Abstract
Levels of gonadotropic hormones in human sera or urine are routinely measured by radioimmunoassay or by enzyme-linked immunoassay (ELISA), which determine the immunoactivity of the hormone, but not its biological activity. We have utilized immortalized stable steroidogenic granulosa cells, which express 5–10 times more of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptors than the primary cells, to develop a biological assay and radioreceptor assay for this hormone. We found that stimulation of cells expressing LH/CG receptor with increasing doses of human LH or human CG resulted in a dose-dependent increase of cAMP and progesterone with an ED50 of 30 and 57 mlU/mL, respectively. These dose-response data served as calibration curves for measuring the gonadotropin bioactivity in human serum samples at concentrations as low as 1–5 mlU/mL. We found a close correlation between LH levels measured by enzyme immunoassay (EIA) and the in vitro bioassay in normal cycling and menopausal women, as well as in normal adult men. Also, a close correlation was found between the EIA and the in vitro biological assay of hCG in pregnant women. In addition, we have developed a radioreceptor assay (RRA) for this hormone using enriched cell membranes of the appropriate cell line, which corresponds well to both the EIA and the bioassay in human sera. Deglycosylated hCG was fully active in RRA, but failed to activated cAMP response in these cells, demonstrating the importance of the bioassay in the biologically inactive form of gonadotropins. We believe this novel in vitro bioassay of gonadotropic hormones will serve as a useful tool for a more comprehensive set of assays that will determine not only the amount, but also the possible modulation in bioactivity of the gonadotropin associated with gonadal failure and miscarriage.
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Aharoni, D., Dantes, A., Oren, M., and Amsterdam, A. (1995).Exp. Cell. Res. 218, 271–282.
Amsterdam, A., Nimrod, A., Lamprecht, S. A., Burstein, Y., and Lindner, H. R. (1979).Am. J. Physiol. 5, E129-E138.
Amsterdam, A., Zauberman, A., Meir, G., Pinhasi-Kimhi, O., Suh, B. S., and Oren, M. (1988).Proc. Natl. Acad. Sci. USA 85, 7582–7586.
Apter, D., Cacciatore, B., Alfthan, H., and Stenman, U. H. (1989).J. Clin. Endocrinol. Metab. 68, 53–57.
Bajpai, P. K., Dash, R. J., Midgley, A. R. J., and Reichert, L. E. J. (1974).J. Clin. Endocrinol. Metab. 38, 721–724.
Bradford, M. M. (1976).Anal. Biochem. 72, 248–254.
Conn, P. M., Conti, M., Harwood, J. P., Dufau, M. L., and Catt, K. J. (1978).Nature 274, 598–600.
Cook, A. S., Webster, B. W., Terranova, P. F., and Keel, B. A. (1988).Fertil. Steril. 49, 704–712.
Costagliola, S., Niccoli, P., and Carayon, P. (1994).J. Endocrinol. Invest. 17, 291–299.
Courte, C. and Willemot, J. (1972).J. Biochem. 247, 4429–4434.
Ding, Y. Q., Ranta, T., Nikkanen, V., and Huhtaniemi, I. (1991).J. Endocrinol. 128, 131–137.
Dufau, M. L. and Veldhuis, J. D. (1987).J. Clin. Endocrinol. Metab. 64, 153–176.
Erlanger, B. F., Borek, F., Beiser, S. M., and Lieberman, S. (1959).J. Biol. Chem. 234, 1090–1094.
Fabbri, A., Jannin, E. A., Ulisse, S., Gnessi, L., Moretti, C., Frajese, G., and Isidori, A. (1988).J. Clin. Endocrinol. Metab. 67, 867–875.
Fraker, P. J. and Speck, J. C. (1978).Biochem. Biophys. Res. Commun. 80, 849–855.
Gilman, A. G. (1970).Proc. Natl. Acad. Sci. USA 67, 305–309.
Green, E. D. and Baenziger, J. U. (1988).J. Biol. Chem. 263, 36–44.
Green, E. D., Boime, I., and Baenziger, J. U. (1986).Mol. Cell. Biochem. 72, 81–100.
Haavisto, A. M., Dunkel, L., Pettersson, K., and Huhtaniemi, I. (1990).Pediatr. Res. 27, 211–214.
Harling, J., Khan, S. A., and Diczfalusy, E. (1986).Fertil. Steril. 46, 1055–1061.
Imse, V., Holzapfel, G., Hinney, B., Kuhn, W., and Wuttke, W. (1992).J. Clin. Endocrinol. Metab. 74, 1053–1061.
Jaakkola, T., Ding, Y. Q., Kellokumpu-Lehtinen, P., Valavaara, R., Tapanainen, J., Martikainen, J., Rönnberg, L., and Huhtaniemi, I. (1990).J. Clin. Endocrinol. Metab. 70, 1496–1505.
