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BOR - Papers in Press, published online ahead of print December 27, 2006.
Biol Reprod 2006, 10.1095/biolreprod.106.053280
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BIOLOGY OF REPRODUCTION 76, 619–627 (2007)
DOI: 10.1095/biolreprod.106.053280
© 2007 by the Society for the Study of Reproduction, Inc.

research-article

Infusion of Exogenous Tumor Necrosis Factor Dose Dependently Alters the Length of the Luteal Phase in Cattle: Differential Responses to Treatment with Indomethacin and L-NAME, a Nitric Oxide Synthase Inhibitor1

Dariusz J. Skarzynski 2 3, Izabela Woclawek-Potocka 3, Anna Korzekwa 3, Mamadou M. Bah 3, Katarzyna Piotrowska 3, Beata Barszczewska 4, and Kiyoshi Okuda 5

Department of Reproductive Immunology,3 Institute of Animal Reproduction and Food Research, PAS, Olsztyn 10-747, Poland Department of Pathology and Pharmacology,4 Faculty of Veterinary Medicine, Warmia and Mazury University in Olsztyn, Olsztyn 10-975, Poland Laboratory of Reproductive Endocrinology,5 Graduate School of Natural Sciences and Technology, Okayama University, Okayama 700-8530, Japan

ABSTRACT

We examined whether prostaglandins (PGs) and nitric oxide (NO) mediate tumor necrosis factor (TNF) actions in the estrus cycle. On Day 14 of the cycle, the following solutions were infused into the aorta abdominalis of a total of 51 heifers (Experiments 1 and 2): saline; 1 or 10 µg of TNF; 480 mg indomethacin (INDO), an inhibitor of prostaglandin H synthase; 800 mg L-NAME, an inhibitor of NO synthase; and TNF (1 or 10 µg) in combination with INDO or L-NAME. TNF at 1 µg infused directly into aorta abdominalis increased the level of PGF2alpha and decreased the level of progesterone (P4) in the peripheral blood and shortened the estrus cycle. The high TNF dose stimulated P4 and PGE2 and prolonged the corpus luteum (CL) lifespan. INDO blocked the effects of both TNF doses on the CL lifespan and hormone output. L-NAME completely blocked the effects of the luteolytic TNF dose, whereas the effects of the luteotropic TNF dose were not inhibited. In Experiment 3 (Day 14), saline or different TNF doses were infused into the jugular vein (n = 9) or into the uterine lumen (n = 18). The CL lifespans of the different groups were not different when TNF was infused into the jugular vein. Although high TNF doses (1 and 10 µg) infused into the uterine lumen prolonged the CL lifespan, low doses (0.01 and 0.1 µg) induced premature luteolysis. We suggest that the actions of exogenous TNF on the CL lifespan depend on PG synthesis stimulated by TNF in the uterus. TNF at low concentrations initiates a positive cascade between uterine PGF2alpha and various luteolytic factors, including NO, to complete premature luteolysis. PGE2 is a good candidate mediator of the luteotropic actions of exogenous TNF action.

corpus luteum function, cytokine, estrus cycle, necrosis factor, ovulatory cycle, progesterone, prostaglandins, tumor, uterus

INTRODUCTION

The mechanisms that control the formation and maintenance of the corpus luteum (CL) and eventual luteolysis in cattle are known to be immune cell- and cytokine-dependent processes [1–5]. The CL lifespan has been found to decrease with increasing doses of tumor necrosis factor (TNF) in vivo [6]. Low doses of TNF infused directly into the aorta abdominalis increase prostaglandin (PG)F2{alpha} and nitric oxide (NO) concentrations and decrease progesterone (P4) levels in the peripheral blood. Consequently, these processes lead to premature luteolysis in cattle [6]. However, high doses of TNF stimulate P4 and PGE2 secretion, which results in prolongation of the estrus cycle [6]. These data, together with the results obtained from earlier in vitro studies [3, 4, 7–11], suggest that TNF at low concentrations plays an important role in luteolysis and consequently, leads to functional and structural regression of the CL in cattle. On the other hand, exogenous TNF at high concentrations prolongs the luteal phase of the estrus cycle in cattle by stimulating PGE2 production and indirectly stimulating P4 secretion [6, 10, 12–15]. Thus, TNF seems to play more than one role in the regulation of the estrus cycle and pregnancy in cattle [16, 17].

Functional tumor necrosis factor receptors (TNFRSF1A and TNFRS1B) have been found in the bovine cyclic endometrium [7], as well as in all the major types of CL cells [3, 4, 12, 18]. Therefore, the ability of TNF to modulate the lifespan of the bovine CL [6] may be a result of its direct action on CL cells and may depend on the effect of TNF on PG secretion in the bovine endometrium. In fact, from Day 13 until Day 18 of the estrus cycle, the expression levels of TNF protein [9] and of TNF and TNFRSF1A mRNAs increase in the bovine CL [3, 9]. TNF is released locally by the bovine CL during PGF2{alpha}-induced and spontaneous luteolysis [19]. Moreover, about 2 h after the start of PGF2{alpha}-induced luteolysis, the level of TNF mRNA increases in the bovine CL [20]. Therefore, TNF has been suggested to serve as a mediator/modulator of the luteolytic action of PGF2{alpha} within the CL [21, 22]. In the ovine CL, TNF interacts with PGF2{alpha} and endothelin-1 (EDN1), and together they directly inhibit local P4 release [21, 22]. During luteolysis, TNF in concert with other cytokines may directly induce apoptosis, resulting in regression of the CL and shortening of the estrus cycle [3, 4, 11, 23, 24]. Moreover, TNF directly stimulates the proliferation of the cells in the follicles of various species [17, 25, 26]. Therefore, it is possible that TNF regulates the estrus cycle [6] by acting locally within the bovine CL.

