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Regular Article |
a Laboratory of Reproductive Endocrinology, Department of Animal Science, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
b Department of Immunology, National Institute of Animal Health, Ibaraki 305-0856, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(IFN; 50 ng/ml) and/or tumor necrosis factor 
(TNF; 50 ng/ml). After 24 h of culture, the expression of Fas mRNA was detected in the cultured cells and was increased by IFN. Moreover, TNF augmented the stimulatory action of IFN, whereas TNF alone did not affect the expression of Fas mRNA. The effects of IFN and TNF on Fas-mediated cell death were also examined. Cells were exposed to IFN and/or TNF for 24 h and were further treated with IFN and/or TNF in the presence or absence of Fas L (100 ng/ml) for 24 h. Treatments of the cells with IFN alone and in combination with TNF resulted in killing of 30% and 50% of the cells (P < 0.05), respectively, whereas TNF alone did not have a cytotoxic effect on the cells. On the other hand, Fas L killed 60% of the cells treated with IFN (P < 0.01) and 85% of the cells treated with the combination of TNF and IFN (P < 0.01), respectively, whereas Fas L showed no effect on the viability of the luteal cells treated with or without TNF. Furthermore, shrunken nuclei and apoptotic bodies were observed in the cells treated with Fas L in the presence of TNF and IFN. The overall results suggest that a Fas-Fas L system is present in bovine CL and that leukocyte-derived TNF and IFN play important roles in Fas-mediated luteal cell death.
apoptosis, corpus luteum, corpus luteum function, cytokines, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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released by the uterus in a pulsatile manner causes regression of the corpus luteum (CL) [1]. A rapid functional regression of CL, characterized by inhibition of progesterone production, is followed by a phase of structural regression. During luteolysis, cells of the CL undergo apoptosis, a process that has been described by morphologic and biochemical parameters in many domestic species including the cow [2, 3], pig [4], and sheep [5]. It is generally accepted that the immune response plays central roles in apoptosis of several tissues and cell types [6]. Moreover, since the number of leukocytes in bovine CL (e.g., T lymphocytes, macrophages) increases at the time of luteolysis [7], it is assumed that leukocytes that infiltrate into CL mediate apoptosis at the time of structural luteolysis. Leukocytes are known to produce a variety of cytokines, including tumor necrosis factor (TNF) and interferon (IFN), which have been shown to affect luteal cell function in vitro [8]. Evidence for the luteolytic actions of these cytokines is supported by a number of in vitro studies wherein treatments of luteal cells with TNF or IFN resulted in elevated levels of PGF2 and inhibition of progesterone synthesis [9, 10]. Recently, Petroff et al. [11] demonstrated that a combination of IFN and TNF reduced the viability of bovine luteal cells and induced DNA fragmentation in the cells, suggesting that these cytokines are involved in apoptotic luteal cell death in bovine CL. However, TNF in the presence of IFN did not reduce the viability of bovine luteal cells until Day 5 of culture but reduced the viability of the cells thereafter [10]. In contrast to this in vitro observation, the appearance of internucleosomal fragmentation of DNA has been observed at 24–48 h after injection of PGF2 in bovine CL in vivo [2]. Thus, we hypothesize that apoptosis in bovine CL is regulated not only by leukocyte-derived cytokines but also by one or more other factors, which transduce apoptotic signals to bovine luteal cells. The Fas antigen (Fas) is a member of the tumor necrosis factor family of cell surface receptors [12], and engagement of Fas with its ligand (Fas ligand; Fas L) induces apoptosis. Fas L is expressed at high levels on activated T lymphocytes [13] and mediates apoptosis of target cells [12]. Expression of Fas and Fas L mRNAs has been demonstrated in CL of mice, rats, and humans [14–16], and their protein expression in the CL increases with maximal levels at the regressing stage in rat CL [15]. Moreover, Fas L has been shown to enhance mouse structural luteolysis in vivo [14]. These results suggest that a Fas-mediated apoptosis pathway exists in CL and that Fas-mediated apoptosis plays an important role in structural luteolysis. In addition, it has been reported that human granulosa lutein cells [17] and mouse luteal cells [18] become sensitive to Fas-mediated apoptosis when the cells are treated with IFN alone or with IFN in combination with TNF. Moreover, IFN alone and in combination with IFN and TNF have been shown to induce Fas expression and Fas-mediated cell death in many ovarian cells [17–20].
