0021-972X/90/7105-1318$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1990 by The Endocrine Society

Vol. 71, No. 5 Printed in U.S.A.

Secretion of Chorionic Gonadotropin by Cultured Human Pituitary Cells* WILLIAM D. ODELL, JEANINE GRIFFIN, HILDEGARDE M. BASHEY, AND PETER J. SNYDER Department of Internal Medicine, University of Utah School of Medicine (W.D.O., J.G.), Salt Lake City, Utah 84132; and the Endocrinology Section, Department of Medicine, University of Pennsylvania School of Medicine (H.M.B., P.J.S.), Philadelphia, Pennsylvania 19104-6149

ABSTRACT. To investigate the possibility that the human pituitary gland secretes CG, we used a highly specific, two-site, double monoclonal immunoradiometric assay to measure CG in the medium in which the dispersed cells of pituitary glands from human fetuses of 20-24 weeks gestation were cultured. The cross-reactivity of immunopurified human LH in the CG assay was less than 0.03%. LH was also measured by a double monoclonal immunoradiometric assay. Secretion of CG by the cultured fetal pituitary cells was readily detectable, although in gradually decreasing amounts, for the 11 days of culture. LH secretion paralleled CG secretion and was much greater in magnitude. Pituitary cells from female


EVERAL publications have offered indirect evidence that a hCG-like material is secreted by the pituitary gland of normal humans. Robertson et al. (1) and Matsuura et al. (2) have detected and partially characterized CG extracted from pituitary glands. Stenman et al. (3) have detected CG in blood of nonpregnant women and men and have shown that this CG is stimulated by GnRH and is suppressed by estrogens. Studies from our laboratory have shown the following. 1) CG is detectable in blood from women during the normal menstrual cycle, from postmenopausal women, and from men (4, 5). 2) CG is secreted in a pulsatile fashion in postmenopausal women and women during the normal menstrual cycle (4, 5). 3) CG is stimulated by GnRH and suppressed by GnRH agonist administration (4). 4) A previously unknown pituitary cell type exists, which stains immunospecifically for CG, but not for LH, FSH, or other pituitary hormones (6). 5) Purified human LH prepared from pituitary glands is contaminated with small amounts of CG (7). These studies provide indirect evidence that the norReceived May 18,1990. Address all correspondence and requests for reprints to: William D. Odell, M.D., Ph.D., Department of Internal Medicine, University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, Utah 84132. * This work was supported by Grant ROl-HD-18986.

fetuses generally secreted more CG as well as more LH than those from male fetuses. Dilutions of the medium showed that the secreted CG exhibited parallelism with the CG standard. Chromatofocusing of the medium across a pH 6-9 gradient yielded several peaks of LH immunoreactivity between pH 6.5 and 8.5, but no peaks of CG. Chromatofocusing of the medium across a pH 3-7 gradient yielded peaks of CG, but not LH, between pH 4.0-5.5. These data indicate that CG immunoreactivity, distinct and separable from LH immunoreactivity, is secreted by the dispersed cells of fetal human pituitary glands. (J Clin Endocrinol Metab 7 1 : 1318-1321, 1990)

mal human pituitary secretes CG. The studies reported herein provide direct evidence that CG is secreted by the normal human pituitary, specifically by human fetal pituitary cells in culture. Materials and Methods Pituitary cell cultures After approval by the University of Pennsylvania Committee on Studies Involving Humans, pituitary glands were obtained from 20- to 24-week-old human fetuses after pregnancy termination. They were transported to the laboratory on ice within 3-6 h. For cultures to be used for chromatofocusing studies, three to five glands from both male and female fetuses were combined and minced together; for studies of the effect of fetal sex on CG secretion, pituitary glands were minced individually. The minced tissue was incubated with 1 g/L trypsin and 0.1 g/ L DNAse in Ca- and Mg-free Tyrode's solution while shaking at 37 C to disperse the cells. The dispersed cells were washed with 45% Dulbecco's Modified Eagle's Medium, 45% Ham's F12, and 10% fetal calf serum (DMEM-F12-FCS); plated (0.5 X 106 cells/well) in 16-mm plastic wells precoated with poly-Llysine; and incubated in DMEM-F12-FCS at 37 C under 5% CO2. The medium was changed after 1, 4, 7, and 11 days. After the medium was harvested, it was centrifuged to sediment any loose cells and then frozen at —20 C. 1318

