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Article |
and Its Receptor in Bovine Corpus Luteum Throughout the Estrous Cycle1
a Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
b Institute of Physiology, Technical University of Munich, D-85350 Freising-Weihenstephan, Germany
c Laboratory of Animal Reproduction, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) expression, the presence of functional TNF- receptors, and expression of TNF receptor type I (TNF-RI) mRNA in the bovine corpus luteum (CL) during different stages of the estrous cycle. Reverse transcription (RT)-polymerase chain reaction (PCR) showed no difference in TNF- mRNA expression during the estrous cycle. Concentrations of TNF- in the CL tissue increased significantly from the mid to the late luteal stage and decreased thereafter (P < 0.05). An RT-PCR analysis showed higher levels of TNF-RI mRNA in CL of Days 3–7 than of other stages (P < 0.05). 125I-TNF- binding to the membranes of bovine CL was maximal after incubation at 38°C for 48 h. The binding was much greater for TNF- than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant: 3.60 ± 0.58–5.79 ± 0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage of the estrous cycle. When cultured cells of all luteal stages were exposed to TNF- (1–100 ng/ml), TNF- stimulated prostaglandin F2 and prostaglandin E2 secretion by the cells in a dose-dependent fashion (P < 0.01), especially during the early luteal phase, although it did not affect progesterone secretion. These results indicate the local production of TNF- and the presence of functional TNF-RI in bovine CL throughout the estrous cycle, and suggest that TNF- plays some roles in regulating bovine CL function throughout the estrous cycle.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) is a nonglycosylated protein with a molecular mass of 17 kDa, which was first described as a tumoricidal factor produced by activated macrophages [1]. Extensive research during the last decade suggests that TNF- plays one or more physiological roles in the corpus luteum (CL) in a variety of species. It has been demonstrated that TNF- inhibits gonadotropin-supported progesterone production by murine [2], porcine [3], and bovine [4] luteal cells and stimulates prostaglandin synthesis by bovine luteal cells [4]. These findings imply that the actions of TNF- on CL function are concerned with luteal regression. Indeed, the interaction of TNF- and inflammatory cells has been postulated to promote the regression of the CL [5]. Hence, if TNF- has one or more roles in luteolysis, functional TNF- receptors should be present in the CL at least by the time of luteal regression.
It has been well demonstrated that TNF- affects steroidogenesis and protein secretion in bovine granulosa and theca cells [6,7]. Similar results have been observed in many species including rats [8], pigs [9], and humans [10]. Furthermore, Wang et al. [11] demonstrated the presence of TNF- in follicular fluid in human ovary and demonstrated that TNF- increases proliferation in granulosa-luteal cells taken from hCG-treated women before ovulation. These results strongly suggest that TNF- plays physiological roles in follicular development and luteal development as well as in luteal regression.
Although, as mentioned above, TNF- modulates the functions of steroidogenic cells in the bovine ovary and CL, the local production of TNF- and the existence of TNF- receptors in the bovine ovary (the CL as well as follicle cells) is not established. Therefore, in the present study, bovine CL from different stages of the estrous cycle were examined to determine whether 1) TNF- mRNA is expressed and TNF- is produced, 2) TNF receptor type I (TNF-RI) mRNA is expressed, 3) specific binding sites for TNF- are present using a radioreceptor assay, and 4) the TNF- receptors in bovine CL are functional. For the last experiment (#4), the effects of TNF- on progesterone, prostaglandin (PG) F2, and PGE2 secretion by cultured cells were studied.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recombinant human TNF- (lot no. HF-13) and recombinant human interleukin-1 (IL-1: lot no. HL-18) were kindly donated by Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). Recombinant bovine interferon (IFN-) was kindly donated by Novartis Pharmaceutical Co. (Basel, Switzerland). Transforming growth factor (TGF-: #GE003) was purchased from Wakunaga Pharmaceutical Co., Ltd. (Osaka, Japan). PGF2 (#P7652), PGE2 (#P0409), calf serum (#C6278), dithiothreitol (DTT: #D0632), aprotinin (#A6279), and PMSF (#P7625) were purchased from Sigma Chemical Co. (St. Louis, MO).
