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BOR - Papers in Press, published online ahead of print August 6, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016212
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BIOLOGY OF REPRODUCTION 69, 1907–1913 (2003)
DOI: 10.1095/biolreprod.103.016212
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Roles of Tumor Necrosis Factor-{alpha} of the Estrous Cycle in Cattle: An In Vivo Study1

Dariusz J. Skarzynski2,3, Mamadou M. Bah3, Katarzyna M. Deptula3, Izabela Woclawek-Potocka3, Anna Korzekwa3, Masami Shibaya4, Wojciech Pilawski3,5, and Kiyoshi Okuda4

Division of Reproductive Endocrinology and Pathophysiology,3 Institute of Animal Reproduction and Food Research, PAS, Olsztyn 10-747, Poland Laboratory of Reproductive Endocrinology,4 Faculty of Agriculture, Okayama University, Tsushima Naka, Okayama 700-8530, Japan Department of Pharmacology,5 Faculty of Veterinary Medicine, Warmia and Mazury University in Olsztyn, Olsztyn 10-718, Poland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have suggested in a previous in vitro study that tumor necrosis factor-{alpha} (TNF{alpha}) plays a role in the initiation of luteolysis in cattle. The aim of the present study was to examine the influence of different doses of TNF{alpha} on the estrous cycle in cattle by observing the standing behavior and measuring peripheral concentrations of progesterone (P4) during the estrous cycle. Moreover, we evaluated the secretion of P4, oxytocin (OT), nitric oxide (NO), and luteolytic (prostaglandin F2{alpha} [PGF2{alpha}] and leukotriene C4 [LTC4]) and luteotropic (PGE2) metabolites of arachidonic acid in peripheral blood plasma as parameters of TNF{alpha} actions. Mature Holstein/Polish black and white heifers (n = 36) were treated on Day 14 of the estrous cycle (Day 0 = estrus) by infusion into the aorta abdominalis of saline (n = 8), an analogue of PGF2{alpha} (cloprostenol, 100 µg; n = 3) or saline with TNF{alpha} at doses of 0.1 (n = 3), 1 (n = 8), 10 (n = 8), 25 (n = 3), or 50 µg (n = 3) per animal. Peripheral blood samples were collected frequently before, during, and up to 4 h after TNF{alpha} treatment. After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. Lower doses of TNF{alpha} (0.1 and 1 µg) decreased the P4 level during the estrous cycle and consequently resulted in shortening of the estrous cycle (18.8 ± 0.9 and 18.0 ± 0.7 days, respectively) compared with the control (22.3 ± 0.3 days, P < 0.05). One microgram of TNF{alpha} increased the PGF2{alpha} (P < 0.001) and NO (P < 0.001) concentrations and decreased OT secretion (P < 0.01). Higher doses of TNF{alpha} (10, 25, 50 µg) stimulated synthesis of P4 (P < 0.001) and PGE2 (P < 0.001), inhibited LTC4 secreton (P < 0.05), and consequently resulted in prolongation of the estrous cycle (throughout 30 days, P < 0.05). Altogether, the results suggest that low concentrations of TNF{alpha} cause luteolysis, whereas high concentrations of TNF{alpha} activate corpus luteum function and prolong the estrous cycle in cattle.

corpus luteum function, cytokines, female reproductive tract, ovulatory cycle, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanisms controlling the development, secretion, and regression of the bovine corpus luteum (CL) involve many factors produced both inside and outside the CL [1–3]. Prostaglandins (PGs), along with sex steroids, are among the most important factors involved in the regulation of the estrous cycle and pregnancy [3–5]. Progesterone (P4) produced by the CL is crucial for determining the duration of the estrous cycle and for achieving a successful pregnancy [1, 2]. PGF2{alpha} released from the uterus has been shown to cause regression of the ruminant CL, and the administration of PGF2{alpha} analogues terminates pregnancy in ruminants [4, 6]. However, the current concept that the regression of CL in cattle is directly brought about by PGF2{alpha} of uterine origin secreted in response to oxytocin (OT) released by the CL is inadequate to explain many events that actually occur at the time of regression [3–8].

