Effects of the estrous cycle, pregnancy, and interferon tau on 2',5'-oligoadenylate synthetase expression in the ovine uterus

The enzymes which comprise the 2',5'-oligoadenylate synthetase (OAS) family are interferon (IFN) stimulated genes which regulate ribonuclease L antiviral responses and may play additional roles in control of cellular growth and differentiation. This study characterized OAS expression in the endometrium of cyclic and pregnant ewes as well as determined effects of IFNtau and progesterone on OAS expression in cyclic or ovariectomized ewes and in endometrial epithelial and stromal cell lines. In cyclic ewes, low levels of OAS protein were detected in the endometrial stroma (S) and glandular epithelium (GE). In early pregnant ewes, OAS expression increased in the S and GE on Day 15. OAS expression in the lumenal epithelium (LE) was not detected in uteri from either cyclic or pregnant ewes. Intrauterine administration of IFNtau stimulated OAS expression in the S and GE, and this effect of IFNtau was dependent on progesterone. Ovine endometrial LE, GE, and S cell lines responded to IFNtau with induction of OAS proteins. In all three cell lines, the 40/46-kDa OAS forms were induced by IFNtau, whereas the 100-kDa OAS form appeared to be constitutively expressed and not affected by IFNtau. The 69/71-kDa OAS forms were induced by IFNtau in the S and GE cell lines, but not in the LE. Collectively, these results indicate that OAS expression in the endometrial S and GE of the early pregnant ovine uterus is directly regulated by IFNtau from conceptus and requires the presence of progesterone.     

Detection of bradykinin and bradykinin-beta(2) receptors in the porcine endometrium during the estrous cycle and early pregnancy

During the period of attachment of the trophectoderm to the uterine lumenal surface in the pig, there is an increase in uterine blood flow and a localized hyperemic response induced by the developing conceptuses. The presence of tissue kallikrein in the porcine uterine lumen suggests that the kallikrein-kinin system may be functional during pregnancy in the pig. The objective of the present study was to determine the concentration of bradykinin within the uterine lumen during the estrous cycle and early pregnancy as well as endometrial gene expression and cellular localization of the bradykinin beta(2) receptor. Concentration of bradykinin in uterine flushings was greatest during estrus (Day 0) and Days 12-18 of the estrous cycle. However, there was a 5- to 10-fold increase in bradykinin content in pregnant uterine flushings on Days 12-18 of pregnancy compared with the estrous cycle. Endometrial bradykinin beta(2) receptor gene expression was greatest on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy as gene expression decreased almost 6-fold on Days 5 and 10. Bradykinin beta(2) receptors were detected in the endometrial surface and glandular epithelium with greatest intensity of staining observed on Days 0, 12, 15, and 18 of the estrous cycle and pregnancy. Results from the present study suggest that the kallikrein-kinin system plays a role in the establishment of pregnancy in the pig.     

Effects of recombinant ovine interferon tau, placental lactogen, and growth hormone on the ovine uterus

Studies were conducted to determine effects of intrauterine administration of recombinant ovine interferon tau (IFNtau), placental lactogen (PL), and growth hormone (GH) on endometrial function. In the first study, administration of IFNtau to cyclic ewes for one period (Days 11-15) resulted in an interestrous interval (IEI) of approximately 30 days, whereas administration for two periods (Days 11-15 and Days 21-25) extended the IEI to greater than 50 days. Administration of IFNtau from Days 11 to 15 and of PL or GH from Days 21 to 25 failed to extend the IEI more than for IFNtau alone. In the second study, effects of IFNtau, PL, and GH on endometrial differentiation and function were determined in ovariectomized ewes receiving ovarian steroid replacement therapy. Endometrial expression of mRNAs for estrogen receptor (ER), progesterone receptor (PR), and oxytocin receptor (OTR) were not affected by PL or GH treatment; however, uterine milk protein mRNA levels and stratum spongiosum gland density were increased by both PL and GH treatments. Collectively, results indicated that 1) PL and GH do not regulate endometrial PR, ER, and OTR expression or affect corpus luteum life span; 2) down-regulation of epithelial PR expression is requisite for progesterone induction of secretory gene expression in uterine glandular epithelium; 3) effects of PL and GH on endometrial function require IFNtau; and 4) PL and GH regulate endometrial gland proliferation and perhaps differentiated function.     

Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium

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.     

