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Biology of Reproduction 64, 1682-1688 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Autocrine/Paracrine Action of Oxytocin in Pig Endometrium1

Jianbo Hu3,a, Tenneille E. Ludwig4,a, Ugur Sallib, Fredrick Stormshakb, and Mark A. Mirando2,a

a Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164-6353 b Department of Animal Science, Oregon State University, Corvallis, Oregon 97331-6702

ABSTRACT

Luminal epithelial cells of porcine endometrium are unresponsive to oxytocin (OT) in vitro although they express the greatest quantity of OT and receptors for OT in vivo. Therefore, the objective of this study was to determine if oxytocin acted in an autocrine manner on luminal epithelial cells to stimulate prostaglandin (PG)F2{alpha} secretion. Treatment of endometrial explants or enriched luminal epithelial cells with OT antagonist L-366,948 decreased (P < 0.05) basal secretion of PGF2{alpha}. Oxytocin increased (P < 0.01) PGF2{alpha} secretion from luminal epithelial cells that were pretreated with 1:5000 or 1:500 OT antiserum for 3 h to immunoneutralize endogenously secreted OT. However, OT only increased (P < 0.05) PGF2{alpha} secretion from glandular epithelial cells when pretreated with 1:500 OT antiserum. Pretreatment with OT antiserum did not alter the ability of OT to induce PGF2{alpha} secretion from stromal cells. Medium conditioned by culture of luminal epithelial cells stimulated (P < 0.05) phospholipase C activity in stromal cells, indicative of the presence of bioactive OT. Oxytocin was secreted by luminal epithelial cells and 33% was released from the apical surface. These results indicate that luminal epithelial cells secrete OT that acts in an autocrine and/or paracrine manner in pig endometrium to stimulate PGF2{alpha} secretion.

endometrium, oxytocin, uterus

INTRODUCTION

Oxytocin (OT) is a peptide hormone synthesized primarily in the hypothalamus and released from the neurohypophysis. Its systemic effects on reproduction (i.e., lactation, parturition, and luteolysis) are well established [1]. Although synthesis of OT by the corpus luteum was suggested very early in the 20th century [2], it was only recently that the synthesis and secretion of OT by the ovary [3, 4], placenta [5, 6], and uterus [6–9] in a variety of species was clearly established. Initial studies with ruminants suggested that most of the circulating OT that contributes to luteolysis came from the ovary rather than the hypothalamus [10–12], although this conclusion has recently been challenged [13]. Although less OT is apparently secreted from the ovary than the neurohypophysis in pigs [14], recent studies clearly demonstrated that OT mRNA was abundantly present in porcine uterine endometrium [7, 15], and OT was secreted in large quantities into the uterine lumen, especially during pregnancy [8, 15].

Oxytocin is the major known stimulus for pulsatile secretion of prostaglandin (PG)F2{alpha}, which is responsible for initiating luteolysis in domestic ungulates [16–18]. Although the role of OT in promoting luteolytic secretion of PGF2{alpha} in cyclic sheep is well established [16, 18], considerable evidence also implicates OT in luteolysis in pigs [17]. For example, intramuscular injections of OT twice daily during late diestrus shortened the estrous cycle of pigs [19, 20].

In the endometrium of ruminants, luminal epithelial cells are most responsive and stromal cells are unresponsive to exogenous OT in terms of PGF2{alpha} secretion [21]. However, the opposite is true for OT-induced PGF2{alpha} secretion from porcine endometrial cells [22], even though luminal epithelial cells possess the greatest numbers of OT receptors and stromal cells possess the least [23]. Because luminal epithelial cells also express OT peptide to the greatest degree [7] and large quantities of OT are secreted into the uterine lumen [8, 15], we hypothesized that OT secreted from porcine luminal epithelial cells may act in an autocrine manner to bind to OT receptors and stimulate PGF2{alpha} secretion from these cells, thereby obscuring any response to exogenous OT treatment. Alternatively, luminal epithelial cells may secrete OT in a basolateral direction, which then acts in a paracrine manner on other cell types within the endometrium. Therefore, the objective of this study was to determine if 1) treatment of endometrium or enriched luminal epithelial cells with OT antagonist or OT antisera would suppress basal PGF2{alpha} secretion or promote a response to subsequent OT treatment, 2) medium conditioned by culture of luminal cells would stimulate phospholipase C activity in stromal cells, and 3) luminal cells secreted products with OT-like activity.

