Journal of Histochemistry and Cytochemistry, Vol. 45, 985-990, Copyright © 1997 by The Histochemical Society, Inc.


ARTICLE

Production and Characterization of Specific Anti-peptide Antiserum Against Free {alpha}-subunit of Rat Pituitary Glycoprotein Hormones

Shigeyasu Tanakaa, Shingo Kurabuchic, Hiroshi Mochidaa, Hiroaki Hayashid, and Katsumi Wakabayashib
a Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
b Biosignal Research Center, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan
c Department of Histology, The Nippon Dental University School of Dentistry at Tokyo, Chiyoda-ku, Tokyo, Japan
d Gunma Prefectural College of Health Science, Maebashi, Japan

Correspondence to: Shigeyasu Tanaka, Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma Univ., Showa-machi 3-39-15, Maebashi 371, Japan.


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

To obtain an antibody specific for the {alpha}-subunit of rat pituitary glycoprotein hormones, we synthesized a peptide corresponding to the sequence 37-53 (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the rat {alpha}-subunit. The polyclonal antiserum against this peptide was generated in rabbits. This region is hydrophilic and highly conserved among several mammalian species. Noncompetitive binding tests showed that the ST-7 antiserum had specific affinity for the rat free {alpha}-subunit, but not for rat intact LH, FSH, and TSH. The ST-7 antiserum immunostained two types of cells in the rat anterior pituitary, i.e., gonadotrophs and thyrotrophs. This was also the case in mouse, cattle, sheep, and pig, which have an identical sequence of ST-7 in their {alpha}-subunit. The pituitary cells of horse (Arg substituted for Lys as residue 48 of the rat {alpha}-subunit), human, and eel (Leu for Ala at residue 45), chicken (Met for Ala at residue 45), and bullfrog (Tyr for Phe at residue 37 and Met for Ala at residue 45) were not stained with the ST-7 antiserum. This study indicated that the ST-7 antiserum is sequence-specific for the {alpha}-subunit and is therefore useful for immunohistochemical studies on the secretory pathway of the free {alpha}-subunit. (J Histochem Cytochem 45:985-990, 1997)

Key Words: {alpha}-subunit, pituitary glycoprotein hormone, anti-peptide antiserum, region-specific antiserum, immunocytochemistry, pituitary gland, rat


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Pituitary glycoprotein hormones (luteinizing hormone, LH; follicle-stimulating hormone, FSH; and thyroid-stimulating hormone, TSH) consist of {alpha}- and ß-subunits. The {alpha}-subunit is considered to be identical in amino acid sequence among all these glycoprotein hormones within one species (Hayashi et al. 1992 ; Pierce and Parsons 1981 ). Recently, several reports have documented that a certain amount of the free {alpha}-subunit of glycoprotein hormones is secreted from the normal pituitary, suggesting that this subunit may possibly play physiological roles (Oguchi et al. 1996 ; Begeot et al. 1983 , Begeot et al. 1984 ). In addition, human neoplastic pituitaries (Beck-Peccoz et al. 1985 , Beck-Peccoz et al. 1992 ; Osamura and Watanabe 1987 ; Landolt and Heitz 1986 ; Capella et al. 1983 ; Klibanski et al. 1983 ; Kourides et al. 1976 ) and human carcinoid tumors (Fukayama et al. 1986 , Fukayama et al. 1987 ; Heitz et al. 1983 ; Wahlstrom and Seppala 1981 ) have also been shown to produce the {alpha}-subunit, but not the ß-subunit, of LH, FSH, and TSH. However, it is not known whether the free {alpha}-subunit is secreted via a constitutive or a regulated secretory pathway.

