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Biology of Reproduction 59, 241-247 (1998)
©Copyright 1998 Society for the Study of Reproduction, Inc.

In Vitro Response to Oxytocin and Interferon-Tau in Bovine Endometrial Cells from Caruncular and Inter-Caruncular Areas1

Eric Asselinc, Patrick Droletc, , and Michel A. Fortier2,c,d

c Département d'Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier de l'Université Laval et Centre de Recherche en Biologie de la Reproduction, Ste-Foy, Québec, Canada G1V 4G2 d Département d'Obstétrique et Gynécologie, Université Laval, Ste-Foy, Québec, Canada G1V 4G2


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Caruncules are differentiated sites of the endometrium in which placentation occurs in ruminants. We investigated whether the response to agents involved at the time of recognition of pregnancy differed in the caruncular (CAR) and inter-caruncular (ICAR) areas of the endometrium in vitro. The specialization in prostaglandin (PG) production previously described in cells from whole endometrium was reproduced in the CAR and ICAR areas: PGF2{alpha} and PGE2 were produced in greater proportions, respectively, in epithelial and stromal cells. The relative production of PGE2 was equivalent in epithelial cells from CAR and ICAR regions, but the production of PGF2{alpha} was higher (p < 0.05) in the ICAR region (2.2 ± 0.5 vs. 4.0 ± 0.2 ng/µg DNA, respectively). In stromal cells, the ICAR area produced more PGE2 than did the CAR area (3.4 ± 0.4 vs. 2.1 ± 0.4 ng/µg DNA, p < 0.05), and the respective PGE2:PGF2{alpha} ratio was significantly higher in the ICAR area (p < 0.05). The production of PGs was measured first in response to oxytocin (OT, 10-9 to 10-5 M) and then to recombinant ovine interferon-tau (roIFN-{tau}, 0.02 to 20 µg/ml) in a separate set of experiments. In epithelial cells, OT stimulated the production of PGF2{alpha} 6.3-fold in the CAR area and more than 33.0-fold in the ICAR area (7.1 ± 3.2 vs. 36.3 ± 9.8 ng/µg DNA, respectively, p < 0.05). Production of PGE2 was also increased in both regions and reached a plateau at 4.1 ± 0.4 ng/µg DNA. In epithelial cells from the ICAR but not the CAR region, the PGE2:PGF2{alpha} ratio was decreased in the presence of OT (p < 0.05). In separate experiments, addition of roIFN-{tau} stimulated PGE2 production significantly (p < 0.05), and no difference (p > 0.8) was observed between CAR and ICAR regions. An increase in PGE2:PGF2{alpha} ratio was observed in epithelial cells from both CAR and ICAR regions, but it was significant only in the CAR region (p < 0.05). In stromal cells, roIFN-{tau} stimulated PGE2 production significantly in cells from the CAR and ICAR regions (35.6 ± 2.9 vs. 24.1 ± 3.8 ng/µg DNA, respectively, p < 0.05). In summary, the ICAR region seems to be the privileged site for regulation of PGF2{alpha} production by OT, but the caruncules may be a preferred site for recognition of the embryonic IFN-{tau} signal. Endometrial cells from the CAR and ICAR areas appear to exhibit specialized responses, with cells from the ICAR region more responsive to OT and those from the CAR region more sensitive to roIFN-{tau}.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In ruminants, prostaglandins (PGs) are primary regulators of the interaction between the embryo and the maternal organism at the time of recognition and establishment of pregnancy. Several factors of maternal origin (oxytocin, [OT]) and embryonic origin (interferon-tau [IFN-{tau}]) influence PG production to favor recognition of pregnancy or the return to estrus. The production of PGF2{alpha} by the uterus is responsible for luteolysis during the estrous cycle (reviewed in [1–3]). OT binds to endometrial OT receptors (OTR) to stimulate cyclooxygenase-2 (COX-2) [4] and PGF2{alpha} production [2, 5]. Several studies in vivo and in vitro have shown that levels of PGF2{alpha} or PGFM, its stable metabolite, are reduced in the presence of a viable conceptus (or its signal) at the time of recognition of pregnancy [6–10]. In contrast, PGE2 may be a luteotropic agent [11] and could be a luteo-protective signal to antagonize potential luteolytic effects of PGF2{alpha}. Before implantation, PGE2 may also be responsible for the increase in vascular permeability and secretion of growth factors and nutrients, and it may be involved in the local regulation of immune responses [12]. Inhibition of PG synthesis with nonsteroidal anti-inflammatory drugs such as indomethacin delays or inhibits these changes and prevents the establishment of pregnancy [3]. Administration of PGE2 protects the corpus luteum (CL) from spontaneous regression [13, 14], as well as luteolysis induced by exogenous PGF2{alpha} [15, 16]. In the cow, intrauterine infusion of PGE2 extends luteal maintenance [17, 18]. In the pregnant ewe, secretion of PGE2 from the uterus is greater during maternal recognition of pregnancy [19–21]. Therefore, it appears to be crucial to control the type of PG that is released and/or the pattern of its release to ensure establishment and maintenance of pregnancy.

