Xanthine oxidase and dehydrogenase in rat ovarian tissues

activities

YAEL MARGOLIN AND HAROLD R. BEHRMAN Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 Margolin, Yael, and Harold R. Behrman. Xanthine oxidase and dehydrogenase activities in rat ovarian tissues. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E173-E178, 1992.The production of reactive oxygen species in the ovary is rapidly inducible, but the nature of the generator is unknown. One possibility is xanthine oxidase (X0), an enzyme that produces superoxide in the presence of hypoxanthine (or xanthine) and oxygen. The objective of the present studies was to measure levels of X0 in follicular and luteal tissue to determine whether X0 may be a source of reactive oxygen species in the rat ovary. Ovarian levels of X0 were about one-fifth of that seen in the liver and adrenal, and X0 levels were about one-third of xanthine dehydrogenase (XDH). Preovulatory ovarian levels of X0 activity were unchanged after induction of ovulation with gonadotropin and in follicles incubated with gonadotropin. Luteal X0 activity was not changed during natural or prostaglandin Fnn (PGF,,) -induced luteolysis. Allopurinol, an inhibitor of X0, did not inhibit ovulation or PGFa,-induced luteal regression. Finally, neither catalase and superoxide dismutase nor oxypurinol altered luteal cell function in the presence of hypoxanthine. Thus, while X0 is present in the ovary, it does not appear that it is a major source of reactive oxygen species in this organ. oxygen radicals;

corpus luteum;

ovulation

XANTHINE OXIDASE (X0) of endothelial origin is thought to mediate injury that is associated with ischemia in some tissues (14, 17, 24). Under normal circumstances, xanthine dehydrogenase (XDH) is the predominant enzyme that metaboli .zes hypoxanthine and xanthine, but ischemia generates x0 by proteolytic cleavage of XDH (3). Sulfhydryl oxidation of XDH also leads to formation of X0 in various rat tissues, which is reversible with dithiothreitol (DTT) (25). Although XDH utilizes NAD+ as an electron acceptor, X0 uses molecular oxygen and consequently generates the superoxide anion (19). Reactive oxygen species are toxic to cells and can also lead to chemotaxis of neutrophils, which further exacerbates the conversion of XDH to X0 in endothelial cells (32). Reactive oxygen species, including the superoxi .de anion and hydrogen peroxide 9 are generated in the ovary (20, 33-35), but their origin is unknown. Although X0 has not been examined in the ovary, the conditions that could lead to X0 formation are extant in this tissue. For example, both follicles and corpora lutea are subject to ischemic episodes, leukocytic infiltration is evident at ovulation and leuteolysis (2, 8, 9, 13, 18, 28, 30), and X0 in the presence of hypoxanthine evokes marked antigonadotropic and antisteroidogenic actions in ovarian cells (12). Proteolytic enzymes, which participate in rupture of the follicular wall at ovulation (lo), may convert XDH into X0, and cytokine-leukocyte interactions release proteolytic enzymes that also generate X0 from XDH (23, 32). Moreover, production of purine substrate is 0193-1849/92

$2.00 Copyright

evident during ovulation and luteolysis. Depletion of ATP leads to an increase in extracellular levels of hypoxanthine and xanthine (l), which occurs in granulosa cells and luteal tissue in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) (29, 36). Interestingly, allopurinol, an inhibitor of X0, was found to produce a modest inhibition of ovulation in the rabbit after induction of ovulation with human chorionic gonadotropin (hCG) (26). The objectives of the present study were therefore to evaluate the possibility that X0 may serve as a source of reactive oxygen species in the ovary. Two approaches were used. One was to directly measure the activities of both XDH and X0 in rat follicular and luteal tissue during ovulation and luteolysis. The second approach was to examine the effects of allopurinol and oxypurinol, inhibitors of X0 and XDH, on ovarian function. Although X0 was found in follicular and luteal tissue at levels that could generate significant levels of reactive oxygen species, the levels of activity of X0, relative to XDH, were not changed during ovulation or luteolysis. In addition, inhibitors of X0 did not influence ovulation or luteolysis, even though enzyme activity was substantially reduced. MATERIALS

