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Female Reproductive Tract |
a Centre de Recherche en Reproduction Animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada J2S 7C6
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is produced by the trophoblast prior to implantation in ruminants. It is involved in maternal recognition of pregnancy, and is a pleiotropic molecule that can alter the synthesis of endometrial proteins and inhibit proliferation of some cells. We have observed that IFN- reduces the DNA content in cultures of bovine endometrial epithelial cells; therefore, the objective of this study was to determine whether IFN- would induce apoptosis in bovine endometrial cells. Epithelial cells were prepared, cultured to confluence, and then incubated for 24 or 48 h in the presence or absence of 10 ng/ml progesterone, 100 ng/ml IFN-, or 10 µg/ml cycloheximide (CHX; an apoptosis inducer used as a positive control). Cells undergoing apoptosis exhibit such characteristics as the appearance of apoptotic bodies and DNA fragmentation. The incidence of apoptosis was assessed by using TUNEL, DNA fragmentation analysis, and Western blot analysis of Bax-
protein expression. The results showed that IFN- and CHX significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment of the cells significantly inhibited the ability of IFN- to induce apoptosis (14.6%) compared with IFN- alone (33.6%; P < 0.05). DNA fragmentation analysis showed that INF- and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures. Western blot analysis showed that IFN- and CHX treatment resulted in a greater expression of the proapoptotic protein Bax- compared with that in control cultures. These data demonstrate that IFN- can induce apoptosis in bovine uterine epithelial cells and that this effect is modulated by progesterone. We speculate that IFN- might play a critical role in the remodeling of the endometrium around the time of implantation.
apoptosis, implantation, pregnancy, progesterone, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) is secreted specifically by the conceptus in ruminants during early pregnancy [1, 2]. IFN-, originally named trophoblastin or trophoblast protein-1, is a type I interferon that is involved in the establishment of early pregnancy in ruminants. Analysis of the cDNA and amino acid sequence revealed 45% to 68% identity between IFN- and IFN- [3]; IFN- competes with IFN- for the type I IFN receptor and possesses potent antiviral, antiproliferative, and immunomodulatory activities [4]. IFN- is released by the bovine conceptus as early as Day 9 of pregnancy [2] and serves as the signal for maternal recognition of pregnancy in domestic ruminants [5] by attenuating the release of prostaglandin F2 and rescuing the corpus luteum from regression [6]. It can also alter the synthesis of endometrial proteins [7, 8], enhance the activity of 2', 5'-oligoadenylate synthetase in endometrial stromal and epithelial cells, induce an antiviral activity in target cells [9], and inhibit the proliferation of lymphocytes [10] and oviductal epithelial cells [11]. These antiproliferative effects have been suggested to slow the growth of the endometrium during the period when the conceptus is elongating to establish contact with a high percentage of the surface epithelium of the uterine lumen [4].
Cell death is classified into two distinct types; namely, necrosis and apoptosis. Apoptosis is a typical form of programmed cell death that eliminates unwanted cells in the development of the immune system, organ formation, and embryogenesis [4]. The characteristic features of apoptosis are the condensation and fragmentation of nuclear chromatin, accompanied by the compaction of cellular organelles, dilatation of the endoplasmic reticulum, and a marked reduction in cell volume, all of which distinguish it from necrosis [12]. The occurrence of apoptosis has been described in many reproductive tissues including the uterine epithelium [13]. The induction of apoptosis in tissue and cells by IFN-
and IFN- as evidenced by studies on DNA fragmentation levels and DNA nick-end labeling has been reported [14–16], and Kim et al. [17] have shown that IFN- induces apoptosis in sheep hepatocytes. However, the activation of apoptosis by IFN- in bovine endometrium remains to be elucidated.
