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Regular article |
and Its Type I Receptor in Luteal Regression: Induction of Programmed Cell Death in Bovine Corpus Luteum-Derived Endothelial Cells
a Sections of Immunology and
b Reproduction, Department of Animal Sciences, Faculty of Agricultural, Environmental and Food Sciences, Hebrew University of Jerusalem, Rehovot, Israel
ABSTRACT
The role of tumor necrosis factor
(TNF) and its type I receptor (TNFRI) in structural luteolysis was investigated. A semiquatitative reverse-transcription polymerase chain reaction (RT-PCR) was used to characterize the pattern of TNFRI mRNA expression within the corpus luteum (CL) throughout the estrous cycle and its cellular distribution. Increase in TNFRI mRNA levels was recorded both in regressed luteal tissue and in CL of cows injected with prostaglandin F2. All three major cell types composing the CL, steroidogenic (large and small) and endothelial cells expressed the TNFRI gene. A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells had significantly higher levels of TNFRI mRNA than steroidogenic luteal cells. The physiological effects associated with TNFRI expression were investigated in the various luteal cell types. TNF-induced programmed cell death (PCD) in dose- and time-dependent manners of cultured luteal endothelial cells (LECs) but not of in vitro luteinized steroidogenic cells. Several lines of evidence are provided to show that progesterone regulates luteal cell survival: 1) CL and LECs express progesterone receptor mRNA, 2) physiological levels of the steroid abolished TNF-induced PCD of LECs, and 3) progesterone-producing cells are protected from PCD. In conclusion, this study suggests that TNF-induced PCD during structural luteolysis is mediated by TNFRI, primarily affects endothelial cells, and that the decline in progesterone, preceding structural luteolysis, is a prerequisite for the initiation of apoptosis in endothelial cells.
corpus luteum, corpus luteum function, cytokines, hormone action, ovulatory cycle, reproductive immunology
INTRODUCTION
The luteolytic cascade of the bovine corpus luteum (CL) is primed by surges of uterine prostaglandin F2 (PGF2). A rapid functional regression [1], characterized by inhibition of progesterone release, is followed by a phase of structural regression [2, 3]. During luteolysis, cells of the CL undergo programmed cell death (PCD), a process that has been morphologically described in humans, cows, pigs, mice, and golden hamsters [2, 4–7]. Unlike the well-characterized histological aspects, the cellular and molecular mechanisms associated with the apoptotic events in the CL are just beginning to become unraveled.
Immune cells and cytokines were shown to play a role in regulating luteal functions [8, 9]. In PGF2-treated pig CL, macrophage numbers progressively increased within 24 h [10]. The probable source for these macrophages was circulation-derived monocytes emigrating in response to local chemotactic factors, such as monocyte chemoattractant protein-1 (MCP-1). Enhanced MCP-1 expression in CL during natural and PGF2-induced luteolysis in domestic ruminants [11, 12] supports the contention that luteolysis involves macrophage recruitment.
Activated macrophages secrete tumor necrosis factor (TNF) in response to various stimuli, including PGF2 [13, 14], and were postulated to be its major source in pig and cow CL [14, 15]. TNF inhibited progesterone production and enhanced PGF2 secretion from bovine and pig luteal cells [16, 17]. High levels of TNF (>100 pg/ml) were detected in the bovine CL 36 h after PGF2-induced luteolysis [18]. Similarly, high TNF bioactivity was recorded in PGF2-treated sheep CL and in LPS-treated rabbit CL [19, 20]. Surprisingly, luteal TNF mRNA expression in bovine CL remained unchanged during PGF2-induced luteolysis [21]. Combinations of interferon 
(IFN-) and TNF decreased the number of viable mouse luteal cells on Days 3–6 of culture [6]. Although TNF binding sites were identified in luteal membranes of various species [22, 23], the localization of these receptors to cell types composing the CL or the receptor subtype were not clearly identified.
The present study was undertaken to characterize the pattern of TNF receptor type I (TNFRI) mRNA expression throughout the bovine estrous cycle. We determined the cellular distribution and temporal expression patterns of the receptor following PGF2 administration and continued to examine the effects of this cytokine on the viability of luteal cell populations.
