ARTICLE |
Correspondence to: Tommi Paukku, Dept. of Physiology, Univ. of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland..
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Summary |
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Biotinylation of antibodies is an established method for producing systems for detection of antigens. We currently aim to develop liposomal targeting vectors for gene transfer into transgenic gonadal tumor cells expressing the luteinizing hormone (LH) receptor (R). We have biotinylated (B) human chorionic gonadotrophin (hCG) to obtain a selective targeting molecule to be attached to biotinylated liposomes via an avidinstreptavidin bridge. The biotinylation was performed by combining biotin isothiocyanate (BITC) and hCG in alkaline reaction buffer in a 100:1 (BITC:hCG) molar ratio. B-hCG maintained its ability to bind specifically to rat testicular membranes and was also bound to streptavidin-coated polypropylene wells. cAMP production was induced in BLT-1 Leydig tumor cells in vitro after stimulation with B-hCG, as a sign of persistent bioactivity. Frozen sections of rat testicular and ovarian tissues and skeletal muscle were labeled by incubating for 2 hr at 37C with 10 ng/µl B-hCG. The binding was subsequently visualized by the avidinbiotinperoxidase system, followed by silver enhancement of Ni-DAB staining. In rat testicular and ovarian sections, labeling was observed in structures known to strongly express the LH-R, i.e., Leydig cells, corpora lutea, and blood vessels. The labeling was blocked by preincubation with a 100-fold excess of the native hormone, and by injecting the rats SC with a high dose of hCG (1000 IU/kg) 48 hr before sacrifice. Skeletal muscle, used as negative control, was not labeled. These data demonstrate that the bioactivity of hCG is relatively well preserved after biotinylation. The biotinylated gonadotropin offers a new nonradioactive alternative for visualization of bioactive LH receptors in tissue sections. (J Histochem Cytochem 46:993998, 1998)
Key Words: testis, ovary, streptavidin, biotinylated human, chorionic gonadotropin, luteinizing hormone receptor
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Introduction |
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Gonadal tumor cells of Leydig cell origin often bear luteinizing hormone (LH) receptors (
The detection of LH receptors in tissue sections has thus far been achieved using radioactively labeled LH/CG (
To create a targeting molecule for certain gonadal tumors and to achieve the detection of functional LH receptors using nonradioactive labeling, we set out to study the feasibility of using biotinylated hCG for these purposes.
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Materials and Methods |
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Biotinylation of hCG and Bovine Serum Albumin (BSA)
Biotin isothiocyanate (BITC) (Wallac; Turku, Finland) (1.33 mmol/liter) was incubated with 13.3 µmol/liter hCG (CR-121, 11,500 IU/mg; National Institutes of Health, Bethesda, MD) or BSA for 4 hr in 50 mmol/liter sodium carbonate buffer (pH 9.8) at 23C. The endproducts were separated from unbound BITC by gel filtration. The biotinylated product was eluted with TBS buffer (trisaminomethane 50 mmol/liter, sodium chloride 154 mmol/liter, pH 7.75) through PD-10 (Pharmacia; Uppsala, Sweden) and NAP-10 (Pharmacia) (because of increased sample volume after the first elution) Sephadex G25 columns, according to the manufacturer's instructions. The degree of biotinylation of hCG was determined with 2-(4'-hydroxyazobenzene) benzoic acid (HABA) (Pierce; Rockford, IL) -reaction according to the manufacturer's instructions, as earlier described (
[125I]-Iodo-hCG Binding to Rat Testicular Membranes
Both B-hCG and intact hCG, at dilutions of 0.1400 ng in 50 µl Dulbecco's PBS with 0.1% BSA (DPBSBSA), were mixed with 10,000 cpm (100 µl) of [125I]-iodo-hCG in DPBSBSA and 100 µl of rat testicular membrane (one testis per 5 ml DPBSBSA) in a total volume of 250 µl/tube, and incubated in triplicate for 24 hr at 23C as described earlier (-spectrometer. All the procedures using rats were approved by the University of Turku Ethical Committee on Use and Care of Animals.
Binding of B-hCG to Coated Microtitration Plates
A time-resolved immunofluorometric Delfia hCG kit (Wallac) was used to determine the binding of 0.2200 ng B-hCG/microtitration plate to monoclonal hCG antibodies according to the instructions of the manufacturer. B-hCG (0200 ng) was applied to streptavidincoated microtitration plates from the same manufacturer to detect the binding of the hormone derivative to both streptavidin and the same europium-labeled second monoclonal antibody as used in the Delfia hCG kit.