Collection of Bovine CL
Ovaries with CL from German Fleckvieh cows were collected at a local abattoir within 10–20 min after exsanguination. The luteal stage was classified as early, mid, late, or regressed by macroscopic observation of the ovary as described previously [12,13]. After determination of the stages, CL were immediately separated from the ovaries, frozen rapidly in liquid nitrogen, and then stored at -80°C until processed for studies of specific binding of TNF- and gene expression. For experiments involving cell culture, the ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory.
RNA Isolation
Total RNA was isolated by the single-step method of Chomczynski and Sacchi [14] using TRIzol reagent (Gibco BRL, Rockville, MD) and spectroscopically quantified at 260 nm. Aliquots were electrophoresed on a 1% denaturing agarose gel to verify the quantity and quality of RNA by ethidium bromide staining.
Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)
Four micrograms of total RNA was used to generate single-strand cDNA in a 60-µl reaction mixture as described previously [15]. Conditions for enzymatic amplification were optimized for each PCR as follows: the TNF- and TNF-RI PCR contained 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.6 µM of each primer, and 0.5 units of thermostable polymerase PrimeZyme (Biometra, Göttingen, Germany) to 5 µl cDNA (final volume 25 µl). Ubiquitin PCR was performed under the same conditions as those for TNF- and TNF-RI, but a higher concentration of primer (1.5 µM) was used. Samples for TNF- and TNF-RI were amplified for 27 and 28 cycles, respectively (one single denaturation step, 94°C for 2 min; each cycle, 94°C for 1 min and 60°C for 1 min; and afterwards one additional elongation step, 72°C for 2 min). Samples for the housekeeping gene ubiquitin were amplified by 22 cycles (one single denaturation step, 94°C for 2 min; each cycle, 94°C for 45 sec and 55°C for 45 sec; and afterwards one additional elongation step, 72°C for 45 sec). To determine the optimal quantity of reverse transcriptase needed for PCR and to verify that the cDNA product depended on the mRNA transcript used for the template, varying quantities of transcriptase were used in the PCR reaction. The RT product from 3 µl was in the linear range of these amounts and produced a visible band. To exclude the possibility of amplification of genomic DNA, all experiments included reactions in which the RT enzyme or cDNA template was omitted. As a negative control, water was used instead of RNA for the RT-PCR to exclude any contamination from buffers and tubes.
The primers encoding the bovine sequences were designed by using the EMBL database or were used as described elsewhere and commercially synthesized (Amersham-Pharmacia, Freiburg, Germany). The primers were chosen using the "Husar" online software package in Heidelberg (http://genome.dkfz-heidelberg.de) and are as follows: TNF- forward 5'-GAAGCTGGAAGACAACCA-3' and reverse 5'-TCCCAAAGTAGACCTGCC-3' (338 bp); TNF-RI forward 5'-CACCACCACCATCTGCTT-3' and reverse 5'-TCTGAACTGGGGTGCAGA-3' (257 bp); ubiquitin forward 5'-ATGCAGATCTTTGTGAAGAC-3' and reverse 5'-CTTCTGGATGTTGTAGTC-3' (189 bp; Gabler et al. [16]).
Aliquots of the PCR reaction products (5 µl) were added to 1 µl bromphenol blue glycerin and fractionated by electrophoresis through a 1.5% agarose gel containing ethidium bromide in a constant 60-V field. To determine the length of the products, a mass ladder and 100-basepair (bp) marker was used. The ethidium bromide-stained gels were evaluated by a video documentation system (Amersham-Pharmacia). Band intensities were analyzed by computerized densitometry using the Image Master program (Amersham-Pharmacia). This method allowed only a relative quantification. To verify each PCR product, double-strand sequencing was performed directly or after subcloning (TopLab, Munich, Germany).
