Ovine endometrial cells fail to lyse K-562 target cells: a preliminary investigation

The ability of unfractionated and fractionated endometrial cells to lyse K-562 target cells was investigated within ewes during the late luteal phase of the estrous cycle (Days 12 to 14) and pregnancy (Days 16 and 19). In separate experiments, lymphokine activated killer (LAK) cells and endometrial cells, both designated as effector cells, were co-cultured with chromium-51 (51Cr)-labeled K-562 target cells in varying effector: target cell ratios. At 22 h, lytic activity was assessed by the release of 51Cr into the culture medium. The LAK cells exhibited lytic activity in a ratio-dependent manner, whereas the unfractionated and fractionated endometrial cells failed to lyse the target cells. For ratios combined, the rate of cytotoxicity for unfractionated endometrial cells recovered from ewes in the cyclic and pregnant (Days 16 and 19 combined) groups was 13.9 and 5.4%, respectively. Although the findings are preliminary, they indicate that ovine endometrial cells recovered during the late luteal phase and early pregnancy failed to exhibit natural killer activity upon K-562 target cells.     

Lymphocyte-mediated lysis of sheep chorion: susceptibility of chorionic cells to third-party and maternal cytotoxic lymphocytes and presence of cells in the endometrium exhibiting cytotoxicity toward natural-killer cell targets

In several species, the trophoblast is resistant to lysis by cytotoxic lymphocytes. Such resistance is believed to contribute to survival of the semiallogenic conceptus. We tested whether ovine chorionic cells are susceptible to lysis by specific and nonspecific cytotoxic lymphocytes in peripheral blood (PBL) and whether cytotoxic cells that can lyse target cells for natural-killer cells are present in the endometrium. Primary chorionic cells from pregnant ewes at Days 51-91 of gestation were labeled with 51Cr and incubated for 20 h at 50:1 and 100:1 ratios with PBL from the pregnant mother or from a third-party ewe. In the absence of interleukin-2 (IL-2), there was no killing of primary chorionic cells by third-party PBL even after infection of chorionic cells with bovine herpes virus-1. Incubation with IL-2-induced cytotoxic action in third-party PBL towards one of six primary chorionic cell preparations only. Primary chorionic cells from two of four placentae were lysed by maternal PBL. Luminal epithelial cells from cyclic ewes and from the pregnant and nonpregnant uterine horns of unilaterally-pregnant ewes were evaluated for the presence of cells capable of killing D17 target cells (a natural-killer cell target). Killing was observed but there was no difference in activity between physiological stages. In contrast, there was intense immunochemical localization of perforin in glandular and luminal endometrial epithelial cells in pregnant ewes, and less intense staining in nonpregnant animals. It is concluded that ovine chorionic cells are generally resistant to killing by natural-killer-like cells and lymphokine-activated killer cells. Generation of maternal cytotoxic lymphocytes against trophoblast can occur in some cases and may contribute to pregnancy loss.     

Natural killer-like cells in the sheep: functional characterization and regulation by pregnancy-associated proteins

Natural killer (NK) cells represent an important component of the innate immune system. In ruminants there are few reports regarding presence or characterization of NK cells. Although absence of expression of major histocompatibility complex proteins on ovine trophoblast makes it potentially a target for NK cells, little is known about regulation of NK cells by products of pregnancy in sheep. Objectives of the present study were to determine whether cells with characteristics of NK cells exist in preparations of ovine peripheral blood lymphocytes (PBL) and endometrial epithelial cells (EEC) and to determine regulation of such cells by two pregnancy-associated molecules with immunoregulatory properties (ovine uterine serpin [OvUS] and interferon-tau [IFN-tau]). Ovine PBL and EEC lysed a putative NK target cell, the BHV-1 infected D17 cell, and lysis by both types of cells was neutralized by antibody against a molecule called function-associated molecule (FAM) expressed on NK cells of several species. Moreover, inhibitors that interfere with perforin-mediated lysis blocked NK-like activity of PBL. The NK-like lytic activity of PBL and EEC was inhibited by OvUS, whereas ovine and bovine IFN-tau significantly enhanced NK-like activity of PBL. In conclusion, NK-like activity present in preparations of ovine PBL and EEC is mediated by FAM(+) cells, is dependent on processes that involve perforin processing, and is regulated by OvUS and IFN-tau. Inhibition of NK-like activity of PBL and EEC by OvUS is consistent with a role for OvUS in protecting the conceptus from maternal cytotoxic lymphocytes. Stimulation of lysis by IFN-tau implies the existence of other inhibitory mechanisms during early pregnancy to prevent NK cell-mediated destruction of the conceptus.     

