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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.008573
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BIOLOGY OF REPRODUCTION 68, 1674–1681 (2003)
DOI: 10.1095/biolreprod.102.008573
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Administration of a Nitric Oxide Synthase Inhibitor Counteracts Prostaglandin F2-Induced Luteolysis in Cattle1

Dariusz J. Skarzynski2,3, Jerzy J. Jaroszewski4, Mamadou M. Bah3, Katarzyna M. Deptula3, Beata Barszczewska4, Barbara Gawronska5, and William Hansel5

Division of Reproductive Endocrinology and Pathophysiology,3 Institute of Animal Reproduction and Food Research, PAS, Olsztyn 10-747, Poland Department of Pharmacology,4 Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, 10-719 Olsztyn, Poland Pennington Biomedical Research Center,5 Louisiana State University, Baton Rouge, Louisiana 70808


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to determine whether nitric oxide (NO) is produced locally in the bovine corpus luteum (CL) and whether NO mediates prostaglandin F2{alpha} (PGF2{alpha})-induced regression of the bovine CL in vivo. The local production of NO was determined in early I, early II, mid, late, and regressed stages of CL by determining NADPH-d activity and the presence of inducible and endothelial NO synthase immunolabeling. To determine whether inhibition of NO production counteracts the PGF2{alpha}-induced regression of the CL, saline (10 ml/h; n = 10) or a nonselective NOS inhibitor (N{omega}-nitro-L-arginine methyl ester dihydrochloride [L-NAME]; 400 mg/h; n = 9) was infused for 2 h on Day 15 of the estrous cycle into the aorta abdominalis of Holstein/Polish Black and White heifers. After 30 min of infusion, saline or cloprostenol, an analogue of PGF2{alpha} (aPGF2{alpha}; 100 µg) was injected into the aorta abdominalis of animals infused with saline or L-NAME. NADPH-diaphorase activity was present in bovine CL, with the highest activity at mid and late luteal stages (P < 0.05). Inducible and endothelial NO synthases were observed with the strongest immunolabeling in the late CL (P < 0.05). Injection of aPGF2{alpha} increased nitrite/nitrate concentrations (P < 0.01) and inhibited P4 secretion (P < 0.05) in heifers that were infused with saline. Infusion of L-NAME stimulated P4 secretion (P < 0.05) and concomitantly inhibited plasma concentrations of nitrite/nitrate (P < 0.05). Concentrations of P4 in heifers infused with L-NAME and injected with aPGF2{alpha} were higher (P < 0.05) than in animals injected only with aPGF2{alpha}. The PGF2{alpha} analogue shortened the cycle length compared with that of saline (17.5 ± 0.22 days vs. 21.5 ± 0.65 days P < 0.05). L-NAME blocked the luteolytic action of the aPGF2{alpha} (22.6 ± 1.07 days vs. 17.5 ± 0.22 days, P < 0.05). These results suggest that NO is produced in the bovine CL. NO inhibits luteal steroidogenesis and it may be one of the components of an autocrine/paracrine luteolytic cascade induced by PGF2{alpha}.

corpus luteum function, female reproductive tract, nitric oxide, ovulatory cycle, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Secretion of progesterone (P4) is crucial for determining the duration of the estrous cycle and for achieving a successful pregnancy. The bovine corpus luteum (CL) grows rapidly and regresses within 2 days at the time of luteolysis. The mechanisms that control the development and secretory function of the bovine CL involve many factors produced inside and outside of the CL [1–4]. Although prostaglandin (PG) F2{alpha} released from the uterus has been shown to cause regression of the CL of ruminant species [5], the neuroendocrine, paracrine, and autocrine mechanisms that regulate luteolysis in the cow are not yet fully understood. Studies examining the direct effects of PGF2{alpha} on pure populations of steroidogenic luteal cells [6–9] show that it does not inhibit basal P4 production by the large luteal cells and stimulates P4 production by the small luteal cells and by a mixture of large and small luteal cells [9, 10]. These results led researchers to postulate that some products of nonsteroidogenic cells of the bovine CL may mediate the luteolytic action of PGF2{alpha} [4, 11–15]. The CL is a heterogenous gland and, in addition to steroidogenic luteal cells and endothelial cells, it contains fibroblasts and immune cells [16]. Pate [11, 12] suggested that the immune cells and their secreted products, cytokines, are involved in the process of luteal regression. Thus, at the time of luteolysis, macrophages invade the bovine CL [17] and cytokines, especially tumor necrosis factor-{alpha} and interferon-{gamma}, participate in the apoptotic events that finally lead to structural luteolysis [18–21].

