Oxytocin-Stimulated Phosphoinositide Hydrolysis and Prostaglandin F Secretion by Luminal Epithelial, Glandular Epithelial, and Stromal Cells from Pig Endometrium. I. Response of Cyclic Pigs on Day 16 Postestrus¹

^a Department of Animal Sciences and Center for Reproductive Biology, Washington State University, Pullman, Washington 99164–6353

	ABSTRACT

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Oxytocin (OT) is the physiological stimulus for pulsatile release of endometrial prostaglandin (PG) F₂ during luteolysis in domestic ungulates, and the cellular mechanism for this appears to involve phosphoinositide (PI) hydrolysis. To determine which endometrial cell type(s) was responsive to OT during luteolysis in swine, luminal epithelial (LEC), glandular epithelial (GEC), and stromal cells (SC) were isolated from endometrium by differential enzymatic digestion and sieve filtration on Day 16 postestrus and cultured. For PI hydrolysis in experiment 1, SC were most responsive to 100 nM OT (p < 0.001), whereas LEC were least responsive and GEC had an intermediate response (p < 0.001). For PGF secretion in experiment 2, the response to OT was greatest for SC, least for LEC, and intermediate for GEC. In experiment 3, 100 nM OT increased PI hydrolysis in SC within 30 min (p < 0.05) and in GEC within 60 min (p < 0.05) but did not increase PI hydrolysis in LEC. In experiment 4, PI hydrolysis in SC was increased (p < 0.05) by 33–333 nM OT but was not increased by 333 nM OT in GEC or LEC after 30 min. However, PGF secretion from SC was increased (p < 0.05) by 10–333 nM OT, and from GEC by 10–333 nM OT, but was not increased from LEC by 333 nM OT. Results of this study indicate that 1) there was differential responsiveness to OT among endometrial cell types, and 2) within cell type, there generally was a similar response to OT for both PI hydrolysis and PGF secretion, further implicating PI hydrolysis as the signaling pathway for OT-stimulated PGF₂ release. The differential response of endometrial cell types may have an important role in the pattern of PGF₂ secretion during luteolysis in swine.

	INTRODUCTION

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In domestic ungulates, luteolysis occurring at the end of the estrous cycle is a uterine-dependent event [1], and pulsatile release of uterine prostaglandin (PG)F₂ is responsible for inducing luteolysis [2, 3]. Lysis of the corpora lutea and concomitant decline in progesterone production permits further follicular development leading to estrus, ovulation, and the opportunity for mating and pregnancy to occur. The physiological and biochemical mechanisms for regulation of endometrial PGF₂ secretion during the estrous cycle in ruminants, especially sheep, have been extensively studied, but the mechanism(s) in swine has not received similar attention.

In sheep, pulsatile release of endometrial PGF₂ is essential to induce luteolysis during late diestrus [4, 5]. The major physiological stimulus for episodic PGF₂ secretion in ruminants is oxytocin (OT), which is also released in a pulsatile fashion [6]. The action of OT in stimulating endometrial PGF₂ secretion is mediated through phospholipase C [7–9], which hydrolyzes phosphoinositides (PI) to produce inositol (1,4,5)-trisphosphate (IP₃) and diacylglycerol second messengers. In turn, diacylglycerol and IP₃ promote protein kinase C activation and release of intracellular Ca²⁺, respectively, resulting in PGF₂ release through a mechanism involving phospholipase A₂ [10] that has not been completely elucidated.

Pulsatile release of luteolytic PGF₂ also occurs in pigs during the late luteal phase [11–13], but the physiological and biochemical mechanisms regulating endometrial pulsatile PGF₂ secretion in swine are poorly understood. Part of the mechanism controlling luteolytic PGF₂ release in pigs [14] may be similar to that which has been established for domestic ruminants. It has been shown that OT stimulates PGF₂ release from endometrium during late diestrus (i.e., Days 14–16) [15, 16] and that responsiveness to OT develops between Days 12 and 14 postestrus, which is before the onset of functional luteolysis [16]. Further, when OT was administered to cyclic gilts during late diestrus, interestrous interval was reduced, and the response was uterine-dependent: hysterectomized gilts did not return to estrus after OT injection [17]. The requirement for the presence of the uterus strongly implies that OT reduced interestrous interval by promoting endometrial PGF₂ release, which in turn induced luteolysis.

The action of OT on pig endometrium appears to occur through specific OT receptors to promote PI hydrolysis [14], thereby activating the IP₃-diacylglycerol second-messenger system [18–21]. However, it is not known which cell types within pig endometrium are responsive to OT. Therefore, the objectives of this study were 1) to ascertain which endometrial cell types (i.e., luminal epithelial [LEC], glandular epithelial [GEC], and stromal cells [SC]) from cyclic pigs were responsive to OT during the time of luteolysis, and 2) to determine whether these cell types responded to OT with increases in both PI hydrolysis and PGF₂ secretion.