We have observed that IFN- decreases the DNA content in cultures of bovine endometrial epithelial cells. This could be due to initiation of apoptosis or to a decrease in cell proliferation. We hypothesize that apoptosis in bovine endometrium epithelial cells could be induced by IFN-. The objective in the present study was to elucidate the effect of IFN- on the induction of apoptosis in bovine endometrial cells. Because progesterone (P4) signaling regulates the function and development of the bovine endometrium, we have examined the possibility that it modulates the effect of IFN-.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture medium (RPMI 1640), Hanks buffered saline solution (HBSS; calcium-free and magnesium-free), newborn calf serum (NBCS), antibiotics, and trypan blue were purchased from Gibco (Grand Island, NY). Collagenase (type II), trypsin (type III, from bovine pancreas), DNase I (type I, from bovine pancreas), gentamicin, BSA and paraformaldehyde (PFA) were purchased from Sigma Chemical Company (St. Louis, MO). A stock solution of P4 was prepared by dissolving the steroid in ethanol. Matrigel was obtained from VWR Canlab (Montreal, PQ, Canada). Protein assay-dye reagent concentrate was obtained from Bio-Rad Laboratories (Mississauga, ON, Canada). The in situ detection kit for programmed cell death (Mebstain Apoptosis Kit Direct) was purchased from Medical & Biological Laboratories Company, Ltd. (Naka-ku Nagoya, Japan). The recombinant bovine IFN- was a generous gift from Dr. R. Michael Roberts, University of Missouri. The activity of IFN- is 10.9 x 107 IU/mg and we have previously shown that it has no detectable endotoxin activity that might induce apoptosis [18].
Preparation and Culture of Cells
The epithelial cells were prepared as previously described [19]. Uteri from cows at Days 1 to 3 of the estrous cycle (ovaries with a corpus hemorrhagicum) were collected at the slaughterhouse and transported on ice to the laboratory. Cells prepared from endometrium at this stage respond to IFN- in a physiological manner; that is, IFN- inhibits the oxytocin stimulation of prostaglandin F2 secretion [20]. Briefly, the two horns of the uteri were placed in sterile HBSS containing 100 U penicillin, 100 µg streptomycin, and 0.25 µg/ml-1 amphotocerin. the myometrial layers were dissected from the two horns and the horns were then everted to expose the epithelium. The everted horns were digested for 2 h in HBSS with 0.3% (w/v) trypsin at 37°C to obtain epithelial cells. At the end of incubation, 10% NBCS in HBSS was added to inhibit the trypsin, the cell suspension was centrifuged at 60 x g for 5 min, and the pellet was washed three more times with HBSS. For further purification, the epithelial cell pellet was suspended in 20 ml of RPMI-1640 medium supplemented with 5% NBCS and 50 mg/ml-1 gentamicin, plated onto 100 x 20 mm Petri dishes (Nunclon, Grand Island, NY), and incubated at 37°C with 5% CO2 and 95% air for 3 h. At the end of incubation, contaminating stromal cells adhered to the dish and the floating epithelial cells were collected. After cell counting and viability determination by trypan blue-exclusion, 2.5 x 106 viable cells per dish were plated onto Matrigel-coated 6-well culture plates (200 µl of 11% Matrigel was added to each well and dried overnight).
Cell Proliferation Experiment
All cells were cultured in phenol red-free RPMI-1640 medium containing 5% fetal calf serum (depleted of steroids by dextran-charcoal extraction) at 37°C in humidified air (5% CO2). After the epithelial cells had reached about 60% confluence, they were cultured for 4 days in medium alone or in medium containing P4 (50 ng/ml), IFN- (10 or 100 ng/ml), or P4 plus IFN-. The medium was changed every 2 days.
