Oxytocin stimulates secretion of prostaglandin F2α from endometrial cells of swine in the presence of progesterone

Domestic Animal Endocrinology 23 (2002) 435–445

Oxytocin stimulates secretion of prostaglandin F2␣ from endometrial cells of swine in the presence of progesterone K.G. Carnahan1 , M. Uzumcu2 , J. Hu3 , G.L. Sample4 , G.T. Braileanu5 , M.A. Mirando∗ Department of Animal Sciences, Center for Reproductive Biology, Washington State University, Pullman, WA 99164-6353, USA Received 20 December 2001; accepted 5 June 2002

Abstract Oxytocin (OT) stimulates endometrial secretion of prostaglandin (PG) F2␣ during corpus luteum regression in swine but there is differential responsiveness to OT among endometrial cell types. To determine if progesterone influenced responsiveness of luminal epithelial, glandular epithelial, and stromal cells to 100 nM OT during luteolysis in swine, cells were isolated from endometrium of 15 gilts by differential enzymatic digestion and sieve filtration on day 16 postestrus and cultured continuously in the presence of 0, 10 or 100 nM progesterone. For phospholipase C (PLC) activity and PGF2␣ secretion, stromal cells were most responsive to OT (P < 0.01) in the absence of progesterone, whereas luminal epithelial cells were unresponsive and glandular epithelial cells displayed an intermediate response to

∗

Corresponding author. Present address: National Research Initiative Grants Program, 1400 Independence Avenue, S.W. Stop 2241, U.S. Department of Agriculture, Washington, DC 20250-2241, USA. Tel.: +1-202-401-4336; fax: +1-202-205-3641. E-mail address: [email protected] (M.A. Mirando). 1 Present address: Department of Animal and Veterinary Science, University of Idaho, Moscow, ID 83844-2330, USA. 2 Present address: School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234, USA. 3 Present address: Department of Animal Science, Texas A&M University, College Station, TX 77843-2471, USA. 4 Present address: College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7010, USA. 5 Present address: Department of Pediatrics, School of Medicine, University of Maryland, Baltimore, MD 21201, USA. 0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 2 ) 0 0 1 7 6 - 5

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OT (P < 0.09). Progesterone enhanced PLC activity linearly in glandular epithelial cells (P < 0.05) and influenced it quadratically in stromal cells (P = 0.05). The effect of OT and progesterone on PLC activity in luminal epithelial cells was not significant, and progesterone did not increase PLC activity in response to OT in any cell type. Culture in the presence of progesterone, enhanced PGF2␣ secretion in response to OT in luminal epithelial cells (P < 0.05) but not in glandular epithelial or stromal cells. Progesterone also increased overall PGF2␣ release from glandular epithelial (P < 0.05) and stromal cells (P < 0.06) across both levels of OT treatment. These results indicate that progesterone enhanced PGF2␣ secretion from luminal epithelial cells in response to OT and increased basal PGF2␣ release from glandular epithelial and stromal cells. © 2002 Elsevier Science Inc. All rights reserved.

