Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture.

The activation of latent transforming growth factor beta (L-TGFbeta) is essential for the action of TGFbeta, which, in turn, is involved in the regulation of expression of some progesterone-responsive genes. One mechanism by which TGFbeta is activated involves thrombospondin (TSP), a protein that binds extracellular proteins. Immunoreactive TSP (irTSP) protein and TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women were localized by immunohistochemistry and in situ hybridization. IrTSP and TSP-1 mRNA were randomly distributed in myometrial smooth muscle cells of some, but not all, tissues of pregnant women at term before labor; but in some areas of most of these tissues, irTSP was intense and commonly localized extracellularly. Intense irTSP and TSP-1 mRNA in myocytes were more common in myometrium during labor. In myometrium from ovulatory women (n = 26), irTSP was localized primarily in vascular smooth muscle cells and was detected occasionally in scattered myocytes. Little TSP-1 mRNA was demonstrable by in situ hybridization in vessels or myocytes of myometrial tissue from ovulatory women (n = 7). By Northern analysis of total RNA, TSP-1 mRNA was detected in myometrial tissue of pregnant women and in human myometrial smooth muscle cells in culture. The levels of TSP-1 mRNA in myometrial tissues of pregnant women during labor (n = 18) were greater than those in myometrium at > 37 wk gestation before labor began (n = 25, p < 0.001). The ratios of TSP-1 to glyceraldehyde 3-phosphate dehydrogenase mRNAs in 3 myometrial tissues during oxytocin-induced labor were not statistically different from those in myometrium during spontaneous labor but were greater than those in myometrium before labor (p < 0.05). The level of TSP-1 mRNA in confluent human myometrial cells in culture was relatively high, was increased by treatment with fetal bovine serum, and was decreased by treatment with platelet-derived growth factor or activators of adenylyl cyclase or protein kinase C. Myometrial cells in culture constitute a useful model for studying the regulation of TSP-1 gene expression in human myometrium.

Endothelin receptor mRNAs in human fetal membranes, chorionic vessels, and decidua parietalis.

Endothelin-1 (ET-1) is synthesized in avascular human amnion; and, immunoreactive ET-1 is present in amniotic fluid in concentrations 10 to 100 times those in blood. ET-1 acts, most commonly, in a local or paracrine manner; therefore, it is possible that amnion/amniotic fluid ET-1 acts on contiguous tissues, namely chorion laeve or placental surface (chorionic) vessels, or in an autocrine fashion on amnion cells. To address these possibilities, the levels of ET(A) and ET(B) receptor mRNAs were evaluated in amnion, chorion laeve, decidua parietalis, placenta, and chorionic vessel tissues. By Northern analysis of total RNA (20 microg), ET(A) and ET(B) receptor mRNAs were detected in decidua (n = 18), placenta (n = 14), and chorionic vessels (n = 13). In chorion laeve (n = 24), ET(B) receptor mRNA but not ET(A) receptor mRNA was detected by Northern analysis of total RNA. Northern analysis of chorion laeve poly(A)+ mRNA (1.5-2.5 microg) revealed ET(A) receptor mRNA at low levels. Neither ET(A) nor ET(B) receptor mRNAs were detected in amnion tissue by Northern analysis of total RNA (n = 30; placental and reflected amnion from 15 pregnancies) or by Northern analysis of poly(A)+ mRNA (1.5-2.5 microg). Moreover, there was no demonstrable dose-dependent effect of ET-1 on prostaglandin E2 production or DNA synthesis in amnion epithelial cells in primary culture. The findings of this investigation are indicative that ET-1 in amniotic fluid or secreted from amnion may act in a paracrine fashion on chorion laeve by way of the ET(B) receptor and on chorionic vessels by way of ET(A) and ET(B) receptors.

Keratinocyte growth factor expression in the mesenchymal cells of human amnion.

Amnion epithelial and mesenchymal cells were separated by differential protease treatment, and the separated cells were maintained in monolayer culture. Keratinocyte growth factor (KGF) messenger RNA (mRNA) was readily detected by Northern analysis of amnion mesenchymal cell total RNA (10 micrograms) but not in amnion epithelial cells. Treatment of the amnion mesenchymal cells in serum-free medium with tetradecanoyl phorbol acetate (1 nM) caused an increase in the level of KGF mRNA. Forskolin treatment also caused an increase in KGF mRNA but not to the levels attained with tetradecanoyl phorbol acetate treatment. Dexamethasone (1 nM) treatment of these cells effected a reduction in the level of KGF mRNA. Prolonged maintenance of mesenchymal cells in serum-free medium also was associated with an increase in the level of KGF mRNA. Treatment with a variety of other agents, viz., interleukin (IL)-1, IL-6 plus or minus IL-6 soluble receptor, IL-11, oncostatin M, epidermal growth factor (EGF), and transforming growth factor-beta and not modify the level of KGF mRNA. Treatment of amnion epithelial cells with KGF caused an increase in the rate of [3H]thymidine incorporation, but the rate of cell replication induced by KGF was less than that induced by treatment with EGF. Transforming growth factor-beta treatment inhibited basal and EGF- and KGF-stimulated amnion epithelial cell replication. The findings of this study are indicative the KGF is expressed in human amnion mesenchymal cells, and that KGF may act on the epithelial cells of this tissue.

