Ovine Uterine Research Articles

Cervical Clamping Does Affect Ovine Uterine Perfusion: A Laser Speckle Contrast Imaging Study

Cervical Clamping Does Affect Ovine Uterine Perfusion: A Laser Speckle Contrast Imaging Study

B-mode and Doppler Ultrasonographic Assessment of Uterine Involution in Ewes Treated with Two Different Doses of ProstaglandinF2α

Background: Several studies consisted of postmortem evaluations and B-mode ultrasonography have been performed on ovine uterine involution. However, researches with Doppler ultrasonography are very limited in ewes. Doppler ultrasonography is a non-invasive method which provides information about vascular dynamics of the tissues which cannot be examined by B-mode ultrasonography. The aim of the study was to evaluate the effects of two different PGF2α doses on uterine measurements by real time B-mode ultrasonography; and on uterine artery hemodynamics by pulsed-wave Doppler analysis during postpartum uterine involution in ewes.Materials, Methods & Results: The study was conducted with 30 primiparous Kiwircik ewes which lambed singleton without any complication. The ewes were randomly divided into three groups (n= 10 for each group). A single i.m. injection of 125 μg/sheep PGF2α, 75 μg/sheep PGF2α, and 1.0 mL/sheep sterile saline solution were administered to Group 1, 2, and 3, respectively. The day of parturition was considered as the first day of the study. Examinations were performed on days 1, 2, 3, 7, 14, 21 and 28. Diameters of previously gravid horn and caruncles were measured by real time B-mod ultrasonography. Presence of lochia was also noted. Uterine artery pulsatility index (PI), resistance index (RI), systolic/diastolic ratio (S/D) values were measured by pulsed-wave Doppler ultrasonography. The one-way analysis of variance and Duncan’s test were used for statistical analysis. The mean previously gravid horn diameters of ewes were 8.30 ± 0.16 cm and 1.53 ± 0.07 cm on day 1 and day 28, respectively. Previously gravid horn and caruncle diameters had a similar declining pattern in all groups as involution period proceeded. Involution was mostly completed by day 21. More than 50% reduction in uterine size was achieved in prostaglandin administered groups by day 7. Caruncles were not able to be identified after the second week postpartum. Lochia was observed for a longer period in control group. Uterine artery PI and RI showed fluctuations throughout involution period with a similar pattern among groups. S/D values progressively increased until day 14, then showed a decreasing pattern.Discussion: The effects of different PGF2α doses on uterine measurements and uterine artery hemodynamics during postpartum period were compared in ewes for the first time. The uterine size reduction in prostaglandin administered groups suggested that both PGF2α doses were effective in uterine involution. The last observation of lochia in uterine cavity were achieved by day 7 in prostaglandin administered groups, suggesting both PGF2α doses trigger uterine contractions which lead to the removal of uterine content. Automatic measurements by Doppler device were taken in order to provide uniformity and to prevent operator-based bias. The significantly higher uterine artery PI value found in Group 1 on day 1 might suggest the stimulatory effect of PGF2α administration on uterine contractility synergitically with already released endogenous PGF2α. Uterine artery PI and RI showed fluctuations throughout involution period and reached their peak values on day 14. The increasing S/D values up to day 14 indicated increasing diastolic flow, increasing resistance and decreasing blood perfusion in prostaglandin administered groups. Prostaglandin administered groups tended to show higher hemodynamic parameters throughout the study which suggested a vasoconstrictor effect of PGF2α. In conclusion, PGF2α administration on the day of parturition might have constrictor effects both on uterine artery and myometrium which leads to a reduction in uterine blood flow and a rapid decline in uterine size especially during early puerperium even with a reduced dose.

Effects of the Catechol and Methoxy Metabolites of 17β-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2β, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2β metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2β metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2β metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2β metabolite-induced NO production. E2β and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-β antagonist) inhibited the rises in NOx levels stimulated by E2β and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (β2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2β, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2β metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy.