MATERIALS AND METHODS
Reagents
RPMI 1640, DMEM-F12, Na-pyruvate, penicillin-streptomycin, L-glutamin and fetal calf serun (FCS) were obtained from Biological Industries (Beit HaEmek, Israel). PGF2 analogue, Cloprostenol-Estrumate was obtained from Coopers (Berkhamsted, England). Forskolin, insulin, MTT [(3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide] and DAPI [4,6-diamidino-2-phenylindole] were obtained from Sigma (St. Louis, MO). SuperScript II RNase H- reverse transcriptase was obtained from Gibco BRL Life Technologies (Gaithersburg, MD). Deoxynucleotide triphosphates (dNTPs), random hexamer oligodeoxynucleotides, Taq DNA polymerase, and BglI were obtained from Farmentas (Vilnius, Lithuania). Oligonucleotide primers were synthesized by Biotechnology General (Kiryat Weizmann, Rehovot, Israel). Recombinant human TNF (rhTNF) and polyclonal rabbit anti-human TNF (IP-300) were obtained from Genzyme (Cambridge, MA).
CL Collection and Luteal Cell Enrichment
Bovine CL were collected at different stages of the luteal phase as previously described [24]. Briefly, CL were collected at slaughter, and CL age was determined macroscopically as described by Fields and Fields [25]. In experiments in which PGF2 was used to induce luteolysis, a PGF2 analogue (500 µg cloprostenol) was injected (i.m.) at mid-cycle. Tissues were snap-frozen in liquid nitrogen and stored at -70°C. Total RNA was extracted from tissues by the guanidinium thiocyanate method [26]. Enriched populations of luteal cell types (large luteal cells [LLCs], small luteal cells [SLCs], and endothelial cells [ECs]) were isolated after collagenase digestion and centrifugal elutriation as previously described [27]. Briefly, collagenase-dissociated cells (100 x 106) were resuspended and subjected to centrifugal elutriation using a Sanderson elutriation chamber and the eluates were collected with continuous flow. The yield and viability of cells in each fraction was determined immediately after elutriation. Fraction 1 contained predominantly ECs (78.2% ± 9.7%) and SLCs (15.2% ± 4.1%; n = 3). Fraction 2 contained 81.6% ± 2.78% SLCs, 0.3% ± 0.22% LLCs, and 15.7% ± 2.08 ECs (n = 3). Fraction 4 contained 59.6% ± 6.62% LLCs, 21.6% ± 5.01% SLCs, and 18.1% ± 1.47% ECs (n = 3). RNA was extracted from fractions 1, 2, and 4 by the guanidinium thiocyanate method.
In Vitro Luteinized Granulosa and Theca Cells
Granulosa and theca cells were isolated from healthy bovine preovulatory follicles as previously described [28, 29]. Cells from individual follicles were cultured in 24-well plates (8 x 104 granulosa and 3 x 104 theca cells per well) for 8 days in DMEM-F12 media (basal media) containing 1% FCS, insulin (2 µg/ml), and forskolin (10 µM), conditions that were previously shown to induce cell luteinization [28, 29]. Luteinized theca and granulosa cells are referred to as LTCs and LGCs, respectively.
Endothelial Cell Culture
Luteal endothelial cells (LECs), derived from microvessels of the bovine CL were kindly provided by Dr. Katharina Spanel-Borowski (University of Leipzig, Germany) [30]. The endothelial cells (1 x 105/well) were grown in RPMI 1640 containing 10% FCS, 1 mM L-glutamin, 10 mM Na-pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin on plates that had been precoated with collagen type I (Vitrogen 100). Experiments were performed on confluent cultures and cells were obtained from passages 4–10.
Semi-Quantitative RT-PCR
Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out with the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH), as an internal standard. G3PDH is constitutively expressed and has been used effectively in studies on the regulation of gene expression in ovarian cells [27, 31, 32]. The RT-PCR amplification was calibrated in order to determine the optimal number of cycles that would allow detection of the appropriate mRNA transcripts while still keeping amplification for these genes in the log phase (primer dropping method [33]). G3PDH oligonucleotide primer pair (sense 5'-TGTTCCAGTATGATTCCACCC-3'; antisense 5'-TCCACCACCCTGTTGCTGTA-3') and P450 side chain cleavage (P450scc) oligonucleotide primer pair (sense 5'-AACGTCCCTCCAGAACTGTACC-3'; antisense 5'-CTTGCTTATGTCTCCCTCTGCC-3') were synthesized according to bovine sequences (GenBank accession numbers AF077815 and Y10214, respectively) [24]. The TNFRI primer pairs (sense 5'-GACCTGGAGAAGAGAGAGAGTC-3'; antisense 5'-CTTAGCATGTCGCTACCA-3') were synthesized according to bovine TNFRI cDNA (GenBank accession number U90937). This set of primers does not amplify TNF receptor type II (TNFRII).