	MATERIALS AND METHODS

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Animals

Crossbred gilts (Yorkshire, Landrace, Chester 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 16 after second or third estrus as previously described [18–21]. Day 16 was chosen because previous and contemporary studies with this population of pigs demonstrated that endometrium was maximally responsive to OT in vivo [16] and in vitro [21], and functional luteolysis is 50% complete by that time as evidenced from plasma progesterone concentrations of 10–14 ng/ml [16, 17, 21]. Endometrium (20–25 g) was collected aseptically from one randomly selected uterine horn and placed in Dulbecco's PBS (2.68 mM KCl, 1.47 mM KH₂PO₄, 0.49 mM MgCl₂, 136.9 mM NaCl, and 8.1 mM Na₂HPO₄).

Separation of Endometrial Cells for Culture

Cell populations were separated using a modification of a procedure previously described [22]. After being washed with incomplete Hanks' balanced salt solution (IHBSS: 5.36 mM KCl, 0.44 mM KH₂PO₄, 4.17 mM NaHCO₃, 0.34 mM Na₂HPO₄, 136.9 mM NaCl, and 5.55 mM glucose), the tissue was digested with 4.8 mg/ml dispase (Boehringer Mannheim, Indianapolis, IN) in IHBSS for 20 min at 20°C. Endometrial tissue was digested further with 4.8 mg/ml dispase and 4-strength pancreatin (Gibco BRL, Grand Island, NY) for 1 h at 20°C with gentle shaking at 20- to 30-min intervals, and then washed twice with 10–15 ml of IHBSS. Luminal epithelial cells released during the first two digestions and from the washes were pooled by centrifugation for 7 min at 200 x g, washed once by resuspending in IHBSS, and repelleted by centrifugation at 300 x g. Red blood cells were removed by layering the LEC suspension in 4 ml RPMI-1640 (GIBCO BRL) containing 10% fetal bovine serum (FBS; Gibco BRL) onto a mixture of 3 ml Ficoll-Paque (Pharmacia Biotech, Inc., Piscataway, NJ) and then centrifuged at 400 x g for 20 min at 20°C. The completely dispersed LEC at the interface were collected, washed twice, resuspended in 10 ml RPMI-1640 containing 20% FBS, counted by hemacytometry, and plated in 24-well culture plates at a density of 2.5–7.5 x 10⁵ cells/well.

Approximately one-fourth of the remaining tissue was minced with scissors and then digested with 0.04% trypsin (Sigma Chemical Co., St. Louis, MO), 0.06% collagenase (Worthington Biochemical Co., Freehold, NJ), and 0.01% DNase-I (Worthington Biochemical Co.) in 25 ml of IHBSS for 50 min at 37°C with gentle shaking at 10-min intervals. Undigested tissue was removed, and the GEC and SC in the remaining suspension were separated by passing the suspension through a sterile 38-µm stainless steel sieve. Stromal cells passed through the sieve, whereas GEC retained on top of the sieve were back-washed from the sieve with IHBSS, pelleted by centrifugation at 200 x g for 7 min, and washed twice with IHBSS before being centrifuged at 250 x g for 5 min. GEC, along with some intact and partially intact glands, were plated in 24-well culture plates at a density of 0.25–1.0 x 10⁶ cells/well (estimated by hemacytometry) in 1 ml RPMI-1640 containing 10% FBS. The SC were pelleted by centrifugation at 200 x g, washed twice with IHBSS, centrifuged at 300 x g, and plated in 24-well culture plates at a density of 0.25–1.0 x 10⁶ cells/well in 1 ml RPMI-1640 containing 10% FBS. All three cell types were incubated at 37°C in a humidified atmosphere of 95% air:5% CO₂. Cellular viability, determined by trypan blue dye exclusion, was approximately 93%, 86%, and 97% for the enriched populations of LEC, GEC, and SC, respectively. Cells were cultured for at least 60 h to allow them to adhere to the plates before experiments were initiated when cells were estimated to be 85–90% confluent.

Experiment 1

For determination of PI hydrolysis in response to OT and LiCl, SC, GEC, and LEC from 6 gilts were cultured for 60–72 h after initial plating. The cultured cells were then washed twice with RPMI-1640 and incubated for 24 h with 5 µCi myo-[2-³H]inositol (sp. act. 20 Ci/mmol; Amersham, Arlington Heights, IL) in RPMI-1640 supplemented with 5% FBS. After 24 h, cells were washed twice with RPMI-1640 containing 10 µM inositol and incubated with 0, 1, 10, 50, 75, or 100 mM LiCl for 10 min before treatment with 0 or 100 nM OT in a 6 x 2 factorial arrangement. Treatment with LiCl was to inhibit degradation of inositol phosphates by Li⁺-sensitive inositol phosphatases, thereby allowing the accumulation of detectable amounts of [³H]inositol phosphates. After 30 min, cell lysates were collected for determination of PI hydrolysis as subsequently described. Potential effects of LiCl and OT on cell growth were monitored in duplicate wells of cultured cells from 3 additional gilts by quantifying total cellular protein [23] as subsequently described for experiment 2.

