Detection of Environmental Androgens: A Novel Method Based on Enzyme-Linked Immunosorbent Assay of Spiggin, the Stickleback (Gasterosteus Aculeatus) Glue Protein

Abstract

We report the development and validation of a novel in vivo biomarker test for waterborne androgens. During breeding, male sticklebacks (Gasterosteus aculeatus) manufacture a glue protein, spiggin, in their kidneys that they use to build their nests. Spiggin production is under the control of androgens. Until now, however, it has only been possible to quantify its production by measurement of the height of kidney epithelial cells. In the present study, we report the development of an enzyme-linked immunosorbent assay (ELISA) for spiggin and demonstrate its application to the measurement of spiggin in the kidneys of female sticklebacks that have been exposed to androgens in water. Results from the ELISA procedure revealed a strong correlation with measurement of kidney epithelial cell height (r2 = 0.93). However, the ELISA was much quicker and had a considerably higher response range (100,000-fold vs fourfold). Clear, graded responses in spiggin production were obtained by exposing intact females to increasing concentrations of 17a-methyltestosterone and 5alpha-dihydrotestosterone over three-week test periods. The lowest effective concentrations for these two steroids were 100 ng/L and 3 micorg/L, respectively. Female sticklebacks that were exposed to pulp mill effluent also produced spiggin in their kidneys. Possession of an androgen-regulated protein by the female stickleback makes it a unique bioassay organism for detecting androgenic contamination in the aquatic environment.

Perchlorate induces hermaphroditism in threespine sticklebacks

“…The threespine stickleback is uniquely suited for research concerning the disruption of sex steroids, because it has both male-and female-specific biomarkers that can be easily and rapidly measured by enzyme-linked immunosorbent assays [17,31]. Reproductive males produce a glue protein called spiggin from the kidneys, which is used to glue together nesting material, and the reproductive female produces the eggyolk protein vitellogenin.…”

“…Reproductive males produce a glue protein called spiggin from the kidneys, which is used to glue together nesting material, and the reproductive female produces the eggyolk protein vitellogenin. Exposure to androgenic compounds can cause the female kidney to produce spiggin, whereas estrogenic compounds can cause the male to produce vitellogenin [17].…”

“…However, hermaphroditism among treated females and oversized testes among treated males suggest that perchlorate has an androgenic endocrinedisrupting role in addition to its known thyroid-disrupting properties. With the knowledge that perchlorate is capable of inducing hermaphroditism, a sensitive assay, such as that described by Katsiadaki et al [17], could be combined with structural studies of reproductive tissues and genetic testing to explore perchlorate's mode of action. Studies should ex-amine whether the masculinization response is mediated through direct activation of androgen receptors or by indirect means, such as alteration of steroid metabolism.…”

“…Endocrine-disrupting chemicals have been used to produce intersex fishes in the laboratory [16,17] and by aquaculturists [18]. However, relatively few studies have described masculinized females [19].…”

Surveys of plasma vitellogenin and intersex in male flounder (Platichthys flesus) as measures of endocrine disruption by estrogenic contamination in United Kingdom estuaries: Temporal trends, 1996 to 2001

“…After this, plasma VTG has been measured by ELISA. The equipment, plate-coating buffer, plate-washing buffer, assay buffer, anti-rabbit IgG alkaline phosphatase conjugate, revelation solution, and pipetting procedures were the same as those described for ELISA measurement of spiggin [15]. A new batch of standard, prepared exactly as described by Scott and Hylland [14], was introduced in 1999 and another in 2001.…”