for post-hoc comparisons of multiple treatments. Treatment differences between experiments were determined by one-way ANOVA, and post-hoc comparisons were made with Dunnett’s method. In all analyses, P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SO generation by preovulatory follicles and granulosa cells
Both preovulatory follicles and freshly isolated granulosa cells produced low basal levels of ROS with values rarely exceeding 1 pmol/6 follicles·min and 0.3 pmol/106 cells·min, respectively (Table 1Go). Addition of the phorbol ester TPA (1 µM) to follicles and granulosa cells resulted in a 20- and 9-fold increases in ROS production, respectively, which were consistent and reached a maximum within 3–4 min (Table 1Go). Basal and TPA-stimulated luminescence were measured in all subsequent experiments and used to calculate TPA-stimulated ROS production as described in Materials and Methods.

The ROS generated by granulosa cells was SO, as TPA-stimulated luminescence (4.35 ± 0.86 pmol SO/3 x 106 cells·min) was reduced to basal levels (0.81 ± 0.22 pmol SO/3 x 106 cells·min) or less after the addition of SOD (0.5 mg/ml; Fig. 1Go) or the NADPH/NADH oxidase inhibitor diphenylene iodonium bisulfate (DPI; 2.5 µM; data not shown), respectively, to the assay medium. SOD had a similar inhibitory effect on TPA-stimulated SO production by preovulatory follicles (data not shown). Catalase (0.127 mg/ml) was without effect on superoxide generation, ruling out hydrogen peroxide as an additional or alternative source of luminescence in both granulosa cells (4.80 ± 0.27 pmol SO/3 x 106 cells/min; Fig. 1Go) and preovulatory follicles (data not shown).

SO generation by ovarian leukocytes
Fractionation of freshly isolated granulosa cells with an antibody against LCA-1 (CD45) resulted in a mean total recovery of 78 ± 3% of the 107 cells used in each experiment. The leukocyte-depleted fraction represented 95.0 ± 0.7% of the recovered cells, whereas the leukocyte-enriched fraction represented 5.0 ± 0.5% of the cells recovered. Leukocyte-enriched cells produced the preponderance of SO (25 ± 9 pmol SO/106 cells·min) compared with leukocyte-depleted cells and unfractionated granulosa cells (0.7 ± 0.4 and 1.7 ± 0.6 pmol SO/106 cells·min, respectively; Fig. 2Go). Repeated fractionation of the same granulosa cell preparation using the same antibody resulted in a further decrease in SO generation by the leukocyte-depleted fraction and enhanced SO generation by the fraction selected for leukocytes (data not shown). Exogenous NADPH (2 mM) was without effect on SO production by the leukocyte-enriched fractions, whereas DPI (2.5 µM) significantly reduced SO generation by these cells compared with the control (1.4 ± 0.6 vs. 10.4 ± 2.7 pmol SO/106 cells·min, respectively).



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  		Figure 2. SO production by unfractionated, leukocyte-depleted, and leukocyte-enriched granulosa cells. Granulosa cells isolated from DES-primed immature female rats were fractionated using an antibody directed against LCA-1 and a magnetic cell sorter as described in Materials and Methods. After sorting, cells were counted and subjected to luminol-amplified chemiluminescence as described for Fig. 1Go. SO generation was calculated by subtracting the summation of the baseline values from that of the TPA-stimulated values for each sample, and results are expressed as picomoles of SO per min/106 cells. Data are the mean ± SEM of at least three independent fractionations.

 
Stimulation of follicular SO production by gonadotropin
Follicles isolated from animals treated with LH in vivo (25 µg sc/animal 48 h after PMSG treatment) showed a 5-fold increase in phorbol ester-stimulated SO generation compared with follicles from saline-treated controls (19.8 ± 3.8 vs. 3.4 ± 0.4 pmol SO/6 follicles·min, respectively). This response was maximal within 4 h of LH administration and sustained at 6 h (Fig. 3Go). Basal values were not significantly different between LH-treated and control follicles (data not shown). Endogenous LH surge suppression with 4 h of phenobarbital treatment (4.5 mg/rat) did not affect subsequent TPA-stimulated SO generation by control and LH-treated animals, which produced SO levels (4.5 ± 0.8 and 18.6 ± 2.6 pmol SO/6 follicles·min, respectively) similar to those in the absence of phenobarbital treatment (3.4 ± 0.4 and 19.8 ± 3.8 pmol SO/6 follicles·min, respectively). There was no time-dependent increase in TPA-stimulated SO production in the absence of LH in vivo, as the 0 and 4 h controls were not significantly different (3.4 ± 0.4 and 4.5 ± 0.8 pmol SO/6 follicles·min, respectively.