Trophoblastic IFN-{tau} released by the conceptus (embryo and associated membranes) appears to be the most likely candidate to trigger the establishment of pregnancy (see [7, 8] for reviews). There is clear evidence of a potent antiluteolytic activity of ovine (o) IFN-{tau} in ewes and of bovine (b) IFN-{tau} in cows, and cross-reactivity of these interferons has been shown in vivo and in vitro. IFN-{tau} is produced during the periimplantation period and has a direct action on endometrial cells by 1) inhibiting the formation of OTR [22] and 2) stimulating COX-2 and PGE2 production [23, 24]. In vitro, low concentrations of IFN-{tau} have been shown to reduce the basal production of PGF2{alpha} [9, 10] and higher concentrations to reduce the response to OT [4, 24]. The life span of the CL is also extended after administration of exogenous bovine trophoblast protein-1 (now named IFN-{tau}) [25]. However, basal PGF2{alpha} levels remain high during pregnancy in the ewe [26]. Thus the resistance of CL in pregnant ewes to luteolytic effects of PGF2{alpha} may be due to inhibition of pulsatile release of PGF2{alpha} and potential luteo-protective effects mediated by PGE2 or other secretions of the conceptus that protect the structure and function of the CL [7]. A role of COX-2 in the maintenance of CL is also supported by results in humans in vitro showing that PGE2 could stimulate progesterone production in situations in which hCG failed to do so [27]. A recent study in sheep by Charpigny et al. [28] demonstrated that COX-2 protein was expressed for an extended period of time in pregnant animals. It is believed that the release of arachidonic acid and the levels of COXs are the major limiting steps in the release of PGs [3, 29]. We have found, however, that increased PGs output in cultured endometrial cells is accompanied by an increase in COX-2 but not in phospholipase A2 mRNA [4, 23]. These results, together with those of Charpigny et al. [28], suggest that increased capacity to produce PGs may be involved during recognition and maintenance of pregnancy.

In ruminants, the uterus has developed specialized areas of the endometrium. These sites are called caruncules and are mushroom-like projections from the inner surface of the uterus of ruminants that allow attachment of the fetal membranes. During pregnancy these caruncles increase in size to accommodate the chorionic villi, which develop in localized areas referred to as cotyledons. Since these areas are more vascularized, they may be privileged sites for PG production or sensitivity to regulating factors.

Cultured epithelial and stromal cells of the bovine endometrium have been well characterized over the past few years in our laboratory. We have demonstrated that these cells are responsive to sex steroids [30], OT [30, 31], thrombin [32], and recombinant ovine (ro) and bovine IFN-{tau} [24]. We have also shown that both OT and roIFN-{tau} regulate COX-2 gene expression [4, 23]. Since caruncules are differentiated sites of the endometrium where placentation occurs in ruminants, the present study was conducted in epithelial and stromal cell isolated from the caruncular (CAR) and inter-caruncular (ICAR) regions. The production of PGE2 and PGF2{alpha} and its regulation by two factors known for their importance in recognition of pregnancy were compared using conditions described in previous publications and optimized for cells from whole endometrium.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Endometrial Cells and Culture

