Endometrial prostaglandin E2-binding sites in the pig: characterization and changes during the estrous cycle and early pregnancy

Because prostaglandins (PGs) are thought to mediate, in part, endometrial responses at implantation in a number of species and because pig conceptuses are capable of producing PGs, we have characterized the binding of [3H]PGE2 to endometrium obtained from pigs during the estrous cycle and early pregnancy. Binding of [3H]PGE2 to a membrane preparation obtained from endometrium was temperature- and pH-dependent, and reversible. Pretreatment of the membrane preparation with proteolytic enzymes, phospholipase A2, or heat reduced subsequent specific binding of [3H]PGE2. PGE1 and PGE2 competed equally for binding, whereas relative cross-reactivity for other prostanoids and compounds tested was less than 5%. Scatchard analysis of specific binding suggested that two classes of binding sites were present, a high-affinity binding site with an apparent dissociation constant (Kd) of approximately equal to 6 pM, and a lower-affinity site with an apparent Kd of approximately equal to 3 nM. For the high-affinity binding site, estimates of apparent Kd did not differ between days of the estrous cycle, but maximum binding capacity did change, being lowest on Day 10 and highest on Days 0-5 of the cycle. For the lower-affinity binding site, estimates of the apparent Kd differed, being lowest on Day 10 and highest on Day 15; numbers of binding sites were lowest on Day 5 and highest on Day 15. Estimates of these variables on Day 15 of pregnancy did not differ significantly from Day 15 of the cycle. These data are consistent with the presence of PGE receptors within the endometrium of the pig.     

Characterization of temporal and cell-specific changes in transcripts for prostaglandin E(2) receptors in pseudopregnant rat endometrium

In the rodent uterus, prostaglandin E(2) (PGE(2)) is believed to have a major role in implantation and decidualization. The present study investigated the temporal and hormonal control of mRNA expression for the four E-prostanoid (EP(1-4)) receptors in the rat endometrium. For Northern blot analysis and in situ hybridization, samples were obtained from rats on Days 1-10 of pseudopregnancy or from rats differentially sensitized for the decidual cell reaction with estradiol. No EP(1) mRNA signal was detected. Endometrial EP(2) and EP(3) mRNA levels increased to a maximum on Day 5, and the mRNAs were localized to the luminal epithelium at the antimesometrial pole, and in the endometrial stroma and glandular epithelium, respectively. Endometrial EP(4) mRNA levels were unchanged on Days 1-5, but the mRNA was concentrated in the antimesometrial endometrial stroma on Day 5. Cell-specific expression of EP(2), EP(3), and EP(4) on Day 5 was dependent upon a dose of estradiol given on Day 4 that induced differential uterine sensitization on Day 5. After the application of a deciduogenic stimulus on Day 5, mRNA levels for these receptors decreased significantly, while in nonstimulated horns they remained elevated. Overall, these results support a role for PGE(2) in the onset of receptivity and initiation of decidualization in the rat.     

Uterine progesterone metabolism during early pseudopregnancy in the rat

We measured uptake and metabolism of progesterone (P4) during the estrous cycle and Days 2-6 of pseudopregnancy (PSP) to determine uterine P4 dynamics during preimplantation. Rats were infused with [3H]P4 for 60 min, blood was obtained, the uterus was removed, and endometrium and myometrium were isolated. Tissue radiolabeled P4 and P4 metabolites (5 alpha-pregnane-3,20-dione, DHP; 20 alpha-hydroxy P4; 17 alpha-hydroxy P4, and hydroxylated DHP derivatives) were extracted and separated by thin-layer chromatography (TLC). Serum P4 was measured by radioimmunoassay (RIA) in another group of rats. Endometrial and myometrial concentrations of [3H]P4 were greater (p less than 0.05) than plasma values. In contrast, [3H]DHP levels in the endometrium were higher (p less than 0.01) than values in myometrium or plasma. Compared to values in the estrous cycle, endometrial ratios of [3H]DHP/[3H]P4 and [3H]metabolites/[3H]P4 decreased (p less than 0.02) on Days 3-5 of PSP. Serum P4 levels during the estrous cycle (13-25 ng/ml) increased (p less than 0.01) to 120 ng/ml on Days 3-5 of PSP. Estimated concentration of P4 in the endometrium during the estrous cycle (90 ng/g) increased (p less than 0.05) to 580 ng/g by Day 5 of PSP. Similar observations were noted for the estimated endometrial concentrations of DHP and all P4 metabolites. We suggest that both endometrium and myometrium take up and metabolize P4 during the estrous cycle and early PSP. However, endometrial P4 metabolism during PSP is greater than during the estrous cycle, in part because of increased ovarian secretion and endometrial concentration of P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mifepristone (RU 486) on concentrations of prostaglandin E-2 binding sites in the rat endometrium

