0021-972X/91/7204-0747$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1991 by The Endocrine Society

Vol. 72, No. 4 Printed in U.S.A.

A Chorionic Gonadotropin- Secreting Human Pituitary Cell* E. HAMMOND, J. GRIFFIN, AND W. D. ODELL Departments of Medicine and Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132

ABSTRACT. Recent data from our laboratory show that hCG is secreted in a pulsatile manner in parallel with human LH (hLH) in nonpregnant normal humans. Furthermore, GnRH stimulates hCG and hLH release. Using a monoclonal antibody specific for the /3-chain of hCG and not reacting with hLH, we have identified a heretofore unknown cell type in human pitui-

taries which stains only for hCG. The light microscopic, imraunocytochemical, and ultrastructural features are described. These data coupled to those from multiple earlier studies indicate that hCG is secreted by these cells in normal nonpregnant humans. (J Clin Endocrinol Metab 72: 747-754, 1991)

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LTHOUGH hCG is often considered a hormone secreted in normal humans exclusively by the trophoblastic cells during pregnancy, considerable data indicate that hCG circulates in small amounts in the blood of nonpregnant women and men. Furthermore, much of the data suggest that the pituitary gland is the source of part or all of this circulating hCG. As part of a series of studies concerning hCG in normal humans, we have immunostained human pituitaries with a specific monoclonal antibody that reacts only with the /3-chain of hCG. We have also used an antibody that reacts only with the carboxyl tail of hCG. We describe a previously unknown cell type that stains for hCG and does not stain for other anterior pituitary hormones. These results were confirmed using double labeling techniques and ultrastructural immunocytochemistry. These findings coupled with previous data from our laboratory concerning control of hCG secretion in normal humans suggest that this new cell type is the source of hCG in normal men and women. Methods Griffin and Odell (1) described a two-monoclonal antibody immunoradiometric assay which reacts only with intact hCG, showing no cross-reaction with the free /?-chain of hCG or with the biochemically closely related glycoprotein hormones, human LH (hLH), human FSH (hFSH), human TSH (hTSH), and the free a-chain of these hormones. In a standard equilib-

rium RIA, one of these monoclonal antibodies (MAB 4) was specific for the /3-chain of hCG and showed no reaction with intact glycoprotein hormones or the free a-chain of these hormones. This antibody was purified, as previously described, in our laboratory (1). Tissue preparation Tissue used for this study consisted of five normal human pituitaries (three men and two women) obtained at autopsy within 8 h of death. Two women and two men died of cardiac failure; one man died of metastatic carcinoma. Sections of normal pituitary fragments obtained at the time of resection of a PRL-secreting pituitary adenoma were also studied. Autopsy pituitaries were removed intact, bisected, and placed in a freshly prepared 4% solution of paraformaldehyde buffered with phosphate (buffered to 300 mosmol) for 1-2 h. This fixative is excellent for both light and electron microscopy (2). After 124 h of washing in phosphate-buffered saline (PBS), half of each pituitary was embedded in paraplast by standard techniques. A 1-mm slice of the other half, through the face adjacent to that used for light microscopy, was postfixed in 1% osmium tetroxide and routinely processed for electron microscopy by standard techniques. The remainder of this half, in two patients, was cut into 50-Aim thick sections using a vibratome (no. 1000, Lancer Co.). Sections were then used for ultrastructural immunocytochemistry. In the case of the surgically removed pituitary, random fragments were used for light microscopic examination and electron microscopy. It was not possible to use adjacent pieces for each type of examination because of the way in which the specimen was received.

August 23, 1989. Address all correspondence and requests for reprints to: William D. Odell, M.D., Ph.D., University of Utah School of Medicine, 50 North Medical Drive, Salt Lake City, Utah 84132. * This work was supported by a grant from the NIH. Presented at the 70th Annual Meeting of The Endocrine Society, New Orleans, LA, June 8-11,1988 (Abstract 49).