MATERIALS AND METHODS

Animals

Crossbred gilts (Yorkshire, Landrace, Large White, Duroc, and Hampshire) were observed daily for estrous behavior in the presence of an intact boar. Onset of standing estrus was designated Day 0. Gilts were hysterectomized on Day 15 (experiment 1) or Day 16 (experiments 2–6) after second or third estrus as previously described [24, 25]. Days 15–16 were chosen because previous studies demonstrated that endometrium was most responsive to OT in vivo [26] and in vitro [27] on these days. Endometrium (20–25 g) was collected aseptically from one randomly selected uterine horn and placed in Krebs-Ringer bicarbonate (KRB) buffer (experiment 1) [24] or incomplete Hanks balanced salt solution (experiments 2–6) [22].

Isolation of Endometrial Cells for Culture

Cell populations were separated as previously described [22, 28]. All three cell types were seeded in 24-well culture plates at a density of approximately 0.5 x 106 cells per well and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2 as described previously [22, 28] for use in experiments 2–5. Cells were cultured for at least 72 h to allow them to adhere to the plates before initiation of experiments when cells were estimated to be 90%–95% confluent. The purity of the enriched populations of luminal epithelial, glandular epithelial, and stromal cells exceeded 97%, 90%, and 99%, respectively.

Luminal epithelial cells also were cultured under polarizing conditions, as described previously [29], for use in experiment 6. Briefly, cells were plated onto 12-mm Millicell-HA inserts (0.45 µm pore size; Millipore, Bedford, MA) at a density of 0.25 x 106 cells/insert in a volume of 400 µl RPMI-1640. Each insert was then placed in 24-well plates in a volume of 600 µl RPMI-1640 per well such that the level of medium inside the insert was approximately equal to that on the exterior of the insert. After 3 days, medium was replaced with fresh medium. Beginning on Day 5, cellular polarity was assessed daily by measuring electrical resistance with a Millicell Electrical Resistance System (Millipore). Cell monolayers were shown to have reached confluence when electrical resistance increased sharply from one day to the next and was >500 {Omega}/cm2 [29].

Experiment 1

Secretion of PGF2{alpha} by endometrial explants from 10 cyclic gilts in experiment 1 was determined as described previously [24, 27]. Endometrium (200 ± 10 mg) was placed in vials with 2 ml ice-cold KRB. Tissues were rinsed with 2 ml KRB and then incubated for 3 h at 39°C under an atmosphere of 95% O2 and 5% CO2. The medium was replaced with fresh KRB 30 min before treatment. During the final 30 min of incubation, samples were treated with 0 µM antagonist (i.e., control), 1 µM OT antagonist (L-366,948; Merck, Sharp and Dohme Research Laboratories, West Point, PA), 1 µM antagonist of type 2 (V2) vasopressin receptors ([ß-mercapto-ß,ß-cyclopentamethylenepropionyl2,-O-Et-Tyr2,Val4,Arg8]-vasopressin; Sigma Chemical Co., St. Louis, MO) or 1 µM lysine-vasopressin (Sigma Chemical Co.) for 40 min. The V2 antagonist was utilized instead of a V1 antagonist to minimize binding to OT receptors; V2 antagonists generally bind to vasopressin and OT receptors in the order of V2 > V1 > OT, whereas V1 antagonists tend to bind to both V2 and OT receptors. Therefore, the V2 antagonist served as a control that was expected to provide very little antagonism of action mediated through OT receptors. Incubation was terminated 30 min after treatment, and KRB samples from the final two (pre- and posttreatment) incubation periods were stored at -20°C until RIA of PGF2{alpha} as described subsequently.