Membrane Preparation
CL were thawed and minced with scissors in ice-cold 25 mM Tris-HCl containing 300 mM sucrose, 2 mM EDTA, 3 mM DTT, 500 kIU/ml aprotinin, and 0.5 mM PMSF pH 7.4. They were then homogenized in the same buffer with a Polytron homogenizer (Kinematica, Lucerne, Switzerland) using three 10-sec bursts separated by a 1-min cooling period in ice. For each luteal stage of the estrous cycle, four pools were prepared. Each pool was made from 5–17 CL.
The homogenate of luteal tissue was subsequently centrifuged at 800 x g for 10 min to remove tissue debris, and the supernatant was collected and centrifuged at 30 000 x g for 20 min to obtain the plasma membrane pellet. The pellets were resuspended and recentrifuged in the same buffer to dissociate TNF- from their binding sites. The pellets were then washed three times by centrifugation for 10 min at 30 000 x g, decanted, and resuspended in 25 mM Tris-HCl containing 10 mM MgCl2 pH 7.4. All steps of the luteal membrane preparations were conducted at 4°C. The protein concentrations of the membrane preparations were determined by the method of Lowry et al. [17] using BSA (#735078, Boehringer Mannheim GmbH, Mannheim, Germany) as a standard. The preparation was diluted to give a protein concentration of 10 mg/ml of luteal membrane with 25 mM Tris-HCl containing 10 mM MgCl2 and 0.5% (w:v) BSA pH 7.4.
Radioreceptor Assay
Recombinant human TNF- was iodinated with carrier-free 125I-Na (IMS 30; Amersham International plc, Buckinghamshire, England) by the iodogen method as described previously [18]. The specific activity of 125I-TNF- ranged between 530 and 540 Ci/mmol, and the maximum bindability was 30%.
Preliminary studies with midluteal (Days 8–12) membranes were carried out to establish the optimal conditions of incubation time and temperature for maximal binding of 125I-TNF- to the membranes. To reduce nonspecific binding, glass microtubes (12 x 75 mm; MLB Culture Tube, Ontario, Canada) were coated overnight with complete calf serum, and then binding assays were initiated. Nonspecific binding was assessed for each level of tracer through coincubation with a 240-fold excess of unlabeled TNF- (120 nM; 10 µl). The incubation mixture consisted of approximately 6 x 104 dpm (0.5 nM) 125I-TNF- (50 µl) and 50 µg protein (50 µl). The total volume of the mixture was 110 µl. The specificity of 125I-TNF- binding was determined by incubating increasing amounts of various unlabeled hormones (IFN-, IL-1, or TGF-) with a constant amount of 125I-TNF- (6 x 104 dpm/tube). All reagents were prepared in 10 mM Tris containing 10 mM MgCl2 (pH 7.5), 3.0 mM NaN3, and 0.1% (w:v) BSA.
The incubation was terminated by transferring the tubes into ice-cold water and by adding the same buffer into the assay tube; bound and free tracers were separated by centrifugation at 3000 x g for 40 min at 4°C. Supernatants were decanted immediately, and the pellets were counted for 125I in a gamma-counter (Pharmacia-Wallac 1282; Compugamma CS, Turku, Finland) at an efficiency of 82%. Nonspecific binding accounted for < 35% of total binding.
