M.H.F. Sullivan

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells.

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues(10-8-10-6 M) on the functional activity (testosterone and cyclic AMP production and [125I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0-90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells. The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10-7 M) and after an initial lag period (2h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10(6) cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (p less than 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased. These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.

Endothelial nitric oxide synthase in the human placenta: regional distribution and proposed regulatory role at the feto-maternal interface.

Feto-placental vessels lack innervation, hence control of this circulation is dependent on locally produced and circulating vasoactive factors. Functional studies have presented evidence that nitric oxide, a potent vasodilator and platelet anti-aggregating agent, may be generated into the feto-placental circulation, contributing to control of vascular tone. In view of the absence of nerves supplying the placenta the source of NO is likely to be endothelial. We have therefore investigated the localization of endothelial constitutive nitric oxide synthase (ecNOS) in human normal full-term placentae, using immunocytochemistry, with rabbit antiserum to a synthetic peptide, corresponding to amino acid residues 1172-1186 of human and bovine ecNOS. On Western blots of partially purified NO synthase extracted from placenta, the peptide antiserum reacted exclusively with a single protein band of approximately 135kDA. Immunoreactivity in tissue sections was localized to endothelium of umbilical artery and vein, and appeared uniform in sections at different levels along the cord. Staining in chorionic vessels was much more variable; it was present mainly in the larger vessels close to the cord where it had a patchy distribution. Staining was not seen in the endothelium of small feto-placental vessels. Strong immunoreactivity was evident in the syncytiotrophoblast of the placenta, although the intensity of staining was variable, being weaker along stem villi and strongest along terminal villi. The differential distribution and intensity of nitric oxide synthase immunoreactivity in the human placenta might indicate that locally produced, and in particular trophoblast-derived nitric oxide may play a pivotal role both in control of feto-placental vascular tone and as a platelet anti-aggregating agent in the utero-placental circulation.

Nitric oxide synthase in human placenta and umbilical cord from normal, intrauterine growth-retarded and pre-eclamptic pregnancies.

1 It has been suggested that a deficiency of nitric oxide (NO) may explain many of the pathophysiological features of pre‐eclampsia (PE) and intra‐uterine (foetal) growth retardation (IUGR). To elucidate further the role of NO in the pathophysiology of pregnancy we have determined the relative amount and activity of NO synthase (NOS) in first trimester and normal‐term placental tissues, as well as in the placenta and umbilical cord in pregnancies complicated by PE and IUGR, using NG‐nitro‐L‐[2,3,4,5‐3H]‐arginine ([3H]‐L‐NOARG) binding, quantitative in vitro autoradiography, [3H]‐arginine to [3H]‐citrulline conversion and Western blotting. 2 Specific, high affinity (KD = 38 nM) [3H]‐L‐NOARG binding was demonstrated in the villous trophoblast of normal‐term placentae. Binding was calcium‐independent, stereoselective and exhibited a rank order of inhibition by NOS inhibitors and substrate (L‐NOARG ≥ L‐NMMA ≥ 7‐NI > L‐NAME > L‐Arg ≥ L‐NIO > ADMA). 3 [3H]‐L‐NOARG binding density and NOS activity were both significantly greater in placental tissues from first trimester and PE or IUGR complicated pregnancies compared to normal‐term placentae. 4 Western blotting, using an endothelial NOS peptide antiserum, demonstrated a ∼ 140 KDa protein band in placental extracts and indicated that the amount of immunoreactive material was significantly greater in first trimester compared to normal‐term placentae. 5 Specific [3H]‐L‐NOARG binding was also localized to the endothelial lining of umbilical arteries and veins, binding density being greater in the artery than the vein. [3H]‐L‐NOARG binding to the umbilical artery endothelium was significantly lower in PE and IUGR complicated pregnancies compared to normal‐term controls. 6 The role of trophoblast‐derived NO in human placental pathophysiology remains to be established, but differences in the amount of placental [3H]‐L‐NOARG binding, NOS activity and immunoreactive material indicate that expression of NOS in the villous trophoblast falls during pregnancy. Conversely, the apparent reduction in NOS in the umbilical artery endothelium in PE and IUGR complicated pregnancies may be indicative of endothelial dysfunction.

Partial characterization of an immortalized human trophoblast cell-line, TCL-1, which possesses a CSF-1 autocrine loop

Many previous studies in both mouse and human placenta have implicated a role for colony stimulating factor-1 (CSF-1) in the regulation of placental development. In this study we have examined CSF-1 production by an immortalized cell line (TCL-1) derived from the choriodecidua, transfected with a retrovirus gene coding for the large-T antigen. TCL-1 cells were uniformly positive by immunocytochemistry for the composite sub-units of human chorionic g gonadotrophin (hCG) but were negative for markers of other cell types localized at the fetal-maternal interface. Gelatinase enzymes were secreted by TCL-1 cells cultured on extracellular matrix in a manner indicative of extra-villous trophoblast. Dot-blot immunoassays and ELISA indicated that CSF-1 was secreted by TCL-1 cells, at levels comparable to primary trophoblast cells and BeWo choriocarcinoma (trophoblast tumour) cells. Reverse transcriptase-polymerase chain reaction analysis confirmed the presence in TCL-1 cells of CSF-1 receptor mRNA (c-fms gene product), indicating that the components of a potential autocrine loop were present in these cells. Proliferation of TCL-1 cells was not affected by the addition of exogenous CSF-1 but was elevated in response to treatment with a CSF-1 neutralizing antibody. The immortalized cell line, TCL-1, provides a potential model in which to investigate regulation of growth and differentiation of trophoblast cells in vitro.

