Production of insulin-like growth factor binding proteins by human ovarian carcinoma cells

Cells of the human ovarian carcinoma lines EFO-21, EFO-27, MFO-35 and MFO-36 secrete binding proteins for insulin-like growth factors (IGFBPs) into their culture media. By sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and ligand blotting, seven groups of IGFBPs with molecular masses of 25, 30 (doublet), 34, 37, 40, 45, and 50 kDa were observed, depending on the cell line under investigation. By Northern blot analyses using cDNAs or oligonucleotides specific for the six types of IGFBP (IGFBP-1 to IGFBP-6), mRNA for all IGFBPs tested except for IGFBP-1 could be detected in the ovarian carcinoma cell extracts. In detail, analysis of EFO-21 protein products by SDS-PAGE yielded IGFBPs of 25, 34, and 50 kDa; extracts of EFO-21 cells contained mRNAs for IGFBP-2, -3, -4, and -6. EFO-27 cells produced IGFBPs of 40 kDa and 45 kDa as determined by SDS-PAGE, and mRNAs for IGFBP-3, -4, and -6 were detected. In the conditioned medium of MFO-35 cells, IGFBPs of 25, 30 (doublet), 34, 37, 40, and 45 kDa were observed by SDS-PAGE, while mRNAs for the five proteins IGFBP-2 to IGFBP-6 were found. MFO-36 cells produced IGFBPs of 34 kDa and 50 kDa as determined by SDS-PAGE, and the cells expressed mRNAs for IGFBP-2, -3, -4, and -6. In relation to published molecular mass data of the known IGFBPs, the size of the secreted proteins could be correlated to the mRNA patterns expressed by the ovarian carcinoma cells. It is concluded that ovarian carcinoma cells frequently express IGFBP-3, -4, and -6 and, to a lesser extent, IGFBP-2; the expression of IGFBP-5 appears as a rather rare event, while IGFBP-1 was not found to be expressed in ovarian carcinoma cells.Abbreviations BSA bovine serum albumin - FCS fetal bovine serum - PBS phosphate-buffered saline - SCC citrate-buffered saline - SFM serum-free medium - TRIS/NaCl TRIS-buffered saline Supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (SFB 215, project Al), and by the Bundesministerium für Forschung und Technologie, Bonn, Germany (BMFT grant 01GA8715/9)     

Luteinizing hormone-releasing hormone induces nuclear factor kappaB-activation and inhibits apoptosis in ovarian cancer cells

More than 80% of human ovarian cancers express LHRH and its receptor as part of a negative autocrine mechanism of growth control. This study was conducted to investigate whether LHRH affects apoptosis in ovarian cancer. EFO-21 and EFO-27 ovarian cancer cells were treated with LHRH agonist Triptorelin or with cytotoxic agent Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow cytometry. Expression of nuclear factor kappa B (NFkappaB) was assessed by RT-PCR and immunoblotting. For determination of Triptorelin-induced NFkappaB activation, cells were transfected with a NFkappaB-secreted alkaline phosphatase reporter gene plasmid (pNFkappaB-SEAP) and cultured for 96 h with or without Triptorelin. The causal relation between Triptorelin-induced NFkappaB activation and Triptorelin-induced protection against apoptosis was investigated using SN50, an inhibitor for nuclear translocation of activated NFkappaB. Apoptosis induction by Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L) for 72 h increased the percentage of apoptotic cells in EFO-21 and EFO-27 ovarian cancer cell lines to 31% or 34%, respectively. In cultures treated simultaneously with Triptorelin (100 nmol/L), the percentage of apoptotic cells was reduced significantly, to 17% or 18%, respectively (P < 0.001). RT-PCR and immunoblotting experiments showed that NFkappaB subunits p50 and p65 were expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21 or EFO-27 cells were transfected with pNFkappaB-SEAP and subsequently treated with Triptorelin (100 nmol/L), NFkappaB-induced SEAP expression increased 5.3-fold or 4.7-fold, respectively (P < 0.001). Triptorelin-induced reduction of Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition of NFkappaB translocation into the nucleus. We conclude that LHRH induces activation of NFkappaB and thus reduces Doxorubicin-induced apoptosis in human ovarian cancer cells. This possibility to protect ovarian cancer cells from programmed cell death is an important feature in LHRH signaling in ovarian tumors, apart from the inhibitory interference with the mitogenic pathway.     