Experiment 2

To determine the time course for PGF₂ secretion in response to OT, SC, GEC and LEC from 7 gilts were cultured for 60–72 h after initial plating and washed, and medium was replaced with RPMI-1640 containing 5% FBS. Cells were then cultured an additional 24 h before treatment with 0 or 100 nM OT in RPMI-1640 for 0, 10, 30, 60, 180, 360, or 720 min in a 7 x 2 factorial arrangement. At the end of each treatment period, media were collected and stored at -20°C until PGF₂ concentrations were quantified by RIA as subsequently described. Cells were then lysed with 0.1 N NaOH, and total cellular protein was measured [23].

Experiment 3

To determine the time course for PI hydrolysis in response to OT, SC, GEC, and LEC from 5 gilts were cultured for 60–72 h after initial plating, washed, labeled with [³H]inositol, washed again, and incubated with 50 mM LiCl for 10 min as described for experiment 1. The concentration of LiCl was based on results from experiment 1, which demonstrated that 50 mM LiCl resulted in maximal accumulation of [³H]inositol phosphates (IP) (see Fig. 1). Cells were then treated with 0 or 100 nM OT in a 2 x 4 factorial arrangement for 0, 15, 30, or 60 min before collection of cell lysates for determination of PI hydrolysis as subsequently described.

View larger version (23K): [in this window] [in a new window]   FIG. 1. Effect of 100 nM OT on PI hydrolysis, as determined by accumulation of [3H]inositol-labeled IP, in endometrial cells from Day 16 cyclic gilts in experiment 1. In SC (A), the OT-x-LiCl interaction (p < 0.001) indicated that OT stimulated IP accumulation only in the presence of 1–100 mM LiCl. In GEC (B), the OT-x-LiCl interaction (p < 0.01) indicated that OT stimulated IP accumulation only in the presence of 50–100 mM LiCl. In LEC (C), accumulation of IP was increased by LiCl (p < 0.001) but was not stimulated by OT.

Experiment 4

To study the concentration-dependent response to OT for PI hydrolysis and PGF₂ secretion, SC, GEC, and LEC from the same 5 gilts used in experiment 3 were cultured and prepared as previously described. For PI hydrolysis, cells were labeled with [³H]inositol, washed, and incubated with 50 mM LiCl as previously described before treatment with 0, 3.3, 10, 33, 100, or 333 nM OT for 30 min. Cell lysates were collected for determination of PI hydrolysis as subsequently described.

For PGF₂ secretion, cells were treated with 0, 3.3, 10, 33, 100, or 333 nM OT, and media were collected after 3 h for quantification of PGF₂ concentration by RIA as subsequently described. The duration of treatment with OT was based on results from experiment 2, which indicated that 3 h was the earliest period at which OT-stimulated increases in PGF₂ secretion were detected for both SC and GEC (see Fig. 2, A and B). Cells were then lysed with 0.1 N NaOH, and total cellular protein was measured [23].

View larger version (21K): [in this window] [in a new window]   FIG. 2. Effect of 100 nM OT on PGF secretion from endometrial cells from Day 16 cyclic gilts in experiment 2. For SC (A), the OT-x-time interaction (p < 0.001) indicated that PGF secretion in response to OT was greatest at 180, 360, and 720 min. For GEC (B), the OT-x-time interaction (p < 0.01) indicated that PGF secretion in response to OT was greatest at 180, 360, and 720 min. For LEC (C), PGF secretion increased over time (p < 0.001) but was not increased by OT or influenced by the OT-x-time interaction. Note that the scales for GEC and LEC are 10-fold greater than for SC.

Determination of Phosphoinositide Hydrolysis

Hydrolysis of PI in cell cultures was determined for experiments 1, 3, and 4 as previously described [7, 14, 18, 21]. Briefly, medium was removed at the end of the respective treatment period, 1 ml ice-cold 15% trichloroacetic acid (TCA) 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 IP (i.e., PI hydrolysis) by anion exchange chromatography as previously described [7, 14, 18, 21]. Data were expressed as [³H]IP (dpm/well).

RIA of PGF₂

Concentrations of PGF₂ in 25-µl cell culture medium from experiments 2 and 4 were quantified by RIA as previously described [7, 14, 18, 21]. Intra- and interassay coefficients of variation were 6.4% and 19.5%, respectively. Values were standardized to cellular protein content and expressed as PGF (pg/µg protein) because of high cross-reactivity with PGF₁ [24].