Progesterone implants in ovariectomized rats increased endometrial concentrations of PGE-2 receptors. The increase was completely inhibited by simultaneous daily injection (7.5 mg/kg) of mifepristone (RU 486). A single injection of mifepristone on the morning of Day 1 of pseudopregnancy (day of oestrus) decreased the amount of PGE-2 receptors found in the endometrium on Day 5 by 64%. This inhibitory effect probably resulted from the antiprogesterone activity of this compound since it was not counteracted by simultaneous treatment with dexamethasone, shown to reverse totally the antiglucocorticoid action of mifepristone. The inhibition by mifepristone lasted only for 1 day; endometrial PGE-2 receptor levels on Day 6 of pseudopregnancy returned to the high values present in controls. Under these conditions, administration of the mifepristone did not affect the plasma oestradiol and progesterone concentrations during the 1st week of pseudopregnancy. The administration of mifepristone on Days 2 and 3 of pseudopregnancy kept the endometrial PGE-2 receptor levels low, even by 4 days after the end of treatment. We therefore concluded that, in the rat, progesterone priming leading to uterine receptivity can be delayed, at least by 1 day. In contrast, interruption of the progesterone action for a longer period later during the early pseudopregnant period resulted in an altered subsequent evolution of the endometrium, in terms of acquisition of the PGE-2 binding sites.     

Failure to detect specific binding sites for prostaglandin F-2 alpha in membrane preparations from rat endometrium

Membrane preparations from endometria of rats in different physiological states (e.g. pseudopregnancy, ovariectomized animals receiving progesterone + oestradiol or oestradiol alone) were studied for [3H]PGF-2 alpha binding by methods which detected PGF-2 alpha binding in ovary preparations and PGE binding in the same endometrial preparations. There was no evidence of high-affinity binding sites for [3H]PGF-2 alpha. Saturable [3H]PGF-2 alpha binding that increased with the onset of uterine sensitivity was detected but this binding does not fulfil all the criteria required for a PGF-2 alpha receptor and is probably due to binding to PG metabolizing enzymes in our preparations, or to binding of [3H]PGF-2 alpha to PGE binding sites. The failure to detect specific PGF-2 alpha binding sites seems to reflect a true absence of these sites in the rat endometrium.     

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 progesterone treatment on the oxytocin-induced prostaglandin F2 alpha response and the levels of endometrial oxytocin receptors in ovariectomized ewes.

The oxytocin-induced uterine prostaglandin (PG) F2 alpha response and the levels of endometrial oxytocin receptors were measured in ovariectomized ewes after they had been given steroid pretreatment (SP) with progesterone and estrogen to induce estrus (day of expected estrus = Day 0) and had subsequently been treated with progesterone over Days 1-12 and/or PGF2 alpha over Days 10-12 postestrus. The uterine PGF2 alpha response was measured after an i.v. injection of 10 IU oxytocin on Days 13 and 14, using the PGF2 alpha metabolite, 13,14-dihydro-15-keto-PGF2 alpha (PGFM), as an indicator for PGF2 alpha release. The levels of oxytocin receptors in the endometrium were measured on Day 14. During the treatment with progesterone, the peripheral progesterone concentrations were elevated and remained above 1.8 ng/ml until the morning of Day 14. The PGFM responses to oxytocin in untreated controls and SP controls were low on both Days 13 and 14 whereas the levels of endometrial oxytocin receptors in the same ewes were high. Treatment with progesterone either alone or in combination with PGF2 alpha significantly (p less than 0.04) increased the PGFM response on Day 14 and reduced the levels of endometrial oxytocin receptors; treatment with PGF2 alpha alone had no effect. It is concluded that progesterone promotes the PGFM response to oxytocin while simultaneously suppressing the levels of endometrial oxytocin receptors. PGF2 alpha treatment had no effect on either the uterine secretory response to oxytocin or the levels of oxytocin receptors in the endometrium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of day of the oestrous cycle, side of the reproductive tract and heat shock on in-vitro protein secretion by bovine endometrium