Interleukin-1β-stimulated PGE2 production from early first trimester human decidual cells is inhibited by dexamethasone and progesterone

Cytokines are known to increase the production of prostaglandins by human decidual cells, but negative regulators have not been identified. We have examined the effects of dexamethasone and progesterone on prostaglandin (PG) E2 synthesis by cultured human first trimester decidual cells. The numbers of cyclooxygenase (COX) enzyme positive cells were visualised by immunocytochemistry, using antibodies specific for COX-1 and COX-2. Interleukin-1 beta stimulated the production of prostaglandins E2 and F2 alpha dose-dependently, and this was associated with increased numbers of COX-2 positive cells. Progesterone (10(-7)-10(-6) M) and dexamethasone (10(-7)-10(-6) M) inhibited basal and interleukin-1 beta-stimulated prostaglandin production, and decreased the numbers of COX-2 positive cells. Neither interleukin-1 beta nor the steroids affected numbers of COX-1 positive cells. COX-2 seems to be the main enzyme controlling the synthesis of PGE2 by human decidual cells, and may be negatively regulated by progesterone.

Intraperitoneal radioimmunotherapy for ovarian cancer

Summary. Twenty‐eight patients with assessable residual ovarian cancer after cytoreductive surgery and chemotherapy received intraperitoneal 1–131 labelled monoclonal antibodies. There was no response in eight patients with tumour nodules >2 cm, a partial response in two of the 15 patients with tumour nodules <2 cm, and a complete response in three of the other five patients with positive peritoneal washings. A further six patients received Y‐90 labelled monoclonal antibodies for residual ovarian cancer. There was no response in one patient with nodules >2 cm, and a partial response in one of the other five patients with tumour nodules <2 cm. The non‐specific radiation dose in the peritoneal cavity from the infused isotope was measured by lithium fluoride thermoluminescent dosimetry (TLD). The radiation dose received by the peritoneal serosa was <500 cGy and was not sufficient to account for the observed tumour response. Significant bone marrow suppression was observed with 1–131 activities greater than 120 mCi and with Y‐90 activities greater than 13 mCi. The haemopoietic bone marrow is the dose‐limiting organ in patients receiving radioimmunotherapy.

Differential localization of endothelin ETa and ETB binding sites in human placenta

1 The localization and differential distribution of endothelin (ET) receptor subtypes (ETA and ETB) was investigated in sections of human placenta by use of quantitative in vitro autoradiography and receptor selective ligands. 2 Specific, high density [125I]‐ET‐1 binding sites were localized to the decidua and foetal membranes as well as to arteries and veins in the chorionic plate and throughout the villous tree. Moderate to low density binding was found in the extravillous and villous trophoblast respectively. 3 [125I]‐ET‐1 binding sites exhibited a rank order of inhibition by unlabelled peptide sequences (ET‐1 > ET‐3 >[Ala3,11,18Nle7]‐ET‐1 > BQ123 ≥ sarafotoxin 6c). However, in contrast to the monophasic inhibition curve of ET‐1, the other sequences produced a significantly better fit to a two component inhibition curve suggesting the presence of a heterogeneous population of ET binding sites. 4 ETA and ETB receptors were distinguished by competitive inhibition of [125I]‐ET‐1 binding with increasing concentrations of unlabelled ET‐3, [Ala3,11,18Nle7]‐ET‐1, sarafotoxin 6c and BQ123 and by incubating sections with the ETB agonist, [125I]‐BQ3020. ET receptor subtypes exhibited a differential distribution in the placenta. ETA type binding sites predominated (∼ 80% of the total) on veins and arteries in the chorionic plate. Veins in stem villi, blood vessels in distal regions of the villous tree and decidual cells displayed a high density (∼ 60–70% of the total) of the ETB receptor subtype. 5 No difference was detected in either the relative density of [125I]‐ET‐1 binding sites or the proportion of ETA to ETB sites in placentae from pregnancies complicated by pre‐eclampsia compared with normal term controls. 6 ET may have a local autocrine or paracrine role in the placenta, acting via specific receptors to influence foetoplacental blood flow and other aspects of placental function.

Limited transfer of prostaglandin E2 across the fetal membrane before and after labor

The transfer of prostaglandin E2 (PGE2) across intact fetal membranes (amnion‐chorion‐decidua) obtained before and after the onset of labor was investigated using a novel system for in vitro fetal membrane culture. Studies using physiological concentrations of PGE2 showed that very little PGE2 will cross the membranes without being metabolised, before or after the onset of labor. It was only when pharmacological concentrations of PGE2 were used that the enzyme activity of the chorion was no longer able to prevent transfer of PGE2 without conversion to inactive metabolites. These results suggest that only small amounts of PGE2 from amnion are normally transferred across the chorio‐decidua before or after the onset of labor, but the metabolism of PGE2 subsequent to transfer across the fetal membranes requires further assessment.

Production of epoxygenase metabolite by human reproductive tissues

Human amnion, trophoblast and umbilical vein endothelial cells synthesise an arachidonic acid metabolite which is neither a lipoxygenase nor a cyclo-oxygenase product. It is sensitive to stimulants and inhibitors of the cytochrome-P450-dependent epoxygenase system and co-migrates on HPLC with 14,15-epoxyeicosatrienoic acid (14,15-EET), which is an epoxygenase product. The function of 14,15-EET in these reproductive tissues is unknown, but it may be involved in the maintenance of vascular function.

Changes in amniotic arachidonic acid metabolism associated with increased cyclo-oxygenase gene expression

Objectives To study the differences in the metabolism of arachidonic acid, to prostaglandins and other eicosanoids, between amnion cells before and after labour. To study the changes in the expression of the type 1 cyclo‐oxygenase gene associated with the changes in arachidonic acid metabolism.