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SECRETION OF CG BY PITUITARY CELLS Chromatofocusing Chromatofocusing across a pH gradient from 6-9 was performed, as previously described (8), using a 23 x 0.9-cm column of Polybuffer Exchanger 94 (Pharmacia, Piscataway, NJ) that had been equilibrated with 15 bed vol 0.025 mol/L ethanolamine titrated with acetic acid to pH 9.9 (starting buffer). Before a sample was applied to the column, it was dialyzed against two changes each of 4 L starting buffer for 2 h. The sample was eluted at 12 mL/h with Polybuffer 96, which had been diluted 1:10 and adjusted to pH 5.9 with acetic acid; 1.7-mL fractions were collected. The pH of the eluate was monitored continuously using a flow-through electrode (Pharmacia). BSA was added to each column fraction to make a final concentration of 10 g/L, and the fractions were frozen. Chromatofocusing across a pH gradient of 3.0-7.5 was also performed using Polybuffer Exchanger 94, which had been equilibrated with imidazole HC1 titrated to pH 7.4. The sample was eluted with Polybuffer 74 titrated to pH 2.8 with HC1. Immunoassays Media were assayed for human (h) LH and hCG, employing the highly specific two-monoclonal antibody sandwich-type assays previously described from our laboratory (9, 10). In our original assays, cross-reactivity of purified LH preparations in the CG assay was 0.012-0.1%. Careful continued assessment of assay performance over the next 3 yr demonstrated changing isotype of one monoclonal antibody from IGGi, to IGG2B- This was associated with increased cross-reactivity of LH to 0.3%. Because of this, we asked if this was true cross-reactivity of LH in the CG assay or to contamination of the LH with CG. When the LH was immunopurified using a monoclonal antibody believed to be entirely specific for CG (7), the crossreaction of LH in the CG assay decreased to less than 0.1% when that assay was a simultaneous type assay (all reagents added at one time) and to less than 0.03% when the assay was performed sequentially. We, therefore, employed the sequential assay for the studies described herein. The sequential assay was performed as follows. All tubes contained 300 nh unknown sample or reference CG, 100 /iL horse serum and 25 ^1 biotinylated monoclonal antibody (no. 4) (9). Number 4 antibody was diluted in 1% horse serum, 0.4 M K2PO4, 0.15 M NaCl, and 0.5% BSA (antibody buffer). The polystyrene-avidin coated bead was incubated with biotinylated antibody 4 and the unknown sample or reference CG for 22 h at room temperature with constant shaking. After incubation the bead was washed three times in 0.15 M NaCl-0.1 M phosphate, pH 7.4, buffer and then incubated with the 125I-labelled second monoclonal antibody no. 9, also dissolved in antibody buffer, for 6 h at room temperature with constant shaking. The bead was then washed three times in 0.15 M NaCl-0.01% Triton X-100, and the counts per min bound were determined in an automatic 7-spectrometer. The reference preparations were highly purified CG (CR121), kindly supplied by Dr. Robert Canfield, and highly purified hLH (AFP-6332B), kindly supplied by Dr. A. F. Parlow.


Results The specificity of the sandwich-type two-monoclonal antibody sequential assay for CG employed in these studies allowed the detection of small amounts of CG in the presence of relatively large amounts of LH. The cross-reactivity of LH in this assay, as shown in Fig. 1, was less than 0.03%. As previously described, FSH, TSH, and the uncombined CG /3-subunit also have very low cross-reactivities in this assay (9). Using this assay we found that the cultured dispersed cells of three female human fetal pituitary glands secreted readily detectable, although gradually decreasing, amounts of CG during the entire 11 days in culture (Fig. 2, upper panel). These cells also secreted LH in a similar pattern, although in much larger amounts (Fig. 2, lower panel). Assay of dilutions of the medium showed that the secreted CG exhibited parallelism, and thus immunologic similarity, to the CG standard (Fig. 3). The amount of CG secreted by cells from female pituitary glands was generally greater than that from male pituitary glands. As shown in Table 1, the cultured cells of four of five female pituitary glands secreted readily detectable amounts of CG, but only one of five male pituitary glands did so. The amounts of LH secreted by these cultured cells showed a similar sex relationship, but were much greater than those of CG. To separate CG from LH, media in which fetal pituitary cells had been cultured were subjected to chromatofocusing, a column chromatographic technique for separating by charge. When medium from cultures of combined male and female pituitary glands was chromatofocused across a pH 6-9 gradient, several peaks LH immunoreactivity were detected between pH 6.5 and 8.5, but no CG was detected (Fig. 4A). Application of a 1-M NaCl solution to the column eluted both CG and LH immunoreactivities. A second chromatofocusing study with the same pH gradient using medium from another culture of combined male and female pituitary 8000 -1


• o

4000 H

hOG hLH hLH affinity purified




100 1000 HORMONE (pg/tube)



FIG. 1. The sequential two-monoclonal antibody sandwich-type assay for CG. A, Reference CG CR-121; • , LH (AFP 0642B); O, LH (AFP 0642B) after immunopurification.

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JCE & M • 1990 Vol 71 • No 5

TABLE 1. Secretion of CG and LH by fetal human pituitary cells, as stratified by sex


LH (pg/24 h)

CG (pg/24 h)








Secretion of chorionic gonadotropin by cultured human pituitary cells.

To investigate the possibility that the human pituitary gland secretes CG, we used a highly specific, two-site, double monoclonal immunoradiometric as...
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