Endometrial explant cultures were prepared from 16 Brahman x Angus cows killed on Days 0, 2, 5 or 8 after oestrus. Cultures proceeded for 24 h at 39 degrees C (homeothermic) or 43 degrees C (heat shock) in a modified Eagle's minimal essential medium supplemented with 50 microCi L-[4,5(-3)H]leucine. Analysis by two-dimensional polyacrylamide gel electrophoresis of de-novo synthesized proteins secreted into the medium indicated that the major types of secreted polypeptides did not change over Days 0-8. Nevertheless, overall endometrial secretion of protein (incorporation of [3H]leucine into non-dialysable radioactivity in culture supernatants) was greatest at Day 0 and declined thereafter. Incorporation of [3H]leucine into TCA-precipitable material in tissue homogenates was also greatest at Day 0. For tissue cultured at 39 degrees C, several individual polypeptides were secreted at greater rates by endometrium from the horn of the uterus ipsilateral to the corpus luteum, with side differences tending to be greatest at Day 0 or Day 2. Overall, secretion of de-novo synthesized protein by endometrium was significantly elevated by heat shock at Day 0, but not affected thereafter. Nonetheless, heat shock reduced secretion of several individual proteins and exhibited interactions with day of the oestrous cycle and with side of the uterus. Secretion of 7 polypeptides was reduced by heat shock in tissue from the ipsilateral horn of the uterus but not in endometrium from the contralateral horn. We suggest that endometrial protein secretion changes quantitatively during the early oestrous cycle. In addition, there is a local influence of the ovary bearing the corpus luteum on endometrial function that may be disrupted by heat shock.     

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.     

Spatial and temporal patterns of deoxyribonucleic acid synthesis and mitosis in the endometrial stroma during decidualization in the pseudopregnant rat

A quantitative study was made of the spatial patterns of stromal cell mitosis and DNA synthesis in the endometrium of the pseudopregnant rat before and during decidualization. A colchicine block was used for mitotic counts, and DNA synthesis was studied by [3H] thymidine autoradiography. Observations were also made on the subsequent fates of [3H] thymidine-labeled stromal cells. Before the onset of decidualization, on Days 3 and 4 (vaginal cornification = Day 0), mitosis was largely confined to the subepithelial stroma along the sides and around the antimesometrial pole of the lumen. [3H] thymidine labeling and stromal mitosis following a decidualizing stimulus at noon on Day 4 of pseudopregnancy were first seen close to the uterine lumen, with subsequent spread to deeper layers of the endometrium. At noon on Day 5, mitotic figures were numerous on all sides of the lumen and at all depths in the endometrium. At later stages, mitosis and the development of polyploidy continued in the decidual tissue, but little DNA synthesis or mitosis occurred in the basal zone of the stroma adjacent to the myometrium. In this zone, many cells in animals given [3H] thymidine 18 to 24 h after induction of decidualization remained heavily labeled throughout the growth and regression of deciduomata. Labeled cells derived from the basal zone and outer edge of the decidual capsule were present in the stroma of the regenerated endometrium following the regression of deciduomata. It was concluded that although cells at all depths in the endometrial stroma undergo DNA synthesis and mitosis in the early stages of response to a decidualizing stimulus, their subsequent behavior and fate depend upon their position in the endometrium.     