1. Introduction Regulation of the estrous cycle is a uterine-dependent event in domestic ungulates [1,2]. Prostaglandin (PG) F2␣ is the luteolysin that is secreted from the uterine endometrium and induces corpus luteum (CL) regression [3–5]. Lysis of the CL and the concomitant decline in circulating progesterone at the end of the estrous cycle permits further follicular development leading to estrus, ovulation and the next opportunity for mating and pregnancy to occur. During early to mid-diestrus, progesterone prevents premature luteolysis by blocking endometrial formation of OT receptors [6], an event referred to as the “progesterone block” [7]. Subsequently, the endometrium spontaneously emerges from this progesterone block, which allows up-regulation of OT receptors [6,8,9] essential for the initiation of endometrial production of luteolytic PGF2␣ [8]. How the endometrium emerges from the progesterone block during late diestrus is unclear, but does not appear to be due to down-regulation of progesterone receptors [10]. In addition to preventing luteolysis during early to mid-diestrus, progesterone also promotes development of the luteolytic mechanism. Progesterone stimulates cells in the endometrium of ewes to increase lipid stores [11] that presumably contain arachidonic acid, the precursor for PGF2␣ synthesis [12], and induces cyclooxygenase expression [13–15] necessary for conversion of arachidonic acid to PGF2␣ . The increase in cyclooxygenase expression after exposure of ovariectomized ewes to progesterone for 10 to 12 days [14,15] is similar to that which occurs just prior to luteolysis [13,15]. Progesterone also may make the uterus more responsive to OT without up-regulating OT receptor expression [16,17]. Thus, progesterone promotes luteolysis in ruminants [18,19] through a uterine-dependent mechanism [19,20] that makes the uterus competent to secrete PGF2␣ [21]. In cultured porcine endometrial cells, stromal cells are most responsive to OT, luminal epithelial cells are least responsive, and glandular epithelial cells exhibit an intermediate response [22–25]. This pattern of responsiveness to OT among endometrial cells is consistent with OT-stimulated endocrine secretion of PGF2␣ during luteolysis [26,27] because the cells nearest the uterine vasculature (i.e., stromal and glandular epithelial cells) are most responsive to OT and those furthest away (i.e., luminal epithelial cells) are least responsive [22–25]. However, endometrial cells from pregnant and estradiol-induced pseudopregnant gilts also display a response to OT that is only moderately different to those of cyclic gilts [28]. Although

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luminal epithelial cells of pregnant gilts exhibit an increased response to OT on day 12, the response of stromal cells from pregnant gilts to OT on day 16 postestrus is only partially reduced [28], which seems to be somewhat contradictory to the results of several studies in which the endometrium of pregnant and pseudopregnant pigs was markedly less responsive to OT than that of cyclic pigs [26,27,29]. It was hypothesized that progesterone promotes PGF2␣ secretion in porcine endometrium and that the absence of progesterone during culture of endometrial cells for 72–96 h resulted in reduced responsiveness to OT of cells from cyclic gilts that was only moderately greater than the relatively small response of cells from pregnant and pseudopregnant gilts [28]. Therefore, the objective of this study was to determine if the presence of progesterone in culture medium enhanced the response of endometrial cells from cyclic gilts to OT. 2. Materials and methods 2.1. Animals Peripubertal crossbred gilts (Yorkshire, Landrace, Large White, Duroc and Hampshire) were observed daily at 7:00–9:00 h for standing estrous behavior in the presence of an intact boar. Fifteen gilts were hysterectomized 16 days after onset of second or third estrus, as described previously [30,31]. Endometrium was collected aseptically from one randomly-selected uterine horn and placed in Dulbecco’s PBS (2.68 mM KCl, 1.47 mM KH2 PO4 , 0.49 mM MgCl2 , 136.9 mM NaCl and 8.1 mM Na2 HPO4 ). 2.2. Separation of endometrial cells for culture Cell populations were separated using a procedure described previously [32] and modified subsequently [22]. Luminal epithelial, glandular epithelial and stromal cells were seeded in 24-well culture plates at a density of 1 × 106 , 0.5 × 106 and 0.5 × 106 cells/well, respectively. Wells of each cell type were cultured in the presence of 0, 10 or 100 nM progesterone at 37◦ C in a humidified atmosphere of 95% air and 5% CO2 . Cellular viability was determined by trypan blue exclusion to be 91, 82 and 95% for the enriched populations of luminal epithelial, glandular epithelial and stromal cells, respectively. 2.3. Removal of endogenous progesterone from FBS The FBS used for culturing endometrial cells during the period of progesterone treatment was stripped with dextran-coated Norit A charcoal to remove endogenous progesterone. Stripping was achieved by adding 1.25 g charcoal and 125 mg dextran to 500 mL FBS and gently stirring the mixture overnight at 4◦ C. Charcoal was removed by centrifugation for 30 min at 8000 × g and the supernatant was treated three more times with charcoal and dextran for 1 h each at 4◦ C. Progesterone concentrations quantified [33] subsequently in several samples of FBS obtained before and after charcoal stripping were below the detectable limits of the assay (i.e., 0.1 ng/mL).