Determination of DNA Content
At the end of the culture period, the medium was removed and the cells were rinsed twice with HBSS and detached with 1 mmol EDTA in HBSS and the use of a rubber scraper. Cells were pelleted by centrifugation at 1000 x g for 5 min, and 100 µl of 0.2% (w/v) SDS in ETN buffer (10 nmol EDTA, 10 mmol Tris-HCl, and 100 mmol NaCl pH 7.0) was added to the pellet. The pellet was sonicated 15 times using a Branson sonifier-450 (VWR Canlab) at 10% power. The DNA content in a 10 µl sonicated cell suspension was determined using the bisbenzimide fluorescent dye method [21]. Calf thymus DNA was used as the standard.
Measurement of Total Protein
Total protein was measured in 10 µl of sonicated cell suspension using the Bradford method (Bio-Rad Laboratories). BSA was used as the standard.
Apoptosis Experiments
After the cells had reached confluence (about 7 days) they were cultured for 24 or 48 h in the presence or absence of P4 (10 ng/ml), cycloheximide (CHX; 10 µg/ml), and IFN- (100 ng/ml). At the end of culture, the medium was collected and centrifuged at 500 x g for 5 min at 4°C to collect the cells. The cells attached to the dish were harvested by adding either 0.25% trypsin for the TUNEL labeling and DNA fragmentation studies or TED sonification buffer (20 mM Tris, 50 mM EDTA, and 0.1 mM diethyldithiocarbamic acid pH 8.0) containing 32 mM octyl glucoside for the analysis of Bax protein expression. Cells harvested from the dish were pooled with the cells from the medium.
DNA 3' End-Labeling and Quantification of Apoptosis
Cells were washed several times with PBS containing 2% NBCS and 0.1% NaN3, centrifuged at 500 x g for 5 min at 4°C, and resuspended in PBS at a concentration of 105 cells/ml. Fifty microliters of cell suspension were applied to ProbePlus slides (Fisher Scientific Co., Nepean, ON, Canada). The ProbePlus slides were air-dried for about 1 h and cells were then fixed at 4°C for 15 min with 4% PFA (in 0.1 mM NaH2PO4 pH 7.4). The cells were permeabilized with 0.5% Tween-20 after removing the PFA and then DNA nick end-labeled, counterstained, and mounted as described in the Mebstain Apoptosis Kit directions. Negative control slides were prepared from cells taken from each treatment group using precisely the same procedures, except that water was added instead of the terminal deoxynucleotidyl transferase (TdT). Positive controls were prepared by treatment with DNase I (1 µg/ml) at 37°C for 1 h prior to end labeling. Using this method, apoptotic cells appear green.
The incidence of apoptosis was evaluated by examining stained cells under a fluorescent microscope (Nikon Eclipse E800, Nikon Canada, Mississauga, ON, Canada) using the green filter set with excitation/emission wavelengths at 475/535 nm. The number of apoptotic epithelial cells was expressed as a percentage of total cells counted. One thousand cells were counted in each of three randomly selected microscopic fields and the average of these fields was used as one data point per experiment.
Gel Electrophoretic Analysis of Internucleosomal DNA Fragmentation
To estimate the degree of internucleosomal DNA fragmentation, the cells were lysed following the instructions provided by the manufacturer (TACS DNA Laddering Kit, R&D Systems, Minneapolis, MN). Genomic DNA was isolated from harvested cells and analyzed for oligonucleosomal DNA fragmentation as a marker for apoptosis. The DNA samples (15 µg) were electrophoretically separated on 1% agarose gels in 1x TAE buffer (90 mM Tris-acetic acid and 1 mM EDTA pH 8.0) at 90 V for 30 min. A 100-base pair (bp) DNA ladder (5 µl) was run with each gel as a molecular weight standard. The gel was stained with ethidium bromide (1 µg/ml) and viewed with UV light, then photographs were taken using the Foto/Analyst (version 1.1) gel documentation system (Fotodyne, Hartland, WI).
Analysis of Bax Protein Expression
Cell extracts were prepared as previously described [22] with minor modifications. After treatment, the harvested cells were sonicated (8 sec/cycle; 3 cycles) in 250 µl of TED sonification buffer. The sonicates were centrifuged at 13 000 x g for 25 min at 47°C and supernatants were stored at -70°C until immunoblotting analysis.