AND

METHODS

Hormones, drugs, and reagents. Ovine LH (National Institute of Diabetes and Digestive and Kidney Diseases; oLH24) was a gift from the National Institutes of Health (Bethesda, MD). Prostaglandin FZLY[ PGF,,; tris(hydroxymethyl)aminomethane (Tris) salt], pterin, isoxanthopterin, methylene blue, allopurinol, DL-DTT, phenylmethylsulfonyl fluoride (PMSF), aprotinin, and X0 were purchased from Sigma Chemical (St. Louis, MO). Prepacked Sephadex G-25 columns (PD IO) were purchased from Pharmacia LKB Biotechnology (Piscataway, NJ). Animals. Two animal models were used, both of which were based on the immature (26-28 days old) female rat (CD strain; Charles Rivers Laboratories, Wilmington, MA). In experiments that addressed ovulation, the animals were treated with 10 IU of pregnant mare serum gonadotropin (PMSG; Gestyl; Oragnon Pharmaceuticals, West Orange, NJ) followed 48 h later with 25 IU hCG (Ayerst Laboratories, Rouses Point, NY) as described earlier (31). Preovulatory follicles were isolated from immature rats injected with PMSG (10 IU) and treated with or without hCG (25 IU) 48 h later as described previously (31). In experiments that addressed the corpus luteum, the animals were superovulated by treatment with 50 IU PMSG followed 54 h later with 25 IU hCG. A luteolytic dose of PGFaa was injected (0.5 mg SC) 7-8 days after hCG. Allopurinol was dissolved in saline, pH 9.5, and injected (10 mg/rat ip) 24 and 3 h before PGF2,. Assay of X0 and XDH actiuity. Ovarian tissue was removed, frozen immediately, and stored at -70°C until used (within 2 wk). Cytosol samples were prepared at 4°C using a modification of a procedure previously described (4). Ovaries (2 ovaries of 1

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rat, 0.1-0.2 g tissue), isolated follicles (0.02-0.04 g tissue), and adrenals (0.05-0.1 g tissue from 3 rats) were homogenized (O.l0.2 g tissue/ml) in 50 mM potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose, 5 mM EDTA, 10 mM DTT, 1 mM PMSF, and 0.2 U/ml aprotinin. The homogenate was centrifuged at 40,000 g for 30 min, and the supernatant fraction was chromatographed on Sephadex G-25 (g-ml bed volume) preequilibrated with 25 ml potassium phosphate buffer, pH 7.4, containing (in mM) 0.1 EDTA, 0.2 PMSF, and 10 DTT. The eluate was collected and stored at -20°C until assay of enzyme activity (within 3 days). Storage of the samples at -20°C did not affect X0 or XDH activities (data not shown). Protein concentration of the cytosol was measured before and after chromatography using the Bradford assay (Bio-Rad Laboratories, Richmond, CA). Protein recovery after Sephadex chromatography was 80-95%. Liver samples (0.1-0.2 g tissue), prepared as described above, were not chromatographed on Sephadex G-25. Brain tissue (0.1-0.2 g tissue) was homogenized (0.2 g tissue/ml) in 50 mM potassium phosphate buffer, pH 7.4, containing (in mM) 0.1 EDTA, 0.2 PMSF, and IO DTT. The homogenate was centrifuged and chromatographed as described for ovarian tissue. Activities of XDH and X0 were assayed by fluorometry, using oxidation of pterin to isoxanthopterin as a measure of enzyme activity (4). A dual-excitation spectrofluorometer (PTI; Brunswick, NJ) was used with 345-nm excitation, 390-nm emission, and a 5-nm bandwidth slit. For assay, the samples were diluted to 2 ml in 50 mM potassium phosphate, 0.1 mM EDTA, pH 7.4, and placed in a cuvette within a thermostated chamber (37°C). A typical assay is illustrated in Fig. 1. In brief, the rate of pterin (10 PM) oxidation was measured in the absence and presence of the electron acceptor methylene blue (10 PM) to measure the activities of X0 and of X0 plus XDH, respectively. Allopurinol (IO PM) was then added to inhibit the reaction. The slope measured in the presence of allopurinol was subtracted from the slopes representing X0 and XDH activities. Isoxanthopterin (0.4-1.5 nmol) was added as an internal standard to calculate the concentration of product formed. The precise concentration of isoxanthopterin was determined spectrally (E = 13 mM-l cm-l). The assay was linear in the range of 0.01-2 nmol . min. ml-l, and the samples were diluted to obtain activity within this range. The total X0 plus XDH activity in ovarian tissues was increased twofold after chromatography on Sephadex G-25. One milliunit (mu) of enzyme activity is defined as the oxidation of 1 nmol pterin min-’ .g tissue? l

