Immunocytochemical Localization of the Human Growth Hormone ...

0021-972X/89/6905-1069$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1989 by The Endocrine Society

Vol. 69, No. 5 Printed in U.S.A.

Immunocytochemical Localization of the Human Growth Hormone Variant in the Human Placenta* CATHERINE S. JARAf, ANNE T. SALUDf, GILLIAN D. BRYANT-GREENWOOD, G. PIRENS, G. HENNEN, AND F. FRANKENNE Department of Anatomy and Reproductive Biology, University of Hawaii, Honolulu, Hawaii 96822; and Endocrinologie Clinique et Experimentale, Institut de Pathologie, Centre Hospitalier Universitaire, B-4000, Sart Tilman/Liege, Belgium

ABSTRACT. Two monoclonal antibodies (Mab 5B4 and K24) were used for the immunocytochemical localization of human GH (hGH) and human placental GH in the human pituitary and placenta. On the basis of prior selection by RIA, Mab 5B4 was known to be directed to the N-terminal of hGH and was able to detect both pituitary and placental hGH, since their primary amino acid sequences are identical in this domain. Mab K24 was directed to an epitope present in hGH, but not in placental hGH, and was able to detect pituitary hGH only; neither the 5B4 or K24 Mab detected human placental lactogen. Five placentas were studied from women at term after elective cesarean section without labor. The cells of the anterior pituitary

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OLECULAR studies on the human GH (hGH) gene family predicted a protein differing from hGH by 13 amino acids (1). Recent work has shown that this protein, called the hGH variant (hGH-V), is expressed in the human placenta (2) and that the sequence of the hGH-V cDNA cloned from a placental library corresponds to the expected hGH-V protein sequence (3). In addition, it has been suggested that alternative splicing of the hGH-V mRNA gives rise to 2 proteins, hGH-V and hGH-V2, with marked structural differences (4). Two monoclonal antibodies (Mab) to hGH, coded 5B4 and K24, have been selected according to their specificity to pituitary and placental extracts (5). Mab 5B4 is directed to the N-terminal region of hGH in common with the placental protein (6). On the other hand, an internal epitope absent in placental hGH is detected by Mab K24 (6). These antibodies have been extensively screened for cross-reactivities with a variety of human and animal GHs and related peptides with the exception of hGH-V2 (6). Neither antibody detects human placental lactogen Received April 7, 1989. Address all correspondence and requests for reprints to: Dr. G. D. Bryant-Greenwood, Department of Anatomy and Reproductive Biology, University of Hawaii, 1960 East-West Road, Honolulu, Hawaii 96822. * This work was supported by Grant RCMl-RR-036-01. t MARC scholars (GM-07684-09).

gland and the syncytiotrophoblast of the placental villi were stained with Mab 5B4, whereas K24 only stained the pituitary cells; results consistent with the RIA screen. Mab 5B4 specifically stained two different cell types in the placental basal plate. Neither antiserum stained any cell in the amniotic or chorionic membranes or the adherent decidua. The results demonstrate that pituitary hGH and placental hGH are expressed uniquely in the pituitary and fetal placenta, respectively. In addition, the placental hGH gene is also expressed in the placental basal plate, a region of mixed fetal and maternal cells. (J Clin Endocrinol Metab 69: 1069, 1989)

(hPL), which allowed them to be used in RIAs to distinguish between hGH and placental hGH in serum during human gestation (5, 6). Up to 20-22 weeks of pregnancy both RIAs yielded similar values, suggesting parallel processing; thereafter, the hGH level measured with the K24 RIA decreased progressively until term, becoming undetectable by week 36. The RIA based upon the Mab 5B4, on the other hand, measured an increase in levels up to weeks 37-38 of gestation. Since K24 RIA levels were decreasing, it may be inferred that placental hGH replaces maternal pituitary hGH in metabolic regulation in the second half of pregnancy (6). Recently, sequence data obtained from placental hGH purified from tissue extracts provided definitive evidence of its identity with the hGH-V protein (to be published). Northern analysis with a specific hGH-V2 probe and mRNAs isolated from different intrauterine tissues has shown the expression of hGH-V2 mRNA in the villous trophoblast, but not in the amnionic or chorionic membranes or decidua (4). We have, therefore, used Mab 5B4 and K24 to specifically localize the placental cells responsible for the production of the hGH-V protein.