Critical steps for initiating an animal uterine transplantation model in sheep: Experience from a case series

BackgroundRecent reports have demonstrated uterus transplantation as a relevant solution to treat absolute uterine infertility. Training on animal models is a prerequisite to set up a uterine transplantation program in humans. Sheep have been used as an optimal model for training and research as they display similar vessels size to human. While the ovine model might seem easy there are many difficulties in performing this complex surgery. In this study we describe through our experience the critical initial steps toward building a learning curve toward an optimal ovine uterine transplantation model. MaterialsWe performed nine orthotopic uterine autotransplantations using end-to-side anastomoses to the external iliac vessels in sheep. We recorded the duration of all surgical steps and pointed out specific difficulties and solution found. ResultsWe were able to perform optimal uterine dissection after the first 5 cases and optimal bilateral arterial and venous anastomoses, after 7 and 9 cases respectively. The main factors associated to success rate were optimal exposure, appropriate equipment, careful vessel preparation and modification of the anastomosis technique. ConclusionAs uterine transplantation research programs are expanding, setting up an ovine model to train and perform research is critical. Such model is complex and requires optimized multidisciplinary approach to build an efficient learning curve.

MicroRNA-210 Targets Ten-Eleven Translocation Methylcytosine Dioxygenase 1 and Suppresses Pregnancy-Mediated Adaptation of Large Conductance Ca2+-Activated K+ Channel Expression and Function in Ovine Uterine Arteries.

Gestational hypoxia inhibits large conductance Ca2+-activated K+ (BKCa) channel expression and function in uterine arterial adaptation to pregnancy. Given the findings that microRNA-210 (miR-210) is increased in hypoxia during gestation and preeclampsia, the present study sought to investigate the role of miR-210 in the regulation of BKCa channel adaptation in the uterine artery. Gestational hypoxia significantly increased uterine vascular resistance and blood pressure in pregnant sheep and upregulated miR-210 in uterine arteries. MiR-210 bound to ovine ten-eleven translocation methylcytosine dioxygenase 1 mRNA 3' untranslated region and decreased ten-eleven translocation methylcytosine dioxygenase 1 mRNA and protein abundance in uterine arteries of pregnant sheep, as well as abrogated steroid hormone-induced upregulation of ten-eleven translocation methylcytosine dioxygenase 1 expression in uterine arteries of nonpregnant animals. In accordance, miR-210 blocked pregnancy- and steroid hormone-induced upregulation of BKCa channel β1 subunit expression in uterine arteries. Functionally, miR-210 suppressed BKCa channel current density in uterine arterial myocytes of pregnant sheep and inhibited steroid hormone-induced increases in BKCa channel currents in uterine arteries of nonpregnant animals. Blockade of endogenous miR-210 inhibited hypoxia-induced suppression of BKCa channel activity. In addition, miR-210 decreased BKCa channel-mediated relaxations and increased pressure-dependent myogenic tone of uterine arteries. Together, the results demonstrate that miR-210 plays an important role in the downregulation of ten-eleven translocation methylcytosine dioxygenase 1 and repression of BKCa channel function in uterine arteries, revealing a novel mechanism of epigenetic regulation in the maladaptation of uterine hemodynamics in gestational hypoxia and preeclampsia.

Hypoxia Represses ER-α Expression and Inhibits Estrogen-Induced Regulation of Ca2+-Activated K+ Channel Activity and Myogenic Tone in Ovine Uterine Arteries: Causal Role of DNA Methylation.

Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-α (ER-α) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-α gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-α gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O2 and 10.5% O2 for 48 hours. Hypoxia significantly increased ER-α promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-α expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2'-deoxycytidine. In addition, hypoxia abrogated steroid hormone-mediated increase in ER-α expression and inhibited the hormone-induced increase in large-conductance Ca(2+)-activated K(+) channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2'-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-α gene repression and ablation of steroid hormone-mediated adaptation of uterine arterial large conductance Ca(2+)-activated K(+) channel activity and myogenic tone in pregnancy.

Gestational hypoxia up-regulates protein kinase C and inhibits calcium-activated potassium channels in ovine uterine arteries.