The present study was conducted to characterize the pattern of Fas mRNA expression throughout the bovine estrous cycle. We also determined the possible actions of TNF and IFN on Fas-mediated luteal cell death.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovaries with CL from Holstein cows were collected at a local abattoir within 10–20 min after exsanguination. The luteal stages were classified as early, mid, late, or regressed by macroscopic observation of the ovary as described previously [21]. After stages were determined, CL were immediately separated from the ovaries, frozen rapidly in liquid nitrogen, and then stored at -80°C until processed for RNA isolation. For experiments involving cell culture, the ovaries with CL were submerged in ice-cold physiological saline and transported to the laboratory.
Semiquantitative Reverse Transcription-Polymerase Chain Reaction
Total RNA was prepared from CL and cultured luteal cells using Isogen (Nippongene, Toyama, Japan) according to the manufacturer's directions. One microgram of each total RNA was reverse-transcribed using a First-strand cDNA synthesis kit (Pharmacia Biotech, Tokyo, Japan, No. 27-9261-01), and one-tenth of the reaction mixture was used in each polymerase chain reaction (PCR) using specific primers for bovine Fas, ß-actin, and glyceraldehyde 3-phosphate dehydrogenase (G3PDH), respectively. The reverse transcription (RT)-PCRs were carried out with the housekeeping genes, ß-actin and G3PDH (luteal tissues) and ß-actin (luteal cells) as an internal standard. The sequence of Fas primers, which were based on a report by Vickers et al. [19], were 5'-ATGGGCTAGAAGTGGAACAAAAC-3' (5' primer, 23 mer) and 5'-CAGGAGGGCCCATAAACTGTTTGC-3' (3' primer, 24 mer). These primers generated a specific 206-base pair (bp) product from all cell types. The primers for ß-actin, which were designed as described by Asselin et al. [22], were 5'-GAGGATCTTCATGAGGTAGTCTGTCAGGTC-3' (5' primer, 30 mer) and 5'-CAACTGGGACGACATGGAGAAGATCTGGCA-3' (3' primer, 30 mer). These primers generated a specific 349-bp product from all cell types. The primers for G3PDH, which were designed as described by Friedman et al. [23], were 5'-TGTTCCAGTATGATTCCACCC-3' (5' primer, 21 mer) and 5'-TCCACCACCCTGTTGCTGTA-3' (3' primer, 20 mer). These primers generated a specific 850-bp product from all cell types. Each PCR yielded only a single amplification product. The PCRs were carried out using an AmpliTaq Gold DNA polymerase (Perkin Elmer, Foster City, CA, No. N888-0240) and a thermal cycler (Takara TP240, Tokyo, Japan). The conditions for the PCRs were as follows: after activation of the DNA polymerase by incubating for 7 min at 94°C, 33 (Fas), 21 (ß-actin), or 24 (G3PDH) cycles of reactions including denaturation for 1 min at 94°C, annealing for 1 min at 60°C, and extension for 2 min at 72°C were performed, followed by an additional extension for 5 min at 72°C. The PCR amplification was calibrated to determine the optimal number of cycles that would allow detection of the appropriate mRNA transcripts while still keeping amplification of these genes in the log phase. A two-fifths aliquot of each reaction mixture was electrophoresed on a 1.5% agarose gel containing ethidium bromide with a known standard (100-bp Ladder; New England BioLabs Inc., Beverly, MA, No. N3231S) and photographed under ultraviolet illumination. The band intensities were analyzed by computerized densitometry using NIH Image (National Institutes of Health, Bethesda, MD). This method allowed only a relative quantification. The amplified cDNA fragments were sequenced directly and/or after being subcloned into pGEM3Zf (+) (PE Applied Biosystems, Chiba, Japan). Dideoxynucleotide sequencing was performed using fluorescent primers and an automated DNA sequencer (Applied Biosystems 373A, Chiba, Japan). Sequence analysis was carried out using GENETYX software (Software Development, Tokyo, Japan) and the Blast program (National Center for Biotechnology Information, National Institutes of Health; http://www.ncbi.nlm.nih.gov/).