Materials and Methods Tissues Placentas with attached fetal membranes were collected at Kapiolani Women's and Children's Medical Center (Honolulu, 1069

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HI), from women after elective cesarean section at term who had not undergone labor (n = 5). These were chosen to avoid changes that could arise from the process of labor and delivery. The tissues were immediately taken to the laboratory on ice and processed within 1 h after removal. Representative slices (10 mm thick) were cut across the placenta to include the basal plates and pieces of fetal membrane with adherent decidua and were rolled and cut into 5-mm lengths. The sampled tissue was immediately fixed in Bouin's solution (16-24 h at 25 C) and then processed for paraplast embedding. Seven-micron sections were cut and mounted on glass slides. A pituitary gland from a 2-day-old infant was kindly provided by Dr. J. Hardman, Department of Pathology, University of Hawaii. This was processed identically to the intrauterine tissues. This pituitary gland was used as a control because of its availability at the time the placental study was in progress. Antisera and antigens Mab 5B4 and K24 were sent to Hawaii in lyophilized form after purification, as previously described (6). The lyophilized powder was reconstituted in water to a final concentration of 1 mg/mL and thereafter diluted in 0.015 M phosphate-0.15 M NaCI, pH 7.2, for use on the sections. These antibodies were found to be extremely susceptible to freezing and once in solution were kept at 4 C. Preabsorption studies were carried out with highly purified hPL, kindly supplied by Dr. A. Parlow, and pituitary hGH prepared in our laboratory and stored lyophilized over drying agent at -20 C (7). Other materials used and their sources were: Vectastain ABC kits (Vector Laboratories, Inc. Burlingame, CA), 30% hydrogen peroxide, (Fisher Scientific, Irvine, CA), and diaminobenzidine (Polysciences, Inc. Warrington, PA). The buffers used in the immunostaining procedure were phosphate-buffered saline (0.075 mol/L with 0.6 mol NaCl/L; pH 7.2; further diluted 1:5 just before use) and Tris-HCl (0.1 M; pH 7.2). Immunocytochemical staining Deparaffinized and rehydrated sections were immunostained using the avidin-biotin immunoperoxidase method. The sections were pretreated with 0.3% hydrogen peroxide for 5 min to remove endogenous peroxidase activity and with 0.5% normal horse serum for 20 min to saturate nonspecific binding sites for immunoglobulin G. The sections were then sequentially exposed to the following solutions at 25 C. Appropriately diluted Mab 5B4 and K24 were used on pituitary sections (1:750) and for placenta (1:100). Identical dilutions of mouse ascites fluid (ICN Immunobiologicals, Inc., Lisle, IL) were used as controls. Incubation with primary Mab was carried out for 60 min, followed by biotinylated horse antimouse immunoglobulin (1:250) for 30 min and avidin-biotin horseradish peroxidase complex (Vectastain ABC reagent) for 45 min. After each step, the sections were thoroughly rinsed in phosphate-buffered saline for 9 min (three changes of 3 min). Peroxidase activity was demonstrated cytochemically by incubation with a peroxidase substrate containing 0.5 mg/mL diaminobenzidine with 0.01% hydrogen peroxide in Tris-HCl buffer. The sections were rinsed

