Robert Benveniste

Ontogenesis and characteristics of epidermal growth factor receptors in human placenta

Epidermal growth factor promotes growth in many cell types. The role of epidermal growth factor during gestation and fetal development is unknown. This study investigates the presence and binding characteristics of epidermal growth factor receptors in placentas throughout gestation. Cell membrane preparations were obtained from first-, second-, and third-trimester placentas and hydatidiform moles. Specific epidermal growth factor receptors were observed as early as 6 weeks of gestation. Throughout gestation, Scatchard analysis of epidermal growth factor binding was curvilinear, and dissociation studies were consistent with site-to-site interaction with negative cooperativity. Affinity constants at high (Ke = 9.9 +/- 0.79 X 10(9) L/mol) and low (Kf = 3.0 +/- 0.25 X 10(9) L/mol) receptor occupancy were unchanged throughout normal gestation. However, the number of receptors per milligram of protein significantly increased with advancing normal gestation (correlation coefficient, r = 0.81). In hydatidiform moles, the number of receptors was reduced when compared to that of normal tissue of similar gestational age. Our study provides a quantitative basis for further evaluation of epidermal growth factor as a possible factor in embryogenesis and fetal development.

Binding characteristics of epidermal growth factor receptors in male and female rat liver cell membrane preparations

The binding characteristics of epidermal growth factor (EGF) receptors were studied in cell membrane preparations from adult male (n = 14) and female (n = 13) rat livers. Results indicate a significant lower (about 65%) number of EGF receptors in female preparations. The possibility that the decrease in EGF receptors was only a reflection of an excess free EGF in female preparations was ruled out by means of acid extraction, ultrafiltration, and measurement of EGF in the acid extracts. In view of the known role of EGF in cell differentiation, it may be important to recognize that the number of its receptors, at least in liver preparations, is markedly different between sexes.

The effect of dehydroepiandrosterone sulfate on de novo and low-density lipoprotein-stimulated progesterone secretion by human chor ocarcinoma JEG-3 cells

Abstract It has been suggested that fetal adrenal steroids affect the secretion of progesterone by the human trophoblast and decrease the progesterone/estrogen secretory ratio at the time of parturition. In the present study, cultured human choriocarcinoma JEG-3 cells were used as an experimental model in order to examine the effect of dehydroepiandrosterone sulfate on both de novo and low-density lipoprotein-stimulated progesterone secretion. In serum-free and cholesterol-free Dulbecoos modified Eagles medium, JEG-3 cell cultures demonstrated a significant secretion of both pregnenolone and progesterone. A 200% to 300% increase in pregnenolone and progesterone secretion was achieved by physiologic low-density lipoprotein concentrations added to Dulbeccos modified Eagles medium while androgens and estrogens remained undetectable. Dehydroepiandrosterone sulfate added to Dulbeccos modified Eagles medium was actively converted into C-19 and C-18 steroids but had no significant effect (up to 20 μg/ml) on basal (de novo) progesterone secretion. In contrast, the addition of dehydroepiandrosterone sulfate (1 to 5 μg/ml) to cultures grown in Dulbeccos modified Eagles medium plus low-density lipoprotein induced a dose-related inhibition of progesterone and a return of that secretion to basal levels.

Characterization of an immune suppressor from transformed human trophoblastic JEG-3 cells☆

Cultured human choriocarcinoma JEG-3 cells secrete an immunosuppressor that inhibits lymphocyte proliferation stimulated by either an antigen or a mitogen. In this study, the immunosuppressive factor was characterized by three methods: ion-exchange and exclusion chromatography, partition in organic solvents, and thin-layer chromatography on silicic acid. This JEG-3 cell factor appeared to be a protein complex of about 150,000-200,000 Da that contained an immunologically active polar lipid. The structural and functional characteristics of JEG-3 cell immunosuppressor are similar if not identical to those of SIF, a suppressor lymphokine derived from T cells. These secretions from transformed trophoblastic cells may correspond to normal placental products or represent a function of malignant cells.

Human chorionic gonadotropin α-subunit in pregnancy

We have validated a direct radioimmunoassay (RIA) which measures the alpha-subunit of human chorionic gonadotropin (hCG) in pregnancy sera in the presence of large amounts of hCG. The levels of alpha-subunit in 124 sera from normal pregnancy women were measured and compared to the levels of hCG measured by a radioreceptor assay (RRA) which utilizes a rat testicular membrane preparation. Unlike hCG, which peaks at the end of the first trimester, the alpha-subunit continues to rise until 36 weeks of pregnancy. During the first half of pregnancy the levels of alpha-subunit remain less than 100 ng/ml whereas in the second half the levels remain less than 200 ng/ml. Three pathologic pregnancies were also studied; on the basis of the hCG determinations, they could not be differentiated from normal pregnancy. In contrast, all determinations of alpha-subunit were abnormally elevated. The concept that placental dysfunctions would cause an early and abnormally high level of alpha-subunit secretion deserves further investigation.