Correlation between the release of ovine trophoblast protein-1 by the conceptus and the production of polypeptides by the maternal endometrium of ewes

We studied the biosynthesis of two proteins, p70 (Mr 70,000; pI 4.0) and p15 (Mr 15,000; pI 5.7), by endometrial tissues from ewes between Days 12 and 24 of pregnancy and between Days 12 and 16 of the oestrous cycle to determine whether production of the two was correlated with the period of biosynthesis of ovine trophoblast protein-1 (oTP-1) by the conceptus. We also compared the protein synthetic activities of endometrium from gravid and non-gravid horns of pregnant ewes at Days 14, 16 and 18 in which the conceptus had been confined to one uterine horn. Proteins p70 and p15 were produced maximally between Days 14 and 20 of pregnancy, but synthesis by endometrial cultures from cyclic ewes was low or absent. Furthermore, synthesis of Protein p70 in particular was much greater by the gravid than non-gravid horn of unilaterally pregnant ewes. We conclude that synthesis of Proteins p70 and p15 by the uterus of sheep coincides with the time of oTP-1 production by the conceptus.     

Endometrial suppressor cells in beef cattle

Endometrial cells were recovered post mortem from cyclic and pregnant crossbred beef cattle (n = 5 each) on Days 16 to 18 after estrus, and were evaluated for their ability to suppress lymphocyte responses and release suppressor factor into the culture medium. The suppressor factor was assessed for transforming growth factor-beta (TGF-beta) activity. In addition, Percoll was used to fractionate endometrial cells from Angus cows (n = 4) on Days 16 to 18 of pregnancy to determine the density of the suppressor cells. Endometrial cells from cyclic and pregnant cows suppressed lymphocyte proliferative responses and released suppressor factor into the culture medium. The suppressor factor exhibited TGF-beta activity. Suppressor activities tended to be greatest for fractionated cells with densities of 1.01 and 1.095 g/mL. In conclusion, the bovine endometrium contains low- and high-density suppressor cells capable of releasing suppressor factor. The factor seems to be associated with TGF-beta.     

Characterization of endometrial receptors for ovine trophoblast protein-1 during the estrous cycle and early pregnancy in sheep

Scatchard analysis was used to determine the distribution, number, and affinity of unoccupied receptors for ovine trophoblast protein-1 (oTP-1) in endometrium of sheep throughout the estrous cycle and early pregnancy. In Experiment I, oTP-1 receptor characteristics were determined in membrane preparations of caruncular and intercaruncular regions of endometrium collected from uterine horns ipsilateral and contralateral to the ovary bearing the corpus luteum. Receptor concentrations and affinity constants for oTP-1 were not different (p greater than 0.1) between the four endometrial regions examined, suggesting that the expression of receptors for oTP-1 occurs uniformly throughout the endometrium. Endometrial receptor characteristics for oTP-1, luteal wet weights, and progesterone contents were determined throughout the estrous cycle and early pregnancy in Experiment II. Concentration of receptors and affinity constants for oTP-1 varied throughout the estrous cycle and early pregnancy (p less than 0.01), with the pattern of change differing between cyclic and pregnant ewes (p less than 0.01). Numbers of receptors for oTP-1 were maximal on Day 4 of the estrous cycle and declined progressively to Day 12 (p less than 0.05) in both cyclic and pregnant ewes. After Day 12, the quantity of unoccupied receptors for oTP-1 increased (p less than 0.05) gradually to Day 16 in cyclic ewes, but declined (p less than 0.05) further in the endometrium of pregnant ewes. The affinity constants of endometrial receptors for oTP-1 were similar in cyclic and pregnant ewes prior to Day 12, increasing threefold from Days 4 to 12 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of the interferon tau inducible ubiquitin cross-reactive protein in the ovine uterus.