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in water and counterstained with hematoxylin. Coverslips were mounted with Pro-texx (Baxter-Scientific, Irvine, CA), and the slides were examined by brightfield microscopy. Preabsorption studies were carried out by incubation of the primary Mab at the concentration used for immunocytochemistry in a range of concentration of antigen (1-2000 ng/mL) for 48 h at 4 C. Results The hGH-producing cells of the anterior pituitary gland were stained with both Mab K24 and 5B4, shown in Fig. 1, A and B, respectively. There was no staining in the posterior pituitary gland treated with these Mab (not shown) or when sections from the anterior pituitary were treated with ascites fluid as a control (Fig. 1C). Preabsorption of both Mab with hGH showed reduced staining in the anterior pituitary gland at 500 SI units1 and complete loss of stain at 1000 SI units (not shown). Preabsorption of these Mab with hPL, however, caused no decrease in pituitary staining at any concentration up to 92.6 SI units. Mab K24 failed to stain any cells in the placenta in any of the tissues studied; the villous trophoblast from one placenta is shown in Fig. ID. However, 5B4 at the same concentration stained the placental syncytiotrophoblast (Fig. IE). This staining was only marginally reduced in intensity when the antibody was preabsorbed with 92.6 SI units hPL (Fig. IF). In contrast, the staining was almost completely reduced to background when this antibody was preabsorbed with 500 SI units hGH (not shown). It should be noted that when a polyclonal antiserum to hPL was used to detect placental hPL by the same method, staining was completely blocked by preabsorption of the antiserum with a 46.3-SI unit solution to hPL (our unpublished data). Mab 5B4 also stained cells of the placental basal plate, as shown in Fig. 1G. As seen in this micrograph, cells of different appearance show cytoplasmic stain. There are rounded or oval cells, some with two nuclei; these are possibly extravillous trophoblast (8). In addition, more elongated decidual-like cells of the basal plate immunostained. With preabsorption using 92.6 SI units hPL, it can be seen that this treatment failed to reduce the staining with 5B4 in either of these cell types (Figs. 1H). It was considerably reduced, however, in both cell types by preabsorption of the Mab with 500 SI units hGH (not shown). A control section treated with mouse ascites fluid is shown in Fig. II and shows no background or nonspecific stain. None of the specimens studied showed any localization to cells of the extravillous trophoblast present in the 1

Conversion factors: hGH, nanograms per mL to SI units (micrograms per L) is 1.0; hPL, micrograms per mL to SI units (nanomoles per L) is 46.3.


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FlG. 1. A-C, Sections of the anterior pituitary gland of a 2-day-old infant; (X200). A, Stained with Mab K24 (1:750 dilution); B, serial section stained with Mab 5B4 (1:750 dilution); C, serial section as control treated with mouse ascites fluid (1:750 dilution). D-F, Sections of the placental villi from tissue collected at term cesarean section without labor (X250). D, Stained with Mab K24 (1:100 dilution), note there is no staining; E, stained with Mab 5B4 (1:100 dilution), note staining in the syncytiotrophoblast of the villi; F, stained with Mab 5B4 (1:100 dilution) preabsorbed with 92.6 SI units hPL, note only slight reduction of stain. G-I, Sections of the placental basal plate from tissue collected at term cesarean section without labor (X250). G, Stained with Mab 5B4 (1:100 dilution), note cytoplasmic staining of rounded oval cells as well as more elongated deciduallike cells; H, serial section stained with Mab 5B4 (1:100 dilution) preabsorbed with 92.6 SI units hPL, note only slight reduction of stain; I, serial section treated as control with mouse ascites fluid (1:100 dilution).

chorionic membrane; likewise, there was no staining in the amnion or maternal decidua attached to the fetal membranes.

Considerable variation in the amount of stain in the five placentas studied, all from term cesarean sections, was noted. Indeed, the photomicrograph shown in Fig.