In general, polyclonal antibodies against the {alpha}-subunit should have a high probability of recognizing intact glycoprotein hormones as well as the {alpha}-subunit (Tanaka et al. 1990 ). To our knowledge there is no available antibody that recognizes only the free {alpha}-subunit. Production of a specific antibody against the free {alpha}-subunit would be a useful tool in morphological and physiochemical research on the mechanism of secretion of this subunit of glycoprotein hormones. In this study, therefore, we generated a polyclonal antiserum against a synthetic peptide (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) corresponding to the conserved region, residues 37-53, of the amino acid sequence of the rat {alpha}-subunit. We showed that this antiserum is specific for the free {alpha}-subunit and immunohistochemically stains gonadotrophs and thyrotrophs in normal rat pituitary. In addition, we demonstrated that this antiserum recognizes only the glycoprotein hormone-secreting cells in pituitary glands of animals possessing the {alpha}-subunit sequence identical to the ST-7 peptide.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Peptide Synthesis
A peptide (ST-7: Phe-Ser-Arg-Ala-Tyr-Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) corresponding to the sequence 37-53 of the {alpha}-subunit of rat pituitary glycoprotein hormones was synthesized by an automated peptide synthesizer (Applied Biosystems model 431A). The protected peptide was assembled on an Fmoc-Val-p-alkoxybenzyl alcohol-resin (Kokusan Chemical Works; Tokyo, Japan). Deprotection and purification of the protected peptide were performed as described previously (Tanaka et al. 1991 ). The purity of the peptide was ascertained using a Hitachi 835 amino acid analyzer after hydrolysis with 6N HCl.

Immunization Procedure
The ST-7 peptide was coupled to bovine serum albumin (BSA) at a peptide/BSA ratio of 40:1 (mol:mol) with 0.05% glutaraldehyde. Rabbits were injected with the BSA-coupled peptide every 2 weeks for 7 months by the multiple site method as described previously (Tanaka et al. 1991 ).

Animals
Pituitaries from adult males of the following species were used for immunohistochemical examination: rat (Wistar), mouse (Balb/c), cattle, pig, sheep, horse, chicken, bullfrog (Rana catesbeiana), human, and eel. All the procedures involving animals were approved by the Animal Care and Use Committee of Gunma University.

Immunohistochemical Procedure
The pituitary glands were fixed by immersion in Bouin- Hollande sublimate or Bouin's fluid for 2 days or longer, dehydrated with an ethanol series, and then embedded in Paraplast. Four-µm-thick sections were mounted on gelatin-coated slides, deparaffinized with xylene, treated with iodine alcohol and sodium pyrosulfite to eliminate the sublimate, and then incubated in a solution of 0.3% H2O2 in methanol for 30 min to inhibit endogenous peroxidase activity. After rinsing with distilled water (DW) and then with PBS (0.01 M phosphate buffer and 0.14 M NaCl, 0.1% sodium azide, pH 7.5), the sections were immunostained by the peroxidase-anti-peroxidase (PAP) method. The sections were incubated sequentially with the following reagents: 1% BSA-PBS for 2 hr; rabbit anti-rat {alpha}-subunit serum (ST-7; 1:10,000), rabbit anti-sheep LHß serum (1:10,000), or rabbit anti-rat TSH serum (1:10,000; Wakabayashi and Tanaka 1988 ) overnight; goat anti-rabbit {gamma}-globulin serum (1:200) for 2 hr; and finally rabbit PAP complex (1:50) for 1.5 hr. After all these steps, the sections were washed three times with PBS, stained with 10 mg of 3,3'-diaminobenzidine tetrahydrochloride (Dojin; Kumamoto, Japan) and 0.005% H2O2 in 100 ml of 0.05 M Tris-HCl buffer, pH 7.6, to detect peroxidase activity, rinsed with DW, stained with 1% methyl green, dehydrated through a graded ethanol series, and mounted in Entellan (Merck; Darmstadt, Germany). The specificity of the ST-7 antibody was examined by conducting an immunohistochemical pre-absorption test. The diluted antiserum was mixed with the ST-7 peptide, rat LH {alpha}-subunit (NIDDK, I-1), rat LH (I-9), rat FSH (I-8), rat TSH (I-9), or bullfrog {alpha}-subunit (Hayashi et al. 1992 ; Hanaoka et al. 1984 ) at a final concentration of 10 µg/ml and pre-absorbed for 12 hr at 4C before its use in the specificity test.