Bovine uteri were collected at the slaughterhouse within 15 min of death, and the physiological status of the tissue was estimated by the examination of ovarian morphology [33]. The tissues were brought to the tissue culture laboratory and dissected under a laminar flow hood. In this study, uteri of the early estrous cycle (Days 1–5) were used for each of the replicate experiments run in triplicate or quadruplicate, and endometrial cells were cultured separately. The epithelial and stromal endometrial cell cultures were prepared as described previously [30] with a slight modification for isolation of caruncles. The two horns of the uteri were placed in sterile Hanks' balanced salt solution (HBSS). Myometrial layers were dissected from the two horns, which were then inverted to expose the epithelium. All caruncles (approximately 90 on each horn) were dissected from the horns with scissors. The first 3 h of digestion in HBSS with trypsin (0.3%) yielded a suspension of CAR or ICAR epithelial cells. All the caruncles and horns were scraped with glass slides to eliminate remaining epithelial cells before a 45-min digestion with collagenase (0.064%), DNase I (0.016%), and trypsin (0.03%) for stromal cell isolation. The cells were plated in 24-well culture plates (Becton Dickinson, Lincoln Park, NJ) and incubated at 37°C in an atmosphere of 5% CO2:95% air. The culture medium used was RPMI-1640 containing 50 µg/ml gentamicin (Sigma, St. Louis, MO) and supplemented with 10% fetal bovine serum (Flow Laboratories, McLean, VA) depleted of steroids by dextran-charcoal extraction. The purity of the stromal preparation was improved by changing the medium 18 h after plating, at which time selective attachment of stromal cells had occurred. The medium was changed every 2 days until confluence was reached. Confluence of epithelial and stromal cells isolated from endometrium in the beginning (Days 1–5) of the estrous cycle is generally reached after 6–7 days in culture, and cultures remains stable for at least 15 days.

Experimental Protocol

In experiment 1, uteri from 3 different animals were used to determine the effect of OT. In experiment 2, three cell preparations from 3 different uteri were used to evaluate the response to roIFN-{tau}, and one dose of OT (10-7 M) was used as a control. After the cells reached confluence, the medium was replaced with 1 ml of fresh serum-free RPMI-1640 containing increasing doses of either OT (0, 10-9 to 10-5 M) or roINF-t (0 to 20 µg/ml) in quadruplicate. The roIFN-{tau} was provided by Dr. Fuller Bazer (Texas A&; University, College Station, TX). It was produced and purified as described previously by Ott et al. [34], and antiviral activity was determined as described by Pontzer et al. [35]. The concentrations of roIFN-{tau} used were in the range reported for these interferons to produce antiproliferative effects [36] and to regulate PGE2 production [23, 24] in vitro. Further, it has been shown that secretion of oIFN-{tau} increases to about 10 000 ng/h on Day 16 for sheep conceptuses [37] and that intrauterine injections of 100 µg/day on Days 11–15 delay luteolysis [34]. The antiviral activity of roIFN-{tau} was 1 x 108 U/mg protein. The cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h. For all experiments, at the end of the incubation, the culture medium was stored at -20°C until further processing. The plates were rinsed with ethanol, and DNA content was determined by 3,5 diamino benzoic acid (DABA) fluorescence according to Fiszer-Szafarz et al. [38]. DNA content was used to estimate the cell number for each well and standardize the results.

Enzyme Immunoassays (EIA) of PGs

For PGE2 and PGF2{alpha} measurement, an EIA technique was used which utilized acetylcholinesterase-linked PG tracers. We used fully characterized rabbit anti-PGE2 [39] and sheep anti-PGF2{alpha} (Bio Quant, Ann Arbor, MI) antisera. The inter- and intraassay coefficients of variation (n = 12) were 16% and 10%, respectively. The EIA technique was used as described previously [30].