Stimulation of cAMP Production by B-hCG
One day before stimulation, the BLT-1 Leydig tumor cells (
Detection of LH Receptors in Rat Tissue Sections by B-hCG
Ten-µm frozen tissue sections of rat testis, ovary, and skeletal muscle were washed and incubated with 10 mg/liter B-hCG in DPBS for 2 hr at 37C. Then the slides were washed and incubated for 30 min at 23C with the ABC reagent of the Vectastain ABC kit according to the instructions of the manufacturer (Vector Labs; Burlingame, CA). After washing, the bound peroxidase enzyme was visualized by a nickeldiaminobenzidine solution (nickel chloride 70 mg/liter, DAB 200 mg/liter, hydrogen peroxide 0.002% in TBS, pH 7.5, 2 min at 23C), followed by a silver enhancement method (silver nitrate 1 g/liter, ascorbic acid 200 mg/liter, sodium acetate 80 g/liter, cetylpyridinium chloride 100 mg/liter, sodium tungstate 5 g/liter, acetic acid 0.56%, and Triton X-100 0.06%, mixed immediately before use, 17 min at 23C) as described previously (
Immunohistochemistry of LH Receptor
Frozen sections of rat ovary semiparallel to those labeled with B-hCG (see above) were Bouin-fixed and preincubated with 4% normal goat serum in TBS for 30 min at 23C. The antiserum (kindly provided by Dr. P. Roche, Mayo Clinic, Rochester, MN), containing polyclonal peptide antibodies raised in a rabbit against the N-terminal peptide sequence 1538 of the rat LH/CG receptor, was applied to the rat ovarian sections at a dilution of 1:500 in 1% BSATBS and incubated for 16 hr at 4C. After washing, biotinylated anti-rabbit IgG antibodies (1:250 dilution of Vectastain ABC kit secondary antibody solution in 1% BSATBS) were placed on sections for 30 min at 23C. After addition of the secondary antibody the procedure was continued as described for labeling with B-hCG (see above).
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Results |
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HABA chemical reaction showed an average biotinylation degree of 3.5 ± 0.2 (SD) biotin molecules per hCG molecule for the biotinylated product. B-hCG was able to compete in parallel fashion with [125I]-iodo-hCG for specific binding to rat testicular membranes (Figure 1). The amount needed for 50% displacement was approximately sixfold higher than that of native hCG.
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After the biotinylation of hCG, the Delfia hCG kit test showed qualitatively the existence of a compound recognizable by the monoclonal anti-hCG antibodies of the kit (data not shown). Similarly, application of B-hCG to the streptavidin-coated microtitration wells indicated binding to streptavidin and thereafter to a monoclonal antibody raised against hCG (data not shown).
cAMP production by BLT-1 cells was induced with B-hCG. The concentration needed for stimulation of a similar level was approximately 10-fold compared to native hCG (Figure 2), which is in good agreement with the binding inhibition data shown in Figure 1.
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Incubation of B-hCG on rat testicular sections showed almost exclusive binding into the interstitial compartment of the tissue (Figure 3A). In ovarian sections, the luteal, thecal, and interstitial cells were labeled, whereas the immature granulosa cells showed no staining (Figure 3B). The skeletal muscle was not labeled by B-hCG (Figure 3C). The control gonadal sections incubated with native hCG or B-BSA instead of B-hCG, or preincubated with excess native hCG (Figure 3E), or sections of the gonads of rats pretreated with high doses of hCG, showed no B-hCG binding (data not shown). In the testis, the low-level punctate intratubular binding was not totally blocked by preincubation with native hCG before B-hCG. The muscular wall of the gonadal blood vessels showed clear labeling with B-hCG (Figure 4).
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Semiserial sections of the rat ovaries showed a similar pattern of labeling when incubated with either the rat LHR antibody or B-hCG. The internal parts of the corpora lutea showed more intense labeling by immunohistochemistry (Figure 5).
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Discussion |
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These results demonstrate that the biotinylated hCG binds to LHR in the binding assay and in rat gonadal tissue sections and that it induces cAMP production in vitro. On the sections, the visualization of LHR was performed by using the avidinbiotinperoxidase system for labeling. The monoclonal antibodies used in Delfia microtitration well kits showed affinity with the biotinylated derivative. At the receptor site, the B-hCG had affinity that was about 10-fold lower than that of native hCG, which is still acceptable for most of the applications, being on the same order as that of native human LH (
The cAMP production and binding affinity of B-hCG were similarly approximately 10-fold lower compared to native hCG. Therefore, it appears that biotinylation has little effect on the regions of hCG important for receptor binding and signal transduction in the cAMP-dependent pathway.
