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Biology of Reproduction 59, 1069-1076 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

Prostaglandin F2{alpha} Treatment In Vivo, but Not In Vitro, Stimulates Protein Kinase C-Activated Superoxide Production by Nonsteroidogenic Cells of the Rat Corpus Luteum1

Raymond F. Aten2,a, Thomas R. Kolodecika, Michael J. Rossib, Claire Debusschera, , and Harold R. Behrmana

a Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520–8063 b Departments of Biology and Environmental Science, University of New Haven, West Haven, Connecticut 06516


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal regression is associated with the generation of reactive oxygen species (ROS). To determine the nature of the ROS generator, cells isolated from luteinized rat ovaries were examined for ROS production using luminol-amplified chemiluminescence (LCL). Cells cultured for 2–48 h exhibited minimal LCL, but there was a significant (30- to 50-fold), rapid (maximum at 3–5 min), and dose-dependent increase in LCL in response to phorbol ester (phorbol 12-myristate 13-acetate; TPA; ED50 = 0.03 µM) and diacylglycerol (1,2-dioctanoyl-glycerol; ED50 = 30 µM). The TPA-induced response was cell number dependent and was virtually abolished by superoxide dismutase, freezing, or heating (95°C for 5 min). Zymosan, known to induce a phagocytic response in leukocytes, stimulated a superoxide (O2-·) response with a slow onset (maximum at 40 to 60 min) and a maximum about one third of that observed for TPA. The response to TPA and zymosan was inhibited by the NADPH/NADH-oxidase inhibitor, diphenylene iodonium (ID50 = 5 µM for TPA), but not by the mitochondrial inhibitors, potassium cyanide, rotenone, or sodium azide. Fractionation of cells by centrifugal elutriation showed that TPA-stimulated O2-· production coeluted with the nonsteroidogenic cells and that little, if any, O2-· generation coeluted with the steroidogenic cells. Cells isolated 1, 2, and 4 h after in vivo treatment with a luteolytic dose of prostaglandin F2{alpha} (PGF2{alpha}) showed a significant increase in TPA-stimulated O2-· production at 2 h, whereas luteal cells or corpora lutea incubated directly with 1 µM PGF2{alpha} did not show any increase in response. Corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated level of TPA-stimulated O2-· production. In conclusion, there is a superoxide generator in luteinized ovaries that is activated through a protein kinase C pathway, localized in nonsteroidogenic cells, transiently increased during PGF2{alpha}-induced luteolysis in vivo, and elevated during natural luteal regression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Both natural and prostaglandin F2{alpha}-induced functional regression of the corpus luteum are associated with a loss of progesterone production and a significant increase in the production of reactive oxygen species (ROS) [1–6]. In addition, ischemia and reperfusion of the pregnant rat ovary decreases serum progesterone levels, and this reduction appears to be ROS mediated [7]. Micromolar concentrations of hydrogen peroxide diminish gonadotropin-stimulated cAMP accumulation and progesterone production by rat luteal, rat granulosa, and human granulosa-luteal cells [8–10], as well as low-density lipoprotein (LDL) uptake and LDL-stimulated progesterone production by porcine luteal cells [11]; superoxide, generated with xanthine oxidase and hypoxanthine, reduces gonadotropin-stimulated cAMP accumulation and progesterone production by rat luteal cells [12]. The ROS-induced inhibition of steroidogenesis appears to be due to a disruption of the intracellular transport of cholesterol to and/or into the mitochondria [13].

Although the effects of ROS on luteal cell function are well documented, the nature of the ROS generator within the ovary has not been determined. Neither the form of the generator nor the mode of activation has been assessed. Leukocytes, endothelial cells, and stromal cells make up a majority of the cells in luteinized ovaries [14–17], and each cell type has been shown to produce ROS [18–23]. Superoxide-producing NADPH/NADH-dependent oxidases have been demonstrated in phagocytic leukocytes and nonleukocytic cells that include fibroblasts, endothelial, vascular smooth muscle, and kidney glomerular cells, the carotid body, and osteoclasts [18–30]. Xanthine oxidase of endothelial cell origin generates superoxide following ischemia reperfusion [19, 31, 32], and ROS-producing microsomal NADPH-cytochrome P450 reductases and plasma membrane-bound NADH/NADPH-dependent oxidases have been demonstrated in hepatocytes, adipocytes, and thyrocytes [19, 33, 34]. A broad array of stimulators for ROS production activate phagocytic NADPH oxidases [18, 19]. These stimulators include opsonized zymosan and formyl-met-leu-phe, complement C5a, platelet activating factor, leukotriene B4, interleukin-8, calcium ionophore (A23187), and phorbol ester (phorbol 12-myristate 13-acetate; TPA). Interleukin-1, tumor necrosis factor {alpha}, transforming growth factor ß, arachidonic acid, complement, opsonized zymosan, epidermal growth factor, insulin, angiotensin II, calcium ionophore, and TPA are also known activators of ROS production in nonphagocytic cells [18–24, 26, 33, 34].