Immunofluorescent Staining for Cytokeratin

At the time of initial plating, cells of each type obtained from four gilts in experiments 1 and 2 were also plated onto SuperCell culture slides (Fisher Scientific, Pittsburgh, PA) and cultured as previously described. Cells were then fixed with ice-cold acetone and stored at -20°C until immunofluorescent staining for cytokeratin was performed. Before staining, the slides were rinsed twice with distilled water and submerged in PBS. Guinea pig anti-cytokeratin (Sigma) was diluted 1:20 in PBS, and 200 µl were placed on slides for 60 min at 37°C in a humidified chamber. Negative control staining was performed using nonimmune guinea pig serum. Sera were then removed, and slides were rinsed with PBS. Fluorescein-isothiocyanate-conjugated rabbit anti-guinea pig immunoglobulin G was applied at dilution of 1:32 for 60 min at 37°C, and slides were rinsed with PBS. Slides were counterstained with Evans blue dye for visualization by light microscopy. Four microscopic fields were examined for each slide using a Zeiss Universal microscope (Carl Zeiss, Inc., Thornwood, NY) with epifluorescent illumination, and the proportions of positively stained (i.e., epithelial) and negatively stained cells (i.e., SC) were quantified [22]. Using this procedure, purity of SC, LEC and GEC was determined to be 94.8 ± 0.8%, 96.6 ± 1.2%, and 90.3 ± 1.9%, respectively.

Statistical Analyses

Data for each cell type were subjected to least-squares analysis of variance (ANOVA) or heterogeneity of regression for randomized block designs using the General Linear Models (GLM) procedure of the Statistical Analysis System [25]. For PI hydrolysis and total cellular protein content in experiment 1, the main effects of LiCl and OT were cross-classified, and pig was the block effect. Data for PI hydrolysis in LEC were also analyzed for heterogeneity of regression when an inexplicable LiCl x OT interaction was detected by ANOVA. For PGF secretion and total cellular protein content in experiment 2 and PI hydrolysis in experiment 3, the main effects of OT and time were cross-classified and pig was the block effect. For PI hydrolysis and PGF secretion in experiment 4, the main effect was concentration of OT and pig was the block effect. All tests of hypotheses were performed using the appropriate error terms according to the expectation of the mean squares [26]. Least-squares means and the appropriate standard errors were generated from the ANOVA using the Least Squares Means statement of the GLM procedure.

	RESULTS

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Experiment 1

Accumulation of [³H]IP was affected (p < 0.001) by LiCl in a concentration-dependent manner due to inhibition of degradation of IP. One millimolar LiCl did not significantly increase accumulation of IP in any cell type, whereas 10–100 mM LiCl increased accumulation of IP in all three cell types (Fig. 1). Maximal accumulation of IP occurred at 50–100 mM LiCl and did not differ significantly among the 50- to 100-mM concentrations for all cell types. Treatment with 100 nM OT stimulated (p < 0.001) PI hydrolysis in SC; however, the OT-x-LiCl interaction (p < 0.001) indicated that OT increased PI hydrolysis in the presence of 1–100 mM LiCl an average of 90% but not in the absence of LiCl (Fig. 1A). The response of GEC to 100 nM OT (p < 0.001) was intermediate to that of SC and LEC, and the OT-x-LiCl interaction (p < 0.001) indicated that OT stimulated PI hydrolysis an average of 20% in the presence of 50–100 mM LiCl (Fig. 1B). Across all concentrations of LiCl, OT did not significantly stimulate PI hydrolysis in LEC (Fig. 1C); however, the OT-x-LiCl interaction (p = 0.05) indicated that OT increased PI hydrolysis in the presence of 1 and 75 mM LiCl, but not in the presence of 0, 10, 50, or 100 mM LiCl. However, analysis for heterogeneity of regression indicated that the regression lines for 0 and 100 nM OT did not differ significantly (p = 0.68). Collectively, this indicated that OT did not stimulate PI hydrolysis in LEC. Total cellular protein content was not significantly influenced by OT, LiCl, or the OT-x-LiCl interaction for any cell type (data not shown).

Experiment 2

There was a time-dependent increase (p < 0.001) in PGF secretion from all three cell types, and the pattern of PGF secretion generally was similar to the pattern of PI hydrolysis seen for experiment 1: the response to 100 nM OT was again greatest for SC (Fig. 2A), intermediate for GEC (Fig. 2B) and least for LEC (Fig. 2C). For SC, 100 nM OT increased (p < 0.001) PGF secretion across all times and the OT-x-time interaction (p < 0.001) indicated that the response to OT was greatest at 180, 360, and 720 min, although the magnitude of the response at 10 and 30 min was proportionally similar to that at 180–720 min. The average OT-stimulated increase for SC was 100% at 10–30 min and was 120% at 180–720 min. Similarly, GEC also responded to 100 nM OT with increased PGF (p < 0.001) secretion, and the OT-x-time interaction (p < 0.001) indicated that the response was greatest 180–720 min after OT treatment. The average OT-stimulated increase for GEC at 180–720 min was 80%. For LEC, however, 100 nM OT did not stimulate PGF secretion, and the lack of an OT-x-time interaction indicated that the response was similar at all times. Basal PGF secretion was approximately 10-fold greater for GEC and LEC than for SC at 180–720 min.

Total cellular protein content for all three cell types was not affected by OT or the OT-x-time interaction, indicating that, at each time point, cellular protein content was similar for cells treated with 0 or 100 nM OT. Total cellular protein content increased (p < 0.01) over time in all three cell types regardless of treatment (results not shown). In SC, protein content increased only 5% between 0 and 360 min, but increased an additional 64% between 360 and 720 min. In GEC, protein content increased only 8% between 0 and 180 min but was increased 21% at 360 min and 71% at 720 min. In LEC, protein content increased only 14% between 0 and 360 min but then increased an additional 58% between 360 and 720 min.