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  		Figure 3. Stimulation of SO production by preovulatory follicles after in vivo LH treatment. PMSG-primed immature female rats were injected with LH (25 µg/animal) and killed 0–6 h later. Preovulatory follicles were isolated and subjected to luminol-amplified chemiluminescence as described in Materials and Methods. SO generation was calculated by subtracting the summation of the baseline values from that of the TPA-stimulated values for each sample, and results are expressed as picomoles of SO per min/6 follicles. Basal data did not differ significantly between treatment groups and ranged from 0.09–2 pmol SO/min·6 follicles. Each data point represents the mean ± SEM of three independent experiments performed in duplicate.

 
In vitro incubation of isolated preovulatory follicles with hCG (20 IU/ml) for up to 6 h did not produce a statistically significant increase in TPA-stimulated SO generation. At 6 h, hCG-treated and control follicles produced 16.7 ± 1.5 and 14.2 ± 1.5 pmol SO/6 follicles·min, respectively. However, in vitro incubation alone, in the absence of hCG, resulted in a time-dependent increase in phorbol ester-stimulated SO generation by preovulatory follicles that was significant at 6 h compared with the 0 h control value (14.2 ± 1.5 and 7.2 ± 1.2 pmol SO/6 follicles·min, respectively).

Indomethacin blocks LH-induced superoxide generation
In vivo treatment of rats with the cyclooxygenase inhibitor indomethacin (20 mg/kg; sc) for 1 h before LH administration significantly blocked subsequent TPA-stimulated follicular superoxide generation compared with that in controls treated with LH alone (10.6 ± 2.0 vs. 18.6 ± 2.9 pmol SO/6 follicles·min, respectively; Fig. 4Go). In vivo indomethacin treatment in the absence of LH was without a significant effect on follicular SO production compared with the control (2.2 ± 0.8 vs. 3.7 ± 0.8 pmol SO/6 follicles·min, respectively; Fig. 4Go). In vitro treatment of follicles with indomethacin (10 µM) for 6 h was without effect on TPA-stimulated SO production in both the presence and absence of 10 IU/ml hCG (14.2 ± 2.1 and 12.6 ± 2.1 pmol SO/6 follicles·min, respectively, vs. control (14.2 ± 1.5 pmol SO/6 follicles·min)).



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  		Figure 4. Inhibition of LH-stimulated SO generation by indomethacin. PMSG-primed, phenobarbital (4.5 mg)-treated rats were injected with indomethacin (I; 20 mg/kg) followed by LH (25 µg) 1 h later in the absence (LH+I) or presence of a PGE2 and PGF2{alpha} mixture (0.25 mg each) administered simultaneously with LH (LH+I+PG). Four hours post-LH treatment, rats were killed, and preovulatory follicles were isolated and subjected to luminometry as described in Materials and Methods. Superoxide generation was calculated by subtracting the summation of the baseline values from that of the TPA-stimulated values for each sample, and results are expressed as picomoles of SO per min/6 follicles. Basal data did not differ significantly between treatment groups and ranged from 0–1 pmol SO/min/6 follicles. Data are expressed as a percentage of the LH plus indomethacin (LH+I)-treated animals, and each bar represents the mean ± SEM of at least three experiments performed in duplicate. TPA-stimulated SO generation by follicles from LH+I-treated animals was 22 ± 3 pmol SO/min·6 follicles, which represents multiple controlled experiments, as not all treatments were carried out in a single experiment.

 
Figure 4Go also shows that the blockade of LH-induced SO production by indomethacin treatment was not reversible by in vivo coadministration of a PGE2 and PGF2{alpha} mixture (0.25 mg each/rat, sc); in fact, PGE2/PGF2{alpha} cotreatment further decreased subsequent TPA-stimulated follicular superoxide production to 66 ± 9% of that in the indomethacin- plus LH-treated animals (Fig. 4Go). In vivo PGE2/PGF2{alpha} (0.25 mg each) alone did not affect SO generation compared with the control (5.3 ± 0.8 vs. 4.5 ± 0.8 pmol/6 follicles·min, respectively; Fig. 4Go).

PGs enhance SO generation by isolated follicles
Isolated preovulatory follicles incubated with PGE2/PGF2{alpha} (1 µM each) produced a time-dependent increase in TPA-stimulated SO generation (Fig. 5Go). PGE2 (1 µM) alone had an identical effect, whereas PGF2{alpha} (1 µM) alone was without significant effect; therefore, results from both PGE2 and PGE2/PGF2{alpha}-treated follicles were combined. The increased capacity for SO generation produced by PG treatment was significantly up-regulated by 4 h compared with the 4 h control value (15.6 ± 1.0 and 11.0 ± 0.9 pmol/6 follicles·min, respectively). There was no significant effect of PGE2 on superoxide generation by freshly isolated granulosa cells or on those enriched or depleted for leukocytes (data not shown).