Exogenous TNF may also affect the lifespan of the bovine CL by stimulating PGF2{alpha} and PGE2 production in endometrial stromal cells, as well as in steroidogenic and endothelial cells of the CL [3, 4, 8, 10, 14, 15, 18, 23] via the activation of phospholipase (PL)A2 [8, 10] and partially via the activation of NO synthase [8, 27]. Nitric oxide has recently been found to be an important mediator of PGF2{alpha} action in cattle [28–31]. Therefore, we hypothesize that endocrine mechanisms, such as utero-ovarian interactions and uterine PG production and action, are the major and primary mechanisms by which TNF affects CL lifespan in the cow. Therefore, the aim of the present in vivo study was to investigate whether the effect of TNF on the estrus cycle is mediated by the actions of PG and NO in cattle. The production of PG and NO in the bovine reproductive tract was inhibited using respective blockers before TNF treatment. To evaluate the actions of exogenous TNF, we measured the concentrations of P4 and the luteolytic and luteotropic metabolites of arachidonic acid (PGF2{alpha} and PGE, respectively) in the peripheral blood, and we looked for signs of estrus. We also examined whether the effect of exogenous TNF on the lifespan of bovine CL depends on utero-ovarian mechanisms, including local PG production in the uterus, rather than on the systemic effects of TNF.

MATERIALS AND METHODS

Animals and Surgical Procedures

Normally cycling Holstein/Polish Black and White (75% and 25%, respectively) heifers (18–20 months of age; Wroblik Animal Farm, Poland) were used for the present studies. Two to three weeks after weighing and choosing the animals for experiments (only animals that weighed 390–400 kg were chosen), the estrus cycle was synchronized using implants of a progesterone analogue (Crestar, Intervet, Holland), as described previously [6, 27]. The onset of estrus, which was confirmed by observing the signs of estrus, was defined as Day 0 of the estrus cycle. All animal procedures were approved by the Polish Local Animal Care and Use Committee (agreement no. 23/2004/N).

Cows were infused with one of four solutions on Day 14 of the subsequent estrus cycle through a catheter inserted into the posterior aorta abdominalis through the coccygeal artery, as described previously [28]. Crestar, cloprostenol, sedazin, polocainum hydrochloricum, and other veterinary drugs and materials used in the present study were gifts from Centrowet (Olsztyn, Poland). The solution was either saline or saline that contained the non-selective prostaglandin H synthase (PTGS) inhibitor indomethacin (INDO; Polpha, Gorzow Wielkopolski, Poland), the non-selective NO synthase inhibitor NG-nitro-L-arginine methyl ester dihydrochloride (L-NAME; Cayman Chemical Co., Ann Arbor, MI) or recombinant human TNF(rhTNF HF-13; kindly donated by Dainippon Pharmaceutical Co., Ltd., Osaka, Japan). The animals were premedicated i.m. with xylazine (Sedazin; Biowet Pulawy, Poland) at a dose of 25–30 mg/animal, and local epidural anesthesia was induced by injecting 4 ml of 2% procaine hydrochloride (polocainum hydrochloricum) between the first and second coccygeal vertebrae. The tip of the cannula was positioned in the aorta 60–65 cm ahead of the point of insertion, just cranial to the origin of the ovarian artery and caudal to the renal artery [28]. This placement allowed infused reagents to be transported by the bloodstream directly into the reproductive tract. A second catheter was inserted into the jugular vein for frequent collection of blood samples.

Experiment 1: A Preliminary Study

Twelve heifers were used to study the effects of L-NAME and INDO on the duration of the estrus cycle. On Day 14 of the estrus cycle, four heifers received an infusion of 20 ml of saline into the aorta abdominalis over a period of 2 h (Control group). Four other animals were infused into the aorta abdominalis with 240 mg/h INDO for 2 h. The remaining four heifers were infused in the same way with 400 mg/h of the NOS inhibitor L-NAME. The doses of inhibitors were established in previous studies [27, 31].

Peripheral blood samples were collected from the jugular vein on every third day after the beginning of the estrus cycle (Days 0, 3, 6, 9, and 12 of the cycle). After Day 14 of the cycle, blood was collected once daily until Day 16 following the first estrus and then every two days (Days 18, 20, and 22 of the cycle). Blood plasma was separated by centrifugation at 2000 x g for 10 min at 4°C, and stored at –20°C until P4 was assayed. Animals were monitored for signs of estrus every 12 h after TNF or saline treatment.

Experiment 2: Influences of INDO and L-NAME on the Activity of TNF on the Estrus Cycle

Three experiments were carried out on Day 14 of the estrus cycle, to test the hypothesis that PGs and NO mediate the effect of TNF on the estrus cycle. Thirty-nine heifers were used in these experiments.

Experiment 2.1. (Control groups): To show both the luteolytic and luteoprotective effects of exogenous TNF on the estrus cycle, saline (20 ml) was infused for 2 h into the aorta abdominalis (n = 12). Thirty minutes after the beginning of saline infusion, 10 ml of saline (n = 4; internal control) or TNF at a luteolytic (1 µg; n = 4) or luteotropic dose (10 µg, n = 4) in 10 ml of saline was slowly injected (over 30 min) into the aorta abdominalis. The doses of TNF were based on our previous study [6]. For the three cows in each group (control, 1 µg TNF and 10 µg TNF infused), the results obtained in the present study reflect those obtained in the previous study [6]. The data for one animal from each group (control, 1 µg TNF, and 10 µg TNF) represent novel data. In total, four cows were used in each experiment and the data ( see Figures 2a, 3a, and 4a) are a combination of new (n = 1) and previous data (n = 3) [6].