Presence by radioimmunoassay of a calcitonin-like substance in porcine pituitary glands

We studied acidic acetone extracts of whole porcine pituitary glands for the presence of immunoreactive calcitonin (CT) using a porcine CT (pCT) RIA which did not react with other known pituitary hormones. Four preparations of porcine pituitary extract contained immunoreactive CT. Three of these displayed inhibition of binding parallel to that of authentic pCT in the pCT RIA and contained a single peak of immunoreactivity similar to pCT when studied by two different gel filtration chromatography systems. One preparation of porcine pituitary extract showed nonparallelism in RIA dose-dilution experiments and multiple immunoreactive species both similar to and larger than pCT on gel filtration in 6 M guanidine HCl. The effect of the reduction of disulfide bonds, followed by carboxymethylation of sulfhydryl groups, on immunoreactivity and apparent molecular size was similar for the CT-like substance in porcine pituitary extract and for authentic pCT. Preliminary immunohistological studies showed cytoplasmic staining in cells of the porcine adenohypophysis. These results demonstrate that the porcine pituitary gland contains a substance which has some of the immunochemical and biochemical properties of thyroidal pCT.     

Isolation of luteinising hormone receptor binding inhibitor from bovine corpus luteum.

A luteinising hormone receptor binding inhibitor (LHRBI) has been purified from bovine corpus luteum (CL). Steroid-free extract of the CL was subjected to successive chromatographies on Sephadex G-50, Q-Sepharose, Orange A dye and metal chelate affinity columns followed by high performance-reverse phase and gel filtration columns. Purification was monitored by the ability of the fractions to inhibit the binding of 125I-human chorionic gonadotropin (hCG) to porcine granulosa cells in vitro. The final isolate showed an 8000-fold enrichment of activity. It was also capable of inhibiting porcine granulosa cell secretion of estradiol and progesterone (P) in vitro. Administration of LHRBI into follicle-stimulating hormone (FSH)-stimulated, immature rats strongly inhibited the ovarian ovulatory response to hCG as revealed by decreased P levels and the number of ova released. The M(r) of LHRBI as assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis was ca. 15 kDa and the pI was between 5.0 and 5.5.     

LHRH might act as a negative autocrine regulator of proliferation of human ovarian cancer

OBJECTIVE: More than 80% of human ovarian cancers express LHRH and its receptor. The proliferation of human ovarian cancer cell lines is reduced by both LHRH agonists and antagonists. This study was designed to further clarify the possible biological function of this LHRH system. DESIGN: As LHRH agonists and antagonists uniformly reduce proliferation of human ovarian cancer in a dose-dependent way, the effect of low concentrations of authentic LHRH was studied. In addition, longer periods of treatment (up to 9 days) were analyzed. To assess the physiological role of LHRH produced by ovarian cancer cells it was neutralized by adequate concentrations of a specific LHRH antiserum. METHODS: Human ovarian cancer cells EFO-21 and EFO-27, which express LHRH and its receptor, were incubated for 1-9 days with increasing concentrations (1pmol/l to 10 micromol/l) of authentic LHRH or with concentrations of LHRH antiserum capable of neutralizing at least 1nmol/l LHRH. Proliferation was assessed by counting cells. RESULTS AND CONCLUSIONS: Authentic LHRH reduced time- and dose-dependently proliferation (by maximally mean+/-s.e.m. 32.7 +/- 4.4%, Newman-Keuls, P < 0.001) of both ovarian cancer cell lines. At very low concentrations (1pmol/l) a marginal reduction of proliferation or no effect was observed. A mitogenic effect of authentic LHRH was never detected. Treatment of ovarian cancer cell cultures with antiserum to LHRH significantly increased (up to mean+/-s.e.m. 121.0 +/- 2.8% of controls, Newman-Keuls P <0.001) proliferation of EFO-21 and EFO-27 cells. These findings suggest that LHRH produced by human ovarian cancer cells might act as a negative autocrine regulator of proliferation.     

Identification of the major mannose-binding proteins from chicken egg yolk and chicken serum as immunoglobulins.