The objective of the present study was to assess whether isolated cells from luteinized rat ovaries produced ROS, to characterize the ROS generator as to activators, inhibitors, and cellular origin, and to determine whether the level of ROS production changed during luteolysis or natural regression. A recent study found that a TPA- and calcium ionophore-stimulated ROS generator was present in bovine luteal tissue, although the magnitude of the response varied markedly and not all preparations responded [35].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Experimental Designs

Follicular development, ovulation, and formation of corpora lutea in immature female rats (26–27 days old, CD strain; Charles River Laboratories, Wilmington, MA) were induced by treatment with 50 IU eCG (s.c., Gestyl; Organon Pharmaceuticals, West Orange, NJ) followed at 56 h by 25 IU hCG (s.c.; Sigma Chemical Company, St. Louis, MO). For studies with isolated corpora lutea, follicular development was induced with 10 IU eCG (s.c.; Gestyl). The animals were housed and cared for in the fully accredited facilities operated by the Division of Animal Care. Treatments and procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and a protocol approved by Yale University Animal Care Committee. Functional regression of luteinized ovaries was induced with prostaglandin F2{alpha} (PGF2{alpha}, 3 mg/kg, s.c., Lutalyse; The Upjohn Company, Kalamazoo, MI) to rats on the morning of the fifth to seventh day after hCG administration [3, 36, 37]. Naturally regressed ovaries were obtained 18 days after hCG administration. 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 (5.0:1.1:3.9, respectively; 1.8 ml/kg BW) and were perfused with 0.9% NaCl through the left ventricle, after incising of the right atrium, until the perfusate was visibly clear of blood. Perfusions were performed to remove blood cells, especially leukocytes—known producers of ROS [18, 19]&; might be loosely trapped in the capillaries of the ovaries. Corpora lutea were microscopically isolated from perfused ovaries by blunt dissection [38].

Preparation of Cells from Luteinized Ovaries, Cell Culture, and Corpora Lutea Culture

Cells were isolated from the perfused luteinized ovaries by collagenase and DNase digestion and EDTA dispersion as described previously [39]. Cells were cultured for less than 8 h in 12 x 75 plastic tubes containing Hepes-buffered Minimum Essential Medium (12360; Gibco, Grand Island, NY) supplemented with BSA (1 mg/ml) and L-glutamine (0.29 mg/ml) under a humidified atmosphere of 95% air:5% CO2. To examine the long-term effects of test agents, cells were cultured overnight by a slight modification of a previously described method [40]. Cells were cultured in laminin (Gibco/BRL or Becton Dickinson, Bedford, MA; 5 µg/tube)-coated 12 x 75 plastic tubes, rather than 24 well culture plates, in Hepes-buffered McCoy's 5a Medium (12330; Gibco) supplemented with BSA (1 mg/ml), Pen/Strep (100 U/ml penicillin/ml and 100 µg/ml streptomycin; Gibco), bovine high-density lipoprotein (5 µg/ml cholesterol; Boehringer Mannheim, Indianapolis, IN), ovine LH (100 pg/ml), and ovine prolactin (1 µg/ml) under a humidified atmosphere of 95% air:5% CO2. Previous studies showed that under these conditions, cells attach, are steroidogenic, and remain viable [40].

Isolated corpora lutea were incubated in Hepes-buffered Minimum Essential Medium (12360; Gibco) supplemented with BSA (1 mg/ml) and L-glutamine (0.29 mg/ml) under a humidified atmosphere of 95% oxygen:5% CO2. Upon completion of the incubation, the corpora lutea were dispersed by collagenase/DNase digestion [39].