Oxytocin receptors in the porcine endometrium during the estrous cycle and early pregnancy

Oxytocin (OT) receptors in the porcine endometrium were investigated at four stages of the estrous cycle (Days (D) 0, 5, 10 and 15, n = 3), and at two stages of early pregnancy (D5 and D15 after mating, n = 3) by a radioreceptor assay using ¹²⁵I-labeled OT antagonist [d(CH₂)₅,Tyr(Me)²,Thr⁴,Tyr-NH⁹₂]-vasotocin. Binding specificity was demonstrated by displacement with four peptides related to oxytocin ([Arg⁷]-vasopressin, [Thr⁴,Gly⁷]-OT, OVT, OT) and two peptides unrelated to oxytocin (luteinizing hormone-releasing hormone, [Ile³]-pressinoic acid (tocinoic acid)). The dissociation constant (K_d) of endometrial OT receptors on D0 (0.59 ± 0.10 nM) was similar to those on D10 and D15 (D10, 0.75 ± 0.21; D15, 0.60 ± 0.14 nM; mean ± SEM). In the early luteal stage (D5), K_d (2.41 ± 0.24 nM) was higher than on D0, D10 and D15 (P < 0.01). In early pregnancy, K_d values were 3.25 ± 0.29 nM on D5 and 2.44 ± 0.44 nM on D15. Binding site concentration (B_max) on D0 (910.0 ± 25.1 fmol mg⁻¹ protein) was significantly higher than on D5 and D10 (D5, 322.5 ± 71.7; D10, 147.5 ± 25.8 fmol mg⁻¹ protein; P < 0.01) of the estrous cycle and D5 and D15 (D5, 302.5 ± 82.6; D15, 315.0 ± 20.1 fmol mg⁻¹ protein; P < 0.01) of early pregnancy. In the two stages of early pregnancy, B_max values were constant and similar to that on D5 of the early luteal stage.Our results reveal the existence of specific OT binding sites in the porcine endometrium during the estrous cycle and early pregnancy. Furthermore, the fluctuation in the binding of OT to the endometrium during the different stages of the estrous cycle suggests that OT plays an important role in regulating the estrous cycle of the pig as seen in other animals.     

Oxytocin-stimulated inositol phosphate turnover in endometrium of ewes is influenced by stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins

Three experiments (Exp) assessed the influence of stage of the estrous cycle, pregnancy, and intrauterine infusion of ovine conceptus secretory proteins (oCSP) on turnover of inositol trisphosphate (the putative second-messenger for oxytocin-stimulated secretion of prostaglandin F2 alpha) in ovine endometrium during luteolysis and maternal recognition of pregnancy. In Exp 1, endometrium was collected from 5 cyclic (Cy) and 6 pregnant (P) ewes on Day 16 after onset of estrus. In Exp 2, endometrium was collected from Day 12 Cy (n = 5), Day 12 P (n = 3), Day 16 Cy (n = 4), and Day 16 P (n = 3) ewes. In Exp 3, 12 Cy ewes were allotted randomly, in a 2 x 2 factorial arrangement, to receive serum protein (SP), or oCSP and estradiol-17 beta (E2), or vehicle treatments. Ewes were injected i.v. with 0.5 mg E2 or vehicle on Day 12 and received twice-daily infusions of 1.5 mg SP or oCSP (containing 25 micrograms ovine trophoblast protein-1 by radioimmunoassay [RIA]) + SP (1.5 mg total protein) into each uterine horn on Days 12, 13, and 14. Blood samples for RIA of plasma progesterone were collected on Days 10-15 (before treatment on each day) and endometrium was collected on Day 15. For each Exp, 100 mg endometrium was incubated, in duplicate, for 2 h with 10 microCi [3H] inositol and treated with 0 or 100 nM oxytocin (OT) for 20 min, then [3H]inositol mono-, bis-, and trisphosphates (IP1, IP2, and IP3, respectively) were quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of intrauterine and subcutaneous administration of recombinant ovine interferon tau on the endometrium of cyclic ewes.

Interferon tau (IFNtau) is the antiluteolytic signal produced by the conceptus of ruminants. Intrauterine administration of recombinant ovine IFNtau suppresses expression of endometrial estrogen receptor (ER) and oxytocin receptor (OTR) in the luminal and superficial glandular epithelia to abrogate the production of luteolytic prostaglandin F(2alpha) (PGF(2alpha)) pulses. Subcutaneous (s.c.) injections of recombinant ovine (o) IFNtau appear to extend the interestrous interval by altering uterine PGF(2alpha) response to oxytocin. The present study tested the hypothesis that antiluteolytic effects of roIFNtau injected into the uterine lumen (paracrine) or s.c. (endocrine) are equivalent in suppressing expression of endometrial ER and OTR and inducing uterine expression of type I IFN-regulated Mx and ubiquitin cross-reactive proteins (UCRP). Sixteen cyclic ewes were fitted with uterine catheters on Day 5 (Day 0 = estrus), were assigned randomly to receive treatment with control proteins or roIFNtau (2 x 10(7) antiviral units/day) by either intrauterine or s.c. injections from Days 11 to 15, and were ovariohysterectomized on Day 16. Results indicated that expression of ER and OTR mRNAs in endometrial epithelium was suppressed by intrauterine but not by s.c. injections of roIFNtau. Intrauterine injections of roIFNtau increased expression of Mx and UCRP mRNA in the endometrium. Subcutaneous injections of roIFNtau increased endometrial Mx mRNA levels but not UCRP mRNA. Unexpectedly, intrauterine and s.c. injections of roIFNtau were equally effective in inducing expression of Mx and UCRP mRNA in the corpus luteum. Although s.c. injections of roIFNtau induced Mx mRNA in the endometrial epithelium, s.c. injections of roIFNtau did not abrogate activation of the uterine luteolytic mechanism by suppressing epithelial ER and OTR expression. Therefore, results of this study failed to support the assumption that endocrine roIFNtau mimics antiluteolytic effects of paracrine IFNtau to improve pregnancy rates in sheep.     

