Release of Free and Conjugated Forms of the Putative Pheromonal Steroid 11-Oxo-etiocholanolone by Reproductively Mature Male Round Goby (Neogobius melanostomus Pallas, 1814)

Abstract

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.

Olfactory sensitivity to steroid glucuronates in Mozambique tilapia suggests two distinct and specific receptors for pheromone detection

“…A similar observation was made of African catfish (Clarias gariepinus) males, where the most potent testicular odorant was found to be 20one-P-3-G (Lambert and Resink, 1991). Androstanes and pregnanes with a 5β,3α configuration are also potent odorants for the round goby (Neogobius melanostomus, Pallas 1814) (Murphy et al, 2001), and recent studies have demonstrated that round goby males release several conjugated forms of these steroids via their urine (Katare et al, 2011), eventually to attract females (Tierney et al, 2013). However, in the round goby, the olfactory receptors detecting ETIO-3-G appear to be less specific than those in tilapia; several unconjugated androstanes, pregnanes and even androsten, are detected by the same (ETIO-3-G) receptor site (Murphy et al, 2001).…”

The Effect of Elevated Steroids Released by Reproductive Male Round Gobies, Neogobius melanostomus, on Olfactory Responses in Females

“…The procedure for generating methanol extracts of water that held reproductive males (RM) has been described elsewhere (Katare et al, 2011). Each RM (chosen on the basis of secondary sexual characters; Belanger et al 2004) was isolated in 1 l of dechlorinated water, which was collected after 4 h ("pre-injection").…”

“…Individual stocks were diluted 100× further in dechlorinated water for EOG testing; thus, tested concentrations of extracted compounds were all below the original concentrations produced by the RMs. We used a custom immunoassay to measure the amount of released immunoreactive unconjugated and conjugated 11-O-ETIO in the methanol extracts (Katare et al, 2011). The release rate of immunoreactive 11-O-ETIO was 2.13±0.64 ng/ml/h in methanol extracts prepared prior to injection.…”

“…The olfactory system of the round goby, Neogobius melanostomus Pallas, responds to steroidal compounds (Murphy et al, 2001), and reproductive females show increased olfactory and behavioral responses to water from reproductive males vs. non-reproductive males (Belanger et al, 2004;Gammon et al, 2005). Katare et al (2011) observed that following injection with salmon GnRH analog (sGnRHa), male N. melanostomus increase the release of several steroids into the water, including 11-oxo-etiocholanolone (11-O-ETIO) and 11-O-ETIO-3-sulfate (11-O-ETIO-3-s), which are novel fish steroids and putative pheromones in this species. We used electro-olfactogram (EOG) recordings of female N. melanostomus to determine how a surge of sGnRHa would affect the olfactory signal released by males.…”

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment

“…Furthermore, steroids were identified as pheromones and adversely affect fish behavior even at extremely low concentrations (Kolodziej et al, 2003). For example, androgens at picomolar concentrations elicit both odorant and pheromonal responses in fish (Adams et al, 1987;Moore and Scott, 1991;Serrano et al, 2008;Katare et al, 2011).…”