Cell Isolation
Dissociation of the luteal tissue and culture of luteal cells were performed as described previously [24]. Briefly, CL were perfused for 15 min with EGTA-buffer (0.1 mM EGTA [Sigma Chemical Co., St. Louis, MO, No. E-4378], 10 mM Hepes [Sigma, No. H-9136], 140 mM NaCl, 7.1 mM KCl; pH 7.4) to remove vascular blood and to loosen the connection between the vascular endothelial cells. Then, CL were perfused for 15 min with wash buffer (10 mM Hepes, 140 mM NaCl, 7.1 mM KCl, 5.0 mM CaCl2; pH 7.4). These perfusion buffers were bubbled with 5% CO2 in 95% O2 during perfusion. The dissociation of the cells was achieved by perfusing the tissue for 30 min with wash buffer containing 0.05% (w/v) collagenase (Sigma, No. C-0130) and 0.1% (w/v) BSA (Boehringer Mannheim GmbH, Mannheim, Germany, No. 735078). The cells were dispersed from the CL matrix with steel combs. Finally, the dissociated luteal cells were pooled and stirred for 30 min in Dulbecco modified Eagle medium (DMEM; Sigma, No. D-1152) containing 0.05% collagenase, 0.005% DNase I (Sigma, No. D-5025), and 0.1% BSA in a water bath at 37°C. After the cells were stirred, they were filtered through metal wire meshes (150 and 80 µm) to remove undissociated tissue fragments. The filtrate was washed three times by centrifugation for 5 min at 150 x g with DMEM supplemented with 60 µg/ml penicillin, 100 µg/ml streptomycin, and 0.1% BSA. After three washes, the cells were resuspended in a culture medium, DMEM and Ham F-12 medium (D/F; 1:1 [v/v]; Sigma, No. D-8900) containing 5% calf serum (CS; Sigma, No. C6278) and 20 µg/ml gentamicin (Gibco BRL, Grand Island, NY, No. 15750-060). Cell viability was higher than 85% as assessed by trypan blue exclusion. The cell suspension contained about 10% endothelial cells or fibrocytes and no erythrocytes and consisted of about 20% large luteal cells and 70% small luteal cells.
Effect of Cytokines on Fas mRNA Expression in Cultured Bovine Luteal Cells
The dispersed luteal cells were seeded at 2.0 x 105 viable cells in 0.5 ml, in 48-well culture dishes (Costar, Cambridge, MA, No. 3524). After 12 h of culture, the medium was replaced with fresh medium. The cells were then exposed to 50 ng/ml recombinant human TNF (kindly donated by Dainippon Pharmaceutical Co., Ltd., Osaka, Japan) and/or 50 ng/ml recombinant bovine IFN (kindly donated by Dr. S. Inumaru, NIAH, Ibaraki, Japan) for 24 h. After the final 24 h of culture, total RNA was prepared from the cells.
Cytotoxic Assays
The dispersed luteal cells were seeded at 2.0 x 104 viable cells in 0.1 ml, in 96-well culture dishes (Iwaki, Chiba, Japan, No. 3860-096). After 12 h of culture, the medium was replaced with fresh medium. The cells were then exposed to TNF and/or IFN (50 ng/ml) for 24 h. After 24 h of culture, the medium was replaced with fresh medium. The cells were then exposed to TNF and/or IFN in the presence or absence of 100 ng/ml soluble recombinant human Fas L (Upstate Biotechnology, Lake Placid, NY, No. 05-351) for 24 h. After the final 24 h of culture, the viability of the cells was determined by a Dojindo Cell Counting Kit including WST-1 (Dojindo, Kumamoto, Japan, No. 345-06463) as described previously [25]. Briefly, WST-1 (Dojindo), a kind of MTT (3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), is a yellow tetrazolium salt that is reduced to formazan by live cells containing active mitochondria. The viability of the cells was assessed using a Dojindo Cell Counting Kit including WST-1 (Dojindo). The culture medium was replaced with 100 µl D/F medium without phenol red, and a 10-µl aliquot (0.3% WST-1, 0.2 mM 1-methoxy PMS in PBS, pH 7.4) was added to each well. The cells were then incubated for 4 h at 37°C. The absorbance (A) was read at 450 nm using a microplate reader (Model 450; Bio-Rad, Hercules, CA). Percentage of cytotoxicity was determined by subtracting the mean A of TNF-, IFN-, and/or Fas L-treated wells (Atest) from the mean A of untreated wells (Acontrol) and then dividing by the mean A of untreated wells (Acontrol). The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells. The percent cytotoxicity was calculated as follows:
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TUNEL and Propidium Iodide Labeling
The dispersed luteal cells were seeded at 5.0 x 104 viable cells in 1 ml on glass slides in six-well cluster dishes (Sumitomo Bakelite, Tokyo, Japan, No. MS-80060). After 12 h of culture, the medium was replaced with fresh medium. The cells were then exposed to TNF and IFN (50 ng/ml) for 24 h. After 24 h of culture, the medium was replaced with fresh medium. The cells were then exposed to TNF and IFN in the presence of Fas L (100 ng/ml) for 24 h. After the final 24 h of culture, the cells were washed twice in 1 ml PBS (Seikagaku Corporation, Tokyo, Japan, No. 05193). The cells were fixed for 1 h at room temperature in PBS containing 4% paraformaldehyde, followed by two washes in PBS before permeabilization with 0.5% Triton X-100 (Bio-Rad) in PBS for 20 min. Cells were then briefly washed twice in PBS. The cells were incubated in 30 µl of fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase (TUNEL reagents; MBL, Nagoya, Japan, No. 8445) for 1 h at 37°C in a dark, moist chamber. After the TUNEL reaction, the cells were washed twice in PBS and once in PBS containing 0.0002% propidium iodide (PI; Sigma, No. P4170). Then, the cells were washed three times in PBS and stored in the dark at 4°C. The cells were observed under florescent illumination using a 470-nm excitation filter and a 515-nm absorption filter for fluorescein isothiocyanate (FITC) and a 545-nm excitation filter and a 610-nm absorption filter for PI.