Activation of adenylyl cyclase in human myometrial smooth muscle cells by neuropeptides.

The levels of intracellular cAMP in human myometrial smooth muscle cells in serum-free medium, or medium that contained FBS (1%, vol/vol), were determined after treatment with the homologous peptides, calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), and amylin, without or with added isobutylmethylxanthine (IBMX). These cells were sensitive to CGRP, responding in a dose-dependent manner, with maximal levels of cAMP being attained with 5 nM CGRP in the presence of IBMX (1 mM). In the absence of IBMX, the level of cAMP attained in cells treated with CGRP (5 nM) (675.3 +/- 58.8 pmol.mg protein.15 min; mean +/- SEM, n = 3) was approximately 90x that in nontreated cells (7.5 +/- 0.4 pmol.mg protein.15 min). The level of cAMP in myometrial cells treated with CGRP (5 nM)+IBMX (1 mM), 1998 +/- 420 pmol.mg protein.15 min, was 29x that in cells treated with IBMX alone (69.2 +/- 10.2). The maximum level of cAMP achieved by treatment with ADM+IBMX was similar to that with CGRP+IBMX, but the dose of ADM required (1 microM) was approximately 200x that of CGRP. Amylin amide also caused an increase in cAMP but with considerably less potency; at a concentration of 500 nM, amylin amide+IBMX effected a 2.3-fold increase in cAMP relative to IBMX alone. CGRP8-37, an antagonist of CGRP via the CGRP1 receptor, inhibited the action of CGRP, ADM, and amylin in myometrial cells. Treatment with [cys(ACM)2-7]-CGRP, a CGRP2 receptor agonist, did not cause an increase in the levels of cAMP in these cells. These findings are indicative that CGRP, ADM, and amylin act via that the CGRP1 receptor in human myometrial cells. Vasoactive intestinal peptide and pituitary adenylate cyclase activating polypetide also caused a dose-dependent increase in cAMP in myometrial cells. The findings of this study are indicative that multiple neuropeptides, acting by way of heptahelical receptors linked to the G alpha s-subunit of the G-proteins, may contribute to the maintenance of uterine quiescence during some period of human pregnancy.

Human saturated steroid 6alpha-hydroxylase.

This study was conducted to evaluate further the reaction catalyzed by the saturated steroid 6alpha-hydroxylase of extrahepatic human tissues. Progesterone and 5alpha-dihydroprogesterone (5alpha-DHP) are plasma-borne precursors of 5alpha-pregnan-3alpha-ol-20-one, an anxiolytic/anesthetic steroid, and 5alpha-pregnan-3beta-ol-20-one in extrahepatic human tissues. These two steroids are metabolized further by a saturated steroid 6alpha-hydroxylase enzyme(s) that is distinct from the cytochrome P450 6alpha-hydroxylase that catalyzes the 6alpha-hydroxylation of delta4-3-ketosteroids such as progesterone, cortisol, and testosterone. Products of this saturated steroid 6alpha-hydroxylase, viz. 3beta/alpha,6alpha-dihydroxy-5alpha-pregnan-20-ones, are major radiolabeled urinary metabolites (excreted as glucuronosides) of i.v. administered tritium-labeled 5alpha-DHP in women and men. T47-D human breast cancer cells, which are rich in saturated steroid 6alpha-hydroxylase activity, were used as the enzyme source in this study. The greatest total and the highest specific activity of saturated steroid 6alpha-hydroxylase were localized in microsome-enriched preparations; enzyme activity was linear with incubation time up to 30 min and with microsome-enriched tissue protein concentrations between 0.05-0.5 mg/mL incubation mixture. The velocity of the reaction was similar in incubations in which the pH was varied from 6.0-8.0, and NADH and NADPH were equally effective in supporting the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one. The more efficient substrates for this enzyme were 5alpha-pregnan-3beta-ol-20-one and 5alpha-pregnan-3alpha-ol-20-one, and the apparent Km (approximately 3.5 micromol/L) and maximum velocity (approximately 150 pmol/min x mg microsome-enriched protein) for these two substrates were indistinguishable. 5alpha-Androstane-3beta,17beta-diol was less efficiently 6alpha-hydroxylated, and 5alpha-androstane-3alpha,17beta-diol was an inefficient substrate. The addition of a variety of inhibitors of cytochrome P450 monooxygenases to the incubation mixtures did not diminish significantly the 6alpha-hydroxylation of 5alpha-pregnan-3beta-ol-20-one, findings consistent with those of other investigators who suggested that human saturated steroid 6alpha-hydroxylase (of human prostate) is not a cytochrome P450.