Luteal Cell Culture
Luteal cells were prepared and cells were cultured as previously described [19]. The CL cells of early (Days 5–7), mid (Days 8–12), and late (Days 15–17) stages were counted with a hemocytometer, and cell viability at each stage was higher than 80% as assessed by trypan blue exclusion. The obtained cell suspension contained very few endothelial cells or fibrocytes (0–5%) and no erythrocytes. Viable cells (5 x 105/well) of CL were cultured in a culture medium (Dulbecco's Modified Eagle's Medium and Ham's F-12 medium, 1:1 [v:v], #D8900; Sigma) supplemented with 10% calf serum and 20 µg/ml gentamicin (#15750-011; Gibco BRL) in 24-well culture plates (Costar, Cambridge, MA) for up to 48 h in a humidified atmosphere of 5% CO2 in air at 37.5°C. In the final 24 h of culture, the cells were exposed to varying concentrations of TNF- (1–100 ng/ml) or bovine LH (USDA-bLH-B6; 10 ng/ml). The conditioned media were collected and stored at -30°C until assayed for progesterone, PGF2, and PGE2.
Enzyme Immunoassay
Concentrations of progesterone were determined directly from the cell culture media with an enzyme immunoassay [20]. The samples for the progesterone assay were diluted 200 times with assay buffer. The standard curve ranged from 0.39 to 100 ng/ml, and the effective dose for 50% inhibition (ED50) of the assay was 9.56 ng/ml. The intra- and interassay coefficients of variation were on average 6.8% and 9.6%, respectively.
The concentrations of PGF2 in the culture medium were determined directly with a double-antibody enzyme immunoassay as described previously [21]. The samples for the PGF2 assay were diluted 50 times with assay buffer. The standard curves ranged from 15.6 to 4000 pg/ml, and the ED50 of the assay was 250 pg/ml. The intra- and interassay coefficients of variation were on average 8.8% and 12.5%, respectively.
PGE2 concentrations were determined with an enzyme immunoassay using peroxidase-labeled PGE2 and anti-PGE2 serum as described previously [22]. Cross-reactivities of the anti-PGE2 serum, validated by comparing the inhibition of binding of peroxidase-labeled PGE2 to antiserum, were as follows: PGE2, 100%; PGE1, 18%; PGA1, 10%; PGA2, 4.6%; PGB2, 6.7%; PGD2, 0.13%; PGF2, 2.8%; PGJ2, 14%; and 15-keto PGE2, 0.05%. The PGE2 standard curve ranged from 0.11 ng/ml to 28.19 ng/ml, and the ED50 of the assay was 0.97 ng/ml. The intra- and interassay coefficients of variation were on average 4.9% and 8.6%, respectively.
TNF- concentrations were determined with a commercial enzyme immunoassay for human TNF- (IBL, Hamburg, Germany). The assay is a solid-phase ELISA based on the sandwich principle. Recombinant human TNF- was used as a standard and was calibrated against WHO Standard 87/650. The sensitivity of the assay is 2.5 pg/ml. Linearity in the bovine tissue samples (CL and liver) were tested by diluting tissue extracts in sample dilution buffer. CL or liver tissue extracts diluted 1:2, 1:4, 1:8 or 1:2, 1:5, 1:10, 1:15 gave concentrations of 1420, 1471, 1535, and 1052, 991, 995, 1062 pg/g wet tissue, respectively. Tissue levels (pg/g wet tissue) were in the range of 999–2464 for liver, 855–1902 for kidney, 36–193 for muscle, and < 25–44 for lung. The percentage of recovery for the human standard added to bovine CL or liver extracts was 85.54 ± 16% (mean ± SD). The intra- and interassay coefficients of variation were on average 6.4% and 9.1%, respectively. Since no bovine TNF- preparation was available for cross-reaction control, the results are expressed as human TNF- immunoreactivity.
Tissue Extraction of TNF-
Tissue (1 g wet w) was transferred into 10 ml of an acidic buffer (pH 2.8) containing 2.54 mg orthophosphoric acid, 22.64 mg NaH2PO4·H2O, 18.6 mg EDTA, 70.1 mg NaCl, 2 mg NaN3, 20 mg BSA, and 1 ml Triton X-100, and was homogenized in an ice bath with Ultra Turrax equipment (Janke and Kunkel, Staufen, Germany). Five bursts of 15 sec at maximum speed with 45-sec intervals of cooling between each burst were applied. The homogenate was subsequently centrifuged at 2000 x g for 15 min at 4°C. The supernatant was directly used for the ELISA.