Noncompetitive Binding Test
Radioiodinated rat LH {alpha}-subunit (NIDDK, I-1), LH (I-9), FSH (I-8), and TSH (I-9) were prepared using the chlora-mine-T method with minor modifications (Greenwood et al. 1963 ). The labeled hormones were separated by gel filtration on a Bio-Gel P-60 (BioRad; Richmond, CA) column with 0.05 M phosphate buffer, pH 7.5, as the eluant. The reaction mixture was composed of 300 µl of 1% BSA-PBS, 100 µl of serially diluted ST-7 antiserum in normal rabbit serum-EDTA-PBS, and 100 µl of radioiodinated hormone in 1% BSA-PBS. After incubation for 1 day, 200 µl of the secondary antiserum, diluted with 3.5% polyethylene glycol 6000 in 0.05 M EDTA-PBS, was added and the mixture was incubated for 2 hr. After centrifugation, the precipitates were counted using a gamma counter.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

As shown in Figure 1, the amino acid sequence of the ST-7 peptide, i.e., residues 37-53 of the {alpha}-subunit, used as the immunogen is hydrophilic and is highly conserved among animal species.



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Figure 1. Comparison of the amino acid sequence of the {alpha}-subunit among several animal species. The sequences are aligned for maximal identity. Amino acid residues different from those of the rat {alpha}-subunit are indicated by a single letter, identical residues by {bullet}, and deletions by -.

Specificity of Anti-rat {alpha}-subunit
In the noncompetitive binding tests, the anti-rat {alpha}-sub-unit serum (ST-7) showed an affinity for the rat {alpha}-subunit but not for rat LH, FSH, or TSH (Figure 2). These data suggest that the ST-7 antiserum is specific for the rat free {alpha}-subunit and does not react with intact rat LH, FSH, and TSH.



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Figure 2. Radioligand binding of anti-ST-7 serum to 125I-labeled rat {alpha}-subunit and intact pituitary glycoprotein hormones. The ST-7 antiserum binds specifically to the {alpha}-subunit.

Distribution of Cells Immunoreactive to Antiserum Against the {alpha}-subunit
Immunoreactive cells revealed with antiserum against rat {alpha}-subunit (ST-7) were distributed throughout the rat anterior pituitary (Figure 3). These positive cells displayed a large polygonal, ovoidal, or polymorphic shape. The entire cytoplasm was immunostained, but the degree of immunoreactivity was somewhat different from cell to cell. When three serial sections of rat pituitary were sequentially stained with anti-sheep LHß, anti-rat {alpha}-subunit, and anti-rat TSH, the {alpha}-subunit-positive cells corresponded almost cell-to-cell with the population of immunoreactive LHß or TSH cells (Figure 3). The immunoreactivity obtained with the anti-rat {alpha}-subunit serum was completely eliminated by addition of 10 µg/ml of the ST-7 peptide or rat {alpha}-subunit. However, when sections were stained with the antiserum preabsorbed with 10 µg/ml of rat LH, FSH, TSH, or bullfrog {alpha}-subunit, the immunoreactivity was not eliminated.



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Figure 3. Three consecutive sections of rat anterior pituitary gland immunostained with antisera against LHß (a), {alpha}-subunit (b), and TSH (c). Cells bearing the {alpha}-subunit also contain LHß (arrows) or TSH (arrowheads). Bar = 50 µm.

Species Specificity of Immunostainability
As shown in Figure 4, the immunoreactivity to the ST-7 antiserum of pituitaries from different species varied, depending on the amino acid sequence 37-53 of the {alpha}-subunit. In addition to binding to cells in the rat pituitary, the ST-7 antiserum also immunostained gonadotrophs and thyrotrophs in the anterior pituitaries of mouse, cattle, sheep, and pig, all of which have an identical sequence of the ST-7 peptide. However, the ST-7 antiserum did not stain any pituitary glycoprotein hormone-producing cells in the pituitary gland of horse, in which there is one amino acid substitution (to 48-Arg) in the 37-53 region. Moreover, pituitaries of human and eel (to 45-Leu), and chicken (to 45-Met) and bullfrog (to 37-Tyr and 45-Met), were not stained.