Parathyroid hormone-related protein and human myometrial cells: action and regulation.

Parathyroid hormone-related protein (PTH-rP), like parathyroid hormone (PTH), acts on myometrial smooth muscle to cause relaxation, and PTH-rP expression has been demonstrated in the myometrium of pregnant and estrogen-treated nonpregnant rats and in human myometrium, leiomyomata and separated myometrial smooth muscle cells in culture. PTH-rP may facilitate the myometrial quiescence characteristic of the first 95% of normal pregnancy and uterine vasorelaxation. This study was conducted to explore further the function and regulation of expression of PTH-rP in human myometrium. Treatment of myometrial smooth muscle cells with analogs of PTH-rP caused an increase the intracellular levels of cAMP and treatment of myometrial cells with forskolin or dibutyryl cyclic AMP caused an increase in the levels of PTH-rP mRNAs. Tetradecanoyl phorbol acetate (TPA) and okadaic acid caused a striking increase in the levels of PTH-rP mRNAs, much greater than that evoked by forskolin, dibutyryl cAMP, or transforming growth factor-beta (TGF-beta). These findings are supportive of the conclusion that myometrial cells respond to PTH-rP with activation of adenylyl cyclase and that PTH-rP gene expression may be modulated by way of a number of distinct intracellular signaling pathways.

Tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 expression in human amnion mesenchymal and epithelial cells.

OBJECTIVE: This study was conducted to define the cellular site of expression of tissue inhibitor of metalloproteinase-1 and tissue inhibitor of metalloproteinase-2 in human amnion by an evaluation of the levels of messenger ribonucleic acids in separated amnion epithelial and mesenchymal cells and to ascertain whether amnion epithelial and mesenchymal cells maintained in culture continue to express tissue inhibitor of metalloproteinase messenger ribonucleic acids. STUDY DESIGN: Human placentas and fetal membranes were obtained immediately after delivery. Amnion tissue was separated from chorion laeve and either frozen immediately (-80 degrees C) or processed by differential enzymatic treatment to separate the epithelial and mesenchymal cells, which were frozen (-80 degrees C) or else plated and maintained in monolayer culture. The levels of tissue inhibitor of metalloproteinase types 1 and 2 messenger ribonucleic acid were evaluated by Northern analyses of total ribonucleic acid extracted from amnion tissue, freshly separated epithelial and mesenchymal cells, and epithelial and mesenchymal cells in monolayer culture. RESULTS: Tissue inhibitor of metalloproteinase types 1 and 2 messenger ribonucleic acids were detected by Northern analysis in freshly isolated amnion tissues from midtrimester and term pregnancies. The major species of tissue inhibitor of metalloproteinase-1 messenger ribonucleic acid was 0.9 kb in length; a minor species of approximately 3.5 kb also was present. Tissue inhibitor of metalloproteinase-2 messenger ribonucleic acids of 3.5 and 1.0 kb and of similar intensity were also detected. The levels of type 1 messenger ribonucleic acid were not different in amnion tissues obtained at term or during the midtrimester of pregnancy. The levels of tissue inhibitor of metalloproteinase type 2 messenger ribonucleic acids in amnion tissue most commonly were greater at term than in tissues obtained during the midtrimester. The level of type 1 messenger ribonucleic acid in mesenchymal cells was appreciably greater than that in epithelial cells, and this difference was maintained during culture of these cells. The level of type 2 messenger ribonucleic acid was similar in both cell types and was maintained during culture. The levels of type 1 or 2 messenger ribonucleic acids were not affected by treatment of amnion epithelial or mesenchymal cells in culture with a variety of test agents, including steroid hormones, cytokines, and growth factors. CONCLUSION: The amnion mesenchymal cells are the primary source of tissue inhibitor of metalloproteinase-1 in human amnion, whereas both cell types have the potential to produce tissue inhibitor of metalloproteinase-2.

Origin of deoxycorticosterone sulfate (DOC-SO4) in plasma of pregnant women: pregnenolone-3,21-disulfate is a placental precursor of DOC-SO4.