Statistical Analysis
All experimental data are shown as the mean ± SEM. The data on binding of TNF- to CL membranes were analyzed with the LIGAND program [23] using nonlinear iterative curve-fitting procedures [24]. The initial parameters were calculated by Scatchard analysis [25] and were then iteratively refined until the weighted sum of squares was minimized. The goodness of fit for the selected model was analyzed by a runs test. Different models (one or two binding sites) were compared using F-test statistics to determine whether a change in the model resulted in a significant reduction in the weighted sum of squares. The criteria for rejecting or accepting a particular model were based on the calculated probability values [23]. The statistical significance of differences in mRNA expression of TNF- and TNF-RI, TNF- concentrations in the CL tissue, the binding parameters of TNF- receptors, and the concentrations of progesterone, PGF2, and PGE2 in culture media were assessed by ANOVA followed by Fisher's protected least-significant difference (PLSD) as a multiple comparison test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and TNF-RI
Specific transcripts for TNF- and TNF-RI were detected in bovine CL. Each PCR product showed 100% homology to the known bovine genes after sequencing. To confirm the integrity of the mRNA templates and RT-PCR protocol, the housekeeping gene ubiquitin was examined in all samples. A representative sample for the ubiquitin-specific RT-PCR products (189 + 417 bp) is shown in Figure 1a. The relative signal intensities for PCR products specific for TNF- and TNF-RI were assessed after correction based on the ubiquitin signal intensities. The ubiquitin was found to be stably expressed in the bovine CL during the estrous cycle. A representative example for the TNF- RT-PCR is given in Figure 1b. There is obviously no difference in the expression between the samples. The results of the densitometric analysis of TNF- mRNA in the CL tissue during the estrous cycle are shown in Figure 2. There was no statistically significant difference between any of the stages examined.
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A representative example for the TNF-RI RT-PCR is shown in Figure 1c. There are clear differences for the receptor during early (Days 3–7) and late (Days 13–18) luteal phases. The results of the densitometric analysis of TNF-RI mRNA are demonstrated in Figure 3. The mRNA expression was high at the very early stage (Days 1–2) and significantly increased during Days 3–7, with a significant decrease thereafter (P < 0.05).
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Tissue Concentration of TNF-
The TNF- concentrations in the CL tissue are given in Figure 4. TNF- in CL tissue was very low during the early and midluteal phases (average range 71–116 pg/g wet tissue), and then increased significantly during the late luteal phase (Days 13–18) and decreased after regression (P < 0.05).
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Binding Characteristics
A preliminary assay of the binding of TNF- to bovine CL membranes was carried out to test the conditions for the radioreceptor assay described in Materials and Methods. It was confirmed that maximal binding was reached after 48 h at 38°C (Fig. 5a). Specific binding increased with increasing protein concentrations. A linear relationship was established in the amount of binding from 1 to 100 µg/50 µl (Fig. 5b). For this reason, further assays were carried out under these conditions.
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Figure 5c shows the displacement curves of 125I-TNF- with three related peptides. The binding was highly specific for TNF-. There was little or no competition for TNF- binding sites by IFN-, IL-1, or TGF-. Scatchard plots of the binding data were linear (Fig. 5d). Analysis with the LIGAND program revealed that the concentrations of TNF- receptors were constant in the estrous cycle except in the late stage (Days 15–17) (Table 1; P < 0.05). The dissociation constant (Kd) values of the CL membranes in the mid and late stages and regressed stage (Days 19–21) were significantly higher than those in the early stages (Table 1; P < 0.05).