In our previous study, although both OT and tumor necrosis factor-{alpha} (TNF{alpha}) affected endometrial PGF2{alpha} output at the follicular stage, only TNF{alpha} affected PGF2{alpha} output at the mid and late luteal stages by acting through specific binding sites present in the bovine endometrium during the whole estrous cycle [9]. TNF{alpha} stimulated PGF2{alpha} production by the endometrial stromal cells via activation of phospholipase A2 (PLA2) and nitric oxide (NO) synthase [10]. We recently showed that NO is a good candidate to serve as a mediator and/or modulator of PGF2{alpha} action on the bovine CL during luteolysis in cattle [11–13]. NO directly inhibits P4 secretion from bovine luteal cells and augments the action of extragonadal PGF2{alpha} on the CL [11]. The inhibition of NO production in the female reproductive tract counteracts both spontaneous [14] and PGF2{alpha}-induced [12, 13] luteolysis in cattle. Moreover, NO stimulates the secretion of leukotriene C4 (LTC4) in the bovine CL [13]. Several products of the lipoxygenase pathway of the arachidonic acid cascade, particularly leukotriene B4 and LTC4, have been demonstrated to play roles in luteolysis [15, 16]. An in vivo microdialysis study showed that LTC4 concentrations in perfusates from the CL rose before the decline in P4 during spontaneous luteolysis in heifers [17].

Complete structural regression of the bovine CL involves the action of products of immune cells [18–22]. At the time of luteolysis, immune cells invade into the bovine CL [18]. Cytokines produced by the immune cells, especially TNF{alpha} and interferon-{gamma} (IFN{gamma}), seem to participate in the regression of the bovine CL [19]. TNF{alpha} and its specific receptors (TNF-RI) are present in the bovine CL during luteolysis [20, 21]. TNF{alpha} in combination with IFN{gamma} reduces P4 production and induces the apoptotic events that finally lead to functional and structural luteolysis [8, 21–23].

Unexpectedly, TNF{alpha} also induces the production and output of luteotropic PGE2 [2, 24] in cultured bovine luteal [20] and stromal-endometrial cells and tissue [25], suggesting luteotropic and luteoprotective roles of TNF{alpha}. After fertilization, a relatively high proportion of luteotropic PGE2 relative to luteolytic PGF2{alpha} (PGE2 > PGF2{alpha}) is needed for proper embryonic development, recognition, and establishment of pregnancy [4, 5, 25, 26]. Thus, TNF{alpha} seems to play one or more roles in the regulation of the estrous cycle and pregnancy in cattle. However, the properties and mechanisms of the opposite TNF{alpha} actions (luteolytic versus luteotropic) during the estrous cycle have not been yet clarified. In the present study, therefore, we examined the influence of TNF{alpha} on the estrous cycle in conscious cattle by observing standing behavior and measuring peripheral concentrations of P4 during the estrous cycle. We evaluated the concentrations of P4, OT, stable metabolites of NO (nitrate/nitrite [NO2-/NO3-]), and luteolytic (PGF2{alpha} and LTC4) and luteotropic (PGE2) metabolites of arachidonic acid in the blood samples as indicators of TNF{alpha} action.


   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 No. 4/2001/N).

Animals and Surgical Procedures

Normally cycling Holstein/Polish black and white (75% and 25%, respectively) heifers (18–20 months of age and final 400–450 kg body weight, n = 36) were used for the present study. Two to three weeks after weighing and choosing the animals for experiments (only animals weighing 390–400 kg were chosen), the estrus was synchronized using implants of a progesterone analogue (Crestar; Intervet, Boxmeer, Holland). Crestar consists of two components: a silicone ear implant containing the progestogen norgestomet (17{alpha}-acetoxy-11-ß-methyl-19-norpregna-4-en-2.20-dione) and an injection containing norgestomet (3 mg/2 ml) with estradiol valerate (5 mg/2 ml). The estrus was synchronized using the standard procedure without any additional treatment with the analogue of PGF2{alpha} treatment, as recommended by the manufacturer for the estrus synchronization of maiden heifers. The Crestar injection was administered i.m., and at the same time the Crestar implant was inserted s.c. at the outer edge of the ear in all animals. After 10 days, the norgestomet implants were removed. The onset of estrus was confirmed by standing behavior and taken as Day 0 of the estrous cycle. The length of the estrous cycle was defined as the number of days between the first days of standing estrus.