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2.4. Determination of phospholipase C (PLC) activity For determination of PLC activity, luminal epithelial, glandular epithelial and stromal cells were cultured for 72–80 h after initial plating, as described previously [22]. The cultured cells were then washed twice with RPMI 1640 and incubated for 24 h with 5 ␮Ci myo-[2-3 H]inositol (specific activity 20 Ci/mmol; Amersham, Arlington Heights, IL, USA) in RPMI 1640 supplemented with 5% charcoal-stripped FBS and 0, 10 or 100 nM progesterone. After 24 h, cells were washed twice with RPMI 1640 containing 10 ␮M inositol but devoid of progesterone and FBS, and incubated with 100 mM LiCl (to inhibit degradation of inositol phosphates) for 10 min before treatment with 0 or 100 nM OT in a 2 × 3 factorial arrangement for 30 min. Accumulation of [3 H]inositol phosphates (i.e., PLC activity) then was determined as described previously [22,30]. Because DNA content could not be quantified in cells used for PLC activity, data were expressed as [3 H]inositol phosphates (dpm) per well. 2.5. Determination of PGF2␣ secretion For determination of PGF2␣ secretion, luminal epithelial, glandular epithelial and stromal cells were cultured for 72–80 h after initial plating, washed twice with RPMI 1640 and cultured for 24 h in RPMI 1640 supplemented with 5% FBS and 0, 10 or 100 nM progesterone. After 24 h, cells were washed twice with RPMI 1640 devoid of progesterone and FBS, and then treated with 0 or 100 nM OT in a 2 × 3 factorial arrangement for 3 h. At the end of the 3-h treatment period, media was collected and stored at −20◦ C until PGF2␣ concentrations were quantified by RIA as described previously [22,30]. Intra and interassay coefficients of variation were 14.0 and 12.9%, respectively. Culture plates of cells were also stored at −20◦ C until DNA content was quantified as described subsequently and data for PGF2␣ secretion were expressed as picogram/microgram DNA. 2.6. Determination of cellular DNA content Cellular DNA content was determined by the method of Burton [34]. Briefly, each well of cells was treated with 0.3 mL 0.5N perchloric acid, heated to 90◦ C in a drying oven for 30 min and cooled to room temperature. Samples were then treated with diphenylamine reagent and incubated at room temperature in the dark for 14–16 h. After incubation, samples were measured spectrophotometrically (Beckman DU 64, Fullerton, CA, USA) for absorbance at 590 nm. 2.7. Statistical analyses Data for each cell type were subjected to least squares analysis of variance (ANOVA) for a randomized block design using the general linear models (GLM) procedure of SAS [35]. The statistical model included main effects of progesterone concentration and OT treatment, pig as the block effect, all appropriate interactions and residual error. All tests of hypotheses were performed using the appropriate error term according to the expectation of the mean squares [36]. Least squares means and standard errors were generated from the ANOVA using the least squares means statement of the GLM procedure.

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3. Results 3.1. PLC activity Progesterone did not alter PLC activity in luminal epithelial cells (Fig. 1). Additionally, 100 nM OT did not stimulate PLC activity in luminal epithelial cells and the response to OT was not influenced by progesterone treatment. The response of glandular epithelial cells (Fig. 1) to 100 nM OT (P < 0.09) was intermediate to that of stromal cells and luminal epithelial cells. Progesterone did not affect the response to OT, but progesterone increased PLC activity linearly (P < 0.05) in glandular epithelial cells. Treatment with 100 nM OT

Fig. 1. Effect of 100 nM oxytocin (OT) on PLC activity, as determined by accumulation of [3 H]inositol labeled inositol phosphates, in luminal epithelial (LEC, top panel), glandular epithelial (GEC, center panel) and stromal cells (SC, bottom panel) in the presence of 0, 10 or 100 nM progesterone. OT increased (P < 0.09) PLC activity in glandular epithelial cells. Activity of PLC was also increased (P < 0.01) by OT in stromal cells. OT did not increase PLC activity in luminal epithelial cells. Progesterone increased PLC activity linearly (P < 0.05) in glandular epithelial cells, and quadratically in stromal cells (P = 0.05), but did not alter it in luminal epithelial cells.