Histological immunocytochemistry Immunoperoxidase. For routine immunocytochemistry, serially labeled 4-jttm thick sections were cut from the paraffin block and affixed to glass slides. Serial sections were stained with 747

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FIG. 1. Photomicrograph of tissue section stained with antibody directed against hCG. Cells similar to those stained in Fig. 2 are shown. Magnification, X250.

monoclonal antibodies directed against hCGjS (1:250 dilution), hLH (1:250 dilution), hTSH (1:250 dilution), and polyclonal antibodies directed against hFSH (1:250 dilution), hPRL (1:500 dilution), ACTH (1:500 dilution), and hGH (1:500 dilution). One of the pituitaries was stained with purified rabbit polyclonal antibody directed against the carboxyl tail of hCG. In these experiments, adjacent serial sections were always stained with hCG, hLH, and hFSH in that order. Blocking experiments were performed on all pituitaries using concentrations of purified hLH and hCG predicted to block by RIA. The hLH preparation was AFP-6332B, kindly supplied by Dr. A. F. Parlow; the hCG preparation was CR121, kindly supplied by Dr. R. Canfield. For hLH, this concentration was 10 ixg/mL; for hCG, it was 1 lig/rnL. Additionally, sections were incubated with mixtures of antibodies and blocking hormone [anti-hLH (1:250) plus hLH (10 Mg/mL) and anti-hCG (1:250), and hCG (1 /ug/mL) and anti-hLH (1:250)] to evaluate the cross-reactivity of antibodies. Immunocytochemistry was performed by a standard peroxidase antiperoxidase technique using goat bio-

J C E & M • 1991 Vol 72 • No 4

FIG. 2. Photomicrograph of tissue section stained with antibody directed against the carboxyl tail of hCG. Cells similar to those in Fig. 1 are stained. Magnification, X250.

tinylated antimouse or antirabbit IgG as a linking antibody and the avidin-biotin-peroxidase complex (3, 4). 3,3-Diaminobenzidine was used as a substrate for the peroxidase reaction. It produced a brown reaction product in the positive cases. The length of the reaction time was gauged by observation of the positive controls. Positive controls for the hCG antibodies consisted of surgical specimens of placental villae obtained at curettage and fixed and processed in an identical fashion; positive controls for other antibodies were not used, since normal pituitaries contain these hormones. Negative controls consisted of serial sections of the pituitary incubated without the primary antibody. Immunofluorescence. Primary monoclonal antibodies directed against hCG and hLH, identical to those used in the immunoperoxidase procedure described above, were conjugated to either fluorescein isothiocyanafe (FITC) or rhodamine isothiocyanate (RITC), as described by Brandtzaeg (5). These labeled antibodies, tested in equilibrium immunoassays, displayed the same binding characteristics as the unlabeled antibodies. Serial sec-

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FIG. 3. Photomicrographs of tissue section stained with anti-hCG (left) and anti-hLH (right) by the immunoperoxidase method. Section is counterstained with hematoxylin. Note the large granules stained dark brown in the hCG-stained cells. The hLH-stained cells have granules of more variable size. Also note that the basement membrane is intact around the acini, adjacent to blood vessels. Autolytic changes are seen, since the tissue is from a postmortem pituitary. Magnification, X400. tions, identical to those used above, were deparaffinized, incubated in PBS (containing 2% BSA) for 24 h, and subsequently incubated in FITC-anti-hLH (undiluted; protein concentration, 385 /ng/mL) and RITC-anti-hCG (1:5 dilution; protein concentration, 39 jig/mL) for 72 h at 4 C. After a thorough wash in PBS, the slides were coverslipped with water-soluble mounting medium and viewed in an Olympus epifluorescence microscope (Olympus Corp., New Hyde Park, NY) equipped with filters to detect the emission spectra of fluorescein and rhodamine. Photographs of identical areas were made using each filter combination on slides subjected to double labeling. Positive and negative controls were identical to those described above.