Amplification of the detected signal by using multiple biotin binding sites of avidin gives the assays surpassing sensitivity. The systems employing biotinylated derivatives are relatively fast and easy to perform (
LHR expression has been reported in interstitial cells in the rat testis (
In an attempt to improve the current method, biotinylation of the common -subunit of the glycoprotein hormone family (TSH, LH, FSH, and CG) would leave the selectivity-determining ß-subunit intact. After coupling the
- and ß-subunits, this method could yield an easily detectable hormone with highly restored binding affinity.
Selective gene transfer into cells expressing a specific receptor has been reported (
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Acknowledgments |
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Supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, and the Finnish Cancer Fund. TP was supported by the Turku University Graduate School for Medical Sciences.
We thank Dr Kati Hakola, Dr Antti Rannikko, and Ms Aila Metsävuori for technical advice.
Received for publication January 27, 1998; accepted April 27, 1998.
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Literature Cited |
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Ascoli M (1981) Characterization of several clonal lines of cultured Leydig tumor cells: gonadotropin receptors and steroidogenic responses. Endocrinology 108:88-95[Abstract]
Ascoli M (1982) Internalization and degradation of receptor-bound human choriogonadotropin in Leydig tumor cells. Fate of the hormone subunits. J Biol Chem 257:13306-13311
Ascoli M, Puett D (1978) Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells. Proc Natl Acad Sci USA 75:99-102[Abstract]
Bukovsky A, Chen TT, Wimalasena J, Caudle MR (1993) Cellular localization of luteinizing hormone receptor immunoreactivity in the ovaries of immature, gonadotropin-primed and normal cycling rats. Biol Reprod 48:1367-1382[Abstract]
Cameron JL, Stouffer RL (1982) Gonadotropin receptors of the primate corpus luteum. I. Characterization of 125I-labeled(human luteinizing hormone and human chorionic gonadotropin binding to luteal membranes from the rhesus monkey. Endocrinology 110):2059-2067
Catt KJ, Ketelslegers J-M, Dufau ML (1976) Receptors for gonadotropic hormones. In Blecher M, ed. Methods in Receptor Research. New York, Marcel Dekker, 176-250
Childs GV, Unabia G (1989) Activation of protein kinase C and L calcium channels enhances binding of biotinylated corticotropin-releasing hormone by anterior pituitary corticotropes. Mol Endocrinol 3:117-126[Abstract]
Childs GV, Yamauchi K, Unabia G (1989) Localization and quantification of hormones, ligands, and mRNA with affinity-gold probes. Am J Anat 185:223-235[Medline]
Cotten M, LangleRouault F, Kirlappos H, Wagner E, Mechtler K, Zenke M, Beug H, Birnstiel ML (1990) Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels. Proc Natl Acad Sci USA 87:4033-4037[Abstract]
Ghinea N, Hai MTV, GroyerPicard M-T, Milgrom E (1994) How protein hormones reach their target cells. Receptor-mediated transcytosis of hCG through endothelial cells. J Cell Biol 125:87-97[Abstract]
Green NM (1965) A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem J 94:23c-24c
Habberfield AD, Dix CJ, Cooke BA (1986) Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells. Biochem J 233:369-376[Medline]
Harper JF, Brooker G (1975) Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'-O-acetylation by acetic anhydride in aqueous solution. J Cyclic Nucleotide Res 1:207-218[Medline]
Huhtaniemi IT, Catt KJ (1981) Differential binding affinities of rat testis luteinizing hormone (LH) receptors for human chorionic gonadotropin, human LH, and ovine LH. Endocrinology 108:1931-1938[Medline]
Jeltsch A, Fritz A, Alves J, Wolfes H, Pingoud A (1993) A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases. Anal Biochem 213:234-240[Medline]
Kananen K, Markkula M, El-Hefnawy T, Zhang F-P, Paukku T, Su J-GJ, Hsueh AJW, Huhtaniemi I (1996) The mouse inhibin -subunit promoter directs SV 40 T-antigen to Leydig cells in transgenic mice. Mol Cell Endocrinol 119:135-146[Medline]
Kananen K, Markkula M, Rainio E, Su J-GJ, Hsueh AJW, Huhtaniemi IT (1995) Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin -subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines. Mol Endocrinol 9:616-627[Abstract]