In addition to immune cells and cytokines, endothelial cells and their main secretory product, endothelin-1 (ET-1), may mediate the luteolytic action of PGF2{alpha} on bovine steroidogenic luteal cells [13–15, 22, 23]. Other studies suggest that ET-1 may be only one of the essential mediators of luteolysis [24, 25]. We previously suggested that NO is a good candidate to serve as a mediator of PGF2{alpha} action during luteolysis in cattle [25, 26]. Nitric oxide (NO) directly inhibits P4 secretion from bovine luteal cells [27, 28] and augments the action of extragonadal PGF2{alpha} on the CL, suggesting that it may be involved in the process of luteolysis [26]. The response of the target tissue (CL) to PGF2{alpha} can be modified by local factors that have autocrine or paracrine actions [10]. Finally, the inhibition of ovarian NO production by perfusion of the CL with a nonselective NO synthase (NOS) inhibitor (N{omega}-nitro-L-arginine methyl ester dihydrochloride, L-NAME) prolonged the duration of the estrous cycle in heifers [25]. Therefore, one might assume that NO is a component of an autocrine/paracrine cascade in the bovine CL and that it plays an important role in the regulation of functional and structural luteolysis. A number of substances produced locally in the CL, including ET-1, cytokines, and NO may be involved in maintaining an equilibrium between luteal development and regression.

The present study was undertaken to determine whether NO mediates PGF2{alpha}-induced regression of the bovine CL in vivo. We also examined whether NO may be produced locally in the bovine CL, as indicated by the presence of NADPH-diaphorase (NADPH-d) activity and distribution of endothelial and inducible NOS isoforms (eNOS and iNOS, respectively) in the CL at different stages of the estrus cycle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All animal procedures were approved by the local animal care and use committee (agreement 4/2001/N).

In Vitro Studies

NADPH-d staining and immunocytochemistry for eNOS and iNOS CL from cyclic crossbred Holstein/Polish Black and White (75%/25%; respectively) heifers (n = 20) were obtained at a local abattoir within 10 min after slaughter. The stages of the estrous cycle were estimated by macroscopic observation of the ovary and uterus, as described by Miyamoto et al. [29]. These observations allowed the classification of the CL into five stages: early I (Days 2–4; n = 4), early II (Days 5–7; n = 4), mid (Days 8–12; n = 4), late (Days 14–17; n = 4), and regressed (Days 19–21; n = 4). Immediately after collection, CL were placed into 4% paraformaldehyde solution (Fluka Chemie; Buchs, Switzerland) in 0.1 M phosphate buffer (PB) for an initial fixation of 2 h (during transport of the tissues from the slaughterhouse to the laboratory). CL were then cut into smaller pieces and fixed in 4% paraformaldehyde for an additional 4 h. Fixed tissues were stored in 18% sucrose (POCH; Gliwice, Poland) in PB with 0.01% sodium azide.

Cryostat sections (8 µm) were used for immunocytochemistry and histochemistry as previously described [30]. To demonstrate NADPH-d activity, cryostat sections of CL were incubated at 37–38°C in a freshly prepared solution of ß-NADPH (5 mg/ml; Sigma, St Louis, MO), nitroblue tetrazolium (0.5 mg/ml; Sigma-Aldrich; Steinhem, Germany), and Triton X-100 (5 µl/ml; Merck, Darmstadt, Germany) in 0.1 M PB pH 7.4 for l h. Control sections were exposed to the staining solution without NADPH. All CL samples were assayed in the same time course and at least three replicates per animal were examined. The activity of NADPH-d in bovine CL was measured as distribution of formazan deposits (the end product of the histochemical reaction). The observations and photographs were made using a light microscope (Olympus IMT-2; Olympus Optical, Tokyo, Japan).

