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BOR - Papers in Press, published online ahead of print April 30, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.010710
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BIOLOGY OF REPRODUCTION 69, 835–842 (2003)
DOI: 10.1095/biolreprod.102.010710
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Possible Role of Cyclooxygenase II in the Acquisition of Ovarian Luteal Function in Rodents1

Toshihiro Sakurai3, Kazuhiro Tamura2,3, Shiki Okamoto4, Takahiko Hara4, and Hiroshi Kogo3

Department of Pharmacology,3 Tokyo University of Pharmacy and Life Science, Hachioji, Horinouchi, Tokyo 192-0392, Japan Tokyo Metropolitan Institute of Medical Science,4 Honkomagome, Bunkyo-ku, Tokyo 113-0032, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of the corpus luteum (CL), which involves angiogenesis, is essential for the establishment of early pregnancy. We investigated the roles of the prostaglandin synthases cyclooxygenase (COX) I and COX-II in angiogenesis and progesterone production in the newly formed CL, using inhibitors of the COX enzymes and the gonadotropin-induced pseudopregnant rat as a model. Injection of indomethacin, a nonselective COX inhibitor, on the day of ovulation and the following day decreased serum levels of progesterone, as did injection of the selective COX-II inhibitor NS-398. In contrast, a selective COX-I inhibitor, SC-560, had no effect on serum progesterone concentrations. None of the inhibitors had any effect on the weight of the superovulated ovaries or on the synthesis of progesterone by cultured luteal cells. To determine whether changes in angiogenesis are responsible for the decrease in progesterone synthesis, we measured hemoglobin and CD34 levels in luteinized ovaries following injection of COX inhibitors and measured the relative frequency of cells positive for platelet-endothelial cell adhesion molecule as a specific marker for endothelial cells. All of these parameters were reduced by the COX-II inhibitors, suggesting that changes in the vasculature are responsible for the decrease in serum progesterone. Histological examination of ovarian corrosion casts indicated that NS-398 inhibited the establishment of luteal capillary vessels following the injection of hCG. The results are consistent with the hypothesis that the activity of COX-II is associated with the formation of functional CL via its stimulation of angiogenesis.

corpus luteum, corpus luteum function, female reproductive tract, ovary, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The corpus luteum (CL), which is formed from the ovulated follicle, plays a critical role in secretion of progesterone for the maintenance of early pregnancy. Angiogenesis (the growth of new capillaries from existing ones) is important for such things as wound healing and embryonic development and is essential for the formation of a steroidogenically functional CL [1–3]. Therefore, the CL is a good model for the study of angiogenesis in vivo. The vascular development of a newly formed CL is regulated by angiogenic factors, including vascular endothelial growth factor (VEGF) [4, 5] and basic fibroblast growth factor (FGF-2, bFGF) [6]. The interaction of these factors for angiogenesis has been discussed by Hyder and Stancel [7]. Luteal-conditioned medium possess mitogenic activity for endothelial cells in rats [8]. Treatment with an anti-VEGF receptor 2 antibody during the preovulatory stage inhibits luteal angiogenesis in mice [9]. The expression of VEGF and of its receptor mRNAs is robust in the early stage of the bovine luteal phase [10].

A large variety of eicosanoids, well known to be important for reproduction, are produced by the actions of cyclooxygenase (COX) enzymes I and II on arachidonic acid [11]. COX-I is constitutively expressed in a variety of cells, but the expression of COX-II requires induction, which can be accomplished by various agents including mitogens, cytokines, and tumor promoters. In the ovary, the expression of COX-II and the synthesis of the prostaglandin (PG) F2{alpha} and PGE2 needed for ovulation are induced after the ovulatory surge in gonadotropins. Both PGF and PGE are produced at higher rates than other PGs immediately after the ovulation of luteinizing follicles [12, 13]. Indomethacin (INDO) inhibits ovulation [14], and administration of INDO to rabbits suppressed corneal angiogenesis [15]. Recent attention has also been focused on the ability of COX-II to modulate the production of angiogenic factors in colon cancer [16]. Enhanced angiogenesis in vivo is particularly sensitive to the actions of E-type PGs (PGE1, PGE2) [17, 18].

The role of the expression of the COX enzymes and their metabolites in the process of CL formation has not been addressed. Therefore, we used the gonadotropin-induced pseudopregnant rat and mouse models to examine the effects of COX inhibitors on the secretion of progesterone in the developing CL and on luteal angiogenesis.


