Fish Physiology and Biochemistry vol. 15 no. 6 pp 459-467 (1996} Kugler Publications, Amsterdam/New York Specific binding of 11-ketotestosterone in an androgen target organ, the kidney of the male three-spined stickleback, Gasterosteus aculeatus Staffan Jakobsson ~, Ian Mayer I, Rfidiger W. Schulz 2, Marinus A. Blankenstein -~and Bertil Borg ~ 1Department of Zoology, UniversiO~ of Stockholm, S- 106 91 Stockholm, Sweden: "Department of Experimen- tal Zoology, Universi O, of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands: ~Department of Endocrinology, Academic Hospital Utrecht, 3584 CX Utrecht, The Netherlands Accepted: March 29, 1996 Keywords: three-spined stickleback, secondary sexual character, kidney hypertrophy, I l-ketotestosterone, testosterone, specific binding, displacement Abstract The kidney of male three-spined stickleback, Gasterosteus aculeatus, hypertrophies during tile breeding season and produces a "glue" which is used in the building of the nest. This hypertrophy is androgen depend- ent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this sec- ondary sexual character. In the present study kidneys were excised from stickleback males that had been castrated two days earlier. The purpose of this gonadectomy was to reduce the endogenous levels of andro- gens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 1 I KT with and without unlabelled steroids at increasing concentrations. Displaceable specific 11KT binding was found in kidney tissue fragments whereas only non-specific binding was observed when liver and muscle were investigated in a similar way. Unlabelled 11KT displaced specifically bound, tritiated 11KT with an ED50-value (50% of displaceable binding) of 28 nM. Similar ED50 values were found for 17[3-hydroxy-5c~- androstane-3,11-dione (29 nM) and 5c~-dihydrotestosterone (20 nM), whereas higher ED50 concentrations were estimated for testosterone (T; 203 nM) and progesterone (69 nM). No displacement of tritiated 11KT was found for the other investigated substances tested; estradiol, 17a,20[3-dihydroxy-4-pregnen-3-one, flutamide or cyproterone acetate. No specific binding to kidney tissue fragments could be detected when labelled T was used instead of labelled 11KT. Specific binding of 11KT or T was not found either in the kidney cytosol or nuclear extracts. However, using the kidney membrane fraction a displacement oftritiated 11KT with unlabelled 1 I KT (10 r M) was observed. In conclusion there is a specific binding of 11KT in the stickleback kidney. The absence of binding in liver and muscle, the ED50 value observed and the displace- ment with some, but not all steroids are consistent with a receptor function. The presence of binding in membrane fractions, but not in cytosol or nuclear extracts suggests that the binding is not related to classic steroid receptors. Introduction In mammals, the major androgens are testosterone (T) and its 5or-reduced derivative, 5o.-dihydro- testosterone (5o~-DHT). In teleost fishes, however, l l-ketotestosterone (IIKT) is generally consid- ered to be the major androgen in males and is highly effective in stimulating secondary sexual characters (for reviews, see Fostier et al. 1982; Borg 1994). Male three-spined sticklebacks, Gas- terosteus aculeatus, display a number of pro- nounced secondary sexual characteristic, amongst which is the hypertrophy of the kidney. This hyper- trophy occurs during the breeding season and re- sults in the production by the kidney tubules of a "glue" that is used in the building of the nest. This male secondary sexual character is androgen-de- pendent, both in vitro (de Ruiter and Mein 1982)