Ubiquitin cross-reactive protein (UCRP) is a 17-kDa protein that shows cross-reactivity with ubiquitin antisera and retains the carboxyl-terminal Leu-Arg-Gly-Gly amino acid sequence of ubiquitin that ligates to, and directs degradation of, cytosolic proteins. It has been reported that bovine endometrial UCRP is synthesized and secreted in response to conceptus-derived interferon-tau (IFNtau). In the present studies, UCRP mRNA and protein were detected in ovine endometrium. Ovine UCRP mRNA was detectable on Day 13, peaked at Day 15, and remained high through Day 19 of pregnancy. The UCRP mRNA was localized to the luminal epithelium (LE), stromal cells (ST) immediately beneath the LE, and shallow glandular epithelium (GE) on Day 13, but it extended to the deep GE, deep ST, and myometrium of uterine tissues by Day 15 of pregnancy. Western blotting revealed induction of UCRP in the endometrial extracts from pregnant, but not cyclic, ewes. Ovine UCRP was also detected in uterine flushings from Days 15 and 17 of pregnancy and immunoprecipitated from Day 17 pregnant endometrial explant-conditioned medium. Treatment of immortalized ovine LE cells with recombinant ovine (ro) IFNtau induced cytosolic expression of UCRP, and intrauterine injection of roIFNtau into ovariectomized cyclic ewes induced endometrial expression of UCRP mRNA. These results are the first to describe temporal and spatial alterations in the cellular localization of UCRP in the ruminant uterus. Collectively, UCRP is synthesized and secreted by the ovine endometrium in response to IFNtau during early pregnancy. Because UCRP is present in the uterus and uterine flushings, it may regulate endometrial proteins associated with establishment and maintenance of early pregnancy in ruminants.     

Endometrial HSD11B1 and cortisol regeneration in the ovine uterus: effects of pregnancy, interferon tau, and prostaglandins

In ruminants, the elongating conceptus secretes interferon tau (IFNT), the pregnancy recognition signal, and prostaglandins (PGs). Progesterone from the ovary induces prostaglandin synthase two (PTGS2) and hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) in the endometrial epithelia, and PTGS2-derived PGs regulate endometrial functions and conceptus elongation. The enzyme HSD11B1 interconverts inactive cortisone and active cortisol. These studies determined the effects of pregnancy, IFNT, and PGs on endometrial HSD11B1 expression and activity in the ovine uterus. Study one found that HSD11B1 activity was present in both the endometrium and conceptus during early pregnancy. In study two, ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PTGS2 inhibitor, from Days 8 to 14 of pregnancy. Endometrial HSD11B1 activity and cortisol in the uterine lumen were substantially lower in MEL-infused ewes. In study three, cyclic ewes received intrauterine infusions of vehicle as a CX, MEL, recombinant ovine IFNT, or IFNT and MEL. Infusion of IFNT increased endometrial HSD11B1 expression and activity and cortisol in the uterine lumen, and this effect was diminished by coinfusion of MEL. In study four, cyclic ewes were infused with vehicle as a CX, IFNT, PGE2, PGF2 alpha, or PGI2. Infusion of all the PGs and IFNT increased endometrial HSD11B1 expression and activity, and IFNT and PGI2 infusion increased cortisol in the uterine lumen. These studies support the idea that IFNT and PGs from the conceptus regulate endometrial HSD11B1 expression and activity that regenerates bioactive cortisol in the ovine uterus during early pregnancy to influence endometrial functions and conceptus elongation.     

Depletion of NK by cellular immunoadsorption.

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.     

Endometrial immunoreactive beta-endorphin increases during mid-estrous cycle and early pregnancy in gilts.

Immunoreactive (ir) beta-endorphin (BEND) was recently identified in porcine uterine fluids. In the study reported here, we examined the hypothesis that porcine endometrium serves as a source of uterine fluid ir-BEND during the estrous cycle and early pregnancy. Endometrial ir-BEND was chromatographically characterized, sites of ir-BEND synthesis were immunocytochemically localized, and concentrations of endometrial ir-BEND during the estrous cycle and early pregnancy were measured. Sephadex G-50 chromatographic profiles of endometrial extracts from Day 15 of the estrous cycle revealed three distinct peaks of ir-BEND, with the first peak occurring near void volume and the second and third peaks coinciding with standard porcine beta-lipotropin and standard porcine BEND, respectively. Reverse-phase HPLC C18 chromatographic profiles indicated that endometrial ir-BEND contained both standard BEND and alpha-N-acetylated BEND. Immunocytochemical studies demonstrated ir-BEND in the surface and glandular epithelial cells of the endometrium, with immunostaining most prominent in the apical portion of epithelial cells. Concentrations of ir-BEND in endometrial tissues were higher on Days 14-15 than on Days 8-12 during the estrous cycle and pregnancy (p less than 0.05); however, values were not different in pregnant and cyclic gilts. Biochemical and immunocytochemical evidence supports our hypothesis that ir-BEND present in uterine fluids is derived from the endometrium. The increase in endometrial ir-BEND concentration during Days 14-15 in cyclic and pregnant gilts indicates that ovarian steroids may influence the synthesis of endometrial ir-BEND.     