Experiment 2.2. To test the hypothesis that inhibition of PG production counteracts the action of exogenous TNF on CL lifespan, INDO (240 mg/h in 20 ml of saline; n = 15) was infused for 2 h into the aorta abdominalis. Thirty minutes after the start of INDO infusion, saline (10 ml, n = 5; internal control) or TNF at a luteolytic (1 µg; n = 5) or luteotropic dose (10 µg, n = 5; both in 10 ml of saline) was administered (over 30 min) into the aorta abdominalis.

Experiment 2.3. To test the hypothesis that inhibition of NO production counteracts the actions of exogenous TNF on the length of the luteal phase, L-NAME (400 mg/h in 20 ml of saline; n = 12) was infused for 2 h into the aorta abdominalis. Thirty minutes after the beginning of L-NAME infusion, saline (10 ml; n = 4; internal control) or TNF in a luteolytic (1 µg, n = 4) or luteotropic dose (10 µg, n = 4; both in 10 ml of saline) was administered into the aorta abdominalis.

Peripheral blood samples were collected from the jugular vein at 10-min intervals (beginning 30 min before and continuing until 2.5 h after the infusions). Moreover, peripheral blood samples were collected from the jugular vein on every third day after the beginning of the estrus cycle (Days 0, 3, 6, 9, and 12 of the cycle). After Day 14 of the cycle, blood was collected once daily until Day 16 following the first estrus and then every two days (Days 18, 20, and 22 of the cycle). The external signs of estrus were checked every 12 h after TNF or saline treatment.

The blood plasma was immediately separated by centrifugation at 2000 x g for 10 min at 4°C and stored at –20°C. The concentrations of P4, PGE2, and 13,14-dihydro 15-keto-prostaglandin F2{alpha} (PGFM) in the plasma samples were measured.

Experiment 3: Influence on the Estrus Cycle of Exogenous TNF Infusion into the Peripheral Vasculature or Directly into the Uterine Lumen

Two experiments were carried out on Day 14 of the estrus cycle to test the hypothesis that PGs that originate directly from the uterus mediate TNF influence on the estrus cycle. Thirty-nine heifers were used for these experiments:

Experiment 3.1. To determine whether TNF modulates the lifespan of the bovine CL, saline (10 ml, n = 3) or TNF in a luteolytic (1 µg; n = 3) or luteotropic dose (10 µg, n = 3; both in 10 ml of saline) was slowly injected (over 30 min) into the jugular vein.

Experiment 3.2. To test the hypothesis that exogenous TNF acts locally on the bovine uterus to regulate the lifespan of the bovine CL, saline (5 ml; n = 3) or 0.001, 0.01, 0.1, 1 or 10 µg TNF (n = 3 per dose; in 5 ml of saline) was administered directly into the uterine lumen.

Peripheral blood samples were collected from the jugular vein at 10-min intervals. Moreover, the blood samples were collected from the jugular vein on every third day after the beginning of the estrus cycle (Days 0, 3, 6, 9, and 12 of the cycle). After Day 14 of the cycle, blood was collected once daily until Day 16 following the first estrus and then every two days (Days 18, 20, and 22 of the cycle). The external signs of estrus were checked every 12 h after TNF or saline treatment.

The blood plasma was immediately separated by centrifugation at 2000 x g for 10 min at 4°C and stored at –20°C. The concentrations of P4, PGE2, and PGFM in the plasma samples were measured.

Hormone Assays

The progesterone concentrations in the plasma samples were assayed using a direct enzyme immunoassay (EIA), as described previously [32]. The progesterone antiserum was a kind gift from Dr. Stanislaw Okrasa (University of Warmia and Mazury, Olsztyn, Poland). 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 were 6.6% and 8.4%, respectively.

The concentrations of PGFM in the plasma samples were determined using a direct EIA, as described previously [28]. The anti-PGFM serum (WS4468–5) was donated by Dr. W.J. Silvia (University of Kentucky, Lexington, KY). 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 7.6% and 10.4%, respectively.

The concentrations of PGE2 were determined by EIA in plasma samples (direct test), as described previously [28]. The anti-PGE2 serum was donated by Dr. S. Ito, Kansai Medical University, Osaka, Japan. The cross-reactivities of the anti-PGE2 serum, validated by comparing the inhibition of binding of peroxidase-labeled PGE2 to the antiserum, were as follows: PGE2, 100%; PGE1, 18%; PGJ2, 14%; PGA1, 10%; 15-keto PGE2, 8.8%; PGB2, 6.7%; PGA2, 4.6%; PGD2, 0.13%; and PGF2{alpha}, 2.8%. The PGE2 standard curve ranged from 0.07 ng/ml to 20 ng/ml and the ID50 of the assay was 1.25 ng/ml. The intraassay and interassay coefficients of variation were 6.9% and 9.7%, respectively.

Statistical Analysis

Least squares means and standard errors were determined. Differences in the length of the estrus cycle were analyzed using one-way analysis of variance followed by the Bonferroni multiple comparison test (ANOVA; GraphPad PRISM ver. 4.00; GraphPad Software, San Diego, CA). P4 and arachidonic acid metabolites (PGE2, PGFM) in the jugular plasma samples were analyzed using a repeated measure design approach in which treatments and time of sample collection (minutes) were fixed effects and all interactions were included [6]. All analyses were carried out using repeated measures ANOVA tests followed by the Bonferroni multiple comparison test (P < 0.05 was considered significant). In Experiment 3, the total amounts of released P4, PGE2, and PGFM were shown by the areas under the curve (relative units; see Table 1) and were analyzed using one-way analysis of variance followed by the Bonferroni multiple comparison test. The baseline was defined on the basis of data from the first hour of the experiment. The area under the curve was measured using data from the last 4 h of the experimental period.