Experiments 2 and 3

Luminal epithelial, glandular epithelial, and stromal cells were isolated from four gilts and cultured until approximately 90%–95% confluent. Cells were then cultured in RPMI-1640 devoid of fetal bovine serum (FBS) for 12 h before pretreatment with 0, 50, or 500 nM OT antagonist L-366,948 for 3 h in experiment 2 or with 0, 1:5000 or1:500 dilutions of rabbit anti-OT serum R-1 [30] or normal rabbit serum for 3 h in experiment 3. Cells were then washed three times with RPMI-1640 and treated with 0 or 100 nM OT for 3 h, after which culture medium was collected and stored at -20°C until PGF2{alpha} concentrations were quantified by RIA as described subsequently.

Experiment 4

To determine if medium, conditioned by culture of luminal epithelial cells, could stimulate phospholipase C activity of stromal cells in a manner similar to OT, luminal epithelial cells were isolated from five gilts and cultured in RPMI-1640 containing 20% FBS until reaching approximately 90%–95% confluence. Cells were washed three times with RPMI-1640, cultured for 24 h in RPMI devoid of FBS, washed again, and cultured for 24 h in RPMI with 20% FBS. Medium from the two 24-h culture periods was collected and stored at -20°C until used to treat stromal cells as described subsequently. Media samples, conditioned from culture of luminal epithelial cells, were subsequently pooled across gilts to create two pools of conditioned medium: that from the first 24-h period devoid of FBS and that from the second 24-h period containing 20% FBS. Stromal cells were isolated from four gilts on Day 16 postestrus. After achieving approximately 90%–95% confluence, cells were washed, cultured in FBS-free RPMI-1640 containing 5 µCi [3H]inositol for 12 h, and washed again, after which 50 mM LiCl was added (to inhibit degradation of inositol phosphates) before treatment. Treatments were 1) 0 nM OT without FBS; 2) 100 nM OT without FBS; 3) 50% luminal epithelial cell-conditioned medium without FBS; 4) 100% luminal epithelial cell-conditioned medium without FBS; 5) 0 nM OT + 20% FBS; 6) 100 nM OT + 20% FBS; 7) 50% luminal epithelial cell-conditioned medium containing 20% FBS; or 8) 100% of luminal epithelial cell-conditioned medium containing 20% FBS. Media for treatment of stromal cells were either devoid of FBS (treatments 1–4) or had FBS added such that the final FBS concentration was 20% (treatments 5–8). After 3 h of treatment, cells were lysed and phospholipase C activity was determined as described subsequently.

Experiments 5 and 6

To determine if luminal epithelial, glandular epithelial, or stromal cells secreted OT during culture in experiment 5, cells were isolated from six gilts and cultured in 24-well plates as described previously. Cells were washed three times with RPMI-1640 and then cultured for 3 h in the presence of 0 or 100 nM OT, after which culture medium was collected and stored at -20°C until concentrations of OT and PGF2{alpha} were quantified by RIA as described subsequently. Concentrations of OT were quantified only in samples from cells treated with 0 nM OT. Plates were then stored at -20°C until cellular content of DNA was determined [31] in order to express OT concentrations as pg/µg DNA for comparison among cell types. Luminal epithelial cells were isolated from an additional four gilts in experiment 6 and cultured under polarizing conditions on Millicell-HA inserts as described previously. Cells were washed three times with RPMI-1640 and then cultured for 3 h, after which culture medium was collected from the luminal (upper) and basal (lower) compartments. Samples of culture medium were stored at -20°C until concentrations of OT and PGF2{alpha} were quantified by RIA as described subsequently.

Radioimmunoassay of PGF2{alpha}

Concentrations of PGF2{alpha} in 10 µl culture medium from experiments 1–6 were quantified by RIA as described previously [22, 27]. Intraassay and interassay coefficients of variation were 6.8% and 13.7%, respectively.

Radioimmunoassay of OT

Concentrations of OT in culture medium from experiments 5 and 6 were quantified by RIA after extracting 350 µl of sample as described previously [32, 33]. Extraction efficiency was 64.2%, and samples were corrected for loss due to extraction. Sensitivity of the assay was 0.5 pg/tube. Intraassay and interassay coefficients of variation were 3.27% and 8.17%, respectively.