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Effects of TNF- on Progesterone, PGF2, and PGE2 Secretion by Bovine Luteal Cells
Bovine LH stimulated progesterone secretion by cultured luteal cells of all stages of the estrous cycle, indicating that the cells cultured according to the present experimental design were reactive (Fig. 6a). As shown in Figure 6a, progesterone secretion by the cells at all luteal stages was not affected by any dose of TNF- (1–100 ng/ml). In contrast, TNF- significantly stimulated PGF2 and PGE2 secretion by the cells of early, mid, and late stages in a dose-dependent fashion (Fig. 6, b and c; P < 0.01). The stimulatory effects of TNF- (100 ng/ml) on PGF2 and PGE2 secretion by the early luteal cells (330–790% vs. control) were higher than those of the other stages (180–230% vs. control).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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concentrations by an enzyme immunoassay, mRNA expression of TNF- and TNF-RI by an RT-PCR analysis, and the specific binding for TNF- by a radioreceptor assay to provide information on TNF- and its receptor in bovine CL. In addition, the fact that TNF- stimulated PGF2 as well as PGE2 secretion by cultured bovine luteal cells of all luteal stages in a dose-dependent fashion confirmed that the receptors for TNF- in bovine CL are functional. It is well known that there are two types of TNF receptors, i.e., TNF-RI and type II receptor [26]. However, since the cDNA sequence of the bovine TNF- receptors has been reported only for the type I receptors [27], the expression of mRNA for the type I receptor in bovine CL was examined by use of RT-PCR in the present study. The mRNA of TNF-RI was clearly expressed in the bovine CL at all stages during the estrous cycle. It is therefore likely that the TNF-RI might contribute to the specific binding of 125I-TNF- to membrane preparations of the bovine CL, as was observed in the present study. Collectively, these findings provide evidence for a local action of TNF- within the bovine CL by receptor-mediated mechanisms.
It is well recognized that bovine CL consists of several cell types, e.g., large and small luteal cells, endothelial cells, and fibroblasts [28]. Since we used membrane homogenates that were obtained from whole CL for the radioreceptor assay in this study, it is not possible to say which cell types have TNF- receptors. Kull et al. [29] showed the presence of high-affinity binding sites for TNF- on bovine aortic endothelial cells. In addition, it has been demonstrated that TNF- stimulated phospholipase A2 activity in bovine aortic endothelial cells [30], providing indirect evidence that functional TNF- receptors are present on endothelial cells derived from CL. Therefore, the results of our mRNA analysis and binding test might indicate the expression of TNF- receptors on endothelial cells in bovine CL. However, most of the endothelial cells as well as fibroblasts and erythrocytes would have been removed with use of our cell culture techniques [19]. Furthermore, TNF- clearly stimulated PGF2 and PGE2 secretion by the cultured luteal cells in the present study. We assume that functional TNF- receptors are present at least in the luteal cells in the bovine CL, although the distribution of TNF- receptors in luteal cells (large or small cells) remains unknown.
It is interesting to note that high-affinity TNF- receptors and TNF-RI mRNA expression were found in bovine CL in the early stage, and that TNF- dose-dependently stimulated both PGF2 and PGE2 secretion by the cells of the early stage. It is well demonstrated that luteal PGF2 and PGE2 have a luteotropic effect; e.g., they stimulate progesterone secretion by bovine CL in vitro [31,32]. Moreover, it is well known that the macrophages [1] and endothelial cells [33] are sources of TNF-, and that these cells infiltrate into newly formed CL concomitant with vascular angiogenesis [34,35]. Therefore, we postulate that TNF- contributes to the production of PGF2 and PGE2 by early CL, and may partly promote the formation of CL. However, although we expected in the present study that TNF- indirectly stimulates progesterone output by the TNF--promoted PGs from the cultured luteal cells, no changes in progesterone secretion were observed. We could not find an appropriate explanation for this phenomenon. Further studies are needed to clarify the roles of TNF- in the early luteal stage.