RESULTS

Experiment 1: Preliminary Study

Infusion of L-NAME and INDO for 2 h did not change the level of P4 during the estrus cycle (Fig. 1). Infusion with L-NAME or INDO on Day 14 of the cycle did not change the cycle length in the heifers (21.5 ± 0.9 days and 21.7 ± 0.8 days, respectively) compared with the control group (22.1 ± 0.7 days; P > 0.05).


Figure 01
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  		FIG. 1. Concentrations of progesterone in the peripheral blood plasma of heifers infused with L-NAME (n = 4) or INDO (n = 4) on Day 14 of the estrus cycle. The control group (Control; n = 4) was infused with saline only. The drugs were administered into the aorta abdominalis.

Experiment 2. Influences of INDO and L-NAME on Estrus Cycle Regulation by TNF

Length of the estrus cycle. Infusion of TNF at a dose of 1 µg shortened (18.4 ± 1.0 days) the estrus cycle compared with that of control heifers infused with saline alone (22.1 ± 0.7 days; P < 0.05) (Fig. 2a). In the heifers infused with 10 µg TNF, spontaneous luteolysis was prevented and the functional lifespan of the CL was prolonged in comparison with those of the control group (more than 30 days vs. 21.8 ± 0.65 days; P < 0.01). The administration of INDO canceled the effects of both doses of TNF on the length of the estrus cycle (P < 0.01; Fig. 2b). The lengths of the estrus cycles in heifers treated with 1 µg (21.1 ± 1.1 days) or 10 µg (22.7 ± 1.2 days) TNF and preinfused with INDO did not differ from that of the group preinfused with INDO alone (internal control; 21.4 ± 0.7) or that of the control group (22.1 ± 0.7 days; P > 0.05). Preinfusion with L-NAME completely blocked the actions of low-dose TNF on the estrus cycle (P < 0.05), whereas the effect of high-dose TNF was not inhibited (P > 0.05) (Fig. 2c). The lengths of the estrus cycles in heifers treated with 1 µg TNF and preinfused with INDO (21.4 ± 0.9 days) were longer than those of heifers preinfused with saline and treated with 1 µg of TNF (18.4 ± 1.0 days; P < 0.05) and were comparable to that of the control group (22.1 ± 0.7 days; P > 0.05). However, when TNF was infused at the high dose (10 µg) after L-NAME application, the estrus cycle was prolonged compared with the control animals (P < 0.05; Fig. 2c).


Figure 02
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  		FIG. 2. Concentrations of progesterone in the peripheral blood plasma of heifers infused with TNF (1 µg and 10 µg) (a), INDO (b) or L-NAME (c) on Day 14 of the estrus cycle. The control group (Control) was treated with saline only. All reagents were infused into the aorta abdominalis. Different superscript letters indicate significant differences (P < 0.05) between the treatment groups, as assessed by repeated-measures ANOVA followed by the Bonferroni multiple comparison test.

P4 secretion. Although 1 µg TNF did not affect the P4 levels in the blood 4 h after treatment (Fig. 3a), it markedly decreased P4 at 24 h after application (Fig. 2a; P < 0.05). The administration of 10 µg TNF increased P4 secretion compared to the levels in control heifers (P < 0.05; Fig. 3a). For the P4 concentrations (Fig. 3a), two-way interactions were found between 10 µg TNF treatment and time of sample collection (P < 0.01). Three-way interactions were found between 10 µg TNF treatment and saline treatment and time of sample collection (P < 0.01), and between the 10 µg and 1 µg TNF treatments and time of sample collection (P < 0.05).


Figure 03
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  		FIG. 3. Concentrations of progesterone in the peripheral blood plasma of heifers infused for 2 h (horizontal line) with saline (a), INDO (b) or L-NAME (c) and concomitantly treated (arrow) with saline (control) or TNF (1 µg or 10 µg/animal). All reagents were infused into the aorta abdominalis. Different superscript letters indicate significant differences (P < 0.05) between treatment groups, as assessed by repeated-measures ANOVA followed by the Bonferroni multiple comparison test.

The level of P4 secretion in heifers infused with INDO followed by infusion with 1 µg or 10 µg TNF was similar to the P4 secretion levels in the groups infused with INDO or saline alone (Fig. 3, a and b; P > 0.05). Infusion of INDO completely inhibited the stimulatory effect of 10 µg TNF (P < 0.01). Three-way interactions were found between 10 µg TNF treatment alone (Fig. 3a) and 10 µg TNF treatment concomitantly with INDO infusion (Fig. 3b) and time of sample collection (P < 0.01).

The P4 concentrations in heifers infused with L-NAME alone or treated with 10 µg TNF following preinfusion with L-NAME were elevated compared with control animals treated with saline alone or with the animals treated with 1 µg TNF alone (P < 0.05; Fig 3, a and c). For the P4 concentrations (Fig. 3c), two-way interactions were found between L-NAME treatment (Fig. 3c) and time of sample collection, and between 10 µg TNF treatment following preinfusion with INDO and time of sample collection (P < 0.01). Three-way interactions were found between L-NAME treatment only (Fig. 3c) and saline treatment (Fig. 3a) and time of sample collection (P < 0.01), and between 10 µg TNF treatment following preinfusion with INDO (Fig. 3c) and saline treatment (Fig. 3a) and time of sample collection (P < 0.05).