Experiment 3

Hydrolysis of PI increased (p < 0.001) in a time-dependent manner for all three cell types (Fig. 3). OT increased PI hydrolysis in SC (p < 0.01) and in GEC (p < 0.05), but the OT-x-time interactions (p < 0.05) indicated that OT increased PI hydrolysis in SC within 30 min (Fig. 3A) and in GEC at 60 min (Fig. 3B). The average OT-stimulated increase for SC at 30–60 min was 60% and for GEC at 60 min was 20%. However, OT did not significantly stimulate PI hydrolysis in LEC during the 60-min treatment period (Fig. 3C).

View larger version (22K): [in this window] [in a new window]   FIG. 3. Effect of 100 nM OT on PI hydrolysis, as determined by accumulation of [3H]inositol-labeled IP, in endometrial cells from Day 16 cyclic gilts in experiment 3. In SC (A), the OT-x-time interaction (p < 0.05) indicated that OT stimulated IP accumulation at 30 and 60 min. In GEC (B), the OT-x-time interaction (p < 0.05) indicated that OT stimulated accumulation of IP only at 60 min. In LEC (C), accumulation of IP was increased over time (p < 0.001) but was not stimulated by OT or influenced by the OT-x-time interaction.

Experiment 4

In SC, OT increased (p < 0.01) PI hydrolysis in a concentration-dependent manner: PI hydrolysis (Fig. 4A) was increased (p < 0.05) by 33–333 nM OT an average of 40%. For GEC and LEC, OT did not significantly stimulate PI hydrolysis at any concentration examined.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Effect of OT concentration on PI hydrolysis (A), as determined by accumulation of [3H]inositol-labeled IP, and PGF secretion (B) by endometrial cells from Day 16 cyclic gilts in experiment 4. In SC, 33–333 nM OT increased (p < 0.05) PI hydrolysis. In GEC and LEC, OT did not significantly stimulate PI hydrolysis at any concentration examined. For SC, 10–333 nM OT increased (p < 0.001) PGF secretion. For GEC, PGF secretion was increased (p < 0.05) by OT only at 100 and 333 nM OT. For LEC, OT did not significantly stimulate PGF secretion at any concentration examined.

Secretion of PGF from SC was increased (p < 0.001) by OT in a concentration-dependent manner: PGF secretion (See Fig. 4B) was increased by 10–333 nM OT an average of 40%. For GEC, PGF release was increased (p < 0.05) an average of 10% but only by 100 and 333 nM OT. For LEC, OT did not significantly stimulate PGF secretion at any concentration examined. Basal PGF secretion from GEC was 50% greater and from LEC was 120% greater than from SC.

	DISCUSSION

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This study is the first to demonstrate that OT stimulates PI hydrolysis and PGF secretion differentially in SC, GEC, and LEC isolated from porcine endometrium. Clearly, SC were most responsive and LEC were least responsive to OT for both PI hydrolysis and PGF secretion. Results from GEC were somewhat less consistent, but the responses of these cells were generally intermediate to those of SC and LEC. These results differ markedly from those using ruminant endometrium, in which responsiveness to OT resided entirely with epithelial cells, and stromal cells were completely unresponsive [27]. Our results are also surprising in that OT receptor expression, as determined by in situ hybridization, appeared to be greater within the epithelium than within the stroma of porcine endometrium [28]. It is not known if GEC and LEC would have been more responsive to OT if grown under polarizing conditions. However, nonpolarized bovine endometrial epithelial cells, grown under conditions similar to those in the present study, were highly responsive to OT [29].

If OT is important for promoting luteolytic release of PGF₂ during late diestrus in pigs, as proposed previously [19, 30], then responsiveness of SC to OT would provide a physiological mechanism for OT-stimulated endocrine secretion of PGF₂ [31], whereby the cells closest to the uterine vasculature (i.e., SC) are most responsive to OT. Although PGF₂ release from SC was less than from GEC and LEC, SC are presumably present in much greater numbers than epithelial cells and may be expected to contribute the greatest amount to endocrine secretion of PGF₂ occurring at the time of luteolysis. However, this speculation cannot be confirmed at the present time because, to our knowledge, a detailed morphometric analysis has not been reported which would provide information on the relative contribution of stromal and epithelial compartments to the composition of pig endometrium.

The present study demonstrated that stimulation of PGF release by OT was associated with stimulation of PI hydrolysis in SC and GEC. In SC, OT stimulated both PI hydrolysis and PGF secretion in all four experiments. Further, OT-stimulated increases in PI hydrolysis in experiment 3 were detectable more rapidly than those for PGF secretion in experiment 2 for both SC and GEC, results that were highly consistent with those previously reported for endometrial explants from Day 16 cyclic gilts [18]. The minimum concentration of OT (i.e., 33 nM) that promoted a detectable increase in PI hydrolysis in SC in experiment 4 also was within one order of magnitude of that which stimulated PGF release (i.e., 10 nM). These results were in close agreement with those obtained for explants of pig endometrium [18]. Finally, OT did not stimulate either PI hydrolysis or PGF release from LEC in any experiment. Collectively, these data are consistent with the concept that the phospholipase C-mediated second messenger pathway is involved in OT-stimulated PGF₂ secretion from pig endometrium [18–21, 30, 32].