For immunocytochemistry, consecutive sections were rinsed in 0.05 M Tris-hydroxylmethyl aminomethane (TBS; Sigma), then placed in ethanol in ascending concentrations (50%, 70%, 96% and absolute alcohol). The sections were treated with 1% H2O2 in methanol (Sigma) for 30 min to block endogenous peroxidases and then in 0.75% glycine (Sigma) in TBS for 30 min to block free aldehyde groups. After rinsing in TBS, the sections were incubated overnight with primary antibody against eNOS (diluted 1:50; a mouse monoclonal antibody directed against amino acids 1030–1209 of human eNOS; Transduction Laboratories, Lexington, KY) or iNOS (diluted 1:50; a mouse monoclonal antibody directed against amino acids 961–1144 of mouse macrophage iNOS; Transduction Laboratories) as previously described [30].

The intensity of histochemical (NADPH-d) and histoimmunological reaction (iNOS, eNOS) in the tissues was estimated by measuring the optical density in a defined area using the PC-IMAGE system (MicroImage; Olympus, Olympus Optical) as described previously [30, 31].

In Vivo Studies

Normally cycling crossbred Holstein/Polish Black and White (75%/25% respectively) heifers (18–20 mo of age and 350–450 kg body weight) were injected i.m. with 500 µg of a PGF2{alpha} analogue, cloprostenol (aPGF2{alpha}; Bioestrophan, Biowet, Gorzow Wielkopolski, Poland) during the luteal phase to induce luteolysis and estrus. The onset of estrus was taken as Day 0 of the estrous cycle.

For infusion of either saline or L-NAME, a catheter was inserted into the posterior aorta abdominalis through the coccygeal artery, as described previously [32] on Day 14 of the subsequent estrous cycle (Day 0 = estrus). The tip of cannula was positioned in the aorta 60–65 cm anterior to the point of insertion and just cranial to the origin of the ovarian artery and caudal to the renal artery (Fig. 1). This placement allowed infused drugs to be transported by the bloodstream directly into the reproductive tract (Fig. 1). A second catheter was inserted into the jugular vein for frequent collection of blood samples.



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  		FIG. 1. Schema of the bovine female tract showing the position of the tip of the cannula in relation to the ovarian artery (adapted from [64]; permission given)

Experiment 1: Preliminary Study

Eighteen crossbred Holstein/Polish Black and White heifers were used to establish the effective dose of L-NAME (Sigma). On Day 15 of the estrous cycle six animals were infused into the aorta abdominalis for 2 h with 20 ml of saline. Thirty minutes after the beginning of saline infusion, three heifers were injected with 2 ml of saline (n = 3) and three with 100 µg of cloprostenol; the dose was based on our previous data [33]. Twelve heifers were infused for 2 h with 50, 100, 200, or 400 mg L-NAME/h (n = 3 per dose) into the abdominal aorta and at 30 min of infusion 100 µg of aPGF2{alpha} was given. Peripheral blood samples were collected from a jugular vein at 10-min intervals (beginning 1 h before and continuing until 1 h after infusions). After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. The plasma was separated by centrifugation (2000 x g; 10 min at 4°C) and stored at -20°C until P4 determinations were made.

Experiment 2: Influence of L-NAME on PGF2{alpha}-Induced Luteolysis

To test the hypothesis that inhibition of NO production may counteract the luteolytic action of PGF2{alpha}, saline alone (10 ml/h; n = 10 animals) or L-NAME (400 mg/h in 20 ml of saline; n = 9 animals) was infused for 2 h into the aorta abdominalis. Thirty minutes after the beginning of saline or L-NAME infusion, saline (2 ml; n = 8 animals) or aPGF2{alpha} (100 µg in 2 ml; n = 11 animals) was injected into the aorta abdominalis. Blood samples were collected every 10 min during the experiment. From Day 15 of the estrous cycle, blood was collected once daily until Day 22 after the first estrus. Concentrations of P4, 3,14-dihydro,15-keto-prostaglandin F2{alpha} (PGFM) and nitrite/nitrate in the plasma samples were measured. All animals were checked for estrus at 12-h intervals after aPGF2{alpha} or saline injection.