PGE2 secretion. Administration of 1 µg TNF did not affect the PGE2 concentration in the blood as compared with the control group (P > 0.05; Fig. 4a). Infusion of 10 µg TNF increased the PGE2 level compared to infusion of saline only (Fig. 4a; P < 0.05). For the PGE2 concentrations (Fig. 4a), a two-way interaction was found between 10 µg TNF treatment and time of sample collection (P < 0.001). Three-way interactions were found between TNF (1 and 10 µg) treatments and saline treatment and time of sample collection (P < 0.001).


Figure 04
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  		FIG. 4. Concentrations of prostaglandin E2 in the peripheral blood plasma of heifers infused for 2 h (horizontal line) with saline (a), INDO (b) or L-NAME (c) and concomitantly treated (arrow) with saline (control) or TNF (1 µg or 10 µg/animal). All reagents were infused into the aorta abdominalis. Different superscript letters indicate significant differences (P < 0.05) between treatment groups, as assessed by repeated-measures ANOVA followed by the Bonferroni multiple comparison test.

There were no differences in PGE2 concentrations between heifers infused with INDO only, treated with both doses of TNF following preinfusion with INDO and control animals (Fig. 4, a and b; P > 0.05). However, preinfusion with INDO completely blocked the effect of a high dose of TNF (10 µg) on the PGE2 output (Fig. 4, a and b; P > 0.05). Three-way interactions were found between 10 µg TNF treatment alone (Fig. 4a) and TNF treatment following preinfusion with INDO (Fig. 4b) and time of sample collection (P < 0.01).

PGE2 output in the animals treated with 1 µg TNF following preinfusion with L-NAME was not changed compared with that of control and L-NAME only treated animals (Fig 4, a and c; P > 0.05). L-NAME preinfusion did not affect the stimulatory effect of the high dose of TNF on PGE2 output (Fig. 4, a and c; P > 0.05). However, in L-NAME-pretreated animals, 10 µg of TNF did not stimulate PGE2 secretion at the following time-points after TNF treatment: 60–90, 110–160 and 170–180 minutes, compared with the control groups (Fig. 4, a and c; P < 0.05). For the PGE2 concentrations (Fig. 5c), two-way interaction was found between 10 µg TNF treatment following preinfusion with L-NAME and time of sample collection (P < 0.001). Three-way interactions were found between 10 µg TNF treatment following preinfusion with L-NAME (Fig. 4c) and saline treatment (Fig. 4a) and time of sample collection (P < 0.001).


Figure 05
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  		FIG. 5. Concentrations of PGFM in the peripheral blood plasma of heifers infused for 2 h (horizontal line) with saline (a), INDO (b) or L-NAME (c) and concomitantly treated (arrow) with saline (control) or TNF (1 µg or 10 µg/animal). All reagents were infused into the aorta abdominalis. Different superscript letters indicate significant differences (P < 0.05) between treatment groups, as assessed by repeated-measures ANOVA test followed by the Bonferroni multiple comparison test.

PGF2{alpha} secretion. Administration of 1 µg TNF induced PGF2{alpha} secretion, as shown by the sharp increase in PGFM in the peripheral blood (P < 0.001; Fig. 5a), whereas TNF at a dose of 10 µg did not change the PGFM concentrations compared with those of control heifers infused with saline alone (P > 0.05; Fig. 5a). For the PGFM concentrations (Fig. 5a), a two-way interaction was found between 1 µg TNF treatment and time of sample collection (P < 0.001). Moreover, three-way interactions were found between 1 µg TNF treatment and saline treatment and time of sample collection (P < 0.01), and between 1 µg and 10 µg TNF treatments and time of sample collection (P < 0.05).

There were no differences in PGFM concentrations between heifers infused with INDO only, treated with both doses of TNF following preinfusion with INDO, and control animals treated with saline alone (P > 0.05; Fig. 5, a and b). However, preinfusion with INDO completely blocked the effect of the lower TNF dose (1 µg) on the PGFM output (P > 0.05; Fig. 5, a and b). Three-way interactions were found between 1 µg TNF treatment alone (Fig. 5a) and TNF treatment following preinfusion with INDO (Fig. 5b) and time of sample collection (P < 0.01).

The PGFM concentrations in the animals treated with 10 µg TNF following preinfusion with L-NAME were not changed compared with those of the control and L-NAME-treated animals (P > 0.05; Fig. 5, a and c). L-NAME preinfusion did not affect the stimulatory effect of the low dose of TNF (1 µg) on PGFM output (P > 0.05; Fig. 5, a and c). For the PGFM concentrations (Fig. 5c), a two-way interaction was found between 1 µg TNF treatment following preinfusion with L-NAME and time of sample collection (P < 0.001). Three-way interactions were found between 1 µg TNF treatment following preinfusion with L-NAME (Fig. 5c) and saline treatment (Fig. 5a) and time of sample collection (P < 0.001).

Experiment 3: Influence on the Estrus Cycle of Exogenous TNF Infusion into the Peripheral Vasculature or Directly into the Uterine Lumen

Effect of systemic infusion of TNF on CL lifespan. The length of the estrus cycle in heifers treated with 1 µg TNF (22.0 ± 0.6 days) or 10 µg TNF (21.3 ± 0.7 days) did not differ from that of heifers in the control group (22.3 ± 0.9) (Fig. 6a). However, in one animal that was infused in the jugular vein with 10 µg TNF, the estrus symptoms were observed earlier (at Day 19). Infusion of TNF at either dose did not affect the concentrations of P4, PGFM or PGE2 during the entire experimental period (P > 0.05; Fig. 6a).