As previously indicated, the response of GEC to OT was less pronounced and more variable than that of SC. For example, OT stimulated PI hydrolysis in GEC within 30 min in experiment 1 and PGF secretion by 3 h in experiments 2 and 4. However, OT did not stimulate PI hydrolysis in GEC until 60 min after treatment in experiment 3 and did not stimulate PI hydrolysis at 30 min in experiment 4. The reason for these latter results is not clear, but it may merely be due to the slightly smaller sample size of experiments 3 and 4, which may have reduced the power of detecting stimulation of PI hydrolysis 30 min after OT treatment. Consistently, OT did not stimulate PI hydrolysis in GEC at 30 min in either experiment 3 or 4, both of which utilized endometrium from the same 5 gilts. Moreover, increased PGF secretion at 3 h after OT in experiment 4 is consistent with OT stimulation of PI hydrolysis at 60 min in GEC from the same gilts in experiment 3.

Immunofluorescent staining for cytokeratin showed that there was approximately 10% SC contamination of GEC. Therefore, a portion of the GEC response to OT may be due directly to the contaminating SC. However, the degree of SC contamination cannot directly account for the entire GEC response to OT because PI hydrolysis and PGF secretion by GEC were increased by more than 10% of the SC response to OT. In contrast, Asselin et al. [27] mathematically demonstrated that contamination by epithelial cells was accountable for the entire response to OT in cultures of bovine SC. There are at least two possible explanations for the magnitude of the response to OT displayed by porcine GEC. First, the response of contaminating SC to OT may have been potentiated by the presence of epithelial cells compared with highly enriched cultures of SC. This explanation is consistent with the fact that epithelial and mesenchymal cells alter the functions of each other in various tissues [33]. A similar interaction between the two cell types also exists in the uterus [34, 35]. Second, GEC may merely have a response to OT which is less than that of SC but greater than that of LEC. In support of this latter possibility, preliminary results from our laboratory indicate that mobilization of intracellular Ca²⁺ in response to OT from epithelial-like cells present in cultures of SC, which probably were GEC, was less than that of stromal-like cells (unpublished results).

It has been previously reported that pig endometrium secretes copious quantities of OT [36] and that epithelial cells produce greater amounts of OT transcripts than do SC [37]. Further, epithelial cells also have a greater abundance of OT receptor transcripts than do SC [28]. Those results may help to explain the present findings that basal PI hydrolysis and PGF production were generally greater for both GEC and LEC than for SC, whereas SC displayed the greatest response to OT. If OT is also produced in high quantities in cultured epithelial cells, then OT acting in an autocrine manner may have elevated basal PI hydrolysis and PGF secretion, thereby obscuring any response to exogenous OT. In contrast, SC express little, if any, OT [37] and may continuously maintain a high degree of responsiveness to exogenous OT in vitro.

As previously indicated, Zhang et al. [22] reported that GEC and SC from Day 13 cyclic sows secreted similar amounts of PGF₂, whereas in the present study, basal PGF secretion from GEC was approximately 10–11 times greater than that from SC after 3–12 h of incubation in experiment 2 and approximately 50% greater after 3 h of incubation in experiment 4. The reason for the difference is not clear, but it may be attributable to any one of several differences in experimental conditions between these two studies. For example, Zhang et al. [22] incubated cells for 24 h (versus 12 h in the present study) before collecting media for determination of PGF₂ secretion. Their culture medium also contained 5% charcoal-stripped FBS compared with our use of nonstripped FBS during initial culture and no FBS during the treatment period. Finally, they obtained cells from cyclic sows on Day 13 compared with Day 16 cyclic gilts in the present study. Any of these factors could have contributed the discrepancy between the current study and that of Zhang et al. [22]. Bovine endometrial epithelial cells also produced substantially more PGF₂ than did SC when expressed on a per cell basis [27, 29, 38, 39]. However, cells in the latter studies were obtained from estrous and metestrous cows and cultured to confluence for 6–7 days in the absence of ovarian steroids before quantification of PGF₂ secretion.

In conclusion, these results generally support the concept that PI-derived second messengers are involved in PGF₂ release from pig endometrium. Additionally, these results indicate that OT differentially regulates PI hydrolysis and PGF₂ secretion within the different cell types of the endometrium. This differential regulation may play a role in regulation of endocrine PGF₂ secretion from the uterine endometrium during the luteolytic period in swine.

	ACKNOWLEDGMENTS

 
The authors are indebted to Dr. Duane L. Davis, Kansas State University, for advice on isolation and culture of endometrial cells and to Drs. Fuller W. Bazer, Texas A&; University, and William W. Thatcher, University of Florida, for supplying the antisera to PGF₂. The authors also are grateful to Mr. W.C. Becker for expert technical assistance 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

 
¹ This work was supported by NIH Grant HD30268.