Supernatant proteins (25 µg) were resolved by one-dimensional SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (Hybond-ECL; Amersham Life Science, Inc., Buckinghamshire, U.K.). The blots were incubated for 18 h at 4°C in the presence of rabbit anti-mouse Bax polyclonal immunoglobulin G (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) diluted to 1:400. Specificity of the reaction was verified by replacing the Bax antiserum with normal rabbit serum. Blots were washed, incubated with secondary antibody, and exposed to chemiluminescent detection substrates as described [23, 24]. The membranes were then scanned using a Storm 840 PhosphorImager scanner (Molecular Dynamics Inc., Sunnyvale, CA) and quantified by densitometry using ImageQuant software (version 1.2, Molecular Dynamics).
The molecular weight of the immunoreactive bands was determined by comparing them with a ladder of biotinylated SDS-PAGE molecular weight standards (Bio-Rad Laboratories, Hercules, CA) applied to a lane in each gel. Prestained standards were also applied to gels to assess the transfer efficiency of samples.
Statistical Analysis
Each treatment was carried out in triplicate using the cells from one uterus and each experiment was repeated with three different uteri. The effect of treatments was evaluated by least-squares ANOVA. The data in the form of percentages were arcsine-transformed before analysis. Individual comparisons between means were made by the Tukey-Kramer test. A probability of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on DNA and Protein Content of Endometrial Epithelial Cells
IFN- at the 100 ng dose, either alone or in the presence of P4, significantly decreased the DNA content of epithelial cells (P < 0.001; Fig. 1A), indicating that a decrease in proliferation or an increase in cell death, or both, occurred. The lower dose of IFN- increased the total protein content of epithelial cells but there was no effect of the high dose of IFN- (Fig. 1B). Progesterone caused an increase in cell protein content but it did not alter the response to IFN-.
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Apoptosis in Endometrial Epithelial Cells
Apoptotic cells and apoptotic bodies were evident in the cultured cells following routine histological staining. Signs of apoptosis included cells with nuclei containing marginated chromatin; cells with a single, small, densely stained nucleus; cells with multiple, densely stained nuclear fragments; and membrane-bound structures containing condensed chromatin or cytoplasm (apoptotic bodies), or both. Cells with the abovementioned apoptotic morphological features were found using the TUNEL method to contain fragmented DNA (Fig. 2). Staining was located predominantly in the nucleus and nuclear fragments. In the TUNEL-positive control (i.e., treated with DNase; Fig. 2E), labeling was observed in all cells, however, in the TUNEL-negative control (no terminal deoxynucleotidyl transferase; Fig. 2F), no labeling was observed, indicating that the labeling procedure was specific.
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To determine the effect of IFN- and P4 on the percentage of cells with apoptotic nuclei, confluent bovine endometrial epithelial cells were treated with or without IFN- and CHX in the presence or absence of P4 for 48 h. The cells were harvested and fixed, and the genomic DNA nick end was labeled with TdT. The results show that IFN- and cycloheximide significantly increased the percentage of cells with apoptotic nuclei (33.6% and 44.8%, respectively) compared with controls (11.7%; P < 0.05). Progesterone treatment alone did not affect the number of dead cells but it significantly inhibited the ability of IFN- to induce apoptosis (14.6%) when compared with IFN- alone (33.6%; P < 0.05; Fig. 3). Progesterone also decreased CHX-induced apoptosis.
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To further confirm the effect of IFN- and P4 on apoptosis in endometrial epithelial cells, genomic DNA was isolated and analyzed for oligonucleosomal DNA fragmentation. DNA fragmentation analysis showed that INF- and CHX treatment resulted in an increase in the appearance of DNA laddering compared with that in untreated control cultures (Fig. 4). Progesterone treatment of cells significantly decreased the effect of IFN- treatment but had no effect on the fragmentation induced by CHX.
