ARTICLE |
Correspondence to: Shigeyasu Tanaka, Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma Univ., Showa-machi 3-39-15, Maebashi 371, Japan.
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Summary |
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To obtain an antibody specific for the -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
-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
-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
-subunit. The pituitary cells of horse (Arg substituted for Lys as residue 48 of the rat
-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
-subunit and is therefore useful for immunohistochemical studies on the secretory pathway of the free
-subunit. (J Histochem Cytochem 45:985-990, 1997)
Key Words:
-subunit, pituitary glycoprotein hormone, anti-peptide antiserum, region-specific antiserum, immunocytochemistry, pituitary gland, rat
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Introduction |
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Pituitary glycoprotein hormones (luteinizing hormone, LH; follicle-stimulating hormone, FSH; and thyroid-stimulating hormone, TSH) consist of - and ß-subunits. The
-subunit is considered to be identical in amino acid sequence among all these glycoprotein hormones within one species (
-subunit of glycoprotein hormones is secreted from the normal pituitary, suggesting that this subunit may possibly play physiological roles (
-subunit, but not the ß-subunit, of LH, FSH, and TSH. However, it is not known whether the free
-subunit is secreted via a constitutive or a regulated secretory pathway.
In general, polyclonal antibodies against the -subunit should have a high probability of recognizing intact glycoprotein hormones as well as the
-subunit (
-subunit. Production of a specific antibody against the free
-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
-subunit. We showed that this antiserum is specific for the free
-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
-subunit sequence identical to the ST-7 peptide.
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Materials and Methods |
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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 -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 (
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 (
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 -subunit serum (ST-7; 1:10,000), rabbit anti-sheep LHß serum (1:10,000), or rabbit anti-rat TSH serum (1:10,000;
-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
-subunit (NIDDK, I-1), rat LH (I-9), rat FSH (I-8), rat TSH (I-9), or bullfrog
-subunit (
Noncompetitive Binding Test
Radioiodinated rat LH -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 (
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Results |
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As shown in Figure 1, the amino acid sequence of the ST-7 peptide, i.e., residues 37-53 of the -subunit, used as the immunogen is hydrophilic and is highly conserved among animal species.
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Specificity of Anti-rat -subunit
In the noncompetitive binding tests, the anti-rat -sub-unit serum (ST-7) showed an affinity for the rat
-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
-subunit and does not react with intact rat LH, FSH, and TSH.
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Distribution of Cells Immunoreactive to Antiserum Against the -subunit
Immunoreactive cells revealed with antiserum against rat -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
-subunit, and anti-rat TSH, the
-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
-subunit serum was completely eliminated by addition of 10 µg/ml of the ST-7 peptide or rat
-subunit. However, when sections were stained with the antiserum preabsorbed with 10 µg/ml of rat LH, FSH, TSH, or bullfrog
-subunit, the immunoreactivity was not eliminated.
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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 -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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Discussion |
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The present study demonstrates that the ST-7 antiserum against the rat -subunit recognizes only the free
-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
-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
-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
-subunit. Based on a recent study of the crystal structure of the human chorionic gonadotropin (
-subunit might be one of the binding sites for the ß-subunit, and that antiserum against this region would therefore recognize only the free
-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 -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
-subunit.
Some recent reports have indicated that the free -subunit is secreted under physiological conditions and has a biological function. For example, the free
-subunit induces differentiation of PRL cells in fetal pituitary cultures (
-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
-subunit may have some growth factor activity (
-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
-subunit of these hormones (
-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
-subunit is secreted via the constitutive secretory pathway (
-subunit and intact glycoprotein hormones are secreted via the regulated secretory pathway (
-subunit.
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Acknowledgments |
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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 -subunit hormone, and to Ms H. Kobayashi for technical assistance.