Statistical Analysis

ANOVA was performed on PGE2 and PGF2{alpha} separately, and on the ratio of PGE2 to PGF2{alpha} from individual wells. For the first experiment (OT), a 2 x 6 x 4 factorial design (area x treatments x cell preparations [cow]) was employed, and for the second experiment (roIFN-{tau}), a 2 x 5 x 3 (area x treatments x cell preparations [cow]) factorial design was employed [40]. Cell variability (epithelial vs. stromal) was not included in the model for roIFN-{tau} experiments, since these cells were not compared, and were cultured and tested separately. Significant differences were analyzed using orthogonal contrasts with the aid of SuperAnova software (Abacus Concept Inc., Berkeley, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Production of PGs by Cells from CAR and ICAR Endometrium

Figure 1 illustrates basal PGE2 and PGF2{alpha} production in the different regions of the endometrium in vitro. The specialization in PG production previously described in cells from whole endometrium was reproduced in the CAR and ICAR areas: PGF2{alpha} was produced in greater proportion in epithelial cells as was PGE2 in stromal cells. The relative production of PGE2 was equivalent in epithelial cells of the CAR and ICAR regions. However, the production of PGF2{alpha} was higher (p < 0.05) in the ICAR region (4.0 ± 0.2 ng/µg DNA) than in the CAR region (2.2 ± 0.5 ng/µg DNA) in epithelial cells. In stromal cells, PGE2 production was higher in the ICAR (3.4 ± 0.4 ng/µg DNA) than in the CAR (2.1 ± 0.4 ng/µg DNA) region (p < 0.05). In these cells, the resulting PGE2:PGF2{alpha} ratio (Fig. 1B) was higher in the ICAR (19.1 ± 3.2) than in the CAR region (6.3 ± 1.9) (p < 0.05).



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  		FIG. 1. Regulation of basal PG production by epithelial and stromal cells from CAR and ICAR regions of the endometrium. A) When epithelial and stromal cells reached confluence, the medium was replaced with fresh RPMI-1640 medium without serum for 24 h, and the medium was recovered for measurement of PGF2{alpha} and PGE2 by EIA. B) Data on PGE2 and PGF2{alpha} production for each quadruplicate well were used to generate the PGE2:PGF2{alpha} ratio. Data are expressed as the mean ± SEM of 4 experiments run in quadruplicate. *p < 0.05 compared to respective controls in the same cell type.

Effect of OT on PG Production by Epithelial Cells from CAR and ICAR Regions

The effect of OT on the production of PGs in epithelial cells is illustrated in Figure 2. We have previously demonstrated that stromal cells do not respond to OT [24, 30, 31]. Thus, these cells were not used for OT experiments. Epithelial cells from the CAR and ICAR regions responded to OT by increasing PGE2 and PGF2{alpha} production in a dose-dependent manner (p < 0.05). However, in the CAR region, stimulation of PGF2{alpha} reached a plateau at a concentration of 10-7 M of OT. OT stimulated PGF2{alpha} production 6.3-fold in the CAR region and more than 33.0-fold in the ICAR region (p < 0.05; Fig. 2B). PGE2 and PGF2{alpha} levels from individual wells used in Figure 2, A and B, were taken to generate the ratio of PGE2 to PGF2{alpha} (Fig. 2C). At the cellular level, the relative production of PGE2 and PGF2{alpha} in vitro may give an indication of the direction of the response at the time of recognition of pregnancy in vivo: an increase may indicate a luteoprotective response whereas a reduction may indicate a luteolytic response. In epithelial cells from the ICAR area, there was a rapid and significant (p < 0.05) decrease of the PGE2:PGF2{alpha} ratio starting at 10-9 M OT and reaching 4-fold at the maximal dose used (10-5 M).



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  		FIG. 2. Influence of OT on PG production by epithelial cells from the CAR and ICAR regions. When the cells reached confluence, the medium was replaced with fresh RPMI-1640 medium without serum. Increasing doses of OT were added for 24 h, and the media were recovered for measurement of PGE2 (A) and PGF2{alpha} (B) by EIA. Data on PGE2 and PGF2{alpha} production for each quadruplicate well were used to generate the PGE2:PGF2{alpha} ratio (C). Data are expressed as the mean ± SEM of 4 experiments run in quadruplicate. *p < 0.05 compared to the same dose. aSignificant dose-dependent increase (p < 0.05). bSignificant dose-dependent decrease (p < 0.05).

