Endothelial vasodilator production by uterine and systemic arteries. VIII. Estrogen and progesterone effects on cPLA2, COX-1, and PGIS protein expression.

During ovine pregnancy, when both estrogen and progesterone are elevated, prostacyclin (PGI2) production by uterine arteries and the key enzymes for PGI2 production, phospholipase A2 (cPLA2), cyclooxygenase 1 (COX-1), and prostacyclin synthetase (PGIS), are increased. This study was conducted to determine whether exogenous estradiol-17beta (E2beta) with or without progesterone (P4) treatment would increase cPLA2, COX-1, and PGIS protein expression in ovine uterine, mammary, and systemic (renal, mental, and coronary) arteries. Nonpregnant ovariectomized sheep received vehicle (n = 10), P(4) (0.9-g controlled internal drug release vaginal implants; n = 13), E2beta (5 microg/kg bolus followed by 6 microg x kg(-1) x day(-1); n = 10), or P4 + E2beta (n = 12). Arteries were procured on Day 10, and cPLA2, COX-1, and PGIS protein were measured by Western immunoblot analysis in endothelial isolated proteins and vascular smooth muscle (VSM). The levels of cPLA2 was increased in uterine artery endothelium in ewes treated with P4 + E2beta but was not altered by any steroid treatment in renal, coronary, mammary, or omental artery endothelium or in VSM of any evaluated artery. Similarly, COX-1 was increased in uterine artery endothelium with P4 + E2beta but was not significantly altered by treatment in other endothelium or VSM. E2beta treatment increased PGIS protein in uterine and renal artery endothelium but did not alter PGIS in other endothelial tissue. P4 increased PGIS expression in the uterine, mammary, omental, and renal artery VSM, and E2beta increased PGIS expression in the uterine and omental artery VSM. Both E2beta and P4 treatments differentially alter protein expression of the key enzymes involved in PGI2 production in different artery types and may play an important role in the control of blood flow redistribution during hormone replacement therapy.     

Endothelial vasodilator production by uterine and systemic arteries. IV. Cyclooxygenase isoform expression during the ovarian cycle and pregnancy in sheep

Uterine artery endothelial production of the potent vasodilator, prostacyclin, is greater in pregnant versus nonpregnant sheep and in whole uterine artery from intact versus ovariectomized ewes. We hypothesized that uterine artery cyclooxygenase (COX)-1 and/or COX-2 expression would be elevated during pregnancy (high estrogen and progesterone) and the follicular phase of the ovarian cycle (high estrogen/low progesterone) as compared to that in luteal phase (low estrogen/high progesterone) or in ovariectomized (low estrogen and progesterone) ewes. Uterine and systemic (omental) arteries were obtained from nonpregnant luteal-phase (LUT; n = 10), follicular-phase (FOL; n = 11), and ovariectomized (OVEX; n = 10) sheep, as well as from pregnant sheep (110-130 days gestation; term = 145 +/- 3 days; n = 12). Endothelial and vascular smooth muscle (VSM) COX-1 protein levels and uterine artery endothelial cell COX-1 mRNA levels were compared. Using immunohistochemistry and Western analysis, the primary location of COX-1 protein was the endothelium; that is, we observed 2.2-fold higher COX-1 protein levels in intact versus endothelium-denuded uterine artery and a 6.1-fold higher expression in the endothelium versus VSM (P < 0.05). COX-2 protein expression was not detectable in either uterine artery endothelium or VSM. COX-1 protein levels were observed to be higher (1.5-fold those of LUT) in uterine artery endothelium from FOL versus either OVEX or LUT nonpregnant ewes (P < 0.05), with substantially higher COX-1 levels seen in pregnancy (4.8-fold those of LUT). Increases in uterine artery endothelial COX-1 protein were highly correlated to increases in the level of COX-1 mRNA (r(2) = 0.66; P < 0.01) for all treatment groups (n = 6-8 per group), suggesting that increased COX-1 protein levels are regulated at the level of increased COX-1 mRNA. No change in COX-1 expression was observed between groups in a systemic (omental) artery. In conclusion, COX-1 expression is specifically up-regulated in the uterine artery endothelium during high uterine blood flow states such as the follicular phase and, in particular, pregnancy.     