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IE shows the most uniform and darkest staining of the five. Two had intermittent staining of the placental syncytiotrophoblast, while the other two had only very light intermittent staining. Discussion This study confirms by immunocytochemistry that both Mab K24 and 5B4 detect pituitary hGH. However, since only 5B4, which recognizes the N-terminal end of hGH and placental hGH, was detected by immunocytochemistry in the syncytiotrophoblast of the placental villi, the presence of placental hGH may only be inferred. Two distinct protein products from the hGH-V gene, designated hGH-V and hGH-V2, are probably produced in the syncytiotrophoblast (3, 4). The antibody used (5B4), which is directed to the N-terminal region of both hGH and placental hGH, would appear likely to detect hGH-V, which is identical to placental hGH (to be published), and the hGH-V2 protein, whose first 1-20 amino acid residues are similar (3). The use of a specific antibody directed to the protein encoded by the fourth intron would be necessary to verify this (4). The immunolocalization studies carried out with the Mab preabsorbed with either hPL or hGH show it to be capable of detecting a specific hGH-related protein in spite of the very high tissue levels of hPL. These studies were carried out because Mab specificity deduced by RIA is not always the same when used in immunocytochemistry. Although hPL has been shown to react only at 3 orders of magnitude less than hGH in RIA based upon Mab 5B4 and K24 (6), we preabsorbed 5B4 with hPL and showed only marginal loss of staining. Thus, specificity of immunocytochemistry was the same as that by RIA. The wide variation in the degree of immunostaining in the different placentas studied, all collected from term elective cesarean section patients, contrasts with our results obtained previously using similar tissues but antisera to PRL (9) and hPL (to be published). It does, however, agree with the observation of increased variability of serum levels detected with this antibody in RIA in the last 4 weeks of gestation (6). This suggests that the regulatory control mechanisms for the production and/or secretion of this protein may be lost in the latter stages of pregnancy. Further studies are needed with placentas obtained at different stages of gestation to

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evaluate immunostaining during the course of pregnancy. The immunostaining of cells in the placental basal plate was unexpected. This region is a junction of maternal and fetal tissues, which is formed by the fusion of the chorion and the decidua basalis, and is considered to be a region where the maternal and fetal cells intermingle most closely (8). It appears that both maternal and fetal cells might produce the hGH-V protein, although the trophoblast cells in the reflective chorion failed to stain. The decidua-like cells in the placental basal plate immunostained with antisera to relaxin and PRL (9) as well as hPL (our unpublished work), but there have been no studies to precisely clarify the cell types involved in the production of these hormones. Clearly, this is an important region of the placenta which needs further study in order to clarify its role in endocrinology. Acknowledgments We thank Mrs. Sandra Yamamoto for her excellent technical assistance, and the staff and nurses of the labor and delivery suite at Kapiolani Women's and Childrens Hospital for their help collecting the placentas.

References 1. Seeburg PH. The human growth hormone gene family: Nucleotide sequences show recent divergence and predict a new polypeptide hormone. DNA 1982;l:239-49. 2. Frankenne F, Rentier-Delrue F, Scippo M-L, Martial J, Hennen G. Expression of the growth hormone variant gene in human placenta. J Clin Endocrinol Metab. 1987;64:635-7. 3. Igout A, Scippo M-L, Frankenne F, Hennen G. Cloning and nucleotide sequence of placental hGH-V cDNA. Arch Int Physiol Biochem 1988;96:63-7. 4. Cooke NE, Ray J, Emery JG, Liebhaber SA. Two distinct species of human growth hormone-variant mRNA in the human placenta predict the expression of novel growth hormone proteins. J Biol Chem. 1988;263:9001-6. 5. Hennen G, Frankenne F, Closset J, Gomez F, Pirens G, Khayat NE. A human placental GH: increasing levels during second half of pregnancy with pituitary GH suppression as revealed by monoclonal antibody radioimmunoassays. Int J Fertil. 1985;30:27-33. 6. Frankenne F, Closset J, Gomez F, Scippo ML, Smal J, Hennen G. The physiology of growth hormones (GHs) in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol Metab. 1988;66:1171-80. 7. Hunter WM, Greenwood FC. A radioimmunoelectrophoretic assay for human growth hormone. Biochem J. 1964;91:43-56. 8. Bulmer JN, Smith J, Morrison L, Wells M. Maternal and fetal cellular relationships in the human placental basal plate. Placenta. 1988;9:237-46. 9. Sakbun V, Koay ESC, Bryant-Greenwood GD. Immunocytochemical localization of prolactin and relaxin C-peptide in human decidua and placenta. J Clin Endocrinol Metab. 1987;65:339-43.