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stimulated (P < 0.01) PLC activity in stromal cells across all progesterone concentrations (Fig. 1). When stromal cells were incubated in the presence of 10 nM progesterone, PLC activity was decreased (P < 0.05) across both concentrations of OT compared to treatment with 0 or 100 nM progesterone. 3.2. PGF2␣ secretion Progesterone enhanced (P < 0.05) the PGF2␣ secretory response to OT in luminal epithelial cells (Fig. 2). In the absence of progesterone, OT did not stimulate PGF2␣ release from luminal

Fig. 2. Effect of 100 nM OT on PGF2␣ secretion from luminal epithelial (LEC, top panel), glandular epithelial (GEC, center panel) and stromal cells (SC, bottom panel) in the presence of 0, 10 or 100 nM progesterone. OT did not stimulate PGF2␣ release from luminal epithelial cells in the absence of progesterone, but the response to OT was enhanced (P < 0.05) in the presence of 10 and 100 nM progesterone. Release of PGF2␣ was increased by OT from glandular epithelial (P < 0.05) and stromal cells (P < 0.01) similarly across all concentrations of progesterone. Secretion of PGF2␣ also was increased by progesterone treatment in glandular epithelial (P < 0.05) and stromal cells (P < 0.06) across both levels of OT.

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epithelial cells, whereas PGF2␣ secretion was stimulated (P < 0.05) by OT in the presence of 10 and 100 nM progesterone. In contrast, OT increased PGF2␣ release from glandular epithelial (P < 0.05, Fig. 2) and stromal cells (P < 0.01, Fig. 2) similarly across all concentrations of progesterone. Secretion of PGF2␣ was increased by progesterone treatment in glandular epithelial (P < 0.05) and stromal cells (P < 0.06) across both levels of OT.

4. Discussion The results of the present study indicated that exposure to progesterone enhanced responsiveness of luminal epithelial cells from porcine endometrium to OT. Such an effect of progesterone in vivo could contribute to the uterine endometrium of cyclic gilts being substantially more responsive to OT than the endometrium of pregnant or pseudopregnant pigs [25,27,29], assuming that the additional PGF2␣ is released in an endocrine direction [37]. In contrast, the response to OT among endometrial cells obtained from cyclic, pregnant and pseudopregnant pigs and cultured in the absence progesterone differed only modestly [28]. The results of the present study are somewhat surprising given that progesterone receptors were undetectable in the luminal surface and superficial glandular epithelium of porcine endometrium after day 12 postestrus [38] and ovine endometrium on days 6–13 postestrus [39]. Moreover, the response of the uterine epithelium to estrogen in mice is mediated through stromal cells [40,41]. How epithelial cells were able to respond to progesterone in the current study is not clear, but several possibilities exist. First, the response of epithelial cells to progesterone may have been mediated through the small amount of stromal cells contaminating the enriched cultures of luminal surface and glandular epithelial cells. However, stromal cells represented less than 4% of the total cells in cultures of luminal epithelium and only 8% of cells in glandular epithelial cultures. Another possibility may be that endometrial epithelial cells in culture regained their progesterone receptors and therefore, became responsive to progesterone. This apparently was the case for polarized luminal epithelial cells isolated from pregnant pigs that were responsive to both estrogen and progesterone [42]. Finally, progesterone receptors may be present in the endometrial epithelium of ungulates at a very low level that still permits epithelial cells to respond to progesterone but is below the sensitivity of the immunocytochemical and in situ hybridization techniques used to assess progesterone receptor status [38,39]. The hypothesis that progesterone would enhance responsiveness of stromal and glandular epithelial cells to OT was not supported by results from the present study. This hypothesis was proposed because endometrium of cyclic gilts developed responsiveness to OT after day 12 [26,27,29], whereas endometrial responsiveness was markedly suppressed by pregnancy [26,27,29] and pseudopregnancy [27]. However, endometrial stromal cells isolated on day 12 of the estrous cycle or on days 12–16 from pregnant and pseudopregnant gilts were almost as responsive to OT as those from cyclic gilts 16 days postestrus [28]. A major difference in the experimental approaches of the studies by Uzumcu et al. [22,28], compared with the present study, was the absence of progesterone for 3 days during culture of endometrial cells. However, the addition of progesterone to the culture medium in the present study generally did not enhance the response of stromal and glandular epithelial cells to OT. One alternative explanation for these observations may be that during pregnancy and pseudopregnancy,