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LuteaL ceZZassay. Luteal cells were isolated from superovulated rats, 5-6 days after hCG, and were enriched over a Percoll density gradient as previously described (6). Cells (2 x 105) were equilibrated 30 min in media (Eagle’s minimum essential medium 2360; GIBCO, Long Island, NY) with 0.1% bovine serum albumin and 2 mM L-glutamine, at 37°C in an atmosphere of 95% air-5% COZ. The final incubation volume was 0.5 ml. Cells were treated with allopurinol (IO PM, 30 min) followed by incubation with PGF2, (1 PM, 30 min) before stimulation with LH (50 ng/0.5 ml, 60 min). In other studies, luteal cells were incubated in the presence and absence of superoxide dismutase (100 U/ml) and catalase (100 U/ml), oxypurinol (1 mM), or hypoxanthine to assess effects on LH-responsive adenosine 3’,5’-cyclic monophosphate (CAMP) and progesterone accumulation. At the end of the incubations, cells were heat treated (95OC, 10 min) and stored at -20°C. Progesterone and CAMP were measured by radioimmunoassay as previously described (16). Statistical analysis. The number of rats in each treatment group (at least 4/group) is indicated (see Figs. l-7). Total X0 and XDH activities were assayed in each animal tissue in duplicate, and the variance (SE) represents the variation between individual animals. Comparison between means was performed by analysis of variance (ANOVA) followed by Newman-Keuls multiple-range test (PC ANOVA; Human Systems Dynamics, Northridge, CA). Isolated cell or follicle experiments were carried out at least twice to verify results of individual experiments (4 replicates/group). Cell and follicle studies were evaluated statistically using a repeated-measures design followed by Newman-Keuls multiple-range test (PC ANOVA; Human Systems Dynamics). Statistical significance is defined as P C 0.05. RESULTS

Xanthine oxidase activity in the ovary. The activities of X0 and XDH in various rat tissues is shown in Table 1. In the ovary, adrenal, and liver -30% of the total enzyme activity consisted of X0. In the brain, the transformation of XDH to X0 appears to be very rapid after death, since most of the enzyme was the X0 type, even though the tissue was removed and frozen within a few minutes. The levels of total X0 plus XDH activity measured in the liver and in the brain are in agreement with values previously reported by others (4, 17). Xanthine oxidase activity in the follicle. The effect of gonadotropin on X0 activity in preovulatory ovaries was studied in both the entire ovary and in follicles. The activities of X0 and XDH were measured in preovulatory ovarian cytosol preparations, after treatment of the animals with an ovulatory dose of hCG, and in isolated preovulatory follicles incubated with hCG. The total X0 plus XDH as well as X0 activities in preovulatory ovaries Table 1. Relative levels of xanthine oxidase and dehydrogenase activities in various rat tissues

I

t

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THE

I”“,.+,,“““1

50

t

I

IN

PT x

ALL0

200 TIME

400

l v*.I 600

IXPT

(SECONDS)

Fig. 1. Fluorometric assay to measure xanthine oxidase (X0) and xanthine dehydrogenase (XDH) activities in rat ovary. Assay was carried out as described in MATERIALS AND METHODS. Pterin (PT), methylene blue (MB), allopurinol (Allo), and isoxanthopterin (IXPT) were added where indicated.