Peritoneal Leukocyte Isolation

Rat peritoneal leukocytes were induced and collected by peritoneal lavage essentially as described previously [41]. Adult female rats (CD strain, Charles River Laboratories) weighing more than 200 g were anesthetized with Metofane (methoxyflurane; Mallinckrodt Veterinary, Inc., Mundelein, IL), and 20 ml of 1.2% casein (Sigma) dissolved in Ca2+, Mg2+-free Dulbecco's PBS (D-PBS; Gibco) was administered i.p. Four days later, the animals were anesthetized with ketamine and xylazine (as described above), and the peritoneal leukocytes were collected by lavage using 50 ml of cold Ca2+-, Mg2+-free D-PBS. Red blood cells were removed by hypotonic lysis. Wright-Giemsa staining indicated that the isolated peritoneal leukocytes consisted of 69 ± 3% monocytes/macrophages and 23 ± 3% eosinophils, the remainder being basophils, lymphocytes, and neutrophils. The peritoneal leukocytes were cultured for less than 8 h in 12 x 75 plastic tubes containing Hepes-buffered Minimum Essential Medium (12360; Gibco) supplemented with BSA (1 mg/ml) and L-glutamine (0.29 mg/ml) under a humidified atmosphere of 95% air:5% CO2.

Centrifugal Elutriation

Cells were isolated from luteinized ovaries by collagenase and DNase digestion and EDTA dispersion as described above; they were then suspended in cold Ca2+- and Mg2+-free D-PBS supplemented with glucose (1 mg/ml), BSA (1 mg/ml), and EDTA (0.1 mM) and fractionated using a Beckman JE-6B elutriator rotor fitted with a standard chamber (Beckman Instruments, Palo Alto, CA) and slight modifications of previously described methods [42, 43]. Nonsteroidogenic (< 12-µm diameter) and steroidogenic (> 12-µm diameter) cell fractions were obtained by elutriation at 2000 rpm and 31 ml/min for 500 ml and 1500 rpm and 75 ml/min for 250 ml, respectively. Previous studies [42, 43] had shown that the former fraction contains nonluteal cells (fibroblasts, endothelial cells, and other small nonsteroidogenic cells) whereas the latter fraction contains small and large luteal cells. The cells were collected by centrifugation and cultured as described above for cells from luteinized ovaries.

Luminol-Amplified Chemiluminescence (LCL) Assay

ROS production was measured by LCL [44, 45]. The assay is based on the principle that luminol, in the presence of one-electron oxidants, forms an excited aminophthalate ion that emits an easily detected photon when it returns to its ground state [44]. Cells were collected by centrifugation and washed with D-PBS (containing Ca2+, Mg2+, and glucose; Gibco) supplemented with BSA (1 mg/ml) (DPBS-BSA). The washed cells were suspended in 0.5 ml DPBS-BSA, and chemiluminescence in the presence of 4.5 µM luminol (Aldrich Chemical Company, Milwaukee, WI) [44] was determined using a luminometer (Turner Designs, Sunnyvale, CA). Luminescence was measured for 1-min intervals with 5-sec delays, and the detection chamber was maintained at 37°C. ROS production was standardized by measuring superoxide production with xanthine oxidase (Sigma) and xanthine (0.5 mM).

Hormones, Drugs, Reagents, and Progesterone RIA

Ovine LH (NIDDK oLH-23) and ovine prolactin (NIDDK oPRL-19) were gifts from the NIH (Bethesda, MD). Human recombinant monocyte chemoattractant protein-1 (MCP-1), interferon-{gamma}, interleukin-1{alpha}, and tumor necrosis factor {alpha} were obtained from Becton Dickinson/Collaborative Biomedical (Bedford, MA). Transforming growth factor ß was a gift from Michael B. Sporn (NCI, NIH, Bethesda, MD). Diphenylene iodonium bisulfate was a gift from A.R. Cross (The Scripps Research Institute, La Jolla, CA). Oxidized human LDL was purchased from Biomedical Technologies Inc. (Stoughton, MA). Phorbol 12-myristate 13-acetate (phorbol ester; TPA), 1,2-dioctanoyl-rac-glycerol (diacylglycerol; DAG), and all other reagents were analytical grade or better and were purchased from Sigma. Serum progesterone levels were determined by RIA as described previously [36, 46].