The plasma levels of deoxycorticosterone sulfate (DOC-SO4) in near-term pregnant women are approximately 100 times those in plasma of men or non-pregnant women. Yet, neither the tissue site of synthesis nor the precursor of DOC-SO4 that enters maternal plasma is known. Several potential sources have been excluded: plasma DOC-SO4 is not derived from plasma DOC; and the secretion of C21-steroids (other than aldosterone) from the maternal adrenals during human pregnancy is not increased. Similarly, the transfer of DOC-SO4 from fetal plasma cannot account for the high level of DOC-SO4 in the maternal compartment, and a reduced clearance of plasma DOC-SO4 during pregnancy cannot account for the high levels of DOC-SO4. Indeed, the rate of clearance of DOC-SO4 from plasma is 10-100 times that of most other steroid sulfates. To address this question further, we evaluated the possibility that fetal plasma pregnenolone-3,21-disulfate serves as a precursor for DOC-SO4 formation in the placenta. The preferential hydrolysis of the 3beta-sulfate of pregnenolone-3,21-disulfate in placenta would give rise to pregnenolone-21-monosulfate, which, if acted upon by placental 3beta-hydroxysteroid dehydrogenase/delta5 --> 4 isomerase, could give DOC-SO4. [3H]Pregnenolone-3,21-disulfate was incubated with minces of human placental tissue for 5, 20, 60 and 120 min. Radiolabelled DOC-SO4, DOC, and pregnenolone-21-monosulfate were isolated from the incubation media and quantified. After a 5 min incubation, 7.5% of substrate was converted to DOC-SO4; and after 20, 60 and 120 min approximately 30% of the [3H]pregnenolone-3,21-disulfate was recovered from the media of these incubations as [3H]DOC-SO4. [3H]DOC was also present in the incubation media and the concentrations of this product increased as a function of incubation time. Therefore, pregnenolone-3,21-disulfate, which is present in very high concentrations in fetal plasma (approximately 1000 ng/ml), is metabolized in the placenta to DOC-SO4. Because of the fetal and maternal vascular arrangements of the hemochorioendothelial placenta of human pregnancy, steroids produced in syncytiotrophoblasts preferentially enter the intervillous space; thus, fetal plasma pregnenolone-3,21-disulfate may serve as a placental precursor of maternal plasma DOC-SO4.

Lysyl oxidase (ras recision gene) expression in human amnion: ontogeny and cellular localization.

The tensile strength of human fetal membranes is attributable to interstitial collagens of the zona compacta of the avascular amnion. Collagen fiber strength and proteolytic resistance is provided by inter and intramolecular cross-links of collagen fibrils, which are formed in a series of reactions initiated by lysyl oxidase. Lysyl oxidase activity in amnion tissues varied by more than 400-fold in a highly significant inverse manner as a function of gestational age (12-43 weeks). At 12-14 weeks gestation, the levels of lysyl oxidase messenger ribonucleic acid, protein, and activity in amnion are very high. During the second trimester of pregnancy, however, these decline abruptly, and a nadir is reached at about 20-24 weeks gestation, which persists to term. The level of lysyl oxidase messenger ribonucleic acid was greater in amnion mesenchymal cells than in amnion epithelial cells. The decline in lysyl oxidase in amnion may be attributable to a correspondent decline in the density of amnion mesenchymal cells with fetal development.

Regulation of metallothionein expression in human amnion epithelial and mesenchymal cells.

OBJECTIVE: In this study the potential for metallothionein expression in amnion epithelial and mesenchymal cells in response to cadmium was evaluated. STUDY DESIGN: Levels of metallothionein messenger ribonucleic acid were evaluated in freshly separated amnion epithelial and mesenchymal cells and in amnion cells in culture. RESULTS: The levels of metallothionein messenger ribonucleic acid in human amnion mesenchymal cells freshly isolated after delivery of term pregnancies were greater than those in epithelial cells of the same tissue. The levels in mesenchymal cells in monolayer culture at confluence also were greater than those in confluent epithelial cells propagated from the same tissue. In response to treatment with cadmium (100 nmol/L to 50 micromol/L), which is inhaled in cigarette smoke, the levels of metallothionein messenger ribonucleic acid in both cell types increased markedly in a dose-dependent manner, but the level was greater in epithelial cells at all concentrations of cadmium chloride tested. With cadmium chloride (10 micromol/L), the level of metallothionein messenger ribonucleic acid increased by as much as 1000-fold in epithelial cells and 10-fold in mesenchymal cells compared with untreated (control) cells. Dexamethasone and tetradecanoyl phorbol acetate also acted to increase the levels in amnion epithelial and mesenchymal cells but not nearly to the levels effected by cadmium. CONCLUSION: These findings are indicative that metallothionein expression in amnion epithelial cells is exquisitely sensitive to cadmium in concentrations similar to those in amniotic fluid of pregnancies of women who smoke cigarettes. We hypothesize that increased levels of metallothionein in amniotic fluid and amnion epithelial cells will bind and thereby may limit the availability of copper to the Cu++-dependent enzyme lysyl oxidase in mesenchymal cells and thereby impair the cross-linking of interstitial collagens, which is effected by this enzyme.