of primary antibodies. The linking antibodies used were polyclonal goat antimouse or antirabbit IgG, and the final incubation was performed in mouse or rabbit peroxidase-antiperoxidase complex. 3,3-Diaminobenzidine was used as a substrate for the peroxidase reaction. After the immunocytochemicai reaction, the sections were fixed in Karnovsky's fixative, routinely dehydrated through graded acetones, and embedded in Embed. No postfixation in osmium was used for these sections. After routine polymerization, thin sections were cut at 800 A and placed on copper grids. Sections were examined with and without the addition of uranyl acetate and lead citrate staining. Electron microscopy

Ultrastructural immunocytochemistry Ultrastructural immunocytochemistry was similar, except the primary antibody was incubated with the tissue sections for 12 h at 4 C. The dilutions of primary antisera and blocking hormones used were identical to those used in the immunoperoxidase experiments described above. Identical types of reactions were carried out on the vibratome sections, including the same blocking experiments and the experiments using mixtures

Sections for electron microscopy from the adjacent face of each pituitary used for routine electron microscopy were sectioned at 1 nm and stained with toluidine blue. Cells with the morphological appearance of those showing immunostaining were thin-sectioned (800 A) for electron microscopy and stained with uranyl acetate and lead citrate by standard techniques (6). Sections were viewed and photographed in a JEOL 100 SX transmission electron microscope (JEOL, Peabody, MA).

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FIG. 4. Photographs of pituitary section incubated with a solution containing both FITC-hLH and RITC-hCG. All photographs were exposed for 60 s, and all magnifications were X400. A, An area containing FITC-hLH-labeled cells. The photograph was taken with a prism used to detect FITC. Cells showing labeling are in the center of the field. B, The same area photographed with a prism used to detect RITC. Arrows mark the area of cells that are labeled with FITC-hLH. They are unlabeled by RITC. C, Another area of the same section, showing one cell labeled with FITC-hLH. The photograph was taken with a prism to detect FITC. Sites of some RITC-labeled cells are marked with arrows. D, The same area shown in C photographed with a prism to detect RITC. Many RITC-hCG-labeled cells are seen. The arrow designates the cell labeled with FITChLH. It shows weak reactivity, which may reflect the overlap in the emission spectra detected by these prisms. RITC-hCG-labeled cells are much more strongly reactive and very abundant in this section. Other areas of the section, as shown in B, showed no RITC-labeled cells.

Results Histology Sections of the pituitaries from the three men and two women stained with hematoxylin and eosin showed no significant morphological abnormalities other than autolysis. Numerous acini were present, with cells showing variable staining characteristics. No inflammation, necrosis, or fibrosis was identified. The same features were present in the surgically resected pituitary, which also showed no visible autolysis. Immunocytochemistry Immunoperoxidase staining of serial sections demonstrated scattered cells in approximately 40% of the acini

of the midportion of the pars distalis of the adenohypophysis, which reacted with antibody directed against hCG/3. An accurate assessment of the percentage of positive cells in the surgically resected pituitary was impossible because the fragments were randomly removed from peripheral portions of the pituitary gland. Sections of these pituitaries were also stained with antibody directed against the carboxyl tail of hCG. The morphology of the cells that were immunostained was identical in both cases (Figs. 1 and 2). These cells contained large granules and occasional neutral fat droplets. They were also distinct morphologically from hLH-containing cells, which showed more variable granularity (Fig. 3). The hCGcontaining cells did not immunostain with antibodies to any of the other glycoprotein hormones or other pituitary hormones, as judged by a careful examination of serial

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FIG. 5. Electron micrographs of cells corresponding to those immunostaining with hCG. A, A low power electron micrograph showing an acinus containing hCG-type cells (shown by arrows). Original print magnification, X5.000. B, The cellular detail of one hCG-type cell. Original print magnification, x 12,500. Sections were counterstained with uranyl acetate and lead citrate.

Electron microscopy

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hCG hLH

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A chorionic gonadotropin-secreting human pituitary cell.

Recent data from our laboratory show that hCG is secreted in a pulsatile manner in parallel with human LH (hLH) in nonpregnant normal humans. Furtherm...
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