Objective: The present study tested the hypothesis that gestational hypoxia up-regulates protein kinase C (PKC) and inhibits calcium-activated potassium channels (KCa)-mediated relaxations of uterine arteries in pregnancy. Study design: Uterine arteries were isolated from nonpregnant (NPUA) and pregnant (PUA) (~140 day gestation) sheep maintained at either sea level or high altitude (3,820 m for 110 days, PaO2: 60 mmHg). Contractions of uterine arteries were determined. Key findings: In normoxic PUA, selective inhibition of large-conductance KCa (BK) channels significantly enhanced PKC activator phorbol 12, 13-dibutyrate (PDBu)-induced contractions. This effect was abrogated by chronic hypoxia in gestation. Unlike BK channels, inhibition of small-conductance KCa (SK) channels had no significant effect on PDBu-mediated contractions. In normoxic PUA, activation of both BK with NS1619 or SK with NS309 produced concentration-dependent relaxations, which were not altered by the addition of PDBu. However, in uterine arteries treated with chronic hypoxia (10.5% O2 for 48 h), both NS1619- and NS309-induced relaxations were significantly attenuated by PDBu. In NPUAs, inhibition of BK channels significantly enhanced PDBu-induced contractions in both normoxic and hypoxic animals. Conclusion: The results suggest that in the normoxic condition BK inhibits PKC activity and uterine vascular contractility, which is selectively attenuated by chronic hypoxia during gestation. In addition, hypoxia induces PKC-mediated inhibition of BK and SK activities and relaxations of uterine arteries in pregnancy.

Chronic Hypoxia During Gestation Causes Epigenetic Repression of the Estrogen Receptor-α Gene in Ovine Uterine Arteries via Heightened Promoter Methylation

Estrogen receptor-α (ERα) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ERα gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ERα gene in uterine arteries via heightened promoter methylation. Ovine ERα promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or hypoxia-inducible factor response elements were found at the promoter. Two transcription factor binding sites, USF −15 and Sp1 −520 , containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ERα through Sp1. Deletion of the Sp1 site abrogated 17β-estradiol–induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1 but not the USF site was significantly decreased in uterine arteries of pregnant as compared with nonpregnant animals. In pregnant sheep exposed to long-term high-altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ERα gene repression in uterine arteries and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Effects of In Vivo Knockdown of Translation of Arginine-Associated Gene Transcripts in Conceptus Trophectoderm on Growth, Development, and Gene Expression by Ovine Conceptuses.

Arginine (Arg), a nutritionally essential amino acid for conceptus development, can be metabolized to nitric oxide (NO) and polyamines. Arg increases 13-fold in the ovine uterine lumen between Days 10 and 15 of pregnancy and activates mechanistic target of rapamycin cell signaling. This study tested the hypothesis that knockdown of translation of Arg-associated gene transcripts inhibits ovine conceptus development. Morpholino antisense oligonucleotides (MAOs) were designed to inhibit translation of mRNAs for: 1) SLC7A1, Arg transporter; 2) NOS3, an enzyme for NO synthesis; 3) ODC1, an enzyme for polyamine synthesis; and 4) mismatch morpholino as MAO-control. On Day 8 post-mating, the uterine horn of ewes (n=7 or 8 /MAO type) ipsilateral to the CL was ligated and MAO injected into the uterine lumen. On Day 16, ewes were hysterectomized to obtain conceptuses, uterine flushings, and uterine tissues from the treatment groups: 1) MAO-SLC7A1 (n=8); 2) MAO-NOS3 (n=7); 3) MAO-ODC1 (n=8); and 4) MAO-control (n=7). Conceptuses in the control uterine horn were healthy, elongated and intact. Conceptuses exposed to the SLC7A1-MAO were small, thin, fragile and fragmented. Conceptuses exposed to the NOS3-MAO were thin and small, but elongated and healthy. In the ODC1-MAO group, half of the conceptus were small, fragile and fragmented, but half were healthy and elongated. Compared to protein concentration (2.1 + 0.5 mg/ml), uterine flush volume recovered (8.2 + 0.8 ml) and total protein in uterine flushing (15.7±3.5 mg) for MAO-control ewes, values did not differ (P>0.05) for treatment MAOs. Slot blot analysis did not detect effects of treatment (P>0.05) on interferon-tau (IFNT) in uterine flushings. However, radioimmunoassay data revealed an increase (P<0.05) in IFNT in MAO-NOS3 (15,057±2,538 ng) compared to MAO-control (9,353±1,768 ng) ewes. Pregnancy rates were not affected by treatment (P>0.2). Thus, knockdown of SLC7A1 protein attenuated conceptus development. Knockdown of NOS3 and ODC1 proteins partially suppressed conceptus development likely due to compensatory effects of NOS1/NOS2, other cationic amino acid transporters and production of putrescine via the arginine decarboxylase and agmatinase pathway. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-20028 from the USDA National Institute of Food and Agriculture.

Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT(2) receptors (AT(2)R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant (n = 16) and term pregnant (n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner (P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM (P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content (P ≤ 0.01). UVSM AT(1)R and AT(2)R expression was similar throughout the UVB and unchanged during pregnancy (P > 0.1). AT(1)R inhibition blocked ANG II-mediated contractions; AT(2)R blockade, however, did not enhance contractions. AT(2)R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

Comprehension of power Doppler quantification and a missed opportunity for landmark research

Comprehension of power Doppler quantification and a missed opportunity for landmark research

Select Nutrients in the Ovine Uterine Lumen. IX. Differential Effects of Arginine, Leucine, Glutamine, and Glucose on Interferon Tau, Ornithine Decarboxylase, and Nitric Oxide Synthase in the Ovine Conceptus1

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.

Prostaglandins Regulate Conceptus Elongation and Mediate Effects of Interferon Tau on the Ovine Uterine Endometrium1

In ruminants, both the endometrium and the conceptus (embryo and associated extraembryonic membranes) trophectoderm synthesizes and secretes prostaglandins (PG) during early pregnancy. In mice and humans, PGs regulate endometrial function and conceptus implantation. In Study One, bred ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PG synthase (PTGS) inhibitor from Days 8-14 postmating, and the uterine lumen was flushed on Day 14 to recover conceptuses and assess their morphology. Elongating and filamentous conceptuses (12 cm to >14 cm in length) were recovered from all CX-treated ewes. In contrast, MEL-treated ewes contained mostly ovoid or tubular conceptuses. PTGS activity in the uterine endometrium and amounts of PGs were substantially lower in uterine flushings from MEL-treated ewes. Receptors for PGE2 and PGF2 alpha were present in both the conceptus and the endometrium, particularly the epithelia. In Study Two, cyclic ewes received intrauterine infusions of CX, MEL, recombinant ovine interferon tau (IFNT), or IFNT and MEL from Days 10-14 postestrus. Infusion of MEL decreased PGs in the uterine lumen and expression of a number of progesterone-induced endometrial genes, particularly IGFBP1 and HSD11B1. IFNT increased endometrial PTGS activity and the amount of PGs in the uterine lumen. Interestingly, IFNT stimulation of many genes (FGF2, ISG15, RSAD2, CST3, CTSL, GRP, LGALS15, IGFBP1, SLC2A1, SLC5A1, SLC7A2) was reduced by co-infusion with MEL. Thus, PGs are important regulators of conceptus elongation and mediators of endometrial responses to progesterone and IFNT in the ovine uterus.

Estradiol-17β-Induced Temporal and Spatial Partitioning of eNOS and Estrogen Receptors in Ovine Uterine Artery Endothelial Cells from Pregnant Sheep.