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

Immature Wistar-Imamichi rats (Imamichi Institute for Animal Reproduction, Ibaraki, Japan) were kept at constant temperature (24°C) and humidity (55% ± 5%) with free access to food and water. All procedures were performed in accordance with institutional guidelines for experimental animal care at the Tokyo University of Pharmacy and Life Science. Animals were injected subcutaneously (s.c.) at 0900 h on Day 23 with 50 IU eCG (Teikoku Hormone MFG Co., Tokyo, Japan). Fifty-four hours later (1500 h, Day 25) they were injected i.p. with 25 IU hCG (Teikoku) to induce superovulation and to produce highly luteinized ovaries. To obtain a normal rate of ovulation, some animals were treated with only 5 IU eCG.

INDO (2.5 mg/kg; Sigma Chemical Co., St Louis, MO), a nonselective COX inhibitor, SC-560 (SC: 5 mg/kg; Cayman Chemical, Ann Arbor, MI), a selective COX-I inhibitor, or NS-398 (NS: 5 mg/kg; Cayman), a selective COX-II inhibitor, was injected at 1000 h on the day of ovulation and the following day (Days 26 and 27). The dosages of SC and NS were determined based upon the work of Smith et al. [19] and of Masferrer et al. [20], respectively, and are sufficient to inhibit COX activity. Ether-anesthetized animals were decapitated at 1000 h on Day 28, and their ovaries were immediately removed for measurement of the hemoglobin content. Blood that was collected from the abdominal aorta was centrifuged. The serum was separated by centrifugation and stored at -80°C until assayed for progesterone. To evaluate the extent of angiogenesis, the numbers of cells positive for platelet-endothelial cell adhesion molecule 1 (PECAM-1, CD31) and the levels of CD34 in the luteinized ovary were analyzed using flow cytometry and immunoblotting, respectively. Some ether-anesthetized animals were infused with a heparin-saline solution followed by a Mercox solution (Okenshoji, Tokyo, Japan) to visualize ovarian vascular vessels. Immature female mice of the C57Br/6 strain were also used to corroborate the data obtained using rats. High doses of gonadotropins (50 IU eCG on Day 23 and 25 IU hCG on Day 25) were injected into immature mice to induce superovulation. The serum levels of progesterone were determined as they were in rats. After a dissociated-cell suspension was prepared from the ovary, the numbers of PECAM-1- and CD45-positive cells were counted with flow cytometry.

Progesterone Assay

The concentration of progesterone in the serum or culture medium was measured by RIA, as described previously [21]. Ovaries were homogenized in PBS at 4°C, and the supernatant was used for RIA after steroid extraction with diethyl ether.

Hemoglobin Assay

The ovarian hemoglobin content was determined with an assay kit that uses the SLS-hemoglobin method (Hemoglobin B test; Wako Pure Chemical Industries, Osaka, Japan). The assay was performed according to the instructions provided by the manufacturer. The hemoglobin was quantified with an absorbance curve at a wavelength of 540 nm using horse erythrocytes as the standard.

Western Blot Analysis

Luteinized ovaries were homogenized in a glass homogenizer in cold buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 150 mM NaCl, 0.1 % Tween-20, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 0.1 mM PMSF, and 0.1 % ß-mercaptoethanol. The insoluble tissue was removed by centrifugation. Samples (150 µg protein) were subjected to electrophoresis on 5–20% gradient SDS-polyacrylamide gels (Nikkyo Technos Co., Tokyo, Japan) and electrotransfered to polyvinylidene difluoride membranes (Immobilon; Millipore, Bedford, MA). The membranes were blocked in a solution of Tris (20 mM, pH 7.5)-buffered saline containing 0.1 % Tween-20 (TBST), 2.5% nonfat dry milk, and 2.5% BSA before being incubated with an anti-CD34 antibody (2 µg /ml) in TBST that contained 0.1 % BSA. After washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (0.4 µg /ml in TBST; Vector Laboratories, Burlingame, CA) followed by a chemiluminescence reagent (NEN Life Science Products, Boston, MA). Signals were visualized by autoradiography on x-ray film (X-OMAT; Kodak, Rochester, NY).