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  		Figure 5. Time-dependent stimulation of follicular SO generation by in vitro PG treatment. Preovulatory follicles dissected from PMSG-primed rats were incubated in the presence and absence of a mixture of PGE2 and PGF2{alpha} (1 µM each) for 2–6 h and then subjected to luminol-amplified chemiluminescence as described in Materials and Methods. SO generation was calculated by subtracting the summation of the baseline values from that of the TPA-stimulated values for each sample, and results are expressed as picomoles of SO per min/6 follicles. Basal data did not differ significantly between treatment groups and ranged from 0.2–0.9 pmol SO/min·6 follicles. Each bar represents the mean ± SEM of at least three experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies demonstrated that rat preovulatory follicles produce SO by a PKC-mediated pathway. Granulosa cells isolated from follicles of DES-primed ovaries also produced SO via activation of PKC; however, cell-sorting studies showed that leukocytes within these granulosa cell preparations were responsible for most, if not all, of the SO generated. A NADPH/NADH oxidase appears to be the source of SO generation, as inhibition of this enzyme complex by DPI decreased ROS production to undetectable levels in granulosa cells. Incubation of follicular cells with SOD also blocked TPA stimulation of SO production, whereas catalase was without effect, indicating that although SO is a direct progenitor of hydrogen peroxide, the latter was not generated in measurable amounts.

The overall level of phorbol ester-stimulated ROS generated by granulosa cells was about 15% of that produced by preovulatory follicles. This finding could reflect the paucity of granulosa cells and, hence, leukocytes assayed or, perhaps, the relative immaturity of the cells studied, as these granulosa cells were isolated from DES-stimulated ovaries and thus were less differentiated than cells obtained from preovulatory follicles. The stage of granulosa cell differentiation determines various parameters, including the response of granulosa cells to gonadotropins (30). Similarly, there were probably differences in leukocyte subsets between the granulosa cells obtained from DES-primed and PMSG-primed ovaries, as the number and type of leukocytes are known to change with follicular maturation (14).

Resident phagocytic leukocytes, probably macrophages, are potential candidates for the SO-producing cell type in the follicle, as most circulating leukocytes were probably removed by saline perfusion at the time of sacrifice. Leukocytes increase in number around the periovulatory period (14), and they are potent generators of SO (31). This was corroborated by the present studies, which demonstrated that as few as 5% of the total number of cells assayed, representing the leukocyte-enriched population, produced essentially all the SO measured. Similarly, a previous study of ROS generation by peritoneal leukocytes and luteal cells showed that 2% of the former were capable of producing the level of ROS observed with the latter (29). Nevertheless, the present studies do not rule out follicular thecal, endothelial, or fibroblastic cells as additional or alternative sources of ROS production. NADPH/NADH-dependent oxidases have been characterized in numerous cell types, including vascular endothelial (32) and smooth muscle cells (33), fibroblasts (34), and adipocytes (35) in addition to phagocytic leukocytes (31).

The ovulatory surge of LH results in leukocyte recruitment to the preovulatory follicle (14), and in the present studies LH also produced an increased capacity for TPA-stimulated follicular SO generation. Increased production of this free radical by the preovulatory follicle may contribute to the depletion of follicular ascorbate observed after LH treatment (36). Taken together with the previously reported findings that administration of long-acting SOD blocks ovulation (21, 22) and that antioxidants inhibit, whereas oxidants promote, oocyte maturation (37, 38), LH-induced SO generation by the preovulatory follicle may serve to promote both ovulation and oocyte maturation, respectively.

The potential physiological significance of LH-induced SO production by the preovulatory follicle is apparent; however, the significance of SO generation by immature granulosa cells is unknown. Furthermore, although there was a clear stimulation of SO generation by leukocytes in the granulosa cell preparations, these data are confounded by immunohistochemical evidence that follicular leukocytes are localized to the thecal layer (14). It is possible that the granulosa cell preparations in the present studies were contaminated by leukocytes from the thecal layer during isolation. As mentioned above, relatively few leukocytes would have been sufficient to generate the amount of SO measured.