The expected PCR product lengths were 850 base pairs (bp) for G3PDH, 395 bp for P450scc, and 604 bp for TNFRI. With the exception of luteal ECs, the number of cycles used for PCR reactions were 23 cycles for G3PDH and P450scc, and 27 cycles for TNFRI. When ECs were used, G3PDH was amplified for 21 cycles and TNFRI for 24 cycles. Computer searches and sequence alignments were performed with software from Genetics Computer Group, Inc. (Madison, WI). To verify the sequence of the TNFRI PCR product, it was cleaved at a unique restriction site using BglI. Figure 1 demonstrates the amplified cDNA and the expected 199- and 405-bp fragments obtained after enzymatic cleavage.
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Detection of Cell Death
In situ apoptosis
LECs, LTCs, and LGCs were grown to confluence on glass slides that had been precoated with collagen type I, and were then exposed to 100 ng/ml rhTNF for 18 h (a dose previously shown to affect luteal steroidogenesis [34]). The cells were fixed with EFA (70% ethanol, 3.7% formaldehyde, 5% glacial acetic acid) for 10 min at -20°C, and then permeabilized with 0.25% Triton X-100 in PBS pH 7.4 for 15 min at room temperature. Apoptosis was determined by immunoperoxidase staining of 3'-OH digoxigenin-labeled ends according to a protocol supplied by the manufacturer (Apoptag S7100 kit, Intergen, Oxford, UK).
Quantification of apoptosis by nuclear morphology
LECs, LTCs, and LGCs were grown to confluence on glass slides that had been precoated with collagen type I, and were then exposed to 100 ng/ml rhTNF for 6, 12, and 24 h. Control cells were cultured for 24 h in the presence of media only. The cells were fixed with EFA for 10 min at -20°C and then permeabilized with 0.25% Triton X-100 in PBS for 15 min at room temperature. The slides were washed in PBS three times and stained with PBS containing 1 µg/ml DAPI at room temperature for 5 min, and then washed 3x with PBS and kept in a dark slide box until photographed under a fluorescence microscope.
Cytotoxic assays
LECs, LTCs, and LGCs were grown to confluence in 96-well plates and were then exposed to rhTNF (12.5–100 ng/ml) for 18 h in the presence or absence of progesterone (10-7 to 10-11 M). Cell viability was determined by MTT uptake as described previously [35]. Viable, stained cells were dissolved in dimethyl sulphoxide and absorbance (A) was measured at 570 nm with the reference filter set at 630 nm. Cells treated with media alone were considered to be an index for 100% viability, whereas cells treated Triton X-100 (4%) were considered as an index for 100% cytotoxicity. The percentage of cytotoxicity was calculated as follows:
Statistical Analysis
Data are presented as means ± SEM. Statistical analysis was carried out using the JMP package (version 3.2; SAS Institute Inc., Cary, NC). Densitometric values of PCR products were analyzed by one-way ANOVA. Percentage of apoptotic cells was analyzed by two-way ANOVA using time of incubation and TNF dose as main effects and their interaction. Percentage of cytotoxicity was also analyzed by two-way ANOVA using progesterone and TNF doses as main effects and their interaction.
RESULTS
TNFRI and P450scc mRNA expression was determined in CL collected during various stages of the estrous cycle (Fig. 2A): early (<Day 5) , mid (Days 5–8), late (Days 12–16), and regressed (>Day 18). High levels of P450scc were expressed through most of the luteal life span, with an increase toward mid cycle. In the regressed CL, a marked decline in the expression of P450scc mRNA was detected. TNFRI mRNA levels, on the other hand, were low throughout most stages of the cycle (early, mid, and late stages) with a significant increase occurring during the time of luteal regression (on Days 18–21 of the bovine cycle). In order to determine whether this up-regulation of TNFRI mRNA resulted from endogenous PGF2, a luteolytic dose of PGF2 was administered to cows at mid-cycle. Indeed, PGF2 induced a significant increase in TNFRI mRNA levels within 24 h of injection (Fig. 2B). The fold increase of TNFRI mRNA as induced by PGF2 was similar to that observed during natural luteolysis (3.49 ± 0.66 and 3.47 ± 0.58, respectively).