For infusion of either saline, an analogue of PGF2{alpha} (aPGF2{alpha}, cloprostenol; Bioestrophan, Biowet, Gorzow Wielkopolski, Poland), or TNF{alpha} (recombinant human TNF{alpha}; HF-13; kindly donated by Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) on Day 13 of the subsequent estrous cycle, a catheter was inserted into the posterior aorta abdominalis through the coccygeal artery, as described previously [12]. The animals were premedicated with i.m. xylazine at a dose of 25–30 mg per animal (Sedazin; Biowet, Pulawy, Poland), and local epidural anesthesia was induced by injecting 4 ml of 2% procaine hydrochloride (Polocainum Hydrochloricum; Biowet, Drwalew, Poland) between the first and second coccygeal vertebrae. The tip of the cannula was positioned in the aorta 65–70 cm ahead of the point of insertion, just cranial to the origin of the ovarian artery and caudal to the renal artery [12]. This placement allowed infused reagents to be transported by the bloodstream directly into the reproductive tract as was established in a previous study [27]. It has been demonstrated using adrenergic drugs [27, 28] that by means of such implanted cannula one can administer drugs into the female reproductive tract more specifically than by administration into the general circulation and that the required dose of experimental factors may be thereby markedly reduced. A second catheter was inserted into the jugular vein for frequent collection of blood samples.

Experiment 1 (Preliminary Study)

Twenty-one heifers were used to establish the effective dose of TNF{alpha}. On Day 14 of the estrous cycle, three heifers received the infusion of 20 ml of saline in the aorta abdominalis throughout a period of 30 min (control group). Another three animals were injected with 100 µg of cloprostenol (aPGF2{alpha}, the dose was chosen based on our previous data [6, 12]). The remaining 15 heifers were infused for 30 min with 0.1, 1, 10, 25, or 50 µg of TNF{alpha} (n =3 per dose) into the aorta abdominalis. Peripheral blood samples were collected from the jugular vein at 10-min intervals (beginning 1 h before and continuing until 3.5 h after the infusion). After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. The blood 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 TNF{alpha} on the Estrous Cycle)

To test the hypothesis that TNF{alpha} differentially affects the duration of the estrous cycle and the secretory function of the CL and uterus, saline as a control (n = 5) and a possible luteolytic dose of TNF{alpha} (1 µg, n = 5) or a possible luteotropic dose of TNF{alpha} (10 µg, n = 5) in 20 ml of saline was infused throughout a period of 30 min into the aorta abdominalis on Day 14 of the estrous cycle. Peripheral blood samples were collected from the jugular vein at 10-min intervals (beginning 1 h before and continuing until 3.5 h after the infusion). After Day 15 of the estrous cycle, blood was collected once daily until Day 22 following the first estrus. The concentrations of P4, OT, PGE2, 3,14-dihydro,15-keto-PGF2{alpha} (PGFM), LTC4, and nitrite/nitrate in the plasma samples were measured. Standing behavior was checked every 12 h after TNF{alpha} or saline treatment to confirm the onset of estrus.

Hormone Determinations

Progesterone concentrations in plasma samples were assayed using a direct enzyme immunoassay (EIA) as described previously [12]. The P4 standard curve was produced for P4 concentrations ranging from 0.39 to 25 ng/ml, and the effective dose for 50% inhibition (ID50) in the assay was 2.85 ng/ml. The intra-assay and interassay coefficients of variation averaged 6.6% and 8.4%, respectively.

The EIA for OT was based on the second antibody method using the biotin-streptavidin-peroxidase technique as described previously [7]. The peptide was extracted from serum as described previously [28]. The coefficient of extraction averaged 89.7%. The standard curve was produced for OT concentrations ranging from 1.95 to 500 pg/ml, and the ID50 of the assay was 34.7 pg/ml. The intra-assay and interassay coefficients of variation were 7.8% and 11.7%, respectively.

The concentrations of PGFM in the plasma samples were determined with a direct EIA, as described previously [12]. The anti-PGFM serum (WS4468-5) was donated by Dr. W. J. Silvia, University of Kentucky, Lexington, KY. The PGFM standard curve was produced for PGFM concentrations ranging from 32.5 to 8000 pg/ml, and the ID50 of the assay was 315 pg/ml. The intra-assay and interassay coefficients of variation were on average 7.6% and 10.4%, respectively.

The concentrations of PGE2 were determined by a direct EIA test as described previously [29]. The anti-PGE2 serum was donated by Dr. S. Ito, Kansai Medical University in Osaka, Japan. Cross-reactivities of the anti-PGE2 serum, determined by measuring the inhibition of binding of peroxidase-labeled PGE2 to this 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 was produced for PGE2 concentrations ranging from 0.07 to 20 ng/ml, and the ID50 of the assay was 1.25 ng/ml. The intra-assay and interassay coefficients of variation were on average 6.9% and 9.7%, respectively.