Corrosion Casting

The procedure was basically performed according to the method of Macchiarelli [22]. Laparotomies and thoracotomies were performed in animals anesthetized with ether. The ovary was exposed, and a small hole was opened in the right atrium of the heart for drainage of fluid. Warmed heparin-saline solution (100 IU/ml) was injected into the left ventricle using a 10-ml syringe with a 26-ga needle. The perfusion was continued until all color was gone from the ovary. Mercox (20–30 ml) was injected via the ventricle and abdominal aorta using a syringe with a 22-ga needle. To allow for complete polymerization, the animals were kept at 55°C for 30 min. The ovaries were carefully removed and placed in warm (60°C) water for 5 h followed by overnight digestion at room temperature in 10% NaOH. Digestion was continued for 24–36 h in fresh 60°C NaOH. Samples were then left for 3 days in 60°C distilled water that was replaced with fresh water three times daily, examined microscopically, and photographed (DC 300 F; Leica Microsystems, Tokyo, Japan).

Flow Cytometry in Rats and Mice

Luteinized ovaries from rats were digested in Ca2+-free and Mg2+-free PBS supplemented with 0.2% collagenase (type I; Sigma) and 250 µg/ml DNase (Sigma) in a shaking water bath at 37°C for 60 min followed by vigorous agitation. Separated cells were incubated on ice for 30 min with monoclonal fluorescein isothiocyanate (FITC)-conjugated anti-PECAM-1 (Rat CD31-FITC, IM3079; Immunotech, Marseille, France) at a concentration of 10 µg/ml in 5% fetal calf serum (FCS). After washing with 20 volumes of 5% FCS-PBS, the cells were stained with biotinylated anti-mouse IgG as a secondary reagent, and the antibody was visualized using FITC-conjugated streptavidin (Pharmingen, San Diego, CA). The cells were resuspended in 0.5 ml of PBS containing propidium iodide (PI; Sigma) and subjected to cell sorting using FACSvantage (EPICS XL; Becton Dickinson, Bedford, MA) as previously described [23]. Mouse luteinized ovaries were also digested into a dissociated cell suspension using the procedure described above. Cells were stained with a biotin-conjugated anti-mouse PECAM-1 monoclonal antibody (1:50, MEC13.3; Pharmingen) followed by incubation with Quantum Red-conjugated streptavidin (1:5; Sigma) and a phycoerythrin-conjugated anti-CD45 monoclonal antibody (1:20, 30F11; Pharmingen). PI-negative viable cells were analyzed by flow cytometry.

Luteal Cell Preparation and Culture

Rats were treated with gonadotropins on Days 23 and 25 as described above, and their ovaries were isolated on Day 26, followed by digestion in collagenase (type I). Highly purified luteal cells were collected from this collagenase-digested suspension using the Percoll gradient method of Luborsky and Behrman [24]. Cell viability was 89% ± 1.7% when tested for the ability of cells to exclude trypan blue. Cultures were started in 24-well plates at a density of 106 cells/ml and maintained for 24 h in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 100 µg/ml penicillin-streptomycin, and 100 µg/ml gentamicin in a CO2 incubator. After incubation for 24 h in serum-free DMEM, the cells were treated with INDO (3 µM), NS (3 µM), or LH (100 ng/ml) for 6 h, and the culture media were collected for the progesterone assay. Progesterone produced by cells exposed to ovine LH (NIDDK, oLH26: AFP-5551B; obtained from Dr. A.F. Parlow, National Hormone and Pituitary Program, Harbor/UCLA Medical Center, Torrance, CA) was used to establish that the cells were steroidogenically responsive. There were no differences in cell viability among the groups after culture.

Statistical Analysis

All experiments were carried out on at least four animals, and values are given as mean ± SEM. The significance of the results was tested using a Dunnett test for multiple comparisons. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of COX Inhibitors on the Production of Progesterone in the Early Luteal Phase

We examined the effects of INDO and NS on the serum levels of progesterone 2 days after ovulation. Progesterone concentrations in COX-II inhibitor-treated animals were significantly reduced compared with those in controls (the value for the control: 227 ± 18.8 ng/ml) (Fig. 1A). Inhibition by NS, a COX-II-specific inhibitor, was greater than that seen with INDO, which inhibits both COXs. Neither INDO nor NS significantly altered the body or ovarian weights of immature rats treated with gonadotropins to induce superovulation (Table 1). Injection of the COX-I-specific inhibitor SC had no effect on serum progesterone concentrations (Fig. 1A).