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progesterone may be required to inhibit the development of endometrial responsiveness to OT as occurs for sheep [43], although this would not account for premature development of endometrial responsiveness to OT in endometrial cells from cyclic gilts on day 12 after estrus [28]. Another explanation may be that the lack of cell to cell contact among cell types, in enriched cultures, prevents expression of the normal phenotypes and cells default to a phenotype that is responsive to OT, regardless of reproductive status or stage of the estrous cycle. If both progesterone and OT are important for the luteolytic release of PGF2␣ during late diestrus in pigs [44,45], as demonstrated in sheep [16,17,46,47], then certain endometrial cell populations may be involved in the physiological mechanism for OT-stimulated secretion of PGF2␣ [37]. In this study, PGF2␣ secretion was highest from luminal epithelial cells, followed by glandular epithelial cells, with stromal cells secreting much lower amounts of PGF2␣ than glandular and luminal epithelial cells. This study indicated that all cells might be contributing to the endocrine secretion of PGF2␣ at the time of luteolysis. Although, PGF2␣ release was less from glandular epithelial and stromal cells, compared to luminal epithelial cells, morphometric analysis of pig endometrium indicates that glandular epithelial and stromal cells are present in a much greater number than are luminal epithelial cells [48]. Therefore, glandular epithelial and stromal cells probably contribute the greatest overall amount of PGF2␣ at the time of luteolysis. Furthermore, PGF2␣ may be continuously secreted at elevated levels from luminal epithelial cells because of the presence of high quantities of OT within the pig endometrium [49,50]. It has been previously reported that epithelial cells produce greater amounts of OT transcripts and have more receptors for OT than do stromal cells [51,52]. If OT and OT receptors are also produced in high quantities in cultured luminal epithelial cells, then OT may be acting in an autocrine and/or paracrine manner to increase the basal secretion of PGF2␣ in these cells while decreasing the responsiveness to exogenous OT [25]. In contrast, stromal cells may maintain a high degree of responsiveness to exogenous OT in vitro because the response to OT in vitro was actually enhanced after OT treatment of cyclic gilts in vivo [53]. The physiological action of OT in stimulating endometrial PGF2␣ secretion is mediated through PLC [30,54,55], which hydrolyzes cell membrane phosphoinositides to produce inositol 1,4,5-trisphosphate [31] and diacylglycerol (DAG) second messengers [56]. In turn, these second messengers stimulate release of intracellular pools of Ca2+ [23,24] and activate protein kinase C [24], thereby inducing a cascade of events involving phospholipase A2 activation [57] to promote release of PGF2␣ . Results obtained for glandular epithelial and stromal cells are consistent with the evidence indicating that the PLC-mediated second messenger pathway is involved in OT-stimulated PGF2␣ secretion from the endometrium of pigs [28,30,31] and sheep [54,55]. Results for luminal epithelial cells are more difficult to explain. The increase in PGF2␣ secretion due to OT stimulation without a corresponding increase in PLC activity is surprising and the reason for this is unclear. However, OT-stimulated increases in PGF2␣ secretion from pig endometrium were always associated with OT-induced stimulation of PLC activity in numerous previous studies [22,28–31,53]. Moreover, OT-stimulated increases in PLC activation preceded that for PGF2␣ secretion [30] and OT-induced IP3 occurred within 30 s [31], consistent with the concept of rapid activation of the PLC-mediated second messenger system. These results are consistent with previous reports that changes in endometrial responsiveness to OT during diestrus in pigs are completely independent of changes in OT receptor binding activity [29] and are mediated, in part, at cellular sites downstream from PLC