Local secretion of TNF- in bovine CL of the late stage was higher than that of the mid stage CL [36], and TNF- concentrations in the CL were dramatically increased from the mid to the late luteal phase in this study. However, the levels of TNF- mRNA expression were similar in the CL throughout the estrous cycle, and similar results were recently reported [37]. The discrepancy between the expression of TNF- mRNA and the concentrations of TNF- in the CL during the estrous cycle could be explained by the fact that macrophages infiltrate into the CL tissue at the time of luteolysis [5,38,39]. Furthermore, another explanation for the discrepancy might be due to post-translational processing. Since it has been well demonstrated that TNF- production is regulated by rate-limiting steps that involve transcription, translation, protein storage, membrane insertion, and ultimate secretion [40,41], the post-translational processing for TNF- could be controlled by an unknown factor(s). Hence, we assume that post-translational processing in the cells might be restricted during the early luteal phase and then start rapidly in the mid and late phases. A related phenomenon has been reported concerning oxytocin and its mRNA expression in bovine CL. Oxytocin mRNA expression was maximal in early CL, although the maximum levels of oxytocin protein was observed in mid stage CL [42].
It has been postulated that TNF- plays some roles in luteolysis [43,44]. Indeed, in the present study, the maximum concentrations of TNF- were observed in the CL of the late stage (Days 13–18). However, the concentration of TNF- receptors in the late CL was significantly lower than the concentrations of the other stages. Since it has been demonstrated that the expression of TNF- receptors was down-regulated by TNF- [45], the low expression of TNF-RI in the late CL in this study might have been due to a down-regulation by locally produced TNF- in the CL. On the other hand, high-affinity binding sites (Kd: 5.5 ± 0.2 nM, concentration: 30.4 ± 2.3 pmol/mg protein) for TNF- were found in the CL of the regressed stage in the present study. It has been clearly demonstrated that TNF- induces a significant increase in the expression of major histocompatibility (MHC) class 1 glycoproteins in cultured bovine luteal cells and that these glycoproteins are recognized by cytotoxic T cells in order for the T cells to devour the luteal cells [4]. Moreover, TNF- was shown to induce apoptosis of cultured mouse luteal cells [46]. All of these findings, along with the high concentrations of TNF- and its specific receptor in bovine CL, suggest that TNF- may play an important role in luteolysis of cattle, in particular at the phase of luteal regression (death and destruction of luteal cells).
In conclusion, the overall results of the present study indicate the production and the presence of local TNF- as well as functional TNF- receptors (at least TNF-RI) in bovine CL during the estrous cycle, and suggest that TNF- plays physiological roles in regulating bovine CL function not only at the time of luteal regression but throughout all luteal phases.
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ACKNOWLEDGMENTS |
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and PGE2; Dr. Akio Miyamoto of Obihiro University for PGE2-horseradish peroxidase; the Dainippon Pharmaceutical Co. Ltd. for recombinant human TNF- and IL-1; the Novartis Pharmaceutical Co. for recombinant bovine IFN-; and the USDA Animal Hormone Program for bovine LH (USDA-bLH-B6). |
FOOTNOTES |
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1 This research was supported by Grants-in-Aid for Scientific Research (No. 11460129) and a Research Fellowship (No. 8763) of the Japan Society for the Promotion of Science (JSPS) from the Ministry of Education, Science, Sports and Culture of Japan; the Japanese-German Cooperative Science Promotion Program of JSPS; and the German Research Foundation (Scha 257/14–1). 
2 Correspondence: Kiyoshi Okuda, Laboratory of Reproductive Endocrinology, Division of Animal Science and Technology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp
3 Current address: Department of Animal Reproduction, College of Agriculture, Osaka Prefecture University, Sakai 599-8531, Japan.
Accepted: August 19, 1999.
Received: December 1, 1998.