Received for publication September 20, 1996; accepted December 31, 1996.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Beck-Peccoz P, Bassetti M, Spada A, Medri G, Arosio M, Giannattasio G, Faglia G (1985) Glycoprotein hormone -subunit response to growth hormone (GH)-releasing hormone in patients with active acromegaly. Evidence for
-subunit and GH coexistence in the same tumoral cell. J Clin Endocrinol Metab 61:541-546[Abstract]
Beck-Peccoz P, Persani L, Faglia G (1992) Glycoprotein hormone -subunit in pituitary adenomas. Trends Endocrinol Metab 3:41-45
Begeot M, Hemming FL, Dubois PM, Combarnous Y, Dubois MP, Aubert ML (1984) Induction of pituitary lactotrope differentiation by luteinizing hormone subunit. Science 226:566-568[Medline]
Begeot M, Hemming FJ, Martnat N, Dubois MP, Dubois PM (1983) Gonadotropin releasing hormone (GnRH) stimulates immunoreactive lactotrope differentiation. Endocrinology 112:2224-2226[Abstract]
Blithe D, Richards RG, Skarulis MC (1991) Free alpha molecules from pregnancy stimulate secretion of prolactin from human decidual cells: a novel function for free alpha in pregnancy. Endocrinology 129:2257-2259[Abstract]
Blomquist JF, Baenziger JU (1992) Differential sorting of lutropin and the free--subunit in cultured bovine pituitary cells. J Biol Chem 267:20798-20803
Capella C, Buffa R, Usellini L, Frigerio B, Jehenson P, Sessa F, Solcia E (1983) Alpha and beta subunits of glycoprotein hormones in argyrophil pituitary tumors with small granule cells. Ultrastruct Pathol 4:35-50[Medline]
Chang YS, Huang CJ, Huang FL, Lo TB (1988) Primary structures of carp gonadotropin subunits deduced from cDNA nucleotide sequences. Int J Pept Protein Res 32:556-564[Medline]
Chin WW, Kronenberg HM, Dee PC, Maloof F, Habener JF (1981) Nucleotide sequence of the mRNA encoding the pre-alpha-subunit of mouse thyrotropin. Proc Natl Acad Sci USA 78:5329-5333[Abstract]
Foster DN, Galehouse D, Giordano T, Min B, Lamb IC, Porter DA, Intehar KJ, Bacon WL (1992) Nucleotide sequence of the cDNA encoding the common alpha subunit of the chicken pituitary glycoprotein hormones. J Mol Endocrinol 8:21-27[Abstract]
Fukayama M, Hayashi Y, Koike M (1987) Human chorionic gonad-otropin in the rectosigmoid colon. Immunohistochemical study on unbalanced distribution of subunits. Am J Pathol 127:83-89[Abstract]
Fukayama M, Hayashi Y, Koike M, Hajikano H, Endo S, Okumura H (1986) Human chorionic gonadotropin in lung and lung tumors. Immunohistochemical study on unbalanced distribution of subunits. Lab Invest 55:433-443[Medline]
Godine JE, Chin WW, Habener JF (1982) Alpha subunit of rat pituitary glycoprotein hormones. Primary structure of the precursor determined from the nucleotide sequence of cloned cDNAs. J Biol Chem 257:8368-8371
Greenwood FC, Hunter WM, Glover JS (1963) The preparation of 131I-labeled human growth hormone of high specific radioactivity. Biochem J 89:114-123
Hanaoka Y, Hayashi H, Takahashi H (1984) Isolation and characterization of bullfrog gonadotropins. Gunma Symp Endocrinol 21:63-77
Hayashi H, Hayashi T, Hanaoka Y (1992) Amphibian lutropin and follitropin from the bullfrog Rana catesbeiana. Complete amino acid sequence of the alpha subunit. Eur J Biochem 203:185-191[Abstract]
Heitz PU, Kasper M, Kloppel G, Polak JM, Vaitukaitis JL (1983) Glycoprotein-hormone alpha-chain production by pancreatic endocrine tumors: a specific marker for malignancy. Immunocytochemical analysis of tumors of 155 patients. Cancer 51:277-282[Medline]
Klibanski A, Ridgway EC, Zervas NT (1983) Pure -subunit-secreting pituitary tumors. J Neurosurg 59:585-589[Medline]