LaPolt P, Jia X, Sincich C, Hsueh A (1991) Ligand-induced down-regulation of testicular and ovarian luteinizing hormone (LH) receptor is preceded by tissue-specific inhibition of alternatively processed LH receptor transcripts. Mol Endocrinol 5:397-403[Abstract]
Lloyd CE, Ascoli M (1983) On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells. J Cell Biol 96:521-526[Abstract]
Loosfelt H, Misrahi M, Atger M, Salesse R, Thi MTVH-L, Jolivet A, GuiochonMantel A, Sar S, Jallal B, Garnier J, Milgrom E (1989) Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science 245:525-528[Medline]
Macielag MJ, Peeters T, Depoortere I (1994) Synthesis and characterization of site-specific biotinylated probes for the motilin receptor. Int J Pept Prot Res 44:582-588
Marcil J, Ravindranath N, Sairam MR (1993) Cytotoxic activity of lutropin-gelonin conjugate in mouse Leydig tumor cells: potentiation of the hormonotoxin activity by different drugs. Mol Cell Endocrinol 92:83-90[Medline]
Merchentaler I, Stancovics J, Gallyas F (1989) A highly sensitive one-step method for silver intensification of the nickelDAB endproduct of the peroxidase reaction. J Histochem Cytochem 37:1563-1565[Abstract]
Michel E, Parsons JA (1988) Characterization of a biotin-conjugated ovine prolactin ligand. Mol Cell Endocrinol 56:71-79[Medline]
Midgley ARJ (1972) Gonadotropin binding to frozen sections of ovarian tissue. In Saxena BB, Beling CG, Gandy HM, eds. Gonadotropins. New York, Wiley, 248-260
Midgley ARJ (1973) Autoradiographic analysis of gonadotropin binding to rat ovarian tissue sections. Adv Exp Med Biol 36:365-378[Medline]
Mullink H, Vos W, Jiwa M, Horstman A, Vander Valk P, Walboomers JMM, Meijer CJLM (1992) Application and comparison of silver intensification methods for the diaminobenzidine and diaminobenzidinenickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization. J Histochem Cytochem 40:495-504
Newman W, Beall LD, Levine MA, Cone JL, Randhawa ZI, Bertolini DR (1989) Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry. J Biol Chem 264:16359-16365
Rajaniemi H, VanhaPerttula T (1972) Specific receptor for LH in the ovary: evidence by autoradiography and tissue fractionation. Endocrinology 90:1-9[Medline]
Rebois RV (1982) Establishment of gonadotropin-responsive murine leydig tumor cell line. J Cell Biol 94:70-76[Abstract]
Richards JS (1980) Maturation of ovarian follicles: actions and interactions of pituitary and ovarian hormones on follicular cell differentiation. Physiol Rev 60:51-89
Segaloff DL, Wang HY, Richards JS (1990) Hormonal regulation of luteinizing hormone/chorionic gonadotropin receptor mRNA in rat ovarian cells during follicular development and luteinization. Mol Endocrinol 4:1856-1865[Abstract]
Singh V, Sairam MR (1989) Hormonotoxins: conjugation of human choriogonadotropin with the ribosome inactivating protein gelonin and comparison with a lutropin conjugate. Mol Cell Endocrinol 67:217-229[Medline]
Sokka T, Hämäläinen T, Huhtaniemi I (1992) Functional LH receptor appears in the neonatal rat ovary after changes in the alternative splicing pattern of the LH receptor mRNA. Endocrinology 130:1738-1740[Abstract]
Visintin I, Luborsky JL (1989) Comparison of monoclonal luteinizing hormone (LH) receptor antibody and LH binding sites in vibratome sections of rat ovary by immunohistochemistry. J Histochem Cytochem 37:1711-1719[Abstract]
VuHai MT, Jolivet A, Jallal B, Salesse R, Bidart JM, Houillier A, GuiochonMantel A, Garnier J, Milgrom E (1990) Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor. Endocrinology 127:2090-2098[Abstract]
Wagner E, Cotten M, Mechtler K, Kirlappos H, Birnstiel ML (1991) DNA-binding transferrin conjugates as functional gene delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety. Bioconjug Chem 2:226-231[Medline]
Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML (1990) Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc Natl Acad Sci USA 87:3410-3414[Abstract]
Wang HY, Ascoli M, Segaloff D (1990) Multiple luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid transcripts. Endocrinology 129:133-138[Abstract]
Wu CH, Wilson JM, Wu GY (1989) Targeting genes: delivery and persistent expression of a foreign gene driven by mammalian regulatory elements in vivo. J Biol Chem 266:16985-16987
Wu GY, Wu CH (1987) Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem 262:4429-4432
Zecchini V, Caucheteux D, Gretry J, Lemaitre M, Dommes J (1995) Detection of nucleic acids in the attomole range using polybiotinylated oligonucleotide probes. Biotechniques 19:286-290[Medline]