Figure 06
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  		FIG. 6. Concentrations of progesterone in the peripheral blood plasma of heifers infused with saline (n = 3) or with several doses of TNF (n = 3 for each dose) into the jugular vein (a) and into the uterine lumen (b) on Day 14 of the estrus cycle. Different superscript letters indicate significant differences (P < 0.05) between treatment groups, as assessed by repeated-measures ANOVA followed by the Bonferroni multiple comparison test.

Effect of intrauterine infusion of TNF on CL lifespan. TNF at a dose of 0.001 µg induced premature regression of the CL in one animal (cycle duration, 18 days), although in two other heifers there were no differences in the length of the estrus cycle compared to the control group (20.67 ± 1.3 vs. 22.0 ± 0.6 days in the control; P > 0.05; Fig. 6b). The other two low doses of TNF (0.01 µg and 0.1 µg) induced premature luteolysis (cycle durations of 17.3 ± 0.3 and 18.3 ± 0.3 days, respectively; Fig. 6b). The length of the estrus cycle was prolonged to more than 30 days by the administration of TNF at doses of 1 and 10 µg as compared to that in control heifers (22.0 ± 0.6 days; P < 0.05).

Although the administration of either dose of TNF (0.01 µg or 0.1 µg) into the uterine lumen did not affect the P4 and PGE2 levels in the blood (Table 1), it strongly induced PGF2{alpha} secretion, as shown by the increased PGFM concentration in the peripheral blood (P < 0.01; Table 1). Intrauterine administration of 1 µg and 10 µg TNF strongly increased the levels of P4 and PGE2 in the peripheral blood (P < 0.01; Table 1), while they did not have any effect on the PGFM concentration (P > 0.01; Table 1).


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  		TABLE 1. Effects of different doses of TNF administered directly into the uterine lumen on the total amounts of released P4, PGFM, and PGE2 in heifers on Day 14 of the estrous cycle.*

DISCUSSION

Indomethacin, which is a nonselective PG synthase inhibitor, blocks the cyclooxygenase pathway of arachidonic acid metabolism and the production of PGE2, PGF2{alpha}, PGD2, PGI2, tromboxanes, and other stable and biologically active metabolites of PTGS [33]. Daily intrauterine administration of INDO at the late luteal stage of the bovine estrus cycle (from Day 14 to Day 21) has been demonstrated to inhibit the secretion of luteolytic PGF2{alpha} and, consequently, to prolong the lifespan of the CL and to prolong the estrus cycle in cows [34]. On the other hand, twice daily intrauterine infusion of INDO on Days 4, 5, and 6 of the estrus cycle in heifers blocked the formation and development of the CL and consequently, shortened the length of the estrus cycle [35]. In addition to luteolytic PGF2{alpha}, luteotropic substances, such as PGI2 and PGE2, may be produced by the bovine uterus and CL during the early and midluteal phase [14, 15, 36–38] and during pregnancy [39]. PGE2 and PGI2 have been found to be the most potent luteotropic substance in the ovine and bovine CL [39, 40]. Moreover, INDO decreases concomitantly PGE2 and P4 secretion by ovine and bovine CL during the estrus cycle and pregnancy [41–44]. Therefore, the shortening of the CL lifespan caused by INDO infusion in the early luteal phase [35] may simply be due to the inhibition of luteotropic PGE2 and/or PGI2 production [36]. In the present study, infusion of INDO at a rate of 240 mg/h for 2 h directly into the aorta abdominalis did not change either the CL lifespan or the duration of the estrus cycle in heifers. However, the same dose of INDO administered into the aorta abdominalis completely blocked PGF2{alpha} secretion induced by oxytocin (OT) over a 6-h period in heifers at Day 17–18 of the cycle [45]. It seems that only chronic, long-term applications of INDO may affect the estrus cycle [34, 35]. However, as demonstrated in the present study, the infusion of INDO over a shorter 2-h period is sufficient to inhibit prostaglandin production during the period of exogenous TNF infusion. Therefore, we believe that the chosen dose of INDO (240 mg/h) is effective on Day 14 of the cycle at inhibiting the stimulatory effect of TNF on the production and output of PG from the uterus and CL (Experiment 2.2).

INDO at high concentrations (10–3-10–6 M) can also inhibit phosphodiesterase activity, resulting in the accumulation of cyclic nucleotides in the cells and tissues [46, 47]. Therefore, the abolition of the effects of INDO on luteolytic and/or luteotropic TNF actions observed in the present study may not depend exclusively on the inhibition of PTGS activity. The inhibition of TNF effects may also involve the accumulation of cAMP (a member of the LH- and PGE2-dependent second messenger cascade) and cGMP (a component of the NO-induced intracellular mechanism of action) in the cells and tissues of the reproductive tract. Moreover, treatment with high concentrations of INDO may increase the production of endogenous TNF, as previously shown [48, 49]. Increasing the local concentration of endogenous TNF may hypothetically desensitize the cells to the action of exogenous cytokines. However, it is unclear whether the dose of INDO used in the present study (240 mg per animal per h for 2 h) results in submicromolar concentrations of INDO in the reproductive tract. The used dosage was relatively low if one considers the prolonged time of application (2 h) and the dilution of INDO in the total blood volume (approximately 7% of the cow body; v:w). Accordingly, we believe that the dose of INDO used in the present study represents nanomolar concentrations at the target tissue. Therefore, the above-mentioned mechanisms should be considered as additional and hypothetical.