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  		FIG. 1. Effects of COX inhibitors on serum and ovarian progesterone concentrations during the early luteal phase in gonadotropin-primed (50 IU eCG on Day 23 + 25 IU hCG on Day 25) immature rats. The COX inhibitors were given on the day of ovulation (Day 26) and on Day 27. A) Blood samples were obtained at 0900 h on Day 28, and progesterone was measured by RIA. Each bar is the mean ± SEM of 12–16 rats in three experiments. Inhibitors were INDO (2.5 mg/kg), NS-398 (5.0 mg/kg), and SC-560 (5.0 mg/kg) **P < 0.01; vs. control, ##P < 0.01; NS vs. INDO. B) Progesterone levels in the extracts from ovarian homogenate were determined as the contents per milligrams of protein. Each bar is the mean ± SEM of five rats from two experiments


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  		TABLE 1. Effects of COX inhibitors on rat body and ovarian weights in a superovulation model.*

In animals in which ovulation was induced by small doses of gonadotropins, similar but not identical results were obtained. Control animals (n = 10) had 74 ± 3.8 ng/ml progesterone, those given INDO (n = 10) had 38 ± 3.8 ng/ml progesterone, and those given NS (n = 8) had 28 ± 2.5 ng/ml progesterone, i.e., about a 50% reduction with either compound. A similar inhibitory effect of NS on progesterone levels was also observed in mice (Fig. 5A). There were no significant differences in CL weight among the groups in this model of normal ovulation, although the weights tended to decrease in NS-treated animals (Table 2). To further evaluate the effect of NS on ovarian progesterone synthesis, we measured the progesterone content in ovaries (Fig. 1B). The progesterone contents of ovaries of NS-treated animals were no different from those of control ovaries. The results indicate that the action of the COX enzymes, particularly COX-II, is needed for progesterone secretion by the CL.



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  		FIG. 5. Effects of NS on the serum levels of progesterone and the number of PECAM-1- and CD45-positive cells during the early luteal phase in gonadotropin-primed immature mice. Mice were treatment with gonadotropin and NS as described in Fig. 1. Ovaries and blood samples were collected on Day 28. Each value shows the mean ± SEM of 4 mice. A) The serum levels of progesterone were determined by RIA. B) Comparison of the number of PECAM- and CD45-positive cells between control animals and those treated with 1.25–5 mg/kg. C and D) Representative data for control (C) and NS 5 mg/kg (D). *P < 0.05 vs. control


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  		TABLE 2. Effects of COX inhibitors on the weight of the rat ovary and CL in a normal ovulation model.*

Effects of COX Inhibitors on Vascular Markers in the Luteinized Ovary

We measured ovarian CD34-positive cells and hemoglobin content to evaluate the effect of COX inhibitors on the blood vessels of ovaries in which ovulation was induced by gonadotropins. The expression of CD34 in the ovaries of rats exposed to INDO or NS but not those exposed to SC was lower than that found in control ovaries (Fig. 2A). Administration of either INDO or NS significantly decreased the ovarian hemoglobin content (Fig. 2B). Decreases in the relative number of capillaries in the ovary were verified by histological examination of vascular plexuses using the corrosion cast method (Fig. 3). The development of capillaries seen in highly luteinized ovaries was missing in ovaries exposed to NS for the inhibition of COX-II. We also examined the levels of another marker of vascularization in the ovary, the proportion of PECAM-1-positive cells, by flow cytometry (Fig. 4). Administration of either INDO or NS decreased the number of PECAM-1-positive cells. In mice, NS (2.5 and 5.0 mg/kg) significantly reduced the number of PECAM-1-positive/CD45-negative cells (Fig. 5, B–D). NS (1.25–5 mg/kg) also tended to reduce the number of PECAM-1-negative/CD45-positive cells in a dose-dependent manner, but this reduction was not significant.