Statistical Analysis
All experimental data are shown as the mean ± SEM. Statistical significance of differences in the expressions of Fas mRNA in the CL tissues and differences between control and treated groups were assessed by ANOVA followed by the Fisher protected least-significant difference procedure (PLSD) as a multiple comparison test. For the statistical analysis of differences in the cellular cytotoxicity, the relative percentages of the control were used.
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RESULTS |
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Specific transcripts for Fas were detected in bovine CL. The PCR product showed 100% homology to the known bovine genes after sequencing. A representative sample for the Fas RT-PCR product is given in Figure 1a. A representative sample for the ß-actin- and G3PDH-specific RT-PCR products (349 and 850 bp) is shown in Figure 1, b and c, respectively. The amplification pattern (level) of G3PDH cDNA was correlated well with that of ß-actin cDNA in each sample. The relative signal intensities for PCR products specific for Fas were assessed after correction based on the ß-actin signal intensities. The results of densitometric analysis of Fas mRNA in the CL tissue during the estrous cycle are shown Figure 2. The expression was significantly higher in the regressed luteal stage (Days 19–21) than in the other stages (P < 0.05).
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Effect of Cytokines on Fas mRNA Expression in Cultured Bovine Luteal Cells
A representative sample of the Fas-specific RT-PCR product is given in Figure 3a. A representative sample for the ß-actin-specific RT-PCR product (349 bp) is shown in Figure 3b. The expression of Fas mRNA was detectable in the cultured luteal cells treated without cytokines (Fig. 3a) and was increased 1.4-fold by IFN (P < 0.05, Fig. 3, bottom panel) and 1.8-fold by the combination of IFN and TNF (P < 0.01, Fig. 3, bottom panel). However, TNF alone did not have any significant effects on the expression of Fas mRNA in the cells.
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Fas L-Mediated Killing of Bovine Luteal Cells
The treatment of cells with IFN alone and in combination with TNF killed 30% and 50% of the cells, respectively (P < 0.05, Fig. 4). However, TNF alone did not have any significant effects on the viability of the cells. On the other hand, there was no cytotoxicity in response to the treatment with Fas L in the absence of cytokines. However, Fas L resulted in the death of 60% of the luteal cells in the presence of IFN (P < 0.01, Fig. 4). Moreover, Fas L resulted in the death of 85% of the cells in the presence of IFN and TNF (P < 0.01, Fig. 4).
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Fas L-Mediated Luteal Cell Death Occurs by Apoptosis
Staining with PI showed that the nuclei of the cells treated with Fas L in the presence of TNF and IFN were condensed and fragmented (Fig. 5). Moreover, TUNEL staining indicated that DNA fragmentation occurred in almost all cells treated with Fas L in the presence of TNF and IFN (Fig. 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, bovine luteal cells became sensitive to Fas L-induced cell death in the presence of IFN and IFN in combination with TNF. Moreover, since shrunken nuclei and apoptotic bodies were observed in the cells treated with Fas L in the presence of TNF and IFN, Fas L-induced bovine luteal cell death in the present study seems to occur by apoptosis. Furthermore, the increased sensitivity to Fas L in bovine luteal cells was correlated with an increase of Fas mRNA expression induced by cytokines, suggesting that Fas L induces cell death of bovine luteal cells mediated via the Fas-Fas L system. On the other hand, the expression of Fas L protein is elevated in the regressing postpartum rat CL [15]. Moreover, injections of an anti-Fas antigen antibody given to mice in the proestrous period make the CL disappear [14], suggesting that the Fas-Fas L system mediates apoptosis of CL cells in structural regression of the CL. As mentioned previously, the number of leukocytes (e.g., T lymphocytes and macrophages) increased at the time of luteolysis in bovine CL. T lymphocytes are known to express Fas L most abundantly and to be a primary source of IFN, whereas macrophages are the main source of TNF. Therefore, we assume that Fas L expressed on T lymphocytes may transduce apoptotic signals to luteal cells in which Fas expression is induced by leukocyte-derived cytokines, and that the Fas-Fas L system may be involved in the physiological process of structural luteolysis in bovine CL.