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[28,29]. A similar phenomenon also occurs, to a lesser extent, in the endometrium of ewes [16,17,55]. In summary, these results demonstrate that progesterone is able to modulate secretion of PGF2␣ in all three major endometrial cell types. In the absence of progesterone, luminal epithelial cells were unresponsive to OT and progesterone promoted responsiveness to OT. In glandular epithelial and stromal cells, progesterone stimulated PGF2␣ secretion but did not significantly influence the response to OT. These results are consistent with the concept that the progesterone promotes luteolytic PGF2␣ release from the endometrium of domestic ungulates. Acknowledgments This research was supported by NIH grant HD30268. The authors are grateful to Dr. W.W. Thatcher, University of Florida, Gainesville, FL for supplying antisera to PGF2␣ . Appreciation is also expressed to the staffs of the Washington State University swine center and Experimental Animal Laboratory building for assistance in care and handling of gilts, and to the members of M.A. Mirando’s laboratory for assistance with surgery. References [1] Anderson LL, Butcher RL, Melampy RM. Uterus and occurrence of oestrus in pigs. Nature 1963;198:311–2. [2] Melampy RM, Anderson LL. Role of uterus in corpus luteum function. J Anim Sci 1968;27(Suppl 1):77–96. [3] Silvia WJ, Lewis GS, McCracken JA, Thatcher WW, Wilson L. Hormonal regulation of uterine secretion of prostaglandin F2␣ during luteolysis in ruminants. Biol Reprod 1991;45:655–63. [4] Mirando MA, Uzumcu M, Carnahan KG, Ludwig TE. A role of oxytocin during luteolysis and early pregnancy in swine. Reprod Domest Anim 1996;31:455–61. [5] McCracken JA, Custer EA, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol Rev 1999;79:263–323. [6] Leavitt WW, Okulicz WC, McCracken JA, Schramm W, Robidoux WF. Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal. J Steroid Biochem Mol Biol 1985;22:687–91. [7] McCracken JA, Schramm W, Okulicz WC. Hormone receptor control of pulsatile secretion of PGF2␣ from ovine uterus during luteolysis and its abrogation in early pregnancy. Anim Reprod Sci 1984;7:31–55. [8] Roberts JS, McCracken JA, Gavagan JE, Soloff MS. Oxytocin-stimulated release of prostaglandin F2␣ from ovine endometrium in vitro: correlation with estrous cycle and oxytocin-receptor binding. Endocrinology 1976;99:1107–14. [9] Sheldrick EL, Flint APF. Endocrine control of uterine oxytocin receptors in the ewe. J Endocrinol 1985;106:249–58. [10] Ott TL, Zhou Y, Mirando MA, Stevens C, Harney JP, Ogle TF, Bazer FW. Changes in progesterone and oestrogen receptor mRNA and protein during maternal recognition of pregnancy and luteolysis ewes. J Mol Endocrinol 1993;10:171–83. [11] Brinsfield TH, Hawk HW. Control by progesterone of the concentration of lipid droplets in epithelial cells of the sheep endometrium. J Anim Sci 1973;36:919–22. [12] Leslie CC. Properties and regulation of cytosolic phospholipase A2 . J Biol Chem 1997;272:16709–12. [13] Huslig RL, Fogwell RL, Smith WL. The prostaglandin forming cyclooxygenase of ovine uterus: relationship to luteal function. Biol Reprod 1979;21:589–600. [14] Eggleston DL, Wilken C, Van Kirk EA, Slaughter RG, Ji TH, Murdoch WJ. Progesterone induces expression of endometrial messenger RNA encoding for cyclooxygenase (sheep). Prostaglandins 1990;39:675–83.

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