Hormone Determinations

P4 concentrations in the plasma samples were assayed using a direct enzyme immunoassay (EIA). P4 labeled by horseradish peroxidase (P4-HRP) was used as a tracer. Cross-reactivities of the anti-P4 serum (donated by Dr. S. Okrasa, University of Warmia and Mazury in Olsztyn, Poland), were determined by comparing the inhibition of binding of P4-HRP to antiserum. Results were as follows: 100% with P4; 38.9% with pregnenolone; 11.1% with 17{alpha}-hydroxy-progesterone; 9.8% with 17ß-estradiol; 1.2% with dihydrotestosterone and testosterone; and less than 0.5% with 11-desoxycortisol, estrone, cortisol, and 4-androsten-3,17-dione. In brief, 25-µl aliquots of standards or serum samples were incubated in the dark at room temperature for 18–24 h with 100 µl of P4 antiserum (1:100 000 final dilution) and with 100 µl of P4-HRP (1:150 000 final dilution) in duplicates in 96-well ELISA plates (Corning Inc., Corning, NY) coated with ovine anti-rabbit secondary antibody. After discarding the reagents, the plates were washed three times with 300 µl of Tween-80 (0.05%) and 150 µl of substrate buffer with 3,3', 5,5' tetramethylbenzidine (Sigma) added to each well. The plates were further incubated at 36°C for 40 min in the dark. The reaction was stopped by adding 50 µl of 2 M H2SO4 to each well. The absorbance was measured at 450 nm with a plate reader (Labsytem, Helsinki, Finland). The P4 standard curve ranged from 0.39 pg/ml to 25 ng/ml and the effective dose for 50% inhibition (ID50) of the assay was 2.85 ng/ml. The intraassay and interassay coefficients of variation averaged 6.6% and 8.4%, respectively.

The plasma concentrations of PGFM were determined with an EIA as previously described for PGF2{alpha} [27] using HRP-labeled PGFM and anti-PGFM serum (WS4468-5; donated by Dr. W.J. Silvia, University of Kentucky, Lexington, KY). Cross-reactivities of the anti-PGFM serum, validated by comparing the inhibition of binding of HRP-labeled PGFM to antiserum, were as follows: PGFM, 100%; PGE2, 0%; PGE1, 18%; PGA1, 10%; PGA2, 4.6%; PGB2, 6.7%; PGD2, 0.13%; PGF2{alpha}, 2.8%; PGJ2, 14%; and 15-keto PGE2, 0.05%. The PGFM standard curve ranged from 32.5 pg/ml to 8000 pg/ml, and the ID50 of the assay was 315 pg/ml. The intraassay and interassay coefficients of variation were on average 7.6% and 10.4%, respectively.

Nitrate/Nitrite Determination in Plasma

Plasma concentrations of nitrite/nitrate, the stable metabolites of NO, were measured by a colorimetric method using the Griess reaction as described by Green et al. [34]. The assay sensitivity was 0.065 µg/ml and the standard curve ranged from 0.05 µg/ml to 6.9 µg/ml. The intraassay and interassay coefficients of variation were on average 8.6% and 15.4%, respectively.

Statistical Analysis

The intensity of NADPH-d activity and histoimmunological reaction, the length of the estrous cycle, and the total amount of released P4, PGFM, and stable metabolites of NO (NO2-/NO3-), represented as the area under the curve (relative units; Table 1) were analyzed using ANOVA with the Bonferroni multiple comparison test (ANOVA; GraphPAD PRISM, San Diego, CA). Least squares means and standard errors of the mean were reported. Plasma concentrations of hormones (P4, PGFM) and stable metabolites of NO (NO2-/NO3-) were analyzed using ANOVA with repeated measures (ANOVA; GraphPAD PRISM).