Effect of lipoxygenase products on choriogonadotropin secretion by cultured human choriocarcinoma JEG-3 cells pre- and post-stimulation with epidermal growth factor and a phorbol ester

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.

Characterization of Glycoprotein Hormone Free α-Subunit from Human Pituitary and Placenta Extracts

Standard alpha-subunit dissociated from glycoprotein hormones differs from individual (free) alpha-subunit found in sera or in cell culture media; secreted free alpha-subunit is larger, more acidic and lacks the ability of recombining in vitro with standard hCG-beta. It is unclear whether the large free alpha-subunit is only a secretory product or whether it is also present in tissue. Herein were studied the molecular size, the isoelectric pH, and the recombining activity of free alpha-subunit obtained from pituitary and placenta extracts. Sephadex exclusion chromatography showed the presence of both a large and a small form, and a changing large/small free alpha-subunit ratio in the various extracts. Most of the large form obtained from placenta extracts electrofocused into two peaks of pI 4.8 and 5.1. The large form showed no recombining activity with standard hCG-beta while the small free alpha-subunit recombined as well as did standard hCG-alpha. The observation of three common characteristics (a larger size, a pI 4.8, and a lack of recombining activity) suggests a similarity between the large secreted form and a fraction of the free alpha-subunit in tissue.

Possible evidence for the existence of a growth hormone fragment in porcine pituitary

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126-180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of 125I-labelled fragment B by procine growth hormone required a 10(3) M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of 125I-labelled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possible representing a growth hormone fragment.

Free α-subunit response to gonadotropin-releasing hormone in women with polycystic ovaries

The response of glycoprotein hormone free α -subunit to gonadotropin-releasing hormone (GnRH) was evaluated in 12 women with polycystic ovaries (PCOs). Six of these women were premedicated for 3 days with micronized 17 β -estradiol before receiving a 100- μ g bolus of GnRH. In nonmedicated PCO patients, GnRH did not significantly alter basal free α -subunit levels. In four of the six PCO patients receiving estrogen premedication, a significant increase in free α -subunit was observed; these four patients had low progesterone levels at the time of the GnRH test. Among the six premedicated patients, two had elevated (>4 ng/ml) progesterone levels, and the GnRH tests showed no significant effect on the levels of free α -subunit. The study revealed a dissociation between the free α -subunit responses to GnRH and the responses of luteinizing hormone; a closer reslationship was observed between free α -subunit and follicle-stimulating hormone responses. It was concluded that (1) the lack of a free α -subunit response to GnRH in PCO patients is not due to a primary inability of the pituitary gonadotroph to produce free α -subunit but is a consequence of an altered estrogenic milieu, and (2) a free α -subunit response to GnRH may reflect the replenishment of both follicle-stimulating hormone and luteinizing hormone in the gonadotrope.

Evaluation of the human gonadotroph free α-subunit secretory pools by administration of gonadotropin hormone-releasing hormone into normal subjects at different phases of the ovarian cycle

The specificity of a monoclonal antibody RIA for the measurement of free α-subunit in plasma is presently documented. This RIA was used to explore the pituitary gonadotroph free α-subunit reserve in normal ovulating women stimulated with gonadotropin hormone-releasing hormone (GnRH). RIA specificity was established by means of competitive inhibition curves with various glycoprotein hormone preparations, and Sephadex G-100 exclusion chromatography of purified hFSH (1–2), hLH (LER 960) and pituitary extract. The 3.8% and 5.5% crossreactivities of hFSH 1–2 and hLH LER 960 were shown by exclusion chromatography to result in part from free α-subunit contamination in these purified preparations. Pituitary extract chromatography indicated hFSH and hLH cross-reactivity below 2.5% and 1.5%, respectively. Normal females were stimulated with GnRH throughout the cycle: 3 tests were performed on Day 7, 1 test on Day 13, 16, 17 and 22, respectively, and 2 tests on Day 24. GnRH stimulation consisted of an initial 100 μg bolus (time 0) followed at 2 h by a 12.5 μg/h constant infusion, and a second 100 μg bolus at 5 h. In all subjects, baseline free α-subunit values were below 2 ng/ml. Total free α-subunit secretion was markedly enhanced in subjects Day 13 and 16, in concert with total hLH and hFSH secretion. In the three subjects Day 7, free α-subunit was released only after the second GnRH bolus. In periovulatory subjects, free α-subunit secretion became apparent after the initial bolus and with constant GnRH infusion. In the three subjects Day 22 and 24, peak levels of free α-subunit were obtained after the second GnRH bolus. Therefore, a first GnRH bolus and GnRH infusion served to document a readily releasable and a reserve free α-subunit pituitary pool, respectively. Following the second GnRH bolus, the presence of a “residual” free α-subunit was observed.