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.     

Select nutrients in the ovine uterine lumen. I. Amino acids, glucose, and ions in uterine lumenal flushings of cyclic and pregnant ewes

Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium, and sodium were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.     

Detection of double target binders at the single cell level: an evaluation of the specificity of NK and MLC-induced NK-like cells

MLC-generated cells were tested on 7 consecutive days in the single cell cytotoxicity assay to determine the kinetics of natural and allospecific killing. Maximum cytotoxicity to the NK-sensitive target, K562, was found on Day 3 of MLC with an increase at that time in both the number of cells binding and the number of cells killing K562. The maximum allospecific response was found on Days 6 and 7 with an increase in cells able to bind and kill the alloantigen-bearing target. To determine whether the anti-K562 and allospecific killing were mediated by the same effector cells or different cell populations, both targets were tested simultaneously in the single cell assay. At no time during the 7 days were cells detected capable of simultaneously binding both K562 and allospecific targets. These data indicate that there are two different cell populations responsible for allospecific cytotoxicity and MLC-induced NK-like cytotoxicity. The cytotoxic specificity of unstimulated and MLC-generated NK-like cells was also investigated. When two different NK-sensitive targets (e.g., K562 and MOLT-4) were tested together in the single cell assay, there was no concurrent binding of targets by either fresh PBL prior to MLC stimulation or Day 3 MLC-generated cells. When unstimulated effector cells were enriched for NK activity by Percoll density gradient centrifugation, only a small number of effector cells simultaneously binding two different NK-sensitive targets was detected in the single cell assay. These results imply that the NK cell population is heterogeneous and composed of subpopulations recognizing diverse target specificities.     

Myometrial activity and plasma progesterone and oxytocin concentrations in cycling and early-pregnant ewes

Myometrial activity and plasma progesterone (P) and oxytocin (OT) were measured in early pregnant (n = 5) and cycling (n = 5) ewes. Electromyography (EMG) leads and jugular and inferior vena cava (IVC) catheters were surgically placed in ewes about 1 wk before data collection. When ewes returned to estrus, they were bred to either an intact or vasectomized ram. Continuous EMG data were collected, and blood samples were collected twice daily from day of estrus (Day 0) until Day 18. Ewes bred with an intact ram were checked surgically for pregnancy on Day 20. Computerized, quantitative analysis of EMG events showed no difference in signal from the right to left uterine horns, and no differences between pregnant and cycling ewes (p less than 0.05) until Days 14-18 when nonpregnant ewes returned to estrus and had increased EMG activity. The mean number of EMG events 180-900 s in length decreased in pregnant ewes, but this difference was not significant (p less than 0.05). Jugular plasma progesterone (P) levels confirmed corpus luteum (CL) formation in all ewes, and no differences in P between pregnant and nonpregnant ewes were measured until Days 14-18, when cycling ewes underwent luteolysis and pregnant ewes maintained CL. IVC plasma oxytocin concentrations were increased in pregnant ewes compared to concentrations in nonpregnant ewes on Days 5-13 (p less than 0.05), and the difference was largest at Day 6 (means +/- SEM pg/ml: pregnant = 68.7 +/- 13.9, nonpregnant = 30.9 +/- 19.9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Gastrin-releasing peptide (GRP) in the ovine uterus: regulation by interferon tau and progesterone

Gastrin-releasing peptide (GRP) is abundantly expressed by endometrial glands of the ovine uterus and processed into different bioactive peptides, including GRP1-27, GRP18-27, and a C-terminus, that affect cell proliferation and migration. However, little information is available concerning the hormonal regulation of endometrial GRP and expression of GRP receptors in the ovine endometrium and conceptus. These studies determined the effects of pregnancy, progesterone (P4), interferon tau (IFNT), placental lactogen (CSH1), and growth hormone (GH) on expression of GRP in the endometrium and GRP receptors (GRPR, NMBR, BRS3) in the endometrium, conceptus, and placenta. In pregnant ewes, GRP mRNA and protein were first detected predominantly in endometrial glands after Day 10 and were abundant from Days 18 through 120 of gestation. Treatment with IFNT and progesterone but not CSH1 or GH stimulated GRP expression in the endometrial glands. Western blot analyses identified proGRP in uterine luminal fluid and allantoic fluid from Day 80 unilateral pregnant ewes but not in uterine luminal fluid of either cyclic or early pregnant ewes. GRPR mRNA was very low in the Day 18 conceptus and undetectable in the endometrium and placenta; NMBR and BRS3 mRNAs were undetectable in ovine uteroplacental tissues. Collectively, the present studies validate GRP as a novel IFNT-stimulated gene in the glands of the ovine uterus, revealed that IFNT induction of GRP is dependent on P4, and found that exposure of the ovine uterus to P4 for 20 days induces GRP expression in endometrial glands.     