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Effect of IFN- and P4 on Expression of Bax-
To determine whether the proapoptotic protein Bax- was expressed following IFN- treatment, proteins extracted from cells were resolved by one-dimensional SDS-PAGE and analyzed by Western blotting using a specific anti-Bax- antibody. The results show that IFN-, and CHX treatment increased the expression of Bax- compared with that of control cultures. Progesterone treatment alone had no effect on Bax-, but it significantly decreased the ability of IFN- to stimulate the expression of Bax- (Fig. 5; P < 0.05). However, P4 did not diminish the induction of Bax- by CHX.
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To define whether the effect of IFN- on expression of Bax- is time-dependent, cellular extracts were prepared from confluent endometrial epithelial cell cultures treated with IFN- at different times (0, 3, 6, 12, and 24 h). Extracted proteins were resolved by one-dimensional SDS-PAGE and analyzed by Western blotting using specific anti-Bax- antibody. The results show that IFN- markedly increased Bax- protein expression at all time points (Fig. 6). IFN- induced Bax- in cultured bovine endometrial epithelial cells as early as 3 h (P < 0.001) and Bax- continued to increase with time (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inhibits epithelial cell proliferation and induces apoptosis in cultured bovine endometrial epithelial cells. Although the involvement of IFN- in maternal recognition of pregnancy has been known for a number of years, this is the first report that this cytokine directly induces a Bcl-2 gene family member, Bax-, and apoptosis in bovine endometrial epithelial cells. The other important finding is that the IFN--induced apoptosis is inhibited by progesterone, which suggests that plasma progesterone concentration during early pregnancy is important for modulating the effect of IFN-. Interferons are cytokines that play a complex and central role in the resistance of mammalian hosts to pathogens [25]. Interferons are widely recognized for their antiviral and antiproliferative effects, and these properties are exploited through their clinical application in the therapy of viral infections or malignant diseases [26]. Recent studies have resulted in a greater appreciation of immunomodulatory responses elicited by IFNs that are distinct from their ability to interfere with cell cycle progression and viral replication [27]. One biological function of IFNs is their ability to manipulate the events that mediate programmed cell death and many studies show that IFNs initiate apoptosis in a variety of cell types [28]. A few studies also illustrate the potential of IFNs to act as negative regulators of programmed cell death but the underlying mechanism remains unidentified [29, 30].
Although Kim et al. [17] have shown that high doses of ovine IFN- induced hepatocyte apoptosis in sheep, there have been no reports of the effect of IFN- on apoptosis in endometrial cells. Internucleosomal DNA fragmentation has been considered to be one of the earliest characteristic events of apoptosis [31] and this was detected after IFN- treatment of endometrial epithelial cells in the present study. These results provide biochemical evidence that IFN- induced endometrial epithelial cell death by apoptosis. In addition to the detection of oligonucleosomes in extracted DNA, the occurrence of apoptosis may also be inferred from the characteristic morphological appearance of degenerating cells, together with the detection of DNA fragmentation in single cells using TUNEL [32]. The role of IFN--induced apoptosis is currently speculative because nothing is known of apoptosis in ruminant endometrium. Apoptosis could be involved in embryo attachment to the endometrium and in placentation. It is known that in primates and rodents, uterine epithelial cells undergo extensive apoptosis with obvious morphological degradation during the early stage of pregnancy [33, 34]. Placentation in ruminants differs from that in primates and rodents in that the embryo does not invade into the endometrium; instead, trophoblast invasiveness in ruminants is limited to fusion of migrating binucleate cells with uterine epithelium. Considerable tissue remodeling and angiogenesis occur, however, within the endometrium at implantation. Thus, IFN--induced apoptosis of endometrial epithelial cells could contribute to the marked endometrial remodeling associated with early placentation. The factors involved in inducing apoptosis in primate and rodent endometrium have not yet been elucidated, however, embryos in these species have been shown to secrete IFN- [35]. Thus, embryonic IFN could be involved in endometrial apoptosis observed at implantation. Because no information is available at present on apoptosis in the endometrium of ruminants, further studies need to be performed in vivo to investigate this possibility.