Statistical Analysis

Values are presented in the tables and figures as means and their associated SE. Statistical significance between values was determined by ANOVA followed by the Student-Newman-Keuls or Dunnett's multiple comparison tests. A p value < 0.05 was considered significant. The experimental unit for statistical purposes was an isolate of cells from a pool of ovaries or an isolate of cells from the corpora lutea of one rat. When SE are not presented, the figure is representative of an experiment that has, at least, been independently replicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cells from luteinized ovaries, freshly isolated or cultured overnight, produced little, if any, ROS. The baseline rarely exceeded the equivalent of 1 pmol superoxide/min for 2.5 x 105 cells (Table 1). However, the addition of phorbol ester (TPA) resulted in a significant (30- to 50-fold), rapid (maximum at 3–5 min), and consistent stimulation of ROS generation (Fig. 1, Table 1). The ROS generated in response to TPA was the superoxide radical, since the inclusion of superoxide dismutase in the assay buffer reduced the response to TPA to nearly baseline whereas catalase alone had no effect (data not shown). The response to TPA was cell number dependent and was linear in the range between 0.05 and 1.0 x 106 for freshly isolated and 0.05 and 0.25 x 106 for cultured ovarian cells, respectively (Fig. 1, inset). The nonlinear increase in response to TPA for cultured ovarian cells at densities greater than 2.5 x 105 cells was most likely due to the surface area and media volume limitations imposed by culturing in 12 x 75 tubes. Studies with cultured ovarian cells were therefore limited to cell densities of 2.5 x 105 or less.


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  		TABLE 1. The effect of phorbol ester on ROS production by freshly isolated and cultured cells from luteinized rat ovaries.



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  		FIG. 1. Time and cell number dependence of TPA-stimulated superoxide production by freshly isolated or cultured cells from luteinized rat ovaries. Cells were isolated, cultured for less than 8 h (circles) or overnight (squares), and assayed for LCL as described in Materials and Methods. Basal luminescence of 0.25 x 106 cells was measured for 10 min (open symbols) and after the addition of 1 µM TPA (TPA, closed symbols). The inset shows the difference between basal and peak TPA-stimulated production as a function of cell number (0.05 to 1.0 x 106 per tube). The main figure is representative of at least six independent experiments. The inset is representative of two independently replicated experiments.

The response to TPA was dose dependent and showed an ED50 of about 30 nM (Fig. 2). Similarly, DAG stimulated a rapid response with a maximum within 3–5 min (data not shown) and an ED50 of about 30 µM (Fig. 2). Zymosan, a particulate material from yeast cell walls that can be phagocytized and induce a respiratory burst [47], stimulated a slow production of superoxide (O2-·) by cells from luteinized ovaries that reached a maximum at 40–60 min (Fig. 3). The flavoprotein inhibitor of NADPH and NADH oxidases, diphenylene iodonium (DPI), significantly inhibited TPA- and zymosan-stimulated O2-· production by cells from luteinized ovaries (Fig. 3). The ID50 for the DPI inhibition of the phorbol response was about 5 µM (Fig. 3, inset). The TPA response was also temperature sensitive. Washed cells that were frozen for 5 min and thawed or incubated at 95°C for 5 min did not generate O2-· after TPA addition (data not shown). Agents that did not inhibit TPA-stimulated O2-· generation included the mitochondrial redox and transport inhibitors, rotenone (0.1 mM), potassium cyanide (1 mM), and sodium azide (1 mM), and two cAMP analogues, dibutyryl cAMP (1 mM) and 8-bromo-cAMP (1 mM, data not shown).



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  		FIG. 2. Concentration-dependent stimulation by TPA or DAG of superoxide production by cultured cells from luteinized rat ovaries. Cells (2.5 x 105 per tube) were isolated from synchronized female rats, cultured overnight, and assayed for TPA-stimulated (0.03–1.0 µM) or DAG-stimulated (1.0–100 µM) LCL as described in Materials and Methods. The difference between basal and peak TPA- or DAG-stimulated production is presented as a function of concentration. Values are means and their associated SE (n = 3).



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  		FIG. 3. Time- and concentration-dependent inhibition by DPI of TPA- or zymosan-stimulated superoxide production by cells from luteinized rat ovaries. Cells (2.5 x 105 per tube) were isolated, cultured overnight, and assayed for TPA-stimulated (1.0 µM, circles and squares) or zymosan-stimulated (0.1 mg/ml, diamonds and triangles) LCL in the absence (circles or diamonds) or presence (squares or triangles) of 1 µM DPI as described in Materials and Methods. The inset shows the difference between basal and TPA-stimulated production as a function of DPI concentration (1, 5, 10 µM). Peak TPA-stimulated production averaged 22 ± 2 pmol/min for controls. An * indicates a significant difference (p < 0.05) from control (no DPI). Both figures are representative of experiments that were at least independently replicated.