Accumulation of interleukin-1beta and interleukin-6 in amniotic fluid: a sequela of labour at term and preterm.

From the finding of micro-organisms or inflammatory mediators, or both, in amniotic fluid (AF), it has been proposed that intrauterine infection is one cause of preterm labour (PTL, intact fetal membranes). This theory, however, remains unproved, i.e. the accumulation of micro-organisms and inflammatory mediators in AF after labour is in progress may be the consequence, not the cause, of labour both at term and preterm. This study was conducted to evaluate this possibility by a comparison of the concentrations of interleukin (IL)-1beta and IL-6 in AFs collected before and during PTL (<34 weeks gestation) with those in AFs collected at term (before labour and from the forebag and upper compartments of the amniotic sac during labour). The concentrations of IL-1beta and IL-6 in AF were also analysed as a function of the duration of labour (term or preterm) before fluid collection. In addition, studies were conducted to define the source of IL-1beta in AF. A total of 666 AFs were evaluated. IL-1beta was not detected (<50 pg/ml) in AFs collected before the onset of labour at any stage of gestation (n = 320), including 170 fluids obtained at term. During labour, IL-1beta was detected (>50 pg/ml) in 58 out of 106 (54.7%), 17 out of 64 (26.6%) and 60 out of 176 (34%) of AF samples obtained during PTL, term labour (upper compartment) and term labour (forebag) respectively. AF sampling, as well as labour and delivery, were completed in <18 h in all term pregnancies. However, labour (with cervical dilation) was in progress for >18 h before AF was collected in 39 out of 106 (37%) PTL pregnancies. The incidence of IL-1beta-positive samples among AFs collected before 18 h of PTL (23 out of 67; 34%) was indistinguishable from that in AFs collected during labour at term. However, in AFs collected after >18 h PTL, the incidence of IL-1beta-positive samples was 35 out of 39 (89.7%) The concentrations of IL-1beta (pg/ml; mean +/- SEM) in AFs collected during PTL (2680 +/- 730; n = 106) were greater than those in AFs collected from the upper compartment and forebag during term labour (436 +/- 244, n = 64; and 468 +/- 119, n = 176) respectively; this difference, however, was attributable to very high concentrations of IL-1beta in AFs in which PTL was in progress for >18 h before AF collection (6021 +/- 1832; n = 39). The concentrations of IL-6 in AF were correlated with those of IL-1beta (P < 0.001). We conclude that IL-1beta and IL-6 accumulate in AF in a similar proportion of pregnancies during the first 18 h of term and preterm labour. Therefore, the accumulation of these cytokines in AF cannot be taken as evidence for a role for infection in the pathogenesis of PTL.

5-Alpha-dihydroprogesterone formation in human placenta from 5alpha-pregnan-3beta/alpha-ol-20-ones and 5-pregnan-3beta-yl-20-one sulfate.

5Alpha-dihydroprogesterone (5alpha-DHP) is the immediate precursor of 5alpha-pregnan-3alpha-ol-20-one, a potent anxiolytic/anesthetic agent in all vertebrate animals tested, including humans. The levels of 5alpha-DHP in the plasma of pregnant women are very high; and during the third trimester of pregnancy, the blood production rate of this steroid may exceed 100 mg/24 h. 5Alpha-DHP in maternal plasma, however, cannot be accounted for totally by the metabolism of maternal plasma progesterone. This study was conducted to evaluate the possibility that 5alpha-DHP is synthesized in placenta from 5alpha-pregnan-3alpha/beta-ol-20-ones delivered to the trophoblast via the fetal umbilical blood. In incubations of placental minces with radiolabelled 5alpha-pregnan-3alpha/beta-ol-20-ones, there is extensive epimerization and the intermediate, 5alpha-DHP, is the major product. In other incubations, 5alpha-pregnan-3beta-ol-20-one-sulfate was hydrolysed and the liberated 5alpha-pregnan-3beta-ol-20-one was converted to 5alpha-DHP by homogenates of placental tissue, but 5alpha-pregnan-3beta-ol-20-one-sulfate was not. The oxidation of 5alpha-pregnan-3alpha/beta-ol-20-ones was concentrated in microsome-enriched preparations of placental tissue and the apparent Kms for 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3beta-ol-20-one were 3.6 microM and 78 nM, respectively. The Vmaxs for 5alpha-DHP formation from 5alpha-pregnan-3alpha-ol-20-one and 5alpha-pregnan-3beta-ol-20-one were, respectively, 336 pmol/min/mg protein and 9.7 nmol/min/mg protein. These oxidation reactions were supported by both NAD+ and NADP+. We suggest that progesterone, which enters the umbilical circulation from its site of synthesis in the syncytiotrophoblast, is metabolized in the fetus to 5alpha-pregnan-3alpha/beta-ol-ones and to 5alpha-pregnan-3alpha/beta-yl-20-one sulfates. These metabolites of progesterone, 5alpha-pregnan-3alpha/beta-ol-20-one and 5alpha-pregnan-3beta-yl-20-one sulfate, formed in the fetus, serve as plasma-borne substrates for trophoblast formation of 5alpha-DHP. Because of the hemochorioendothelial nature of human placentation, 5alpha-DHP secreted from the trophoblast will preferentially enter the maternal compartment, thus constituting a maternal plasma progesterone-independent source of 5alpha-DHP.