The concentrations of LTC4 were determined in plasma samples using a commercially available EIA kit (Cayman Chemical Co., Ann Arbor, MI) according to the instructions of the manufacturer. The intra-assay and interassay coefficients of variation were on average 4.9% and 7.4 %, respectively.

NO2-/NO3- Determination in Plasma

Plasma concentrations of NO2-/NO3-, the stable metabolites of NO, were measured by a colorimetric method using the Griess reaction as described by Green et al. [30] and adapted by us for serum samples [12]. The assay sensitivity was 0.065 µg/ml, and the standard curve was produced for NO2-/NO3- concentrations ranging from 0.05 to 6.9 µg/ml. The intra-assay and interassay coefficients of variation were on average 7.4% and 11.2%, respectively.

Statistical Analysis

Least squares means and SEMs were determined. The length of the estrous cycle and the total amount of released hormones (P4, OT), arachidonic acid metabolites (LTC4, PGE2, PGFM), and stable metabolites of NO represented as the area under the curve (relative units, Table 1) were analyzed using one-way ANOVA followed by the Bonferroni Multiple Comparison Test (ANOVA; GraphPAD PRISM Version 4.00; GraphPad Software, San Diego, CA). The analysis of hormones (P4, OT), arachidonic acid metabolites (LTC4, PGE2, PGFM), and stable metabolites of NO (NO2-/NO3-) in the jugular plasma samples, collected before, during, and after administration of different doses of TNF{alpha} and saline on Day 14 of the cycle, was performed using a repeated-measure design approach with treatments and time of sample collection (minutes) being fixed effects with all interactions included. All analyses were performed using repeated-measures ANOVA tests followed by the Bonferroni Multiple Comparison Test (GraphPAD PRISM; P < 0.05 was considered statistically significant).


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  		TABLE 1. Effects of 30-min infusion of saline (control) or TNF{alpha} on total amounts of released P4, OT, PGFM, PGE2, LTC4, and stabile metabolites of NO in heifers on Day 14 of the estrous cycle.*


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1 (Preliminary Study)

Injection of aPGF2{alpha} shortened (17.7 ± 0.3 days) the estrous cycle length compared with that of control heifers injected with saline only (22.3 ± 0.3 days), whereas infusion of TNF{alpha} had a dose-dependent effect on the duration of the estrous cycle (Fig. 1, P < 0.05). Two low doses of TNF{alpha} (0.1 and 1 µg for 30 min) induced regression of CL (cycle durations, 18.8 ± 0.9 and 18.0 ± 0.7 days, respectively). The length of the estrous cycle was prolonged to more than 30 days by infusion of TNF{alpha} at a dose of 10 µg for 30 min compared with that in control heifers (22.3 ± 0.3 days, P < 0.05). Higher doses of TNF{alpha} (25 and 50 µg for 30 min) also lengthened the estrous cycle to 30 days (Fig. 1). During infusion of TNF{alpha} at these doses (25 and 50 µg for 30 min), clinical symptoms, including rapid increases in pulse and respiration rate, rise of blood pressure, and muscle contractions, were observed. Therefore, on the basis of this preliminary experiment, two doses of TNF{alpha}, 1 (luteolytic) and 10 (luteotropic) µg for 30 min, were chosen for further studies.



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  		FIG. 1. Concentration of progesterone in peripheral blood plasma of heifers infused with saline (n = 3) or with several doses of TNF{alpha} (each dose, n = 3) or cloprostenol (aPGF2{alpha}, 100 µg; n = 3) on Day 14 of the estrous cycle. The control group (control) was injected with saline only. Drugs were administered for 30 min into the aorta abdominalis. Different subscript letters indicate significant differences (P < 0.05) between treated groups

Experiment 2 (Influence of TNF{alpha} on the Estrous Cycle)

There were significant differences in the length of the estrous cycle between animals infused with 1 and 10 µg of TNF{alpha} (P > 0.05, Fig. 2). Infusion of 1 µg of TNF{alpha} shortened (17.5 ± 0.44 days) the cycle length compared with that of the group injected with saline (21.8 ± 0.65 days). In the heifers infused with 10 µg of TNF{alpha}, spontaneous luteolysis was prevented and the functional life of the CL was prolonged compared with those of the control group (more than 30 days versus 21.8 ± 0.65 days, P < 0.01).