Chicken egg yolk contains a mannose-binding protein that could be purified with a modification of the procedure of affinity chromatography and gel filtration used for chicken serum mannose-binding protein. The yolk protein was indistinguishable from the serum protein with respect to apparent molecular masses (169 +/- 7 kDa), subunits (74 kDa and 27 kDa, in approximately 1:1 ratio) produced after denaturation in the presence of mercaptoethanol, immunoreactivity with antibody against the chicken serum mannose-binding protein, amino acid composition, pH optimum for binding, calcium independence of binding, sugar-binding specificity, and specific-binding activity. Moreover, the chicken mannose-binding proteins cross-reacted with gamma-chain-specific antibody against chicken IgG. The binding proteins were identified as IgGs by several other criteria: identical electrophoresis pattern when subjected to reducing and nonreducing NaDodSO4/polyacrylamide gel electrophoresis, cochromatography on a Fractogel TSK HW-55(F) column, similar amino acid composition, and isolation of mannose-binding protein from purified serum IgG at a yield comparable to whole serum. These results support the notion that the mannose-binding proteins from the chicken serum and egg yolk are similar, if not identical, and are a subset of chicken IgG.     

Epidermal growth factor-induced down-regulation of receptor does not occur in HeLa cells grown in defined medium.

125I-Labeled epidermal growth factor (EGF) binds to HeLa cells in culture under completely defined conditions in a specific and saturable manner. When the cultures are maintained in serum, all the reported phenomena of down-regulation occur. In contrast, in cultures maintained in serum-free hormone-supplemented medium, binding is maximal after 50 min and remains constant for at least 18 hr. EGF retains its mitogenic activity in this defined system; as little as 4 pM EGF elicits a 2-fold increase in cell number in 5 days. EGF appears to be in reversible equilibrium with a constant number of surface receptors, and gel filtration analysis indicates a decrease in the rate of degradation and release of bound 125I-labeled EGF. These results indicate that EGF can retain its mitogenic activity in the absence of concomitant down-regulation.     

Subunits of human chorionic gonadotrophin: immunological and biological studies.

The alpha and beta subunits of human chorionic gonadotrophin (hCG) were prepared by incubation in 8 M urea, pH 4.5. The separation of the two subunits was obtained by DEAE-Sephadex A-25 chromatography and purification was carried out by gel filtration on Sephadex G-100. The beta subunit obtained was biologically active and was therefore further purified by affinity chromatography using as immuno-adsorbent the alpha antibodies coupled to Sepharose 4B. The beta subunit so purified showed a biological activity less than 1 IU/mg. The immunological and biological properties of the hCG subunits have been studied. It was found that the anti HCG beta serum can discriminate between hCG and hLH and that in the 125I-hCG + anti-beta serum radioimmunoassay, the cross-reactivity of pituitary hLH was lower than that of urinary hLH. Moreover, it was observed that the less purified was the urinary LH preparation, the higher was the cross-reactivity. Therefore we considered the hypothesis that during the purification of human menopausal gonadotrophin (hMG) some LH subunits or smaller immunoreactive fragments could have been discarded with the waste fractions. In order to test the validity of this hypophysis, all the protein fractions obtained during the purification of the hMG were gel-filtered on Sephadex G-100. The immunoreactivity of the effluents from the gel filtration was tested by hCG, hCG-beta, hCG-alpha and hLH radioimmunoassays. While the alpha reactive material was found in some fractions as a peak having the same Ve/Vo value as hCG-alpha, the beta reactive material presenude hMG fractions was not observed in other fractions. The cross-reactivity with the anti beta serum was very low and was found in the LH region of the gel chromatogram. Furthermore, the neutralization of the biological activity of hCG and of urinary and pituitary LH by the anti hCG beta serum was studied by incubating a fixed amount of the three hormones with increasing volumes of antiserum and measuring the LH ACTIVITY AFTER INCUBATION BY THE OADD test. It was observed that the anti hCG beta serum inhibits hCG more than urinary or pituitary LH.     

Comparisons of various acidic treatments of bovine serum on insulin-like growth factor-I immunoreactivity and binding activity.