When luteinized ovaries were dispersed with collagenase and DNase and the cells were separated by centrifugal elutriation into nonsteroidogenic (Fig. 4, fraction I) and steroidogenic (Fig. 4, fraction II) populations, the nonsteroidogenic cells generated levels of O2-· that were similar to those of nonfractionated cells, whereas the steroidogenic cells generated little O2-· (Fig. 4).



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  		FIG. 4. Separation of TPA-stimulated superoxide-producing cells from LH-stimulated progesterone-producing cells by centrifugal elutriation. Cells were isolated and fractionated by centrifugal elutriation, and the fractions were cultured as described in Materials and Methods. After a 2-h incubation the fractions were assayed for TPA (1 µM)-stimulated LCL or LH (1 µg/ml)-stimulated progesterone production as described in Materials and Methods. The results are expressed as a percentage of the response of cells that were not fractionated. Unfractionated cells produced 56 ± 8 pmol/min and 7.8 ± 0.6 ng/105 cells for TPA-stimulated superoxide production and LH-stimulated progesterone production, respectively (n = 4). Values are means and their associated SE (n = 3). An * indicates a significant difference (p < 0.05) from the respective control.

The addition of TPA to freshly isolated rat peritoneal leukocytes resulted in a significant (50-fold) and rapid (maximum at 3 min) stimulation of O2-· generation (data not shown). The response to TPA stimulation was cell number dependent, linear in the range between 0.5 and 50 x 103 cells, and substantially greater than that produced by an equivalent number of luteal cells (data not shown). A similar level of O2-· production by cells from luteinized ovaries was achieved with 50-fold fewer peritoneal leukocytes (data not shown).

Cells isolated from the ovaries of rats 2 h after in vivo treatment with a luteolytic dose of PGF2{alpha} showed a significant, approximately 3-fold, increase in TPA-stimulated O2-· production (Fig. 5). Cells or corpora lutea isolated from luteinized ovaries of untreated rats and incubated with 1 µM PGF2{alpha} for 2–24 h did not show an increase in TPA-stimulated O2-· production (data not shown); however, corpora lutea isolated from naturally regressed ovaries (18 days after ovulation) showed a significantly elevated TPA response (Fig. 6).



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  		FIG. 5. Increased TPA-stimulated superoxide production by cells after induction of luteal regression by the administration of PGF2{alpha} in vivo. PGF2{alpha} (3 mg/kg, s.c.) or vehicle was administered to eCG- and hCG-synchronized immature female rats 5 to 7 days after the induction of ovulation; 1, 2, and 4 h later, cells were isolated, cultured, and assayed for TPA (1.0 µM)-stimulated LCL as described in Materials and Methods. Values are means and their associated SE (n = 3). An * indicates a significant difference (p < 0.05) from the respective control.



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  		FIG. 6. Increased TPA-stimulated superoxide production by corpora lutea from functionally regressed ovaries. Corpora lutea were isolated from eCG- and hCG-synchronized immature female rats 8 or 18 days after the induction of ovulation, dispersed, and assayed for TPA (1.0 µM)-stimulated LCL as described in Materials and Methods. Values are means and their associated SE for independent isolates of corpora lutea (six corpora lutea per rat) from three rats per group.

Superoxide generation was not stimulated by several agents known to maximally inhibit or stimulate luteal cell function. These included PGF2{alpha} (1 µM) [48], GnRH (1 µM) [39], LH (1 µg/ml) [49], 22-hydroxy-cholesterol (10 µg/ml) [50], and oxidized LDL (100 µg/ml) [51] (data not shown). Ceramide (100 µM; known to induce apoptosis in cultured granulosa and endothelial cells [52, 53]), lysophosphatidylcholine (100 µM), and MCP-1 (10 ng/ml) did not stimulate O2-· production (data not shown). Lysophospholipids are generated by activation of phospholipase A2 during PGF2{alpha}-stimulated luteal regression [54], and MCP-1 increases following PGF2{alpha}- or prolactin-induced luteolysis or during parturition [55–58].