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TNFRI mRNA levels were then determined in enriched bovine luteal cell types. All three major cell populations comprising the CL (i.e., LLCs, SLCs, and endothelial cells) expressed the TNFRI gene (Fig. 3A). A densitometric analysis of TNFRI mRNA expression revealed that resident endothelial cells expressed significantly (P < 0.05) higher levels of TNFRI (2.1 ± 0.11) than did LLCs or SLCs (1.04 ± 0.02 and 1.66 ± 0.12, respectively, Fig. 3A). The TNFRI gene was also expressed in in vitro-luteinized granulosa cells (LGCs) and theca cells (LTCs) and in LECs in amounts similar to those found in cells enriched from bovine CL (Fig. 3B).
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The effect of rhTNF on LECs and steroidogenic luteal cells (LTCs and LGCs) was studied next. rhTNF had a cytotoxic effect on these endothelial cells, which was both time-dependent (P < 0.001) and dose-dependent (P < 0.001; Figs. 4 and 5). Cell death was shown to be caused by PCD, as demonstrated by direct detection of 3' ends in the genomic DNA (Fig. 4A). A significant cytotoxic effect of TNF was induced by 1.56 ng of TNF/ml (P< 0.02; as compared with control untreated cells) and increased with increasing TNF dosages (Fig. 4B). Cytotoxicity was TNF-specific as it was abolished by polyclonal anti-rhTNF antibodies (P < 0.001; Fig. 4B). In order to characterize PCD temporally, LECs were treated with 100 ng/ml rhTNF at various time points and stained with DAPI (Fig. 5). Apoptotic nuclei in LEC cultures increased over a 24-h culture period. In contrast to LECs, steroidogenic luteal cells (LGCs and LTCs) did not undergo PCD following TNF treatment (100 ng/ml) up to 48 h of culture (results not shown).
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As TNFRI expressing progesterone-producing cells appeared to be protected from TNF-induced PCD, we investigated the effect of physiological concentrations of progesterone on LECs (Fig. 6). Progesterone itself did not affect the viability of endothelial cells. Addition of progesterone to rhTNF-treated cells showed that this steroid prevented LECs from undergoing apoptosis (Fig. 6). The protective effect of progesterone was dependent on the rhTNF doses tested (12.5–100 ng/ml), and a significant interaction of rhTNF dose by progesterone dose was detected (P < 0.003). At 100 ng of rhTNF/ml, 10-11 to 10-7 M of progesterone significantly reduced cytotoxicity by 40% to 55% (P<0.05), while at 50 ng of rhTNF/ml, 10-7 and 10-9 M of progesterone abolished PCD (P<0.05) and 10-11 M reduced cytotoxicity by 40% (P <0.05) compared with cells that had been treated with rhTNF alone. At lower rhTNF concentrations, cytotoxicity was abolished by all doses of progesterone tested (P <0.05).
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As progesterone protected cultured endothelial cells from undergoing apoptosis, we investigated whether LECs expressed the progesterone receptor (PR) mRNA. To do so we used PR-specific primers described by Rueda et al. [36]. As shown in Figure 7, PR mRNA was expressed in CL tissue as well as in LECs.
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DISCUSSION
The results described in this study show that 1) TNFRI mRNA was present in the major cell types (endothelial and steroidogenic) present in the bovine CL, 2) TNFRI expression increased during natural or PGF2-induced luteal regression, 3) TNFRI-expressing endothelial cells underwent PCD in response to TNF, and 4) progesterone protected PR-expressing endothelial cells from TNF mediated PCD.
The CL is a transient gland that is composed of parenchymal steroidogenic cells (large and small, about 40% of the total cells in the gland) and stromal cells consisting mainly of endothelial cells, a major cell population within the CL (>50%) [37]. Many studies have indicated capillary regression and endothelial cell death as a hallmark of PGF2-induced luteal regression; these include studies in the cow [2], pig [5], guinea pig [38], sheep [39], and human CL [40]. Steroidogenic luteal cells were found to undergo apoptosis at a later stage, when a massive degeneration of capillaries had already occurred [2, 5]. Our findings demonstrating that endothelial cells express high levels of TNFRI and that they are sensitive to TNF-induced PCD in vitro, together with those demonstrating high levels of TNF in regressing CL [18–20], contribute to the understanding of the molecular mechanisms controlling apoptosis in the regressing CL.