Constitutive expression of uterine receptors for insulin-like growth factor-I during the peri-implantation period in the pig.

Insulin-like growth factor-I (IGF-I), synthesized by the uterine endometrium of cyclic and early pregnant gilts, accumulates in the uterine luminal fluid, where it comes in contact with the developing conceptus and the rapidly growing uterus. The uterus and the conceptus thus represent potential target sites for the biological effects of IGF-I, provided high-affinity Type I receptors are present. This study was undertaken to evaluate the expression of functional IGF-I receptors in the endometrium and myometrium of pregnant (Day 10, 12, and 15) gilts and in the endometrium of cyclic (Day 15) and pseudopregnant (Day 15) gilts and to correlate levels of these receptors with temporally regulated uterine production of IGF-I. Specific binding of 125I-IGF-I to endometrial membranes pretreated with MgCl2 (4 M) at 4 degrees C for 16 h, was saturable and membrane concentration-dependent. Competition of 125I-IGF-I binding to endometrial membranes was highest with unlabeled IGF-I greater than IGF-II much greater than insulin, whereas porcine relaxin was noncompetitive. Affinity cross-linking of endometrial membranes with 125I-IGF-I followed by SDS-PAGE and autoradiography revealed two labeled bands of Mr greater than 200,000 and Mr 135,000, with the major band being the Mr 135,000 species. Scatchard analysis of 125I-IGF-I binding to endometrial membranes from Day 12 pregnant gilts revealed a single class of binding sites with a dissociation constant (Kd) = 4.08 +/- 0.09 nM. Membranes prepared from endometrium of Day 10, 12, and 15 pregnant gilts exhibited comparable 125I-IGF-I binding (p greater than 0.05) that was higher (p less than 0.001) than that for the corresponding myometrial membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endometrial surface and secretory alterations associated with embryonic mortality in gilts administered estradiol valerate on days 9 and 10 of gestation

Administration of estrogen to gilts on Days 9 and 10 of pregnancy results in total embryonic loss by Day 18. The present study examined changes in the uterine endometrial surface and secretion during conceptus attachment in control and estrogen-treated (Days 9 and 10) pregnant gilts. Gilts were unilaterally hysterectomized on either Days 12 and 14 or Days 16 and 18 of gestation. Uterine horns were flushed with saline and conceptuses were evaluated. Intact conceptuses were recovered from all control gilts, whereas estrogen-treated gilts contained normal intact conceptuses only on Day 12 of gestation. Antiviral activity, which reflects conceptus viability, was reduced (p less than 0.01) in uterine flushings after Day 14 in estrogen-treated gilts. Culture of endometrial explants with [3H]glucosamine revealed several glycoproteins that are synthesized during the period of conceptus attachment; however, no difference in glycoprotein synthesis between treatment groups was detected by analysis with two-dimensional PAGE and fluorography. Analyses of the uterine epithelium by scanning and transmission electron microscopy demonstrated that estrogen administration caused an alteration in the uterine surface, a thinning of the uterine epithelial glycocalyx, and a reduction of cationic ferritin binding to the microvilli of the uterine epithelium. Results indicate that conceptus mortality after early administration of estrogen is associated with alterations in the uterine endometrial surface during the period of conceptus attachment in the pig.     

Steroid metabolism by endometrial and conceptus tissues during early pregnancy and pseudopregnancy in gilts