Agents that are known to stimulate O2-· production in phagocytic leukocytes such as leukotriene B4 (1 µM), platelet activating factor-C18 (10 µM), calcium ionophore (A23187, 20 µM), and N-formyl-met-leu-phe (100 µM) [18, 19] had no effect on O2-· production by cells from luteinized ovaries (data not shown). Furthermore, agents that are known to stimulate O2-· production in nonphagocytic cells (endothelial cells, fibroblasts, vascular smooth muscle cells, and adipocytes) and/or phagocytic cells, such as arachidonic acid (150 µM) [21, 59], interleukin-1{alpha} (100 ng/ml) [19, 24], bradykinin (1 µM) [60], angiotensin II (1 µM) [20], and insulin (1 µM) [34], had no effect (data not shown).

Overnight culture in the presence of tumor necrosis factor {alpha} (100 ng/ml), interferon-{gamma} (50 ng/ml), transforming growth factor ß (25 ng/ml), or tumor necrosis factor {alpha} plus interferon-{gamma} (100 and 50 ng/ml, respectively)—cytokines known to prime leukocyte responses to activators such as TPA [19, 23]—did not significantly enhance TPA-stimulated O2-· generation by cultured cells from luteinized ovaries (data not shown). Priming of phagocytic leukocytes is known to reduce the lag time and amplify the production of O2-· during subsequent activation, but primers alone do not stimulate O2-· production [18]. Furthermore, overnight culture in the presence of pentoxifylline (1 mM), a known inhibitor of leukocyte responses [61], did not diminish the level of TPA-stimulated ROS production (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present results support the hypothesis that superoxide is produced by the nonsteroidogenic cells of luteinized ovaries via a protein kinase C-activated pathway, that the enhanced capacity for superoxide generation is an early and integral part of PGF2{alpha}-induced and natural luteal regression, and that the enhanced capacity to generate superoxide occurs only after in vivo treatment with PGF2{alpha}.

While the present results do not rule out superoxide production by luteal cells, the most likely candidates for nonsteroidogenic, superoxide-producing cells are phagocytic leukocytes. There is ample evidence that leukocytes are resident in the ovary and that the number and types of leukocytes change during the cycle and during luteal development and regression [17, 62–65]. There is a 5- to 8-fold increase in the number of macrophages and neutrophils present in the theca during ovulation; most types of leukocytes (lymphocytes, monocytes/macrophages, neutrophils, and eosinophils) are found to migrate into newly forming corpora lutea; and the proportion of macrophages increases as the corpus luteum matures [62]. Also, phagocytic leukocytes are powerful generators of superoxide, and the level produced by peritoneal leukocytes in the present study suggests that relatively few leukocytes, as few as 2%, could produce the level of superoxide observed with cells from luteinized ovaries. Furthermore, in the present studies several agents known to stimulate phagocytic leukocytes such as TPA and zymosan [18, 19] similarly stimulated superoxide production by ovarian cells.

Although the pathway leading to the enhanced capacity for superoxide production following luteal regression remains to be determined, an early step most likely involves steroidogenic luteal cells that have receptors for PGF2{alpha} [42], since leukocytes do not have PGF2{alpha} receptors (T.R. Kolodecik and H.R. Behrman, unpublished data). Subsequent steps may include the release by luteal cells of a chemoattractant that would encourage the infiltration of circulating leukocytes. Studies that examined the number of eosinophils in ovine luteal tissue after administration of PGF2{alpha} support such a possibility [66]. Conditioned media from incubations of luteal tissue obtained 2 h after treatment with PGF2{alpha} showed a greater potential to attract eosinophils in a chemotaxis assay than conditioned media from vehicle-treated controls [66].

Studies of MCP-1 also suggest chemoattract production. The induction of luteolysis by administration of PGF2{alpha} increased detectable levels of MCP-1 mRNA in midcycle corpora lutea of sheep and cows by as early as 1 h, with a maximum at 4 h, but had no effect on MCP-1 expression in early corpora lutea [57, 58]. Similarly, the induction of structural regression by prolactin treatment of hypophysectomized female rats increased the levels of immunodetectable MCP-1 in corpora lutea by 24 h [55]. Although an increase in MCP-1 may increase the capacity for superoxide production, it is also possible that the appearance of MCP-1 is a result of superoxide production. On the basis of the time when each reached a maximum, the superoxide generator (maximum at 2 h) preceded the appearance of MCP-1 (maximum at 4 h) [57]. Furthermore, as has been discussed by Tsai et al. [57], MCP-1 is produced by endothelial cells as well as fibroblasts, macrophages, and lymphocytes; and MCP-1 production by endothelial cells is stimulated by exposure to neutrophil products such as superoxide and ROS such as hydrogen peroxide [67, 68].