Phospholipase C Activity

Phospholipase C activity in experiment 4 was determined as described previously [22]. Briefly, medium was removed at the end of the treatment period, 1 ml ice-cold 15% trichloroacetic acid was added, and cells were placed on ice for 20 min. Cell lysates were then collected and analyzed for incorporation of [3H]inositol into total inositol phosphates by anion-exchange chromatography as described previously [22, 24]. Data were expressed as dpm/well [3H]inositol phosphates.

Statistical Analyses

Data were subjected to least-squares analysis of covariance (experiment 1) or ANOVA (experiments 2–6) for randomized block designs using the general linear models procedure of the Statistical Analysis System (SAS) [34]. Data from experiment 1 were analyzed for a one-way treatment structure of main effects with pig as the block effect and PGF2{alpha} secretion during the 30-min pretreatment as the covariate. Data from experiment 2 were analyzed for a two-way treatment structure of main effects. Antagonist treatment was cross-classified with OT treatment and pig was the block effect. Data from experiment 3 were analyzed as the ratio of response of OT antiserum treatment to that of normal rabbit serum treatment (i.e., percentage of control) to avoid the confounding effects of serum. Analysis was performed for a one-way treatment structure of main effects (dilution of OT antiserum) with pig as the block effect. In experiment 4, data were log transformed to alleviate statistical problems associated with heterogeneity of variance. A two-way ANOVA was used with treatment and concentration of FBS as main effects, and pig was the block effect. In experiment 5, data for OT and PGF2{alpha} secretion from the three endometrial cell types were standardized for cellular DNA content and then compared by one-way (OT secretion) or two-way ANOVA (PGF2{alpha} secretion). For OT secretion, cell type was the main effect and pig was the block effect. For PGF2{alpha} secretion, cell type and OT treatment were cross-classified and pig was the block effect. For direction of OT and PGF2{alpha} from polarized luminal epithelial cells in experiment 6, one-way ANOVA were performed with cellular surface of secretion as the main effect and pig as the block effect. Tests of hypotheses were performed using the appropriate error terms according to the expectation of the mean squares [35].

RESULTS

Experiment 1

Secretion of PGF2{alpha} from endometrial explants was inhibited (P < 0.05) by treatment with OT antagonistL-366,948 for 40 min in experiment 1 (Fig. 1). However, PGF2{alpha} release was unaffected by treatment with lysine vasopressin or the V2 antagonist (Fig. 1).



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  		FIG. 1. Effects of 1 µM OT antagonist L-366,948 (OTa), lysine vasopressin (LVP), and V2 receptor antagonist (VPa; [ß-mercapto-ß,ß-cyclopentamethylenepropionyl2,-O-Et-Tyr2,Val4,Arg8]-vasopressin) on PGF2{alpha} secretion (mean ± SEM) from endometrial explants. Asterisk above bars indicates that OTa treatment decreased (*P < 0.05) PGF2{alpha} secretion

Experiment 2

In the absence of OT antagonist, luminal epithelial cells were unresponsive to 100 nM OT, and pretreatment of luminal epithelial cells with OT antagonist L-366,948 did not affect the response to subsequent treatment with OT (Fig. 2). However, PGF2{alpha} release was decreased (P < 0.01) by 500 nM L-366,948, regardless of OT treatment. For glandular epithelial cells, the interaction of OT antagonist x OT (P < 0.07) indicated that OT stimulated (P = 0.01) PGF2{alpha} secretion in the absence of antagonist, but the response to OT was blocked by pretreatment with antagonist (Fig. 2). For stromal cells, the OT antagonist x OT interaction (P < 0.05) indicated that L-366,948 abrogated the stimulatory effect of OT (P < 0.05) that occurred in the absence of antagonist (Fig. 2).