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  		TABLE 1. Effects of 2-h of saline or L-NAME infusion and saline or cloprostenol injection on total amounts of released progesterone, stable metabolites of NO, and PGFM in heifers on Day 15 of the estrous cycle.*


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Studies: NADPH-d Histochemistry and NOS Immunohistochemistry

Bovine CL showed NADPH-d activity (a marker for NO synthase) in luteal tissue and in blood vessels during the entire estrous cycle (Fig. 2a). However, the intensity of the histochemical reactions differed among stages of the estrous cycle (Fig. 2d). The intensity of reaction (arbitrary units, optical density analysis, MicroImage) increased from early I until the late luteal phase of the estrous cycle and than decreased (Fig. 2d). Figure 2b shows eNOS-like immunoreactivity in the endothelium of blood vessels and in luteal cells. Immunostaining of iNOS was also observed in luteal cells and, in lesser amounts, in the endothelium of blood vessels (Fig. 2c). Endothelial NOS and iNOS increased greatly in late CL (Fig. 2d).



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  		FIG. 2. Histochemical activity of NADPH-d (a), eNOS (b), and iNOS (c) in the bovine CL at the late luteal phase (asterisks indicate blood vessels, arrows indicate luteal cells); (d) a schematic illustration of the activity of enzymes (relative units; image analysis, MicroImage, Olympus). Different subscript letters indicate significant differences (P < 0.05). Original magnification x20

Experiment 1: Preliminary Study

There was a dose-dependent effect of L-NAME on aPGF2{alpha}-stimulated luteolysis (Fig. 3; P < 0.05). Injection of aPGF2{alpha} shortened the cycle length (17.7 ± 0.3 days) compared with that of a control heifer injected with saline (22.3 ± 0.3 days). Three low doses of L-NAME (50, 100, and 200 mg/h) failed to inhibit regression of the CL induced by injection of aPGF2{alpha} (cycle durations of 17.3 ± 0.3, 18.0 ± 0.6, and 18.6 ± 0.9 days, respectively). The luteolytic action of aPGF2{alpha} was blocked by 400 mg/h of L-NAME, as shown by the longer cycle duration (23.0 ± 0.9 days). On the basis of this preliminary experiment, the dose of 400 mg/h of L-NAME was chosen for further study.



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  		FIG. 3. Concentrations of P4 in peripheral blood plasma of heifers infused with saline or several doses of L-NAME (an inhibitor of NOS) for 2 h and injected at 30 min of infusion with saline or 100 µg cloprostenol (a [aPGF] PGF2{alpha}) on Day 15 of the estrous cycle. The control group (control) was infused and injected with saline only. Saline, L-NAME, and cloprostenol were administered into the aorta abdominalis

Experiment 2: Influence of L-NAME on PGF2{alpha}-Induced Luteolysis

Administration of 100 µg of aPGF2{alpha} significantly increased plasma nitrite/nitrate concentrations in heifers infused with saline (P < 0.05; Fig. 4, Table 1). Although aPGF2{alpha} temporarily increased P4 secretion in heifers infused with saline, it was decreased within 60 min after treatment compared with the pretreatment values or with the controls injected and infused with saline (Fig. 5; P < 0.05). Infusion of L-NAME inhibited plasma concentrations of nitrite/nitrate (P < 0.05; Fig. 4; Table 1) and concomitantly stimulated P4 secretion (P < 0.001; Fig. 5; Table 1).



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  		FIG. 4. Effect of 2-h infusion of saline or L-NAME (400 mg/h) and injection of saline or 100 µg cloprostenol (aPGF) at 30 min of infusion on P4 concentrations in peripheral blood plasma of heifers on Day 15 of the estrous cycle. Different subscript letters indicate significant differences (P < 0.05)



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  		FIG. 5. Effect of 2-h infusion of saline or L-NAME (400 mg/h) and injection of saline or 100 µg cloprostenol (aPGF) at 30 min of infusion on stabile NO metabolite (NO3-/NO2-) concentrations in peripheral blood plasma of heifers on Day 15 of the estrous cycle. Different subscript letters indicate significant differences (P < 0.05)

The concentrations of PGFM before treatments were similar in all groups and ranged from 60 to 90 pg/ml (Fig. 6). Injection of aPGF2{alpha} during saline infusion evoked a sharp increase in plasma concentrations of PGFM (Fig. 6; Table 1). Infusion of L-NAME temporarily mitigated the stimulatory effect of aPGF2{alpha} on PGFM compared with that of the group infused with saline and injected with aPGF2{alpha} (P > 0.05). Treatment of heifers with L-NAME and saline, or saline and saline, had no effect on plasma concentrations of PGFM (P > 0.05).