² Correspondence. FAX: 509 335 1074; mirando{at}wsu.edu

³ Current address: Department of Biological and Medical Research, MBC-03 King Faisal Specialist Hospital and Research Center, P.O. Box: 3354, Riyadh, 11211, Saudi Arabia.

⁴ Current address: Department of Animal Health and Biomedical Sciences, 1655 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706–1581.

Accepted: July 10, 1998.

Received: July 9, 1997.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Melampy RM, Anderson LL. Role of uterus in corpus luteum function. J Anim Sci 1968; 27(suppl 1):77–96.
2. McCracken JA, Schramm W, Okulicz WC. Hormone receptor control of pulsatile secretion of PGF₂ from the ovine uterus during luteolysis and its abrogation in early pregnancy. Anim Reprod Sci 1984; 7:31–55.
3. Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson LJ. Hormonal regulation of uterine secretion of prostaglandin F₂ during luteolysis in ruminants. Biol Reprod 1991; 45:655–663.[Abstract]
4. Schramm W, Bovaird L, Glew ME, Schramm G, McCracken JA. Corpus luteum regression induced by ultra-low pulses of prostaglandin F₂. Prostaglandins 1983; 26:347–364.[CrossRef][Medline]
5. Zarco L, Stabenfeldt GH, Basu S, Bradford GE, Kindahl H. Modification of prostaglandin F₂ synthesis and release in the ewe during the initial establishment of pregnancy. J Reprod Fertil 1988; 83:527–536.[Abstract]
6. Hooper SB, Watkins WB, Thorburn GD. Oxytocin, oxytocin-associated neurophysin, and prostaglandin F₂ concentrations in the utero-ovarian vein of pregnant and nonpregnant sheep. Endocrinology 1986; 119:2590–2597.[Abstract]
7. Mirando MA, Becker WC, Whiteaker SS. Relationships among endometrial oxytocin receptors, oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F₂ secretion in vitro, and plasma concentrations of ovarian steroids before and during corpus luteum regression in cyclic heifers. Biol Reprod 1993; 48:874–882.[Abstract]
8. Silvia WJ, Lee J-S, Trammell DS, Hayes SH, Lowberger LL, Brockman JA. Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F₂ in response to oxytocin: role of phospholipase C and diacylglycerol. J Endocrinol 1994; 141:481–490.[Abstract]
9. Graf GA, Burns PD, Hayes SH, Silvia WJ. Oxytocin receptor coupling to phospholipase C regulates the timing of oxytocin-induced uterine PGF₂ secretion during the ovine estrous cycle. Biol Reprod 1996; 54(suppl 1):192 (abstract).
10. Lee J-S, Silvia WJ. Cellular mechanisms mediating the stimulation of ovine endometrial secretion of prostaglandin F₂ in response to oxytocin: role of phospholipase A₂. J Endocrinol 1994; 141:491–496.[Abstract]
11. Gleeson AR, Thorburn GD, Cox RI. Prostaglandin F concentrations in the utero-ovarian venous plasma of the sow during the late luteal phase of the oestrous cycle. Prostaglandins 1974; 5:521–529.[CrossRef][Medline]
12. Moeljono MPE, Thatcher WW, Bazer FW, Frank M, Owens LJ, Wilcox CJ. A study of prostaglandin F₂ as the luteolysin in swine: II. Characterization and comparison of prostaglandin F₂ estrogens and progestins concentrations in utero-ovarian vein plasma of nonpregnant and pregnant gilts. Prostaglandins 1977; 14:543–555.[CrossRef][Medline]
13. Rampacek GB, Kraeling RR, Kiser TE, Barb CR, Benyshek LL. Prostaglandin F concentrations in utero-ovarian vein plasma of prepuberal and mature gilts. Prostaglandins 1979; 18:247–255.[CrossRef][Medline]
14. Whiteaker SS, Mirando MA, Becker WC, Hostetler CE. Detection of functional oxytocin receptors on endometrium of pigs. Biol Reprod 1994; 51:92–98.[Abstract]
15. Kieborz KR, Silvia WJ, Edgerton LA. Changes in uterine secretion of prostaglandin F₂ and luteal secretion of progesterone in response to oxytocin during the porcine estrous cycle. Biol Reprod 1991; 45:950–954.[Abstract]
16. Carnahan KG, Prince BC, Mirando MA. Exogenous oxytocin stimulates uterine secretion of prostaglandin F₂ in cyclic and early pregnant swine. Biol Reprod 1996; 55:838–843.[Abstract]
17. Prince BC, Mirando MA, Becker WC, Hostetler CE. Exogenous oxytocin decreases interestrous interval of cyclic gilts. J Anim Sci 1995; 73:3681–3686.[Abstract]
18. Whiteaker SS, Mirando MA, Becker WC, Peters DN. Relationship between phosphoinositide hydrolysis and prostaglandin F₂ secretion in vitro from endometrium of cyclic pigs on day 15 postestrus. Domest Anim Endocrinol 1995; 12:95–104.[CrossRef][Medline]