Interstitial collagen synthesis and processing in human amnion: a property of the mesenchymal cells.

This study was conducted to evaluate the ontogeny and cellular site of interstitial collagen formation in amnion, the tissue that provides the tensile strength of the human fetal membranes. The levels of procollagen alpha 1(I), alpha 2(I), and alpha 1(III) subunit mRNAs and the specific activities of the enzymes prolyl 4-hydroxylase and lysyl hydroxylase were greatest in human amnion tissue early in gestation, decreasing rather abruptly after 12-14 wk gestation to a nadir that persisted to term. To evaluate these findings further, amnion epithelial and mesenchymal cells were separated by differential proteinase treatment. The freshly separated cells as well as epithelial and mesenchymal cells maintained in culture were evaluated to assess the cellular site of interstitial collagen synthesis. By Northern analysis of total RNA, large amounts of procollagens alpha 1(I), alpha 2(I), and alpha 1(III) mRNAs were found in mesenchymal cells (freshly separated and in culture), but only negligible amounts of these transcripts were detected in RNA of freshly separated or cultured epithelial cells. The level of prolyl 4-hydroxylase alpha-subunit mRNA in mesenchymal cells was somewhat greater than that in epithelial cells. Radiolabeled collagen I synthesis from [3H]proline and [3H]glycine was not detected in amnion epithelial cells, whereas [3H]collagen alpha 1(I) and alpha 2(I) subunits were synthesized in large amounts by mesenchymal cells. A very small amount of [3H]collagen alpha 1(III) was synthesized in epithelial cells, but large amounts were formed in mesenchymal cells. These findings indicate that the mesenchymal cells are the site of interstitial collagen synthesis and processing in amnion. The density of mesenchymal cells in human amnion declines after the first trimester of pregnancy, and a significant decline in DNA content per unit of amnion tissue dry weight as pregnancy progressed was demonstrated in this study. Since the epithelial cells form an uninterrupted epithelium throughout gestation, the decline in DNA content probably corresponds to the decrease in the density of mesenchymal cells that commences after the first trimester pregnancy. Thus, the increase in the ratio of epithelial to mesenchymal cells as a function of gestational age may explain, at least in part, the decline in the levels of collagen mRNAs and the specific activities of lysyl and prolyl 4-hydroxylase in amnion tissue during human pregnancy.

Transforming growth factor-beta inhibits progesterone-induced enkephalinase expression in human endometrial stromal cells.

The specific activity of enkephalinase in endometrial tissue of nonpregnant ovulatory women is correlated in a highly significant, positive manner with the plasma level of progesterone. The specific activity and levels of enkephalinase messenger ribonucleic acid and immunoreactive protein also are increased in human endometrial stromal cells in culture by treatment with a synthetic progestin, medroxyprogesterone acetate (MPA), in a time- and dose-dependent manner. From an analysis of the temporal relationship between the specific activity and half-life of enkephalinase in endometrial tissue and the level of progesterone in plasma, it appeared highly likely that some mechanism, in addition to progesterone withdrawal, was operative to reduce enkephalinase activity in endometrium during the late luteal phase of the ovarian cycle before progesterone levels had declined below those known to be effective for progesterone action. In stromal cells previously (and concurrently) treated with MPA (10(-9) mol/L), the addition of transforming growth factor-beta 1 (TGF beta 1) or TGF beta 2 (1 ng/mL) to the medium caused a decrease in enkephalinase specific activity despite the continued presence of MPA. The half-life of enkephalinase (activity) in stromal cells treated with MPA plus TGF beta 1 was 2.8 days, which is similar to the computed half-life for enkephalinase in endometrial tissue during the mid- to late secretory phase of the endometrial cycle (2.5 days). Simultaneous treatment of endometrial stromal cells with MPA (10(-9) mol/L) and TGF beta 1 (1 ng/ mL) prevented the progestin-induced increase in enkephalinase specific activity and immunoreactive enkephalinase protein. Thus, TGF beta acts to oppose the progesterone-induced increase in enkephalinase expression in endometrial stromal cells, even in the continued presence of MPA.