Untreated serum exhibited two forms of insulin-like growth factor-I (IGF-I)-binding protein complexes during gel chromatography: one of Mr 150,000 and the other of Mr 40,000-45,000. The majority of the immunoreactive IGF-I was associated with the Mr 150,000 complex. Following acid-ethanol extraction of serum, the binding activity at Mr 150,000 disappeared and a reduced binding activity appeared in the albumin size range. Acid incubation of serum was slightly less effective than acid-ethanol extraction in reducing the binding activity. Acid-ethanol-extracted or acid-incubated serum were parallel to IGF-I standard in the dose-response displacement of iodinated IGF-I. Gel filtration of serum with 1 mol acetic acid/l almost completely separated IGF-I and the binding proteins. Binding-protein fractions from gel filtration interfered with the immunoreactivity of IGF-I with its antibodies, causing a non-parallel displacement curve in the radioimmunoassay (RIA). Serum IGF-I could be isolated as a single peak by high performance C18 reverse-phase liquid chromatography (HPLC). The concentrations of IGF-I measured in bovine sera by RIA were similar between acid gel filtration and HPLC; the concentrations by acid-ethanol extraction and acid incubation being about 30% smaller than those measured with former methods. The lower concentration of IGF-I measured in bovine serum with acid-ethanol extraction or acid incubation appears to be due to interference of IGF-binding proteins not removed by either treatment.     

Circulating antagonist of luteinizing hormone in association with infertility in stallions

Using a LH radioligand receptor assay (RRA) previously validated for use in serum and an equine monoclonal RIA, we have distinguished a subset of subfertile stallions with an elevated RRA/RIA ratio. After purification of the active moiety by anion exchange chromatography and immunoprecipitation with the equine LH (eLH) monoclonal antibody, RRA activity remained in the supernatant. This activity was also recognized by a polyclonal LH antibody (GDN 15) with wide cross-species recognition. This active fraction was further purified by gel filtration chromatography and shown to displace labeled eLH in a dose-dependent fashion in the RRA with an inhibition slope of 2.8 compared with a slope of 1.1 for native eLH. This fraction also inhibited the LH-stimulated steroidogenesis of Leydig cells in vitro in a dose-dependent fashion, but had no effect on basal (minus LH) steroid production. Polyacrylamide gel electrophoresis and electroelution of this material demonstrated RRA activity in a fraction with a mol wt between 45-66 kDa. We conclude that this substance 1) competitively inhibited binding of eLH and hCG to the LH receptor, 2) antagonized LH-stimulated steroidogenesis in vitro, and 3) may represent a LH isoform found in association with infertility in these animals.     

Morphological and functional characterization of interstitial cells from mouse testes fractionated on Percoll density gradients

Cell fractions were obtained by separation on Percoll density gradients after dissociation of mature mouse testes by mechanical or collagenase dispersion, and the ultrastructure, hCG receptor properties, and hCG-stimulated testosterone (T) production of these fractions were compared. Gradients were fractionated according to specific gravity, and all cell types were quantitated using morphometric techniques. Three peaks of specific [125I]iodo-hCG binding corresponding to densities of 1.0667 g/cm3 (fractions 2-3), 1.045 g/cm3 (fractions 6-7), and 1.0365-1.0215 g/cm3 (fraction 9) were obtained after collagenase dispersion, but the second peak of binding (fractions 6-7) was not observed after mechanical dispersion. Morphometric studies were performed by light microscopy on the cells present in the fractions corresponding to the first and second hCG binding peaks obtained by either mechanical or collagenase dispersion; the third representing germ cells and membranous debris was not studied further. Regardless of the method of preparation, morphologically intact Leydig cells represented 60-80% and 7-10% of the cells in fractions 2-3 and 6-7 that were associated with the first and second peaks of hCG binding Leydig cells obtained from fractions 6-7 contained greater numbers of lipid inclusions than Leydig cells from fractions 2-3. Mechanically dispersed Leydig cells exhibited similar numbers of hCG receptors in the dense and light Leydig cells, but hCG stimulated T production per Leydig cell was significantly greater in the dense Leydig cells containing few lipid inclusions. T production by dense and light collagenase-dispersed Leydig cells was not significantly different. The second hCG binding peak in collagenase-dispersed cell fractions 6-7 was associated with an increase in the number of an indeterminate connective cell type released from the testes by collagenase treatment, whose ultrastructure and limited hCG-binding capacity suggested that they may represent Leydig cell precursors. It is concluded that the identification and quantitation of different cell types in isolated testicular cell fractions is, therefore, of fundamental importance in the interpretation of receptor and secretory capacities of enriched preparations of Leydig cells.     

Chromatographic characterization of insulin-like growth factor-binding proteins in human pregnancy serum.