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T.P. Neuvians, D. Schams, B. Berisha, and M.W. Pfaffl Involvement of Pro-Inflammatory Cytokines, Mediators of Inflammation, and Basic Fibroblast Growth Factor in Prostaglandin F2{alpha}-Induced Luteolysis in Bovine Corpus Luteum Biol Reprod, February 1, 2004; 70(2): 473 - 480. [Abstract] [Full Text] [PDF]
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M Ohtani, S Takase, M P B Wijayagunawardane, M Tetsuka, and A Miyamoto Local interaction of prostaglandin F2{alpha} with endothelin-1 and tumor necrosis factor-{alpha} on the release of progesterone and oxytocin in ovine corpora lutea in vivo: a possible implication for a luteolytic cascade Reproduction, January 1, 2004; 127(1): 117 - 124. [Abstract] [Full Text] [PDF]
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D. J. Skarzynski, M. M. Bah, K. M. Deptula, I. Woclawek-Potocka, A. Korzekwa, M. Shibaya, W. Pilawski, and K. Okuda Roles of Tumor Necrosis Factor-{alpha} of the Estrous Cycle in Cattle: An In Vivo Study Biol Reprod, December 1, 2003; 69(6): 1907 - 1913. [Abstract] [Full Text] [PDF]
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 R. Sakumoto, E. Kasuya, T. Komatsu, and T. Akita Central and peripheral concentrations of tumor necrosis factor-{alpha} in Chinese Meishan pigs stimulated with lipopolysaccharide J Anim Sci, May 1, 2003; 81(5): 1274 - 1280. [Abstract] [Full Text] [PDF]
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M. Abdo, S. Hisheh, and A. Dharmarajan Role of Tumor Necrosis Factor-Alpha and the Modulating Effect of the Caspases in Rat Corpus Luteum Apoptosis Biol Reprod, April 1, 2003; 68(4): 1241 - 1248. [Abstract] [Full Text] [PDF]
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W. A. Ricke, G. W. Smith, E. W. McIntush, and M. F. Smith Analysis of Luteal Tissue Inhibitor of Metalloproteinase-1, -2, and -3 During Prostaglandin F2{alpha}-Induced Luteolysis Biol Reprod, May 1, 2002; 66(5): 1387 - 1394. [Abstract] [Full Text]
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C.L. Keefer, R. Keyston, A. Lazaris, B. Bhatia, I. Begin, A.S. Bilodeau, F.J. Zhou, N. Kafidi, B. Wang, H. Baldassarre, et al. Production of Cloned Goats after Nuclear Transfer Using Adult Somatic Cells Biol Reprod, January 1, 2002; 66(1): 199 - 203. [Abstract] [Full Text]
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B. C. Reggio, A. N. James, H. L. Green, W. G. Gavin, E. Behboodi, Y. Echelard, and R. A. Godke Cloned Transgenic Offspring Resulting from Somatic Cell Nuclear Transfer in the Goat: Oocytes Derived from Both Follicle-Stimulating Hormone-Stimulated and Nonstimulated Abattoir-Derived Ovaries Biol Reprod, November 1, 2001; 65(5): 1528 - 1533. [Abstract] [Full Text] [PDF]
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C.L. Keefer, H. Baldassarre, R. Keyston, B. Wang, B. Bhatia, A.S. Bilodeau, J.F. Zhou, M. Leduc, B.R. Downey, A. Lazaris, et al. Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes Biol Reprod, March 1, 2001; 64(3): 849 - 856. [Abstract] [Full Text]
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A. Friedman, S. Weiss, N. Levy, and R. Meidan Role of Tumor Necrosis Factor {alpha} and Its Type I Receptor in Luteal Regression: Induction of Programmed Cell Death in Bovine Corpus Luteum-Derived Endothelial Cells Biol Reprod, December 1, 2000; 63(6): 1905 - 1912. [Abstract] [Full Text]
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Y. Miyamoto, D. J. Skarzynski, and K. Okuda Is Tumor Necrosis Factor {alpha} a Trigger for the Initiation of Endometrial Prostaglandin F2{alpha} Release at Luteolysis in Cattle? Biol Reprod, May 1, 2000; 62(5): 1109 - 1115. [Abstract] [Full Text]
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