Comparison of the effects of early pregnancy with human interferon, alpha 2 (IFNA2), on gene expression in bovine endometrium

Interferon tau (IFNT), a type I IFN similar to alpha IFNs (IFNA), is the pregnancy recognition signal produced by the ruminant conceptus. To elucidate specific effects of bovine IFNT and of other conceptus-derived factors, endometrial gene expression changes during early pregnancy were compared to gene expression changes after intrauterine application of human IFNA2. In experiment 1, endometrial tissue samples were obtained on Day (D) 12, D15, and D18 postmating from nonpregnant or pregnant heifers. In experiment 2, heifers were treated from D14 to D16 of the estrous cycle with an intrauterine device releasing IFNA2 or, as controls, placebo lipid extrudates or PBS only. Endometrial biopsies were performed after flushing the uterus. All samples from both experiments were analyzed with an Affymetrix Bovine Genome Array. Experiment 1 revealed differential gene expression between pregnant and nonpregnant endometria on D15 and D18. In experiment 2, IFNA2 treatment resulted in differential gene expression in the bovine endometrium. Comparison of the data sets from both studies identified genes that were differentially expressed in response to IFNA2 but not in response to pregnancy on D15 or D18. In addition, genes were found that were differentially expressed during pregnancy but not after IFNA2 treatment. In experiment 3, spatiotemporal alterations in expression of selected genes were determined in uteri from nonpregnant and early pregnant heifers using in situ hybridization. The overall findings of this study suggest differential effects of bovine IFNT compared to human IFNA2 and that some pregnancy-specific changes in the endometrium are elicited by conceptus-derived factors other than IFNT.     

Soybean-derived phytoestrogens regulate prostaglandin secretion in endometrium during cattle estrous cycle and early pregnancy

Phytoestrogens acting as endocrine disruptors may induce various pathologies in the female reproductive tract. The purpose of this study was to determine whether phytoestrogens present in the soybean and/or their metabolites are detectable in the plasma of cows fed a diet rich in soy and whether these phytoestrogens influence reproductive efficiency and prostaglandin (PG) synthesis during the estrous cycle and early pregnancy in the bovine endometrium. In in vivo Experiment 1, we found significant levels of daidzein and genistein in the fodder and their metabolites (equol and p-ethyl-phenol) in bovine serum and urine. The mean number of artificial inseminations (AIs) and pregnancy rates in two kinds of herds, control and experimental (cows fed with soybean 2.5 kg/day), were almost double in the soy-diet herd in comparison with the control animals. In in vivo Experiment 2, three out of five heifers fed soybean (2.5 kg/day) became pregnant whereas four out of five heifers in the control group became pregnant. The concentrations of a metabolite of PGF2alpha (PGFM) were significantly higher in the blood plasma of heifers fed a diet rich in soybean than those in the control heifers throughout the first 21 days after ovulation and AI. The higher levels of PGFM were positively correlated with equol and p-ethyl phenol concentrations in the blood. In in vitro experiments, the influence of isoflavones on PG secretion in different stages of the estrous cycle was studied. Although all phytoestrogens augmented the output of both PGs throughout the estrous cycle, equol and p-ethyl-phenol preferentially stimulated PGF2alpha output. The results obtained lead to the conclusion that soy-derived phytoestrogens and their metabolites disrupt reproductive efficiency and uterus function by modulating the ratio of PGF2alpha to PGE2, which leads to high, nonphysiological production of luteolytic PGF2alpha in cattle during the estrous cycle and early pregnancy.     

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.     

Presence of the acute phase protein, bikunin, in the endometrium of gilts during estrous cycle and early pregnancy

Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-alpha-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12-18). However, bikunin unbound to the inter-alpha-trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.