L-NAME is a nonselective inhibitor of NOS activity and inhibits the production of NO from L-arginine by all known NOS isoforms in several tissues, including the reproductive tract [29, 50, 51]. Intraluteal infusion of this NOS inhibitor at either the middle or late phase of the estrus cycle increases the secretion of both P4 and OT in cows [29]. Moreover, L-NAME administered intraluteally on Days 17–18 of the cycle prolongs the lifespan of the CL [29] and inhibits PGF2{alpha}-induced luteolysis [28, 30]. These findings strongly suggest that NO acts as a significant auto/paracrine factor in the midluteal and late luteal phases in cows and plays an important role in the regulation/mediation of luteal regression. In the present study, L-NAME (400 mg/h) infused directly into the aorta abdominalis over 2 h on Day 14 of the cycle did not change either the CL lifespan or the duration of the estrus cycle in heifers. However, this dose completely blocked PGF2{alpha}-induced luteolysis and inhibited PGF2{alpha}-induced NO production in heifers on Day 14 of the cycle [28]. It seems that only chronic, long-term application of L-NAME affects the spontaneous estrus cycle [29]. However, the infusion of L-NAME over a 2-h period in the present experiments completely inhibited NO production, which appears to correspond to the time of exogenous TNF infusion and action. Therefore, we assume that an L-NAME dose of 400 mg/h is also effective at protecting against the stimulatory effect of TNF on NO production in the bovine reproductive tract (Experiment 2.3).

The data obtained in the current in vivo study support and extend our previous in vitro [7, 8, 13, 27] and in vivo [6] observations and provide new concepts regarding the mechanisms of TNF action in the regulation of the estrus cycle in cattle. As shown previously [6], the higher dose of exogenous TNF (10 µg) stimulated P4 and PGE2 secretion, resulting in the prolongation of the luteal phase of the estrus cycle in cattle. These data suggest that TNF plays luteotropic and luteoprotective roles. Exogenous TNF strongly stimulates luteotropic PGE2 secretion in the bovine CL in vitro [9] and in pregnancy [12]. In ewes, TNF also plays bifunctional roles (luteolytic or luteotropic) in the regulation of CL function during the estrus cycle [21, 22]. In in vivo-microdialyzed ovine CL, TNF at a high dose (2 µg/ml) stimulated P4 and PGE2 secretion and delayed the time to onset of the P4-mediated decrease in PGF2{alpha}-induced luteolysis [22]. Moreover, exogenous TNF has been demonstrated to stimulate PGE synthase mRNA expression [14, 15] and PGE2 production [13–15] in cultured bovine endometrial stromal cells. The increased PGE2 synthase expression and activity may change the PGE2/PGF2{alpha} ratio and contribute to prolongation of the luteal phase of the estrus cycle and/or the establishment of pregnancy. In the present study, treatment with INDO inhibited TNF-induced PG production (including luteotropic PGE2) in both the CL and uterus and completely abolished the effect of the higher dose of exogenous TNF on the prolongation of the lutal phase of the estrus cycle and the stimulatory effect on P4 secretion.

Exogenous TNF has been shown to stimulate not only luteotropic PGE2 [13, 15, 26, present study] but also luteolytic PGF2{alpha} secretion in vitro [7, 8, 27] and in vivo [6], which suggests that TNF also plays a luteolytic role. Although both OT and TNF affect PGF2{alpha} output in the bovine endometrium at the follicular stage of the cycle, TNF, in contrast to OT, also affects PGF2{alpha} secretion at the midluteal and late luteal stages [7]. Indeed, we confirmed our previous findings that the infusion of the lower dose of TNF (1 µg) during the luteal phase (Day 14 of the cycle) increases the plasma concentrations of PGFM (metabolite of luteolytic PGF2{alpha}) and inhibits P4 production, resulting in shortening of the estrus cycle [7]. Furthermore, the inhibition of PG production in the bovine uterus and/or CL by INDO preinfusion completely inhibited the luteolytic action of the lower dose of TNF (Experiment 2). It has been shown that during luteolysis, exogenous TNF is capable of decreasing P4 release if the CL is pretreated with PGF2{alpha} and/or ET-1 [22]. Moreover, at the same time, TNF is able to stimulate ET-1 and PGF2{alpha}, thereby establishing a local positive feedback that can accelerate the luteolytic cascade in the CL [21, 22]. Therefore, based only on the data from Experiment 2 (infusion of TNF into the whole reproductive tract), it is unclear whether the ability of exogenous TNF to modulate the lifespan of the bovine CL is a result of its direct effect on endometrial PG secretion or its participation in the local luteolytic cascade within the CL. However, only uterine PGF2{alpha} has been clearly linked to luteolysis [52, 53], while luteal PGF2{alpha} may be luteotropic during the development and maintenance of the bovine CL [2, 37, 40, 53–56]. Moreover, the data from Experiment 3.2 clearly show that TNF infused locally into the uterine lumen at Day 14 of the cycle directly modulates local PG secretion. High concentrations of TNF administered directly into the uterus stimulated PGE2 production resulting in prolongation of the cycle. However, low TNF doses stimulated PGF2{alpha}, inhibited P4, and eventually led to premature luteolysis. Based on the above findings, we propose that the luteolytic or luteotropic action of TNF in the luteal phase of the estrus cycle in cattle depends on uterine PGF2{alpha} or PGE2 output and action.