Effect of roIFN-{tau} on PG Production

Endometrial epithelial and stromal cells from the CAR and ICAR regions were cultured separately to study the effect of roINF-{tau} on PG production. The effect of cell variability was not included in the statistical model because these cells were not compared together. In epithelial cells, addition of roIFN-{tau} stimulated PGE2 production in both the CAR and ICAR regions (13.8-fold and 7.1-fold, respectively) and PGF2{alpha} production was increased 2.4-fold and 3.6-fold, respectively (Fig. 3, A and B). The PGE2:PGF2{alpha} ratio was increased in both regions, but the increase was significant only in the CAR area (p < 0.05). In stromal cells, roIFN-{tau} stimulated PGE2 production in a dose-dependent manner in the CAR and ICAR regions (p < 0.05; Fig. 4A). PGF2{alpha} production was also stimulated significantly (p < 0.05) but to a much lesser extent (Fig. 4B). In these cells (Fig. 4C), the PGE2:PGF2{alpha} ratio appeared to increase in response to roIFN-{tau} in cells from the CAR area but did not reach statistical significance.



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  		FIG. 3. Influence of roIFN-{tau} on PG production by epithelial cells from the CAR and ICAR regions. After the cells reached maximal density, the medium was replaced with fresh RPMI-1640 medium without serum. Increasing doses of roIFN-{tau} were added for 24 h, and the media were recovered for measurement of PGE2 (A) and PGF2{alpha} (B) by EIA. Data on PGE2 and PGF2{alpha} production for each quadruplicate well were used to generate the PGE2:PGF2{alpha} ratio (C). Data are expressed as the mean ± SEM of 3 experiments run in quadruplicate. In this experiment, OT (10-7 M) was used as a control in each cell type, and PGF2{alpha} production was increased to 37.7 ± 13.3 ng/µg DNA (CAR) and 93.6 ± 21.2 ng/µg DNA (ICAR). aSignificant dose-dependent increase (p < 0.05).



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  		FIG. 4. Influence of roIFN-{tau} on PG production by stromal cells from the CAR and ICAR regions. When the cells reached confluence, the medium was replaced with fresh RPMI-1640 medium without serum. Increasing doses of roIFN-{tau} were added for 24 h, and the medium was recovered for measurement of PGE2 (A) and PGF2{alpha} (B) by EIA. Data on PGE2 and PGF2{alpha} production for each quadruplicate well were used to generate the PGE2:PGF2{alpha} ratio (C). Data are expressed as the mean ± SEM of 3 experiments run in quadruplicate. aSignificant dose-dependent increase (p < 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During the reproductive process at the beginning of pregnancy, communication between the embryo and the maternal genital tract must be established to ensure recognition of pregnancy. Over the years, we have developed an in vitro system using cultured endometrial cells [24, 30–32, 41], and we have found that these cells possess distinct properties that are well preserved among species [42, 43]. In the present study, CAR and ICAR epithelial and stromal endometrial cells were used and cultured separately to study the effect of two important factors involved at the time of recognition of pregnancy: IFN-{tau} (pregnancy recognition) and OT (luteolysis).

Caruncles are the sites of placental attachment in ruminants, and the hypothesis is that they may be privileged sites for embryo-maternal communication. Although endometrial cells from the CAR and ICAR areas exhibit different responses, it is still possible to identify the characteristic morphological and functional properties that we have already described for epithelial and stromal cells of the whole endometrium: a preferential production of PGF2{alpha} sensitive to OT stimulation in epithelial cells, and a production of PGE2 not responsive to OT in stromal cells [30, 31, 41]. We know that PGF2{alpha} is a luteolysin released by the endometrium at the end of a normal estrous cycle in response to OT when no viable conceptus is present. However, in the presence of a viable embryo, the effect of OT on pulsatile PGF2{alpha} production must be prevented [3]. Although a definite obligatory role for PGE2 at the time of recognition of pregnancy has not been proven conclusively, several observations suggest beneficial effects of this prostanoid on establishment of pregnancy. The presence of PGE2 can induce an immunomodulation that helps to prevent rejection of the conceptus semi-allograft [12, 44], and the administration of PGE2 protects the CL from spontaneous regression [14]. It is now well established that PGF2{alpha} is produced preferentially by epithelial cells, while PGE2 production is higher in stromal cells. In the present study, the same properties are found, and the results show, in addition, that the production of PGF2{alpha} in epithelial cells and of PGE2 in stromal cells is higher in ICAR compared to CAR.