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  		FIG. 2. Concentration of progesterone in peripheral blood plasma of heifers infused for 30 min with TNF{alpha} (1 or 10 µg per animal; each dose, n = 3) or saline (n = 3) on Day 14 of the estrous cycle. Saline or TNF{alpha} was infused into the aorta abdominalis. Different subscript letters indicate significant differences (P < 0.05) between treated groups

Although a lower dosage of TNF{alpha} (1 µg for 30 min) did not affect the P4 concentration in the blood (Fig. 3a , Table 1), it strongly reduced the OT concentration (P < 0.001; Fig. 3b, Table 1). The administration of 1 µg of TNF{alpha} induced PGF2{alpha} production, as shown by the increased PGFM concentration in the peripheral blood (P < 0.001, Fig. 4a) but did not affect PGE2 production (P > 0.05, Fig. 4b). Moreover, 1 µg of TNF{alpha} induced NO production, as shown by increase in the NO2-/NO3- level in the blood (P < 0.001; Fig. 5a , Table 1). The increase in the PGFM concentration showed a strong positive correlation with the NO2-/NO3- elevation (P < 0.001, r = 0.73). Furthermore, the elevation in the NO2-/NO3- concentration was inversely correlated with the OT concentration (P < 0.001, r = -0.71). One microgram of TNF{alpha} did not influence the LTC4 concentration in the blood during the experimental period (P > 0.05; Fig. 5b, Table 1).



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  		FIG. 3. Effect of TNF{alpha} (1 or 10 µg per animal; each dose, n = 3) or saline (n = 3) infusions on Day 14 of the estrous cycle on (a) progesterone and (b) oxytocin concentrations in peripheral blood plasma of heifers. Saline or TNF{alpha} was infused for 30 min into the aorta abdominalis. Different subscript letters indicate significant differences (P < 0.05) between treated groups



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  		FIG. 4. Effect of TNF{alpha} (1 or 10 µg per animal; each dose, n = 3) or saline (n = 3) infusions on Day 14 of the estrous cycle on (a) PGFM and (b) PGE2 concentrations in peripheral blood plasma of heifers. Saline or TNF{alpha} was infused for 30 min into the aorta abdominalis. Different subscript letters indicate significant differences (P < 0.05) between treated groups



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  		FIG. 5. Effect of TNF{alpha} (1 or 10 µg per animal; each dose, n = 3) or saline (n = 3) infusion on Day 14 of the estrous cycle on (a) stabile metabolites of NO (NO2-/NO3-) and (b) LTC4 concentrations in peripheral blood plasma of heifers. Saline or TNF{alpha} was infused for 30 min into the aorta abdominalis. Different subscript letters indicate significant differences (P < 0.05) between treated groups

Infusion of 10 µg of TNF{alpha} strongly elevated the concentration of P4 and PGE2 in peripheral blood (P < 0.001; Figs. 3a and 4b, Table 1) without any effect on the PGFM concentration (P > 0.05; Fig. 4a, Table 1). A strong correlation has been found between doses of TNF{alpha} and PGE2 levels (r = 0.86, P < 0.001). Although 10 µg of TNF{alpha} temporarily decreased the OT secretion at the time of the infusion (Fig. 3b), the OT concentration immediately returned to the basal level compared with the pretreatment value or the value in control animals infused with saline only (P > 0.05; Fig. 3b, Table 1). Although the higher dose of TNF{alpha} increased the concentration of NO2-/NO3- in the blood (P < 0.01, Fig. 5a), the effect was weaker than that in the lower dose of TNF{alpha} (1 µg) (P < 0.001, Table 1). Moreover, infusion of 10 µg of TNF{alpha} decreased the LTC4 concentration in the blood (P < 0.001; Fig. 5b, Table 1).

