Susan Kadner

Plasma estriol and its conjugates following oral and vaginal administration of estriol to postmenopausal women: Correlations with gonadotropin levels

A study was designed to compared the metabolic fate and the biologic effects of 4 mg of estriol (E3) administered either orally or vaginally to six postmenopausal women. Blood samples were collected every hour for 6 hours and five different estriol fractions as well as gonadotropins were measured. Vaginal E3 administration resulted in a decline of 45% in luteinizing hormone (LH) levels and 17% in follicle-stimulating hormone (FSH) levels at 6 hours after treatment (p < 0.05). In contrast, the administration of 4 mg of E3 orally did not produce a decline of LH and FSH, despite the fact that the serum levels of E3-3-sulfate, E3-3-sulfate-16-glucosiduronate, estriol-3-glucosiduronate, and estriol-16-glucosiduronate were all fourfold to 24-fold higher after oral administration than after vaginal estriol administration. However, since the levels of unconjugated E3 were higher after the vaginal than after the oral administration of estriol, we conclude that only unconjugated E3 suppresses gonadotropins.

Renal clearances of estriol conjugates in normal human pregnancy at term

In late human pregnancy more than 90 per cent of the total estriol (E3) in body fluids consists of four conjugates, estriol-3-sulfate (E3-3S), estriol-16-glucosiduronate (E3-16G), estriol-3-glucuronide (E3-3G), and estriol-3-sulfate-16-glucosiduronate (E3-3S-16G). Since the relative amounts of E3 in blood and urine would be determined by the kidney, the renal clearance of each conjugate was determined and compared with inulin and p-aminohippuric acid (PAH) clearance, as measures of glomerular filtration rate (GFR) and the effective renal plasma flow. Five women were studied in the lateral decubitus position with inulin and PAH infusion. Samples of blood and urine were collected at 40 minute intervals and analyzed. The method for E3 conjugates involved separation of the four conjugates on Sephadex LH-20, enzyme hydrolysis, and radioimmunoassay. Renal clearances for E3-3S and E3-3S-16G were below inulin. E3-3G approximated inulin; E3-16G exceeded inulin and approached PAH. In plasma E3-3S-16G represented 48.4 +/- 7.2 per cent; in urine E3-16G represented 69.5 +/- 7.3 per cent of total E3. Thus, different conjugates predominate in blood and urine.

Glucocorticoid Enhances Transforming Growth Factor-β Effects on Extracellular Matrix Protein Expression in Human Placental Mesenchymal Cells

Abstract Extracellular matrix (ECM) proteins synthesized by human placental mesenchymal cells (PMCs) provide structural support for the villus. Aberrant expression of ECM proteins by PMCs has been associated with intrauterine growth restriction (IUGR). To provide insight into the mechanisms of ECM protein regulation in the stroma of the placental villus, in the current study, we examined the interaction of glucocorticoid (GC) and transforming growth factor-β (TGFβ) in the modulation of ECM proteins in cultures of PMCs isolated from human term placentas. Initial results obtained by ELISA showed that combined treatment with dexamethasone (DEX) and TGFβ enhanced oncofetal fibronectin (FFN) protein levels in serum-free culture medium severalfold in a dose-dependent manner. Northern blotting and real-time polymerase chain reaction (PCR) analyses revealed a similar enhancement in levels of FN mRNA in cells treated with TGFβ and DEX. Real-time PCR results also revealed that DEX and TGFβ enhanced collagen (Col) I and Col IV expression, but did not affect levels of Col III or laminin, indicative of selective stimulation of ECM proteins. Hypoxic treatment moderately enhanced FFN levels in control cells but not in those treated with DEX and TGFβ. In contrast with the results obtained with PMCs, we noted that DEX treatment suppressed FFN levels in untreated and TGFβ-treated cytotrophoblasts, suggesting that GC and TGFβ modulate FFN expression in placenta in a cell-type-specific manner. We conclude that GC and TGFβ are key regulators of ECM protein synthesis in PMCs, suggesting a role in modulating placental architecture in uncomplicated pregnancies and those associated with aberrant ECM protein expression.

Estradiol esters can replace 17β-estradiol in the stimulation of DNA and esterase synthesis by MCF-7 cells: A possible role for the estrogen-sensitive MCF-7 cell esterase