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  		FIG. 2. Effects of COX inhibitors on ovarian CD34 and hemoglobin concentrations during the early luteal phase in gonadotropin-primed immature rats. Treatment was as indicated for Figure 1. A) Representative Western blot of CD34 protein in homogenates of luteinized ovaries. Density of the blots for individual animals is expressed relative to the density of the control band (100%). Results from two independent experiments (n = 6–8) are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. control. B) Ovarian homogenates were prepared on Day 28 and hemoglobin (Hb) content was measured. Each bar is the mean ± SEM of six to eight rats from two experiments. *P <0.05; vs. control



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  		FIG. 3. Corrosion casts of ovaries from control rats and those treated with 5 mg/kg NS. Treatment was as indicated for Figure 1. Mercox was infused into the ventricle and abdominal aorta on Day 28. The lower picture is an enlargement of the boxed area in the upper picture. The arrow indicates the capillary vessels in the corpus luteum



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  		FIG. 4. Number of PECAM-1-positive (CD31 positive) cells in the ovaries of immature rats treated with exogenous gonadotropins to induce superovulation. Treatment was as indicated for Figure 1. The dispersed cells were prepared on Day 28 and measured using flow cytometory. A) Comparison of the number of PECAM-1-positive cells in ovaries in control animals, those treated with 2.5 mg/kg INDO, or those treated with 5 mg/kg NS. The percentage of PECAM-1-positive cells relative to the total number of ovarian cells was determined using 4 rats in each group. The variation of values was within 20 % in each group. B–D) Representative data in each group: control (B), INDO 2.5 mg/kg (C), NS 5 mg/kg (D). The number of dots in the rectangle defined by a broken line indicates the number of PECAM-1-positive cells

Effects of COX Inhibitors on the Production of Progesterone from Highly Purified Luteal Cells In Vitro

We examined the effects of COX inhibitors on progesterone secretion in the absence or presence of LH using an in vitro culture system of ovarian luteal cells. Neither INDO nor NS at 3 µM affected the basal level of progesterone (Fig. 6). Progesterone production was significantly stimulated by the addition of LH (100 ng/ml). However, neither inhibitor had an effect on the elevation of progesterone levels induced by LH. Thus, COX inhibitors do not directly influence progesterone production in steroidogenic luteal cells.



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  		FIG. 6. Failure of COX-II inhibitors to alter progesterone production by cultured luteal cells. Cells (106) were cultured for 24 h in DMEM supplemented with 10% FBS before the addition of INDO or NS. The culture was continued for 6 h after adding 3 µM INDO or 3 µM NS. Control cells were incubated with medium alone. The steroidogenic capacity of the cells was tested by the addition of 100 ng/ml of LH. Each bar is the mean ± SEM of three cultures. A similar result was obtained in an additional experiment using different doses of both inhibitors (1–10 µM). **P <0.01; vs. control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The use of mice lacking either COX-I or COX-II has shown that absence of COX-II but not of COX-I results in a loss of ovulation [25, 26]. This effect of COX-II appears to involve the production of PGE2 because ovulation, and particularly fertilization, is defective in mice lacking the EP-2 isoform of the PG receptors [27]. Hizaki et al. [27] proposed that the action of PGs is focused on the expansion of the cumulus in the follicle. Because ovarian stimulation by gonadotropin in the mouse is quite similar to that in the rat, we expected that the inhibition of COX-II activity would produce similar ovulatory defects in the rat. Defects in ovulation would be expected to result in a decreased number of CL, and therefore one might expect a decrease in the serum concentrations of progesterone. However, in the present study in which superovulation was induced by treatment with exogenous gonadotropins, the control ovary weight did not differ from weights of ovaries treated with COX-I or COX-II inhibitors. This finding suggests that a deficiency in ovulation was not the basis for the reduction in serum progesterone. The COX inhibitors did not alter progesterone production in cultured luteal cells, which suggests that PGs are not directly involved in progesterone synthesis by luteal cells. The finding of no change in progesterone in NS-398-treated cells is consistent with the findings reported by Mikuni et al. [28] from in vitro preovulatory follicles, indicating that PGs are not directly involved in progesterone synthesis. Armstrong et al. [29], using an eCG-primed immature rat model, found that INDO treatment, which prevented the LH-induced increase in ovarian PGF levels, produced no significant inhibition of progesterone levels. We also observed that, in addition to no effects on progesterone secretion by luteal cells, both inhibitors did not interfere with LH secretion during the early phase of CL formation (data not shown). These results, therefore, suggested that the expression of ovarian COX-II is associated with the acquisition of enhanced capacity for progesterone secretion. Therefore, we must look to other explanations for the loss of progesterone associated with inhibition of COX-II activity.