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  		FIG. 6. Effect of 2-h infusion of saline or L-NAME (400 mg/h) and injection of saline or 100 µg cloprostenol (aPGF) at 30 min of infusion on PGFM concentrations in peripheral blood plasma of heifers on Day 15 of the estrous cycle. Different subscript letters indicate significant differences (P < 0.05)

The effects of L-NAME and aPGF2{alpha} on the concentration of P4 during the estrous cycle are shown in Figure 7. Injection of aPGF2{alpha} during saline infusion shortened (17.5 ± 0.22 days; P < 0.05) the cycle length compared with that of the group infused and injected with saline (21.5 ± 0.65 days). In the group infused with 400 mg/h of L-NAME and injected with aPGF2{alpha}, the luteolytic effect of aPGF2{alpha} was inhibited compared with that of animals infused with saline and injected with aPGF2{alpha} (22.6 ± 0.65 days versus 17.5 ± 0.22 days; P < 0.01). The length of the cycle in the group infused with 400 mg/h of L-NAME and injected with saline was 21.5 ± 0.85 days. There were no differences in the length of the estrous cycle (P > 0.05) between animals infused with L-NAME and injected with saline or aPGF2{alpha} and heifers treated with saline only.



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  		FIG. 7. Concentrations of P4 in peripheral blood plasma of heifers infused for 2 h with saline or L-NAME (400 mg/h) and injected with or saline or an 100 µg cloprostenol (aPGF) on Day 15 of the estrous cycle


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data obtained in the current study support and extend our previous observations [25–27] and provide new evidence that NO may be one of the important factors involved in the regulation of luteolysis in cattle. Infusion of L-NAME stimulated P4 secretion, decreased plasma concentrations of nitrite/nitrate, prevented aPGF2{alpha}-induced luteolysis, and extended the functional life of the CL. We recently demonstrated that NO directly inhibits P4 secretion from bovine luteal cells in vitro [27, 28]. Infusion of L-NAME by a microdialysis system in vivo caused a marked increase in P4 release at Days 17 and 18 of the cycle and prolonged the functional life of CL to at least 25 days [25]. The direct inhibitory effect of NO on P4 production in bovine luteal cells [27, 28], the stimulatory effect of L-NAME on P4 secretion, and the stimulatory effect of PGF2{alpha} on nitrite/nitrate concentrations in conscious heifers all suggest that NO plays an important role as an autocrine/paracrine factor in the regulation of luteal steroidogenesis.

We observed NADPH-d activity in bovine luteal cells and endothelial cells. The most intense staining was observed at the mid and late luteal stages. NADPH-d co-localizes with all known NOS isoforms [35, 36] and serves as a marker for NOS. NOS exists in three major isoforms. One of them, the constitutive-neuronal isoform, has been identified in bovine ovarian nerves [37]. In the rodent and human ovary, NO is produced by the other two isoforms, eNOS and iNOS [38, 39]. Our data demonstrate that eNOS is expressed in bovine luteal cells, as well as in the endothelium of the blood vessels penetrating the CL. The endothelial isoform of NOS was observed with the most intensive immunolabeling in the late luteal phase of the cycle. These observations agree with previous data showing that calcium-dependent eNOS is strongly expressed in ovaries and CL of many species in a stage-dependent manner [39–42]. Moreover, we showed that iNOS is also expressed in bovine CL, with the highest intensity at the late luteal stage (Fig. 2c and d). This reaction was found not only in and around blood vessels, but also in the luteal cells. In contrast to our data, iNOS was observed only in endothelium and around blood vessels in porcine CL [43–45]. However, in situ hybridization studies localized iNOS to granulosa and luteal cells of rat ovaries [38]. In mouse ovaries, iNOS expression was found in the external layers of CL and strong staining was observed in degenerating CL in nonparenchymal cells in the entire CL [41].