Insulin-like growth factor-binding proteins (IGFBPs) in maternal and umbilical cord sera have been analysed by combinations of gel filtration chromatography, affinity cross-linking and electrophoresis. On gel filtration chromatography, the majority of circulating IGF-I in non-pregnant and pregnant women was present in the large molecular mass (150 kDa) binding proteins (IGFBP-3). In umbilical cord serum, by contrast, most IGF-I was present in the 40 kDa binding proteins (consisting of IGFBP-1 and IGFBP-2). Western blots demonstrated an apparent progressive attenuation of IGFBP-3 and IGFBP-2 in serum from pregnant women with an increase in IGFBP-1. After prior cross-linking with disuccinimidyl suberate, the 150 kDa fractions (IGFBP-3) from non-pregnant and pregnant serum showed a similar pattern on SDS-PAGE (several bands at different molecular masses). However, IGFBP-2 (one of the components of the 40 kDa fractions) was undetectable, even after cross-linking, in serum from pregnant women later than 8 weeks of gestational age and in a mixture of maternal serum at term delivery and serum from non-pregnant women. This suggests that serum IGFBP-2 was degraded by specific proteases present in pregnancy serum. Following acid treatment, the 150 kDa fractions from pregnancy serum were split into smaller subunits or fragments while the 40 kDa fractions remained unchanged, suggesting that the 40 kDa binding proteins, are acid-stable. The present data demonstrate that IGFBP-3 is the principal IGFBP in pregnancy serum even though there is an apparent reduction in serum IGFBP-3 activity as revealed on Western blots. The absence of IGFBP-2 in serum from pregnant women may be due to degradation by proteases. In the fetal circulation IGFBP-1 and IGFBP-2 appear to be the major binding proteins for IGF-1.     

Fate of receptor-bound human chorionic gonadotrophin in pseudopregnant rat ovaries perfused in vitro

Pseudopregnant rats were injected with [125I] hCG, anaesthesized 1 h later and after cannulation of the aorta the ovaries were isolated and perfused with Gey & Gey buffer containing 0.2% BSA. The release of radioactivity was monitored for 2 h and analyzed by gel filtration. Five to ten per cent of the radioactivity was released within 2 h and represented small molecular weight peptides and iodotyrosine and [125I]hCG. Analysis of the ovarian radioactivity prior to and after perfusion revealed that virtually all hCG was receptor-bound. Loading the medium with unlabelled hCG displaced [125I]hCG from the receptor but did not enhance its degradation. Histological examination showed that the ovarian tissues were still intact after the 2 h perfusion. Immunohistochemical studies revealed a localization of the hCG at the cell periphery both prior to and after perfusion. These results provide evidence showing that the rate of internalization of receptor-hCG complexes in rat luteal cells is slow in vivo.     

Physical characteristics of the gonadotropin receptor-hormone complexes formed in vivo and in vitro.

The physical properties of detergent-solubilized gonadotropin receptor-hormone complexes, determined by density gradient centrifugation and gel filtration, were compared after in vivo and in vitro labeling of specific ovarian binding sites with radioiodinated human chorionic gonadotropin (hCG). Following intravenous administration of biologically active 125I-labeled hCG, up to 50% of the gonadotropin tracer was bound to the luteinized ovaries of immature female rats treated with pregnant mare serum/human chorionic gonadotropin. Comparable binding of 125I-labeled hCG was observed after equilibration of ovarian particles with the labeled hormone in vitro. The sedimentation properties of the solubilized receptor-hormone complexes formed in vivo were identical with those derived for the corresponding complexes formed in vitro and extracted with Triton X-100 and Lubrol PX, with sedimentation constants of 8.8 S for the Triton-solubilized complex and 7.0 S for the complex extracted with Lubrol PX. During analytical gel filtration of the Triton-solubilized receptor-hormone complex on Sepharose 6B in 0.1% Triton X-100, the partition coefficient (Kav) of the "in vivo" complex (0.32) was not significantly different from that of the complex formed in vitro (0.29). Gel filtration of the Lubrol-solubilized ovarian particles on Sepharose 6B in 0.5% Lubrol PX gave Kav values for the "in vivo" and "in vitro" labeled complexes of 0.36 and 0.32, respectively. These findings demonstrate that the physical properties of size and shape which determine the partition coefficient and sedimentation characteristics of detergent-solubilized gonadotropin receptor-hormone complexes formed in vitro are not distinguishable from those of the complexes extracted after specific interaction of the ovarian gonadotropin receptors with radioiodinated hCG in vivo.     