For the P4 concentration (Fig. 3a), two-way interactions were found between the 10-µg TNF{alpha} treatment and time of sample collection (P < 0.01). Three-way interactions were found among the 10-µg TNF{alpha} treatment, saline treatment, and time of sample collection (P < 0.01) and among the 10-µg TNF{alpha} treatment, the 1-µg TNF{alpha} treatment, and time of sample collection (P < 0.05). For the OT concentration (Fig. 3b), two-way interaction was found between the 1-µg TNF{alpha} treatment and time of sample collection (P < 0.05, Fig. 3b). Moreover, three-way interaction was found among TNF{alpha} (1 µg) treatment, saline treatment, and time of sample collection (P < 0.05). For the PGFM, concentrations (Fig. 4a), two-way interaction was found between the 1-µg TNF{alpha} treatment and time of sample collection (P < 0.001). For the PGE2 concentration (Fig. 3b), two-way interaction was found between the 1-µg TNF{alpha} treatment and time of sample collection (P < 0.001). Three-way interactions were found for both PGs among TNF{alpha} (1 and 10 µg) treatment, saline treatment, and time of sample collection (P < 0.001). For the NO2-/NO3- concentrations, two-way interactions were found between TNF{alpha} treatments (both 1 and 10 µg) and time of sample collection (P < 0.01). Three-way interaction was found among TNF{alpha} treatment, saline treatment, and time of sample collection (P < 0.01). For the LTC4 concentrations, three-way interaction was found among TNF{alpha} (10 µg), saline treatment, and time of sample collection (P < 0.05) and between both doses of TNF{alpha} and time of sample collection (P < 0.01).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present in vivo study showed that the effects of TNF{alpha} on the estrous cycle and the function of the CL in cattle varied dramatically, depending on the dose. At low doses (0.1 and 1 µg), TNF{alpha} induced luteolysis and shortened the estrous cycle, whereas at high doses it prevented luteal regression and prolonged the luteal lifespan. We did not measure the blood plasma concentration of TNF{alpha} achieved by the infusion of TNF{alpha}. However, based on the total amount of infused TNF{alpha} and the total amount of circulated blood, one could calculate that the lower doses of TNF{alpha} used in the present study only slightly raised the concentration of this cytokine in the peripheral blood, and the concentrations could be around the level previously observed during the estrous cycle in healthy cows [31]. On the other hand, the high doses of TNF{alpha} were calculated to lead to peripheral blood concentrations around those observed during inflammatory processes such as mastitis [32, 33]. Thus, the concentrations of TNF{alpha} used in the present study may not correspond to the local concentrations physiologically present in the female reproductive tract in cattle. However, the doses tested in the present study may be useful for possible applications of TNF{alpha} in the manipulation of luteal function either to promote or prevent luteolysis. Moreover, the data obtained in the present study may help explain some of the inconsistencies observed in in vitro studies [10, 20–23, 25, 34, 35].

We assume that the direct action of TNF{alpha} on the CL is one of the major mechanisms by which the effects of TNF{alpha} on the luteal lifespan vary depending on the dose. Shaw and Britt [31] used the in vivo microdialysis system to show that luteal concentrations of TNF{alpha} were detectable in the dialysates of cows exhibiting spontaneous or PGF2{alpha}-induced luteolysis only after the onset of P4 decline. These findings suggest that TNF{alpha} plays an important role in structural luteolysis. It has been shown that macrophages and other immune cells invade the bovine CL at the time of luteolysis [18, 34, 36, 37], and TNF{alpha} participates in the apoptotic events there [21–23]. Thus, lower doses of TNF{alpha} may directly induce apoptosis, resulting in regression of the CL and shortening of the estrous cycle.

However, it is also known that TNF{alpha} is involved in regulating normal ovarian function, including the proliferation of the cells in the CL and follicles of various species [38, 39]. It has been shown that in the rat ovary TNF{alpha} has a mitogenic effect on theca-interstitial cells and preferentially increases the proportion of steroidogenic cells [40]. Thus, TNF{alpha} has a diverse spectrum of biological activities, including stimulation of cell proliferation and differentiation and induction of cell apoptotic death [40, 41]. It has recently been shown that the effects of TNF{alpha} on the rat ovary varied, depending on the dose; although lower doses of TNF{alpha} induced apoptosis in oocytes and follicles, higher concentrations showed no effect [41]. The ability of TNF{alpha} to exert a wide variety of effects is likely due to actions exerted via multiple signaling pathways involving two distinct receptors, i.e., TNF-RI (high affinity, responsible for the transduction of cell death signaling) and TNF-RII (low affinity, implicated in cell proliferation) [42–44]. In addition to TNF-RI mRNA [20], mRNA for TNF-RII is also highly expressed in the bovine CL (unpublished data). Therefore, it is possible that the lower concentrations of TNF{alpha} (0.1 and 1 µg) in the present study induced cytotoxicity in the bovine CL by preferential binding to TNF-RI, whereas higher concentrations of TNF{alpha} (10, 25, and 50 µg) bound to both receptors or bound preferentially to TNF-RII, stimulating a survival pathway.