Chemoattractant production within the ovary is also suggested by the observation that lipid peroxidation increased shortly after the induction of luteolysis and during regression [36, 69]. Oxidized lipoproteins, products of lipid peroxidation, have been shown to be chemoattractants and to stimulate macrophage metabolism [70, 71]. Since ovarian lipid peroxides, detected as malonaldehyde levels, increase significantly after PGF2{alpha}-induced luteolysis in the rat [36] and since lipid peroxidation-derived protein epitopes, detected by immunocytochemical staining, stain intensely in regressing pig corpora lutea [69], it is possible that the increase in lipid peroxides generated by PGF2{alpha} stimulates leukocyte infiltration into the ovary. However, it is also possible that the increase in lipid peroxides is, like that of MCP-1, a result of leukocyte infiltration and superoxide production. Lipid peroxides levels are significantly elevated at 4 h after PGF2{alpha} administration [36], well after the peak for the O2-· generator, suggesting that other chemoattractant candidates need to be considered.

Based on the inhibition by DPI, the ovarian superoxide generator probably includes a flavoprotein component and is an NADPH/NADH oxidase [18, 21, 23]. In both phagocytic and nonphagocytic cells, NADPH/NADH oxidases include multiple protein components (cytosolic p47phox, cytosolic p67phox, membrane-bound heterodimeric cytochrome b558 consisting of p22phox and gp91phox, and a rac family G-protein) that become a membrane-bound complex when activated [72–75]. The lack of a TPA response after freezing, which permeabilizes cells and may have dispersed the cytosolic p47phox and p67phox components, also supports the proposition that the luteal ROS generator is an NADPH/NADH oxidase.

While phagocytic leukocytes are a likely source of the superoxide generator found in luteinized ovaries, it is possible that nonphagocytic cells such as endothelial cells or fibroblasts might also be sources. However, many agents known to activate superoxide production by nonphagocytic cell oxidases do not stimulate ovarian cell superoxide generation. Arachidonic acid, insulin, angiotensin II, interleukin-1{alpha}, or calcium ionophore (A23187) did not stimulate luteal ROS production, yet each is a known activator of endothelial cells, fibroblasts, or vascular smooth muscle cells [20–30]. Similarly, agents known to stimulate phagocytic leukocytes such as formyl-met-leu-phe, platelet activating factor, leukotriene B4, complement C5a, interleukin-1{alpha}, or calcium ionophore (A23187), or agents known to prime phagocytic leukocytes such as tumor necrosis factor {alpha} or interferon-{gamma}, were inactive [18, 19]. While these results suggest that leukocytes, endothelial cells, or fibroblasts may not be the cellular origin of the superoxide generator in the luteinized ovary, it is likely that one or several of these cell types are the source and that endogenous inhibitors may be present that prevent responses to agents other than phorbol ester, the most effective, but nonphysiologic, activator of NADPH/NADH oxidases [18, 19]. The identity of this possible endogenous inhibitor, as well as of the endogenous agent that activates the superoxide generator in luteinized ovaries, remains to be determined.

In conclusion, there is an NADPH/NADH-like superoxide generator present in nonsteroidogenic cells of luteinized ovaries that is activated through a protein kinase C pathway and whose levels increase shortly after the induction of luteolysis and after natural luteal regression. The cellular origin of the generator is most likely leukocytic, but the pathway leading from the induction of luteolysis in luteal cells to the activation of the leukocytic superoxide generator remains to be determined.


   ACKNOWLEDGMENTS
 
The authors thank Sandy Preston for her excellent technical assistance and Cindy Kolodecik for help in manuscript preparation.


   FOOTNOTES
 
1 This work was supported by NIH grant HD-10718. Back

2 Correspondence: Raymond F. Aten, Reproductive Biology Section, Department of Obstetrics and Gynecology, Yale University School of Medicine, P.O. Box 208063, New Haven, CT 06520–8063. FAX: 203 785 7134; raymond.aten{at}yale.edu Back

Accepted: June 19, 1998.

Received: March 31, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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