Growth stimulation of A431 cells by epidermal growth factor: identification of high-affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody.

Epidermal growth factor (EGF) at 3 nM maximally inhibits the proliferation of A431 epidermoid carcinoma cells. We show that at lower concentrations, in the range of 3-100 pM, EGF has a mitogenic effect on A431 cells. In the presence of 100 nM anti-EGF-receptor monoclonal IgG (designated 528), which inhibits A431 cell proliferation and blocks greater than 95% of EGF binding, EGF becomes mitogenic for A431 cells at concentrations up to 3 nM. These results suggest that a minor population of high-affinity EGF receptors may be involved in stimulation of A431 cell proliferation. Saturation binding assays with 125I-labeled EGF indicate that approximately equal to 0.1-0.2% of receptors for EGF are high-affinity receptors that bind EGF with an estimated Kd of 7 X 10(-11) M. This affinity is nearly 2 orders of magnitude higher than that of the remaining EGF receptors. Although A431 cell proliferation is maximally inhibited by nonsaturating amounts of EGF (3 nM), maximal inhibition by 528 IgG (approximately equal to 70% of maximal inhibition by EGF) requires saturating concentrations of antibody (approximately equal to 15 nM). Unlike EGF, rapid down-regulation is not observed with 528 IgG. These results indicate different mechanisms of growth inhibition of A431 cells by EGF and 528 IgG.     

N-Acetyl-D-galactosamine-inhibitable adherence lectin of Entamoeba histolytica. II. Mitogenic activity for human lymphocytes

A soluble protein preparation from virulent axenic amoebae (strain HM1-IMSS) has been demonstrated to be mitogenic for normal human lymphocytes; mitogenesis was optimal at 100 micrograms/ml when incubation was maintained for five to seven days with monocytes (P less than .001). After gel filtration chromatography mitogenic activity of fractions was associated with N-acetyl-D-galactosamine-inhibitable lectin activity (agglutination of Chinese hamster ovary cells; P less than .001). Lymphocyte proliferation induced by these active fractions was specifically inhibited by asialofetuin (P less than .001), which contains three terminal beta 1-4 linked galactose residues. In four strains of axenic amoebae mitogenic activity of soluble protein preparations correlated with in vitro virulence and lectin activity for Chinese hamster ovary cells. The amoebic N-acetyl-D-galactosamine-inhibitable lectin appears to be responsible for the mitogenic activity of soluble amoebic protein preparations; alteration of cell-mediated immunity by this amoebic moiety could potentiate in vivo virulence of the parasite.     

Hormonal control of epidermal growth factor receptors by gonadotropins during granulosa cell differentiation

The hormonal induction of epidermal growth factor (EGF) receptor formation was analyzed during the maturation of granulosa cells obtained from diethylstilbestrol-implanted immature rats. In the absence of FSH, EGF receptors (as measured by the binding of [125I]iodo-EGF to the intact cells) rose by 50% at 6 h of culture, but then declined to about 25-40% of their initial levels at 24-96 h of culture. Scatchard analyses demonstrated the presence of high affinity EGF-binding sites in both freshly prepared cells and after FSH treatment. FSH stimulated a dose-dependent increase in the EGF receptor content of granulosa cells during a 96-h culture period. Concentrations of FSH as low as 2.5-5 ng/ml elevated EGF receptor levels 2- to 3-fold compared to those in untreated control cells, and 30 ng/ml FSH caused a maximal 15-fold rise. FSH increased EGF receptor levels approximately 2-fold in the first 6 h of culture and by up to 7-fold at 96 h compared to levels in freshly prepared cells. FSH treatment did not change the binding affinity (Kd = 5-6 X 10(-11) M) of the EGF receptor, but increased the total number of EGF-binding sites. The stimulatory effects of FSH on EGF receptor expression were mimicked by other cAMP-inducing ligands, including 8-bromo-cAMP, forskolin, and choleragen. Ligands known to inhibit granulosa cell function, including GnRH agonists and the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate, reduced the stimulation of EGF receptors by FSH. However, only 12-O-tetradecanoyl-phorbol 13-acetate suppressed the induction of EGF receptors by 8-bromo-cAMP. In granulosa cells cultured for 48 h with FSH, subsequent treatment with hCG for 24 h reduced EGF receptor content by 25%. Autoradiographic studies with [125I]iodo-EGF in ovarian thin sections demonstrated that EGF-binding sites were uniformly dispersed throughout the ovaries of diethylstilbestrol-implanted rats. Treatment with PMSG markedly increased EGF receptors in the outer walls of the growing follicles, while hCG treatment after PMSG caused a general decline in ovarian labeling. These results indicate that FSH maintains and increases the number of EGF receptors during granulosa cell differentiation, while LH/hCG reduces EGF-binding sites. Such changes in EGF receptors in the presence of endogenous growth factors may influence the number and selection of follicles destined for ovulation.     