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  		FIG. 2. Effect of preincubation with OT antagonist L-366,948 on PGF2{alpha} secretion (mean ± SEM) from luminal epithelial (LEC, top panel), glandular epithelial (GEC, middle panel), and stromal cells (SC, bottom panel) in response to 0 nM OT (open bars) or 100 nM OT (solid bars) in experiment 2. Asterisks above open bars indicate that OT antagonist L-366,948 decreased (**P < 0.01) basal PGF2{alpha} release, whereas asterisks above solid bars indicate that OT stimulated (*P < 0.05) PGF2{alpha} secretion (mean ± SEM)

Experiment 3

In the absence of antisera, OT increased PGF2{alpha} secretion from glandular epithelial and stromal cells (P < 0.01) but not from luminal epithelial cells (Fig. 3). The interaction of OT x antisera dilution (P < 0.01) indicated that PGF2{alpha} secretion was enhanced from luminal epithelial cells after pretreatment with 1:5000 or 1:500 OT antiserum for 3 h (Fig. 3). The enhanced response to OT at 1:5000 anti-OT was due primarily to decreased (P < 0.01) basal PGF2{alpha} release, whereas at 1:500 anti-OT, it was due to both decreased (P < 0.05) basal PGF2{alpha} secretion and increased (P < 0.05) OT-induced PGF2{alpha} secretion. For glandular epithelial cells, the interaction of OT x antiserum dilution (P < 0.05) indicated that release of PGF2{alpha} in response to OT was enhanced (P < 0.05) from glandular epithelial cells pretreated with 1:500 OT antiserum (Fig. 3). There was no effect of OT antiserum on PGF2{alpha} release from stromal cells in response to OT (Fig. 3).



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  		FIG. 3. Mean (±SEM) response of luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) to 0 nM OT (open bars) or 100 nM OT (solid bars) after pretreatment with normal rabbit serum (top panel) or rabbit anti-OT serum (lower 3 panels) in experiment 3. Asterisks above solid bars indicate that OT stimulated (**P < 0.01, *P < 0.05) PGF2{alpha} secretion

Experiment 4

Phospholipase C activity (Fig. 4) was increased (P < 0.01) by treatment with OT, 50% conditioned medium, 100% conditioned medium, and 20% FBS. However, the interaction of treatment x concentration of FBS (P < 0.05) indicated that all effects of treatment (OT and conditioned medium) were more pronounced in the absence of FBS.



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  		FIG. 4. Mean (±SEM) phospholipase C activity in stromal cells in response to treatment with 100 nM OT or medium conditioned by culture of luminal epithelial cells. Asterisks above bars indicate that treatments stimulated (**P < 0.01) phospholipase C activity in experiment 4. Phospholipase C activity also was increased (P < 0.01) in the presence of 20% FBS, regardless of treatment

Experiments 5 and 6

In experiment 5, secretion of OT from luminal epithelial and stromal cells was greater (P = 0.05) than from glandular epithelial cells (Fig. 5) but did not differ between luminal epithelial and stromal cells. The increase in PGF2{alpha} secretion in response to OT (data not shown) was greatest for stromal cells (P < 0.01), least for luminal epithelial cells (P > 0.20) and intermediate for glandular epithelial cells (P < 0.10) and indicated clearly that these cells differed in functional phenotype. In experiment 6, secretion of PGF2{alpha} from polarized luminal epithelial cells (Fig. 6) tended to be less (P < 0.07) from the apical (31%) than the basolateral surface (69%). Although secretion of OT from the apical (33%) and basolateral surfaces (67%) was proportionally similar to that of PGF2{alpha}, rate of OT secretion did not differ significantly (P > 0.20) between surfaces due to the greater variability compared with that of PGF2{alpha} secretion.