Jia, X. C., Perlas, E., Su, J. G. J., Moran, F., Lasley, B. L., Ny, T., and Hsueh, A. J. W. (1993).Biol. Reprod. 49, 1310–1316.
Kalyan, N. K. and Bahl, O. P. (1985).J. Biol. Chem. 258, 67–74.
Keel, B. A. and Grotjean, E. H. (1984).Anal. Biochem. 142, 267–270.
Keren-Tal, I., Dantes, A., Sprengel, R., and Amsterdam, A. (1993).Mol. Cell. Endocrinol. 95, R1-R10.
Keutmann, H. T., McIlroy, P. J., Bergert, E. R., and Ryan, R. J. (1983).Biochemistry 22, 3067–3072.
Kohen, F., Bauminger, S., and Lindner, H. R. (1975). In:Steroid Immunoassay. Cameron, E. H. D., Hillier, S. G., and Griffiths, K. (eds.). Alpha Omego: Cardiff, Wales, pp. 11–23.
Merz, W. E. (1988).Biochem. Biophys. Res. Commun. 156, 1271–1278.
Moyle, W. R., Bahl, O. P., and Marz, L. (1975).J. Biol. Chem. 250, 9163–9169.
Pierce, J. G. and Parsons, T. F. (1981).Ann. Rev. Biochem. 50, 465–495.
Reader, S. C. J., Robertson, W. R., and Disczfalusy, E. (1983).Clin. Endocrinol. 19, 355–363.
Rodgers, M., McLoughlin, J. D., Lambert, A., Robertson, W. R., and Mitchell, R. (1995).Hum. Reprod. 10, 1982–1986.
Sairam, M. R. (1989).FASEB J. 3, 1915–1926.
Sairam, M. R. and Bhargavi, G. N. (1985).Science 229, 65–67.
Salomon, Y. (1991).Methods Enzymol. 195, 22–28.
Schwarz, S., Krude, H., Klieber, R., Dirnhofer, S., Lottersberger, C., Merz, W. E., Wick, G., and Berger, P. (1991).Mol. Cell. Endocrinol. 80, 33–40.
Selvaraj, N. and Moudgal, N. R. (1993).J. Reprod. Fertil. 98, 611–616.
Seron-Ferre, M., Vergara, M., Garcia-Huidobro, V., Huhtaniemi, I., and Diaz, S. (1995).Hum. Reprod. 10, 2849–2853.
Soules, M. R., McLachlan, R. I., Ek, M., Dahl, K. D., Cohen, N. L., and Bremner, W. J. (1989).J. Clin. Endocrinol. Metab. 69, 804–812.
Strollo, F., Harlin, J., Hernandez-Montes, H., Robertson, D. M., Zaidi, A. A., and Diczfalusy, E. (1981).Acta Endocrinol. (Copenh.) 97, 166–175.
Suginami, H. Y., Yano, M., Hamada, K., Ito, T., Yano, K., and Matsuura, S. (1985).Endocrinol. Jpn. 32, 583–587.
Suh, B. S., Sprengel, R., Keren-Tal, I., Himmelhoch, S., and Amsterdam, A. (1992).J. Cell Biol. 119, 439–450.
Van Damme, M. P., Robertson, D. M., and Dicfalusy, E. (1974).Acta Endocrinol. (Copenh.) 77, 655–671.
Weiss, J., Axelrod, L., Whitcomb, R. W., Harris, P. E., Crowley, W. F., and Jameson, J. L. (1992).N. Engl. J. Med. 326, 179–183.
Wennink, J. M. B., Delemarre-van de Waal, A., van Kessel, H., Mulder, G. H., Foster, J. P., and Schowmaker, J. (1988).J. Clin. Endocrinol. Metab. 67, 924–928.
Wide, L. (1982).J. Clin. Endocrinol. Metab. 55, 682–686.
Wide, L. (1985).Acta Endocrinol. (Copenh.) 109, 181–187.
Wilson, C. A., Leigh, A. J., and Chapmann, A. J. (1989).J. Clin. Endocrinol. Metab. 125, 3–14.
Zohar, M. and Salomon, Y. (1992).Brain Res. 576, 49–58.
Zor, U., Shentzer, P., Azrad, A., Sairam, M. R., and Amsterdam, A. (1984).Endocrinology 114, 2143–2149.
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Selvaraj, N., Dantes, A., Limor, R. et al. Establishment of an in vitro bioassay and radio receptor assay for LH/CG in human sera using immortalized granulosa cells transfected with LH/CG receptor. Endocr 5, 275–283 (1996). https://doi.org/10.1007/BF02739060
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DOI: https://doi.org/10.1007/BF02739060