1

Tissue

?a

Ovary Adrenal Liver Brain

6 4 5 4

XDH + X0, nmol . mine1 - g-l

33.8t2.0 16l.lt41.1 314.0t45.4 3.1t0.3

nmol

x0, - min-’

9.5k2.7 45.1t6.4 65.9t3.1 2.5tO.l

- g-l

x0, %

28 28 21 79

Values are means t SE; n, no. of animals. Rat tissues were prepared and enzyme activity was measured as described in MATERIALS AND METHODS. X0, xanthine oxidase; XDH, xanthine dehydrogenase.

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XANTHINE

OXIDASE

were not significantly changed (Fig. 2). Similarly, in isolated follicles, the total X0 plus XDH and X0 activities were also unchanged up to 24 h after incubation with hCG (Fig. 2). Effect of inhibitors of xanthine oxidase on ovulation. Allopurinol and oxypurinol are potent and specific inhibitors of X0; allopurinol inactivates X0 in the absence of substrate, and oxypurinol inactivates X0 in the presence of substrate (15). If oxygen radicals derived from X0 action played a role in ovulation, one might expect that allopurinol would inhibit ovulation. To examine this possibility, rats were treated with allopurinol before induction of ovulation with LH (Table 2). No effect on ovulation by allopurinol was found. Xanthine oxidase activity in the corpus luteum and effect of inhibitors. The levels of X0 and XDH activities

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2. Effect of allopurinol

on ovulation

in rats

Treatment

n

Ova/Rat

Control Allopurinol

6 6

18.7k4.3 17.5k2.7

Values are means t SE; n, no. of animals. Animals were injected with 100 mg/kg ip allopurinol 1 h before and again 3 h after ovulation induction with LH (20 pg SC).

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n

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Days

2 x 20 & G 2 10 a"

12

14

16

18

Post-Ovulation

Fig. 3. X0 and XDH activities in luteal tissue during pseudopregnancy and luteal regression. Rats were superovulated (see MATERIALS AND METHODS), and luteinized ovaries were removed and frozen (-70°C). Cytosol samples were prepared, and X0 and XDH activity were measured as described in MATERIALS AND METHODS. Data are means & SE of 5 rats/group.

hCG

8 (hrs)

16

B

3

hCG

6

24

(hrs)

Fig. 2. Effect of ovulation induction with human chorionic gonadotropin (hCG) on X0 and XDH activities in preovulatory rat ovary in vivo (A) and in isolated follicles incubated with hCG in vitro (B). A: immature rats were treated with pregnant mare serum gonadotropin (10 IU SC) followed 48 h later with hCG (25 IU SC). Rats were killed at times indicated, and ovaries were removed and frozen. Cytosol samples were prepared, and X0 and XDH activities were measured as described in MATERIALS AND METHODS. Results are means t SE of 4 rats in each group. B: preovulatory follicles were isolated as described in MATERIALS AND METHODS and were incubated (30-40 mg tissue, 30-35 pieces) in 2.5 ml media including hCG (20 IU/ml) for time indicated. At end of incubation, follicles and media were frozen (-70°C) until assayed for X0 and XDH activity (within 1 wk). Results are means t SE (n = 4).

were examined during natural luteolysis S-20 days after ovulation with hCG. In this animal model, serum progesterone levels are at a maximum -8 days after ovulation. Luteolysis, as determined from serum progesterone and 20a-dihydroprogesterone analysis, is evident I2 days after ovulation, and progesterone levels are almost completely suppressed 16-18 days after ovulation. As shown in Fig. 3, no change in the relative level of X0 or total (X0 plus XDH) enzyme activities were seen up to 20 days after ovulation. Similar results were also observed when luteolysis was induced with PGF2, in the midluteal phase (Fig. 4). Although PGFZa induced a significant (P < 0.01) decrease in serum levels of progesterone 4 and 24 h after treatment, no significant change in the activities of X0 or XDH were observed. In addition, treatment of animals with allopurinol did not prevent luteolysis induced by PGF2, (Fig. 5). PGFZ, produced a significant (P c 0.01) decrease in the serum level of progesterone, both in the absence and presence of allopurinol. Enzyme activity was measured after allopurinol treatment to verify that effective levels were achieved in the luteinized ovaries. The activities of X0 and XDH were found to be significantly inhibited (P < 0.01) in ovarian tissue of the allopurinol-treated rats. Sephadex chromatography of cytosol from allopurinol-treated rats increased X0 plus XDH activity 3.3 t O.&fold (n = 8) but did not change the relative levels of X0 compared with XDH or remove the inhibitory effect of allopurinol. The effect of allopurinol was also studied in cultured luteal cells. As shown in Fig. 6, PGF2, induced a signifi-