Regulation of insulin-like growth factor binding protein-6 expression in the reproductive tract throughout the estrous cycle and during the development of the placenta in the ewe

Insulin-like growth factors (IGF-I and IGF-II) are essential for normal uterine development and have been particularly implicated in fetal and placental growth. A family of six IGF binding proteins enhance or attenuate IGF-stimulated cell proliferation. In this study we have used in situ hybridization to map the distribution of IGFBP-6, one of the lesser known of the IGFBPs, in sections of the uterus collected from cyclic, anestrous, and ovariectomized nonpregnant ewes and from the uterus and placenta of early pregnant (13-55 days) and unilaterally pregnant ewes. In nonpregnant ewes IGFBP-6 mRNA (measured as arbitrary optical density units from autoradiographs) was abundant in the periepithelium and caruncles, with lower levels in the endometrial stroma and myometrium. In most regions IGFBP-6 mRNA showed cyclic variations with concentrations maximal around ovulation and the early luteal phase. In addition, 16 out of 25 ewes expressed IGFBP-6 mRNA in their endometrial glands between estrus and Day 2. Measurements of IGFBP-6 mRNA were high in anestrous ewes (equivalent values to ovulation) but low in ovariectomized ewes (equivalent values to mid to late luteal phase). In pregnant ewes IGFBP-6 mRNA was found in similar regions to those recorded during the cycle. In the periepithelium and caruncular stroma IGFBP-6 mRNA levels were higher during early pregnancy than in the midluteal phase. In the unilateral pregnant ewes there was no difference in IGFBP-6 mRNA measured between pregnant and nonpregnant horns. In conclusion, IGFBP-6 mRNA is differentially regulated during the estrous cycle and pregnancy and may be functionally important in modulating IGF activity in the uterus and placenta by virtue of its strong affinity and ability to regulate IGF-II mediated actions.     

Effects of pregnancy, oxytocin, ovine trophoblast protein-1 and their interactions on endometrial production of prostaglandin F2 alpha in vitro in perifusion chambers

Pregnancy and intrauterine infusion of ovine trophoblast protein one (oTP-1) decrease oxytocin-induced secretion of prostaglandin F2 alpha (PGF) from the uterus. In the present study, effects of oTP-1 and pregnancy on endometrial secretion of PGF were examined in an in vitro perifusion system. In Experiment 1, endometrium from day 14 pregnant and cyclic ewes was perifused sequentially on both the lumenal and myometrial sides with Krebs Ringers Bicorbonate solution (KRB), KRB plus oxytocin (1 IU/ml) and KRB alone. Endometrium from pregnant ewes secreted more PGF from both lumenal and myometrial sides than endometrium from cyclic ewes (P less than 0.05). Oxytocin stimulated secretion of PGF from both sides of endometrium regardless of status. Secretion of PGF was greater from the lumenal surface of endometrium compared to myometrium (P less than 0.05) for pregnant and cyclic ewes. For Experiment 2, endometrium was collected from day 15 cyclic ewes and perifused sequentially with KRB, KRB plus 300 ng/ml of either Bovine Serum Albumin (BSA) or oTP-1, KRB with or without BSA or oTP-1 plus oxytocin (1 IU/ml) and then KRB alone. Oxytocin stimulated greater release of PGF from oTP-1-treated than BSA-treated endometrium. Pretreatment of endometrium with oTP-1 had the same effect on oxytocin-induced PGF secretion as cotreatment with oTP-1 and oxytocin. In Experiment 3, uterine horns of cyclic ewes were catheterized on day 10 of the estrous cycle, and infused with either oTP-1 or day 16 pregnant sheep serum proteins on days 12, 13 and 14. Endometrium was collected on day 15 and perifused sequentially with KRB, KRB plus oxytocin (1 IU/ml) and then KRB alone. Treatment of ewes with oTP-1 attenuated endometrial secretion of PGF in response to oxytocin. Results of this study indicate that: (1) pregnancy stimulates basal secretion of PGF from endometrium and has no effect on oxytocin-induced secretion of PGF in vitro; (2) short-term oTP-1 treatment enhances oxytocin-induced PGF secretion from day 15 cyclic endometrium and (3) long-term oTP-1 treatment in vivo inhibits oxytocin-induced PGF secretion in ewes.