The influence of reproductive phase on lipopolysaccharide stimulation of prostacyclin production and cyclooxygenase-2 protein concentrations in reproductive and systemic arteries of the sheep

Cyclooxygenase, the enzyme that converts arachidonate to prostaglandins, plays a regulatory role in vasodilation under normal and pathological conditions. Studies were conducted to determine the effects of reproductive phase and lipopolysaccharide (LPS) on production of PGI2 and amounts of cyclooxygenase protein in uterine, mammary, mesenteric, and renal arteries. Arteries were collected from ewes during the follicular (Day 0 = estrus) or luteal (Day 10) phase of the estrous cycle and were cultured in the presence of LPS. After 24 h, media were collected and analyzed for 6-keto-PGF1alpha, the stable metabolite of PGI2. In addition, arteries were collected and homogenized and the relative concentration of cyclooxygenase was determined via Western analysis. Lipopolysaccharide stimulated PGI2 production in all four-artery types from both follicular and luteal phase ewes (p < 0.001). Upon LPS stimulation, uterine and mammary arteries produced more PGI2 compared to mesenteric and renal arteries (p = 0.04). The phase of estrous cycle did not affect PGI2 production by any of the artery populations exposed to LPS (p = 0.35). There was no cyclooxygenase-2 in untreated uterine and mammary arteries and no cyclooxygenase-2 was detected in untreated or LPS-treated mesenteric and renal arteries. In contrast, LPS-treated uterine and mammary arteries from luteal phase ewes had higher (p = 0.064) cyclooxygenase-2 concentrations than those from follicular phase ewes. These results suggest that the hormone conditions of the follicular (high estrogen) and luteal (high progesterone) phases of the ovarian cycle play a role in regulating uterine and mammary artery but not mesenteric and renal artery response to LPS.     

Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS

Uterine blood flow (UBF) and uterine artery endothelial nitric oxide synthase (eNOS) expression are greatest during the follicular vs. luteal phase. 17 beta-Estradiol (E(2)beta) increases UBF and elevates eNOS in ovine uterine but not systemic arteries; progesterone (P(4)) effects on E(2)beta changes of eNOS remain unclear. Nonpregnant ovariectomized sheep received either vehicle (n = 10), P(4) (0.9 g Controlled Internal Drug Release vaginal implants; n = 13), E(2)beta (5 microg/kg bolus + 6 microg x kg(-1) x day(-1); n = 10), or P(4) + E(2)beta (n = 12). Reproductive (uterine/mammary) and nonreproductive (omental/renal) artery endothelial proteins were procured on day 10, and eNOS was measured by Western analysis. P(4) and E(2)beta alone and in combination increased (P < 0.05) eNOS expression in uterine artery endothelium (vehicle = 100 +/- 16%, P(4) = 251 +/- 59%, E(2)beta = 566 +/- 147%, P(4) + E(2)beta = 772 +/- 211% of vehicle). Neither omental, renal, nor mammary artery eNOS was altered, demonstrating the local nature of steroid-induced maintenance of uterine arterial eNOS. In the myometrial microvasculature, eNOS was increased slightly (P = 0.06) with E(2)beta and significantly with P(4) + E(2)beta. Systemic NO(x) was increased with P(4) and P(4) + E(2)beta, but not E(2)beta, suggesting differential regulation of eNOS expression and activity, since P(4) increased eNOS in uterine artery endothelium while E(2)beta and the combination further increased eNOS protein.     

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.     

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.     

Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia

We tested the hypothesis that chronic high-altitude (3,820 m) hypoxia during pregnancy was associated with the upregulation of endothelial nitric oxide (NO) synthase (eNOS) protein and mRNA in ovine uterine artery endothelium and enhanced endothelium-dependent relaxation. In pregnant sheep, norepinephrine-induced dose-dependent contractions were increased by removal of the endothelium in both control and hypoxic uterine arteries. The increment was significantly higher in hypoxic tissues. The calcium ionophore A23187-induced relaxation of the uterine artery was significantly enhanced in hypoxic compared with control tissues. However, sodium nitroprusside- and 8-bromoguanosine 3',5'-cyclic monophosphate-induced relaxations were not changed. Accordingly, chronic hypoxia significantly increased basal and A23187-induced NO release. Chronic hypoxia increased eNOS protein and mRNA levels in the endothelium from uterine but not femoral or renal arteries. In nonpregnant animals, chronic hypoxia increased eNOS mRNA in uterine artery endothelium but had no effects on eNOS protein, NO release, or endothelium-dependent relaxation. Chronic hypoxia selectively augments pregnancy-associated upregulation of eNOS gene expression and endothelium-dependent relaxation of the uterine artery.     

Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function

The last 10 years has seen a dramatic increase in our understanding of the mechanisms underlying the pregnancy-specific adaptation in cardiovascular function in general and the dramatic changes that occur in uterine artery endothelium in particular to support the growing fetus. The importance of these changes is clear from a number of studies linking restriction of uterine blood flow (UBF) and/or endothelial dysfunction and clinical conditions such as intrauterine growth retardation (IUGR) and/or preeclampsia in both humans and animal models; these topics are covered only briefly here. The recent developments that prompts this review are twofold. The first is advances in an understanding of the cell signaling processes that regulate endothelial nitric oxide synthase (eNOS) in particular (Govers R and Rabelink TJ. Am J Physiol Renal Physiol 280: F193-F206, 2001). The second is the emerging picture that uterine artery (UA) endothelial cell production of nitric oxide (NO) as well as prostacyclin (PGI2) may be as much a consequence of cellular reprogramming at the level of cell signaling as due to tonic stimuli inducing changes in the level of expression of eNOS or the enzymes of the PGI2 biosynthetic pathway (cPLA2, COX-1, PGIS). In reviewing just how we came to this conclusion and outlining the implications of such a finding, we draw mostly on data from ovine or human studies, with reference to other species only where directly relevant.     

Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.     

Uteroplacental production of eicosanoids in ovine pregnancy

Dramatic cardiovascular alterations occur during normal ovine pregnancy which may be associated with increased prostaglandin production, especially of uteroplacental origin. To study this, we examined (Exp 1) the relationships between cardiovascular alterations, e.g., the rise in uterine blood flow and fall in systemic vascular resistance, and arterial concentrations of prostaglandin metabolites (PGEM, PGFM and 6-keto-PGF1 alpha) in nonpregnant (n = 4) and pregnant (n = 8) ewes. To determine the potential utero-placental contribution of these eicosanoids in pregnancy, we also studied (Exp 2) the relationship between uterine blood flow and the uterine venous-arterial concentration differences of PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and TxB2 in twelve additional late pregnant ewes. Pregnancy was associated with a 37-fold increase in uterine blood flow and a proportionate (27-fold) fall in uterine vascular resistance (p less than 0.01). Arterial concentrations of PGEM were similar in nonpregnant and pregnant ewes (316 +/- 19 and 245 +/- 38 pg/ml), while levels of PGFM and PGI2 metabolite 6-keto-PGF1 alpha were elevated 23-fold (31 +/- 14 to 708 +/- 244 pg/ml) and 14-fold (12 +/- 4 to 163 +/- 78 pg/ml), respectively (p less than 0.01). Higher uterine venous versus uterine arterial concentrations were observed for PGE2 (397 +/- 36 and 293 +/- 22 pg/ml) and 6-keto-PGF1 alpha (269 +/- 32 and 204 +/- 32 pg/ml), p less than 0.05, but not PGF2 alpha or TxB2. Although PGFM concentrations appeared to be greater in uterine venous (1197 +/- 225 pg/ml) as compared to uterine arterial (738 +/- 150 pg/ml) plasma, this did not reach significance (0.05 less than p less than 0.1). In normal ovine pregnancy arterial levels of PGI2 are increased, which may in part reflect increased uteroplacental production. Moreover the gravid ovine uterus also appears to produce PGE2 and metabolize PGF2 alpha.     