Distribution of the beta-core human chorionic gonadotrophin fragment in human body fluids.

The origins of a fragment of the human chorionic gonadotrophin (hCG) molecule, beta-core (beta C-hCG) were studied by analysis of beta C-hCG concentrations in biological fluids. In addition, the ability of the placenta to produce the fragment and the metabolism of hCG to beta C-hCG by human granulosa cells was determined in tissue culture. Finally the conversion of exogenous hCG to beta C-hCG was studied in vivo. The fragment was present in pregnancy urine as well as that from premenopausal and postmenopausal subjects. The highest concentrations were found in pregnant women. Ratios of beta C-hCG to intact hCG were higher in pregnancy urine when radioimmunoassay (RIA) was used compared with immunoradiometric assay (IRMA) (0.67 and 0.37 respectively). Concentrations of beta C-hCG were higher in postmenopausal urine than in premenopausal specimens. A significant amount of a high molecular weight beta C-hCG immunoreactive material was found in serum samples after size separation, and the molar ratio of beta C-hCG/hCG was estimated as 0.019. Amniotic fluid also contained small quantities of two forms of immunoreactive beta C-hCG and the ratio of 0.01 for authentic beta C-hCG/hCG increased to 0.026 when the high molecular weight form was considered. Cultured trophoblastic tissue released material with beta C-hCG immunoreactivity in the medium and chromatographic separation revealed that the majority of this material was of higher molecular weight compared with the authentic beta C-hCG form. beta C-hCG was the principal glycoprotein found in follicular fluid after hyperstimulated folliculogenesis and intramuscular injection of 5000 IU hCG. We also demonstrated that 26% of follicular fluid samples (n = 50) were positive for beta C-hCG; levels ranged from 5.2 to 23.0 pmol/l (13.1 +/- 5.7); S.D.) when a specific IRMA was used. The RIA could detect beta C-hCG in 48 samples (96%), levels ranging from 7.0 to 28.5 pmol/l (19.4 +/- 5.2). Moreover, granulosa cells cultured in the presence of hCG were able to degrade the intact molecule to both high molecular weight and authentic immunoreactive forms of beta C-hCG. After gel filtration, material of molecular weight over a wide range and immunoreactive for beta C-hCG was present in human seminal plasma. Assaying 74 samples of this fluid by IRMA, beta C-hCG was detected in 42 (56.7%), levels ranging between 5.5 and 59.5 pmol/l (24.9 +/- 15.2).(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of reversed-phase high-performance liquid chromatography to detect proteolytic activity from human pancreas in a radioimmunoassay for corticotrophin-releasing factor

Degradation of tracer during a radioimmunoassay (RIA) can result in false-positive concentrations of immunoreactivity being reported in a biological sample. A technique has been developed using reversed-phase high-performance liquid chromatography (HPLC) to detect proteolytic degradation of corticotrophin-releasing factor-41 (CRF-41) during incubation with tissue extracts under RIA conditions. Human pancreatic tissue was extracted in HCl or urea and incubated with 125I-labelled CRF-41 at neutral pH for 18 h. When samples were analysed by HPLC and fractions counted for radioactivity, tracer was extensively degraded. Heating extracts at 85 degrees C or adding lima bean trypsin inhibitor to the medium prevented degradation. Pancreatic tissue extracted in HCl was analysed by gel filtration and HPLC, and fractions were subjected to RIA for CRF-41. A peak of immunoreactivity was detected by both chromatographic methods. However, when this material was incubated with tracer and analysed by HPLC, the tracer was degraded, indicating that proteolytic activity remained after acid extraction and two forms of chromatography.