One of the most definitive examples of a vertebrate extraorganismal structural protein can be found in three-spined sticklebacks (Gasterosteus aculeatus). In the breeding male the kidney hypertrophies and synthesizes an adhesive protein called "spiggin," which is secreted into the urinary bladder from where it is employed as a structural thread for nest building. This paper describes the first molecular characterization of spiggin and demonstrates that this adhesive is a protein complex assembled from a potential of three distinct subunits (␣, ␤, and ␥). These subunits arise by alternative splicing, and 11-ketoandrogens induce their expression in stickleback kidneys. Analysis of the predicted amino acid sequence of each subunit reveals a modular organization whose structural elements display a similarity to the multimerization domains found within von Willebrand Factor-related proteins. These results implicate that spiggin utilizes a conserved multimerization mechanism for the formation of a viscous agglutinate from its constituent subunits in the urinary bladders of male sticklebacks. This novel extraorganismal structural protein is therefore ideally suited to its function as an adhesive thread.Extrorganismal proteins are common among invertebrates. Examples range from the fibroin threads forming spider webs and silkworm cocoons (1, 2) to the collagen-based matrixes of the byssus threads of Mytilus and the Drosophila sgs family (3, 4).

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The kidney of male three-spined stickleback,Gasterosteus aculeatus, hypertrophies during the breeding season and produces a "glue" which is used in the building of the nest. This hypertrophy is androgen dependent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this secondary 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 androgens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 11KT 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\-hydroxy-5α-androstane-3,11-dione (29 nM) and 5α-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, 17α,20β-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 of tritiated 11KT with unlabelled 11KT (10(-6)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 displacement 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.

Mature Atlantic salmon (Salmo salar) male parr were implanted in June and August with Silastic capsules filled with the progestin 17α,20β‐dihydroxy‐4‐pregnene‐3‐one (17,20β‐P) or with empty capsules. Experiments were terminated in September, when natural androgen levels are at their highest. Radioimmunoassay (RIA) showed that treatment raised plasma levels of 17,20β‐P, whereas the content of gonadotropic hormones (GTH I and II) in the pituitary and plasma levels of the androgens testosterone (T) and 11‐ketotestosterone (11‐KT) were not influenced. Testicular fragments from mature salmon parr were incubated with different levels of 17,20β‐P in combination with T or 17α‐hydroxy‐4‐pregnene‐3‐one as precursors. A very high level of 17,20β‐P (3,000 ng/ml) suppressed 11‐KT production, whereas 3, 30, or 300 ng/ml were without effect. Thus, in these experiments physiological levels of 17,20β‐P did not suppress androgen 11‐KT production or alter pituitary gonadotropin levels, suggesting that some other mechanism is responsible for the natural decline in androgen levels when 17,20β‐P levels rise at spawning. J. Exp. Zool. 277:66–71, 1997. © 1997 Wiley‐Liss, Inc.

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