Distribution and cellular localization of prostacyclin synthase in human brain.

Prostacyclin (PGI(2)) is a labile, lipid-derived metabolite of arachidonic acid synthesized through the sequential action of cyclo-oxygenase (COX) and prostacyclin synthase (PGIS). In addition to its well-characterized vasodilatory and thrombolytic effects, an increasing number of studies report an important role of PGI(2) in nociception in various animal species. In this study we investigated the regional distribution of PGIS in human brain by immunohistochemistry and in situ hybridization. PGIS-immunoreactive (ir) protein was localized to blood vessels throughout the brain. Neuronal cells and glial cells, such as microglia and oligodendrocytes, also showed intense labeling. The strongest expression of PGIS was seen in large principal neurons, such as pyramidal cells of the cortex, pyramidal cells of the hippocampus, and Purkinje cells of the cerebellum. Abundance of PGIS mRNA was observed in blood vessels and large neurons and correlated well with the immunohistochemical findings. The expression of PGIS in human brain was further demonstrated by immunoblotting and detection of 6-keto-PGF (1alpha), the stable degradation product of prostacyclin in human brain homogenate. These results demonstrate a widespread expression of PGIS in the central nervous system and suggest a potentially important role of prostacylin in modulating neuronal activity in human brain.     

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.     

Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NO(x)

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110-130 days gestation; term = 145 +/- 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n = 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO(2)-NO(3) (NO(x)). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT < or = FOL < PREG (P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG (P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls (P < 0.01). Circulating NO(x) levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 +/- 1.1 microM in LUT to 12 +/- 4, 22 +/- 3, and 41 +/- 3 microM at 110, 120, and 130 days gestation (P < 0.01). Systemic NO(x) levels in singleton (12.5 +/- 1.6 microM) were less (P < 0.01) than in multiple (twin 27.6 +/- 6.5 microM; triplet = 46 +/- 10 microM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NO(x) levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.     

Ca2+]i signaling vs. eNOS expression as determinants of NO output in uterine artery endothelium: relative roles in pregnancy adaptation and reversal by VEGF165

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca(2+)](i)) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca(2+)](i) bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca(2+)](i) cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF(165). While treatment of luteal NP-UA Endo and P-UA Endo with VEGF(165) acutely stimulates a very modest [Ca(2+)](i) and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca(2+)](i) and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia.     

Select nutrients in the ovine uterine lumen. ii. glucose transporters in the uterus and peri-implantation conceptuses

Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not the estrous cycle; however, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses (embryo/fetus and associated membranes) are not established. This study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3, and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy, as well as in conceptuses from Days 10 to 20 of pregnancy. The SLC2A1 and SLC5A1 mRNAs and proteins were most abundant in uterine luminal epithelia and superficial glandular epithelia (LE/sGE), whereas SLC2A4 was present in stromal cells and glandular epithelia (GE). SLC5A11 mRNA was most abundant in endometrial GE, whereas SLC2A3 mRNA was not detectable in endometria. SLC2A1, SLC2A3, SLC2A4, SLC5A1, and SLC5A11 were expressed in the trophectoderm and endoderm of conceptuses. Steady-state levels of SLC2A1, SLC5A1, and SLC5A11 mRNAs, but not SLC2A4 mRNA, were greater in endometria from pregnant than from cyclic ewes. Progesterone increased SLC2A1, SLC5A11, and SLC2A4 mRNAs in the LE/sGE and SLC5A1 in the GE of ovariectomized ewes. Expression of SLC5A1 was inhibited by ZK136,317 (progesterone receptor antagonist), and the combination of ZK136,317 and IFNT further decreased expression in GE. In constrast, P4 induced and IFNT stimulated expression of SLC2A1 and SLC5A11, and these effects were blocked by ZK136,317. Results of this study indicate differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses for transport and uptake of glucose, and that P4 or P4 and IFNT regulate their expression during the peri-implantation period of pregnancy.     