The data obtained in the current in vivo study support and extend our previous in vitro observations [9, 10, 25] and suggest the new concept that TNF{alpha} may be one of the crucial factors involved in the regulation of luteolysis at the uterine level in cattle. Infusion of the lower dose of TNF{alpha} (1 µg) increased the production and output of two important luteolytic factors, PGF2{alpha} [3, 4] and NO [10, 12–14], resulting in shortening of the estrous cycle. TNF{alpha}, in contrast to OT, affected PGF2{alpha} output from the bovine endometrium not only at the follicular stage of the estrous cycle but also before the P4 decline at the mid and late luteal stages [9]. Interestingly, TNF{alpha} stimulates PGF2{alpha} synthesis in the stromal cells but not in the epithelial cells of bovine endometrium [10]. The density of the populations of leukocytes and macrophages increases markedly in luminal and glandular epithelia of the bovine uterus during the mid to late luteal phases [45]. Thus, TNF{alpha} may originate from the immune cells that infiltrate the bovine uterus during luteolysis. Moreover, we have recently found by an in situ hybridization study that TNF{alpha} mRNA is expressed in bovine endometrial tissue during the estrous cycle and that TNF{alpha} may be preferentially produced by the epithelial endometrial cells in cattle (unpublished data). These findings suggest that TNF{alpha} induces autoamplification of the PGF2{alpha} synthesis loop in the bovine endometrium [3, 4, 6] in an autocrine and/or paracrine manner and that uterine TNF{alpha} initiates a positive cascade between uterine PGF2{alpha} and various luteolytic factors to complete luteolysis [4, 6, 8, 9, 19, 21, 34, 38, 46].

NO is the most likely candidate for an important component of a luteolytic cascade induced by TNF{alpha} following uterine PGF2{alpha}. NO may mediate the luteolytic actions of both TNF{alpha} and uterine PGF2{alpha} [12–14, 46]. TNF{alpha} at a dose of 1 µg induced a two-phase pattern of NO output. The first peak of NO concentration was observed during TNF{alpha} infusion and was followed by a long-term increase in NO production, as measured by NO2-/NO3- concentrations in the blood. The NO production was highly correlated with the increase in PGFM concentration. Therefore, in addition to the direct effect of TNF{alpha} on the NO synthase activity in the uterus [10, 47], TNF{alpha} may have induced the increase of NO production in the ovary via effects on PGF2{alpha} [12, 46] in the present study. Although we expected that both TNF{alpha} and NO would stimulate the secretion of LTC4 [13], no stimulatory effect of the luteolytic dose (1 µg) of TNF{alpha} on LTC4 output was observed. In fact, peaks of luteolytic leukotrienes (types B4 and C4) [15, 16] have been demonstrated in bovine CL 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 [17]. Therefore, the release of LTC4 from the CL may be one of the last results of the activation of the luteolytic cascade induced by uterine TNF{alpha} and PGF2{alpha}.

TNF{alpha} at a luteolytic dose (1 µg) strongly decreased the concentration of OT in the peripheral blood. The inverse correlation between NO and OT concentrations after TNF{alpha} treatment suggests that inhibitory effects of TNF{alpha} on OT secretion may be mediated by NO. In support, NO donors strongly inhibited the OT production by cultured bovine luteal cells [11] and the OT secretion in microdialyzed bovine CL in vivo [48], but the local inhibition of NO in microdialyzed bovine CL in vivo stimulated OT secretion [14]. Altogether, these findings suggest that the luteal concentration of OT is down-regulated during TNF{alpha}- and NO-dependent luteolysis. In fact, the concentrations of OT in the blood [49] and in intact [47] and microdialyzed in vivo CL [17, 50] are extremely low at the time of spontaneous luteolysis. Moreover, the blockade of uterine OT receptors with a specific OT antagonist from Day 15 to Day 22 of the cycle affected neither luteolysis nor the duration of the estrous cycle in heifers [51]. These findings raise serious questions as to whether OT of luteal origin plays a significant role in initiating luteal regression in cattle, as suggested previously [4, 6, 9, 17, 50, 51]. Therefore, PGF2{alpha} secretion by the endometrium during luteolysis in cattle may be regulated not only by OT but also by one or more other factors, including TNF{alpha} [4, 9, 10].