The acquisition of CL function is dependent upon the growth of new capillary vessels [1–4], angiogenesis. This process, which is associated with normal physiology, occurs in the ovarian CL and is believed to play a role in the growth of luteal cells and the supply of large amounts of cholesterol needed for progesterone synthesis and delivery to the circulation. In the present study, the inhibition of progesterone production by COX-II inhibitors was accompanied by reduction in the formation of ovarian vasculature. Administration of COX-II inhibitors, either selective or nonselective, decreased the ovarian content of hemoglobin and CD34 when compared with vehicle-treated animals. Hemoglobin and CD34 concentrations are indirect indicators of angiogenesis because the concentrations of these proteins can be correlated with the extent of vascular development. Further evidence for reduced angiogenesis in CL exposed to COX-II inhibitors was found using histological analyses of ovarian corrosion casts and the number of PECAM-1-positive cells. COX-II inhibitors significantly decreased the number of PECAM-1-positive cells and the density of vascular vessels in the luteinized ovary. PGs produced in response to COX-II expression probably act on the vasculature of the CL, and without this action there may be insufficient blood perfusion for normal progesterone secretion. Our results are consistent with recent reports that COX-II is related to angiogenesis induced by pharmacological methods in a wide variety of tissues [30, 31]. Administration of rofecoxib, a selective COX-II inhibitor, decreased the number of microvessels and FGF-2 protein expression in the ulcer base during gastric ulcer healing [32]. FGF-2-induced neovascularization of cornea was inhibited by treatment with COX-II inhibitor, which was associated with induction of apoptosis and a decrease in proliferation of endothelial cells [33]. COX-II modulates the production of an undefined angiogenic factor in colon cancer cells [16]. However, the mechanism by which COX-II enhances angiogenic activity remains unknown. INDO and NS-398 have inhibitory effects on in vitro angiogenesis in the rat aortic endothelial cells and human dermal microvascular endothelial cells [34]. Both inhibitors inhibited the activity of the VEGF-induced mitogen-activated protein kinase (ERK2), which is involved in angiogenesis in vitro. Addition of PGE2 alone or PGE2 in combination with prostacyclin reversed the inhibition of in vitro angiogenesis caused by NS-398. Daniel et al. [35] recently suggested that thromboxane A2 is a possible mediator of COX-II-dependent endothelial cell migration and angiogenesis. VEGF stimulated COX-II expression in human umbilical vein endothelial cells and induced COX-dependent proliferation of endothelial cells [36]. PGE2 reportedly stimulates VEGF expression with increased ERK2 and c-Jun N-terminal Kinase1 (JNK1) activation in rat gastric microvascular endothelial cells [37]. However, in this study VEGF expression, as determined by reverse transcription polymerase chain reaction analysis, did not differ between COX-II inhibitor-treated animals and controls (data not shown). In support of the previous reports, we suggest that COX-II expression in the CL is partially associated with angiogenesis in the process of luteinization. Angiogenesis in CL after follicular rupture may be partly modulated by arachidonic acid metabolites, which are abundant in newly formed CLs.

The results of the present study demonstrated that the nonselective COX enzyme inhibitor INDO reduces progesterone production by ovaries superovulated by exogenous eCG and hCG treatment. The specific COX-II inhibitor NS-398 was even more effective at reducing the serum levels of progesterone, but the specific COX-I inhibitor SC-560 had no effect. Lack of effect of inhibitors on in vitro progesterone synthesis by isolated luteal cells further confirmed this finding. However, reduced ovarian content of hemoglobin in combination with reduced capillary development as indicated by corrosion casts and endothelial cell markers indicated that lack of stimulation by factors controlling angiogenesis was the basis for loss of progesterone secretion that accompanied COX-II inhibition. Several agents, including VEGF, bFGF, and the angiopoietins [38] must be considered in future investigations evaluating ovarian PG control systems.


   ACKNOWLEDGMENTS
 
The authors are grateful to D.C. Johnson (University of Kansas Medical Center, Kansas City, KS) for critical reading of the manuscript.


   FOOTNOTES
 
1 This work was partially supported by a Grant-in-Aid for Scientific Research from the Promotion and Mutual Aid Corporation for Private Schools of Japan. Back

2 Correspondence: Kazuhiro Tamura, Department of Pharmacology, Tokyo University of Pharmacy and Life Science, Hachioji, Horinouchi 1432-1, Tokyo 192-0392, Japan. FAX: 81 426 76 4529; hiro{at}ps.toyaku.ac.jp Back

Received: 1 September 2002.

First decision: 25 September 2002.

Accepted: 23 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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