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XANTHINE I-1

XO+XDH

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-

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OXIDASE

IN

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OVARY

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0

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Fig. 4. X0 and XDH activities in luteal tissue and serum progesterone levels after PGFZLY treatment in vivo. Superovulated rats were injected with PGFz, 8 days after ovulation and were killed 4 or 24 h later. Rats in time 0 group were not injected with PGFZ,. After decapitation, trunk blood was collected, and ovaries were quickly removed and frozen. Cytosol samples were prepared, and X0 plus XDH activity was measured as described in MATERIALS AND METHODS. Data are means t SE of 4 rats in 4-h group and 8 rats in each of O-h and 24-h PGFzcy groups.

*P


60

-

-

300

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50

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-

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Fig. 5. Effect of allopurinol and PGFz, on ovarian X0 and XDH activities and serum progesterone levels. Superovulated rats were injected with allopurinol and PGFZLY as described in MATERIALS AND METHODS. Rats were killed 24 h after PGFz, treatment, trunk blood was collected, and ovaries were removed and frozen. All cytosol samples were chromatographed over Sephadex (see MATERIALS AND METHODS). Results are means ? SE of 4 rats/group. * P < 0.05.

cant decrease (P c 0.01) in LH-stimulated CAMP accumulation, but the cell response to LH or PGF2, was not influenced by allopurinol. Progesterone production also was not significantly changed by allopurinol. Incubation of luteal cells with low levels of X0 and hypoxanthine markedly inhibits LH-sensitive CAMP and progesterone accumulation, and these effects are reversed by superoxide dismutase and catalase (12). To test whether endogenous X0 in luteal cells may influence functional activity, the cells were incubated with hypo-

-

-

-

-

+

+ -

+ +

+ -

Fig. 6. Effect of allopurinol on CAMP and progesterone accumulation in isolated luteal cells in response to luteinizing hormone (LH) and PGFZa. Results are means t SE of 2 experiments, each carried out with 4 replicates for each treatment group.

xanthine in the absence and presence of superoxide dismutase and catalase or oxypurinol (Fig. 7). Although hypoxanthine produced a severalfold amplification of LH-sensitive CAMP accumulation, like that reported earlier (7), there was no effect on this response by superoxide dismutase and catalase or oxypurinol. Similarly, these same treatments had no effect on progesterone production in response to LH. DISCUSSION

The studies in this report show that X0 is present in both follicular and luteal tissue. The rat ovary contains about one-fifth the X0 activity as that of the liver and adrenal, and X0 comprises -30% of the total X0 plus XDH activity, like that of the liver and adrenal. Although X0 produces a marked inhibition of ovarian cell responses to LH in the presence of hypoxanthine (12), evidence from the present studies does not support a role for X0 in ovulation or luteolysis. For example, no increase in X0 was found in follicular or luteal tissue associated with ovulation or luteolysis. Allopurinol, an inhibitor of X0 (15), had no effect on ovulation induced by LH or on luteolysis induced by PGFza. Also, no evidence for an inhibitory role of endogenous X0 in luteal cells was found, since coincubation with hypoxanthine amplified rather than inhibited cellular responses to LH, and this response was not influenced by superoxide dismutase and catalase or oxypurinol. Nevertheless, significant levels of reactive species may be produced by X0 in ovarian tissue, since the level of X0 found in the ovary is comparable with the level of X0 that inhibits the action of LH in luteal cells (12). About 5 mU of X0 produced half-maximal antigonadotropic effects in luteal cells (12). Ovarian tissue was found to contain -14 mU/mg protein (1.5 mU/mg tissue) of X0 based on the units of activity of the commercial preparation of X0 that inhibits luteal cell responses (12). Thus the ovary may generate reactive oxygen species via X0, which could possibly influence ovarian cell function