Kourides IA, Weintraub BD, Rosen SW, Ridgway EC, Kliman B, Maloof F (1976) Secretion of alpha subunit of glycoprotein hormones by pituitary adenomas. J Clin Endocrinol Metab 43:97-106[Abstract]
Landolt AM, Heitz PU (1986) Alpha-subunit-producing pituitary adenomas. Immunocytochemical and ultrastructural studies. Virchows Arch [A] 409:417-431
Lapthorn AJ, Harris DC, Littlejohn A, Lustbader HW, Canfield RE, Machin KJ, Morgan FJ, Isaacs NW (1994) Crystal structure of human chorionic gonadotropin. Nature 369:455-461[Medline]
Liao TH, Pierce JG (1971) The primary structure of bovine thyrotropin. II. The amino acid sequences of the reduced, S-carboxymethyl alpha and beta chains. J Biol Chem 25:850-865
Liu WK, Nahm HS, Sweeney CM, Lamkin WM, Baker HN, Ward DN (1972) The primary structure of ovine luteinizing hormone. I. The amino acid sequence of the reduced and S-aminoethylated S-subunit (LH-). J Biol Chem 247:4351-4364
Maghuin-Rogister G, Combarnous Y, Hennen G (1973) The primary structure of the porcine luteinizing-hormone alpha-subunit. Eur J Biochem 39:255-263[Medline]
Moy E, Kimzey LM, Nelson LM, Blithe DL (1996) Glycoprotein hormone -subunit functions synergistically with progesterone to stimulate differentiation of cultured human endometrial stromal cells to decidualized cells: a novel role for free
-subunit in reproduction. Endocrinology 137:1332-1339[Abstract]
Oguchi A, Tanaka S, Yamamoto K, Kikuyama S (1996) Release of -subunit of glycoprotein hormones from the bullfrog pituitary: possible effect of
-subunit on prolactin cell function. Gen Comp Endocrinol 102:141-146[Medline]
Osamura RY, Watanabe K (1987) Immunohistochemical colocalization of growth hormone (GH) and subunit in human GH secreting pituitary adenomas. Vichows Arch [A] 411:323-330
Pankov YA, Karasyov VS (1986) Primary structure of sperm whale luteinizing hormone. Int J Pept Protein Res 28:124-129[Medline]
Pierce JG, Parsons TF (1981) Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465-495[Medline]
Rathnam P, Fujiki Y, Landefeld TD, Saxena BB (1978) Isolation and amino acid sequence of the alpha-subunit of follicle-stimulating hormone from equine pituitary glands. J Biol Chem 253:5355-5362[Abstract]
Rathnam P, Saxena BB (1975) Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. Alpha subunit. J Biol Chem 250:6735-6746[Abstract]
Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS, Rosenfeld MG, Swanson LW (1990) Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors. Gene Dev 4:695-711[Abstract]
Tanaka S, Mizutani F, Yamamoto K, Kikuyama S, Kurosumi K (1992) The alpha-subunit of glycoprotein hormones exists in the prolactin secretory granules of bullfrog (Rana catesbeiana) pituitary gland. Cell Tissue Res 267:223-231[Medline]
Tanaka S, Nomizu M, Kurosumi K (1991) Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in the rat pituitary. J Histochem Cytochem 39:809-821[Abstract]
Tanaka S, Park MK, Hayashi H, Hanaoka Y, Wakabayashi K, Kurosumi K (1990) Immunocytochemical localization of the subunit of glycoprotein hormones (LH, FSH and TSH) in the bullfrog pituitary gland using monoclonal antibodies and polyclonal antiserum. Gen Comp Endocrinol 77:88-97[Medline]
Wakabayashi K, Tanaka S (1988) Assessment of specificity of antiserum for immunohistochemistry. Acta Histochem Cytochem 21:221-229
Wahlstrom T, Seppala M (1981) Immunological evidence for the occurrence of luteinizing hormone-releasing factor and the -subunit of glycoprotein hormones in carcinoid tumors. J Clin Endocrinol Metab 53:209-212[Abstract]
Weiss J, Duca KA, Crowley WF (1990) Gonadotropin-releasing hormone-induced stimulation and desensitization of free -subunit secretion mirrors luteinizing hormone and follicle-stimulating hormone in perfused rat pituitary cells. Endocrinology 127:2364-2371[Abstract]