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  		FIG. 5. Mean (±SEM) secretion of OT from luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) in experiment 5. Bars bearing different letters indicate that cells differed (P = 0.05) in rate of OT secretion



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  		FIG. 6. Mean (±SEM) secretion of OT and PGF2{alpha} from the apical (open bars) and basolateral surfaces (solid bars) of polarized luminal epithelial cells in experiment 5. Letter above bar indicates that PGF2{alpha} secretion was greater (P < 0.07) from the basolateral surface than from the apical surface

DISCUSSION

Previous results indicated that luminal epithelial cells of pig endometrium were unresponsive to OT, whereas stromal cells were most responsive to OT [22, 28]. This pattern of response occurred even though OT receptors were present in greatest abundance on luminal epithelial cells, and stromal cells possessed the least amount of receptors [23]. The reason for this discrepancy is not completely clear. Because luminal epithelium also contained the greatest quantity of OT peptide [7], was the only cell type to secrete detectable amounts of OT in culture (D.L. Davis and W.E. Trout, personal communication), and secreted amounts of PGF2{alpha} that were substantially greater than those of glandular epithelial and stromal cells [22], it was hypothesized that OT may act in an autocrine manner to stimulate PGF2{alpha} release from luminal epithelial cells. Such a continuous action of OT could also obscure any response to exogenous OT treatment of cultured cells [22, 28]. Results of the present studies supported this hypothesis, for the most part. Removal of endogenous OT with OT antisera enhanced the subsequent response to OT of luminal epithelial cells but not of glandular epithelial or stromal cells in experiment 3. Treatment of endometrial explants in experiment 1 with OT antagonist decreased PGF2{alpha} secretion. Similarly, treatment of luminal epithelial cells with OT antagonist in experiment 2 also decreased PGF2{alpha} secretion, although it did not enhance the response to subsequent OT treatment. This latter effect may be attributed to continued binding of antagonist to the OT receptor beyond the pretreatment period, a suggestion that is supported by the observation that OT antagonist completely abolished the response to subsequent OT treatment of glandular epithelial and stromal cells. In experiment 4, medium conditioned by culture of luminal epithelial cells stimulated phospholipase C activity of stromal cells similar to that promoted by OT. Finally, luminal epithelial cells in culture secreted OT from both the apical and basolateral surfaces. Collectively, these results indicate that OT was secreted from luminal epithelial cells and that these luminal epithelial cells may be responsive to OT acting in an autocrine manner. However, secretion of OT from the basolateral surface of luminal epithelial cells and the ability of luminal epithelial cell-conditioned medium to stimulate phospholipase C activity in stromal cells also is indicative of a paracrine effect of OT. Thus, both autocrine and paracrine actions of OT secreted by porcine endometrium are possible.

Although pig endometrium secretes OT into the uterine lumen in vivo [8, 15] and luminal epithelial cells secreted immunoreactive OT into culture (D.L. Davis and W.E. Trout, personal communication), it was not known if this OT was bioactive. Results from the present study showed that luminal epithelial cells secreted OT in apical and basolateral directions. Further, medium conditioned by culture of luminal epithelial cells stimulated phospholipase C activity in stromal cells. These results are consistent with the proposal that luminal epithelial cells secrete bioactive OT. However, the possibility that other substances secreted by luminal epithelial cells were responsible for stimulating phospholipase C activity in stromal cells cannot be excluded completely at the present time.

As previously discussed, luminal epithelial cells were completely unresponsive to OT in vitro [22, 28]. Presently, it is not clear whether OT, endogenously secreted from luminal epithelial cells, downregulates the response of luminal epithelial cells to subsequent OT treatment or if it stimulates these cells continuously, thereby elevating basal PGF2{alpha} release from these cells and obscuring any apparent response to exogenous OT. Evidence is available to support both possibilities. Treatment with OT antiserum or OT antagonist suppressed basal PGF2{alpha} release in the absence of subsequent OT treatment. Both of these effects are consistent with continuous stimulation of luminal epithelial cells by endogenous OT. If luminal epithelial cells were markedly desensitized by endogenous OT, then blocking the action of endogenous OT in these two experiments would not have been expected to decrease PGF2{alpha} secretion. On the other hand, treatment of luminal epithelial cells with 1:500 OT antiserum also increased PGF2{alpha} secretion after OT treatment, an effect that is consistent with some reduced desensitization of luminal epithelial cells. Moreover, stromal cells secreted OT in culture at levels that were similar to those released by luminal epithelial cells, but stromal cells were always the cell type most responsive to OT. Future studies will be required to resolve fully the issue of whether continuous exposure to OT upregulates basal PGF2{alpha} secretion from luminal epithelial cells or desensitizes their response to OT.