Ovine osteopontin: I. Cloning and expression of messenger ribonucleic acid in the uterus during the periimplantation period.

Trophoblast-derived interferon tau (IFNtau) acts on the endometrium to increase secretion of several proteins during the pregnancy recognition period in ruminants. One of these is a 70-kDa acidic protein that has not been identified. Our hypothesis was that the 70-kDa acidic protein is osteopontin (OPN). OPN is an acidic glycoprotein that fragments upon freezing and thawing or treatment with proteases including thrombin. OPN contains a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins to promote cell-cell attachment and cell spreading. Using antisera to recombinant human OPN, both 70-kDa and 45-kDa proteins were identified in uterine flushings from pregnant ewes by Western blotting. A clone containing the entire ovine OPN cDNA coding sequence was isolated by screening a Day 15 pregnant ovine endometrial cDNA library with a partial ovine OPN cDNA. In pregnant ewes, steady-state levels of OPN endometrial mRNA increased (P < 0. 01) after Day 17. In both cyclic and pregnant ewes, in situ hybridization analysis showed that OPN mRNA was localized on unidentified immune cells within the stratum compactum of the endometrium. In pregnant ewes, OPN mRNA was also expressed by the glandular epithelium. Results suggest that progesterone and/or IFNtau induce expression and secretion of OPN by uterine glands during the periimplantation period and that OPN may induce adhesion between luminal epithelium and trophectoderm to facilitate superficial implantation.     

Cellular source in ewes of prostaglandin-endoperoxide synthase-2 in uterine arteries following stimulation with lipopolysaccharide.

Prostaglandin-endoperoxide synthase (PTGS) (also known as cyclooxygenase) converts arachidonic acid into several prostaglandins, many of which have roles in vasodilation and vasoconstriction under normal and pathological conditions. There are two isoforms of PTGS: PTGS-1 and PTGS-2; PTGS-1 is constitutively expressed in many tissues and is believed to be involved in the homeostatic maintenance of the body. In contrast, PTGS-2 is believed to have a "differentiative" role in the cells and is highly inducible during inflammation and in response to lipopolysaccharide (LPS). Endothelial cells as well as vascular smooth muscle cells can be a source of PTGS within the artery. The objective of this study was to determine the cell population(s) in uterine arteries that respond to LPS with an increase in PTGS-2 protein expression. Uterine arteries collected from ewes during the follicular (Day 0, Day 0 = estrus, n = 4) or luteal (Day 10, n = 4) phase were treated in vitro with LPS as intact artery segments, cut-open artery segments, or cut-open and denuded (endothelial cells absent) artery segments. After 24 h of LPS treatment, intact, cut-open, and denuded uterine artery segments were collected into homogenization buffer for determination of PTGS-2 protein levels by Western blot analysis. The culture medium was collected and used for detection of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolite of prostacyclin, using an enzyme immunoassay. In addition, the location of PTGS-2 after LPS treatment was analyzed by immunohistochemistry in intact artery segments. Denuded arteries (endothelium absent) did not show increases in PTGS-2 protein in the homogenates or 6-keto-PGF(1alpha) in the culture medium after LPS exposure. In contrast, cut uterine arteries responded to LPS stimulation with a significant increase in PTGS-2 protein in homogenates and 6-keto-PGF(1alpha) in culture medium. Immunohistochemical staining for PTGS-2 was associated with both endothelial cells and vascular smooth muscle cells. These results suggest that while both endothelial cells and vascular smooth muscle cells are associated with PTGS-2, after LPS exposure it is the endothelial cells that are essential in uterine artery increases in PTGS-2 and prostacyclin in response to LPS stimulation.     

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI₂) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF_1α (a PGI₂ metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF_1α concentration. Regulation of PGI₂ synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF_1α in the endometrium. The concentration of 6-keto PGF_1α in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI₂ metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI₂ secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI₂ synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI₂ release during the implantation period.