In addition to PG stimulation, exogenous TNF has been shown to augment NO production in the bovine reproductive tract [6, 24, 27]. The cellular mechanism of TNF action has been demonstrated to consist of NOS activity induction, which results in NO generation and subsequent cGMP production [27, 50]. NO is considered to be an important mediator of PGF2{alpha}-induced and spontaneous luteolysis in cattle [28, 29]. These findings allow us to hypothesize that NO is involved in the exogenous TNF-regulated secretory function of the bovine CL and uterus, and consequently, it mediates the action of TNF throughout the luteal phase of the estrus cycle. Preinfusion with L-NAME (a non-selective NOS inhibitor) completely blocked the luteolytic effect of 1 µg TNF. During luteolysis, NO increases LTC4 and PGF2{alpha} secretion [57, 58], inhibits P4 secretion [28, 57–59], and may increase the blood flow in the CL [31, 60]. Finally, NO is involved in structural apoptosis and activates apoptosis in the CL cells [24]. Although preinfusion of L-NAME did not affect the stimulatory effect of TNF on PGF2{alpha} output, it partially abolished TNF-stimulated PGE2 secretion from 60 to 180 minutes after treatment. In fact, our previous in vitro study has shown that TNF only partially acts on PG production in the bovine endometrium via induction of NOS, with subsequent stimulation of NO-cGMP formation [27]. Although NO can stimulate the production of both PGs in the bovine endometrium, it preferentially stimulates PGE2 secretion [27]. It has been shown that S-NAP (an NO donor) favorably stimulates PGE2 production in endometrial stromal cells (6.4-fold increase) in comparison to the stimulative effect on the production of PGF2{alpha} (3.1-fold increase) [27]. Moreover, although S-NAP strongly stimulates PGE2 production in epithelial cells, it does not affect PGF2{alpha} production [27]. Therefore, the inhibition of NO production (by L-NAME pre-infusion) may partially and temporarily inhibit the stimulatory effect of exogenous TNF on PGE2 output from the bovine uterus.

TNF at the luteolytic dose of 1 µg induces a two-phase pattern of NO output [6]. The first peak of NO concentration is observed during TNF infusion and is followed by a long-term increase in NO production, as measured by nitrite/nitrate concentrations in the blood [6]. NO production is highly correlated with increased PGFM concentration [6]. PGF2{alpha} induces dramatic increases in NO production in the bovine CL and uterus, as shown by the high concentrations of NO–2/NO–3 detected in vitro [61] and in vivo [28]. Moreover Petroff et al. [4] have shown that cytokine (TNF, interferon-{gamma})-induced apoptosis of bovine CL cells is not mediated by NO delivery or action. Taken together, these data suggest that PGF2{alpha}, not TNF, is the main factor that induces NO output in the bovine CL during luteolysis. Our findings suggest that the administration of exogenous TNF in luteolytic doses initiates a positive cascade between uterine PGF2{alpha} and various luteolytic factors/intraluteal mediators, including NO, to complete the premature regression of the bovine CL, which leads to a shortening the luteal phase of the estrus cycle.

In summary, INDO completely blocked the effects of both luteolytic (lower) and luteotropic (higher) doses of TNF on the CL lifespan. L-NAME completely blocked the actions of the lower (luteolytic) dose of exogenous TNF, whereas the effects of the higher dose (luteotropic) of TNF were not inhibited. Thus, these data suggest that PG, which is mainly of uterine origin, mediates the actions of exogenous TNF on the lifespan of bovine CL. Uterine or luteal PGE2 is a good candidate mediator of the luteotropic action of exogenous TNF. The luteolytic action of exogenous TNF on the bovine CL depends on uterine PGF2{alpha} output and action. Luteolytic concentrations of TNF may initiate the positive cascade between uterine PGF2{alpha} and various intraluteal factors, including NO, to complete premature luteolysis in cattle and to shorten the luteal phase of the estrus cycle. However, further in vivo studies using TNF antagonists or antibodies are needed to clarify whether endogenous TNF, produced locally in the uterus, plays a role in triggering luteolysis under normal physiologic conditions.

ACKNOWLEDGMENTS

We thank our colleague Dr. Katarzyna Deptula for technical support and assistance in preparing this manuscript. We thank Dr. Seiji Ito of Kansai Medical University, Osaka, Japan for antisera against prostaglandin E2; Dr. W.J. Silvia, University of Kentucky, Lexington, KY for PGFM antisera, Dr. Stanislaw Okrasa of University of Warmia and Mazury for progesterone antisera, and Dainippon Pharmaceutical Co., Ltd., Osaka, Japan for recombinant human TNF (HF-13). The authors are grateful to Centrowet, Olsztyn, Poland for the gifts of Crestar, cloprostenol, sedazin, polocainum hydrochloricum and other veterinary drugs used in the present study. The authors also thank Gennuas France, the owner of Animal Farm (Spólka Rolna "Wroblik" Sp. z o.o, Lidzbark Warminski, Poland) for their excellent cooperation and for allowing us to use the animals for the present experiments.

FOOTNOTES

1Supported by the Grants-in-Aid for Scientific Research from the Polish Ministry of Sciences and Informative Technology (2 P06K 025 29) and the Japan Society for Promotion of Sciences (JSPS; 18658114). D.J.S. was supported under the JSPS Invitation Fellowship Program for Research in Japan for Senior Scientists. I.W.-P. was supported by the Domestic Grants for Young Scientists of the Foundation for Polish Science (FNP Programme 2006). Back

Correspondence: 2FAX: 48 89 535 74 38; e-mail: skadar{at}pan.olsztyn.pl

Received: 18 April 2006.

First decision: 19 May 2006.

Accepted: 19 December 2006.

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