Pregnancy-induced elevations in uterine blood flow are closely correlated with concurrent increases in plasma estradiol-17β levels and the expression/activity of eNOS. Indeed during late ovine gestation a physiologic cause and effect relationship has been established in vivo in that unilateral blockade of estrogen receptors (w/ICI 182,780) or nitric oxide synthase (w/L-NAME) locally and by a similar magnitude reduces uteroplacental blood flow. Estradiol-17β binds to estrogen receptors alpha and beta (ERα/β) and binding to these receptors results in either rapid effects (non-classical, non-genomic) and/or chronic effects (classical-genomic). Interestingly, numerous proteins that are involved in the estradiol-17β-induced rapid eNOS activation and nitric oxide production are located in specialized membrane domains called the caveolae of uterine artery endothelial cells (UAECs). Furthermore, treatment with the calcium mobilizing agonist ATP induced rapid eNOS activation which is accompanied by eNOS repartitioning with alterations in its multi-site phosphorylation state. We hypothesized that estradiol-17β induces eNOS activation via the non-classical ER receptor and initiates the sequential and coordinated re-partitioning and activation of eNOS with altered multi-site phosphorylation. UAECs were isolated from uterine arteries of pregnant ewes (gestational day 120-130; term 147d). Confluent passage 4 UAECs were treated with vehicle (Control) or with estradiol-17β (10 nM) for 10 min. The caveolae were fractionated from UAECs using sucrose density gradient centrifugation, and immunoblotting was utilized to study the multi-site phosphorylation state of eNOS relative to the domain specific levels of ERs. In control UAECs, total eNOS was predominantly located in the caveolar domain, whereas it was detectable both in the caveolar and non-caveolar domains in estradiol-17β-treated cells. In control UAECs, stimulatory P635eNOS and P1177eNOS were both not detected in any of the fractions whereas inhibitory P114eNOS was detected strictly in the non-caveolar domain. In response to estradiol-17β, stimulatory P635eNOS was detected in all cellular domains whereas stimulatory P1177eNOS was detected mainly in the caveolar domain and barely visible in the non-caveolar domain. The inhibitory P114eNOS was decreased by estrogen to low levels in the non-caveolar domain and was not detected in the caveolar domain. In control UAECs, ERs were detected in a higher abundance in the non-caveolar domain compared to the caveolar domain. estradiol-17β did not alter the distribution of the ERs. Estradiol-17β produces temporal and spatial re-partitioning of eNOS from the caveolar to non-caveolar domains and alters its multi-site phosphorylation state and their distributions in UAECs. Furthermore, since estradiol-17β actions are not calcium mediated, the striking similarities of these results to the ATP-responses illustrate alternative activation pathways of calcium mobilizing agonists. These findings may be critical to understand estradiol-17β-induced gestational vascular adaptations. NIH HL49210, HD38843, HL87144, HL70562, R25GM083252. (platform)

Unilateral Effects of Pregnancy on Differential Expression of the Gap Junction Proteins Connexin 43 and 37 in Ovine Uterine Artery Endothelium.

Unilateral Effects of Pregnancy on Differential Expression of the Gap Junction Proteins Connexin 43 and 37 in Ovine Uterine Artery Endothelium.

Select Nutrients in the Ovine Uterine Lumen. VII. Effects of Arginine, Leucine, Glutamine, and Glucose on Trophectoderm Cell Signaling, Proliferation, and Migration1

Histotroph is required for survival and development of ovine conceptuses (embryo and extraembryonic membranes). Results from our laboratory indicate that arginine (Arg), leucine (Leu), glutamine (Gln), and glucose increase in the uterine lumen between Days 10 and 15 of pregnancy, coincident with increases in expression of amino acid and glucose transporters by uterine epithelia as well as trophectoderm and yolk sac of conceptuses and elongation of the conceptus trophectoderm. Therefore, we hypothesized that Arg, Leu, Gln, and glucose have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary ovine trophectoderm (oTr) cells isolated from Day 15 conceptuses were serum-starved for 24 h in a customized medium, deprived of select nutrients, and then treated with either Arg, Leu, Gln, or glucose. Western blot analyses of whole oTr cell extracts revealed that Arg, Leu, and glucose, but not Gln, increased phosphorylated AKT1 by 2.8-, 2.5-, and 1.8-fold, respectively, within 15 min, and the increase was maintained to 60 min. Arg, Leu, and glucose also stimulated increases in phosphorylated ribosomal protein S6K (pRPS6K) by 4.2-, 4.7-, and 2.3-fold, respectively, within 15 min, as well as increases in phosphorylated ribosomal protein S6 (pRPS6) between 0 and 30 min posttreatment, that were sustained to 60 min. When oTr cells were treated with Arg, pRPS6K protein increased in nuclei, but this was not observed in nuclei of oTr cells treated with Leu and glucose. Immunocytochemical analyses also revealed abundant amounts of pRPS6 protein in the cytoplasm of oTr cells treated with Arg, Leu, and glucose. Furthermore, Arg and Leu increased proliferation and migration of oTr cells. Collectively, these results indicate that Arg, Leu, and glucose, but not Gln, in histotroph coordinately activate AKT1-mechanistic target of rapamycin and RPS6K-RPS6 cell signaling pathways to stimulate hypertrophy, hyperplasia, and migration of oTr cells.

Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function1

Endothelium-mediated vasodilation is specifically enhanced in uterine circulation during pregnancy, and production of nitric oxide (NO) is increased in response to a wide array of agonists. Uterine artery endothelial cells from nonpregnant (NP-UAECs) or pregnant (P-UAECs) ewes maintained in culture still show a pregnancy-enhanced difference in ATP-stimulated endothelial NO synthase (eNOS; official symbol NOS3) activation, even though NOS3 protein, purinergic receptors, and associated cell signaling proteins are expressed at equal levels. We have also shown that the pregnancy-enhanced endothelial cell NO response to ATP requires an enhanced and sustained capacitative entry phase that is likely mediated via canonical transient receptor potential protein/inositol 1,4,5-trisphosphate receptor type 2 interaction. In this study, we now show by simultaneous video imaging of individual Fura-2-loaded cells that the pregnancy-enhanced capacitative entry phase is not continuous and equal in all cells, but is in fact mediated as a series of periodic [Ca(2+)](i) bursts within individual cells. Not only does pregnancy increase the number of bursts over a longer time period in individual cells, but also a greater proportion of cells exhibit this burst activity, and at high cell density this occurs in a synchronous manner. The mediator of cell synchronization is connexin 43 (Cx43) gap junctions because 1) Cx43 is readily detectable by Western blot analysis in UAECs, whereas Cx40 and Cx37 are weakly detected or absent, and 2) pregnancy-specific enhancement of [Ca(2+)](i) bursts by ATP is blocked by inhibitory loop peptides selective to Cx43 ((43,37)GAP27) but not by a scrambled control peptide or (40)GAP27 or (40,37)GAP26 peptides, which are specific to Cx40 or Cx37. The relationship between Ca(2+) bursts and NOS3 activation is further established by the finding that (43,37)GAP27 inhibits ATP-stimulated NOS3 activation but has no effect on cell mitogenesis. We conclude that it is pregnancy-enhanced gap junction communication between cells that underlies pregnancy enhancement of capacitative entry via TRPC3 and, in turn, NOS3 activation. Such improved gap junction function allows greater and more sustained [Ca(2+)](i) responses to agents such as ATP within a single cell, as well as the additional recruitment of greater numbers of cells to the response in a coordinated and synchronous manner to support enhanced NO production.

Changes in expression of cell‐cycle‐related genes in PC‐3 prostate cancer cells caused by ovine uterine serpin

The hormonal-regulated serpin, ovine uterine serpin (OvUS), also called uterine milk protein (UTMP), inhibits proliferation of lymphocytes and prostate cancer (PC-3) cells by blocking cell-cycle progression. The present aim was to identify cell-cycle-related genes regulated by OvUS in PC-3 cells using the quantitative human cell-cycle RT(2) Profiler PCR array. Cells were cultured +/-200 microg/ml recombinant OvUS (rOvUS) for 12 and 24 h. At 12 h, rOvUS increased expression of three genes related to cell-cycle checkpoints and arrest (CDKN1A, CDKN2B, and CCNG2). Also, 14 genes were down-regulated including genes involved in progression through S (MCM3, MCM5, PCNA), M (CDC2, CKS2, CCNH, BIRC5, MAD2L1, MAD2L2), G(1) (CDK4, CUL1, CDKN3) and DNA damage checkpoint and repair genes RAD1 and RBPP8. At 24 h, rOvUS decreased expression of 16 genes related to regulation and progression through M (BIRC5, CCNB1, CKS2, CDK5RAP1, CDC20, E2F4, MAD2L2) and G(1) (CDK4, CDKN3, TFDP2), DNA damage checkpoints and repair (RAD17, BRCA1, BCCIP, KPNA2, RAD1). Also, rOvUS down-regulated the cell proliferation marker gene MKI67, which is absent in cells at G(0). Results showed that OvUS blocks cell-cycle progression through upregulation of cell-cycle checkpoint and arrest genes and down-regulation of genes involved in cell-cycle progression.