In most organs, including ovaries, iNOS is strongly expressed in response to an immune stimulus, such as infection or trauma [44, 45]. The physiological relevance of the expression of iNOS in normal ovaries of several species and at all stages of the estrous cycle is unclear [35]. However, the mechanisms that control luteolysis in cattle are described as immune cell-dependent and immune response-dependent processes [19–21]; therefore, all these findings provide evidence that luteal cell-derived, vascular endothelium-derived, or immune cell-derived NO (or a combination of these) plays an important role in CL function, especially with regard to regulation of steroidogenesis and PG secretion [25–27, 35].

An inhibitor of NOS, L-NAME, did not affect the viability of cultured midcycle luteal cells [19], nor were stable metabolites of NO detected in cells undergoing apoptosis after cytokine treatment [19]. However, in several tissues and organs, including the ovary, the expression of iNOS is correlated with cytotoxic/cytostatic events and results in a sustained synthesis of NO, which in turn induces apoptotic cell death [35, 46]. In fact, [Ca2+]i-mobilizing agents and cytokines elicit an apoptotic response in the vascular endothelial cells through mechanisms that require NO synthesis [47]. We have also found that spermine NONOate (an NO donor) strongly reduces the viability of late luteal cells, whereas L-NAME (an NOS inhibitor) had opposite effects (Korzekwa, Okuda, Jaroszewski, Skarzynski, unpublished data). Therefore, the greater concentrations of both eNOS and iNOS observed in late-stage bovine CL in our study suggest that NO produced by NOS isoforms may be involved in both the structural and functional changes that occur in the CL at the time of luteolysis.

Although injections of PGF2{alpha} or its analogues induced several parameters of functional and structural luteolysis [32, 48, 49], direct exposure of the bovine CL to PGF2{alpha} via the microdialysis system stimulated P4 secretion [28, 50]. These results suggest that PGF2{alpha} is most effective as a luteolytic factor when it reaches the CL through blood vessels [51]. Several studies suggest that ET-1 may play a role in PGF2{alpha}-induced luteolysis [4, 13–15, 22, 23, 50]. Other studies suggest that ET-1 may not be the only essential mediator of luteolysis in the ewe [24, 25, 52]. Although ET-1 and angiotensin-II are elevated 2 h after PGF2{alpha} treatment [4, 51], the effects of PGF2{alpha} action on the bovine CL are observed at 30 min after treatment [32, 49]. Recently, Acosta et al. [23] showed that blood flow within the bovine CL initially increased at 0.5–2 h, decreased at 4 h to the level observed at 0 h, and then decreased to a lower level from 8 h after PGF2{alpha} treatment. In our study, the plasma concentrations of nitrite/nitrate were elevated during the first 2 h after aPGF2{alpha} injection; after that they slowly decreased, indicating that the first factor released by PGF2{alpha} may be NO. NO is a potent vasodilator and increases blood flow. Thus, our findings can explain the recent data of Acosta et al. [23] showing that blood flow in the bovine CL is increased just after PGF2{alpha} treatment, not decreased, as is commonly assumed. During the first 2 h of PGF2{alpha} action, NO may act directly or via PGE2 [27] to relax vascular smooth muscle cells and maintain the luteal blood flow required for invasion of immune cells into the CL during luteolysis [17, 20].

In the ewe, a single injection of ET-1 administered systemically at midcycle increased concentrations of P4 in plasma 4 h after treatment and then reduced mean concentrations of plasma P4 for the remainder of the cycle [52]. These findings suggest that ET-1 action during luteolysis may represent an indirect effect mediated by other factors [52]. Therefore, an alternative model of CL regression may include a sharp ET-1 elevation just after PGF2{alpha} action and then release of some factors that are directly responsible for the early luteolytic events. ET-1 and NO may interact on several regulatory levels [53, 54], but the precise relationship between these components is not clear. ET-1 is a potent factor in inducing NO production in bovine [55] and rat endothelial cells [56]. Therefore, it is possible that NO may be released by ET-1 during regression of bovine CL.