OT experiments were carried out using epithelial cells only, because we had previously demonstrated that OT response was not present in stromal cells [30, 31]. When OT was added, a dose-dependent increase in the production of both PGs was observed in the CAR and ICAR regions. However, the production of PGF2{alpha} remained higher than that of PGE2 up to the highest dose used (36.3 ± 9.8 and 4.0 ± 0.4 ng/µg DNA, respectively). Interestingly, OT preferentially stimulated PGF2{alpha} in epithelial cells from the ICAR compared to the CAR region (33.0-fold vs. 6.3-fold, respectively). These results are in accordance with a previous study done in the ovine species using intrauterine devices to induce luteolysis [45]. The authors did not test whether contents differed between CAR and ICAR tissues, but their data showed roughly 50–300% more PGF in the ICAR area than in the CAR area. In the present study, OT caused a reduction in the PGE2:PGF2{alpha} ratio in the ICAR region. If this were reproduced in vivo, it would induce a return to estrus, given that PGF2{alpha} is responsible for CL regression and that PGE2 has an opposite effect in favor of pregnancy. In our model in vitro, a higher ratio could translate in vivo into promotion of pregnancy, whereas a decrease could indicate a return to estrus. This reduction is detected only in epithelial cells of the ICAR region, in which OT preferentially stimulates PGF2{alpha} production. In vivo, the combined effect of a denser population of OT receptors in the ICAR region of the epithelium and a larger surface area would give a stronger response in terms of PGF2{alpha} production. We have demonstrated previously that OT up-regulates COX-2 gene expression in vitro [4], and this was supported by a study done in sheep in vivo [28]. It is therefore possible that COX-2 may be differentially regulated by OT in the ICAR and CAR regions.

The second part of this study was conducted to investigate the effect of roIFN-{tau} on endometrial cells of the CAR and ICAR regions. IFN-{tau} is a factor produced by the conceptus during the periimplantation period and serves as a signal for recognition of pregnancy [8, 11]. Many studies carried out in vivo and in vitro have been published in the past few years to define the role of IFN-{tau} in maternal recognition of pregnancy [9, 10, 24]. In general, published observations from in vivo and in vitro studies support the accepted hypothesis that a reduction of PGF2{alpha} production is effected at the time of recognition of pregnancy. In cultured bovine endometrial cells, Danet-Desnoyers et al. [10] have shown a 30% reduction in the production of PGF2{alpha}. Under these circumstances, our initial observation that roIFN-{tau} and rbIFN-{tau} could stimulate PGE2 production by an overwhelming factor of 3000% was confusing [24]. Indeed, the huge stimulatory effect observed at high doses of roIFN-{tau} and the expression of the results as a percentage of control did hide a modest 30% inhibition of PGF2{alpha} production (unpublished observations), similar to that found by others. We have thus demonstrated, using bovine epithelial and stromal cells, that roIFN-{tau} increased PGE2 production in both cell types [24], and up-regulated COX-2 gene expression in a dose-dependent manner, within the range of IFN-{tau} concentration that can be reached in the vicinity of the conceptus [23]. The present study was conducted to compare endometrial cells from the CAR and ICAR regions, using roIFN-{tau} concentrations that in our hands consistently stimulated PGE2 production and COX-2 expression. The results indicate that roIFN-{tau} stimulates PG production in epithelial cells of both CAR and ICAR regions. This production of PGs was not significantly different between regions, but the PGE2:PGF2{alpha} ratio was increased significantly only in CAR area, indicating a possible preference of PGE2 regulation in this region. The effect of roIFN-{tau} on PG production in epithelial cells is a preferential stimulation of the production of PGE2 over that of PGF2{alpha}, and this is in agreement with our previous studies [23, 24]. In epithelial cells from whole endometrium, the effect was such that it changed the primary PG produced from PGF2{alpha} to PGE2, causing an increase in the PGE2:PGF2{alpha} ratio. In the present study, the PGE2:PGF2{alpha} ratios were higher in epithelial cells from the second experiment (Figs. 2C and 3C). This may reflect higher cell densities in the second experiment, in which we measured DNA levels twice as high as in the first experiment (results not shown). In stromal cells of the CAR and ICAR regions, roIFN-{tau} stimulated both PGs. In keeping with the response observed in epithelial cells, a significant dose-dependent increase of both PGs was observed in stromal cells. However, the PGE2:PGF2{alpha} ratio was also increased in stromal cells but was not significant. A recent study done by Charpigny et al. [28] demonstrated that in the ovine endometrium in vivo, COX-2 was up-regulated at the time of recognition of pregnancy and also during early pregnancy. These results are in accordance with our previous studies, in which we demonstrated that 1) OT up-regulates COX-2 gene expression and PGF2{alpha} production in epithelial cells [4] and 2) the presence of roIFN-{tau} in both cell types stimulates PGE2 production and up-regulates COX-2 gene expression [23].