Endometrial and conceptus tissues were obtained on Days 10.5, 11, 12, 16 and 25 of pregnancy and Day 25 of pseudopregnancy of gilts and incubated for 6 h in Minimal Essential Medium (5 ml) containing 35 ng [3H]progesterone. Metabolism of [3H]progesterone to oestrone, oestradiol and oestriol was determined by gas and high-pressure liquid chromatography and successive recrystallizations with unlabelled standards. Conceptuses collected between Days 10.5 and 12 were spherical, tubular or filamentous and incubated with 500 mg endometrium and [3H]progesterone. Production of oestrone by spherical conceptuses was not detected, but was 44-47 pg/tubular conceptus and 21 pg/filamentous conceptus. A similar trend was observed for oestradiol. Conceptus tissues from Days 16 and 25 (chorion) were most active in producing oestrone (123 and 520 pg/mg tissue, respectively) and oestradiol (277 and 876 pg/mg tissue, respectively). Endometrial oestrogen production was less than that for conceptus tissue for oestrone and oestradiol on Days 16 and 25 of gestation. Coincubations of endometrium and conceptus tissues had lower oestrogen production than conceptus alone. Endometrium from Day 25 of pseudopregnancy metabolized [3H]progesterone to several non-polar metabolites, but no oestrogens were detected. An unidentified phenolic metabolite of [3H]progesterone was detected in higher quantities than either oestrone or oestradiol; 445 to 461 pg/conceptus at the tubular stage. These results indicate temporal changes in the conversion of [3H]progesterone to oestrogens by conceptus and endometrial tissue from pregnant gilts, but not endometrium from pseudopregnant gilts.     

Prostaglandin translocation from the lumen of the rabbit uterus in vitro in relation to day of pregnancy or pseudopregnancy

Transport of 3H-labeled prostaglandins (PGs) E2 and F2 alpha from the uterine lumen across the uterine wall has been studied in rabbit uteri in vitro in incubations lasting up to 180 min, in relation to sexual state of the rabbit, incubation temperature, intraluminal PG concentration, addition of metabolic inhibitors and time of incubation. PG accumulation by the tissue increased rapidly up to 30 min and then remained relatively constant. By 30 min, radioactivity was found in the external incubation medium, and this increased linearly with time. The translocation of PGF2 alpha was significantly greater in pseudopregnant than in pregnant animals on Day 6, whereas that of PGE2 was significantly higher in pregnant than in pseudopregnant animals on Day 6.8. In pregnant animals, both PGF2 alpha and PGE2 were translocated to the exterior more rapidly on Day 6.8 than on Days 5 or 6. Transport of PGs was reduced by low temperature, unaffected by metabolic inhibitors and only that of PGE2 increased with increased (5 microM) intraluminal concentrations. During incubation, the tissue remained viable as judged by T/M ratios (dpm tissue/dpm medium) for 204 thallium. Transport of [14C] sucrose was much slower than that of [14C] urea, which was greater than the fastest rates exhibited by the PGs. In general, amounts of radioactivity found in antimesometrial, mesometrial and lateral portions of the uterine wall, or in implantation and interimplantation areas did not differ, but more was found in the endometrium than the myometrium. PGF2 alpha was translocated unmetabolized to the external medium, while only two-thirds of the PGE2 was translocated unchanged, and one-third converted to PGF2 alpha. It is concluded that the rabbit uterus shows some selectivity in handling PGs in relation to stage of pregnancy.     

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA-Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR-15b, which inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR-15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.     

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.     

Effect of oestrous cycle and early pregnancy on uterine production and expression of immune regulatory factors in gilts

This study was undertaken to characterize uterine immune factors involved in the establishment of pregnancy in gilts. Thirty crossbred Yorkshire-Landrace gilts of similar age and weight were observed twice a day for oestrous behaviour with intact boars. On the day of first standing oestrus (Day 0) and 12h later, 15 gilts were inseminated with pooled semen from Duroc boars of proven fertility. Pregnant gilts were slaughtered either on Days 10, 15 or 25 of gestation (n=5 per day). The other 15 gilts were not inseminated and were slaughtered on either Days 0, 10 or 15 of the oestrous cycle (n=5 per day). Immediately after slaughter, endometrial tissue samples from the mesometrial side were removed for gene expression using RNase protection assay and in situ hybridization methodologies. The other uterine horn was flushed with 20 ml of PBS to collect the uterine fluid. In pregnant gilts, endometrial interleukin (IL)-6 mRNA expression was higher on Day 15 than on Days 10 and 25 (P<0.01 and P<0.1, respectively). On Day 15, IL-6 expression was also significantly higher (P<0.01) in pregnant gilts than in cyclic gilts. In both pregnant and cyclic gilts, transforming growth factor (TGF)-beta2 in uterine fluid was significantly higher (P<0.0001) on Day 15 than on Day 10. At the gene expression level, TGF-beta2 also increased between Days 10 and 15 in both cyclic and pregnant gilts but differences were not significant. On Day 15, concentrations of interferon-gamma and prostaglandin E(2) (PGE(2)) in uterine fluid were markedly higher (P<0.001) in pregnant gilts than in cyclic gilts, whereas the total amount of TGF-beta2 in uterine fluid and its endometrial expression were approximately 70% higher although this increase was not significant. Finally, tumour-necrosis factor-alpha and granulocyte-macrophage/colony-stimulating factor mRNA expressions were undetectable in all endometrial samples. In conclusion, production and/or expression of uterine TGF-beta2, IL-6 and PGE(2) increased during the embryonic attachment period and are coincidental with embryonic interferon-gamma production.     