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XANTHINE

1-1

G 4 -

m

30

Control

rq

OXIDASE

Hypoxanthine

TT

jj 25

0

IN

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regimen on ovulation in rats, although we only examined the animals 24 h after induction of ovulation with LH (Table 2). A DTT-reversible conversion of XDH to X0 was demonstrated in rat liver and kidney (25). Because the cytosol preparations of tissues in the present studies were carried out in the presence of DTT, this type of X0 could not be detected. Thus the present studies do not preclude the possibility that a reversibly oxidized X0 was generated in ovarian tissues after various treatments. However, no tissue produces only reversible X0, which makes it unlikely that the ovary generates only reversible, but not irreversible, X0. The lack of effect of allopurinol on ovarian function also supports this conclusion. A rapid increase in ovarian hydrogen peroxide and superoxide occurs in response to a luteolytic challenge with PGF2, and at natural luteal regression in rats (33, 35). Superoxide dismutase inhibits ovulation in the perfused rabbit ovary (27), and superoxide and hydrogen peroxide evoke marked antigonadotropic and luteolytic responses in rat ovarian cells (5, 12, 22). These findings indicate that reactive oxygen species play important functional roles in the ovary. However, the results of the present studies do not support a major role for X0 as an endocrine-regulated generator of reactive oxygen species in the ovulatory follicle or the regressing corpus luteum of rats. This work was supported by National Institutes of Health Grant HD-10718. Address for reprint requests: H. R. Behrman, Dept. of Ob/Gyn, Yale Univ. School of Medicine, PO Box 3333, New Haven, CT 06510.

0 Control

SOD and Catalase

Received

31 July

1991; accepted

in final

form

3 October

1991.

Oxypurinol

Fig. 7. Effect of hypoxanthine in absence and presence of oxypurinol on LH-stimulated CAMP and progesterone accumulation in isolated luteal cells. Cells were incubated simultaneously where indicated with hypoxanthine (50 PM), superoxide dismutase (SOD; 100 U/ml), catalase (100 U/ml), and oxypurinol (1 mM). All incubations were in presence of LH (100 rig/ml). CAMP and progesterone accumulation in absence of LH was 0.08 rf~ 0.02 pmol and 4.9 & 0.3 ng/lO” cells, respectively. Results are means t SE (n = 4/group).

if substrate such as hypoxanthine or xanthine was present. Both FSH and LH deplete the level of ATP in granulosa and luteal tissue, respectively (29, 36), effects that are associated with an increase in hypoxanthine and xanthine in other tissues (1). Interestingly, adenine purines are depleted in the regressing corpus luteum (36), and the level of hypoxanthine in human follicular fluid is significantly higher after hCG treatment to induce ovulation (21). Possibly, an increase in ovarian hypoxanthine and xanthine may increase the rate of oxygen radical production by X0, without a change in the relative level of X0. One report showed no effect of allopurinol on ovulation in rabbits (11). In another report, allopurinol (50 mg/kg) was shown to produce a modest inhibition of ovulatory efficiency in rabbits, which was evident 12 h, but not 24 h, after induction of ovulation with LH (26). Yet we found no effect of twice this dose of allopurinol administered under a similar treatment