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Figure 4. Immunoreactivity of the pituitaries of several animals to ST-7 antiserum. +, positive staining; -, no staining.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The present study demonstrates that the ST-7 antiserum against the rat {alpha}-subunit recognizes only the free {alpha}-subunit of pituitary glycoprotein hormones (LH, FSH, and TSH), and not the intact hormones. The amino acid sequence of the ST-7 peptide used as immunogen is highly conserved among the species thus far examined. The specificity of the ST-7 antiserum was confirmed by noncompetitive binding tests, indicating that this antiserum specifically binds to the free rat {alpha}-subunit. In the present immunoabsorption test, moreover, the antiserum pre-absorbed with the intact LH, FSH, or TSH clearly stained glycoprotein hormone-producing cells; whereas the antiserum pre-adsorbed with the rat {alpha}-subunit or ST-7 peptide did not stain any pituitary cells. These results, too, suggest that the ST-7 antiserum is specific for the free {alpha}-subunit. Based on a recent study of the crystal structure of the human chorionic gonadotropin (Lapthorn et al. 1994 ), we presume that the region of residues 37-53 in the {alpha}-subunit might be one of the binding sites for the ß-subunit, and that antiserum against this region would therefore recognize only the free {alpha}-subunit.

In the present immunohistochemical study, the ST-7 antiserum stained gonadotrophs and thyrotrophs in rat pituitary glands. Similarly, the ST-7 antiserum stained the gonadotrophs and thyrotrophs in other animals with an amino acid sequence identical to that of the ST-7 peptide. However, no pituitary cells of horse (48-Arg in place of 48-Lys of the rat sequence), human or eel (45-Leu in place of 45-Ala), chicken (45-Met in place of 45-Ala), or bullfrog (37-Tyr in place of 37-Phe and 45-Met in place of 45-Ala) pituitary glands were stained with the antiserum. Therefore, the nature of the side chain of residue 45 in the {alpha}-subunit is important for recognition of the subunit. We concluded that the ST-7 antiserum recognizes a restricted three-dimensional structure from 45-Ala to 48-Lys in the rat {alpha}-subunit.

Some recent reports have indicated that the free {alpha}-subunit is secreted under physiological conditions and has a biological function. For example, the free {alpha}-subunit induces differentiation of PRL cells in fetal pituitary cultures (Begeot et al. 1983 ), stimulates synergistically with progesterone the differentiation of cultured human endometrial stromal cells to decidualized cells (Moy et al. 1996 ), stimulates secretion of prolactin from human decidual cells in pregnancy (Blithe et al. 1991 ), and stimulates PRL release from the bullfrog pituitary (Oguchi et al. 1996 ; Tanaka et al. 1992 ). Moreover, the crystalline structure of the {alpha}-subunit reveals that its tertiary structure is a cystine knot formed by three disulfide bridges. This motif is shared by the glycoprotein hormone ß-subunits and by three growth factors: nerve growth factor, transforming growth factor, and platelet-derived growth factor, suggesting that the free {alpha}-subunit may have some growth factor activity (Lapthorn et al. 1994 ). In addition, the free {alpha}-subunit was found to be expressed in the placode of Rathke's pouch several days before the appearence of the ß-subunit of glycoprotein hormones in the pituitary gland, suggesting that it may play a role other than the {alpha}-subunit of these hormones (Simmons et al. 1990 ). As part of the research on the physiological roles of the {alpha}-subunit, it is important to know the secretion mechanisms of the subunit. Results thus far are contradictory, with one biochemical study showing that the free {alpha}-subunit is secreted via the constitutive secretory pathway (Blomquist and Baenziger 1992 ), whereas the other suggests that the free {alpha}-subunit and intact glycoprotein hormones are secreted via the regulated secretory pathway (Weiss et al. 1990 ). Therefore, immunocytochemical approaches using the ST-7 antiserum would be useful to define the intracellular secretory pathway of the free {alpha}-subunit.


  Acknowledgments

Supported by grants-in-aid from the Japanese Ministry of Education, Science, and Culture (ST).

We are grateful to Dr A.F. Parlow of the Pituitary Hormones and Antisera Center and National Pituitary Agency, NIDDK, for supplying rat pituitary hormones, to Dr Y. Hanaoka of our institute for supplying bullfrog {alpha}-subunit hormone, and to Ms H. Kobayashi for technical assistance.

Received for publication September 20, 1996; accepted December 31, 1996.


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Introduction
Materials and Methods
Results
Discussion
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