19. Mirando MA, Uzumcu M, Carnahan KG, Ludwig TE. A role of oxytocin during luteolysis and early pregnancy in swine. Reprod Domest Anim 1996; 31:455–461.
20. Tysseling KA, Uzumcu M, Hoagland TA, Crain RC, Mirando MA. The role of phosphoinositide-derived second messengers in oxytocin-stimulated prostaglandin F₂ release from endometrium of pigs. Domest Anim Endocrinol 1996; 13:411–420.[CrossRef][Medline]
21. Ludwig TE, Sun B-C, Carnahan KG, Uzumcu M, Yelich JV, Geisert RD, Mirando MA. Endometrial responsiveness to oxytocin during diestrus and early pregnancy in pigs is not controlled solely by changes in oxytocin receptor population density. Biol Reprod 1998; 58:769–777.[Abstract/Free Full Text]
22. Zhang Z, Paria BC, Davis DL. Pig endometrial cells in primary culture: morphology, secretion of prostaglandins and proteins, and effects of pregnancy. J Anim Sci 1991; 69:3005–3015.[Abstract]
23. Lowry OH, Rosebrough NJ, Farr AL. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193:265–275.[Free Full Text]
24. Dubois DH, Bazer FW. Effect of porcine conceptus secretory proteins on in vitro secretion of prostaglandin F₂ and E₂ from luminal and myometrial surfaces of endometrium from cyclic and pseudopregnant gilts. Prostaglandins 1991; 41:283–301.[CrossRef][Medline]
25. SAS. PC-SAS, Propriety Software Release 6.11 System for Windows. Cary, NC: Statistical Analysis System Institute, Inc.; 1995.
26. Snedecor GW, Cochran WG. Factorial experiments. In: Statistical Methods, 7th ed. Ames, IA: Iowa State University Press; 1980: 298–333.
27. Asselin E, Goff AK, Bergeron H, Fortier MA. Influence of sex steroids on the production of prostaglandins F₂ and E₂ and response to oxytocin in cultured epithelial and stromal cells of bovine endometrium. Biol Reprod 1996; 54:371–379.[Abstract]
28. Boulton MI, McGrath TJ, Gilbert CL. Oxytocin receptor mRNA expression in the porcine uterus during the oestrous cycle, and pregnancy. J Reprod Fertil 1995; abstract series 16:21.
29. Kim JJ, Fortier MA. Cell type specificity and protein kinase C dependency on the stimulation of prostaglandin E₂ and prostaglandin F₂ production by oxytocin and platelet-activating factor in bovine endometrial cells. J Reprod Fertil 1995; 103:239–247.[Abstract]
30. Mirando MA, Prince BC, Tysseling KA, Carnahan KG, Ludwig TE, Hoagland TA, Crain RC. A proposed role for oxytocin in regulation of endometrial prostaglandin F₂ secretion during luteolysis in swine. Adv Exp Med Biol 1995; 413:421–433.
31. Bazer FW, Thatcher WW. Theory of maternal recognition of pregnancy in swine based on estrogen controlled endocrine versus exocrine secretion of prostaglandin F₂ by the uterine endometrium. Prostaglandins 1977; 14:397–401.[CrossRef][Medline]
32. Carnahan KG, Prince BC, Ludwig TE, Uzumcu M, Mirando MA. Does oxytocin up-regulate its own receptor in endometrium of pigs? Proc West Sect Am Soc Anim Sci 1996; 47:19–22.
33. Skinner MK, Parrot JA. Growth factor-mediated cell-cell interactions in the ovary. In: Findlay JK (ed.), Molecular Biology of the Female Reproductive System. New York: Academic Press; 1994: 67–81.
34. Giudice LC. Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine. Fertil Steril 1994; 61:1–17.[Medline]
35. Everett LM, Caperell-Grant A, Bigsby RM. Mesenchymal-epithelial interactions in an in vitro model of neonatal mouse uterus. Proc Soc Exp Biol Med 1997; 214:49–53.[Abstract]
36. Trout WE, Smith GW, Gentry PC, Galvin JM, Keisler DH. Oxytocin secretion by the endometrium of the pig during maternal recognition of pregnancy. Biol Reprod 1995; 52(suppl 1):189 (abstract).
37. Boulton MI, McGrath TJ, Goode JA, Broad KD, Gilbert CL. Changes in content of mRNA encoding oxytocin in pig uterus during the oestrous cycle, pregnancy, at parturition and in lactational anoestrus. J Reprod Fertil 1996; 108:219–227.[Abstract]
38. Asselin E, Bazer FW, Fortier MA. Recombinant ovine and bovine interferons tau regulate prostaglandin production and oxytocin response in cultured bovine endometrial cells. Biol Reprod 1997; 56:402–408.[Abstract]
39. Fortier MA, Guilbault LA, Grasso F. Specific properties of epithelial and stromal cells from the endometrium of cows. J Reprod Fertil 1988; 83:239–248.[Abstract]