On the other hand, TNF{alpha} also induced the production and output of luteotropic PGE2 in cultured bovine luteal [20] and endometrial stromal cells and tissue in vitro [25]. Those in vitro observations were confirmed by the present in vivo data. A strong correlation has been found between doses of TNF{alpha} and PGE2 levels. The higher dose of TNF{alpha} (10 µg) stimulated P4 and PGE2 secretion and inhibited LTC4 secretion, resulting in the prolongation of the estrous cycle in cattle. These data suggest that TNF{alpha} has luteotropic and luteoprotective roles. In fact, TNF{alpha} gene expression and very high TNF{alpha} protein production have been found in embryo, placenta, and pregnant uterus and oviduct [38, 52]. It has recently been shown that TNF{alpha} stimulates PGE synthase mRNA expression in cultured bovine endometrial stromal cells [53]. Increased PGE synthase production and activity may change the PGE2/PGF2{alpha} ratio and contribute to establishing pregnancy [5, 25, 26, 54]. Since PGF2{alpha} and PGE2, which are synthesized by the bovine endometrium, are assumed to play opposite roles, the relative proportions of PGF2{alpha} and PGE2 synthesis may be more important than the absolute levels of each individual PG. The mechanisms and properties of features regulation of PGF2{alpha} and PGE2 synthesis in the bovine endometrium by different doses of TNF{alpha} are not known. However, it has been shown that TNF{alpha} induced a switch from the PGD2 to PGE2 synthesis pathway via the regulation of PGE synthase expression and activity in murine macrophages [55]. Both PGD2 and PGE2 may be converted enzymatically to PGF2{alpha} by PGF synthases (11-keto-PGD reductase, 9-keto-PGE reductase, and/or other aldo-keto-reductases) [25, 56]. However, Madore et al. [57] recently found that AKR1C family members (to which all the currently known PGFs belong) are not expressed in the bovine endometrium. Alternatively, an aldose reductase known for its 20{alpha}-hydroxysteroid dehydrogenase activity, AKR1B5, is a likely candidate enzyme for controlling the sufficient and timely production of PGF2{alpha} directly from PGH2 in the bovine endometrium [57]. Thus, modulation of the activity of PGF-converting enzymes may be a mechanism by which TNF{alpha} switches prostanoid metabolism in the bovine endometrium from production of luteolytic PGF2{alpha} to luteotropic PGE2. Further studies are needed to test these possibilities.

The mechanisms controlling the formation and maintenance of CL and, finally, luteolysis in cattle are known to be immune cell- and cytokine-dependent processes [8, 19, 21–23, 34]. The present in vivo study showed that TNF{alpha} inversely regulates the lifespan of CL in cattle, depending on its concentrations. These data together with results obtained from earlier in vitro studies suggest the concept that TNF{alpha} at low concentrations plays an important role in luteolysis, especially with regard to stimulation of PGF2a production in the uterus, and consequently leads to both functional and structural regression of CL in cattle. On the other hand, TNF{alpha} at high concentrations prolongs the estrous cycle in cattle by inducing a survival pathway in the CL and by contributing to P4 production.


   ACKNOWLEDGMENTS
 
We thank Dr. Genowefa Kotwica and Dr. Stanislaw Okrasa of the Warmia and Mazury Univeristy in Olsztyn, Poland, for OT and P4 antiserum, respectively; Dr. William Silvia of the University of Kentucky, Lexington, KY, for antiserum against PGFM; Dr. Seiji Ito of Kansai Medical University, Osaka, Japan, for PGE2 antiserum; and Dainippon Pharmaceutical Co., Ltd., Osaka, Japan for recombinant human TNF{alpha} (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 Mrs. Hanna Kostuch and Mr. Jerzy Kostuch of the Animal Farm "Farmer" in Zalesie for their excellent cooperation and agreement to let us use the animals for the present experiments.


   FOOTNOTES
 
1 This research was supported by the Grants-in Aid for Scientific Research (KBN 5P06K 003 21; JSPS 14360168) and Polish-Japanese Joint Research Project under the agreement between the Polish Academy of Sciences and the Japan Society for the Promotion of Sciences. Back

2 Correspondence: Dariusz J. Skarzynski, FAX: 48 89 524 03 47; skadar{at}pan.olsztyn.pl Back

Received: 18 February 2003.

First decision: 18 March 2003.

Accepted: 31 July 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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