Select Nutrients in the Ovine Uterine Lumen. VI. Expression of FK506-Binding Protein 12-Rapamycin Complex-Associated Protein 1 (FRAP1) and Regulators and Effectors of mTORC1 and mTORC2 Complexes in Ovine Uteri and Conceptuses1

FRAP1 (FK506-binding protein 12-rapamycin complex-associated protein 1), a component of the nutrient-sensing cell signaling pathway, is critical for cell growth and metabolism. The present study determined expression of FRAP1 and associated members of the mTORC1 and mTORC2 cell signaling pathways in uteri of cyclic and pregnant ewes and conceptuses, as well as effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression. The mRNAs for FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR, TSC1, TSC2, RHEB, and EIF4EBP1 were localized to luminal, superficial glandular, and glandular epithelia and stromal cells of uteri from cyclic and pregnant ewes, as well as trophectoderm and endoderm of conceptuses between Days 13 and 18 of pregnancy. The abundance of FRAP1, RAPTOR, RICTOR, TSC1, and TSC2 mRNAs in endometria was unaffected by pregnancy status or by day of the estrous cycle or pregnancy; however, levels of LST8, MAPKAP1, RHEB, and EIF4EBP1 mRNA increased in endometria during early pregnancy. In ovariectomized ewes, P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Total endometrial FRAP1 protein and phosphorylated FRAP1 protein levels were affected by pregnancy status and by day after onset of estrus, and phosphorylated FRAP1 protein was detected in nuclei of uterine epithelia and conceptuses. In endometria of pregnant ewes, increases in abundance of mRNAs for RICTOR, RHEB, and EIF4EBP1, as well as RHEB protein, correlated with rapid conceptus growth and development during the peri-implantation period. These results suggest that the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptuses and that P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1.

Select Nutrients in the Ovine Uterine Lumen. V. Nitric Oxide Synthase, GTP Cyclohydrolase, and Ornithine Decarboxylase in Ovine Uteri and Peri-Implantation Conceptuses1

Nitric oxide (NO) and polyamines are critical for implantation and development of conceptuses (embryo and extraembryonic membranes), but mechanisms regulating their biosynthesis in uteri and conceptuses are largely unknown. This study determined the effects of the estrous cycle, pregnancy, progesterone, and interferon tau (IFNT) on expression of NO synthases (NOS1, NOS2, and NOS3), guanosine triphosphate (GTP) cyclohydrolase (GCH1, the key enzyme in de novo synthesis of tetrahydrobiopterin, a cofactor for NO production), and ornithine decarboxylase (ODC1) in uterine endometria in cyclic ewes (Days 10-16) and pregnant ewes (Days 10-20). The mRNAs and proteins for NOS1 and ODC1 were most abundant in uterine luminal (LE) and superficial glandular (sGE) epithelia, and abundance was affected by day of estrous cycle and early pregnancy. NOS2, GCH1, and NOS3 mRNAs were detected in very low abundance in uterine epithelia and stromal cells in both cyclic and pregnant ewes. NOS1 mRNA also was expressed very weakly in conceptuses, whereas NOS3 mRNA was abundant in the trophectoderm and endoderm of conceptuses, as were total NOS1 and NOS3 proteins, inhibitory p-NOS1 protein, and stimulatory p-NOS3 protein. GCH1 mRNA was abundant in the trophectoderm and endoderm of conceptuses between Days 13 and 15 of pregnancy and then decreased thereafter, whereas ODC1 mRNA abundance increased in conceptuses between Days 13 and 18 of pregnancy. GCH1 protein was localized primarily in the nuclei of trophectoderm and endoderm, and its abundance decreased after Day 14 of pregnancy, whereas ODC1 protein was more abundant in the trophectoderm than in the endoderm between Days 13 and 18 of pregnancy. Progesterone stimulated NOS1 and GCH1 expression in LE/sGE and glandular epithelia, whereas IFNT inhibited NOS1 expression in these cell types. Thus, biosynthesis of NO and polyamines in ovine uterine endometria and conceptuses is potentially regulated at transcriptional, translational, and posttranslational levels to favor conceptus development and implantation.