REFERENCES 1. Arch, J. R. S., and E. A. Newsholme. The control of the metabolism and the hormonal role of adenosine. In: Essays in Biochemistry, edited by P. S. Campbell and S. Dickens, New York: Academic, 1980, p. 82-123. R. C. Wiggins, and P. L. 2. Bagavandoss, P., S. L. Kunkel, Keyes. Tumor necrosis factor-a (TNF-(u) production and localization of macrophages and T lymphocytes in the rabbit corpus luteum. EndocrinoZogy 122: 11851187, 1980. 3. Battelli, M. G., E. Lorenzoni, and F. Stripe. Milk xanthine oxidase type D (dehydrogenase) and type 0 (oxidase). Purification, interconversion and some properties. Biochem. J. 131: 191-198, 1973. 4. Beckman, J. S., D. A. Parks, J. D. Pearson, P. A. Marshall, and B. A. Freeman. A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues. Free Radical Biol. Med. 6: 607-6X,1989. 5. Behrman, H. R., and S. L. Preston. Luteolytic actions of peroxide in rat ovarian cells. Endocrinology 124: 2895-2900, 1989. 6. Behrman, H. R., S. L. Preston, and A. K. Hall. Cellular mechanism of the antigonadotropic action of LHRH in the corpus luteum. Endocrinology 107: 656-664, 1980. 7. Brennan, T. J., R. Ohkawa, S. D. Gore, and H. R. Behrman. Adenine-derived purines increase ATP levels in the luteal cell: evidence that cell levels of ATP may limit the stimulation of cyclic AMP accumulation by LH. Endocrinology 112: 499-508, 1983. 8. Bulmer,’ D. The histochemistry of ovarian macrophages in the rat. J. Anat. Land. 98: 313-319, 1964. 9. Cavender, J. L., and W. J. Murdoch. Morphological studies of the microcirculatory system of periovulatory ovine follicles. BioZ. Reprod. 39: 989-997, 1988. 10. Espey, L. L. Ovarian proteolytic enzymes and ovulations. Biol.

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Reprod. 10: 216-235, 1974. 11. Espey, L. L., V. I. Stein, and J. Dumitrescu. Survey of antiinflammatory agents and related drugs as inhibitors of ovulation in the rabbit. Fertil. Steril. 38: 238-247, 1982. 12. Gatzuli, E., R. F. Aten, and H. R. Behrman. Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells. Endocrinology 128: 2253-2258, 1991. 13. Gillim, S., K. Christensen, and C. McLennan. Fine structure of the human menstrual corpus luteum at its stage of maximum secretory activity. Am. J. Anat. 126: 409-428, 1969. 14. Granger, D. M., M. E. Hoellwarth, and D. A. Parks. Ischemia-reperfusion injury: role of oxygen-derived free radicals. Acta Physiol. Stand. Suppl. 548: 47-62, 1986. 15. Hitchings, G. H. Allopurinol, an inhibitor of xanthine oxidase: physiological and biochemical studies. FEBS Symp. 16: 11-22, 1969. 16. Jaffe, B. M., and H. R. Behrman. Prostaglandins and prostaglandin metabolites. In: Methods of Hormone Radioimmunoassay, edited by B. M. Jaffe and H. R. Behrman, New York: Academic, 1979, p. 19-42. 17. Kinuta, Y., M. Kimura, Y. Itokawa, M. Ishikawa, and H. Kikuchi. Changes in xanthine oxidase in ischemic rat brain. J. Neurosurg. 71: 417-420, 1989. 18. Kirsch, T., A. Friedman, R. Vogel, and G. Flickinger. Macrophages in corpora lutea of mice: characterization and effects on steroid secretion. Biol. Reprod. 25: 629-638, 1981. 19. Kuppusamy, P., and J. L. Zweier. Characterization of free radical generation by xanthine oxidase: evidence for hydroxyl radical generation. J. Biol. Chem. 264: 9880-9884, 1989. 20. Laloraya, M., G. P. Kumar, and M. Laloraya. Changes in the levels of superoxide anion radical and superoxide dismutase during the estrous cycle of Rattus norvegicus and induction of superoxide dismutase in rat ovary by lutropin. Biochem. Biophys. Res. Commun. 157: 146-153,1988. 21. Lavy, G., H. R. Behrman, and M. L. Polan. Purine levels and metabolism in human follicular fluid. Human Reprod. Eynsham 5: 529-532,199o. 22. Margolin, Y., R. F. Aten, and H. R. Behrman. Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. Endocrinology 127: 245-250, 1990. T., and M. Ziff. Increased superoxide anion release 23. Matsubara, from human endothelial cells in response to cytokines. J. Immunol. 137: 3295-3298,1986. J. M. Oxygen-derived free radicals in postischemic tissue 24. McCord, injury. N. Engl. J. Med. 312: 159-163, 1985.

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Xanthine oxidase and dehydrogenase activities in rat ovarian tissues.

The production of reactive oxygen species in the ovary is rapidly inducible, but the nature of the generator is unknown. One possibility is xanthine o...
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