Metabolism of 5 alpha-dihydroprogesterone in women and men: 3 beta- and 3 alpha-,6 alpha-dihydroxy-5 alpha-pregnan-20-ones are major urinary metabolites.

The metabolism of 5 alpha-dihydroprogesterone (5 alpha-DHP) in women and men was evaluated by defining the pattern and identity of selected metabolites excreted in urine after the iv infusion of radiolabeled 5 alpha-DHP. Virtually all of the radioactivity in urine (approximately 37% of the administered dose) was excreted within 72 h. Quantitatively, the 2 major urinary metabolites of 5 alpha-DHP in each of 13 studies conducted in 7 women and 2 men were 3 beta,6 alpha-dihydroxy-5 alpha-pregnan-20-one and 5 alpha-pregnane-3 alpha,20 alpha-diol, which could be extracted after beta-glucuronidase, but not solvolysis, treatment of the urine. Radiolabeled 3 alpha,6 alpha dihydroxy-5 alpha-pregnan-20-one (glucuronoside), in lesser amounts, also was identified in the urine of each subject. The 3 alpha/beta, 6 alpha-dihydroxy-5 alpha-pregnan-20-ones arise through specific extrahepatic pathways of progesterone/5 alpha-DHP metabolism. These metabolites are not the products of the enzyme reaction catalyzed by the cytochrome P450 steroid 6 alpha-hydroxylase of human liver (and other tissues), which affects the 6 alpha-hydroxylation of C19- and C21-delta 4-3-ketosteroids (e.g., progesterone, testosterone, and cortisol), but does not act upon 5 alpha-reduced steroids. Moreover, the steroid 5 alpha-reductases do not act upon 6 alpha-hydroxy-delta 4-3-ketosteroids. In addition, the 6 alpha-hydroxylation of 5 alpha-reduced-3 alpha/beta-hydroxysteroids is not demonstrable in adult liver tissue. Rather, the formation of 6 alpha-hydroxylated-5 alpha-pregnane-3 alpha/beta-ol-20-ones is indicative of an extrahepatic pathway of progesterone metabolism, viz. progesterone-->5 alpha-DHP-->5 alpha-pregnan-3 beta/alpha-ol-20-one(s)-->3 beta/alpha,6 alpha-dihydroxy-5 alpha-pregnan-20-one(s), in which 5 alpha-pregnan-3 alpha/beta-ol-20-ones are metabolized by an enzyme(s) that catalyzes the 6 alpha-hydroxylation of saturated substrates. There are important differences among mammalian species in the enzymes that catalyze the C-6-hydroxylation of 5 alpha-reduced C19- and C(21)-3 beta/alpha-hydroxysteroids, but in all species studied, these enzymatic reactions are the final steps in the extrahepatic inactivation of 5 alpha-reduced bioactive metabolites of progesterone (or testosterone).

The endothelin-parathyroid hormone-related protein vasoactive peptide system in human endometrium: modulation by transforming growth factor-beta.

Endothelin-1 (ET-1), a potent vasoconstrictor, and parathyroid hormone-related protein (PTH-rP), a potent vasorelaxant, are present and synthesized in human endometrium. The production of these vasoactive peptides in stromal cells of the human endometrium is modulated by sex steroid hormones. In addition, neutral endopeptidase 24.11 (also called enkephalinase), which degrades ET-1, is expressed in the stromal cells of the endometrium, and the levels of this enzyme (mRNA, protein and specific activity) are increased in response to progesterone/progestin. The sex steroid hormone-mediated regulation of vasoactive peptide formation and action in the human endometrium is believed to serve a key role in modulating spiral artery tone/blood flow, and thus endometrial bleeding, whether progestin induced or naturally occurring. Moreover, the expression of ET-1, PTH-rP and enkephalinase is modulated by transforming growth factor-beta. We hypothesize that the inappropriate expression of ET-1, enkephalinase and PTH-rP in endometrial stromal cells during synthetic progestin administration may be one cause of abnormal endometrial development and erratic endometrial bleeding.

Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-beta 1.