It has been demonstrated that exogenous PGF2{alpha} stimulates the utero-ovarian release of PGF2{alpha} in ewes [57, 58] and cows [32, 59]. In our study, L-NAME inhibited PGF2{alpha} luteolytic action in bovine CL. However, it did not influence the PGF2{alpha} autoamplification system in the uterus or the ovary. Although aPGF2{alpha} evoked a sharp increase in PGFM level in the blood, infusion of L-NAME only temporarily mitigated the stimulatory effect of aPGF2{alpha} on PGFM. As mentioned previously, PGF2{alpha} did not prove to be directly luteolytic on the luteal cells [7], or in microdialyzed bovine CL [60]. Therefore, it has been suggested in several studies that PGF2{alpha} may play a role as a luteotropic agent in an autocrine/paracrine manner [6, 7, 9, 10]. Our study showed that L-NAME inhibited aPGF2{alpha}-induced luteolysis but did not influence the PGF2{alpha} autoamplification loop. Thus, a direct luteotropic effect of PGF2{alpha} on bovine CL may be observed when luteolytic (vascular, NO-dependent) mechanisms are blocked.

In summary, our previous data showed that NO may directly inhibit P4 secretion in cattle [27, 28]. The greater effects of PGF2{alpha} on luteal cells previously exposed to NO suggests that priming of bovine CL by NO is needed for complete luteal regression [26]. This suggestion is confirmed by data showing that an inhibition of ovarian NO production by perfusion of the CL with an NOS inhibitor prolonged the duration of the estrous cycle [25]. The present results demonstrate that the inhibition of NO production (administration of L-NAME) abolished the luteolytic action of aPGF2{alpha}. Moreover, we directly showed that NO may be produced in bovine CL. The highest concentrations of NOS activity are found in CL of the late luteal phase (Days 14–17 of the estrous cycle), before uterine luteolytic PGF2{alpha} is released (Days 18–20) [61, 62]. The first subluteolytic pulses of PGF2{alpha}, which are responsible for the early luteolytic events, may be observed at 12–24 h before P4 decline in cows (Days 17–19 of the estrous cycle) [61, 62]. Moreover, peaks of luteal PGF2{alpha} and luteolytic leukotrienes (types B and C) have been demonstrated on Day 18 in heifers undergoing spontaneous luteolysis and their frequency increased within the 12-h period during which the onset of P4 decline occurred [63]. However, there might be no overlap between CL from Days 14–17 (highest concentrations of NOS activity) and Days 18–20 of the estrous cycle, when functional and structural luteolysis occurs. Therefore, a complete explanation of the role of NO in initiation of functional luteolysis and the precise relationship between PGF2{alpha}, ET-1, and NO in the process of luteal regression in the bovine CL remains to be determined.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Marek Bogacki (Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, LA) for his peer review and editorial assistance. We thank Dr. Stanislaw Okrasa of the University of Warmia and Mazury in Olsztyn, Poland, for the P4 antiserum; Dr. William Silvia of University of Kentucky, Lexington, KY, for antiserum of PGFM. The authors also thank Mrs. Hanna and Mr. Jerzy Kostuch of the Animal Farm "Farmer" in Zalesie, Poland, for excellent cooperation and agreement to use animals for in vivo experiments.


   FOOTNOTES
 
1 This research was supported by grants KBN 5P06K012 19 and KBN 5P06K 003 21 from the State Committee for Scientific Research and by The Gordon and Mary Cain Foundation. Back

2 Correspondence: Dariusz J. Skarzynski, Division of Reproductive Endocrinology and Pathophysiology, Institute of Animal Reproduction and Food Research, PAS, Tuwima-St 10, Olsztyn 10-747, Poland. FAX: 48 89 524 03 47; skadar{at}pan.olsztyn.pl Back

Received: 22 June 2002.

First decision: 8 July 2002.

Accepted: 25 November 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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