Treatment of glandular epithelial cells and stromal cells with the highly specific OT antagonist completely blocked the response to subsequent treatment with OT. The results of the present research are in agreement with previous studies that indicated that L-366,948 displaced binding of [3H]OT from pig endometrial membrane preparations [24] and inhibited the response of endometrial explants to OT [36]. The present work is also consistent with the action of OT occurring through specific OT receptors on these cells and not through V1 receptors. Type-1 vasopressin receptors are apparently present in pig endometrium but are not coupled to PGF2{alpha} secretion [24, 36]. Although these results provide evidence for the presence of OT receptors on glandular epithelial cells and stromal cells, they do not exclude the possibility that these cells also possess receptors for vasopressin.

In the present study, luminal epithelial and stromal cells secreted approximately equal quantities of OT on a per cell basis. Although both cell types secreted more OT than glandular epithelial cells, these results are somewhat inconsistent with those of Boulton et al. [7] who reported that luminal epithelium of pig endometrium expressed the greatest amount of OT and stromal cells expressed the least. Similarly, medium from culture of porcine luminal epithelial cells contained detectable concentrations of OT, whereas that from culture of stromal cells did not (D.L. Davis and W.E. Trout, personal communication). An explanation for this discrepancy between the results of the current study and those previously reported [7] is not readily apparent. Moreover, the high level of OT secretion from stromal cells compared with both epithelial cell types apparently did not influence stromal cell responsiveness to OT because stromal cells were always most responsive to exogenous OT and immunoneutralization of endogenous OT in experiment 3 did not enhance the response of stromal cells to OT.

In summary, these results confirmed that luminal epithelial cells were unresponsive to OT, stromal cells were most responsive to OT and glandular epithelial cells displayed an intermediate response. Treatment of endometrial explants or luminal epithelial cells with an OT antagonist decreased secretion of PGF2{alpha}, whereas pretreatment with anti-OT enhanced the subsequent response of luminal epithelial cells to OT. Medium conditioned by culture of luminal epithelial cells contained substantial amounts of OT and stimulated phospholipase C activity in stromal cells in a manner equivalent to that of OT. These results indicate that luminal epithelial cells secrete OT, which acts in an autocrine and/or paracrine manner to promote PGF2{alpha} secretion.

ACKNOWLEDGMENTS

The authors are indebted to Dr. Duane L. Davis, Kansas State University, for advice on isolation and culture of endometrial cells, to Dr. William W. Thatcher, University of Florida, for supplying the antisera to PGF2{alpha}, to Drs. Genowefa and Jan Kotwica, Polish Academy of Sciences, Olsztyn-Kortowo, Poland, for supplying the antisera to OT used in experiment 3, and to Dr. Dieter Schams, Technical University of Munich, Germany, for supplying the OT antibody used in experiments 5 and 6. The authors also are grateful to the members of M.A. Mirando's laboratory for assistance with surgery and to the staffs of the Washington State University Swine Center and the Experimental Animal Laboratory Building for assistance in care and handling of animals.

FOOTNOTES

First decision: 20 December 2000.

1 This research was supported by NIH grant HD30268. Back

2 Correspondence and current address: Mark A. Mirando, National Research Initiative Competitive Grants Program, Stop 2241, 1400 Independence Ave. SW, Washington, DC 20250-2241. FAX: 202 205 3641;mmirando{at}reeusda.gov Back

3 Current address: Oregon Regional Primate Research Center, Beaverton, OR 97006. Back

4 Current address: Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI 53706-1581. Back

Accepted: January 18, 2001.

Received: November 7, 2000.

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A. Blitek and A. J Ziecik
Effect of LH on prostaglandin F2{alpha} and prostaglandin E2 secretion by cultured porcine endometrial cells
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