In this communication we extend our earlier observations on estrogen-sensitive carboxyl esterases in MCF-7 human breast cancer cells able to hydrolyze esters of estradiol. Using either estradiol acetate or p-nitrophenyl hexanoate as substrates, esterase activity was found to increase 2-3-fold in MCF-7 cells maintained in the presence of 10(-8) M estradiol. Following sucrose density centrifugation, over 85% of total esterase activity was found in the cytoplasmic fraction. No esterase activity was found in spent media from growing cells. By size exclusion chromatography, estradiol acetate esterase activity exhibited a mol. wt of 45-50 kDa. Attempts to demonstrate incorporation of [3H]estradiol into estradiol fatty acid esters by the above MCF-7 cell line (203P) were unsuccessful, although, such incorporation could be demonstrated in two other MCF-7 cell sublines. Incubation of the 203P cells with 10 nM [3H]estradiol in the presence of 0.5 mM radioinert estradiol acetate resulted in the incorporation of 35 +/- 12% of the label into the estradiol acetate in 10 min. In the absence of radioinert estradiol acetate, no incorporation was observed. When MCF-7 cells were incubated with [3H]estradiol in the presence of a large excess of radioinert estradiol valerate, label was found only in estradiol valerate. Similarly, when the incubation was carried out in the presence of a mixture of radioinert estradiol acetate and valerate, label was incorporated into both esters. We conclude that the apparent formation of radiolabeled estradiol esters by MCF-7 cells incubated under the above conditions, results at least in part, from an esterase-catalyzed exchange reaction. Under conditions where no ester hydrolysis could be detected in the absence of cells, valerate and stearate esters of estradiol were found to be as effective as unesterified estradiol in stimulating esterase synthesis and the incorporation of [3H]thymidine into DNA. These results are consistent with a model in which an intracellular esterase in MCF-7 cells can generate estradiol from an exogenous lipoidal steroid and elicit an estrogen response.

Hypoxia Stimulates ecNOS mRNA Expression by Differentiated Human Trophoblasts

Cytotrophoblasts isolated from normal human placenta cultured under normoxic conditions (20% O2, pO2 = 130 mmHg) for 48-72 h differentiate to a form which expresses high levels of hCG and which morphologically resembles syncytiotrophoblast. We had previously shown that hypoxia (0-1% O2, pO2 = 12-14 mmHg) blocks this differentiation process, although trophoblasts exposed to hypoxia for up to 96 h were completely viable. In this article we showed that trophoblast responds to hypoxia by expressing the hypoxia-sensitive DNA binding protein HIF-1. We also showed that in trophoblast cultured under normoxic conditions, expression of endothelial cell nitric oxide synthase (ecNOS) mRNA increases with time, reaching a maximum in 48-72 h. However, in trophoblast maintained under hypoxic conditions for 48 h (after an initial 24 h in normoxia), expression of ecNOS mRNA is greatly reduced. These observations are consistent with the expression of ecNOS by syncytiotrophoblast but not by cytotrophoblast. In contrast, exposure of differentiated trophoblasts to hypoxia for 24 h (after 48-72 h in normoxia) significantly stimulates expression of ecNoS mRNA over that of cells maintained continuously in normoxia. These results suggest that in differentiated trophoblast hypoxia can stimulate ecNOS expression.

Intermediary metabolism of estriol in pregnancy.

Estriol (E3), the most abundant estrogen in pregnancy is produced predominantly in the placenta from androgen precursors of fetal origin. The estriol so formed is secreted efficiently into the maternal circulation where it is converted to 4 conjugates--estriol-3-sulfate (E3-3S), estriol-16-glucosiduronate (E3- 16G ), estriol-3-glucosiduronate (E3- 3G ) and estriol-3-sulfate-16-glucosiduronate (E3-SG). The order of renal clearances is E3- 16G greater than E3- 3G greater than E3-3S approximately E3-SG. Unconjugated E3 and E3- 3G differ from the other forms of estriol in that their removal from the blood compartment is essentially irreversible. E3-3S, E3- 16G and E3-SG undergo interconversions during enterohepatic circulation and eventual partial conversion to E3- 3G . Following delivery of the fetus and placenta, unconjugated E3 is no longer detectable in the maternal serum within 1-2 h, whereas the concentrations of the conjugates decline more slowly, the rates being determined by the rates of renal clearance and enterohepatic interconversions. E3- 3G levels were dramatically elevated in a case of Group C polycystic kidney disease, providing evidence that this conjugate is indeed an end-product of estriol metabolism.

Synthesis of α 1 -Antichymotrypsin and α 1 -Antitrypsin by Human Trophoblast

ABSTRACT: α1-Antichymotrypsin (α1-ACHY) and α1-an-titrypsin (α1-AT) are closely related glycoprotein protease inhibitors, present in plasma and other extracellular fluids, that neutralize proteases released by leukocytes in response to trauma and inflammatory stimuli. Both inhibitors are synthesized primarily by hepatocytes, although lower levels of synthesis by monocytes and breast and intestinal epithelial cells have been demonstrated. Recently, the immunohistochemical localization of α1-AT and α1-ACHY in intrauterine and extrauterine human trophoblastic tissue has been reported. In the present study, we have sought to determine whether human trophoblast is also able to synthesize α1-AT and α1-ACHY. Messenger RNA for both inhibitors was found by Northern blotting in chorionic villi obtained from first trimester and term placenta. Substantial differences in messenger levels for both inhibitors among individual placentas were noted. α1-ACHY and α1-AT messenger was also present in trophoblast cells in primary culture. Synthesis of α1-AT and α1-ACHY protein was demonstrated by SDS-PAGE after immunoprecipitation of [35S]-labeled α1-AT and α1-ACHY from conditioned media of trophoblast cells in culture metabolically labeled with [35SJ-methionine. It is of some interest that the Mr of the α1-AT and α1-ACHY secreted by trophoblast were 50 000 and 49 000, respectively, compared with 54 000 and 68 000 for these proteins in plasma (or secreted by HepG2 human hepatoma and MCF-7 human breast cancer cells). After enzymatic deglycosylation, the Mr of the α1-AT and α1-ACHY secreted by trophoblast and HepG2 cells were all approximately 46 000, suggesting incomplete glycosylation of the inhibitors released by trophoblast.