Present and previous observations by our laboratory raise questions that will have to be addressed both in vivo and in vitro. It is generally accepted that regulation of PGF2{alpha} alone would be sufficient for recognition of pregnancy. The effect of PGE2 as a luteotropic hormone is not as well documented as that of PGF2{alpha} as a luteolysin. The role of PGF2{alpha} in the cow has been documented mainly by measurement of circulating levels of PGFM, the stable metabolite of PGF2{alpha}. We do not know if levels of the PGE metabolite are affected as well. Furthermore, the metabolism of PGF2{alpha} into PGFM may also be affected at the time of recognition of pregnancy. Similarly, we will have to verify if indeed endometrial cells from the CAR and ICAR regions play distinct roles in terms of production of PGs at the time of establishment of pregnancy. In support of the observation showing an increase in PGE2 production in response to roIFN-{tau}, it should be remembered that the maintenance of CL functions and production of progesterone are necessary but not sufficient to ensure the successful outcome of pregnancy. The embryo must also implant and develop. The action of PGE2 on vascular permeability [46, 47], vasodilation [48], modulation of the immune system [12], and regulation of uterine contractility [49] may be more important within the uterus than at the level of CL. In the hamster, PGE2 has even been reported to protect the embryo against lethal levels of radiation [50]. The results that we have obtained showing increased PG production in response to roIFN-{tau} are surprising and challenge the current hypothesis, but they are reproduced consistently in different cell cultures using different preparations of rbIFN-{tau} and roIFN-{tau}.

Collectively, these results support previous studies from our laboratory using cells from whole endometrium in vivo and in vitro and suggest that caruncles may be a privileged site for recognition of pregnancy since roIFN-{tau} increases the PGE2:PGF2{alpha} ratio preferentially in these regions of the epithelium. On the other hand, in the absence of a viable conceptus, OT would act preferentially in the ICAR regions to stimulate PGF2{alpha} production and induce luteolysis. It will be necessary to investigate in more detail the molecular properties of endometrial cells from the CAR and ICAR regions in order to attribute specific functions to each tissue during maternal recognition of pregnancy.


   ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Fuller W. Bazer for kindly providing roIFN-{tau}, Dr. Thomas G. Kennedy for generously donating the PGE2 antiserum for the ELISA technique, and Serge Lapointe for his help in statistical analysis.


   FOOTNOTES
 
1 This work has been supported by Natural Sciences and Engineering Research Council of Canada grant (NSERC) grant #OGPIN030 (M.A.F.) and an NSERC scholarship (E.A.). Back

2 Correspondence: M.A. Fortier, Ontogénie et Reproduction, Centre de Recherche du Centre Hospitalier, de l'Université Laval, 2705 Boul. Laurier, Ste-Foy, PQ, Canada G1V 4G2. FAX: (418) 654–2765; mafortier{at}crchul.ulaval.ca Back

Accepted: March 11, 1998.

Received: July 7, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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