Bcl-associated X protein (Bax) is a member of the Bcl-2 gene family and has extensive amino acid sequence homology with Bcl-2 protein [36]. It is known that overexpression of Bax protein induces apoptotic cell death, and the action of Bax appears to be neutralized when heterodimerized with Bcl-2 and some other members of the Bcl-2 protein family that function as suppressors of cell death [36]. It has been proposed that the ratio of Bax to Bcl-2 and other antiapoptotic Bcl-2 family proteins appears to predetermine the life or death response of a cell to an apoptotic stimulus [36]. In the present study we sought to determine the expression of Bax- protein in bovine endometrial epithelial cells and to evaluate Bax expression in relationship to DNA fragmentation and the apoptotic phenotype. The present results demonstrate that immunoreactivity for Bax- increases after IFN- treatment of the cells in a time-dependent manner. Thus it appears that IFN--induced apoptosis is mediated by increased intracellular levels of Bax-.
The corpus luteum is a transient endocrine organ that synthesizes P4 to act on the uterus and support the developing embryo. In vivo, P4 acts on an estradiol-primed uterus to stimulate growth of the endometrium and it is believed that cell death may be as important as cell proliferation in the regulation of normal uterine epithelial growth. There is mounting evidence to suggest that P4 can inhibit apoptosis in a variety of P4 receptor-positive tissues [37–41]. Nawaz et al. [42] reported that treatment of rabbits with P4 dramatically decreased cell death in uterine epithelial cells. Results of the present study provide evidence of P4 modulation of IFN--induced apoptosis in bovine endometrial epithelial cells in vitro. The physiological significance of this remains to be established. Because IFN- is secreted from early blastocyst stage embryos and IFNs in general induce apoptosis, the role of P4 could simply be to prevent IFN--induced apoptotis, especially before the time of placentation. If IFN--induced apoptosis is indeed involved in placentation, then it is possible that local P4/IFN- concentrations at the sites of attachment determine whether there is an effect on the endometrium. Progesterone has been shown to alter the apoptotic threshold of endometrial cells in rats [43]. This suggests that production of IFN- by the embryo and P4 by corpus luteum might be important for the establishment of pregnancy in addition to their role in preventing the secretion of the uterine luteolysin. Alterations in endometrial apoptosis, due to abnormal production of P4, IFN-, or both may therefore represent an alternative cause of early pregnancy failure in cows.
In summary, our results demonstrate that IFN- significantly increased the percentage of endometrial epithelial cells with apoptotic nuclei, the appearance of DNA laddering, and the expression of preapoptotic protein Bax-. An interesting observation was that treating cells with P4 significantly inhibited the ability of IFN- to induce apoptosis. These findings support the need for a balanced apoptosis regulatory process during the establishment of pregnancy. We speculate that the action of IFN- in bovine uterine epithelial cells plays a critical role in implantation and placentation during early pregnancy and that abnormal P4, IFN-, or both could result in pregnancy failure for reasons other than failure to maintain the corpus luteum.
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ACKNOWLEDGMENTS |
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and Dr. B.D. Murphy's group for their assistance. |
FOOTNOTES |
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2 Correspondence: Alan K. Goff, Université de Montréal, Centre de Recherche en Reproduction Animale, Faculté de médecine vétérinaire, 3200 rue Sicotte, St-Hyacinthe, QC, Canada J2S 7C6. FAX: 450 778 8103; goffak{at}medvet.umontreal.ca
Received: 25 April 2002.
First decision: 17 May 2002.
Accepted: 14 September 2002.
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