Estriol conjugates in body fluids in late human pregnancy

Abstract A new method was developed for the assay of estriol (E 3 ) conjugates in amniotic fluid, urine and plasma of third trimester human pregnancy. Tritiated estriol-3-sulfate (E 3 -3S), estriol-16-glucosiduronate (E 3 -16G) estriol-3-glucosiduronate (E 3 -3G) and estriol-3-sulfate-16-glucosiduronate (E 3 -3S-16G) are added to the fluid. The conjugates are separated as their triethylammonium salts on Sephadex LH-20. Each conjugate is hydrolyzed enzymatically and the estriol is purified by solvent extractions. The estriol is quantitated by radioimmunoassay. In the amniotic fluid E 3 -16G and E 3 -3S-16G comprised about 75% of the total E 3 conjugates. In normal pregnancy the E 3 -16G/E 3 -3S-16G ratio increased markedly with gestational age. In Rh-isoimmunization disease the ratio increased less dramatically and the patterns were erratic. In the urine E 3 -16G predominated (70–80%) whereas in the plasma E 3 -3S-16G comprised about 45% and E 3 -16G about 25% of the total. The concentrations of E 3 -3S and E 3 -16G were variable. The renal clearances in ml/min of the E 3 conjugates measured in 2 subjects are as follows: E 3 -16G, 343–508 (similar to that of p-aminohippuric acid); E 3 -3G, 64—149; E 3 -3S, 13–34; and E 3 -3S-16G, 21—29. These methods appear applicable to the study of a variety of conditions in pregnancy in which pathology may be reflected in abnormal profiles of E 3 conjugates.

Differential Effects of Lipopolysaccharide and Thrombin on Interleukin‐8 Expression in Syncytiotrophoblasts and Endothelial Cells: Implications for Fetal Survival

Abstract: Syncytiotrophoblasts (SCTs) are directly bathed by maternal blood and, as such, are in direct contact with proinflammatory stimuli present in the maternal circulation. The extent and nature of cytokine responses induced in SCTs play a central role in the maintenance of pregnancy. Thrombin is a critical mediator of tissue factor‐initiated blood coagulation. Thrombin has been more recently demonstrated to induce cytokine expression and inflammation in several cell types. To dissect the patterns of regulation of cytokine production in the placental villus, we compared the effects of thrombin and lipopolysaccharide (LPS) treatments on cytokine expression in SCTs and endothelial cells. For studies, primary cultures of cytotrophoblasts from human term placentas were differentiated to SCTs. We observed that the presence of thrombin only modestly enhanced interleukin‐8 (IL‐8) levels in SCTs in a manner that was not dose‐dependent. Conversely, SCTs were exquisitely sensitive to LPS, the presence of which induced approximately a 10‐fold increase in IL‐8 levels with an EC50∼ 1 ng/mL. Northern blotting and real‐time PCR results indicated that LPS (but not thrombin) treatment induced a >4‐fold increase in levels of IL‐8 mRNA. The addition of the anti‐inflammatory steroid, dexamethasone, significantly reduced the LPS‐mediated increase in levels of IL‐8 in SCTs. Conversely, in human umbilical vein endothelial cells, thrombin and LPS treatments induced 10‐ and 20‐fold increases in IL‐8 expression, respectively. These results indicate that LPS, but not thrombin, promotes proinflammatory processes in SCTs, with cell‐type specificity. The inability of thrombin in the intervillous space to evoke inflammatory responses in SCTs may constitute an important aspect of fetal survival. Conversely, our results suggest that SCTs do play a key role in infection‐associated changes in placental cytokine expression.

cDNA cloning of esterase 1, the major esterase activity in mouse plasma.

We report here the cloning of a partial cDNA for Esterase 1, the major esterase activity in mouse plasma. A 470 base pair insert was isolated from a lambda gt11 cDNA library constructed from mouse liver poly A+ RNA, and identified by hybrid selected translation. We show that the sexual dimorphism displayed in the plasma levels of this protein is caused by a difference at the level of transcription. In addition, RFLP data using mouse recombinant inbred strains mapped this clone at the Es-1 locus on mouse chromosome 8.