Interestingly, although bovine luteal cells expressed Fas mRNA in the absence of cytokines, Fas L alone did not induce cell death in the present study. Similar results have been shown in bovine granulosa cells cultured in a medium containing fetal bovine serum [19]. Recently, Quirk et al. [26] have demonstrated that bovine granulosa cells become sensitive to Fas-mediated apoptosis when the cells are cultured in serum-free media, although Fas mRNA expression in bovine granulosa cells is not different when the cells are cultured with or without serum. They also have demonstrated that growth factors such as insulinlike growth factor I (IGF-I) blocks Fas-mediated cell death in a serum-free medium, suggesting that the IGF-I contained in serum prevents Fas-mediated cell death. Thus, since we cultured bovine luteal cells in a medium containing 5% CS in the present study, IGF-I in CS might prevent the cytotoxic action of Fas L on the cells in the control groups. On the other hand, IGF-I is produced locally within the bovine CL [27] and is known to be a potent luteotropic factor in bovine luteal cells [28]. In addition, luteal concentrations of mRNA encoding IGF-I and the receptor for IGF-I increase during luteal development [29]. Thus, one could speculate that Fas L fails to induce apoptosis in luteal cells under conditions in which the cells are regulated by survival factors such as IGF-I. However, Fas L induced the death of midluteal cells in the presence of TNF and IFN in a medium containing 5% CS in the present study. Moreover, it has been demonstrated that IFN overcomes the inhibitory effects of serum and IGF on Fas L-induced cell death in bovine granulosa cells [26]. Therefore, we assume that TNF and IFN may conquer the inhibitory effects of growth factors contained in serum (e.g., IGF-I) on Fas-mediated apoptosis in bovine CL.
In the present study, IFN alone reduced the viability of bovine luteal cells and TNF augmented the cytotoxic action of IFN observed at 48 h of culture. These results support previous findings [10] and suggest that both TNF and IFN are potent mediators of bovine luteal cell death. However, Benyo and Pate [10] have demonstrated that TNF in the presence of IFN does not reduce the viability of bovine luteal cells until Day 5 of culture. We could not find an appropriate explanation for these different conditions needed to induce cell death in cultured bovine luteal cells. The different culture systems and evaluation system for cell death could be the reasons. Further studies are needed to clarify these points. In the present study, TNF alone did not show any effect on bovine luteal cell death, whereas TNF and IFN synergistically induced it. Since it has been demonstrated that IFN up-regulates TNF receptors in a variety of cell types [30–32], the bovine luteal cells might become sensitive to the cytotoxic action of TNF in the presence of IFN. Based on these findings, we postulate that TNF and IFN also induce apoptosis in bovine CL by a mechanism not mediated via the Fas-Fas L system.
In conclusion, the overall results of the present study demonstrate the presence of the Fas-Fas L system in bovine CL and suggest that TNF and IFN play essential roles in Fas-mediated bovine luteal cell death.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (No. 11460129) and Grants-in-Aid for the Recombinant Cytokine Project provided by Ministry of Agriculture, Forestry and Fisheries of Japan. 
2 Correspondence. FAX: 81 86 251 8388; kokuda{at}cc.okayama-u.ac.jp
Accepted: October 25, 2001.
Received: July 23, 2001.
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alters bovine luteal cell synthesis capacity and viability. Endocrinology 1992; 130:854-860[Abstract] a trigger for the initiation of endometrial prostaglandin F2 release at luteolysis in cattle?. Biol Reprod 2000; 62:1109-1115 production in endometrial epithelial cells in vitro: role of cyclooxygenase-2. Endocrinology 1997; 138::4798-4805 and its type I receptor in luteal regression: induction of programmed cell death in bovine corpus luteum-derived endothelial cells. Biol Reprod 2000; 63:1905-1912 and TNF- on cultured cells. J Neuroimmunol 1995; 62:161-167[CrossRef][Medline]This article has been cited by other articles:
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K. Nagaosa, A. Shiratsuchi, and Y. Nakanishi Determination of Cell Type Specificity and Estrous Cycle Dependency of Monocyte Chemoattractant Protein-1 Expression in Corpora Lutea of Normally Cycling Rats in Relation to Apoptosis and Monocyte/Macrophage Accumulation Biol Reprod, November 1, 2002; 67(5): 1502 - 1508. [Abstract] [Full Text] [PDF]
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