Selected functions of uterine endometrium of ovulatory women before and during pregnancy appear to be modulated by cytokines and other paracrine-acting factors. Some of these functions are regulated, in turn, by cyclic changes in ovarian steroid secretion or by pregnancy-induced endocrine and paracrine factors. The recruitment of specific types and numbers of bone marrow-derived cells into the endometrium occurs in a predictable manner with hormonal changes of the ovarian cycle, during the process of endometrial decidualization, at the time of blastocyst implantation, and during pregnancy, parturition, and the puerperium. As part of an investigation of the regulation of the leukocyte population of endometrium/decidua, this study was conducted to evaluate further the regulation of interleukin-8 (IL-8) gene expression by transforming growth factor-beta (TGF beta). IL-8 is a neutrophil chemoattractant/activating and T cell chemotactic factor as well as a chemotactic factor for fibroblasts. IL-8 is produced by mesenchymal cells of many tissues, including human endometrial stromal cells in culture. The level of IL-8 messenger ribonucleic acid (mRNA) in endometrial stromal cells and the accumulation of immunoreactive IL-8 in medium are increased by TGF beta 1 treatment of these cells. This response to TGF beta 1 is attributable primarily to an increase in the stability of IL-8 mRNA through a process that is dependent on protein synthesis. Transcription of the IL-8 gene in endometrial stromal cells is not increased, but, rather, is slightly decreased, by treatment with TGF beta 1. The findings of this study indicate that TGF beta may act in endometrial stroma to modulate the stability of IL-8 mRNA.

Progestin regulation of interleukin-8 mRNA levels and protein synthesis in human endometrial stromal cells.

The migration of leukocytes into the human endometrium is a normal occurrence that appears to be linked to the hormonal events of the ovarian cycle. In addition, the appearance of large numbers of leukocytes in decidua accompanies normal human pregnancy and puerperal endometrial repair during uterine involution. These leukocytic investments in endometrial-decidual function are also affected by external stimuli, e.g. infection. The formation of cytokines and the response of the uterine endometrium and decidua to cytokines is likely to be an important function of this tissue before, during and soon after pregnancy. Because interleukin-8 (IL-8) is a chemo-attractant activating factor for neutrophils and T cells, the possibility was considered that IL-8 may participate in endometrial leukocytic infiltration in a manner that is hormonally regulated. Previously, we found that IL-8 mRNA is present in the endometrium and decidual tissues; and, using human endometrial stromal cells in monolayer culture as a model system, we found that IL-1 and tumor necrosis factor-alpha act in a time- and concentration-dependent manner to increase IL-8 mRNA and the accumulation of immunoreactive (ir)IL-8 protein. In this study, we found that progesterone and a synthetic progestin, medroxyprogesterone acetate (MPA) act to enhance the action of IL-1 to increase the level of IL-8 mRNA; this action of progesterone/MPA appears to be affected principally by the stabilization of IL-8 mRNA, together with a small increase in IL-8 gene transcription.

Modulation of the levels of transforming growth factor beta messenger ribonucleic acids in human endometrial stromal cells.

We speculate that transforming growth factor beta (TGF beta) is involved in the predecidualization of human endometrial stromal cells (the progenitors of the decidual cells) during the late secretory phase of nonfertile, ovulatory cycles and in the completion of decidualization during fertile cycles after blastocyst implantation. In this study, the regulation of the levels of TGF beta mRNA levels in human endometrial stromal cells in culture was evaluated. TGF beta 1 and TGF beta 3 mRNAs were demonstrable by Northern analysis of total RNA from endometrial stromal cells maintained in serum-containing culture medium. TGF beta 2 mRNA was not detected by similar analyses of total RNA. The levels of TGF beta 3 mRNA in endometrial stromal cells increased in a time-dependent fashion after these cells were changed to serum-free medium. Platelet-derived growth factor acted, in a dose-dependent manner, to increase the levels of both TGF beta 1 and TGF beta 3 mRNAs in these cells. TGF beta 1 treatment caused an increase in the levels of TGF beta 1 mRNA and a decrease in the level of TGF beta 3 mRNA. The effects of platelet-derived growth factor and TGF beta 1 to increase the levels of TGF beta 1 mRNA were at least additive. Treatment of endometrial stromal cells with estradiol-17 beta caused an increase in the levels of TGF beta 1 and TGF beta 3 mRNAs. Treatment of stromal cells with medroxyprogesterone acetate (MPA, a synthetic progestin) also effected a small increase in the level of TGF beta 1 mRNA. The level of TGF beta 3 mRNA in endometrial stromal cells, however, was decreased by MPA treatment. The level of TGF beta 3 mRNA was greater in proliferative phase endometrial tissue than in secretory phase tissue. The levels of TGF beta 3 mRNA in decidua of the first trimester of pregnancy, in atrophic endometrium (from women treated with a GnRH agonist), and in endometrium from women ingesting oral progestin (MPA), were approximately one-fourth that in proliferative phase endometrium. These findings support the potential for modulation of TGF beta 1 and TGF beta 3 gene expression in endometrial stromal cells.