Regulation of heat shock-induced alterations in the release of prostaglandins by the uterine endometrium of cows

Maternal heat stress in cattle may disrupt pregnancy by elevating uterine prostaglandin F(2alpha) (PGF(2alpha)) secretion. The objectives of this study were to determine the effects of elevated temperature (42 degrees C) in vitro upon 1) prostaglandin secretion by endometrial tissue; 2) the actions of extracellular regulators of uterine PGF [conceptus secretory proteins (bCSPs) and platelet-activating factor, (PAF)]; 3) the activity of the cyclooxygenase-endoperoxidase enzyme complex (PG synthetase); and 4) the activity of the endometrial PG synthesis inhibitor present in the endometrium from pregnant cattle. Endometrial explants at Day 17 of the estrous cycle produced more PGF than PGE(2) while elevated temperature caused increased PGF secretion but did not affect PGE(2) secretion. Elevated temperature did not reduce the ability of bCSPs or PAF to suppress release of PGF. The heat shock-induced increase in PGF at Day 17 was not due to the direct effects on PG synthetase, because PGF production from a cell-free cotyledonary microsomal enzyme preparation was reduced at elevated temperature. The activity of the cytosolic inhibitor of cyclooxygenase present in the endometrium of Day-17 pregnant cows could be reduced but not eliminated at 42 degrees C. We conclude that in vitro heat stress induces PGF secretion from the bovine uterine endometrium at Day 17 after estrus. This increase is not accompanied by the loss of regulatory capacity of conceptus products or increased activity of PG synthetase.     

Insulin-like growth factor-I expression during early conceptus development in the pig

Porcine conceptuses (embryo and associated membranes) in utero undergo developmental morphological transformations coincident with structural and biochemical changes in the uterine endometrium during early gestation. To elucidate a possible role for insulin-like growth factor-I (IGF-I) in these events, porcine endometrial (Days 8, 10, 11, 12, 14, and 30) and conceptus (Days 12, 14, and 16) tissues were characterized for the presence of IGF-I peptide and mRNAs. The corresponding uterine luminal fluids (ULF) at these stages of pregnancy were also analyzed for immunoreactive IGF-I concentration. ULF IGF-I was lowest on Day 8, highest on Day 12, and declined by Day 14. In contrast, endometrial tissue IGF-I content remained constant during this period. Conceptus tissues contained less IGF-I than endometrial tissues; however, conceptus IGF-I values were maximum on Day 12 coincident with peak values for ULF IGF-I. Dot-blot hybridization analyses revealed temporal variation in steady-state levels of IGF-I mRNAs in endometrium. Highest levels of endometrial IGF-I mRNA were detected on Day 12 and were about 4-fold greater than on Day 30 of pregnancy. IGF-I mRNA expression in conceptus tissues on Days 12, 14, and 16 was the same and was significantly less than that in endometrium on Day 12. These results demonstrate the temporal variation of IGF-I mRNA abundance in uterine endometrium and of immunoreactive IGF-I in ULF and in conceptus tissues, with the developmental processes occurring in the conceptuses at early pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)