Although recent studies have implicated the PKC pathway in the actions of LH associated with ovulation (39, 40), basal levels of SO production were not affected by LH, indicating that this gonadotropin does not directly activate the follicular SO generator in vitro. At present, there is no known physiological activator of the SO generator in our model system. This is consistent with previous studies in luteal cells where only phorbol ester was able to activate the ROS generator in vitro (29). The consistent stimulation of SO production by TPA was expected, as phorbol esters are the most effective, although nonphysiological, activators of NADPH/NADH oxidases (31, 41). In vivo, LH may act indirectly as a trigger that sets into motion a cascade of secondary mediators and events, resulting in follicular SO generation. Alternatively, an intact follicle may be required for physiological stimulation of the SO generator or, as previously suggested (29), there may be an endogenous, intraovarian inhibitor of NADPH/NADH that prevents activation of SO generation in vitro, although this endogenous inhibitor(s) remains uncharacterized.

In vitro treatment of follicles for up to 6 h with hCG did not produce a significant increase in TPA-stimulated SO generation, suggesting the importance of leukocyte infiltration for enhanced follicular SO generation. On the other hand, the in vitro studies may have been limited by confounding ROS generation from prolonged incubation, as suggested by the 2-fold stimulation of SO production by incubation alone, which did not occur with the in vivo control follicles. Ischemia is a known stimulator of ROS generation, and oxidative stress leads to apoptosis in cultured follicles within a few hours of isolation (42, 43).

Intrafollicular levels of PGs, such as PGE2 and PGF2{alpha}, rise in response to the LH surge (6), and these eicosanoids mediate various LH actions (44). LH induction of cyclooxygenase-2 expression by granulosa cells peaks at 4 h (45), and this enzyme catalyzes the first rate-limiting step in prostanoid synthesis (46). The LH-induced increase in follicular SO generation also peaked at 4 h, and in vivo administration of the cyclooxygenase inhibitor indomethacin significantly blocked this increase in SO generation, suggesting a potential role for PGs as mediators of LH-induced follicular SO production.

Although administration of PGE2/PGF2{alpha} in vivo did not reverse the indomethacin blockade, preovulatory follicles incubated with PGE2 in vitro demonstrated an increased capacity for TPA-stimulated SO generation that was time-dependent and significant within 4 h. That in vitro PGE2 alone, and not PGF2{alpha}, induced SO production is consistent with the finding that ovarian leukocytes do not have PGF2{alpha} receptors (Kolodecik, T. R., and H. R. Behrman, unpublished data). PGE2 did not affect SO generation by leukocytes separated from freshly isolated granulosa cell preparations, which may have been due to the fact that these cells were obtained from immature follicles that consisted of different leukocyte subtypes than those found in preovulatory follicles. Furthermore, intercellular communication between ovarian cells and leukocytes within an intact follicle may be required for PGE2 to induce an enhanced capacity for TPA-activated SO generation.

The lack of reversal of the indomethacin blockade by in vivo PG treatment may have been secondary to the pharmacokinetics of PGs. These eicosanoids are very rapidly and efficiently inactivated in vivo by widely distributed degradation enzymes (47). Alternatively, the opposite effects observed with in vivo and in vitro PG treatment on follicular SO generation may have been secondary to the state of activation of the leukocytes involved. Pleiotropic effects of PGs have been reported previously; for instance, PGE2 stimulates tumoricidal activity by resident or elicited macrophages, but inhibits that of activated macrophages through a negative feedback system involving the up-regulation of cAMP (48). In the present studies, exogenous PG treatment may have down-regulated SO production by recruited leukocytes in LH-treated follicles, but stimulated SO production by resident leukocytes in isolated follicles incubated in vitro where gonadotropin-stimulation of the SO generator did not occur.

The possibility also exists that indomethacin blockade of LH-stimulated SO production involved a mechanism(s) distinct from the inhibition of PG synthesis. Murdoch and McCormick (49) demonstrated in sheep that high, but not low, concentrations of indomethacin blocked ovulation by inhibiting collagenolysis and secretion of a leukocyte chemotactic agent, whereas both concentrations of the drug completely blocked PG synthesis. Thus, in the present studies, indomethacin may have decreased LH-induced follicular SO generation by blockade of leukocyte recruitment to the follicle, perhaps through the inhibition of a chemotactic agent.

In conclusion, there is a PKC-activated, NADPH/NADH oxidase-type SO generator in the preovulatory follicle that is leukocytic in origin and stimulated by gonadotropin. The significance of endocrine-regulated superoxide generation by the preovulatory follicle may lie in a potential role for this ROS during the ovulatory cascade.


   Footnotes
 
1 This work was supported by NIH Grant HD-35663. Back

Received July 12, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Espey LL 1980 Ovulation as an inflammatory reaction: a hypothesis. Biol Reprod 22:73–106[CrossRef][Medline]
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