TNF plays a role in a variety of immunological and physiological functions [41], and many of the biological effects of soluble TNF are mediated via TNFRI [42], including the initiation of PCD [43]. TNF receptors are widely distributed in different tissues. Many cells express both TNF receptor types, but TNFRI is more commonly expressed, and especially so in cells of nonhematopoietic lineages (reviewed by Krakauer et al. [41]). Specific TNF binding sites were previously demonstrated in porcine CL by the use of labeled cytokine [22] and, more recently, Sakumoto et al. [23] identified TNFRI in mRNA from bovine CL. Our data support and extend these findings in that all three major luteal cell populations (steroidogenic and endothelial cells) were found to contain mRNA for TNFRI, with endothelial cells expressing the highest levels of the receptor. The overall increase of TNFRI mRNA in the regressed CL, as observed in this study, could have resulted from a relative increase within each luteal cell type. Our data would imply that such an increase in endothelial cells may be physiologically relevant, as the other cell types did not undergo PCD in response to TNF. Nevertheless, this implication requires further research.
In contrast to our results, a recent study did not find an increase in TNFRI toward the end of the bovine cycle [23]. The cause for this discrepancy is presently unresolved, particularly because the increase we show herein was not only observed in late cycle (natural luteolysis), but also in PGF2-induced luteolysis.
Our data showing that progesterone protected CL-derived endothelial cells from TNF-induced apoptosis, that these cells express the PR, and that progesterone-producing cells were resistant to apoptosis, all suggest that progesterone may serve as a regulator of luteal cell survival. This proposition is supported by the recent report of Rueda et al. [36] who demonstrated that inhibition of P450scc activity reduced progesterone synthesis and increased oligo-nucleosomal DNA fragmentation. The expression of the PR by endothelial cells as described herein supports the notion that progesterone may directly affect endothelial cell function as was previously indicated by studies demonstrating progesterone-dependent suppression of endothelin-1 (ET-1) production [44] and modulation of cell proliferation [45].
If progesterone blocks luteal cell death, it can explain why apoptosis during luteal regression always proceed the decline of progesterone secretion. This is analogous to the physiological sequence of events occurring in uterine epithelial cells where the fall in progesterone is followed by PCD of these cells [46]. Pecci et al. [47] demonstrated that removal of progesterone, or the addition of antiprogestins both increased the percentage of apoptotic cells in rat endometrial cell lines. The direct antiapoptotic effect of progesterone in these cells was achieved by increasing the amount of the apoptosis-inhibiting form of Bcl-X (Bcl-X long) relative to that of the apoptosis promoting form (Bcl-X short). In contrast to the endometrium, Bcl-X short was not detected in bovine luteal tissue (during pregnancy or regression), and the occurrence of apoptosis in the CL was associated with an increase in BAX, which is another proapoptotic member of the Bcl family [48]. Whether or not progesterone regulates the expression Bcl family members in various CL cell types remains to be determined.
We have previously shown that endothelial cell-derived ET-1 has a vital role in functional luteolysis; namely, in its initial step involving a decline in steroidogenic activity [49]. The findings reported herein and in our previous study [49] suggest that ET-1 may also regulate PCD during structural luteolysis as outlined in Figure 8. ET-1 may act by initially decreasing progesterone production and then by stimulating TNF secretion from macrophages [49], thereby promoting the apoptotic cascade in CL cells. Moreover, elevated TNF may induce a further increase in ET-1, as demonstrated by Woods et al. [50], thus suggesting that ET-1 and TNF may act in a paracrine, positive feedback loop at the time of luteolysis.
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ACKNOWLEDGMENTS
The authors are grateful to Dr. John S. Davis of the University of Kansas, Wichita, for cDNA prepared from enriched bovine luteal cell types, and to Dr. Katharina Spanel-Borowski of the University of Leipzig, Germany, for bovine luteal microvascular endothelial cells.
FOOTNOTES
First decision: 17 November 1999.
1 Correspondence: Rina Meidan, Department of Animal Sciences, POB 12, Rehovot, 76100, Israel. FAX: 972 8 9465763; rina{at}agri.huji.ac.il 
2 Both authors made equal contribution to this study.
Accepted: August 3, 2000.
Received: October 18, 1999.
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